AMG510

European Journal of Medicinal Chemistry 226 (2021) 113816

Targeting mutated GTPase KRAS in tumor therapies
Guangjin Fan 1, Linlin Lou 1, Zhendong Song**, Xiaolei Zhang***, Xiao-Feng Xiong*
Guangdong Key Laboratory of New Drug Design and Evaluation, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, China

a r t i c l e i n f o

Article history:
Received 8 July 2021 Received in revised form 24 August 2021
Accepted 29 August 2021
Available online 4 September 2021

Keywords: KRAS
GTPase
Covalent inhibitors Tumor therapies

a b s t r a c t

Kirsten rat sarcoma virus oncogene (KRAS) mutation accounts for approximately 85% of RAS-driven cancers, and participates in multiple signaling pathways and mediates cell proliferation, differentiation and metabolism. KRAS has been considered as an “undruggable” target due to the lack of effective direct inhibitors, although high frequency of KRAS mutations have been identified in multiple carcinomas in the past decades. Encouragingly, the KRASG12C inhibitor AMG510 (sotorasib), which has been approved for treating NSCLC and CRC recently, makes directly targeting KRAS the most promising strategy for cancer therapy. To better understand the current state of KRAS inhibitors, this review summarizes the biological functions of KRAS, the structure-activity relationship studies of the small-molecule inhibitors that directly target KRAS, and highlights the therapeutic agents with improved selectivity, bioavailability and physicochemical properties. Furthermore, the combined medication that can enhance efficacy and overcome drug resistance of KRAS covalent inhibitors is also reviewed.
© 2021 Elsevier Masson SAS. All rights reserved.

Contents
1. Introduction 2
2. The structure and function of KRAS protein 2
3. KRAS downstream signaling pathways 2
3.1. RAF-MEK-ERK signaling pathway 2
3.2. PI3K-AKT-mTOR signaling pathway 2
3.3. RalGDS-Ral signaling pathway 3
3.4. Tiam1-RAC1 signaling pathway 4
3.5. PLCε/PKC signaling pathway 4
4. KRAS mutations 4
5. Roles of KRAS in cancers 4
5.1. Lung cancer 5
5.2. Pancreatic cancer 5
5.3. Colorectal cancer 5
5.4. KRAS in other cancers 5
5.5. Tumor immune microenvironment (TME) 6
6. Overview of small molecular KRAS inhibitors 7
6.1. Direct and covalent inhibitors of KRASG12C 7
6.1.1. AMG510 (sotorasib) 7
6.1.2. MRTX849 (adagrasib) 8
6.1.3. ARS3248 9
6.2. Direct and covalent KRASG12C/D inhibitors in pre-clinical trial 9

* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: [email protected] (Z. Song), [email protected] (X. Zhang), [email protected] (X.-F. Xiong).
1 These authors equally contributed to the present work.

https://doi.org/10.1016/j.ejmech.2021.113816
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

6.2.1. Tetracyclic quinolines 9
6.2.2. ARS853 10
6.2.3. ARS1620 10
6.2.4. Other direct KRASG12C inhibitors 11
6.2.5. PROTACs 11
6.3. Protein-protein interactions (PPIs) inhibitors 11
6.3.1. Abd-7 11
6.3.2. Thioureas 11
6.3.3. BI3406 12
6.3.4. Indoles 13
6.3.5. DCAI 13
7. Conclusion and perspective 13
Declaration of competing interest 14
Acknowledgements 14
Abbreviations 15
References 15

1. Introduction

RAS is the first oncogene identified in human cancer. Among the three isoforms of RAS gene family (KRAS, HRAS, and NRAS), KRAS harbors the most frequent mutations in solid tumors, and is asso- ciated with 85% of RAS-driven cancers, including pancreatic carci- noma, colorectal carcinoma, and lung adenocarcinoma. KRAS is a promising therapeutic target for human cancers [1,2].
KRAS is a membrane-bound protein with GTPase activity and plays essential roles in cell proliferation, differentiation, and metabolism through the nucleotide exchange between GDP-bond inactive state and GTP-bond active state. It also mediates multiple signaling pathways including RAF-MEK-ERK, PI3K-AKT, RalGDS- Ral, Tiam1-Rac1, and PLCε-PKC pathways, etc. Moreover, oncopro- tein KRAS is associated with tumor-promoted inflammation and immune modulation, emerging as a major driver in tumor micro- environment [3].
In the past decades, KRAS has been considered as an “undrug- gable” target owing to the relatively smooth protein surface and lack of hydrophobic pockets for compound binding. Besides, the picomolar binding affinity between KRAS and GDP or GTP is quite challenging for the competitive binding of inhibitor [4e6]. Inter- rupting the KRAS post-translational modifications, synthetic lethality and autophagy have made significant progress in tumor therapy, however, the abovementioned strategies are still suffered from the unsatisfied therapeutic effects and drug resistance. Encouragingly, Shokat team pioneering reported a novel strategy to directly target the KRASG12C mutation covalently for cancer therapy. Subsequently, a number of covalent KRASG12C inhibitors have been developed and under different phases of clinical trials, such as MRTX849 (adagrasib) and ARS1620. Very recently, AMG510 (sotorasib) has been approved for treating NSCLC and CRC, sug- gesting that directly targeting KRASG12C is one of the most prom- ising strategies for cracking KRAS. In this review, we summarize the functions and roles of KRAS in human cancer and the structure- activity relationship studies of KRAS inhibitors.

2. The structure and function of KRAS protein

KRAS gene locates on chromosome 12p12 with the molecular weight of approximately 21 kDa. KRAS is composed of two splice variants KRAS4A and KRAS4B, among which KRAS4B is the major isoform. KRAS proteins consist of G-domain, the membrane-targeting domain and C-terminal hypervariable region (Fig. 1). The G-domain consists of p-loop, switch I and II loops, and is involved in nucleotide

exchange [6]. The structural changes of the switch I and II regions driven by the phosphorylation of KRAS, regulates the exchange be- tween GDP and GTP cycle [7]. The cycle between the GTP-bound active state and the GDP-bound inactive state start to modulate signaling cascades. Guanine nucleotide exchange factors (GEFs) catalyze the loading of GTP; GTPase activating proteins (GAPs) accelerate the hydrolysis of GTP. GEFs and GAPs corporately regulate the exchange of GDP-bound (inactive) and GTP-bound (active).
KRAS is essential for maintaining physiological functions. Acti- vated KRAS participates in a number of signaling cascades including RAF-MEK-ERK, PI3K-AKT, RalGDS-Ral, Tiam1-Rac1, and PLCε-PKC
pathways, etc. Then, the front-line sensor KRAS allows the trans- mission of signals from cell surface to nucleus. Moreover, KRAS mutations lead to the continuous activation of KRAS and result in initiation of various types of human cancers.

3. KRAS downstream signaling pathways

KRAS plays crucial roles in various signaling pathways and reg- ulates multiple physiological processes through downstream ef- fectors. Upon activation, KRAS will mediates a number of intracellular signaling pathways by post-translational modification, plasma membrane-localization and activation of downstream ef- fectors (Fig. 2).

3.1. RAF-MEK-ERK signaling pathway

Mitogen-activated protein kinase (MAPK) signal pathway is involved in cancer progression and drug resistance. RAF consists of RAS-binding domain (RBD) [8,9], Cys-rich domain [10,11] and ki- nase domain [12e14], which play an essential role in membrane translocation and enzyme activation. In 1986, Smith et al. [15] found that serine/threonine-protein kinase RAF is the downstream of RAS and could bind to activated RAS. Then, the dimerization of RAF induces the catalytic activity and leads to phosphorylation of mitogen-activated protein kinase kinases MEK at Ser218 and Ser222. Finally, the activated MEKs transmit signals to the down- stream extracellular signal-regulated kinases ERKs, thereby regu- lating the tumor proliferation through the ERKs phosphorylation and activation [16e18].

3.2. PI3K-AKT-mTOR signaling pathway

The class I family of phosphoinositide 3-kinase (PI3K) contains regulatory subunit p85, catalytic subunit p110 [19,20] and RBD. In

Fig. 1. Structural basis of KRAS and its interaction with GDP. (A) Crystal structure of GDP-bound Human KRAS (PDB ID: 4OBE). (B) Major domain of KRAS protein. The conformation of switch I and switch II regions participate in GDP-GTP exchange cycle.

Fig. 2. Regulation of KRAS GDP-GTP cycle and KRAS downstream signaling. Codon mutations will cause KRAS to be locked in the KRAS-GTP state and no longer dependent on the stimulation of the superior signal, thus continuously activating the downstream signal pathway leading to the occurrence of tumor. GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor.

general, PI3K signaling pathway could be activated when the RBD directly interacts with RAS in a GTP-dependent manner [21e24], followed by the phosphorylation of protein kinase B (AKT) at resi- dues Thr308 and Ser473, and promotes the activation of mammalian target of rapamycin (mTOR) via direct and indirect manners [25].

3.3. RalGDS-Ral signaling pathway

Ral guanine nucleotide dissociation stimulator (RalGDS) pos- sesses guanine-nucleotide exchange factor activity, serving as one

of the KRAS effectors. Highly conserved RAS binding domain in RalGDS plays a major role for the recruitment of RalGDS to the plasma membrane, which is mediated by the switch I region of RAS [26]. Multiple regions and domains of RAS and RalGDS demonstrate synergistic effects in cellular signaling, regulating cellular mem- brane trafficking and cytoskeletal remodeling [27]. The member of the RAS superfamily RalA signaling plays a critical role in RAS- induced transformation and tumorigenesis, while the activation of RalB appears to be involved in invasion and metastasis [28].

3.4. Tiam1-RAC1 signaling pathway

In 1994, T-lymphoma invasion and metastasis-inducing protein- 1 (Tiam1) was identified in T-lymphoma cells for the first time [29]. Tiam1 belongs to the Dbl family of GEFs which all share a combined Dbl homology (DH)epleckstrin homology (PH) domain and regu- late the GDP/GTP exchange [30]. Increasing evidences demon- strated that Tiam1 is involved in tumor migration and invasion [31]. Similar to the majority of RAS effectors, Tiam1 has a RAS binding domain that could bind with RAS-GTP, and function as the effector that directly associated with RAC1 which is a small GTPase RAS- related C3 botulinum toxin substrate 1 [32]. Thus, RAC1 promotes cell migration and division in cancers, including pancreatic cancer and lymphocytic leukemia [33,34].

3.5. PLCε/PKC signaling pathway

Phospholipase Cε (PLCε) is a novel RAS effector. The N-terminal GRF CDC25-like domain of PLCε is homologous to GEF of RAS. PLCε hydrolyzed phosphatidylinositol 4,5-bisphosphate in a Ca2þ- dependent manner, which activates protein kinase C (PKC) [35,36]. Once activated, PKCs phosphorylate a number of proteins central to calcium signaling and regulate their activity [37].

4. KRAS mutations

KRAS is the most frequently mutated gene in human cancer, and 85% of RAS-driven cancer cases are associated with KRAS mutation [38,39]. Moreover, the frequency and distribution of KRAS gene mutations are distinct in different cancer types (Fig. 3). Hyper- activated mutations of KRAS have been identified in 90% of pancreatic cancers, 40% of colorectal cancers, and 30% of lung

cancers [40]. G12 is the most frequent codon mutation, and ac- counts for approximately 80% of all KRAS mutants. KRAS mutations also occur in G13 and Q61. Among two types of deadly cancers, G12 mutations are the most primary codon mutations in pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LADC), and account for 94% and 91% of all mutations, respectively. G13 mutations have higher percentage in LADC (6%) than that in PDAC (1%). Moreover, the predominant mutation in colorectal cancer (CRC) is also G12 (76%), G13 mutations are less frequent with 20% in all CRC cases. In three cancer types mentioned above, Q61 muta- tions represent a similar proportion, with 1e2% of all mutations. Otherwise, KRAS mutations also occur in codons 63, 117, 119 and 146, with a very low probability of less than 1% [41].
KRAS codon 12, 13 and 61 mutations occur frequently in KRAS mutated cancers, and are clustered in either exon 2 (codons 12 and 13), or exon 3 (codon 61). Real-time NMR showed that different KRAS mutations caused the alterations in nucleotide exchange ki- netics and effector binding capacity [42,43]. Oncogenic G12/G13 mutation impairs both GAP protein binding and GTP hydrolysis after GAP stimulation [44]. In contrast, Q61 mutation plays a direct role in catalysis through the interaction with a water molecule, and attributes to the stabilization of the transition states for GTP hy- drolysis [45]. Therefore, targeted therapeutics must be developed according to different KRAS mutations.

5. Roles of KRAS in cancers

KRAS mutations account for approximately 85% of RAS-driven cancers [47]. Quinlan et al. [48] demonstrated that the activated KRAS regulates the expansion of endothelial stem/progenitor cells, and promotes the occurrence of endodermal originated tumors (lung, pancreas, and colon). In addition to potential influence on

Fig. 3. KRAS alteration frequency in different tumor types. Annotated mutation distribution in MSK-IMPACT clinical sequencing cohort [46].

tumor initiation, oncogenic KRAS also mediates cancer cell growth, invasion, metastasis, energy metabolism and chemoresistance. Moreover, different KRAS mutations showed particular codon preference, and participate in different downstream signaling pathways. Therefore, the roles of different KRAS mutations in tumorigenesis and therapy need further clarification.

