RAS Mutant Cancers Research Articles

Dependence of NPPS creates a targetable vulnerability in RAS-mutant cancers.

RAS is the most frequently mutated oncoprotein for cancer driving. Understanding of RAS biology and discovery of druggable lynchpins in RAS pathway is a prerequisite for targeted therapy of RAS-mutant cancers. The recent identification of KRASG12C inhibitor breaks the "undruggable" curse on RAS and has changed the therapy paradigm of KRAS-mutant cancers. However, KRAS mutations, let alone KRASG12C mutation, account for only part of RAS-mutated cancers. Targeted therapies for cancers harboring other RAS mutations remain the urgent need. In this study we explored the pivotal regulatory molecules that allow for broad inhibition of RAS mutants. By comparing the expression levels of nucleotide pyrophosphatase (NPPS) in a panel of cell lines and the functional consequence of increased NPPS expression in RAS-mutant cells, we demonstrated that cancer cells with various kinds of RAS mutations depended on NPPS for growth and survival, and that this dependence conferred a vulnerability of RAS-mutant cancer to treatment of NPPS inhibition. RAS-mutant cells, compared with RAS-wildtype cells, bored and required an upregulation of NPPS. Transcriptomics and metabolomics analyses revealed a NPPS-dependent hyperglycolysis in RAS-mutant cells. We demonstrated that NPPS promoted glucose-derived glycolytic intermediates in RAS-mutant cells by enhancing its interaction with hexokinase 1 (HK1), the enzyme catalyzing the first committed step of glycolysis. Pharmacological inhibition of NPPS-HK1 axis using NPPS inhibitor Enpp-1-IN-1 or HK1 inhibitor 2-deoxyglucose (2-DG), or genetic interfere with NPPS suppressed RAS-mutant cancers in vitro and in vivo. In conclusion, this study reveals an unrecognized mechanism and druggable lynchpin for modulation of pan-mutant-RAS pathway, proposing a new potential therapeutic approach for treating RAS-mutant cancers.

Development and safety of investigational and approved drugs targeting the RAS function regulation in RAS mutant cancers.

The RAS gene family holds a central position in controlling key cellular activities such as migration, survival, metabolism, and other vital biological processes. The activation of RAS signaling cascades is instrumental in the development of various cancers. Although several RAS inhibitors have gained approval from the United States Food and Drug Administration (FDA) for their substantial antitumor effects, their widespread and severe adverse reactions significantly curtail their practical usage in the clinic. Thus, there exists a pressing need for a comprehensive understanding of these adverse events, ensuring the clinical safety of RAS inhibitors through the establishment of precise management guidelines, suitable intermittent dosing schedules, and innovative combination regimens. This review centers on the evolution of RAS inhibitors in cancer therapy, delving into the common adverse effects associated with these inhibitors, their underlying mechanisms, and the potential strategies for mitigation.

IκBα kinase inhibitor BAY 11-7082 promotes anti-tumor effect in RAS-driven cancers

BackgroundOncogenic mutations in the RAS gene are associated with uncontrolled cell growth, a hallmark feature contributing to tumorigenesis. While diverse therapeutic strategies have been diligently applied to treat RAS-mutant cancers, successful targeting of the RAS gene remains a persistent challenge in the field of cancer therapy. In our study, we discover a promising avenue for addressing this challenge.MethodsIn this study, we tested the viability of several cell lines carrying oncogenic NRAS, KRAS, and HRAS mutations upon treatment with IkappaBalpha (IκBα) inhibitor BAY 11-7082. We performed both cell culture-based viability assay and in vivo subcutaneous xenograft-based assay to confirm the growth inhibitory effect of BAY 11-7082. We also performed large RNA sequencing analysis to identify differentially regulated genes and pathways in the context of oncogenic NRAS, KRAS, and HRAS mutations upon treatment with BAY 11-7082.ResultsWe demonstrate that oncogenic NRAS, KRAS, and HRAS activate the expression of IκBα kinase. BAY 11-7082, an inhibitor of IκBα kinase, attenuates the growth of NRAS, KRAS, and HRAS mutant cancer cells in cell culture and in mouse model. Mechanistically, BAY 11-7082 inhibitor treatment leads to suppression of the PI3K-AKT signaling pathway and activation of apoptosis in all RAS mutant cell lines. Additionally, we find that BAY 11-7082 treatment results in the downregulation of different biological pathways depending upon the type of RAS protein that may also contribute to tumor growth inhibition.ConclusionOur study identifies BAY 11-7082 to be an efficacious inhibitor for treating RAS oncogene (HRAS, KRAS, and NRAS) mutant cancer cells. This finding provides new therapeutic opportunity for effective treatment of RAS-mutant cancers.

Clinical and Genomic Landscape of RAS Mutations in Gynecologic Cancers.

We aimed to describe RAS mutations in gynecologic cancers as they relate to clinicopathologic and genomic features, survival, and therapeutic implications. Gynecologic cancers with available somatic molecular profiling data at our institution between February 2010 and August 2022 were included and grouped by RAS mutation status. Overall survival was estimated by the Kaplan-Meier method, and multivariable analysis was performed using the Cox proportional hazard model. Of 3,328 gynecologic cancers, 523 (15.7%) showed any RAS mutation. Patients with RAS-mutated tumors were younger (57 vs. 60 years nonmutated), had a higher prevalence of endometriosis (27.3% vs. 16.9%), and lower grades (grade 1/2, 43.2% vs. 8.1%, all P < 0.0001). The highest prevalence of KRAS mutation was in mesonephric-like endometrial (100%, n = 9/9), mesonephric-like ovarian (83.3%, n = 5/6), mucinous ovarian (60.4%), and low-grade serous ovarian (44.4%) cancers. After adjustment for age, cancer type, and grade, RAS mutation was associated with worse overall survival [hazard ratio (HR) = 1.3; P = 0.001]. Specific mutations were in KRAS (13.5%), NRAS (2.0%), and HRAS (0.51%), most commonly KRAS G12D (28.4%) and G12V (26.1%). Common co-mutations were PIK3CA (30.9%), PTEN (28.8%), ARID1A (28.0%), and TP53 (27.9%), of which 64.7% were actionable. RAS + MAPK pathway-targeted therapies were administered to 62 patients with RAS-mutated cancers. While overall survival was significantly higher with therapy [8.4 years [(95% confidence interval (CI), 5.5-12.0) vs. 5.5 years (95% CI, 4.6-6.6); HR = 0.67; P = 0.031], this effect did not persist in multivariable analysis. RAS mutations in gynecologic cancers have a distinct histopathologic distribution and may impact overall survival. PIK3CA, PTEN, and ARID1A are potentially actionable co-alterations. RAS pathway-targeted therapy should be considered.

