The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers.

Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.

Chromatin-bound RB targets promoters, enhancers, and CTCF-bound loci and is redistributed by cell-cycle progression.

The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.

KRASG12C-independent feedback activation of wild-type RAS constrains KRASG12C inhibitor efficacy.

Although KRAS has long been considered undruggable, direct KRASG12C inhibitors have shown promising initial clinical efficacy. However, the majority of patients still fail to respond. Adaptive feedback reactivation of RAS-mitogen-activated protein kinase (MAPK) signaling has been proposed by our group and others as a key mediator of resistance, but the exact mechanism driving reactivation and the therapeutic implications are unclear. We find that upstream feedback activation of wild-type RAS, as opposed to a shift in KRASG12C to its active guanosine triphosphate (GTP)-bound state, is sufficient to drive RAS-MAPK reactivation in a KRASG12C-independent manner. Moreover, multiple receptor tyrosine kinases (RTKs) can drive feedback reactivation, potentially necessitating targeting of convergent signaling nodes for more universal efficacy. Even in colorectal cancer, where feedback is thought to be primarily epidermal growth factor receptor (EGFR)-mediated, alternative RTKs drive pathway reactivation and limit efficacy, but convergent upstream or downstream signal blockade can enhance activity. Overall, these data provide important mechanistic insight to guide therapeutic strategies targeting KRAS.

CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.

We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.

Clinical Acquired Resistance to KRASG12C Inhibition through a Novel KRAS Switch-II Pocket Mutation and Polyclonal Alterations Converging on RAS-MAPK Reactivation.

Mutant-selective KRASG12C inhibitors, such as MRTX849 (adagrasib) and AMG 510 (sotorasib), have demonstrated efficacy in KRAS G12C-mutant cancers, including non-small cell lung cancer (NSCLC). However, mechanisms underlying clinical acquired resistance to KRASG12C inhibitors remain undetermined. To begin to define the mechanistic spectrum of acquired resistance, we describe a patient with KRAS G12C NSCLC who developed polyclonal acquired resistance to MRTX849 with the emergence of 10 heterogeneous resistance alterations in serial cell-free DNA spanning four genes (KRAS, NRAS, BRAF, MAP2K1), all of which converge to reactivate RAS-MAPK signaling. Notably, a novel KRAS Y96D mutation affecting the switch-II pocket, to which MRTX849 and other inactive-state inhibitors bind, was identified that interferes with key protein-drug interactions and confers resistance to these inhibitors in engineered and patient-derived KRAS G12C cancer models. Interestingly, a novel, functionally distinct tricomplex KRASG12C active-state inhibitor RM-018 retained the ability to bind and inhibit KRASG12C/Y96D and could overcome resistance. SIGNIFICANCE: In one of the first reports of clinical acquired resistance to KRASG12C inhibitors, our data suggest polyclonal RAS-MAPK reactivation as a central resistance mechanism. We also identify a novel KRAS switch-II pocket mutation that impairs binding and drives resistance to inactive-state inhibitors but is surmountable by a functionally distinct KRASG12C inhibitor.See related commentary by Pinnelli and Trusolino, p. 1874.This article is highlighted in the In This Issue feature, p. 1861.

Vertical Pathway Inhibition Overcomes Adaptive Feedback Resistance to KRASG12C Inhibition.