5.1. Lung cancer

Lung cancer is the most common cause of cancer-related mor- tality worldwide, accounting for 18% of all cancer-related deaths. Non-small cell lung cancer (NSCLC) encompasses over 80e85% of lung cancers, and LADCs is the main subtype of NSCLC. KRAS mu- tations account for approximately 30% of LADCs in western coun- tries, especially in patients with smoking history [2,49]. Conversely, in Asian patients with LADCs, EGFR mutations are more common compared to KRAS mutations which account for only 10% [50]. KRAS codon mutations in lung cancer usually occur in exon 2 and exon 3. The mutation rate is found predominantly on exon 2 at codon 12 (more than 90%), less frequently at codon 13 (3e5%), and rarely on exon 3 at codon 61 (less than 1%) [2,6,49].
The genetic alterations with KRAS mutations is a critical bio- logical feature for lung cancer. Investigation of 330 patients with KRAS-mutant lung cancers indicated that KRAS-mutation in NSCLC co-occurs with three most frequently mutations: TP53 (42%), STK11 (29%), and KEAP1/NFE2L2 (27%) [51]. TP53 mutations are most common in NSCLC with KRAS mutations. TP53 and KRAS co- mutations activate NF-kB signaling to regulate chemoresistance and promote tumorigenesis. Moreover, the co-mutations of STK11 and KRAS are more frequent in NSCLC, compared with those with only KRAS mutation. Several studies showed that the loss of STK11 promotes the invasion, differentiation and metastasis of NSCLC [52,53]. Arbour et al. [51] observed that KRAS-mutated NSCLC have significantly shorter overall survival from initiation of a combina- tional platinum/pemetrexed therapy in patients with co- mutational KEAP1/NFE2L2. It was also reported that KEAP1/NFE2L2 mutation led to the increase of cancer cytoprotective enzymes, thus promoted cisplatin resistance [54,55].
The heterogeneous behavior of mutant KRAS represents another important biological feature for tumorigenesis [56]. KRASG12D could activate PI3K and MEK signaling, while KRASG12C or KRASG12V acti- vates Ral signaling and suppresses the growth factor-dependent AKT activation in NSCLC cells [56]. The sensitivity of specific mu- tations to chemotherapy also reflects the heterogeneity of specific codon mutations. KRASG12C mutation in NSCLC increased the sensitivity to taxol and pemetrexed, whereas led to a reduced response to cisplatin. Meanwhile, KRASG12D is associated with the resistance to taxol treatment and sensitivity to sorafenib. In addi- tion, the G12V mutant is sensitive to cisplatin, but with slightly resistance to pemetrexed [57].

5.2. Pancreatic cancer

In the past decades, the survival rate of PDAC was not signifi- cantly improved [58]. KRASG12D mutation occurs approximately 41% in all KRAS-driven PDAC [59]. More and more evidences suggested that cancer is a metabolic disease, and multiple metabolic alter- ations, including anabolic glucose metabolism [60], autophagy [61], glutaminolysis and micropinocytosis, have been discovered in KRAS-driven PDAC [62]. The reprogramming of metabolism plays important roles in maintaining the cellular redox balance and meeting the demands of biosynthesis.
KRAS-driven PDAC modulates metabolic reprogramming through regulation of metabolic genes. Enrichment of HIF1a pro- moter elements was found in KRASG12D-mediated transcriptional

regulation, and promote metabolism reprogramming via MAPK and Myc pathways [60]. Oncogene KRAS-directed enhanced Nrf2 tran- scriptional activity, and thereby reducing reactive oxygen species (ROS) and promoting tumorigenesis and proliferation of PDAC [63]. Humpton et al. [64] demonstrated that KRAS-Nix mitophagy axis modulates glycolysis and redox robustness to promote PDAC development. In addition, KRAS mutation regulates branched-chain amino acid BCAA-BCAT2 axis by the inhibition of spleen tyrosine kinase (SYK) and E3 ligase TRIM21, and upregulation of trans- aminase 2 (BCAT2) in the early stage of KRAS-driven PDAC [65].
Accumulating study revealed that PDAC is feature with high autophagy to meet the high metabolic demands [66,67]. Autophagy substrate p62/SQSTM1 [68], Syndecan 1 (SDC1) [69] and Myc [70] are critical for regulation of macropinocytosis in KRAS-driven PDAC. Recently, Bryant et al. [61] reported that inhibition of KRAS en- hances autophagic flux. It was also found that the combination of MEK or ERK inhibitor with autophagy inhibitor has significant synergistic antitumor activity in animal model. Consequently, RAF- MEK-ERK pathway is the main regulator for RAS-mediated meta- bolic changes.

5.3. Colorectal cancer

CRC is the second most deadly cancer worldwide with more than 900 000 deaths annually. KRAS mutations occur approxi- mately 30e40% in CRC, with G12D being the most common mu- tation [71]. Accumulated evidences indicated that KRAS could be an independent cancer driver in CRC.
KRAS mutations are associated with poor prognosis and therapy resistance in mCRC [72]. Differential expression analysis showed that invasive and metastatic tumors transited into adenomas after deletion of KRAS mutations in mouse model of mCRC [73]. Knock- down of KRAS in CRC cells can effectively reduce the activation of ERK, but has no obvious effect on AKT phosphorylation. Besides, RTKs are critical for PI3K signaling and the activation of AKT in KRAS-mutant CRC [74]. Different KRAS mutation types impact on drug sensitivity. Compared with KRASG12C mutation, KRASG13D is highly correlated with the sensitivity of CRC cells to cetuximab in vitro and in vivo. Both progression-free survival and overall sur- vival times of patients with chemotherapy-refractory colorectal cancer and KRASG13D mutation were extended by cetuximab [75]. Significant variations after the treatment with cetuximab were also found in KRASG13D-mutated mCRC, whereas the remarkable effect was not found for G12V and other mutations [76].

5.4. KRAS in other cancers

In addition to the solid tumors mentioned above, KRAS muta- tions are also critical for progression of other types of cancers including endometrial cancer, multiple myeloma, gastric cancer and biliary tract cancer.
Endometrial cancer (EC), originated from the epithelial malig- nant tumors in endometrium, is the most common gynecologic cancer. KRAS mutations account for 10e30% of type I EC [77,78]. The recent clinical evidence suggested an important role of KRAS mu- tations in transition of early endometrial cancer to late-stage invasive tumors [79]. KRAS is associated with EC biological fea- tures including microsatellite instability (MSI) and mucinous dif- ferentiation [77,80,81]. Moreover, Tu et al. [82] demonstrated that KRAS mutations participate in the regulation of transcriptional ac- tivities of estrogen receptor ER, and a combination treatment of MEK inhibitors and anti-estrogen agents could alter the estrogen signaling in KRAS-mutant EC to enhance the response rate [83].
KRAS mutations occur in approximately 20% of multiple myeloma (MM) [84]. KRAS mutations in exon-4 regulate MEK/ERK

and PI3K/AKT signaling in MM [85], and constitutively activated AKT could promote KRAS-driven MM cell survival independently [86]. In terms of the mechanism, selective COX-2 expression in KRAS-transfected MM cells mediates adhesion to fibronectin and chemoresistance [87]. Another novel target Germinal center kinase (GCK) is identified in MM harboring RAS mutations, and knock- down of GCK could inhibit MM cell growth by downregulation of MKK4/7-JNK phosphorylation and degradation of IKZF1/3, BCL-6, and c-Myc [88].
Up to 11% of gastric cancer (GC) cases harbor KRAS mutations, and the most common mutations locates at codon 12 and codon 13 [89]. Helicobacter pylori infection, a primary causative factor of gastric cancer, is related to higher frequency of KRAS mutations in GC [90]. Furthermore, based on a large multicenter study, KRAS activation occurs in the early stages of GC before phenotype determination [91]. Meanwhile, KRAS activation is associated with MSI status [92], EMT [93] and promotes poor clinical outcomes [94]. KRAS mutations was found in 26% of biliary tract cancer (BTC).
Though the activation of KRAS alone almost has no effect on biliary epithelium, combination of KRAS mutation and PTEN loss stimu- lates a hyperplasia of the biliary epithelium to invasive carcinoma [95]. Besides, KRAS activation and homozygous deletion of Ink4a/ Arf participate in stemness maintenance and tumorigenicity of BTC initiating cells [96].

5.5. Tumor immune microenvironment (TME)

KRAS-driven tumor microenvironment that supports tumor growth and metastasis has drawn increasing attention (Fig. 4). Oncogenic KRAS expression triggers further immune reaction to exacerbate the cancer progression. KRASG12D mutations in mouse pancreas induce ductal lesions and recapitulate the full spectrum of pancreatic intraepithelial neoplasias (PanINs) which is a major precursor lesion of PDAC, thereby representing cancer initiation with gain-of-function KRAS mutations [97]. Low KRAS activation in PanINs induces a strong fibroinflammatory reaction consisting of stromal and immune cells. Tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg) are the three major leukocyte subtypes that could lead to

failure of immune surveillance in preinvasive lesions and play roles in KRAS-driven tumor microenvironment [98].
Researchers reported that KRAS might be responsible for pro- moting inflammatory environment within tumors. KRAS-driven cancer cells secrete cytokines and other factors to mediate sur- rounding stromal cells, including fibroblasts, innate and adaptive immune cells. KRAS signaling activation could promote the pro- duction of chemokines including interleukin-8 (CXCL-8/IL-8), MIP- 2, KC, MCP-1 and LIX [99,100]. Otherwise, sonic hedgehog, interleukin-6 (IL-6), and prostaglandin E2 (PGE2) are expressed in a KRAS-dependent manner [101e103]. Inflammatory mediators Src, EGR1, STAT3, NF-kB and Cox-2 are activated to maintain KRAS oncogenic activation through positive feedback loops [104]. Meanwhile, IL-6-dependent STAT3/SOCS3 activation is critical for progression and development of PanINs and PDAC through the generation of ROS via MAPK signaling [102,105] Inhibition of IL-6/ JAK/STAT3 axis reduces the number of M2-type macrophages [106]. Besides, recent study revealed that combination of tumor- derived CSF2 and lactate contributes to TAMs in KRAS-driven CRC [107]. KRAS and Myc cooperatively promote highly proliferative and infiltrative adenocarcinomas through stromal reprogramming sig- nals CCL9 and IL-23. CCL9 is capable of driving macrophage recruitment, angiogenesis and PD-L1 dependent expulsion of T and B cells, while IL-23 could strongly suppress T and B cells and lead to rapid exclusion of NK cells [108]. CXCL-8 plays a role in RasV12- driven tumor due to inhibition of CXCL-8 function, and results in an impairment of tumor angiogenesis and tumor necrosis [99]. Inflammatory responses could promote lung tumorigenesis partly by the activation of IL-8/CXCR2, and KRAS/CXCR2 signaling could lead to alterations of CAF phenotype [109,110]. Taken together, IL-8/ CXCR2 may play an important function in KRAS-induced inflam- mation in cancer. Studies indicated that oncogenic KRAS induces enhanced granulocyte-macrophage colony stimulating factor (GM- CSF) production and promotes the infiltration of MDSCs. Moreover, GM-CSF promotes carcinogenesis with immunosuppressive Gr1þCD11bþ cells [111]. KRAS-driven cancers could also induce Tregs to regulate TME. Mechanistically, IL-10 and TGF-b1 are secreted by mutant KRAS via MEK-ERK-AP1 pathway, thereby suppressing the activation of T cells [112].

Fig. 4. Oncogenic KRAS affects tumor microenvironment (TME).

6. Overview of small molecular KRAS inhibitors

Although extensive efforts have been tried to find effective drugs for KRAS-driven tumors in past decades, only AMG510 (sotorasib)has been approved for the treatment for NSCLC until very recently, probably due to the complexity of KRAS signaling path- ways. Initially, scientists expected to find drugs by interrupting KRAS post-translational modifications, synthetic lethality, inhibi- tion of the metabolism or autophagy [113,114]. However, the results were not satisfactory for a long time. Excitingly, strategies including the development of directly covalent inhibitors and protein-protein interactions (PPIs) inhibitors recently renewed hopes for the treatment of KRAS-driven tumors. These inhibitors interrupt the aberrant KRAS by locking KRAS into the inactive state or inter- rupting the crucial PPIs with GEF and its downstream effectors. In addition to AMG510, more than five small molecular KRAS in- hibitors are currently in clinical trials including MRTX849, ARS3284, GDC-6036, BI-1701963 and JAB-3312, which are sum-
marized in Table 1.

6.1. Direct and covalent inhibitors of KRASG12C

Essential challenge to target KRAS is the G12C mutation which is one of the frequent KRAS mutation at codon 12 in RAS-driven lung adenocarcinomas (~50%) [115]. Although the strategy that directly targets KRAS had been impeded by lacking binding pocket and high affinity to GTP/GDP, an allosteric pocket recently identified in KRASG12C provided opportunities to develop small molecule in- hibitors. In 2013, Shokat and coworkers [116] found a novel switch II pocket (SIIeP) by a “tethering” approach, and developed covalent inhibitors to target the reactive Cys12 of KRASG12C. These discov- eries boosted the development of direct and covalent inhibitors of KRASG12C.