Abstract LB053: A novel BRAF-specific allosteric inhibitor that effectively targets acquired RAFi-resistant BRAF(V600E) cancers and RAS-mutated cancers in vitro and in vivo

Abstract Targeting hyperactive RAS-RAF-MEK-ERK signaling for treating cancers has achieved a promising outcomes, and a number of RAS/RAF inhibitors have been applied to clinical cancer treatment. However, these inhibitors have only a short-term efficacy and their effectiveness is abrogated by acquired drug resistance in less than one year. For those resistant/refractory RAS/RAF-mutated cancers, there’s no effective therapeutics available in clinic. In this study, we developed a novel BRAF-specific inhibitor that docks on an allosteric site on BRAF kinase domain. This inhibitor effectively killed both primary and drug-resistant cancer cell lines with RAS mutation or BRAF(V600E) mutation in vitro and inhibited the growth of xenografted cancers derived from these cell lines in vivo without apparent toxicity. Mechanistically, we found that although this inhibitor blocked the catalytic activity of BRAF in vitro kinase assay, it triggered a super high ERK signaling through improving BRAF/CRAF heterodimerization and hence induced cellular apoptosis of cancer cells with RAS mutation or BRAF(V600E) mutation. Surprisingly, there’s no drug resistance occurring when treating cancer cells with RAS mutation or BRAF(V600E) mutation at a low concentration. To further evaluate the efficacy of this BRAF against RAS/RAF-mutated cancers in vivo, we constructed a series of pancreatic patient-derived xenograft cancer models with Kras G12D or G12V mutations, and found that our BRAF inhibitor exhibited a comparable or much better efficacy than Kras G12D-specific inhibitor on theses PDX models. Altogether, our data indicated that our BRAF-specific allosteric inhibitor may serve as a good therapeutic drug for treating BRAF(V600E)/RAS- cancers, particularly those resistant/refractory cancers, though it still need further evaluations via clinical trials. Citation Format: Jiancheng Hu. A novel BRAF-specific allosteric inhibitor that effectively targets acquired RAFi-resistant BRAF(V600E) cancers and RAS-mutated cancers in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB053.

Abstract LB220: Farnesyltransferase inhibitors show synergistic anticancer effects in combination with novel KRAS G12C inhibitors

Abstract Background. Farnesyltransferase inhibitors (FTI) failed as monotherapies for various cancer types until the discovery of their potency in HRAS mutant human head and neck and bladder cancers. On the other hand, RAS mutant cancers have been a great challenge for cancer research until the discoveries and approvals of several KRAS-G12C allele specific inhibitors. However, G12C inhibitors were effective mainly in lung cancer and de novo and acquired resistance prompted the development of various combinational modalities including immunotherapy as well as SOS- and SHP2-inhibitors. Based on in-silico analysis of drug sensitivities of KRAS mutant cancer cell lines treated with various drugs including FTIs, we have explored the possibility of the combination of FTIs with KRAS inhibitors.Methods. Clinically approved farnesyl-transferase inhibitors (tipifarnib and lonafarnib) were combined with, novel KRAS G12C inhibitors (sotorasib and adagrasib) using human lung-, pancreatic and colorectal- adenocarcinoma cells in vitro and in vivo. Antitumoral effects were evaluated for cell proliferation, apoptosis and migratory activity. Mechanisms of action were investigated by immunoblot analyzes of various farnesylated proteins, RAS activation and signaling, videomicroscopy and also by histopathology of xenograft tumors. Results. Synergistic anticancer effects were observed upon the combination of FTIs with G12C inhibitors in KRAS G12C mutant human cancer cell lines in vitro. We found that the combination interfered with the compensatory re-activation of HRAS, farnesylation of RHEB in the PI3K/mTOR pathway and lamin. Furthermore, we observed enhanced efficacy of sotorasib upon combination with tipifarnib in the xenograft models of lung adenocarcinoma affecting mitotic- and apoptotic rates and inducing necrosis.Discussion. Our findings suggest the potential clinical applicability of the combination of KRAS-G12C inhibitors and farnesyl-transferase inhibitors. Furthermore, our preliminary data suggest that the synergistic effect of FTIs on KRAS-G12C inhibitors can be projected to G12D inhibitors as well. Citation Format: Jozsef Timar, Marcell Baranyi, Eszter Molnar, Ivan Randelovic, Mihaly Cserepes, Jozsef Tovari, Balazs Hegedus. Farnesyltransferase inhibitors show synergistic anticancer effects in combination with novel KRAS G12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB220.

