RAF Inhibitors Research Articles

Abstract PR03: Cooperative anti-tumor effects of combined inhibition of KRASG12C plus autophagy in preclinical models of KRASG12C-driven lung cancer

Abstract Approximately 25% of lung cancers are driven by mutationally-activated KRAS. A major obstacle in treating lung cancer is resistance to current therapeutic treatments. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, the twin problems of primary or acquired resistance to direct pharmacological inhibition of KRASG12C remain major obstacles in sustaining the deepest and most durable patient responses. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. Moreover, combined inhibition of RAS pathway signaling plus autophagy had potent anti-tumor effects in preclinical models of melanoma or pancreatic cancer. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagic flux in response to treatment with KRASG12C inhibitors. Moreover, combined treatment of such cells with a specific, selective and potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy, led to superior anti-tumor effects in human cell line xenograft models. Previous work to understand why inhibiting autophagy sensitizes KRAS mutant cancer cells to targeted therapy has been confounded by the use of non-specific lysosomal inhibitors such as hydroxychloroquine. Using selective autophagy inhibitors, such as ULK1/2i, provides a unique opportunity to investigate the precise mechanism as to why inhibition of autophagy sensitizes cells to KRASG12C inhibition. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in regulation of nutrient sensing and autophagy. Patients with KRAS-mutated lung cancer whose tumors also lack LKB1 expression are characterized by aggressive behavior and resistance to standard treatment. In other KRAS-driven cancers, the LKB1>AMPK>ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data suggests that LKB1 is dispensable for sotorasib-induced autophagy in KRASG12C-driven lung cancer cells. To this end, KRASG12C-driven lung cancer cell lines with loss of LKB1 expression significantly increased autophagic flux after treatment with either a KRASG12C inhibitor or inhibitors of RAF>MEK>ERK signaling suggesting that the mechanism(s) of autophagy induction in KRASG12C-driven may be different to those detected in other KRAS mutated cancers. Consequently, we have generated new genetically engineered mouse models of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. Citation Format: Phaedra C. Ghazi, Conan Kinsey, Madhumita Bogdan, Bryan D. Smith, Daniel L. Flynn, Martin McMahon. Cooperative anti-tumor effects of combined inhibition of KRASG12C plus autophagy in preclinical models of KRASG12C-driven lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR03.

Discovering potential inhibitors of Raf proto-oncogene serine/threonine kinase 1: a virtual screening approach towards anticancer drug development

Raf proto-oncogene serine/threonine kinase 1 (RAF1 or c-Raf) is a serine/threonine protein kinase crucial in regulating cell growth, differentiation, and survival. Any disruption or overexpression of RAF1 can result in neoplastic transformation and other disorders such as cardiomyopathy, Noonan syndrome, leopard syndrome, etc. RAF1 has been identified as a potential therapeutic target in drug development against various complex diseases, including cancer, due to its remarkable role in disease progression. Here, we carried out a multitier virtual screening study involving different in-silico approaches to discover potential inhibitors of RAF1. After applying the Lipinski rule of five, we retrieved all phytocompounds from the IMPPAT database based on their physicochemical properties. We performed a molecular docking-based virtual screening and got top hits with the best binding affinity and ligand efficiency. Then we screened out the selected hits using the PAINS filter, ADMET properties, and other druglike features. Eventually, PASS evaluation identifies two phytocompounds, Moracin C and Tectochrysin, with appreciable anti-cancerous properties. Finally, all-atom molecular dynamics simulation (MDS) followed by interaction analysis was performed on the elucidated compounds in complex with RAF1 for 200 ns to investigate their time-evolution dynamics and interaction mechanism. Molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) and Dynamical Cross-Correlation Matrix (DCCM) analyses then followed these results from the simulated trajectories. According to the results, the elucidated compounds stabilize the RAF1 structure and lead to fewer conformational alterations. The results of the current study indicated that Moracin C and Tectochrysin could serve as potential inhibitors of RAF1 after required validation. Communicated by Ramaswamy H. Sarma