6.1.1. AMG510 (sotorasib)
AMG510 is developed by Amgen in August 2018 (NCT04185883) and has been approved for treating NSCLC and CRC in 2021. In preclinical study, AMG510 exhibited the capability of inhibiting p- ERK with the IC50 value of 0.068 mM in MIA PaCa-2 cell line [117]. Besides, it exhibited strong anti-tumor activity with the tumor growth inhibition (TGI) of 86% under 10 mg/kg concentration in MIA PaCa-2 T2 (p.G12C) xenograft [118].
The discovery of AMG510 started from identifying the “bait” which is a linker containing an acryloyl group and a reactive site binding to various fragments, based on Chemotype Evolution developed by Carmot Therapeutics. As a result, compound 1 (Fig. 5) with azetidine exhibited preference in intrinsic reactivity and shape complementarity with KRASG12C and was identified with the kobs/[I] (a constant evaluating conversion of covalent adduct for- mation between inhibitors and KRASG12C) of 2 M—1 s—1. The coc- rystal structure of compound 1 with KRASG12C confirmed the covalent bond between 1 and Cys12. Besides, the amide nitrogen within compound 1 forms hydrogen bonds with Ala59 and Tyr96, and the isoxazole-amide forces His95 to get away from Tyr96 and Gln99 to provide a new gap. Further screening led to the discovery of an indole analogue 2 with the improved kobs/[I] value of 230 M—1 s—1. Compound 2 displayed the IC50 value of 11.4 mM in MIA PaCa-2 cells, and the cocrystal structure of 2 with KRASG12C showed that the 5-methoxy-tetrahydroisoquinoline within com- pound 2 is able to occupy the Tyr96/His95/Gln99 cryptic pocket and interacts with His95 through p p interactions. To improve the cellular potency, researchers initially modified tetrahy- droisoquinoline region, however the modification didn’t improve the cellular activity. Subsequently, they focused on the indole scaffold, and C7-methyl, C2-cyclopropyl and C5-chloro were introduced into the indole moiety to generate compound 3, which showed the IC50 value of 0.219 mM in MIA PaCa-2 cells. The intro- duction of C2-cyclopropyl restricts compound 3 in a more suitable

Table. 1
Six small molecular KRAS inhibitors in clinical trials.
ID Structure Company Research status Adaptation disease

AMG510 (sotorasib) Amgen Approved NSCLC CRC

MRTX849 (adagrasib)

Mirati Therapeutics Phase III PDAC
CRC NSCLC

ARS3284 UNKNOWN Wellspring Biosciences and Janssen Phase I CRC
NSCLC
GDC-6036 UNKNOWN Genentech Phase I NSCLC
BI-1701963 UNKNOWN Boehringer Ingelheim RCV GmbH Phase I CRC
JAB-3312 UNKNOWN Jacobio Pharmaceutical Co Ltd Phase I Solid tumor

Fig. 5. The optimization strategies of AMG510.

conformation, and C7-methyl interacts with Glu58, Tyr71 and Gly60 through van der Waals interactions. Although compound 3 displayed good cell activity and selectivity, its’ high rat hepatocytes clearance and low oral bioavailability profiles in vivo restricted compound 3 to enter further study. To further improve the phar- maceutical properties, researchers merged indole scaffold with the quinazoline skeleton of ARS-1620 to make inhibitors reach the cryptic pocket groove while maintaining the occupancy to SIIeP. Compound 4 was subsequently designed and synthesized, and exhibited significant inhibition potency on SOS1-catalyzed GDP/ GTP exchange and ERK phosphorylation with the IC50 value of
0.101 mM and 0.335 mM, respectively [118]. The cocrystal structure of 4 and KRASG12C showed similar binding model to 3, and the isopropylphenyl group orients the molecule into the cryptic pocket with the Tyr96, His95, and Gln99 residues. However, 4 was wit- nessed to have poor membrane permeability and aqueous solubi- lity. Further study led to the discovery of compound 5. By formation of the internal hydrogen-bond between nitrogen atom at C-8 po- sition and phenolic, the membrane permeability of 5 was well improved compared to compound 4. Meanwhile, the introduction of a methyl group at C-2 position not only contributed to the improvement of oral bioavailability (33%), but also enhanced cell activity. Compound 5 was synthesized and effectively inhibited the MIA PaCa-2 cells with the IC50 value of 0.044 mM. To avoid the interconversion of the R- and S-atropisomers, the bis-ortho substituted methyl isopropyl phenyl ring was introduced into compound 6 by increasing the biaryl rotational barrier from
26.0 kcal/mol to more than 30 kcal/mol, and 6 showed the improved activity with the IC50 value of 0.028 mM in MIA PaCa-
2 cells but with the poor oral bioavailability (4e12%). Finally, AMG510 was developed by replacing the C6-position chorine with fluorine as well as introducing the nitrogen atom in the isopropyl benzene to reduce the lipophilicity and increase the polar surface area.
The co-crystallization of AMG510 with KRASG12C showed that AMG510 covalently binds to the mutated Cys12 through the acryloyl group and occupies the critical SIIeP by quinazolinone

ring. The C2-methyl of the (S)-methylpiperazine ring interacts with Cys12 and Tyr96, and the hydroxyl substituent at fluorophenol forms hydrogen bonds with Glu63 and Arg68. In addition, the isopropyl pyridine ring is oriented into the cryptic Tyr96/His95/ Gln99 with the nitrogen atoms interacting with the His95 mediated by a water molecule.

6.1.2. MRTX849 (adagrasib)
MRTX849 is another KRASG12C covalent inhibitor with potent anti-tumor activity (the IC50 value of 0.014 mM in H358) developed by Mirati Therapeutics and Array Biopharma in 2019 [119]. Currently, MRTX849 is under phase III evaluation (NCT04685135). The discovery of MRTX849 started from the lead compound 7 (Fig. 6), which was identified by Array BioPharma through a protein modification assay. Compound 7 exhibited 13% modification of KRASG12C under 3 h/5 mM in the assay but did not inhibit the phosphorylation of ERK in H358 cells. The cocrystal structure of compound 7/KRASG12C showed a critical covalent bond is generated between the acrylamide group and Cys12, together with a non- covalent attachment with Lys16. The nitrogen atom at 1-position of pyrimidine interacts with His95 through hydrogen bond. The naphthyl ring of compound 7 occupies a hydrophobic pocket and the replacement of naphthyl ring with phenyl group dramatically reduced KRAS modification (2%) and cell activity (the IC50 > 16 mM). Additionally, there is a potential interaction between naphthyl ring and Asp69. By introducing a hydroxyl group at naphthyl ring to form a hydrogen bond, compound 8 was obtained and displayed 99% protein modification under 3 h/5 mM and the IC50 value of
7.6 mM in H358 cells. Further modification at the C2 position of the
pyrimidine ring by introducing a N-methyl pyrrolidine group to access the carboxylate of Glu62 and His95 generated compound 9, which resulted in 84% modification under 15 min/3 mM and the IC50 value was 0.070 mM in H358 cells. Nevertheless, compound 9 showed low permeability, high hepatic clearance rate in mice (46 mL/min/kg) and low oral bioavailability (2.4%). Given the hy- droxyl group was the hotspot of metabolism, researchers attemp- ted to decrease Glutathione (GSH) conjugation by replacing

Fig. 6. The optimization strategies of MRTX849.

naphthol with indazole groups while maintaining the hydrogen bond with Asp69. Unfortunately, these compounds with good hu- man hepatocyte and microsome stability exhibited reduced cell reactivity and permeability. Subsequently, removing the hydroxyl moiety led to the discovery of compound 10, which exhibits lower CL (25.6 mL/min/Kg in CD-1 mice) and better bioavailability (13.9%). Nevertheless, the cellular activity was dramatically reduced (p-ERK IC50 value of 4.4 mM) compared to compound 9 because of lacking additional interaction with Asp69. Given that the additional interaction with Gly10 could potentially improve the activity, introducing a R-cyanomethyl substituent on piperazine ring made compound 11 boost not only in cellular potency (p-ERK IC50 value of
0.01 mM)but also in improved ADME properties. To obtain more
potential candidates, modification of compound 11 occurs at 8- position of the naphthalene ring by introducing several hydro- phobic groups including methoxy, cyano, ethyl, trifluoromethyl, chloride, and methyl substituents. These groups could orient naphthalene ring into a narrow pocket comprised of Val9, Thr58, Met72, and Tyr96. As a result, compound 12 with chloric naph- thalene ring displayed significant and improved cellular activity with the IC50 value of 0.001 mM. It worth noting that compound 12 exhibited 31.1% bioavailability and could significantly induce tumor regression. However, Compound 12 was predicted with high hu- man hepatic clearance (145 mL/min/Kg). According to the analysis of metabolic experiments data, compound 12 was identified mainly metabolized by GSH and GST mediated GSH conjugation. To further impair the reactive activity with GST/GSH conjugation, the electron-withdrawing groups were introduced and led to the dis- covery of MRTX849, which exhibits excellent stability in the whole blood assay (>50 h across mouse/dog/human) and a slightly decrease in activity (p-ERK IC50 value of 0.014 mM). Further in vivo efficacy studies demonstrated MRTX849 with low clearance value of 19.9/44.2/29.6 mL/min/kg under 3 mg/kg iv in mouse/rat/dog.
According to the co-crystal complex of MRTX849 with KRASG12C, the acrylamide, piperazine ring, naphthalene ring and the N-methyl pyrrolidine group of MTRX849 are the major binding sites. The acrylamide forms pivotal covalent bond with the mutated Cys12, as well as hydrogen bonds with the amine nitrogen of Lys16. The cyanomethyl at piperazine ring replaces water molecule to form hydrogen bond with Gly10 and Thr58. The chlorinated atom orients the hydrophobic naphthalene ring into a narrow hydrophobic cleft formed by residues Val9, Thr58, Met72, and Tyr96. Meanwhile, a

salt-bridge between N-methyl pyrrolidine and the carboxylate of Glu62 was observed.

6.1.3. ARS3248
Based on previous KRASG12C covalent inhibitor ARS1620, ARS3248 developed by Wellspring Biosciences and Janssen is under clinical phase I (NCT04006301) for the treatment of solid tumors. To date, none of detailed structure information about ARS3284 has been released.

6.2. Direct and covalent KRASG12C/D inhibitors in pre-clinical trial

6.2.1. Tetracyclic quinolines
In 2020, Kettle et al. reported a novel direct and covalent KRASG12C inhibitor 14 with tetracyclic quinoline scaffold [120]. 14 exhibited significant cellular potency with the IC50 value of less than 0.005 mM on inhibiting KRASG12C and decent ADME properties (bioavailability 94%, rat hepatic clearance 4 mL/min/Kg). Addi- tionally, the proliferation assays demonstrated that 14 inhibits the growth of KRASG12C cells and leads to dose-dependent tumor regression in KRASG12C MIA PaCa-2 T2 xenografts.
The lead compound 13 (Fig. 7) was initially obtained from the reported literature (KRASG12C IC50 value of 0.496 mM) [120]. The crystal structure between 13 and KRASG12C showed the similar co- valent combination to the previous covalent compounds through acrylamide moiety interacting with Cys12. The nitrogen atom at 1- position of quinazoline forms a hydrogen bond with His95, as well as the side indazole group with Asp69 and Ser65. Later relative
energy profile for torsion of 4-piperazinoquinazoline showed that 4-piperazinoquinazoline torsion at 312○ exhibits the lowest bind- ing free energy, and close to the bioactive conformation. The dis- covery guided the introduction of an additional ring to lock 4-
piperazinoquinazoline into the bioactive conformation. Tetracyclic quinoline was incorporated and compound 14 inhibited the KRASG12C with the IC50 value of 0.081 mM. However, the introduc- tion of morpholine increased the log D (4.2) and resulted in high clearance in both rat hepatocytes and human microsomal systems in vitro. Replacing the indole with fluorophenol resulted in com- pound 15 with lower lipophilicity (log D 3.6) and rat hepatic clearance. Compound 15 exists as a mixture of atropisomers, and the introduction of a small group at C8-position of quinazoline successfully stabilizes the inhibitor in a single active atropisomer to

Fig. 7. The optimization strategies of Tetracyclic quinolines.

generate compound 16 with the IC50 value of 0.016 mM. To simplify the synthetic process, further SAR discussion based on the lead compound 14 without 8-F substituent. Subsequent modification by introducing an additional tertiary lactam ring led to the discovery of compound 17 with significant improvement in inhibition po- tency with the IC50 value of 0.150 mM and log D of 3.0 compared to compound 15. Finally, the incorporation of methyl group on piperazine and 8-F group improved the activity of compound 18, as well as metabolic stability by increasing the steric hindrance to reduce glutathione reactivity.
The crystal structure of compound 18/KRASG12C complex dis- played the primary small molecule-protein interactions, including the formation of a crucial covalent bond between acrylamide moiety and Cys12, and the hydrogen bonds between quinazoline N1, C7 and His95, Asp69, respectively. Besides, a water-mediated hydrogen bond between quinazoline C7 and Ser65 was observed.

6.2.2. ARS853
As the first covalent inhibitor targeting KRASG12C with cellular potency (H358 IC50 value of 1.7 mM), ARS853 was developed by the cooperation between Janes et al. and Wellspring Biosciences [121]. Based on previous compound 19 (Fig. 8) which targets the switch II pocket [122], Patricelli et al. employed LC/MS-MSebased assay to find that compound 19 has low engagement with KRASG12C in H358 cells (the IC50 value is 100 mM). By iterative structure- activity relationship study and biochemical hits, ARS107 was discovered to have strong engagement with KRASG12C (the IC50 value is ~50 mM). Considering the importance of substituent at C5- position for activity, replacing the chlorine with cyclopropyl group

led to the discovery of ARS853 with more than 600 folds improvement on cellular engagement rate (the IC50 value is 1.6 mM) compared to compound 19. Studies towards ARS853 demonstrated that there is a rapid cycle between active and inactive state of KRASG12C in cancer cells, and ARS853 could trap KRASG12C into the GDP-bound state. Additionally, ARS853 completely inhibited the downstream MAPK and PI3K signaling pathways for 72 h under the concentration of 10 mM in H358 cells. Consistent with this, ARS853 inhibits the growth of H358 cells under both 2-D and 3-D prolif- eration conditions.
The binding mode of ARS853 with KRASG12C was revealed by the co-crystal complex. The acrylamide warhead covalently binds to Cys12 and forms hydrogen bonds with Lys16. The benzene ring occupies a hydrophobic pocket, the chloro- and methylcyclopropyl substituents form van der Waals interactions to enhance the occu- pation. In addition, the hydroxyl group and piperazine linker form hydrogen bonds with residues Asp69, Arg68 and Gln63.

6.2.3. ARS1620
In 2018, Janes et al. [123] and Wellspring Biosciences developed another direct KRASG12C inhibitor ARS1620 (Fig. 9), which displayed 10-fold improvement of activity in H358 cells with the IC50 value of
0.12 mM compared to ARS853.
The development of ARS853 was stopped because the metabolic hotspots phenol core and glycine linker caused the poor metabolic stability and oral bioavailability of ARS853. To overcome the drawbacks, researchers initially truncated side linker with acryl- amide warhead for adjusting distance to make sure that inhibitors suitably occupy SIIeP, as well as improving ADME/PK properties.