Abstract 2494: RAS-Bio a unique pan-cancer biobank for RAS-driven tumors

Abstract Background: RAS-driven cancers represent a significant unmet clinical need, while breakthroughs in treatments targeting KRASG12C driven-NSCLC have invigorated drug-development for other RAS mutant cancers. Most RAS mutant cancers are not driven by KRASG12C, leaving an enormous amount of work needed to establish the contextual treatment vulnerabilities of cancers driven by different RAS mutant subtypes. RAS-Bio was pioneered within The Cancer Research UK Lung Cancer Centre of Excellence in Manchester. RAS-Bio is a uniquely curated resource of unparalleled clinical, genomic, translational and biological detail. RAS-Bio was established with the aim of generating novel model systems along with clinical observation to facilitate RAS precision medicine breakthroughs. Method: RAS-Bio recruits patients affected by epithelial cancers with RAS pathway mutations, aiming to i) optimize sample collection &amp; processing protocols (FFPE/fresh/frozen) for multi-omic profiling, ii) validate preclinical hypothesis-generating findings in a clinical cohort, iii) generate novel RAS-mutant organoid, PDX and other models for analyses and drugging experiments, iv) foster academic/industry collaboration using a unique dataset including demographics, pathology/imaging details, treatment types and survival outcomes. Results: In two years, we have recruited 190 cancer patients with cancers harboring KRASG12C (n=71), KRASG12D(17), KRASG12V(23), KRASG12A (8), KRASG12S (2), KRASG12F (3), KRASG12R (1) and KRASG12E (1). A further 20 harboring KRAS codon 13 mutations and 12 KRASQ61 cancers and an additional 32 patients with mutations in other key signaling nodes of the MAPK pathway have been recruited. Seventeen PDX models have been attempted; lung cancer (14) and colorectal cancer (3). Four lung cancer cases are currently growing (KRASG12C treatment naïve, KRASG12C inhibitor resistance, KRASG12C inhibitor refractory, KRASG13C treatment naïve) and two colorectal cancer cases (KRASG12C and KRASG12V). We will present two deep-dive lung patient cases i) KRASG12C ‘OFF’ state inhibitor (sotorasib) resistance where multiple tissue samples, CTCs and a successful PDX were derived, and ii) sotorasib resistance where paired biopsies and PDXs were derived pre- and post-treatment. Both cases demonstrate the clinical utility of longitudinal sampling in understanding clinically relevant resistance mechanisms to targeted treatment. Conclusion: RAS-Bio represents a comprehensive biobank of clinical, pathological, and genomic detail in RAS-mutant lung cancers. Optimizing collaborative potential with academia/industry to facilitate prospective sampling of patients at different timepoints and integrating colorectal and pancreas cancers in our protocol. Citation Format: Mathew Carter, Katherine D. Brown, Helen Adderley, George Morrissey, Laura Woodhouse, Jamie Weaver, Kathryn Simpson, Jordan Roebuck, Jane Rogan, Joseph Mercer, Anshuman Chaturvedi, David Wedge, Claus Jorgensen, Angeliki Malliri, Caroline Dive, Colin R. Lindsay. RAS-Bio a unique pan-cancer biobank for RAS-driven tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2494.

Abstract 1925: Wild-type RAS signaling is an essential therapeutic target in RAS-mutated cancers

Abstract Single-agent targeting of RAS-mutated cancers using RAF/MEK/ERK or PI3K/AKT pathway inhibitors is ineffective; blocking one pathway relieves negative feedback control of RTK signaling and hyperactivates parallel effector pathways. While combined MEK and PI3K inhibition is effective in preclinical models, toxicity of this combination prevents its clinical use. Alternative strategies for treating RAS-mutated cancers are essential. Therapeutics directly targeting mutated RAS proteins have the potential to spare normal cellular function thereby lessening overall toxicity. Unfortunately, direct RAS inhibition as a monotherapy does not fully block RAS effector signaling. RAS proteins show differential activation of RAF and PI3K pathways: KRAS potently activates RAF but poorly activates PI3K, whereas HRAS effectively activates PI3K but poorly activates RAF. Further, mutant RAS inhibition relieves negative feedback controls leading to rapid hyperactivation of RTK−WT RAS signaling. A more comprehensive understanding of the interplay between mutant RAS and RTK−WT RAS signaling is essential to developing rational therapeutic approaches to treat RAS-mutated cancers. We found that WT RAS enhanced mutant RAS-driven transformation by activating RAS effectors poorly engaged by mutated RAS. Further, inhibition of RAS effectors activated poorly by mutant RAS synergized with and limited resistance to mutant HRAS and KRAS inhibitors. The mutant HRAS inhibitor tipifarnib blocked PI3K signaling and synergized with MEK inhibitors in HRAS-mutated cancer cell lines; covalent KRASG12C inhibitors blocked MEK signaling and synergized with PI3K inhibitors in KRASG12C- mutated cell lines. Synergy between inhibitors of mutant RAS and RAS effectors was dependent on intact RTK/WT RAS signaling and was lost in both RASless or SOSless MEFs. Dual knock-out of WT RAS isoforms in KRAS- and HRAS-mutated cancer cell lines reduced PI3K signaling in KRAS-mutant cell lines and MAPK signaling in HRAS-mutant cell lines and reduced proliferation, confirming our findings in MEF cells. Overall, our data highlight the critical role of WT RAS isoforms in supporting mutant RAS signaling and should be considered when designing combination therapies in RAS-mutated cancers. Citation Format: Nancy E. Sealover, Bridget Finniff, Jacob Hughes, Hyun Lee, Robert L. Kortum. Wild-type RAS signaling is an essential therapeutic target in RAS-mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1925.

