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Inactive state-selective KRAS(G12C) inhibitors1–8 demonstrate a 30–40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials. Multiple treatment-emergent alterations appear in patients with advanced-stage cancer who were treated with a KRAS inhibitor.

The recently approved KRASG12C inhibitor sotorasib induces durable responses of KRASG12C-mutant non-small cell lung cancers (NSCLCs), however, some patients do not derive benefit. Identification of specific vulnerabilities conferred by co-occurring mutations may enable the development of biomarker-driven combination therapies in distinct subsets of patients. We report that co-occurring loss of STK11/LKB1 is associated with a drug-induced vulnerability of KRAS-mutant NSCLCs to MCL-1 inhibition. In LKB1-deficient cells, inhibition of KRAS-MAPK signaling leads to hyperactivated JNK, which phosphorylates BCL-XL and impairs its ability to sequester BIM, thus creating a dependency on MCL-1 for survival. In LKB1-proficient cells, LKB1 suppresses drug-induced JNK hyperactivation in a NUAK-dependent manner. Ex vivo treatment of tumors from LKB1-deficient but not LKB1 wild-type KRAS-mutant NSCLC patients with sotorasib or trametinib increased MCL-1 dependence. These results uncover a novel role for the LKB1-NUAK axis in regulation of apoptotic dependency and suggest a genotype-directed therapeutic approach for KRAS-LKB1 mutant NSCLC.

Sotorasib is an approved KRASG12C-selective inhibitor for the treatment of KRAS p.G12C-mutant advanced and previously treated non-small cell lung cancers (NSCLC). Acquired resistance due to genomic alterations following sotorasib treatment has been observed in 28% of lung cancer patients (CodeBreaK100, ASCO 2022) and can include bypass signaling, inactivation of negative feedback loops, or compensatory mutations in KRASG12C including amplification of KRAS. Preclinical studies on the mechanisms of acquired resistance are critical for understanding these resistance patterns and can reveal new therapeutic or combination strategies in the clinic. For that reason, we developed an NCI-H358 lung cancer model of acquired resistance to KRASG12C inhibition in vitro. NCI-H358 cells were continuously cultured in the presence of elevated concentrations of sotorasib (IC90) until resistance to KRASG12C inhibitor was achieved. Expression analyses of the resistant cells by ddPCR and immunoblotting showed increases in both KRAS gene and protein levels, resulting in enhanced MAPK signaling. siRNA knockdown of KRAS G12C expression re-sensitized the resistant cells to an analog of sotorasib. Further characterization of this cell line by whole exome sequencing (WES) and fluorescence in situ hybridization (FISH) showed that resistance was not due to genetic co-alterations but amplification of the mutant allele specifically. In comparison to DMSO-treated and parental controls, the sotorasib-resistant NCI-H358 cells contained an approximately 50-fold increase in KRAS G12C mutant allele copy numbers. Preclinical data generated using a KRASG12C inhibitor-resistant lung cancer cell line is consistent with clinical observations of acquired KRAS amplification following KRASG12C inhibitor treatment as a mechanism of resistance. This model further provides an opportunity to study acquired KRAS amplification and investigate combination treatment options in vitro. Citation Format: Deanna Mohn, Anne Y. Saiki, Pragathi Achanta, Andres Plata Stapper, J. Russell Lipford, Rati Verma. KRAS G12C mutant allele amplification drives resistance to sotorasib in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3871.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3–5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking. Treatment of KRASG12C-mutant cancer cells with the KRAS(G12C) inhibitor AMG 510 leads to durable response in mice, and anti-tumour activity in patients suggests that AMG 510 could be effective in patients for whom treatments are currently lacking.

Pim kinases are a family of constitutively active serine/threonine kinases that are partially redundant and regulate multiple pathways important for cell growth and survival. In human disease, high expression of the three Pim isoforms has been implicated in the progression of hematopoietic and solid tumor cancers, which suggests that Pim kinase inhibitors could provide patients with therapeutic benefit. Herein, we describe the structure-guided optimization of a series of quinazolinone-pyrrolodihydropyrrolone analogs leading to the identification of potent pan-Pim inhibitor 28 with improved potency, solubility, and drug-like properties. Compound 28 demonstrated on-target Pim activity in an in vivo pharmacodynamic assay with significant inhibition of BAD phosphorylation in KMS-12-BM multiple myeloma tumors for 16 h postdose. In a 2-week mouse xenograft model, daily dosing of compound 28 resulted in 33% tumor regression at 100 mg/kg.