Fig. 8. The optimization strategies of ARS853.

Fig. 9. The optimization strategy of ARS1620.

Meanwhile, screening of the quinazoline-based compounds led to the discovery of ARS1620, which contains a fluorophenol hydro- phobic moiety and two stereoisomers. The activity of the R-ste- reoisomer is 1000 times lower than the S-stereoisomer.
Consistent with model of action for ARS853, ARS1620 inhibits the KRAS signaling pathways in H358 by binding to the inactive KRAS. Importantly, ARS1620 showed more than 75% KRASG12C oc- cupancy and significant dose-dependent anti-tumor activity in patient-derived tumor xenograft models. ARS1620 exhibited improved drug-like properties, which not only clarified the thera- peutic efficacy of KRASG12C inhibitors in vivo, but also advanced the development of KRASG12C inhibitors.
Similar to ARS853, the cocrystal structure of ARS1620/KRASG12C showed that the acrylamide warhead interacts with Cys12 and Lys16, and the hydrophobic pocket occupied by fluorobenzene comprises of Asp69, Arg68 and Gln99. The hydrogen bonds be- tween N1 nitrogen of quinazoline and His95 are the key in- teractions, which contribute to the rigid conformation that beneficial for the covalent inhibition. Besides, the fluorine atom at quinazoline moiety forms hydrogen bond with His95.

6.2.4. Other direct KRASG12C inhibitors
Other direct KRASG12C inhibitors include LLK10 (Fig. 10) (MIA PaCa-2 IC50 value of 0.79 mM) developed by Li et al. [124], and compound 20 (Fig. 10) (H358 IC50 value of 2.34 mM) developed by Xiao et al. [125]. In addition to the two inhibitors, several inhibitors were also protected by different companies, including WO 2021055728 (Merck sharp & Dohme), CN 112552295 (Jacobio Pharmaceutical), CN 112430234 (Innovent Biologics), CN 112047939 (Jiangsu Sincere Pharmaceutictical), CN 112047948 (Shandong Xuanzhu) and WO 2021058018 (BeiGene).

6.2.5. PROTACs
PROTAC, the abbreviation of proteolysis targeting chimera, is a novel technology that accelerates the protein degradation by the ubiquitin-protease system. As a promising strategy, PROTAC was applied in RAS-driven tumors therapy and stimulated extensive research interests.
The Gray group chose ARS1620 as the KRASG12C warhead and thalidomide as the cereblon (CRBN) warhead, and they designed a KRASG12C degrader library to find the proper linker and measured KRASG12C degradation using GFP-KRAS G12C reporter cells. Though the attempt resulted in proteasomal degradation of KRASG12C in vitro, it failed to achieve endogenous KRASG12C degradation [126]. Bond et al. reported the development of LC-2, which covalently attaches KRASG12C to the MRTX849 warhead and recruits E3 ligase VHL. LC-2 selectively binds to and degrade KRASG12C protein, and regulates the downstream p-ERK, despite MRTX849 warheads could covalently connect to KRAS, therefore preventing LC-2 from
participating in the catalytic cycle of degradation [127].

6.3. Protein-protein interactions (PPIs) inhibitors

Apart from the known KRASG12C inhibitors that showed prom- ising therapeutic potential in cancer treatment, other KRAS mutants including G12V, G12D and Q61H are lacking of the effective in- hibitors. Intriguingly, PPIs are involved in a plethora of signaling pathways for many diseases, the strategies that interrupt the abnormal PPIs are considered as precious therapeutic strategy for RAS-driven tumors [128]. Although small molecules generally fac- ing the hurdle of low affinity and surface area interactions in inhibiting PPIs [129], several inhibitors with potent antitumor ac- tivity and high affinity for targeting mutated RAS have been developed.

6.3.1. Abd-7
In 2018, Quevedo et al. employed the intracellular antibody and identified the compound Abd-1 which bound to RAS proteins at the concentration of 200 mM from a chemical fragment library [129]. Abd-1 (Fig. 11) bound to GST-HRASG12V-GTPgS dose-dependently, but did not bind to inactive or antibody-bounded HRAS. To improve the biophysical properties, replacing the pyridine with furan ring and extended side chain led to discovery of Abd-2, which exhibited the Kd value of 235 mM on KRASG12V-GppNHp. The coc- rystal structure of Abd-2 and KRASQ61H-GppNHp proteins revealed that the binding site locates in the region between KRAS switch I and switch II, and only van der Waals interactions were observed between Abd-2 and KRAS. Further crystallography study of series of analogues suggested that the ligand electron density is mainly around the benzodioxane core and the side chain at C3-position is lack of interactions with HRASG12V. Meanwhile, additional sub- stituents were introduced at C8-position like benzene ring forming interactions with Lys5, Asp54 and Ser39, which led to the discovery of Abd-7. The additional pyridine ring at C8-position of Abd-7 strengthened the interactions with switch I region, and improved van der Waals interactions with different amino acid residues (Lys5, Leu6, Val7, Ser39, Tyr40, Arg41, Asp54, IIe55, Leu56, Gly70, Tyr71, Thr74 and Gly75). Abd-7 showed the preferential affinity to KRASG12V-GppNHp with the Kd value of 0.051 mM in the protein- binding assay. Meanwhile, cell assays confirmed that Abd-7 tar- gets RAS rather than other kinase proteins, resulting in the decreased endogenous phosphorylation of AKT and ERK. Consistent with the on-target effects, Abd-7 inhibited the proliferation of DLD- 1 (KRASG13D) and HT1080 (NRASQ61K) in a period of 72 h with the IC50 value of 8 mM and 10 mM, respectively.

6.3.2. Thioureas
Based on the X-ray structure of HRAS and its complex with GppNHp, Shima et al. [130] applied structure-based drug design technique to find small molecules that bind to the surface pocket of HRAS in 2013. The docking screening led to the discovery of two small molecules Kobe0065 and Kobe2602 with thioureas scaffold (Fig. 12) from virtual libraries. Both compounds displayed potent activity on inhibiting RAS-GTP binding and various downstream effectors including Raf and GEF. Kobe0065 and Kobe2602 showed the significant inhibition on HRASG12Vetransformed NIH 3T3 cells growth with the IC50 value of 0.5 mM and 1.4 mM, respectively. Additionally, Kobe0065 displayed the inhibition rate of 40e50% on the tumor growth with the dose of 80 mg/kg in a xenograft model of SW480 cells.
Because of the poor water solubility, the cocrystal structures of a
Kobe0065 analogue and Kobe2601 with HRasT35—GppNHp revealed a hydrophobic surface pocket near switch I which comprised of residues Lys5, Leu56, Met67, Gln70, Tyr71, and Thr74, and this pocket is occupied by the fluorobenzene moiety. On the other side of the Kobe0065, the dinitrobenzene moiety forms interactions

Fig. 10. Other directly targeting KRASG12C inhibitors.

Fig. 11. The optimization strategies of Abd-7.

Fig. 12. The structures of Kobe series.

with the nearby switch II. The thioureas inhibitors are valid struc- ture in common RAS family members, since the binding regions are conserved in multiple RAS.

6.3.3. BI3406
As a GEF, Son of sevenless (SOS) plays key roles in the transition between the active and inactive state of RAS, thus exerting the related antitumor efficacy. In 2021, Boehringer Ingelheim reported

BI306 as a potent and selective inhibitor with the IC50 value of
0.005 mM in inhibiting SOS1-KRAS interaction [131]. BI3406 binds to the catalytic pocket of SOS1, thereby disrupts the interaction with KRAS.
Initially, Hofmann et al. [131] identified the lead compound BI- 68BS (Fig. 13) with inhibition activity in a GDP-dependent KRAS- SOS1 displacement assay (the IC50 value of 1.3 mM) through high- throughput screening of 1.7 million compounds. Further

optimization of BI-68BS led to the discovery of BI3406 by intro- ducing additional methyl, tetrahydrofuryl, trifluromethyl and amino groups. BI-68BS was originally developed for targeting EGFR, however the introduction of methyl could improve selectivity for KRAS rather other kinases. The trifluromethyl and amino groups could provide addition interactions, as well as tetrahydrofuryl im- proves the solubility and metabolic stability. Different from other G12C covalent inhibitors that restricted to the variants harboring mutated cysteine, BI3406 exhibited the inhibition of GTP-bound RAS accumulation and p-ERK levels in broad cell lines with several KRAS mutations including G12C, G12V, G12S, G12A, and G13D. Meanwhile, BI3406 significantly inhibits the tumor growth in KRASG12C-mutated MIA PaCa-2 xenografts model in vivo. Further studies demonstrated that the combination of BI-3406 with MEK inhibitor was beneficial for improving the antitumor activity, as well as reducing the acquired resistance to increase the effective- ness and duration of treatment for MEK inhibitors. Collectively, BI3406 is not only a promising inhibitor for KRAS-driven tumors, but also provides new strategy that overcomes the MEK inhibitoredriven drug resistance.
The cocrystal X-ray structure of BI-3406 bound to the catalytic pocket of SOS1 indicated that the trifluoromethyl and the amino substituent form hydrogen bonds with M787SOS1, and the tetra- hydrofuryl substituent improves the interaction with Tyr884SOS1.

6.3.4. Indoles
In 2012, Sun et al. [122] identified several fragments which trap KRAS into inactive state by inhibiting the interaction with SOS from 11 000 fragments by using 15N-labeled GDP-bound KRASG12D and NMR-spectroscopy-based screen. The representative molecules include indoles 20, phenols 21 and sulfonamides 22 (Fig. 14). All these fragment hits can bind both KRASG12D and other variants, which suggested that there is a common pocket in different KRAS isoforms. Meanwhile, the analysis of many crystal structures showed that the hydrogen-bond donor and positive charged group are critical for interaction. Subsequently, several indole derivates have been designed, and all compounds described the inhibition of SOS-catalyzed nucleotide exchange. Among all the analogues, compound 23 containing an isoleucine moiety (Fig. 14) with the Kd value of 190 mM showed the inhibition rate of about 78% in nucleotide exchange assay.
According to the cocrystal structure of these fragments with KRAS, a special pocket between switch I and switch II that only exists in the “open” station of KRAS was discovered. The indole

moiety of compound 23 occupied the primary hydrophobic region comprised of Val7, Leu56, and Tyr71, and forming a hydrogen bond between NH group and Asp54. On the other side of 23, the imi- dazopyridine moiety which interacts with Ser39, is mediated by a water molecule. Meanwhile, the isoleucine substituent group is involved in the interaction with Glu37 and Asp38 in another binding cleft.

6.3.5. DCAI
In 2012, Maurer et al. [132] identified several compounds ben- zamidine (BZDN), benzimidazole (BZIM), and 4,6-dichloro-2- methyl-3-aminoethyl-indole (DCAI) (Fig. 15) by fragment-based lead discovery (FBLD) approach. Based on these fragments, re- searchers attempted to develop inhibitors directly targeting RAS and inhibiting the Ras/SOS interactions. Subsequently, the analysis of the co-crystal structures of KRAS and above inhibitors revealed a new binding pocket which comprised of Lys5, Leu6, Val7, Ile55, Leu56, and Thr74, and the pocket is located in between the helix a2 and the core b-sheet. Importantly, the cocrystal structure between DCAI and KRASm-GMPPCP showed that the binding region with high plasticity could be expanded upon compounds. The chloro- substituent at C6-position of DCAI occupies a hydrophobic pocket, and the 4-chloro substituent induces conformational changes of Arg41 and Asp54, which disrupts the interactions be- tween RAS and SOS. Further nucleotide exchange assays demon- strated that DCAI binds to KRAS displayed the increasing steric hindrance to interrupt interactions between KRAS and SOS. Consistent with this effect, DCAI inhibited RAS activation with the EC50 value of 15.8 mM in HEK-293T cells.

7. Conclusion and perspective

KRAS is a major oncogene that participates in multiple signaling pathways and mediates cell proliferation, differentiation and metabolism. Specific point mutations in KRAS, especially those at position 12, 13 and 61, maintaining KRAS-GTP active form and consequently result in tumor initiation and progression. With the discovery of a new allosteric pocket with mutant cysteine 12 in KRAS by Shokat et al., it lifted the curtain on the development of covalent inhibitors against KRASG12C. So far, several G12C covalent inhibitors were successfully developed and showed significantly anti-tumor activity in vivo and vitro. With extensive efforts to develop KRAS inhibitors over the past three decades, encouragingly, AMG510 (sotorasib) has recently been approved for market. These

Fig. 13. The development of BI-3406.

Fig. 14. The structures of indoles.

Fig. 15. The structures of BZIM, INDL and DCAI.

inhibitors with profound significance break the myth of KRAS undruggable and boost the research interests for KRAS inhibitors. Nevertheless, due to these compounds are limited to tumors car- rying the G12C mutation, other strategic inhibitors like PPIs in- hibitors that applied to different mutant situation in RAS have also drawn attentions.
In addition to lacking of compounds targeting KRAS mutations other than G12C, another noteworthy problem is acquired resis- tance of KRASG12C inhibitors [133]. For example, adagrasib showed good efficacy in preclinical models of KRAS-driven NSCLC, CRC and other solid tumors. Gene set enrichment analysis (GSEA) by Hallin et al. reported that adagrasib selectively inhibited multiple genes which are directly associated with KRAS signaling. MAPK pathway negative regulators, such as DUSP, SPRY and Pleckstrin Homology Like Domain Family A Member (PHLDA) family gene, was down- regulated following adagrasib treatment, and thereby resulting in reactivation ERK -dependent signaling [134]. Otherwise, adaptive feedback activation of KRAS signaling contribute to inhibit the ef- ficacy of KRASG12C inhibitors, and this might trigger drug resistance [135].
Besides, partial clinical responses were investigated in lung cancer patients following KRASG12C inhibitor. Xue et al. [136] reasoned that drug treatment might lead to the uneven distribution of cells with active or inactive KRASG12C, and the adaptive reac- tivation during G12C inhibitors treatment is caused by transition from KRASG12C equilibrium to active/drug insensitive state. RTK- triggered nucleotide-exchange together with effector activation and cell cycle progression through AURKA signaling promotes such transition. Thus, multifactorial and non-uniform adaptive process must be inhibited for complete and durable responses of conformation-specific KRASG12C inhibition.