PURPOSE: Although KRAS represents the most commonly mutated oncogene, it has long been considered an "undruggable" target. Novel covalent inhibitors selective for the KRASG12C mutation offer the unprecedented opportunity to target KRAS directly. However, prior efforts to target the RAS-MAPK pathway have been hampered by adaptive feedback, which drives pathway reactivation and resistance. EXPERIMENTAL DESIGN: A panel of KRASG12C cell lines were treated with the KRASG12C inhibitors ARS-1620 and AMG 510 to assess effects on signaling and viability. Isoform-specific pulldown of activated GTP-bound RAS was performed to evaluate effects on the activity of specific RAS isoforms over time following treatment. RTK inhibitors, SHP2 inhibitors, and MEK/ERK inhibitors were assessed in combination with KRASG12C inhibitors in vitro and in vivo as potential strategies to overcome resistance and enhance efficacy. RESULTS: We observed rapid adaptive RAS pathway feedback reactivation following KRASG12C inhibition in the majority of KRASG12C models, driven by RTK-mediated activation of wild-type RAS, which cannot be inhibited by G12C-specific inhibitors. Importantly, multiple RTKs can mediate feedback, with no single RTK appearing critical across all KRASG12C models. However, coinhibition of SHP2, which mediates signaling from multiple RTKs to RAS, abrogated feedback reactivation more universally, and combined KRASG12C/SHP2 inhibition drove sustained RAS pathway suppression and improved efficacy in vitro and in vivo. CONCLUSIONS: These data identify feedback reactivation of wild-type RAS as a key mechanism of adaptive resistance to KRASG12C inhibitors and highlight the potential importance of vertical inhibition strategies to enhance the clinical efficacy of KRASG12C inhibitors.See related commentary by Yaeger and Solit, p. 1538.

WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop.

ß-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.

Therapeutic strategies to target RAS-mutant cancers.

RAS genes are the most commonly mutated oncogenes in cancer, but effective therapeutic strategies to target RAS-mutant cancers have proved elusive. A key aspect of this challenge is the fact that direct inhibition of RAS proteins has proved difficult, leading researchers to test numerous alternative strategies aimed at exploiting RAS-related vulnerabilities or targeting RAS effectors. In the past few years, we have witnessed renewed efforts to target RAS directly, with several promising strategies being tested in clinical trials at different stages of completion. Important advances have also been made in approaches designed to indirectly target RAS by improving inhibition of RAS effectors, exploiting synthetic lethal interactions or metabolic dependencies, using therapeutic combination strategies or harnessing the immune system. In this Review, we describe historical and ongoing efforts to target RAS-mutant cancers and outline the current therapeutic landscape in the collective quest to overcome the effects of this crucial oncogene.

ERK/MAPK Signaling Drives Overexpression of the Rac-GEF, PREX1, in BRAF- and NRAS-Mutant Melanoma.

Recently, we identified that PREX1 overexpression is critical for metastatic but not tumorigenic growth in a mouse model of NRAS-driven melanoma. In addition, a PREX1 gene signature correlated with and was dependent on ERK MAPK activation in human melanoma cell lines. In the current study, the underlying mechanism of PREX1 overexpression in human melanoma was assessed. PREX1 protein levels were increased in melanoma tumor tissues and cell lines compared with benign nevi and normal melanocytes, respectively. Suppression of PREX1 by siRNA impaired invasion but not proliferation in vitro PREX1-dependent invasion was attributable to PREX1-mediated activation of the small GTPase RAC1 but not the related small GTPase CDC42. Pharmacologic inhibition of ERK signaling reduced PREX1 gene transcription and additionally regulated PREX1 protein stability. This ERK-dependent upregulation of PREX1 in melanoma, due to both increased gene transcription and protein stability, contrasts with the mechanisms identified in breast and prostate cancers, in which PREX1 overexpression was driven by gene amplification and HDAC-mediated gene transcription, respectively. Thus, although PREX1 expression is aberrantly upregulated and regulates RAC1 activity and invasion in these three different tumor types, the mechanisms of its upregulation are distinct and context dependent. IMPLICATIONS: This study identifies an ERK-dependent mechanism that drives PREX1 upregulation and subsequent RAC1-dependent invasion in BRAF- and NRAS-mutant melanoma. Mol Cancer Res; 14(10); 1009-18. ©2016 AACR.

Targeting RAS-mutant cancers: is ERK the key?

The three RAS genes comprise the most frequently mutated oncogene family in cancer. With significant and compelling evidence that continued function of mutant RAS is required for tumor maintenance, it is widely accepted that effective anti-RAS therapy will have a significant impact on cancer growth and patient survival. However, despite more than three decades of intense research and pharmaceutical industry efforts, a clinically effective anti-RAS drug has yet to be developed. With the recent renewed interest in targeting RAS, exciting and promising progress has been made. In this review, we discuss the prospects and challenges of drugging oncogenic RAS. In particular we focus on new inhibitors of RAS effector signaling and the ERK mitogen-activated protein kinase cascade.