The recent approval of the KRAS G12C inhibitor sotorasib (AMG 510) for non-small cell lung cancer (NSCLC) marked a milestone in the development of targeted therapies for KRAS mutant cancers. While sotorasib and other KRAS G12C inhibitors have demonstrated rapid and durable responses in the clinic, some patients do not achieve responses. The identification of specific vulnerabilities conferred by recurrent co-occurring mutations may enable the development of biomarker-driven combination therapies with enhanced activity in distinct subsets of patients. We screened a panel of KRAS-mutant NSCLC cell lines as well as patient-derived xenograft (PDX) mouse models and observed that loss of the tumor suppressor STK11/LKB1 is associated with increased sensitivity to combined MAPK (either the KRAS G12C inhibitor sotorasib or MEK inhibitor trametinib) and MCL-1 inhibition (AMG 176). Restoration of LKB1 expression in LKB1-deficient cell lines and PDX tumors blunted the apoptotic response to MAPK + MCL-1 inhibition; conversely, deletion of LKB1 in LKB1 wild-type models increased sensitivity. Mitochondrial apoptotic cell death is regulated by interactions between pro- (e.g., BIM) and anti-apoptotic (e.g., MCL-1, BCL-XL) BCL-2 family members. MAPK inhibition increases BIM, while MCL-1 inhibition prevents BIM sequestration by MCL-1, resulting in apoptosis. LKB1 deficient cells exhibit increased association of BIM and MCL-1 upon MAPK inhibition, effectively priming cells for death upon inhibition of MCL-1. Mechanistically, LKB1 deficiency and associated loss of NUAK phosphorylation leads to hyperactivation of the JNK phospho-kinase network. JNK phosphorylates MCL-1 at S64 and T163, which enhances BIM: MCL-1 protein-protein interaction. Conversely, JNK phosphorylates BCL-XL at S62 and prevents sequestration of BIM. This series of phosphorylation events increases MCL-1 dependence and creates a specific vulnerability of KRAS-LKB1 tumors to MAPK + MCL-1 inhibition. Consistent with this mechanism, ex vivo treatment of tumor tissue from a KRAS-LKB1 mutant NSCLC patient with sotorasib or trametinib increased MCL-1 dependent priming. These results reveal a novel link between LKB1 and the regulation of BCL-2 family proteins and provide preclinical rationale for evaluation of combined KRAS G12C + MCL-1 inhibitors for KRAS-LKB1 mutant NSCLC. Citation Format: Chendi Li, Mohammed Usman Syed, Yi Shen, Audris Oh, Cameron Fraser, Johannes Kreuzer, Christopher Nabel, Kaitlyn Webster, Robert Morris, Sean Caenepeel, Anne Y. Saiki, Karen Rex, J. Russell Lipford, Wilhelm Hass, Kristopher Sarosiek, Paul E. Hughes, Aaron Hata. LKB1 loss rewires JNK-induced apoptotic protein dynamics through NUAKs and sensitizes KRAS-mutant non-small cell lung cancers to combined KRAS G12C + MCL-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2150.

Sotorasib is a first-in class KRAS

BACKGROUND: No therapies for targeting KRAS mutations in cancer have been approved. The KRAS p.G12C mutation occurs in 13% of non–small-cell lung cancers (NSCLCs) and in 1 to 3% of colorectal cancers and other cancers. Sotorasib is a small molecule that selectively and irreversibly targets KRAS(G12C). METHODS: We conducted a phase 1 trial of sotorasib in patients with advanced solid tumors harboring the KRAS p.G12C mutation. Patients received sotorasib orally once daily. The primary end point was safety. Key secondary end points were pharmacokinetics and objective response, as assessed according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. RESULTS: A total of 129 patients (59 with NSCLC, 42 with colorectal cancer, and 28 with other tumors) were included in dose escalation and expansion cohorts. Patients had received a median of 3 (range, 0 to 11) previous lines of anticancer therapies for metastatic disease. No dose-limiting toxic effects or treatment-related deaths were observed. A total of 73 patients (56.6%) had treatment-related adverse events; 15 patients (11.6%) had grade 3 or 4 events. In the subgroup with NSCLC, 32.2% (19 patients) had a confirmed objective response (complete or partial response) and 88.1% (52 patients) had disease control (objective response or stable disease); the median progression-free survival was 6.3 months (range, 0.0+ to 14.9 [with + indicating that the value includes patient data that were censored at data cutoff]). In the subgroup with colorectal cancer, 7.1% (3 patients) had a confirmed response, and 73.8% (31 patients) had disease control; the median progression-free survival was 4.0 months (range, 0.0+ to 11.1+). Responses were also observed in patients with pancreatic, endometrial, and appendiceal cancers and melanoma. CONCLUSIONS: Sotorasib showed encouraging anticancer activity in patients with heavily pretreated advanced solid tumors harboring the KRAS p.G12C mutation. Grade 3 or 4 treatment-related toxic effects occurred in 11.6% of the patients. (Funded by Amgen and others; CodeBreaK100 ClinicalTrials.gov number, NCT03600883.)