Multiple RTKs have been identified with adaptive feedback resistance to KRASG12C inhibition, encouragingly, RTKs/SHP2 in- hibitors combined with KRASG12C inhibitors enhance the efficacy and overcome the resistance. Importantly, study on combination of SHP2 and KRASG12C inhibitors identified prominent suppression of adaptive feedback and improvement of activity in vivo and vitro, which provide an important strategy for future application. Hallin et al. [119] also identified the effect of combination strategies of MRTX849 with other inhibitors of upstream or downstream signaling pathways like SHP2、EGFR、mTOR and CDK4/6. These combinations all exhibited a broader antitumor activity in KRASG12C-mutant tumor models and caused more effectively tumor regression compared to the single MRTX849 therapy. Several studies have proved the combination effect of anti-PD-1 and MEK inhibitor on solid tumors [137e139]. Thus, combination of immune checkpoint blockade therapy and a direct KRAS covalent inhibitor has emerged as a promising therapeutic strategy. With the further research, it is believed that GTPase KRAS may be a meaningful target for cancers.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors acknowledge financial support by National Natural Science Foundation of China (No. 22077144, 819733598), Guang- dong Natural Science Funds for Distinguished Young Scholar (No.

2018B030306017), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2018), The Fundamental Research Funds for the Central Universities (20ykzd15), Guangdong Basic and Applied Basic Research Foundation (2019A1515011215), Guangzhou Basic and Applied Basic Research Foundation (202002030408).

Abbreviations

KRAS Kirsten rat sarcoma virus oncogene RAS rat sarcoma
HRAS Harvey rat sarcoma
NRAS neuroblastoma rat sarcoma GTP guanosine triphosphate GDP guanosine diphosphate RTKs receptor tyrosine kinases
EGFR epidermal growth factor receptor GEFs guanine nucleotide exchange factors GAPs GTPase activating proteins
MAPK Mitogen-activated protein kinase RAF serine/threonine-protein kinase RBD RAS-binding domain
MEKs mitogen-activated protein kinase kinases ERKs extracellular signal-regulated kinases PI3K phosphoinositide 3-kinase
AKT protein kinase B
mTOR mammalian target of rapamycin
RalGDS Ral guanine nucleotide dissociation stimulator REM RAS exchange motif
Tiam1 T-lymphoma invasion and metastasis-inducing protein- 1
DH Dbl homology
PH pleckstrin homology
Ral RAS-like GTPase
RAC1 RAS-related C3 botulinum toxin substrate 1 PLCε phospholipase Cε
PKC protein kinase C
PDAC pancreatic ductal adenocarcinoma LADC lung adenocarcinoma
CRC colorectal cancer
NSCLC non-small cell lung cancer TP53 tumor protein p53
STK11 serine/threonine kinase 11
KEAP1 Kelchlike ECH associated protein 1
NFE2L2, Nrf2 nuclear factor erythroid 2-related factor 2 EZH2 enhancer of zeste homolog 2
Asf1a anti-silencing function protein 1 homolog A HDAC6 Histone deacetylase 6
Myc myelocytomatosis oncogene ROS reactive oxygen species
Nix NIP3-like protein X
BCCA branched-chain amino acids
BCAT2 branched-chain aminotransferase 2 TRIM21 tripartite motif-containing 21
SYK spleen tyrosine kinase
SDC1 Syndecan 1
mCRC metastatic colorectal cancer
Nudt7 nudix (nucleoside diphosphate linked moiety X)-type motif 7
EMT epithelial-mesenchymal transition EC endometrial cancer
MSI microsatellite instability
MM multiple myeloma
PI proteasome inhibitors
POMP proteasome maturation protein

ER endoplasmic reticulum COX-2 cyclooxygenase-2
GCK germinal center kinase
GC gastric cancer
BTC biliary tract cancer
PTEN phosphatase and tensin homolog TME tumor immune microenvironment PanINs pancreatic intraepithelial neoplasias TAM tumor-associated macrophages MDSC myeloid-derived suppressor cells Treg regulatory T cells
IL-8 interleukin-8
IL-6 interleukin-6
PGE2 prostaglandin E2
GM-CSF granulocyte-macrophage colony stimulating factor PPIs protein-protein interactions
SIIeP switch II pocket
SOS son of sevenless

References

[1] B. Bournet, C. Buscail, F. Muscari, P. Cordelier, L. Buscail, Targeting KRAS for diagnosis, prognosis, and treatment of pancreatic cancer: hopes and realities, Eur. J. Canc. 54 (2016) 75e83.
[2] J. Tím´ar, The clinical relevance of KRAS gene mutation in non-small-cell lung cancer, Curr. Opin. Oncol. 26 (2014) 138e144.
[3] P.D. Carvalho, A.L. Machado, F. Martins, R. Seruca, S. Velho, Targeting the tumor microenvironment: an unexplored strategy for mutant KRAS tumors, Cancers 11 (2019) 2010.
[4] A. Ou, J.W. Schmidberger, K.A. Wilson, C.W. Evans, J.A. Hargreaves, M. Grigg,
M.L. O’Mara, K.S. Iyer, C.S. Bond, N.M. Smith, High resolution crystal structure of a KRAS promoter G-quadruplex reveals a dimer with extensive poly-A p- stacking interactions for small-molecule recognition, Nucleic Acids Res. 48 (2020) 5766e5776.
[5] T.E. Mattox, X. Chen, Y.Y. Maxuitenko, A.B. Keeton, G.A. Piazza, Exploiting RAS nucleotide cycling as a strategy for drugging RAS-driven cancers, Int. J. Mol. Sci. 21 (2019) 141.
[6] A.D. Cox, S.W. Fesik, A.C. Kimmelman, J. Luo, C.J. Der, Drugging the undruggable RAS: mission possible? Nat. Rev. Drug Discov. 13 (2014) 828e851.
[7] Y. Kano, T. Gebregiworgis, C.B. Marshall, N. Radulovich, B.P.K. Poon, J. St- Germain, J.D. Cook, I. Valencia-Sama, B.M.M. Grant, S.G. Herrera, J. Miao,
B. Raught, M.S. Irwin, J.E. Lee, J.J. Yeh, Z.Y. Zhang, M.S. Tsao, M. Ikura, M. Ohh, Tyrosyl phosphorylation of KRAS stalls GTPase cycle via alteration of switch I and II conformation, Nat. Commun. 10 (2019) 224.
[8] N. Nassar, G. Horn, C. Herrmann, A. Scherer, F. McCormick, A. Wittinghofer, The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue, Nature 375 (1995) 554e560.
[9] E. Chuang, D. Barnard, L. Hettich, X.F. Zhang, J. Avruch, M.S. Marshall, Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues, Mol. Cell Biol. 14 (1994) 5318e5325.
[10] H.R. Mott, J.W. Carpenter, S. Zhong, S. Ghosh, R.M. Bell, S.L. Campbell, The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phos- pholipid binding site, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 8312e8317.
[11] T.R. Brtva, J.K. Drugan, S. Ghosh, R.S. Terrell, S. Campbell-Burk, R.M. Bell,
C.J. Der, Two distinct Raf domains mediate interaction with Ras, J. Biol. Chem. 270 (1995) 9809e9812.
[12] G. Heidecker, M. Huleihel, J.L. Cleveland, W. Kolch, T.W. Beck, P. Lloyd,
T. Pawson, U.R. Rapp, Mutational activation of c-raf-1 and definition of the minimal transforming sequence, Mol. Cell Biol. 10 (1990) 2503e2512.
[13] J.R. Fabian, I.O. Daar, D.K. Morrison, Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase, Mol. Cell Biol. 13 (1993) 7170e7179.
[14] R. Marais, Y. Light, H.F. Paterson, C.J. Marshall, Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation, EMBO J. 14 (1995) 3136e3145.
[15] M.R. Smith, S.J. DeGudicibus, D.W. Stacey, Requirement for c-ras proteins during viral oncogene transformation, Nature 320 (1986) 540e543.
[16] T.G. Boulton, S.H. Nye, D.J. Robbins, N.Y. Ip, E. Radziejewska,
S.D. Morgenbesser, R.A. DePinho, N. Panayotatos, M.H. Cobb,
G.D. Yancopoulos, ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF, Cell 65 (1991) 663e675.
[17] C. Montagut, J. Settleman, Targeting the RAF-MEK-ERK pathway in cancer therapy, Canc. Lett. 283 (2009) 125e134.
[18] A.A. Samatar, P.I. Poulikakos, Targeting RAS-ERK signalling in cancer:

promises and challenges, Nat. Rev. Drug Discov. 13 (2014) 928e942.
[19] D.A. Fruman, H. Chiu, B.D. Hopkins, S. Bagrodia, L.C. Cantley, R.T. Abraham, The PI3K pathway in hhuman ddisease, Cell 170 (2017) 605e635.
[20] K. Okkenhaug, Signaling by the phosphoinositide 3-kinase family in immune cells, Annu. Rev. Immunol. 31 (2013) 675e704.
[21] J.M. Backer, The regulation of class IA PI 3-kinases by inter-subunit in- teractions, Curr. Top. Microbiol. Immunol. 346 (2010) 87e114.
[22] C.H. Huang, D. Mandelker, O. Schmidt-Kittler, Y. Samuels, V.E. Velculescu,
K.W. Kinzler, B. Vogelstein, S.B. Gabelli, L.M. Amzel, The structure of a human p110alpha/p85alpha complex elucidates the effects of oncogenic PI3Kalpha mutations, Science 318 (2007) 1744e1748.
[23] E.H. Walker, O. Perisic, C. Ried, L. Stephens, R.L. Williams, Structural insights into phosphoinositide 3-kinase catalysis and signalling, Nature 402 (1999) 313e320.
[24] P. Rodriguez-Viciana, P.H. Warne, R. Dhand, B. Vanhaesebroeck, I. Gout,
M.J. Fry, M.D. Waterfield, J. Downward, Phosphatidylinositol-3-OH kinase as a direct target of Ras, Nature 370 (1994) 527e532.
[25] V. Asati, D.K. Mahapatra, S.K. Bharti, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways inhibitors as anticancer agents: structural and pharma- cological perspectives, Eur. J. Med. Chem. 109 (2016) 314e341.
[26] R. Yoshizawa, N. Umeki, M. Yanagawa, M. Murata, Y. Sako, Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral, Biophys. Physicobiol. 14 (2017) 75e84.
[27] E. Ferro, L. Trabalzini, RalGDS family members couple Ras to Ral signalling and that’s not all, Cell. Signal. 22 (2010) 1804e1810.
[28] K.H. Lim, K. O’Hayer, S.J. Adam, S.D. Kendall, P.M. Campbell, C.J. Der,
C.M. Counter, Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells, Curr. Biol. 16 (2006) 2385e2394.
[29] G.G. Habets, E.H. Scholtes, D. Zuydgeest, R.A. van der Kammen, J.C. Stam,
A. Berns, J.G. Collard, Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins, Cell 77 (1994) 537e549.
[30] J. Collard, Signaling pathways regulated by Rho-like proteins, Int. J. Oncol. 8 (1996) 131e138.
[31] P. Boissier, U. Huynh-Do, The guanine nucleotide exchange factor Tiam1: a Janus-faced molecule in cellular signaling, Cell. Signal. 26 (2014) 483e491.
[32] J.M. Lambert, Q.T. Lambert, G.W. Reuther, A. Malliri, D.P. Siderovski,
J. Sondek, J.G. Collard, C.J. Der, Tiam1 mediates Ras activation of Rac by a PI(3) K-independent mechanism, Nat. Cell Biol. 4 (2002) 621e625.
[33] X. Guo, M. Wang, J. Jiang, C. Xie, F. Peng, X. Li, R. Tian, R. Qin, Balanced Tiam1- rac1 and RhoA drives proliferation and invasion of pancreatic cancer cells, Mol, Canc. Res. 11 (2013) 230e239.
[34] S.W. Hofbauer, P.W. Krenn, S. Ganghammer, D. Asslaber, U. Pichler,
K. Oberascher, R. Henschler, M. Wallner, H. Kerschbaum, R. Greil,
T.N. Hartmann, Tiam1/Rac1 signals contribute to the proliferation and che- moresistance, but not motility, of chronic lymphocytic leukemia cells, Blood 123 (2014) 2181e2188.
[35] G.G. Kelley, S.E. Reks, J.M. Ondrako, A.V. Smrcka, Phospholipase C(epsilon): a novel Ras effector, EMBO J. 20 (2001) 743e754.
[36] C. Song, C.D. Hu, M. Masago, K. Kariyai, Y. Yamawaki-Kataoka, M. Shibatohge,
D. Wu, T. Satoh, T. Kataoka, Regulation of a novel human phospholipase C, PLCepsilon, through membrane targeting by Ras, J. Biol. Chem. 276 (2001) 2752e2757.
[37] D.B. van Rossum, R.L. Patterson, PKC and PLA2: probing the complexities of the calcium network, Cell Calcium 45 (2009) 535e545.
[38] I.A. Prior, P.D. Lewis, C. Mattos, A comprehensive survey of Ras mutations in cancer, Canc. Res. 72 (2012) 2457e2467.
[39] A.D. Cox, C.J. Der, Ras history: the saga continues, Small GTPases 1 (2010) 2e27.
[40] P. Liu, Y. Wang, X. Li, Targeting the untargetable KRAS in cancer therapy, Acta Pharm. Sin. B. 9 (2019) 871e879.
[41] Y. Pylayeva-Gupta, E. Grabocka, D. Bar-Sagi, RAS oncogenes: weaving a tumorigenic web, Nat. Rev. Canc. 11 (2011) 761e774.
[42] M.J. Smith, B.G. Neel, M. Ikura, NMR-based functional profiling of RASo- pathies and oncogenic RAS mutations, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 4574e4579.
[43] J.C. Hunter, A. Manandhar, M.A. Carrasco, D. Gurbani, S. Gondi,
K.D. Westover, Biochemical and structural analysis of common cancer- associated KRAS mutations, Mol. Canc. Res. 13 (2015) 1325e1335.
[44] J.M. Ostrem, K.M. Shokat, Direct small-molecule inhibitors of KRAS: from structural insights to mechanism-based design, Nat. Rev. Drug Discov. 15 (2016) 771e785.
[45] G. Buhrman, G. Holzapfel, S. Fetics, C. Mattos, Allosteric modulation of Ras positions Q61 for a direct role in catalysis, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 4931e4936.
[46] A. Zehir, R. Benayed, R.H. Shah, A. Syed, S. Middha, H.R. Kim, P. Srinivasan,
J. Gao, D. Chakravarty, S.M. Devlin, M.D. Hellmann, D.A. Barron, A.M. Schram,
M. Hameed, S. Dogan, D.S. Ross, J.F. Hechtman, D.F. DeLair, J. Yao,
D.L. Mandelker, D.T. Cheng, R. Chandramohan, A.S. Mohanty, R.N. Ptashkin,
G. Jayakumaran, M. Prasad, M.H. Syed, A.B. Rema, Z.Y. Liu, K. Nafa, L. Borsu,
J. Sadowska, J. Casanova, R. Bacares, I.J. Kiecka, A. Razumova, J.B. Son,
L. Stewart, T. Baldi, K.A. Mullaney, H. Al-Ahmadie, E. Vakiani, A.A. Abeshouse,
A.V. Penson, P. Jonsson, N. Camacho, M.T. Chang, H.H. Won, B.E. Gross,
R. Kundra, Z.J. Heins, H.W. Chen, S. Phillips, H. Zhang, J. Wang, A. Ochoa,
J. Wills, M. Eubank, S.B. Thomas, S.M. Gardos, D.N. Reales, J. Galle, R. Durany,