Abstract 620: Screening of small-molecule MAPK pathway inhibitors on a panel of 130 human cancer cell lines identifies determinants of response in RAS-mutant subgroups

Abstract Oncogenic mutations in members of the RAS family, HRAS, NRAS, and KRAS, are frequently identified in various cancers and often lead to increased signaling of the mitogen-activated protein kinase (MAPK) pathway. KRAS is the most frequently mutated RAS isoform, with a mutation incidence of &amp;gt;10% across all cancers, and up to 70% and 20% in pancreatic and non-small cell lung cancer (NSCLC), respectively. RAS has long been considered to be undruggable, and direct inhibition of RAS with small molecule inhibitors has only recently been achieved, with the approval of the KRAS G12C-mutant selective inhibitors sotorasib and adagrasib for treatment of NSCLC. Indirect targeting of RAS-mutated cancers by inhibitors of downstream components such as MEK and ERK has shown variable responses. Novel insights into MAPK signaling have renewed interest into targeting the MAPK pathway in RAS-mutant cancer, with pan-RAF dimer inhibitors and dual RAF/MEK inhibitors reaching clinical trials. To improve the chances of success, it is important to determine which patient populations are most likely to respond to these inhibitors. To gain more insight into the cellular targeting of MAPK pathway inhibitors, eighteen inhibitors of the MAPK components KRAS, RAF, MEK, and ERK were profiled on a panel of 130 cell lines in cell viability assays (the Oncolines® panel). The cell lines represented a diverse range of tumor tissues and had different genomic backgrounds. Many of the cell lines had mutations in KRAS, NRAS, or BRAF. Drug responses were determined by exposing the cell lines to nine-point duplicate dose ranges of the inhibitors. Using bioinformatics, cell line responses to the inhibitors were related to genomic and transcriptomic features, to identify determinants of drug response in both tissue-agnostic and tissue-specific contexts. The results of our analyses revealed variable responses of KRAS-mutant cell lines to the inhibitors, reflecting clinical responses. Tissue-focused analyses showed that the different responses could be traced back to the different KRAS amino acid substitutions. For instance, in pancreatic cancer cell lines, the KRAS Q61H mutation predicted increased sensitivity to MAPK pathway inhibitors compared to other KRAS amino acid substitutions. Additionally, in tissue groups with a more heterogenous mutation profile, co-mutations explained the decreased effectivity of MAPK pathway inhibitors in KRAS-mutant cell lines, such as PIK3CA mutations in colorectal cancer cell lines. Lastly, a gene expression-based signature of MAPK activity predicted response to MAPK pathway inhibitors, independent of mutation status. Our findings have uncovered determinants of response to MAPK pathway inhibitors in RAS-mutant cell lines. These determinants can serve as markers to select patients who are most likely to benefit from MAPK inhibitors. Citation Format: Jeffrey J. Kooijman, Awan Al Koerdi, Daphne J. Kluitmans, Esmee van den Bossche, Jelle Dylus, Jeroen A. de Roos, Janneke J. Melis, Guido J. Zaman. Screening of small-molecule MAPK pathway inhibitors on a panel of 130 human cancer cell lines identifies determinants of response in RAS-mutant subgroups [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 620.

Abstract 1654: Anti-tumor activity of orally-available cyclic peptide LUNA18 through direct RAS inhibition in RAS-altered tumors

Abstract RAS is the one of the most frequently altered genes in human cancer and contributes to the growth of tumors. Recently, KRAS G12C inhibitors were approved as the first molecular targeting agents for RAS, but there are still huge unmet medical needs in RAS-mutated cancers because drugs targeting non-KRAS G12C tumors have not yet been approved and efficacy by even approved KRAS G12C inhibitors is not satisfactory. LUNA18 is the cyclic peptide that binds to RAS mutants and wildtype, including KRAS, NRAS and HRAS, thereby inhibiting protein-protein interaction (PPI) between the inactive form of RAS and GEFs (guanine nucleotide exchange factors). LUNA18 decreased the active form of KRAS and phosphorylated ERK and AKT, and cell proliferation in RAS-mutated cancer cells with nM order of cellular IC50s irrespective of tumor types and amino acid substitutions except for Q61 mutants. On the other hand, LUNA18 did not inhibit proliferation of cells with active mutations downstream of RAS (BRAF and MEK), suggesting LUNA18 selectively inhibited growth of RAS-dependent cells. Consistent with in vitro cellular activity, oral administration of LUNA18 showed durable RAS signal inhibition up to 24-48 hours and tumor regression in RAS-mutated xenograft models. In addition to KRAS-mutated cells, LUNA18 also showed in vitro and in vivo robust efficacy in KRAS-amplified cells. These data indicated LUNA18 is the versatile compound that selectively suppressed cellular signaling from RAS G12/G13 mutants as well as RAS wildtype amplification through inhibition of PPI between the inactive form of RAS and GEFs, and it can be applicable to a wide range of RAS-altered tumors. Rebound of the MAPK pathway is reported to be the one of the cause for attenuating efficacy of KRAS G12C inhibitors. We also observed the rebound of ERK activation 24-72 hours after the treatment with a KRAS G12C inhibitor in cells with the KRAS G12C mutation though suppression of KRAS activity was maintained. On the other hand, LUNA18 persistently suppressed ERK activity as well as KRAS activity up to 72 hours. The combination of the KRAS G12C inhibitor with siRNA for RAS wildtype showed durable inhibition of ERK activity, suggesting that RAS wildtype plays a key role for the rebound of ERK activity and inhibitory activity to RAS wildtype by LUNA18 contribute to the persistent inhibition of RAS signaling by LUNA18. We orally administered LUNA18 and a KRAS G12C inhibitor to a xenograft model, which showed temporal response and then acquired resistance to a KRAS G12C inhibitor, and found that LUNA18 significantly suppressed emergence of resistance to a KRAS G12C inhibitor. These results supported that the combination of the KRAS G12C inhibitor with LUNA18 could be a promising option to patients with KRAS G12C mutation by suppressing resistance to a KRAS G12C inhibitor through the suppression of rebound of the MAPK pathway. Citation Format: Hitoshi Sase, Saki Michisaka, Yukako Tachibana, Masami Hasegawa, Toshihiko Fujii, Kana Takei, Tomoko Kanei, Naoaki Murao, Shino Kuramoto, Norihito Shibahara, Atsushi Ohta, Mikimasa Tanada, Takuya Shiraishi, Hitoshi Iikura, Takehisa Kitazawa, Hiroshi Tanaka. Anti-tumor activity of orally-available cyclic peptide LUNA18 through direct RAS inhibition in RAS-altered tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1654.