KRAS is the most frequently mutated oncogene in cancer and encodes a key signaling protein in tumors. The p.G12C mutation of KRAS is present in approximately 13% of lung adenocarcinoma, 3% of colorectal cancer, and 2% of other solid tumors. Sotorasib (formerly known as AMG 510), the first KRASG12C inhibitor to reach clinical testing in humans, has demonstrated evidence of clinical activity as a single agent in patients with non-small cell lung (NSCLC), colorectal (CRC), endometrial, and appendiceal carcinoma. Preclinically, sotorasib has shown significant tumor growth inhibition as a single agent in multiple CDX and PDX models. The clinically-validated strategy of combining multiple inhibitors in the MAPK pathway suggests that combination strategies could yield even better outcomes for patients. Specifically, the combination of sotorasib and other inhibitors in the MAPK and AKT signaling pathways might further enhance tumor cell killing and overcome potential resistance. To test this hypothesis, in vitro combination experiments were conducted in multiple KRAS p.G12C cell lines with combination matrices of sotorasib and inhibitors of HER kinases, EGFR, SOS1, SHP2, MEK, PI3K, or mTOR, as well as an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6). The combination of sotorasib with multiple agents resulted in robust synergistic cell killing of KRAS p.G12C tumor cells in vitro. To understand whether these observations translated in vivo, we assessed combinations of sotorasib with a SHP-2 inhibitor or a HER kinase inhibitor in pharmacodynamic assays and efficacy models in tumor xenografts. Consistent with the synergy observed in vitro, sotorasib in combination with a HER kinase inhibitor (afatinib) or a SHP2 inhibitor (RMC-4550) in vivo resulted in enhanced inhibition of MAPK signaling as measured by p-ERK in NCI-H358 tumors. In efficacy studies using the NCI-H358 xenograft model, significantly enhanced anti-tumor activity was observed with a minimally efficacious dose of sotorasib in combination with afatinib, RMC-4550, or a CDK4/6 inhibitor (palbociclib). Furthermore, enhanced anti-tumor activity was observed with sotorasib in combination with a MEK inhibitor or with the anti-EGFR monoclonal antibody panitumumab in a CRC KRAS p.G12C PDX model. Taken together, these data support the clinical evaluation of combination treatment of sotorasib with analogous agents in patients with KRAS p.G12C tumors. Citation Format: Karen Rex, Anne Y. Saiki, Tyler Holt, Alla Verlinsky, Patricia L. McElroy, Tao Osgood, Ji-Rong Sun, Marwan G. Fakih, Upendra P. Dahal, Bernd Bruenner, Victor J. Cee, Brian A. Lanman, Jude Canon, J. Russell Lipford. Combination of the KRASG12C inhibitor sotorasib with targeted agents improves anti-tumor efficacy in KRAS p.G12C cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1057.

Sotorasib (formerly known as AMG 510), the first-in-class KRASG12C inhibitor, has demonstrated promising clinical efficacy in KRAS p.G12C mutant cancers. Sotorasib binds to KRASG12C through a unique interaction with a surface groove created by side-chain rotation of histidine 95 (His95). Characterization of sotorasib and other His95-groove binders revealed enhanced potency and selectivity as compared to other KRASG12C inhibitor scaffolds, which bind in the P2 pocket via hydrogen bonding with His95. The novel binding mode of sotorasib also translated to similar biochemical and cellular potencies against both NRASG12C and HRASG12C, which encode leucine and glutamine at position 95, respectively. In contrast, other KRASG12C inhibitor scaffolds demonstrated a dramatic loss of potency against NRASG12C and HRASG12C, suggesting that the alternate residues impacted the binding of these molecules in the P2 pocket. To extend characterization of the cellular effects of RAS ‘His95-groove' binders, we analyzed the expression of major histocompatibility complex (MHC) class I proteins and other inflammatory markers in multiple human and murine KRAS p.G12C cell lines. These studies revealed a partial dependency on the cytosolic DNA-sensing (cGAS/STING) pathway for the effects observed with some markers. Finally, His95-groove binders were evaluated for potential mechanisms of resistance to this class of KRASG12C inhibitors. In the mouse syngeneic Lewis Lung Carcinoma (LL/2) cell line, which carries both KRAS p.G12C and NRAS p.Q61H mutations, intrinsic resistance to KRASG12C inhibition was observed, but combination treatment with the MEK inhibitor trametinib demonstrated synergistic improvement in the effects on viability. MIA PaCa-2 and NCI-H358 models of acquired resistance to KRASG12C inhibition were also developed through long-term exposure to high concentrations of sotorasib. Characterization of these resistant cell lines indicated a requirement for constant exposure to sotorasib and also showed that the resistance was not due to genetic alterations but involved either overexpression of KRAS or bypass signaling through alternative pathways. Taken together, these data demonstrate that His95-binders like sotorasib display superior potency and off-target selectivity, as well as unique activity against all versions of RASG12C. In addition, characterization of potential resistance mechanisms to sotorasib will inform combination strategies in the clinic. Citation Format: Anne Y. Saiki, Deanna Mohn, Yu Li, Tao Osgood, Karen Rex, Hui-Ling Wang, Ivonne Archibeque, Christopher Mohr, Pragathi Achanta, Andres Plata Stapper, Aaron S. Rapaport, Jude Canon, Victor J. Cee, Brian A. Lanman, J. Russell Lipford. In vitro characterization of sotorasib and other RAS ‘His95-groove' binders and investigation of resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1285.

Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and β-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.

Proteolysis targeting chimeras (PROTACs) are bispecific molecules containing a target protein binder and an ubiquitin ligase binder connected by a linker. By recruiting an ubiquitin ligase to a target protein, PROTACs promote ubiquitination and proteasomal degradation of the target protein. The generation of effective PROTACs depends on the nature of the protein/ligase ligand pair, linkage site, linker length, and linker composition, all of which have been difficult to address in a systematic way. Herein, we describe a "click chemistry" approach for the synthesis of PROTACs. We demonstrate the utility of this approach with the bromodomain and extraterminal domain-4 (BRD4) ligand JQ-1 (3) and ligase binders targeting cereblon (CRBN) and Von Hippel-Lindau (VHL) proteins. An AlphaScreen proximity assay was used to determine the ability of PROTACs to form the ternary ligase-PROTAC-target protein complex and a MSD assay to measure cellular degradation of the target protein promoted by PROTACs.

Two recent studies identified CDK12 inhibitors that bind to CDK12–cyclin K complexes and act as molecular glues to stabilize an interaction with the ubiquitin ligase CUL4–DDB1, leading to cyclin K degradation.

The high expression of proviral insertion site of Moloney murine leukemia virus kinases (Pim-1, -2, and -3) in cancers, particularly the hematopoietic malignancies, is believed to play a role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms of Pim protein appear largely redundant in their oncogenic functions. Thus, a pan-Pim kinase inhibitor is highly desirable. However, cell active pan-Pim inhibitors have proven difficult to develop because Pim-2 has a low Km for ATP and therefore requires a very potent inhibitor to effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors. In particular, compound 17 is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing.

KRAS is one of the most frequently mutated oncogenes in human cancer, with KRASp.G12D, p.G12V, and p.G12C constituting the major mutational subtypes across lung, colon, and pancreatic cancers. Despite over three decades of research, indirect approaches targeting KRAS mutant cancers have largely failed to show clinical benefit, and direct approaches have been limited by the apparent “undruggable” nature of KRAS. Recently, remarkable progress has been reported for targeting KRASG12C with small molecules that bind to the shallow, highly flexible P2 pocket of KRAS and form covalent adducts with the adjacent mutant cysteine. This class of inhibitors has demonstrated robust blockade of KRAS signaling and cell viability with substantial selectivity for KRASp.G12C tumors. Clinical validation of this approach is eagerly awaited. We have used iterative covalent library design and screening to develop unique inhibitors of KRASG12C. In one series, co-crystal structures of improved hit molecules revealed that side-chain motion exposed an undescribed shallow groove that was occupied by ligand atoms. This series was optimized to generate inhibitors with promising cellular activities (cellular IC50 ~100-200 nM and selectivity >200-fold vs. non-G12C lines). To further enhance potency, selectivity, and drug-like properties, scaffold hopping was employed. After extensive optimization, potent and selective inhibitors were identified that potently suppressed KRAS-MAPK signaling (cellular phospho-ERK IC50 ~30 nM) and impaired cell viability (IC50 ~2 nM). In mouse xenograft tumor models, these inhibitors induce tumor regressions when dosed orally, once daily at a dose of 30 mg/kg. The results obtained with these inhibitors have critically informed our efforts to develop KRASG12C inhibitors suitable for clinical testing. Citation Format: J. Russell Lipford, Victor Cee, Brian Lanman, Anne Saiki, Karen Rex, Laurie Volak, Roman Shimanovich, Beth Hinkle. Unlocked groove—developing covalent inhibitors of KRASG12C [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA20.

The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to tumorigenesis. As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Herein, we describe our efforts toward the development of a potent, pan-Pim inhibitor. The synthesis and hit-to-lead SAR development from a 3-(pyrazin-2-yl)-1H-indazole derived hit 2 to the identification of a series of potent, pan-Pim inhibitors such as 13o are described.

PIM kinases are a family of Ser/Thr kinases that are implicated in tumorigenesis. The discovery of a new class of PIM inhibitors, 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines, is discussed with optimized compounds showing excellent potency against all three PIM isoforms.