R. Cambria, W. Abida, A. Cercek, D.R. Feldman, M.M. Gounder, A.A. Hakimi,
J.J. Harding, G. Iyer, Y.Y. Janjigian, E.J. Jordan, C.M. Kelly, M.A. Lowery,
L.G.T. Morris, A.M. Omuro, N. Raj, P. Razavi, A.N. Shoushtari, N. Shukla,
T.E. Soumerai, A.M. Varghese, R. Yaeger, J. Coleman, B. Bochner, G.J. Riely,
L.B. Saltz, H.I. Scher, P.J. Sabbatini, M.E. Robson, D.S. Klimstra, B.S. Taylor,
J. Baselga, N. Schultz, D.M. Hyman, M.E. Arcila, D.B. Solit, M. Ladanyi,
M.F. Berger, Mutational landscape of metastatic cancer revealed from pro- spective clinical sequencing of 10,000 patients, Nat. Med. 23 (2017) 703e713.
[47] G.A. Hobbs, C.J. Der, K.L. Rossman, RAS isoforms and mutations in cancer at a glance, J. Cell Sci. 129 (2016) 1287e1292.
[48] M.P. Quinlan, S.E. Quatela, M.R. Philips, J. Settleman, Activated KRAS, but not Hras or Nras, may initiate tumors of endodermal origin via stem cell expansion, Mol. Cell Biol. 28 (2008) 2659e2674.
[49] A. Ghimessy, P. Radeczky, V. Laszlo, B. Hegedus, F. Renyi-Vamos, J. Fillinger,
W. Klepetko, C. Lang, B. Dome, Z. Megyesfalvi, Current therapy of KRAS- mutant lung cancer, Cancer Metastasis, Rev 39 (2020) 1159e1177.
[50] L. Cheng, R.E. Alexander, G.T. Maclennan, O.W. Cummings, R. Montironi,
A. Lopez-Beltran, H.M. Cramer, D.D. Davidson, S. Zhang, Molecular pathology of lung cancer: key to personalized medicine, Mod. Pathol. 25 (2012) 347e369.
[51] K.C. Arbour, E. Jordan, H.R. Kim, J. Dienstag, H.A. Yu, F. Sanchez-Vega, P. Lito,
M. Berger, D.B. Solit, M. Hellmann, M.G. Kris, C.M. Rudin, A. Ni, M. Arcila,
M. Ladanyi, G.J. Riely, Effects of co-occurring genomic alterations on out- comes in patients with KRAS-mutant non-small cell lung cancer, Clin. Canc. Res. 24 (2018) 334e340.
[52] S. Matsumoto, R. Iwakawa, K. Takahashi, T. Kohno, Y. Nakanishi, Y. Matsuno,
K. Suzuki, M. Nakamoto, E. Shimizu, J.D. Minna, J. Yokota, Prevalence and specificity of LKB1 genetic alterations in lung cancers, Oncogene 26 (2007) 5911e5918.
[53] H. Ji, M.R. Ramsey, D.N. Hayes, C. Fan, K. McNamara, P. Kozlowski, C. Torrice,
M.C. Wu, T. Shimamura, S.A. Perera, M.C. Liang, D. Cai, G.N. Naumov, L. Bao,
C.M. Contreras, D. Li, L. Chen, J. Krishnamurthy, J. Koivunen, L.R. Chirieac,
R.F. Padera, R.T. Bronson, N.I. Lindeman, D.C. Christiani, X. Lin, G.I. Shapiro,
P.A. J€anne, B.E. Johnson, M. Meyerson, D.J. Kwiatkowski, D.H. Castrillon,
N. Bardeesy, N.E. Sharpless, K.K. Wong, LKB1 modulates lung cancer differ- entiation and metastasis, Nature 448 (2007) 807e810.
[54] B. Padmanabhan, K.I. Tong, T. Ohta, Y. Nakamura, M. Scharlock, M. Ohtsuji,
M.I. Kang, A. Kobayashi, S. Yokoyama, M. Yamamoto, Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer, Mol. Cell. 21 (2006) 689e700.
[55] T. Ohta, K. Iijima, M. Miyamoto, I. Nakahara, H. Tanaka, M. Ohtsuji, T. Suzuki,
A. Kobayashi, J. Yokota, T. Sakiyama, T. Shibata, M. Yamamoto, S. Hirohashi, Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth, Canc. Res. 68 (2008) 1303e1309.
[56] N.T. Ihle, L.A. Byers, E.S. Kim, P. Saintigny, J.J. Lee, G.R. Blumenschein, A. Tsao,
S. Liu, J.E. Larsen, J. Wang, L. Diao, K.R. Coombes, L. Chen, S. Zhang,
M.F. Abdelmelek, X. Tang, V. Papadimitrakopoulou, J.D. Minna, S.M. Lippman,
W.K. Hong, R.S. Herbst, Wistuba II, J.V. Heymach, G. Powis, Effect of KRAS oncogene substitutions on protein behavior: implications for signaling and clinical outcome, J. Natl. Cancer Inst. 104 (2012) 228e239.
[57] M.C. Garassino, M. Marabese, P. Rusconi, E. Rulli, O. Martelli, G. Farina,
A. Scanni, M. Broggini, Different types of K-Ras mutations could affect drug sensitivity and tumour behaviour in non-small-cell lung cancer, Ann. Oncol. 22 (2011) 235e237.
[58] V. Jentzsch, J.A.A. Davis, M.B.A. Djamgoz, Pancreatic cancer (PDAC): intro- duction of evidence-based complementary measures into integrative clinical management, Cancers 12 (2020) 3096.
[59] A.M. Waters, C.J. Der, KRAS: the critical driver and therapeutic target for pancreatic cancer, Cold Spring Harb. Perspect. Med. 8 (2018) a031435.
[60] H. Ying, A.C. Kimmelman, C.A. Lyssiotis, S. Hua, G.C. Chu, E. Fletcher-San- anikone, J.W. Locasale, J. Son, H. Zhang, J.L. Coloff, H. Yan, W. Wang, S. Chen,
A. Viale, H. Zheng, J.H. Paik, C. Lim, A.R. Guimaraes, E.S. Martin, J. Chang,
A.F. Hezel, S.R. Perry, J. Hu, B. Gan, Y. Xiao, J.M. Asara, R. Weissleder,
Y.A. Wang, L. Chin, L.C. Cantley, R.A. DePinho, Oncogenic KRAS maintains pancreatic tumors through regulation of anabolic glucose metabolism, Cell 149 (2012) 656e670.
[61] K.L. Bryant, C.A. Stalnecker, D. Zeitouni, J.E. Klomp, S. Peng, A.P. Tikunov,
V. Gunda, M. Pierobon, A.M. Waters, S.D. George, G. Tomar, B. Papke,
G.A. Hobbs, L. Yan, T.K. Hayes, J.N. Diehl, G.D. Goode, N.V. Chaika, Y. Wang,
G.F. Zhang, A.K. Witkiewicz, E.S. Knudsen, E.F. Petricoin 3rd, P.K. Singh,
J.M. Macdonald, N.L. Tran, C.A. Lyssiotis, H. Ying, A.C. Kimmelman, A.D. Cox,
C.J. Der, Combination of ERK and autophagy inhibition as a treatment approach for pancreatic cancer, Nat. Med. 25 (2019) 628e640.
[62] C. Commisso, S.M. Davidson, R.G. Soydaner-Azeloglu, S.J. Parker,
J.J. Kamphorst, S. Hackett, E. Grabocka, M. Nofal, J.A. Drebin, C.B. Thompson,
J.D. Rabinowitz, C.M. Metallo, M.G.V. Heiden, D. Bar-Sagi, Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells, Nature 497 (2013) 633e637.
[63] G.M. DeNicola, F.A. Karreth, T.J. Humpton, A. Gopinathan, C. Wei, K. Frese,
D. Mangal, K.H. Yu, C.J. Yeo, E.S. Calhoun, F. Scrimieri, J.M. Winter,
R.H. Hruban, C. Iacobuzio-Donahue, S.E. Kern, I.A. Blair, D.A. Tuveson, Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis, Nature 475 (2011) 106e109.
[64] T.J. Humpton, B. Alagesan, G.M. DeNicola, D. Lu, G.N. Yordanov,