Interactome dynamics of RAF1-BRAF kinase monomers and dimers

RAF kinases play major roles in cancer. BRAFV600E mutants drive ~6% of human cancers. Potent kinase inhibitors exist but show variable effects in different cancer types, sometimes even inducing paradoxical RAF kinase activation. Both paradoxical activation and drug resistance are frequently due to enhanced dimerization between RAF1 and BRAF, which maintains or restores the activity of the downstream MEK-ERK pathway. Here, using quantitative proteomics we mapped the interactomes of RAF1 monomers, RAF1-BRAF and RAF1-BRAFV600E dimers identifying and quantifying >1,000 proteins. In addition, we examined the effects of vemurafenib and sorafenib, two different types of clinically used RAF inhibitors. Using regression analysis to compare different conditions we found a large overlapping core interactome but also distinct condition specific differences. Given that RAF proteins have kinase independent functions such dynamic interactome changes could contribute to their functional diversification. Analysing this dataset may provide a deeper understanding of RAF signalling and mechanisms of resistance to RAF inhibitors.

Analysis of the ERK Pathway Cysteinome for Targeted Covalent Inhibition of RAF and MEK Kinases

The ERK pathway is one of the most important signaling cascades involved in tumorigenesis. So far, eight noncovalent inhibitors of RAF and MEK kinases in the ERK pathway have been approved by the FDA for the treatment of cancers; however, their efficacies are limited due to various resistance mechanisms. There is an urgent need to develop novel targeted covalent inhibitors. Here we report a systematic study of the covalent ligandabilities of the ERK pathway kinases (ARAF, BRAF, CRAF, KSR1, KSR2, MEK1, MEK2, ERK1, and ERK2) using constant pH molecular dynamics titration and pocket analysis. Our data revealed that the hinge GK (gate keeper)+3 cysteine in RAF family kinases (ARAF, BRAF, CRAF, KSR1, and KSR2) and the back loop cysteine in MEK1 and MEK2 are reactive and ligandable. Structure analysis suggests that the type II inhibitors belvarafenib and GW5074 may be used as scaffolds for designing pan-RAF or CRAF-selective covalent inhibitors directed at the GK+3 cysteine, while the type III inhibitor cobimetinib may be modified to label the back loop cysteine in MEK1/2. The reactivities and ligandabilities of the remote cysteine in MEK1/2 and the DFG-1 cysteine in MEK1/2 and ERK1/2 are also discussed. Our work provides a starting point for medicinal chemists to design novel covalent inhibitors of the ERK pathway kinases. The computational protocol is general and can be applied to the systematic evaluation of covalent ligandabilities of the human cysteinome.

RAF1 contributes to cell proliferation and STAT3 activation in colorectal cancer independently of microsatellite and KRAS status

More than 30% of all human cancers are driven by RAS mutations and activating KRAS mutations are present in 40% of colorectal cancer (CRC) in the two main CRC subgroups, MSS (Microsatellite Stable) and MSI (Microsatellite Instable). Studies in RAS-driven tumors have shown essential roles of the RAS effectors RAF and specifically of RAF1, which can be dependent or independent of RAF’s ability to activate the MEK/ERK module. In this study, we demonstrate that RAF1, but not its kinase activity, plays a crucial role in the proliferation of both MSI and MSS CRC cell line-derived spheroids and patient-derived organoids, and independently of KRAS mutation status. Moreover, we could define a RAF1 transcriptomic signature which includes genes that contribute to STAT3 activation, and could demonstrate that RAF1 ablation decreases STAT3 phosphorylation in all CRC spheroids tested. The genes involved in STAT3 activation as well as STAT3 targets promoting angiogenesis were also downregulated in human primary tumors expressing low levels of RAF1. These results indicate that RAF1 could be an attractive therapeutic target in both MSI and MSS CRC regardless of their KRAS status and support the development of selective RAF1 degraders rather than RAF1 inhibitors for clinical use in combination therapies.