Abstract 593: DCC-3084, a brain penetrant RAF dimer inhibitor, broadly inhibits BRAF class I, II, and III alterations leading to growth inhibition of intracranially implanted tumors in preclinical models

Abstract Background: Mutations in the RAS/MAPK pathway are a frequent driver of cancer, with oncogenic RAS or RAF mutations occurring in &amp;gt;30% of all cancers. First generation BRAF inhibitors are approved for use for tumors with Class I BRAF mutations (V600X). However, these drugs are not efficacious in RAF dimer mutant and RAS mutant cancers due to paradoxical activation of RAF dimers. Herein, we describe DCC-3084, a brain penetrant, potent and selective investigational switch-control inhibitor of BRAF and CRAF kinases, shown preclinically to target all relevant aberrant signaling mechanisms (monomers, heterodimers and homodimers) and inhibits growth of subcutaneously and intracranially implanted tumors in preclinical models. Methods: Inhibition of RAF kinases was measured using recombinant enzymes. Cellular proliferation was measured using resazurin to monitor cell viability. Inhibition of ERK or RSK phosphorylation was measured by AlphaLISA or ELISA. Pharmacokinetics (PK) in the plasma, brain and CSF compartments were measured following oral dosing in Wistar rats. Subcutaneous or intracranial BRAF mutant mouse xenograft models were used to assess PK, pharmacodynamics (PD), and efficacy. Results: DCC-3084 treatment resulted in potent single-agent inhibition of MAPK pathway signaling, and cellular proliferation in a wide range of Class I, II, III BRAF and BRAF fusion altered cell lines. DCC-3084 was demonstrated to be CNS penetrable and exhibited dose dependent oral exposure with robust inhibition of the MAPK pathway in PK/PD models. Oral treatment of DCC-3084 as a single agent resulted in tumor regression in subcutaneously implanted BRAF Class I and BRAF fusion xenograft models. Additionally, DCC-3084 resulted in tumor regression or growth inhibition in intracranially implanted BRAF Class I and BRAF fusion tumor models. Conclusions: The switch-control inhibitor DCC-3084 broadly inhibits Class I, II and III BRAF mutations, BRAF fusions, and BRAF/CRAF heterodimers leading to tumor regression in preclinical models. DCC-3084 CNS penetration enables potential for further research in brain metastases or primary brain cancer. Citation Format: Gada Al-Ani, Stacie L. Bulfer, Jefferey D. Zwicker, Chase K. Crawley, Kristin M. Elliot, Salim Javed, Qi Groer, Molly M. Hood, Joshua W. Large, Kylie Luther, Yeni K. Romero, Forrest A. Stanley, Daniel C. Tanner, Hanumaiah Telikepalli, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DCC-3084, a brain penetrant RAF dimer inhibitor, broadly inhibits BRAF class I, II, and III alterations leading to growth inhibition of intracranially implanted tumors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 593.

Abstract 1215: DIRAS3 inhibits oncogenic RAS signaling and RAS-dependent cell growth driven by prevalent KRAS hot spot mutations

Abstract RAS mutations are found in 30% of human cancers and are characterized by hotspot mutations at codons G12, G13, and Q61. This results in constitutively activated RAS proteins. Despite recent advances in drug design and the emergence of covalent inhibitors, effectively targeting mutant RAS remains challenging. Our group previously identified the tumor suppressor protein DIRAS3, which directly binds Ras and inhibits its function by disrupting KRAS dimers/nanoclusters and blocking effector activation. DIRAS3 expression reduced cancer cell growth in pancreatic and ovarian cancers. To determine whether DIRAS3 plays a definitive role in inhibiting mutant KRAS, we comprehensively characterized DIRAS3's RAS-inhibitory effects using RASless mouse embryo fibroblast (MEF) cells as a model system lacking endogenous RAS isoforms. We ectopically expressed various KRAS mutations in these cells and then examined DIRAS3's effect on each KRAS mutant clone. We analyzed downstream RAS signaling, colony formation, cell viability, and toxicity. Our results showed that DIRAS3 significantly inhibited RAS-dependent colony formation in KRAS mutant (G12C, G12D, G12V, G13D, G12R, Q61R, Q61L) but not wild-type KRAS clones. However, when we added epidermal growth factor (EGF) to the medium, DIRAS3 expression inhibited wild-type KRAS activity, decreasing colony formation. This suggests that DIRAS3 selectively affects the active, GTP-bound form of KRAS and downstream signaling. Additionally, DIRAS3 expression decreased Erk1/2 phosphorylation in MEF cells with mutant KRAS, but not in the cells with a BRAFV600E mutation. This indicates that DIRAS3 specifically inhibits the KRAS-mediated MEK/ERK signaling, cell proliferation, and cytotoxicity. Moreover, to determine the critical region for DIRAS3's RAS-inhibitory function, we compared wild-type DIRAS3 and deletions (ΔNTE, ΔCTE, ΔNCTE). Our results showed both termini are required to effectively suppress clonogenic growth, emphasizing the requirement for DIRAS3 to anchor to the plasma membrane and interact with the RAS dimer interface to inhibit RAS-dependent tumor growth. In conclusion, our findings underscore the potent and specific inhibitory effects of DIRAS3 on RAS-driven oncogenesis, positioning it as a promising pan-RAS inhibitor for RAS-mutant cancers. Citation Format: Gamze Bildik, Junchen Liu, Weiqun Mao, Hailing Yang, John F. Hancock, Robert C. Bast, Zhen Lu. DIRAS3 inhibits oncogenic RAS signaling and RAS-dependent cell growth driven by prevalent KRAS hot spot mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1215.