C.S. Leonhardt, M.A. Yao, P. Alagesan, M.N. Zaatari, Y. Park, J.N. Skepper,
K.F. Macleod, P.A. Perez-Mancera, M.P. Murphy, G.I. Evan, K.H. Vousden,
D.A. Tuveson, Oncogenic KRAS induces NIX-mediated mitophagy to promote pancreatic cancer, Canc. Discov. 9 (2019) 1268e1287.
[65] M. Falcone, O.D.K. Maddocks, The KRAS-BCAA-BCAT2 axis in PDAC devel- opment, Nat. Cell Biol. 22 (2020) 139e140.
[66] S. Yang, X. Wang, G. Contino, M. Liesa, E. Sahin, H. Ying, A. Bause, Y. Li,
J.M. Stommel, G. Dell’antonio, J. Mautner, G. Tonon, M. Haigis, O.S. Shirihai,
C. Doglioni, N. Bardeesy, A.C. Kimmelman, Pancreatic cancers require auto- phagy for tumor growth, Genes Dev. 25 (2011) 717e729.
[67] J.Y. Guo, H.Y. Chen, R. Mathew, J. Fan, A.M. Strohecker, G. Karsli-Uzunbas,
J.J. Kamphorst, G. Chen, J.M. Lemons, V. Karantza, H.A. Coller, R.S. Dipaola,
C. Gelinas, J.D. Rabinowitz, E. White, Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis, Genes Dev. 25 (2011) 460e470.
[68] J. Todoric, L. Antonucci, G. Di Caro, N. Li, X. Wu, N.K. Lytle, D. Dhar,
S. Banerjee, J.B. Fagman, C.D. Browne, A. Umemura, M.A. Valasek, H. Kessler,
D. Tarin, M. Goggins, T. Reya, M. Diaz-Meco, J. Moscat, M. Karin, Stress- activated NRF2-MDM2 cascade controls neoplastic progression in pancreas, Canc. Cell 32 (2017) 824e839, e8.
[69] W. Yao, J.L. Rose, W. Wang, S. Seth, H. Jiang, A. Taguchi, J. Liu, L. Yan,
A. Kapoor, P. Hou, Z. Chen, Q. Wang, L. Nezi, Z. Xu, J. Yao, B. Hu,
P.F. Pettazzoni, I.L. Ho, N. Feng, V. Ramamoorthy, S. Jiang, P. Deng, G.J. Ma,
P. Den, Z. Tan, S.X. Zhang, H. Wang, Y.A. Wang, A.K. Deem, J.B. Fleming,
A. Carugo, T.P. Heffernan, A. Maitra, A. Viale, H. Ying, S. Hanash, R.A. DePinho,
G.F. Draetta, Syndecan 1 is a critical mediator of macropinocytosis in pancreatic cancer, Nature 568 (2019) 410e414.
[70] G.A. Hobbs, N.M. Baker, A.M. Miermont, R.D. Thurman, M. Pierobon,
T.H. Tran, A.O. Anderson, A.M. Waters, J.N. Diehl, B. Papke, R.G. Hodge,
J.E. Klomp, C.M. Goodwin, J.M. DeLiberty, J. Wang, R.W.S. Ng, P. Gautam,
K.L. Bryant, D. Esposito, S.L. Campbell, E.F. Petricoin 3rd, D.K. Simanshu,
A.J. Aguirre, B.M. Wolpin, K. Wennerberg, U. Rudloff, A.D. Cox, C.J. Der, Atypical KRAS(G12R) mutant is impaired in PI3K signaling and macro- pinocytosis in pancreatic cancer, Canc. Discov. 10 (2020) 104e123.
[71] H.T. Li, Y.Y. Lu, Y.X. An, X. Wang, Q.C. Zhao, KRAS, BRAF and PIK3CA muta- tions in human colorectal cancer: relationship with metastatic colorectal cancer, Oncol. Rep. 25 (2011) 1691e1697.
[72] Z.N. Li, L. Zhao, L.F. Yu, M.J. Wei, BRAF and KRAS mutations in metastatic colorectal cancer: future perspectives for personalized therapy, Gastro- enterol. Rep (Oxf). 8 (2020) 192e205.
[73] A.T. Boutin, W.T. Liao, M. Wang, S.S. Hwang, T.V. Karpinets, H. Cheung,
G.C. Chu, S. Jiang, J. Hu, K. Chang, E. Vilar, X. Song, J. Zhang, S. Kopetz,
A. Futreal, Y.A. Wang, L.N. Kwong, R.A. DePinho, Oncogenic KRAS drives in- vasion and maintains metastases in colorectal cancer, Genes Dev. 31 (2017) 370e382.
[74] H. Ebi, R.B. Corcoran, A. Singh, Z. Chen, Y. Song, E. Lifshits, D.P. Ryan,
J.A. Meyerhardt, C. Benes, J. Settleman, K.K. Wong, L.C. Cantley, J.A. Engelman, Receptor tyrosine kinases exert dominant control over PI3K signaling in human KRAS mutant colorectal cancers, J. Clin. Invest. 121 (2011) 4311e4321.
[75] W.D. Roock, D.J. Jonker, F.D. Nicolantonio, A. Sartore-Bianchi, D. Tu, S. Siena,
S. Lamba, S. Arena, M. Frattini, H. Piessevaux, E.V. Cutsem, C.J. O’Callaghan,
S. Khambata-Ford, J.R. Zalcberg, J. Simes, C.S. Karapetis, A. Bardelli, S. Tejpar, Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetux- imab, J. Am. Med. Assoc. 304 (2010) 1812e1820.
[76] S. Tejpar, I. Celik, M. Schlichting, U. Sartorius, C. Bokemeyer, E.V. Cutsem, Association of KRAS G13D tumor mutations with outcome in patients with metastatic colorectal cancer treated with first-line chemotherapy with or without cetuximab, J. Clin. Oncol. 30 (2012) 3570e3577.
[77] D. Llobet, J. Pallares, A. Yeramian, M. Santacana, N. Eritja, A. Velasco,
X. Dolcet, X. Matias-Guiu, Molecular pathology of endometrial carcinoma: practical aspects from the diagnostic and therapeutic viewpoints, J. Clin. Pathol. 62 (2009) 777e785.
[78] K. Banno, M. Yanokura, M. Iida, K. Masuda, D. Aoki, Carcinogenic mechanisms of endometrial cancer: involvement of genetics and epigenetics, J. Obstet. Gynaecol. Res. 40 (2014) 1957e1967.
[79] M. Sideris, E.I. Emin, Z. Abdullah, J. Hanrahan, K.M. Stefatou, V. Sevas, E. Emin,
T. Hollingworth, F. Odejinmi, S. Papagrigoriadis, S. Vimplis, F. Willmott, The role of KRAS in endometrial cancer: a mini-review, Anticancer Res. 39 (2019) 533e539.
[80] J. Xiong, M. He, K. Hansen, C.L. Jackson, V. Breese, M.R. Quddus, C.J. Sung,
M.M. Lomme, W.D. Lawrence, The clinical significance of K-ras mutation in endometrial “surface epithelial changes” and their associated endometrial adenocarcinoma, Gynecol. Oncol. 142 (2016) 163e168.
[81] M. He, C.L. Jackson, R.B. Gubrod, V. Breese, M. Steinhoff, W.D. Lawrence,
J. Xiong, KRAS mutations in mucinous lesions of the uterus, Am. J. Clin. Pathol. 143 (2015) 778e784.
[82] Z. Tu, L. Gui, J. Wang, X. Li, P. Sun, L. Wei, Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor, Gynecol. Oncol. 101 (2006) 274e279.
[83] K.L. Ring, M.S. Yates, R. Schmandt, M. Onstad, Q. Zhang, J. Celestino,
S.Y. Kwan, K.H. Lu, Endometrial cancers with activating KRAS mutations have activated estrogen signaling and paradoxical response to MEK inhibition, Int. J. Gynecol. Canc. 27 (2017) 854e862.
[84]
B.A. Walker, E.M. Boyle, C.P. Wardell, A. Murison, D.B. Begum, N.M. Dahir,
P.Z. Proszek, D.C. Johnson, M.F. Kaiser, L. Melchor, L.I. Aronson, M. Scales,
C. Pawlyn, F. Mirabella, J.R. Jones, A. Brioli, A. Mikulasova, D.A. Cairns,
W.M. Gregory, A. Quartilho, M.T. Drayson, N. Russell, G. Cook, G.H. Jackson,
X. Leleu, F.E. Davies, G.J. Morgan, Mutational spectrum, copy number changes, and outcome: results of a sequencing study of patients with newly diagnosed myeloma, J. Clin. Oncol. 33 (2015) 3911e3920.
[85] S. Weißbach, S.C. Heredia-Guerrero, S. Barnsteiner, L. Großhans, J. Bodem,
H. Starz, C. Langer, S. Appenzeller, S. Knop, T. Steinbrunn, S. Rost, H. Einsele,
R.C. Bargou, A. Rosenwald, T. Stühmer, E. Leich, Exon-4 mutations in KRAS affect MEK/ERK and PI3K/AKT signaling in human multiple myeloma cell lines, Cancers 12 (2020) 455.
[86] T. Steinbrunn, T. Stühmer, S. Gattenlo€hner, A. Rosenwald, A. Mottok,
C. Unzicker, H. Einsele, M. Chatterjee, R.C. Bargou, Mutated RAS and constitutively activated Akt delineate distinct oncogenic pathways, which independently contribute to multiple myeloma cell survival, Blood 117 (2011) 1998e2004.
[87] B. Hoang, L. Zhu, Y. Shi, P. Frost, H. Yan, S. Sharma, S. Sharma, L. Goodglick,
S. Dubinett, A. Lichtenstein, Oncogenic RAS mutations in myeloma cells selectively induce cox-2 expression, which participates in enhanced adhe- sion to fibronectin and chemoresistance, Blood 107 (2006) 4484e4490.
[88] S. Li, J. Fu, J. Yang, H. Ma, D. Bhutani, M.Y. Mapara, C. Marcireau, S. Lentzsch, Targeting the GCK pathway: a novel and selective therapeutic strategy against RAS-mutated multiple myeloma, Blood 137 (2021) 1754e1764.
[89] H. Ayatollahi, A. Tavassoli, A.H. Jafarian, A. Alavi, S. Shakeri, S.F. Shams,
M. Sheikhi, N. Motamedi Rad, M.H. Sadeghian, A. Bahrami, KRAS codon 12 and 13 mutations in gastric cancer in the northeast Iran, Iran, J. Pathol. 13 (2018) 167e172.
[90] R. Jabini, S.A. Eghbali, H. Ayatollahi, M. Sheikhi, M. Farzanehfar, Analysis of KRAS gene mutation associated with helicobacter pylori infection in patients with gastric cancer, Iran, J. Basic Med. Sci. 22 (2019) 529e533.
[91] L.C. Hewitt, Y. Saito, T. Wang, Y. Matsuda, J. Oosting, A.N.S. Silva, H.L. Slaney,
V. Melotte, G. Hutchins, P. Tan, T. Yoshikawa, T. Arai, H.I. Grabsch, KRAS status is related to histological phenotype in gastric cancer: results from a large multicentre study, Gastric Cancer 22 (2019) 1193e1203.
[92] K. Polom, K. Das, D. Marrelli, G. Roviello, V. Pascale, C. Voglino, H. Rho, P. Tan,
F. Roviello, KRAS mutation in gastric cancer and prognostication associated with microsatellite instability status, Pathol. Oncol. Res. 25 (2019) 333e340.
[93] X.H. Fu, Z.T. Chen, W.H. Wang, X.J. Fan, Y. Huang, X.B. Wu, J.L. Huang,
J.X. Wang, H.J. Lin, X.L. Tan, L. Wang, J.P. Wang, KRAS G12V Mutation is an adverse prognostic factor of Chinese gastric cancer patients, J. Canc. 10 (2019) 821e828.
[94] Z. Qian, G. Zhu, L. Tang, M. Wang, L. Zhang, J. Fu, C. Huang, S. Fan, Y. Sun, J. Lv,
H. Dong, B. Gao, X. Su, D. Yu, J. Zang, X. Zhang, J. Ji, Q. Ji, Whole genome gene copy number profiling of gastric cancer identifies PAK1 and KRAS gene amplification as therapy targets, Genes Chromosomes Cancer 53 (2014) 883e894.
[95] V. Marsh, E.J. Davies, G.T. Williams, A.R. Clarke, PTEN loss and KRAS activa- tion cooperate in murine biliary tract malignancies, J. Pathol. 230 (2013) 165e173.
[96] A. Kasuga, T. Semba, R. Sato, H. Nobusue, E. Sugihara, H. Takaishi, T. Kanai,
H. Saya, Y. Arima, Oncogenic KRAS-expressing organoids with biliary epithelial stem cell properties give rise to biliary tract cancer in mice, Canc. Sci. 112 (2021) 1822e1838.
[97] S.R. Hingorani, E.F. Petricoin, A. Maitra, V. Rajapakse, C. King, M.A. Jacobetz,
S. Ross, T.P. Conrads, T.D. Veenstra, B.A. Hitt, Y. Kawaguchi, D. Johann,
L.A. Liotta, H.C. Crawford, M.E. Putt, T. Jacks, C.V. Wright, R.H. Hruban,
A.M. Lowy, D.A. Tuveson, Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse, Canc. Cell 4 (2003) 437e450.
[98] C.E. Clark, S.R. Hingorani, R. Mick, C. Combs, D.A. Tuveson, R.H. Vonderheide, Dynamics of the immune reaction to pancreatic cancer from inception to invasion, Canc. Res. 67 (2007) 9518e9527.
[99] A. Sparmann, D. Bar-Sagi, Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis, Canc. Cell 6 (2004) 447e458.
[100] H. Ji, A.M. Houghton, T.J. Mariani, S. Perera, C.B. Kim, R. Padera, G. Tonon,
K. McNamara, L.A. Marconcini, A. Hezel, N. El-Bardeesy, R.T. Bronson,
D. Sugarbaker, R.S. Maser, S.D. Shapiro, K.K. Wong, K-ras activation generates an inflammatory response in lung tumors, Oncogene 25 (2006) 2105e2112.
[101] S.P. Thayer, M.P. di Magliano, P.W. Heiser, C.M. Nielsen, D.J. Roberts,
G.Y. Lauwers, Y.P. Qi, S. Gysin, C. Ferna´ndez-del Castillo, V. Yajnik, B. Antoniu,
M. McMahon, A.L. Warshaw, M. Hebrok, Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis, Nature 425 (2003) 851e856.
[102] M. Lesina, M.U. Kurkowski, K. Ludes, S. Rose-John, M. Treiber, G. Klo€ppel,
A. Yoshimura, W. Reindl, B. Sipos, S. Akira, R.M. Schmid, H. Algül, Stat3/Socs3 activation by IL-6 transsignaling promotes progression of pancreatic intra- epithelial neoplasia and development of pancreatic cancer, Canc. Cell 19 (2011) 456e469.
[103] C. Charo, V. Holla, T. Arumugam, R. Hwang, P. Yang, R.N. Dubois, D.G. Menter,
C.D. Logsdon, V. Ramachandran, Prostaglandin E2 regulates pancreatic stel- late cell activity via the EP4 receptor, Pancreas 42 (2013) 467e474.
[104] M.P. di Magliano, C.D. Logsdon, Roles for KRAS in pancreatic tumor devel- opment and progression, Gastroenterology 144 (2013) 1220e1229.
[105] Y. Zhang, W. Yan, M.A. Collins, F. Bednar, S. Rakshit, B.R. Zetter, B.Z. Stanger,
I. Chung, A.D. Rhim, M.P. di Magliano, Interleukin-6 is required for pancreatic cancer progression by promoting MAPK signaling activation and oxidative