The structure-based design and optimization of a novel series of selective PERK inhibitors are described resulting in the identification of 44 as a potent, highly selective, and orally active tool compound suitable for PERK pathway biology exploration both in vitro and in vivo.

The kinase/endonuclease inositol requiring enzyme 1 (IRE1α), one of the sensors of unfolded protein accumulation in the endoplasmic reticulum that triggers the unfolded protein response (UPR), has been investigated as an anticancer target. We identified potent allosteric inhibitors of IRE1α endonuclease activity that bound to the kinase site on the enzyme. Structure–activity relationship (SAR) studies led to 16 and 18, which were selective in kinase screens and were potent against recombinant IRE1α endonuclease as well as cellular IRE1α. The first X-ray crystal structure of a kinase inhibitor (16) bound to hIRE1α was obtained. Screening of native tumor cell lines (>300) against selective IRE1α inhibitors failed to demonstrate any effect on cellular viability. These results suggest that IRE1α activity is not essential for viability in most tumor cell lines, in vitro, and that interfering with the survival functions of the UPR may not be an effective strategy to block tumorigenesis.

Activating mutations in RAS represent the most common oncogenic driver mutation in cancer. The single amino acid substitution of cysteine for glycine at position 12 (KRASG12C) is frequently found in solid malignancies, particularly in lung adenocarcinoma (~13%), colorectal adenocarcinoma (3%), and pancreatic adenocarcinoma (~1%). Recently it has been demonstrated that KRASG12C can be targeted with covalent small molecule inhibitors which react with the mutant cysteine adjacent to the switch II pocket (SIIP), locking KRAS in its inactive GDP-bound state. We describe here the discovery and in vitro characterization of AMG 510, a covalent inhibitor of KRASG12C possessing potent biochemical and cellular activity, as well as robust in vivo efficacy. AMG 510 inhibited SOS1-catalyzed nucleotide exchange of recombinant mutant KRASG12C/C118A but had minimal effect on KRASC118A, which is wildtype at position 12. The observed rate constant (kinact/Ki) of covalent modification of KRASG12C by AMG 510 was determined biochemically by mass spectrometry as well as in the cellular context (kobs/[I]). Cysteine proteome analysis of cells treated with AMG 510 revealed that only the G12C-containing peptide of KRAS was covalently modified. AMG 510 inhibited KRAS signaling as measured by ERK phosphorylation in all KRAS p.G12C cell lines tested, but did not inhibit phosphorylation of ERK in cell lines lacking the KRAS p.G12C mutation. Cellular occupancy of KRASG12C by AMG 510 was determined by mass spectrometry and correlated well with inhibition of ERK phosphorylation. AMG 510 also selectively impaired the viability of KRAS p.G12C mutant lines. Combination treatment of AMG 510 with inhibitors of other cellular signaling pathways exhibited evidence for synergistic effects on cell viability. Treatment of KRAS p.G12C lines with covalent KRASG12C inhibitors increased the expression of HLA. To test the impact of KRASG12C inhibition on immune surveillance in vivo, we generated a syngeneic tumor cell line that is suitable for testing AMG 510 in combination with checkpoint inhibitor therapies and characterized this line in vitro. AMG 510 is currently being evaluated in a Phase I study in patients with solid tumors harboring KRAS p.G12Cmutations. Citation Format: Anne Y. Saiki, Kevin Gaida, Karen Rex, Pragathi Achanta, Tisha San Miguel, Neelima Koppada, Dhanashri Bagal, Brian A. Lanman, Robert S. Foti, John D. McCarter, Laurie P. Volak, Jude Canon, Victor J. Cee, J. Russell Lipford. Discovery and in vitro characterization of AMG 510–a potent and selective covalent small-molecule inhibitor of KRASG12C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4484.