Abstract 1947: Studies on pMEK inhibitors for the treatment of RAS-mutant cancers as a single agent or combinations

Abstract RAS mutations are the most common oncogene in human cancers and KRAS has the highest frequency in non-small cell lung cancer (NSCLC) and pancreatic cancer (PDAC). KRAS G12C inhibitors have been approved to treat NSCLC patients. However, drug-resistance is eventually developed for these treatments. Preclinical studies suggested MAPK pathway reactivation is one possible mechanism. Among many MEK inhibitors in clinical development, CH5126766 binds to MEK1 inactive conformation and blocks its phosphorylation and activation. We believe this compound should prevent MAPK “paradoxical activation” commonly observed in most RAF and MEK inhibitors such as vemurafenib and cobimetinib. We studied this compound in various in vitro assays and found its potential in combating the paradoxical activation, which might be the cause of drug-resistance for MAPK signal pathway inhibitors, including RAS-, RAF- MEK- and ERK-inhibitors, as a single agent or in combination with above-mentioned inhibitors. In addition, we are developing a series of compounds based on CH5126766, by improving the inhibition of MEK1/2 kinase activity in addition to its activation. In summary, studies on the pMEK inhibitor demonstrated that this class of compounds might mitigate the MAPK pathway reactivation and prolong drug efficacies in clinical. Detailed research results will be discussed. Citation Format: Xiaobing Lv, Min Xu, Yong Hu, Lanjiao Zhao, Xiuquan Chen, Youqin Chen, Chen Yang, Jizhi Li, Chen Chen. Studies on pMEK inhibitors for the treatment of RAS-mutant cancers as a single agent or combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1947.

Erratum for the Research Article "Rational combination therapy with PARP and MEK inhibitors capitalizes on therapeutic liabilities in RAS mutant cancers," by C. Sun et al.

Erratum for the Research Article "Rational combination therapy with PARP and MEK inhibitors capitalizes on therapeutic liabilities in RAS mutant cancers," by C. Sun et al.

Phase I/II Study of Combined BCL-xL and MEK Inhibition with Navitoclax and Trametinib in KRAS or NRAS Mutant Advanced Solid Tumors.

MEK inhibitors (MEKi) lack monotherapy efficacy in most RAS-mutant cancers. BCL-xL is an anti-apoptotic protein identified by a synthetic lethal shRNA screen as a key suppressor of apoptotic response to MEKi. We conducted a dose escalation study (NCT02079740) of the BCL-xL inhibitor navitoclax and MEKi trametinib in patients with RAS-mutant tumors with expansion cohorts for: pancreatic, gynecologic (GYN), non-small cell lung cancer (NSCLC), and other cancers harboring KRAS/NRAS mutations. Paired pretreatment and day 15 tumor biopsies and serial cell-free (cf)DNA were analyzed. A total of 91 patients initiated treatment, with 38 in dose escalation. Fifty-eight percent had ≥3 prior therapies. A total of 15 patients (17%) had colorectal cancer, 19 (11%) pancreatic, 15 (17%) NSCLC, and 32 (35%) GYN cancers. The recommended phase II dose (RP2D) was established as trametinib 2 mg daily days 1 to 14 and navitoclax 250 mg daily days 1 to 28 of each cycle. Most common adverse events included diarrhea, thrombocytopenia, increased AST/ALT, and acneiform rash. At RP2D, 8 of 49 (16%) evaluable patients achieved partial response (PR). Disease-specific differences in efficacy were noted. In patients with GYN at the RP2D, 7 of 21 (33%) achieved a PR and median duration of response 8.2 months. No PRs occurred in patients with colorectal cancer, NSCLC, or pancreatic cancer. MAPK pathway inhibition was observed in on-treatment tumor biopsies. Reductions in KRAS/NRAS mutation levels in cfDNA correlated with clinical benefit. Navitoclax in combination with trametinib was tolerable. Durable clinical responses were observed in patients with RAS-mutant GYN cancers, warranting further evaluation in this population.

Abstract B089: Oncogenic signaling and responses to treatment in RAS mutant cancers are mutation-specific

Abstract The KRAS, NRAS, and HRAS genes are frequently mutated in cancer, often at hotspots altering codons 12, 13, and 61. Yet how different RAS isoforms and mutations precisely regulate oncogenic signaling is still largely unknown. For example, the role of mutant RAS in driving overactivation of ERK signaling in cancer has long been elucidated, but its involvement in the oncogenic activation of PI3K is still controversial, and different studies have reported conflicting findings regarding the requirement of RAS for physiological and oncogenic PI3K signaling. One of the reasons for this lack of understanding has been the inability to directly target RAS, which has historically been circumvented through genomic perturbation and murine models. However, these approaches are limited because it is not possible to distinguish between the direct effects of RAS on PI3K and compensatory events that occur as an adaptation to the oncogene. With the discovery of RAS inhibitors, we can now directly interrogate the biological effects of RAS isoforms and hotspot mutations in cancer cells with improved time resolution. Here, we use RM-042, a first-in-class broad-spectrum RAS·GTP inhibitor (RASMULTI(ON)), to characterize the features and therapeutic vulnerabilities of mutant RAS signaling in human cancers. Through a detailed biochemical analysis of the signaling response to RAS and MEK inhibition, we reveal that RAS-mediated oncogenic signaling is largely independent of the mutant RAS isoform, rather depending on the mutant codon. Specifically, RASQ61X is exquisitely sensitive to MAPK pathway inhibition, confers near-complete independence of upstream regulation, and is impaired in its signaling to PI3K/Akt/mTOR. Conversely, RASG12X enables oncogenic signaling to both MAPK and PI3K/Akt/mTOR by amplifying upstream input from receptor tyrosine kinases (RTKs). Simultaneous RAS and RTK inhibition selectively impairs the growth of RASG12X-mutant tumors, irrespective of RAS isoform and tumor type, and we investigate the molecular mechanisms behind this synergistic effect. Additionally, we have generated a genetically modified mouse model (GEMM) harboring liver tumors induced by RASG12D or RASQ61R. In cell lines derived from these respective models, RASG12D but not RASQ61R activates PI3K upon growth-factor stimulation. Furthermore, in this system, RASQ61R is less oncogenic than RASG12D, an effect that can be rescued by the concurrent deletion of PTEN. These findings provide mechanistic insights into how mutant RAS interacts with upstream and downstream partners to maximize oncogenic signaling. Furthermore, our results shed light on the selective pressure driving the emergence of RASQ61X mutations as a mechanism of resistance against RTK inhibition: we show that colorectal cancer cells that acquire RASQ61X mutations to overcome EGFR inhibition become more independent of upstream signaling than those that acquire RASG12X. Citation Format: Michelangelo Marasco, Dinesh Kumar, Riccardo Mezzadra, Wei Luan, Ilinca Aricescu, Rona Yaeger, Vladimir Markov, Yu Zhu, Elisa de Stanchina, Scott Lowe, Sandra Misale, Neal Rosen. Oncogenic signaling and responses to treatment in RAS mutant cancers are mutation-specific [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B089.