Abstract 549: Characterization of the selective inhibitory effect of KRas inhibitors in different cellular assay formats

Abstract The proto-oncogene KRas is a well-described small GTPase that functions as a molecular switch for major physiological signaling pathways involved in cell proliferation, differentiation and survival. It has been shown that activating mutations in KRas are among the most common oncogenic drivers of tumorigenesis. Missense mutations of KRas result in constitutive activation due to impaired hydrolysis of GTP which enhances tumor-promoting downstream signaling pathways. Most KRas mutations are located in exon 2 or 3 including the most frequently changed glycine 12 which is present in most pancreatic cancers as well as in colorectal cancers and lung adenocarcinomas. Although direct inhibition of KRas is challenging due to its small size, smooth surface and limited druggable pockets on its surface, first promising drug candidates for selected KRas mutants have been developed in recent years (e.g. Sotorasib, MRTX1133, BI-2852). It was the aim of this study to characterize the selective inhibitory effects of KRas inhibitors in a panel of cancer cell lines harboring different KRas mutations like G12C, G12V or G12D. So, we applied different cellular techniques including phosphorylation assays as well as 2D proliferation and 3D spheroid assays to screen KRas inhibitors for their effect on distinct KRas mutants. Firstly, inhibitors were tested in a cellular assay monitoring phosphorylation of the downstream target ERK at residues Thr202/Tyr204. Analysis applying the cellular pERK AlphaLisa assay showed a high selectivity of several inhibitors for specific KRas mutants, while RAF and MEK inhibitors showed no significant selectivity in the panel of cancer cell lines tested here. Next, KRas inhibitors were tested in a cellular 2D proliferation and a 3D spheroid assay to determine the inhibitory effect on cell growth in both settings. In line with published data, for some inhibitors we could observe a high selectivity for a specific KRas mutant which was even more pronounced in the 3D format as compared to the 2D setup. Most likely due to the fact that 3D growth assays mimic physiological conditions more closely than 2D settings. Interestingly, in the 2D and 3D assays we could not find such a clear inhibitory effect of the MEK and RAF inhibitors as compared to the cellular pERK assay. These results indicate that the described cellular pERK AlphaLisa assay established for selected cancer cell lines could be a useful tool to screen newly developed KRas inhibitors for their selective inhibition of specific KRas mutants. Additionally, the application of not only 2D proliferation but also 3D growth studies can further characterize the inhibitory potential of these KRas inhibitors. Citation Format: Franziska Fimm-Todt, Ezgi Dikici, Katharina Schaich, Oliver Siedentopf, Joachim Lauterwasser, Daniel Feger, Jan Erik Ehlert. Characterization of the selective inhibitory effect of KRas inhibitors in different cellular assay formats [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 549.

Abstract 5734: Designing RAF and MEK inhibitor combinations based on their biochemical properties to effectively target MAPK-driven cancers