The RAS gene family encodes the small GTPase proteins NRAS, HRAS, and KRAS, which play an essential role in cellular growth and proliferation. KRAS is one of the most frequently mutated oncogenes in human cancer, with KRAS p.G12D, p.G12V, and p.G12C constituting the major mutational subtypes across lung, colon, and pancreatic cancers. Despite more than three decades of research, indirect approaches targeting KRAS mutant cancers have largely failed to show clinical benefit, and direct approaches have been stymied by the apparently ‘undruggable’ nature of KRAS. Cysteine-12 of KRASG12C has recently emerged as a unique vulnerability in KRAS-mutant cancers, and a small number of cysteine-reactive inhibitory tool molecules have been disclosed. We here report independent efforts to identify cysteine-reactive molecules capable of selectively inhibiting KRASG12C. Through iterative screening and structural biology efforts, we identified a novel Cys12-reactive inhibitor scaffold that derived its potency from occupancy of a previously unknown cryptic pocket induced by side-chain motion of the His95 residue of KRAS. Employing a scaffold-hopping approach, we leveraged knowledge of this cryptic pocket to design a series of N-aryl quinazolin-2(1H)-one-based inhibitors that demonstrated significantly enhanced potency relative to prior tool compounds. Extensive optimization of these leads led to the identification of a highly potent, selective, and well-tolerated inhibitor of KRASG12C, which was nominated for clinical development as AMG 510. In preclinical tumor models, AMG 510 rapidly and irreversibly binds to KRASG12C, providing durable suppression of the mitogen-activated protein kinase (MAPK) signaling pathway. Dosed orally (once daily) as a single agent, AMG 510 is capable of inducing tumor regression in mouse models of KRASG12C cancer. AMG 510 is, to the best of our knowledge, the first direct KRASG12C therapeutic to reach human clinical testing and is currently in a Phase I clinical trial evaluating safety, tolerability, PK, and efficacy in subjects with solid tumors bearing the KRAS p.G12C mutation (NCT03600883). Citation Format: Brian A. Lanman, Jian Jeffrey Chen, Longbin Liu, Patricia Lopez, Alexander J. Pickrell, Anthony B. Reed, Hui-Ling Wang, Pragathi Achanta, Jude Canon, Daniel A. Erlanson, Raymond V. Fucini, Joon Won Jeong, Christopher Mohr, Anne Y. Saiki, Victor J. Cee, J. Russell Lipford, Karen Rex, Laurie P. Volak. Discovery of AMG 510, a first-in-human covalent inhibitor of KRASG12C for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4455.

Somatic activating mutations of RAS family members are tumor driver mutations found in an estimated 21% of all cancers. Oncogenic KRAS mutations at residues G12, G13, and Q61 represent the most common RAS mutations found in solid malignancies. The prevalence of KRAS p.G12C tumors is ~13% of lung adenocarcinoma (including NSCLC), 3% of colorectal carcinoma (CRC), and 1% to 2% of numerous other solid tumors, representing an unmet medical need. We have developed AMG 510, an orally bioavailable, covalent inhibitor of KRASG12C with potent biochemical and cellular activity, and robust in vivo efficacy. AMG 510 inhibited SOS-catalyzed nucleotide exchange of recombinant mutant KRASG12C/C118A but had minimal effect on KRASC118A, which is wildtype at position 12. In cellular assays, AMG 510 covalently modified KRASG12C and inhibited KRASG12C signaling as measured by phosphorylation of ERK1/2 (p-ERK) in all KRAS p.G12C-mutant cell lines tested but did not inhibit p-ERK in cell lines with various other KRAS mutations. AMG 510 also selectively impaired viability of KRAS p.G12C mutant cell lines but did not affect cell lines with other KRAS mutations. In vivo pharmacodynamic assays demonstrated dose- and time-dependent inhibition of KRASG12Csignaling in human pancreatic and NSCLC tumor xenografts. Covalent modification of KRASG12C by AMG 510 was measured by mass spectrometry and correlated with p-ERK inhibition in tumors. AMG 510 significantly inhibited the growth of KRAS p.G12C xenografts and resulted in tumor regression. Combination treatment of AMG 510 with standard-of-care and targeted agents demonstrated enhanced tumor growth inhibition compared to either single agent. In a syngeneic model of KRAS p.G12C mutant cancer, AMG 510 treatment significantly inhibited tumor growth and caused regression. AMG 510 is currently in Phase 1, first-in-human clinical trials for patients with advanced solid tumors harboring a KRAS p.G12C mutation. Citation Format: Karen Rex, Anne Y. Saiki, Ji-Rong Sun, Tyler Holt, Neelima Koppada, Brian A. Lanman, Laurie P. Volak, Robert S. Foti, Victor J. Cee, J. Russell Lipford, Jude Canon. In vivo characterization of AMG 510 - a potent and selective KRASG12Ccovalent small molecule inhibitor in preclinical KRASG12Ccancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3090.

The identification of Pim-1/2 kinase overexpression in B-cell malignancies suggests that Pim kinase inhibitors will have utility in the treatment of lymphoma, leukemia, and multiple myeloma. Starting from a moderately potent quinoxaline-dihydropyrrolopiperidinone lead, we recognized the potential for macrocyclization and developed a series of 13-membered macrocycles. The structure–activity relationships of the macrocyclic linker were systematically explored, leading to the identification of 9c as a potent, subnanomolar inhibitor of Pim-1 and -2. This molecule also potently inhibited Pim kinase activity in KMS-12-BM, a multiple myeloma cell line with relatively high endogenous levels of Pim-1/2, both in vitro (pBAD IC50 = 25 nM) and in vivo (pBAD EC50 = 30 nM, unbound), and a 100 mg/kg daily dose was found to completely arrest the growth of KMS-12-BM xenografts in mice.