stress resistance, Canc. Res. 73 (2013) 6359e6374.
[106] M.S. Caetano, H. Zhang, A.M. Cumpian, L. Gong, N. Unver, E.J. Ostrin, S. Daliri,
S.H. Chang, C.E. Ochoa, S. Hanash, C. Behrens, I.I. Wistuba, C. Sternberg,
H. Kadara, C.G. Ferreira, S.S. Watowich, S.J. Moghaddam, IL6 blockade re- programs the lung tumor microenvironment to limit the development and progression of K-ras-mutant lung cancer, Canc. Res. 76 (2016) 3189e3199.
[107] H. Liu, Z. Liang, C. Zhou, Z. Zeng, F. Wang, T. Hu, X. He, X. Wu, X. Wu, P. Lan, Mutant KRAS triggers functional reprogramming of tumor-associated mac- rophages in colorectal cancer, Signal Transduct. Target Ther. 6 (2021) 144.
[108] R.M. Kortlever, N.M. Sodir, C.H. Wilson, D.L. Burkhart, L. Pellegrinet, L. Brown Swigart, T.D. Littlewood, G.I. Evan, Myc cooperates with Ras by programming inflammation and immune suppression, Cell 171 (2017) 1301e1315, e14.
[109] M. Awaji, S. Saxena, L. Wu, D.R. Prajapati, A. Purohit, M.L. Varney, S. Kumar,
S. Rachagani, Q.P. Ly, M. Jain, S.K. Batra, R.K. Singh, CXCR2 signaling promotes secretory cancer-associated fibroblasts in pancreatic ductal adenocarcinoma, Faseb. J. 34 (2020) 9405e9418.
[110] L. Gong, A.M. Cumpian, M.S. Caetano, C.E. Ochoa, M.M.D. la Garza, D.J. Lapid,
S.G. Mirabolfathinejad, B.F. Dickey, Q. Zhou, S.J. Moghaddam, Promoting ef- fect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase, Mol. Canc. 12 (2013) 154.
[111] Y. Pylayeva-Gupta, K.E. Lee, C.H. Hajdu, G. Miller, D. Bar-Sagi, Oncogenic KRAS-induced GM-CSF production promotes the development of pancreatic neoplasia, Canc. Cell 21 (2012) 836e847.
[112] S. Zdanov, M. Mandapathil, R. Abu Eid, S. Adamson-Fadeyi, W. Wilson,
J. Qian, A. Carnie, N. Tarasova, M. Mkrtichyan, J.A. Berzofsky, T.L. Whiteside,
S.N. Khleif, Mutant KRAS conversion of conventional T cells into regulatory T cells, cancer immunol, Res. 4 (2016) 354e365.
[113] S.A. Holstein, R.J. Hohl, Is there a future for prenyltransferase inhibitors in cancer therapy? Curr. Opin. Pharmacol. 12 (2012) 704e709.
[114] A. Matikas, D. Mistriotis, V. Georgoulias, A. Kotsakis, Targeting KRAS mutated non-small cell lung cancer: a history of failures and a future of hope for a diverse entity, Crit. Rev. Oncol. Hematol. 110 (2017) 1d12.
[115] A.G. Stephen, D. Esposito, R.K. Bagni, F. McCormick, Dragging Ras back in the ring, Canc. Cell 25 (2014) 272e281.
[116] J.C. Hunter, D. Gurbani, S.B. Ficarro, M.A. Carrasco, S.M. Lim, H.G. Choi, T. Xie,
J.A. Marto, Z. Chen, N.S. Gray, K.D. Westover, In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 8895e8900.
[117] J. Canon, K. Rex, A.Y. Saiki, C. Mohr, K. Cooke, D. Bagal, K. Gaida, T. Holt,
C.G. Knutson, N. Koppada, B.A. Lanman, J. Werner, A.S. Rapaport, T. San Miguel, R. Ortiz, T. Osgood, J.R. Sun, X. Zhu, J.D. McCarter, L.P. Volak,
B.E. Houk, M.G. Fakih, B.H. O’Neil, T.J. Price, G.S. Falchook, J. Desai, J. Kuo,
R. Govindan, D.S. Hong, W. Ouyang, H. Henary, T. Arvedson, V.J. Cee,
J.R. Lipford, The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity, Nature 575 (2019) 217e223.
[118] B.A. Lanman, J.R. Allen, J.G. Allen, A.K. Amegadzie, K.S. Ashton, S.K. Booker,
J.J. Chen, N. Chen, M.J. Frohn, G. Goodman, D.J. Kopecky, L. Liu, P. Lopez,
J.D. Low, V. Ma, A.E. Minatti, T.T. Nguyen, N. Nishimura, A.J. Pickrell,
A.B. Reed, Y. Shin, A.C. Siegmund, N.A. Tamayo, C.M. Tegley, M.C. Walton,
H.L. Wang, R.P. Wurz, M. Xue, K.C. Yang, P. Achanta, M.D. Bartberger,
J. Canon, L.S. Hollis, J.D. McCarter, C. Mohr, K. Rex, A.Y. Saiki, T. San Miguel,
L.P. Volak, K.H. Wang, D.A. Whittington, S.G. Zech, J.R. Lipford, V.J. Cee, Dis- covery of a covalent inhibitor of KRAS(G12C) (AMG 510) for the treatment of solid tumors, J. Med. Chem. 63 (2020) 52e65.
[119] J.B. Fell, J.P. Fischer, B.R. Baer, J.F. Blake, K. Bouhana, D.M. Briere, K.D. Brown,
L.E. Burgess, A.C. Burns, M.R. Burkard, H. Chiang, M.J. Chicarelli, A.W. Cook,
J.J. Gaudino, J. Hallin, L. Hanson, D.P. Hartley, E.J. Hicken, G.P. Hingorani,
R.J. Hinklin, M.J. Mejia, P. Olson, J.N. Otten, S.P. Rhodes, M.E. Rodriguez,
P. Savechenkov, D.J. Smith, N. Sudhakar, F.X. Sullivan, T.P. Tang, G.P. Vigers,
L. Wollenberg, J.G. Christensen, M.A. Marx, Identification of the clinical development candidate MRTX849, a covalent KRAS(G12C) inhibitor for the treatment of cancer, J. Med. Chem. 63 (2020) 6679e6693.
[120] J.G. Kettle, S.K. Bagal, S. Bickerton, M.S. Bodnarchuk, J. Breed, R.J. Carbajo,
D.J. Cassar, A. Chakraborty, S. Cosulich, I. Cumming, M. Davies, A. Eatherton,
L. Evans, L. Feron, S. Fillery, E.S. Gleave, F.W. Goldberg, S. Harlfinger,
L. Hanson, M. Howard, R. Howells, A. Jackson, P. Kemmitt, J.K. Kingston,
S. Lamont, H.J. Lewis, S. Li, L. Liu, D. Ogg, C. Phillips, R. Polanski, G. Robb,
D. Robinson, S. Ross, J.M. Smith, M. Tonge, R. Whiteley, J. Yang, L. Zhang,
X. Zhao, Structure-based design and pharmacokinetic optimization of co- valent allosteric inhibitors of the mutant GTPase KRAS(G12C), J. Med. Chem. 63 (2020) 4468e4483.
[121] M. P Patricelli, M. R Janes, L.S. Li, R. Hansen, U. Peters, L. V Kessler, Y. Chen,
J. M Kucharski, J. Feng, T. Ely, J. H Chen, S. J Firdaus, A. Babbar, P. Ren, Y. Liu, Selective inhibition of oncogenic KRAS output with small molecules target- ing the inactive state, Canc. Discov. 3 (2016) 316e329.
[122] Q. Sun, J.P. Burke, J. Phan, M.C. Burns, E.T. Olejniczak, A.G. Waterson, T. Lee,
O.W. Rossanese, S.W. Fesik, Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation, Angew. Chem., Int. Ed. Engl. 51 (2012) 6140e6143.
[123] M.R. Janes, J. Zhang, L.S. Li, R. Hansen, U. Peters, X. Guo, Y. Chen, A. Babbar,
S.J. Firdaus, L. Darjania, J. Feng, J.H. Chen, S. Li, S. Li, Y.O. Long, C. Thach, Y. Liu,
A. Zarieh, T. Ely, J.M. Kucharski, L.V. Kessler, T. Wu, K. Yu, Y. Wang, Y. Yao,

X. Deng, P.P. Zarrinkar, D. Brehmer, D. Dhanak, M.V. Lorenzi, D. Hu-Lowe,
M.P. Patricelli, P. Ren, Y. Liu, Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor, Cell 172 (2018) 578e589, e17.
[124] L. Li, H. Zhao, H. Liao, J. Chen, J. Liu, J. Chen, Discovery of novel quinazoline- based covalent inhibitors of KRAS G12C with various cysteine-targeting warheads as potential anticancer agents, Bioorg. Chem. 110 (2021) 104825.
[125] X. Xiao, M. Lai, Z. Song, M. Geng, J. Ding, H. Xie, A. Zhang, Design, synthesis and pharmacological evaluation of bicyclic and tetracyclic pyridopyr- imidinone analogues as new KRASG12C inhibitors, Eur. J. Med. Chem. 213 (2021) 113082.
[126] M. Zeng, Y. Xiong, N. Safaee, R.P. Nowak, K.A. Donovan, C.J. Yuan, B. Nabet,
T.W. Gero, F. Feru, L. Li, S. Gondi, L.J. Ombelets, C. Quan, P.A. J€anne, M. Kostic,
D.A. Scott, K.D. Westover, E.S. Fischer, N.S. Gray, Exploring targeted degra- dation strategy for oncogenic KRAS(G12C), Cell Chem. Biol. 27 (2020) 19e31, e16.
[127] M.J. Bond, L. Chu, D.A. Nalawansha, K. Li, C.M. Crews, Targeted degradation of oncogenic KRAS(G12C) by VHL-recruiting PROTACs, ACS Cent. Sci. 6 (2020) 1367e1375.
[128] T. Tanaka, R.L. Williams, T.H. Rabbitts, Tumour prevention by a single anti- body domain targeting the interaction of signal transduction proteins with RAS, EMBO J. 26 (2007) 3250e3259.
[129] C.E. Quevedo, A. Cruz-Migoni, N. Bery, A. Miller, T. Tanaka, D. Petch,
C.J.R. Bataille, L.Y.W. Lee, P.S. Fallon, H. Tulmin, M.T. Ehebauer, N. Fernandez- Fuentes, A.J. Russell, S.B. Carr, S.E.V. Phillips, T.H. Rabbitts, Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment, Nat. Commun. 9 (2018) 3169.
[130] F. Shima, Y. Yoshikawa, M. Ye, M. Araki, S. Matsumoto, J. Liao, L. Hu,
T. Sugimoto, Y. Ijiri, A. Takeda, Y. Nishiyama, C. Sato, S. Muraoka, A. Tamura,
T. Osoda, K. Tsuda, T. Miyakawa, H. Fukunishi, J. Shimada, T. Kumasaka,
M. Yamamoto, T. Kataoka, In silico discovery of small-molecule Ras in- hibitors that display antitumor activity by blocking the Ras-effector inter- action, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 8182e8187.
[131] M.H. Hofmann, M. Gmachl, J. Ramharter, F. Savarese, D. Gerlach,
J.R. Marszalek, M.P. Sanderson, D. Kessler, F. Trapani, H. Arnhof, K. Rumpel,
D.A. Botesteanu, P. Ettmayer, T. Gerstberger, C. Kofink, T. Wunberg,
A. Zoephel, S.C. Fu, J.L. Teh, J. Bo€ttcher, N. Pototschnig, F. Schachinger,
K. Schipany, S. Lieb, C.P. Vellano, J.C. O’Connell, R.L. Mendes, J. Moll,
M. Petronczki, T.P. Heffernan, M. Pearson, D.B. McConnell, N. Kraut, BI-3406, a potent and selective SOS1-KRAS interaction inhibitor, is effective in KRAS- driven cancers through combined MEK inhibition, Canc. Discov. 11 (2021) 142e157.
[132] T. Maurer, L.S. Garrenton, A. Oh, K. Pitts, D.J. Anderson, N.J. Skelton,
B.P. Fauber, B. Pan, S. Malek, D. Stokoe, M.J. Ludlam, K.K. Bowman, J. Wu,
A.M. Giannetti, M.A. Starovasnik, I. Mellman, P.K. Jackson, J. Rudolph,
W. Wang, G. Fang, Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 5299e5304.
[133] H. Chen, J. B Smaill, T. Liu, K. Ding, X. Lu, Small-Molecule inhibitors directly targeting KRAS as anticancer therapeutics, J. Med. Chem. 63 (2020) 14404e14424.
[134] J. Hallin, L.D. Engstrom, L. Hargis, A. Calinisan, R. Aranda, D.M. Briere,
N. Sudhakar, V. Bowcut, B.R. Baer, J.A. Ballard, M.R. Burkard, J.B. Fell,
J.P. Fischer, G.P. Vigers, Y. Xue, S. Gatto, J. Fernandez-Banet, A. Pavlicek,
K. Velastagui, R.C. Chao, J. Barton, M. Pierobon, E. Baldelli, E.F. Patricoin 3rd,
D.P. Cassidy, M.A. Marx, I.I. Rybkin, M.L. Johnson, S.I. Ou, P. Lito,
K.P. Papadopoulos, P.A. Ja€nne, P. Olson, J.G. Christensen, The KRAS(G12C) inhibitor MRTX849 provides insight toward therapeutic susceptibility of KRAS-mutant cancers in mouse models and patients, Canc. Discov. 10 (2020) 54e71.
[135] M.B. Ryan, F. Fece de la Cruz, S. Phat, D.T. Myers, E. Wong, H.A. Shahzade,
C.B. Hong, R.B. Corcoran, Vertical pathway inhibition overcomes adaptive feedback resistance to KRAS(G12C) inhibition, Clin. Canc. Res. 26 (2020) 1633e1643.
[136] J.Y. Xue, Y. Zhao, J. Aronowitz, T.T. Mai, A. Vides, B. Qeriqi, D. Kim, C. Li, E. de Stanchina, L. Mazutis, D. Risso, P. Lito, Rapid non-uniform adaptation to conformation-specific KRAS(G12C) inhibition, Nature 577 (2020) 421e425.
[137] P.J.R. Ebert, J. Cheung, Y. Yang, E. McNamara, R. Hong, M. Moskalenko,
S.E. Gould, H. Maecker, B.A. Irving, J.M. Kim, M. Belvin, I. Mellman, MAP ki- nase inhibition promotes T cell and anti-tumor activity in combination with PD-L1 checkpoint blockade, Immunity 44 (2016) 609e621.
[138] J.W. Lee, Y. Zhang, K.J. Eoh, R. Sharma, M.F. Sanmamed, J. Wu, J. Choi,
H.S. Park, A. Iwasaki, E. Kaftan, L. Chen, V. Papadimitrakopoulou, R.S. Herbst,
J.S. Koo, The combination of MEK inhibitor with immunomodulatory anti- bodies targeting programmed death 1 and programmed death ligand 1 re- sults in prolonged survival in KRAS/p53-driven lung cancer, J. Thorac. Oncol. 14 (2019) 1046e1060.
[139] L. Liu, P.A. Mayes, S. Eastman, H. Shi, S. Yadavilli, T. Zhang, J. Yang,
L. Seestaller-Wehr, S.Y. Zhang, C. Hopson, L. Tsvetkov, J. Jing, S. Zhang,
J. Smothers, A. Hoos, The BRAF and MEK inhibitors dabrafenib and trame- tinib: effects on immune function and in combination with immunomodu- latory antibodies targeting PD-1, PD-L1, and CTLA-4, Clin. Canc. Res. 21 (2015) 1639e1651.