Abstract Oncogenic alterations of the RAS/RAF/MEK/ERK signaling pathway drives growth of over 40% of human tumors, frequently due to activating mutations of components of the pathway, such as RAS or BRAF. Targeting the pathway using BRAF inhibitors (BRAFi) in combination with MEK inhibitors (MEKi) is now the standard of care for metastatic melanoma patients harboring the BRAF(V600E) mutation. However, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including those with RAS activating mutations. The effectiveness of current clinical BRAFi relies on the fact that they bind selectively and inhibit the mutationally activated monomeric form of the BRAF(V600E) kinase in tumors but not wild-type BRAF in normal tissue where BRAF signals in its dimeric form. Yet, the development of adaptive resistance, frequently due to RAF dimerization, limits BRAFi therapeutic effectiveness. Another class of BRAFi equipotent for both monomeric and dimeric BRAF have been developed, but are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues as well. Recently, we identified and characterized a novel class of BRAFi that preferentially binds and inhibits dimeric BRAF over monomeric BRAF(V600E) (i.e. RAF dimer-selective inhibitors). Furthermore, we rationally designed a combinatorial approach utilizing a BRAF monomer-selective plus a BRAF dimer-selective inhibitor plus a MEK inhibitor, that potently disrupts the BRAF/MEK complex, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index. This triple combination potently suppressed tumor growth of multiple BRAF(V600E) colorectal cancer and melanoma models resistant to the current clinical BRAFi and MEKi combinations both in vitro and in vivo. To design effective therapeutic approaches for RAS-mutated tumors, we also assessed the effectiveness of RAF and MEK inhibitors belonging to diverse biochemical and structural classes, either alone or in combination in RAS-mutant tumor models and normal cells with wild-type RAS signaling. We found that RAF dimer-selective inhibitors potently antagonized MAPK signaling and growth in various RAS mutant cancer models as compared to normal cells. We found further that RAF and MEK inhibitor combinations belonging to certain biochemical classes resulted in more potent MAPK suppression in RAS-mutant tumor as compared to wild-type RAS normal cells (“therapeutic synergy”) and should be prioritized for clinical testing. In contrast, those combinations which are more potent in normal over tumor cells (“adverse synergy”) are unlikely to be clinically successful. Thus, rationally designed combinations of selected RAFi and MEKi, based on their distinct biochemical properties, can uncover new therapeutic opportunities tailored for more effective targeting of BRAF or RAS mutant tumors. Citation Format: Ana Orive-Ramos, Christos Adamopoulos, Beau Baars, Jason Lam, Ankita Punetha, Jason Huang, Vasileios I. Petrou, Stuart A. Aaronson, Poulikos I. Poulikakos. Designing RAF and MEK inhibitor combinations based on their biochemical properties to effectively target MAPK-driven cancers. [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 5734.

Abstract 3415: Preclinical characterization of a brain penetrant RAF inhibitor, BDTX-4933, targeting oncogenic BRAF Class I/II/III and RAS mutations

Abstract Mutations in BRAF and RAS are often oncogenic and lead to a constitutively active MAPK pathway that promotes aberrant cell proliferation and tumor growth. Currently approved BRAF inhibitors are selective against monomeric BRAF V600 mutants. These drugs are largely inactive against non-V600 dimeric BRAF mutants and have poor brain penetration. Although there is an FDA-approved KRAS G12C mutant-selective inhibitor, there are no approved inhibitors for cancer patients who harbor other (non-G12C) KRAS and NRAS mutations which promote tumor growth likely through constitutively active RAF dimers. There remains a high unmet clinical need for a highly CNS penetrant oral RAF inhibitor that targets a broad spectrum of BRAF mutations and constitutively active RAF dimers without paradoxical activation of the MAPK signaling pathway. BDTX-4933 is a potent, reversible, CNS penetrant RAF MasterKey inhibitor designed to target a large family of oncogenic BRAF mutations including BRAF monomers and RAF dimers. The compound inhibits not only all classes (I, II, and III) of BRAF mutations but also targets constitutively active RAF dimers promoted by upstream oncogenic MAPK pathway alterations, such as RAS mutations. In a panel of cancer cell lines that endogenously express BRAF or RAS mutations, BDTX-4933 demonstrates inhibition of the MAPK pathway signaling without paradoxical activation, resulting in potent inhibition of cellular proliferation. BDTX-4933 shows target engagement, inhibiting ERK phosphorylation, in tumor models in vivo, achieving strong anti-tumor efficacy and tumor regression across tumor models driven by either BRAF or RAS mutations. Furthermore, BDTX-4933 exhibits high CNS exposure leading to dose-dependent tumor growth inhibition, and survival benefit in mice implanted intracranially with xenograft BRAF mutant tumors. BDTX-4933 has a best-in-class profile to treat cancer patients harboring BRAF mutations or RAF dimer-promoting upstream genetic alterations. IND-enabling studies for BDTX-4933 are on-going. Citation Format: Yoon-Chi Han, Pui-Yee Ng, Luisa Shin Ogawa, Shao Ning Yang, Miao Chen, Noboru Ishiyama, Tai-An Lin, Elizabeth Buck. Preclinical characterization of a brain penetrant RAF inhibitor, BDTX-4933, targeting oncogenic BRAF Class I/II/III and RAS mutations [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 3415.