The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10Delta cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10Delta) and identified 54 proteins that were uniquely recovered from rpn10Delta cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely in abundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS.

A growing literature points to a fundamental role for the ubiquitin-proteasome degradation system (UPS) in transcription. Four recent publications add significant insight to our understanding of the connections between these processes. Each provides evidence that some aspect of the UPS can stimulate the activity of transcriptional activators. UPS might promote transcription by several mechanisms, and in some cases, even the final step of the UPS - proteolysis - might enhance activator function.

High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers. These findings suggest that inhibition of Pim signaling by a small molecule Pim-1,2 inhibitor could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal starting from the highly Pim-selective indole–thiadiazole compound ( 1 ), which was derived from a nonselective hit identified in a high throughput screening campaign. Optimization of this compound’s potency and its pharmacokinetic properties resulted in the discovery of compound 29 . Cyclopropane 29 was found to exhibit excellent enzymatic potency on the Pim-1 and Pim-2 isoforms ( K i values of 0.55 nM and 0.28 nM, respectively), and found to inhibit the phosphorylation of BAD in the Pim-overexpressing KMS-12 cell line (IC 50 = 150 nM). This compound had moderate clearance and bioavailability in rat (CL = 2.42 L/kg/h; % F = 24) and exhibited a dose-dependent inhibition of p-BAD in KMS-12 tumor pharmacodynamic (PD) model with an EC 50 value of 6.74 μM (18 μg/mL) when dosed at 10, 30, 100 and 200 mg/kg po in mice.

Transactivator-promoter complexes are essential intermediates in the activation of eukaryotic gene expression. Recent studies of these complexes have shown that some are quite dynamic in living cells owing to rapid and reversible disruption of activator-promoter complexes by molecular chaperones, or a slower, ubiquitin-proteasome-pathway-mediated turnover of DNA-bound activator. These mechanisms may act to ensure continued responsiveness of activators to signalling cascades by limiting the lifetime of the active protein-DNA complex. Furthermore, the potency of some activators is compromised by proteasome inhibition, leading to the suggestion that periodic clearance of activators from a promoter is essential for high-level expression. Here we describe a variant of the chromatin immunoprecipitation assay that has allowed direct observation of the kinetic stability of native Gal4-promoter complexes in yeast. Under non-inducing conditions, the complex is dynamic, but on induction the Gal4-promoter complexes 'lock in' and exhibit long half-lives. Inhibition of proteasome-mediated proteolysis had little or no effect on Gal4-mediated gene expression. These studies, combined with earlier data, show that the lifetimes of different transactivator-promoter complexes in vivo can vary widely and that proteasome-mediated turnover is not a general requirement for transactivator function.

Pim-1,-2, and -3 are constitutively active serine-threonine kinases which are partially redundant and regulate multiple pathways important for tumor growth and survival. One or more of the human Pims are over-expressed in multiple hematological tumor types (e.g. multiple myeloma (MM), NHL and AML) and in some solid tumors (e.g. prostate and SCLC). Pim over-expression correlates with malignancy and poor prognosis in several indications. Our goal was to generate a pan Pim kinase inhibitor with acceptable physical chemical properties and in vivo anti-tumor efficacy. Here we present data on two ATP-competitive, orally bioavailable pan Pim inhibitors, Compound I and Compound II. These inhibitors have potent enzymatic and cellular activity, acceptable pharmacokinetic properties (PK) and robust in vivo efficacy. In a kinase enzyme assay Compound I inhibits Pim-1 and Pim-2 activity with 0.4 nM and 0.7 nM IC50s, respectively, while Compound II is even more potent with Pim-1 and Pim-2 IC50s of 0.1 nM/0.1 nM. In a cellular assay which measures inhibition of the Pim downstream substrate phospho-BAD (p-BAD), compounds I and II demonstrate IC50s of 56 and 16 nM, respectively. In an in vivo pharmacodynamic assay (PD) to demonstrate on-target Pim activity, compounds I and II significantly inhibited p-BAD in KMS-12-BM multiple myeloma tumors for 16 hours post dose. Treatment of KMS-12-BM tumor xenografts with Compound I demonstrated robust in vivo anti-tumor efficacy resulting in 23% tumor regression at 50 mg/kg BID and tumor stasis at 100 mg/kg QD. Compound II demonstrated improved PK properties leading to greater anti-tumor efficacy of 33% tumor regression at 100 mg/kg QD and tumor stasis at 50 mg/kg QD. Compound II showed efficacy in an orthotopic model of multiple myeloma and in models of AML and DLBCL. Combination treatment of Compound II and the standard of care Dexamethasone in the multiple myeloma RPMI-8226 xenograft model demonstrated enhanced tumor growth inhibition compared to either single agent activity. In summary, Compound I and II are potent and selective inhibitors of Pim kinases with excellent in vivo properties. Pim kinase inhibitors, either as monotherapy or in combination with dexamethasone, may be effective clinical strategies for certain cancer patients. Citation Format: Bethany Mattson, Christine. E. Sastri, Nadia Guerrero, Dean Hickman, Jie Chen, Tian Wu, Hui-Ling Wang, Andrew Taskar, Brian Lanman, Anthony B. Reed, Jude Canon, J. Russell Lipford, Karen Rex. In vivo development of pan-Pim kinase small molecule inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5398. doi:10.1158/1538-7445.AM2015-5398