Mediating kinase activity in Ras-mutant cancer: potential for an individualised approach?

It is widely acknowledged that there is a considerable number of oncogenic mutations within the Ras superfamily of small GTPases which are the driving force behind a multitude of cancers. Ras proteins mediate a plethora of kinase pathways, including the MAPK, PI3K, and Ral pathways. Since Ras was considered undruggable until recently, pharmacological targeting of pathways downstream of Ras has been attempted to varying success, though drug resistance has often proven an issue. Nuances between kinase pathway activation in the presence of various Ras mutants are thought to contribute to the resistance, however, the reasoning behind activation of different pathways in different Ras mutational contexts is yet to be fully elucidated. Indeed, such disparities often depend on cancer type and disease progression. However, we are in a revolutionary age of Ras mutant targeted therapy, with direct-targeting KRAS-G12C inhibitors revolutionising the field and achieving FDA-approval in recent years. However, these are only beneficial in a subset of patients. Approximately 90% of Ras-mutant cancers are not KRAS-G12C mutant, and therefore raises the question as to whether other distinct amino acid substitutions within Ras may one day be targetable in a similar manner, and indeed whether better understanding of the downstream pathways these various mutants activate could further improve therapy. Here, we discuss the favouring of kinase pathways across an array of Ras-mutant oncogenic contexts and assess recent advances in pharmacological targeting of various Ras mutants. Ultimately, we will examine the utility of individualised pharmacological approaches to Ras-mediated cancer.

Ascorbic acid in cancer management – time for a second look

Introduction and aim. Over the past decades, the hypotheses that ascorbic acid (AA) can play a role as an anti-neoplastic therapy have generated many conflicting reports. Despite the controversies, mounting evidence has shown that AA has the potential to play a role as an anti-neoplastic agent. Recent studies have unraveled its pharmacokinetics and various mechanism of action on cancer cells. This has spawned different preclinical studies with reports of good activities against various cancers. Material and methods. A review of the literature regarding ascorbic acid in the management of cancer was performed using the PubMed database. The research was limited to abstracts and available full-text articles. Analysis of the literature. Clinical trials have also demonstrated its safety and tolerability across different dosages. AA has been noted as a multitargeting agent that acts as a pro-oxidative cytotoxic agent, anti-cancer epigenetic regulator and immune modulator. AA has also been shown act synergistically with standard chemotherapy regimens in different cancers. Despite its potentials, phase III clinical trials are seriously lacking. The recent phase III VITALITY study shows that AA may play a role as an adjunct targeted therapy for ras-mutated cancers. Therefore, there is need to for more standardized clinical trials to help identify cancer subtypes and AA combination regimens that can show the most benefits. In this review, the pleiotropic mechanism of action of AA was explored as well as various preclinical and clinical studies in cancer therapy. In addition, recommendations were also made for effective strategies towards an AA and standard cancer regimens in treatment as well as future directions. Ascorbic acid has been shown to induce cell death in various cancer types through different mechanisms of action. Several clinical trials and case reports have shown its efficacy in combination chemotherapy, and the pharmacological route of action can be either intravenous or oral. However, it can impair the actions of some drugs when given in combination. Also, dosage should be determined for maximal pharmacologic action. Conclusion. Ascorbic acid has the potential to provide safe and cost-effective antineoplastic treatment option especially in combination therapy. Its potential needs to be further investigated through clinical trials.

Abstract PR012: Novel strategy for RAS-pathway targeting: Initial results from a phase 1b/2 clinical trial of the oral HDAC inhibitor bocodepsin (OKI-179) combined with binimetinib in patients with RAS-pathway mutated solid tumors and NRAS-mutated melanoma

Abstract Background: Activating RAS pathway mutations occur in &amp;gt; 30% of human cancers, including NRAS mutations in 15-20% of advanced melanoma. MEK inhibitors have modest single agent activity in NRAS-mutated melanoma, showing a 15% overall response rate and 2.8 month progression free survival. Bocodepsin (OKI-179) is a novel Class I-selective, oral histone deacetylase inhibitor (HDACi). The combination of bocodepsin and binimetinib is preferentially synergistic in RAS pathway mutated tumor models, leading to the unique mechanism of double strand DNA breaks and cellular apoptosis. Here we present the initial results of the Nautilus phase 1b/2 trial of bocodepsin and binimetinib (NCT05340621). Materials and Methods: In Phase 1b, patients with previously treated advanced solid tumors with any activating RAS pathway mutation were treated with binimetinib oral 45 mg twice daily with bocodepsin 200 mg (cohort 1) or 300 mg (cohort 2) oral daily 4 days on 3 days off in 28-day cycles. In Phase 2, patients with NRAS-mutated melanoma previously treated with immunotherapy were treated with bocodepsin 300 mg with binimetinib in a Simon optimal 2-stage study with a primary endpoint of objective response rate. Secondary endpoints included safety and PK. Results: As of July 6, 2023, 14 patients were enrolled in Phase 1b in 2 cohorts and across multiple tumor types (colorectal, n=4; melanoma, n=3; NSCLC, n=2; pancreatic, n=2; other, n=3) and RAS-pathway mutations (KRAS, n=9; NRAS, n=3; BRAF, n=1; NF1, n=1). The median age was 70 (41-76) and median number of prior therapies was 6 (1-13). No DLTs were observed. Bocodepsin 300 mg was identified as the RP2D in combination with standard dose binimetinib. The most common treatment-emergent adverse events were consistent with either binimetinib or bocodepsin alone known adverse events and included diarrhea (N=11, 78.57%), nausea (N=9, 64.29%), anemia (N=8, 57.14%), and dermatitis acneiform (N=8, 57.14%). The most common grade 3/4 events included anemia (N=4, 28.57%) and syncope (N=2, 14.29%). Of the 14 evaluable patients in Phase 1, the best response was partial response (PR) in 1 pt (NRAS-mutated melanoma) and stable disease (SD) in 6 pts, with a median time on study of 120 days (50-260). Phase 2 enrollment is ongoing. At the data cutoff, in the 9 response evaluable NRAS melanoma patients (between Ph1 and Ph2), 3 PR and 3 SD were observed. Conclusions: The combination of bocodepsin and binimetinib in patients with RAS-mutated advanced cancers is tolerable with manageable AEs. Initial response data in NRAS melanoma are supportive of potential combinatorial activity of binimetinib and bocodepsin. The data for Phase 2 will be updated at the time of presentation. Citation Format: Rodabe N Amaria, Herbert Duvivier, Katy Tsai, Robert Galamaga, Parisa Momtaz, Evan Pisick, Natalie Langr, Harish Dave, Duncan Walker, Jennifer Diamond, Kevin Litwiler, Ryan Sullivan. Novel strategy for RAS-pathway targeting: Initial results from a phase 1b/2 clinical trial of the oral HDAC inhibitor bocodepsin (OKI-179) combined with binimetinib in patients with RAS-pathway mutated solid tumors and NRAS-mutated melanoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR012.

Macropinocytosis As a Means of Selectively Targeting RAS-Mutant Multiple Myeloma

Introduction Roughly, 50% of multiple myeloma (MM) is driven by mutant RAS, both at presentation and more at relapse. Currently, in MM there are no clinical approaches to target this disease subset and ongoing strategies to inhibit RAS function or downstream effectors have had limited clinical success in other tumors. This has prompted our team to look into other more indirect modes of RAS-selective targeting. Oncogenic RAS-driven cancers use a unique actin-driven endocytosis pathway, termed macropinocytosis (MP), to increase the nutrient supply and sustain proliferation. To create a strategy for targeting RAS mutant MM, we have investigated whether RAS-mutant MM cells undergo MP and whether this process can be exploited as a therapeutic vulnerability, by utilizing this pro-oncogenic biochemical mechanism to selectively deliver a toxin to the MM clone using a MP selective monobody-drug conjugate (MDC). Methods The MDC relies on a proprietary fibronectin-based scaffold (monobody) identified based on its physiochemical properties that facilitates MP selective internalization, and therefore targets RAS-mutant MM tumor cells, with a very favorable pharmacokinetic profile. MMAE was attached to the monobody with a cathepsin-cleavable linker and a maleimide group for conjugation to the C-terminal free cysteine on the monobody protein (MDC-MMAE). MDC-MMAE was pre-clinically evaluated both in-vitro and in-vivo, in human MM cell lines, patient cells and a doxycycline inducible mutant KRAS cell system. Results In-vitro cell line and patient cell studies demonstrated that RAS-mutant MM cells undergo MP whereas wild type (WT) cells do not. To understand the role of mutant RAS in driving uptake of the MDC, a doxycycline-inducible mutant KRAS V12 KMS11 system was established. The functionality of the inducibility of the cell system was verified using protein expression and membrane ruffling in the presence of doxycycline. Fluorescently-labeled monobody (MDC-Cy5.5) and TMR-Dextran underwent a high level of uptake in the presence of induced mutant RAS. In contrast, in conditions where MP was absent, no uptake of MDC-Cy5.5 and TMR-Dextran was seen. Furthermore, when KMS11 KRASV12 and WT KMS11 were treated with MDC-MMAE, a 2-fold increase in cell death was observed in the mutant RAS cell line compared to wild type RAS. In-vivo studies showed that the pharmacological properties of the drug when conjugated to the monobody enabled faster serum clearance compared to antibody drug conjugates (ADCs) and nanomaterials, while maintaining cellular impermeability allowing enhanced drug uptake via MP. Importantly, in-vivo the MDC-MMAE technology had an improved toxicity profile compared to ADC-MMAE, increasing tolerable drug delivery by upwards of 15-fold. To test the anti-tumor effects in-vivo, the L363 (NRAS Q61), WT KMS11, and KMS11 KRASV12 cell lines were subcutaneously injected into MM murine models. After tumors were established, 0.5mg/kg MDC-MMAE was administered every 7 days. Mutant RAS L363 and KMS11 KRASV12 tumors treated with MDC-MMAE exhibited roughly a 6-fold decrease in tumor size after 21 days while WT KMS11 tumors showed no significant difference in tumor volume. MDC-MMAE at 5mg/kg showed complete tumor regression as a single agent in L363 tumors. Improved outcomes were seen with MDC-MMAE in combination with standard of care options at lower doses. Additionally, MDC-MMAE has displayed enhanced immunogenic cell death that has not been seen with other chemotherapeutic options, making it an ideal partner for immunotherapy combination. Conclusion Limited clinical options exist for the treatment of mutant RAS MM tumors. We show that by taking advantage of MP, a process upregulated in RAS-mutant cancers, MDC-MMAE specifically targets and inhibits MM growth and improves survival in murine models. It can also be combined with other MM standard of care drugs and with immunotherapy drugs to have a potentially important clinical effect. In collaboration with Tezcat Biosciences, the MDC-MMAE technology has achieved a phase II SBIR for advancement to clinical application.