The three members of the Pim kinase family, Pim-1, -2, and -3, are established oncogenes and are attractive targets in hematological malignancies. We have developed multiple potent and selective scaffolds of pan-Pim inhibitors with picomolar enzymatic potency and nanomolar cellular potency. Using these agents, we have observed that the abolishment of Pim activity impairs tumor cell viability in multiple settings, both in vitro and in vivo. We have developed numerous assays to measure Pim protein levels and activity, including Pim downstream markers p-PDCD4 and p-BAD, that might be broadly applicable to human tissues. These assays have allowed us to establish a correlation between Pim protein levels and sensitivity to Pim inhibition across multiple tumor settings, in vitro. We have observed that all tested multiple myeloma (MM) cell lines express high levels of Pim-2 protein and that pan-Pim inhibition impairs viability in 90% of these lines and induces apoptosis in a subset. In acute myelogenous leukemia (AML) cell lines, sensitivity to Pim inhibition significantly correlates with Pim-1 protein expression. Numerous diffuse, large B-cell lymphoma (DLBCL) cell lines have high Pim levels and many are sensitive to Pim inhibition. We have also assessed Pim expression and activity in human tumor and normal tissues. Studies performed with myeloma cells isolated from patient bone marrow aspirates have revealed elevated Pim-2 protein levels as well as sensitivity to ex vivo dosing with Pim inhibitors, as evidenced by inhibition of PDCD4 phosphorylation. Primary patient samples from numerous other hematological tumors have also been found to have high Pim-1 or Pim-2 protein levels. To expand the possible utility of Pim inhibitors in the clinic, we have combined our molecules with numerous clinical agents, including dexamethasone, carfilzomib, and PI3K inhibitors, across multiple settings, in vitro. In all indications surveyed, we have observed that the combination of Pim molecules and these agents can lead to synergistic effects on cell viability, apoptosis and pathway signaling. In some cases, cell lines that show mild or no response to either single agent alone are sensitive to combination treatment. Collectively, our data provide a rationale for the development of Pim kinase inhibitors for use either as monotherapy or in combination with other agents in diverse tumor settings. Citation Format: Christine E. Sastri, Nadia Guerrero, Dongyin Yu, Bethany Mattson, Ken Dellamaggiore, Yajing Yang, Paul Hughes, Hui-Ling Wang, Victor Cee, Brian A. Lanman, Liping Pettus, Anthony B. Reed, Bin Wu, Ryan Wurz, Andrew Tasker, Li-Ya Huang, Daniel Branstetter, Karen Rex, Jeffrey Winston, Teresa L. Burgess, Richard Kendall, J Russell Lipford. Characterization of small molecule inhibitors of the PIM kinases in in vitro models of hematological malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5396. doi:10.1158/1538-7445.AM2015-5396

The ubiquitin-proteasome system (UPS) promotes the destruction of target proteins by attaching to them a ubiquitin chain that is recognized by the 26S proteasome. The UPS influences most cellular processes, and its targets include transcriptional activators that are primary determinants of gene expression. Emerging evidence indicates that non-proteolytic functions of the UPS might stimulate transcriptional activity. Here we show that the proteolysis of some transcriptional activators by the UPS can stimulate their function. We focused on the role of UPS-dependent proteolysis in the function of inducible transcriptional activators in yeast, and found that inhibition of the proteasome reduced transcription of the targets of the activators Gcn4, Gal4 and Ino2/4. In addition, mutations in SCF(Cdc4), the ubiquitin ligase for Gcn4 (ref. 5), or mutations in ubiquitin that prevent degradation, also impaired the transcription of Gcn4 targets. These transcriptional defects were manifested despite the enhanced abundance of Gcn4 on cognate promoters. Proteasome inhibition also decreased the association of RNA polymerase II with Gcn4, Gal4 and Ino2/4 targets, as did mutations in SCF(Cdc4) for Gcn4 targets. Expression of a stable phospho-site mutant of Gcn4 (ref. 7) or disruption of the kinases that target Gcn4 for turnover alleviated the sensitivity of Gcn4 activity to defects in the UPS.