Abstract 1709: MAPK pathway alterations are a targetable vulnerability in bladder cancer

Abstract Bladder cancer (BC) is the fourth most common cancer in males and accounts for about 80,000 new cases in the US. Approximately 25% patients with BC will present with muscle-invasive bladder cancer (MIBC) and for the past several decades the standard-of-care for MIBC and metastatic bladder cancer has been cisplatin-based chemotherapy. More recently, multiple immunotherapeutic agents have been approved but only a subset of patients respond to these treatments and therefore novel therapeutic strategies are needed to expand treatment options and improve clinical outcomes for BC patients. Large-scale genomic studies of MIBC have identified numerous recurrent genomic alterations including mutations, copy number alterations, and chromosomal translocations. Using publicly available patient datasets, we identified ~20% of MIBC patients harboring alterations in genes involved in the mitogen-activated protein kinase (MAPK) pathway and their upstream activators. RAF1 copy number amplification was the most predominant alteration followed by activating HRAS mutations. Leveraging our institutional cohorts of bladder cancer specimens, we defined the frequency of these alterations and validated RAF1 amplification in a subset of cases using fluorescence in situ hybridization. To functionally define the role of MAPK pathway alterations in BC, we tested activity of RAF and MEK inhibitors in BC cell line models with diverse MAPK pathway alterations including RAF1 amplification and HRAS/NRAS mutations. We found that these MAPK pathway altered models displayed increased sensitivity to RAF/MEK inhibition compared to MAPK-unaltered models. We further demonstrated the feasibility of preclinically targeting these alterations in mouse models of BC using cell line and patient-derived-xenografts. Additionally, we tested the impact of MAPK pathway modulation on tumor extrinsic properties including changes in the tumor microenvironment that could potentially affect responses to immunotherapeutic agents. Taken together, we have identified MAPK pathway alterations as a novel dependency in BC that can be targeted with emerging RAF inhibitors. Citation Format: Raie Bekele, Amruta Samant, Timothy Hanlon, Kent Mouw. MAPK pathway alterations are a targetable vulnerability in bladder cancer [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 1709.

Abstract 3878: Targeting Hippo-YAP, BRD4 and RAS-MAPK interplay in lung cancer to forestall drug resistance

Abstract Introduction: Identifying parallel survival pathways or compensatory mechanisms that limit the response to targeted therapy is critical to discover effective combinatorial strategies for the treatment of lung cancer and to prevent emergence of drug resistance Results: Using combination drug screening in a panel of cell line models of non-small cell lung cancer (NSCLC), we identified the tumor suppressor STK11 (aka LKB1) as a molecular biomarker of sensitivity to BET and RAS-MAPK pathway inhibitors combination in NSCLC. We found a small molecule inhibitor of BET (bromodomain and extra terminal) family proteins (BRD4, 3, 2), PLX51107, suppressed BRAF and KRAS mutant NSCLC cell proliferation and in combination with RAS (KRAS G12C), RAF or MEK inhibitor led to pronounced loss in cell viability with potent MYC downregulation and synergistic apoptosis induction in vitro and potent tumor regression or stasis in vivo in several KRAS mutant CDX and PDX mouse models of NSCLC. However, BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 were resistant to BET and RAS, RAF or MEK co-inhibition. To unravel the underlying mechanism of resistance to BET and RAS-MAPK inhibitors upon STK11 loss we modeled STK11 deficiency in BRAF and KRAS mutant NSCLC cells and tumors. We found that STK11 loss creates an unprecedented dependence on YAP/TEAD TFs. STK11 silencing in BRAF and KRAS mutant NSCLC cells resulted in nuclear transfer/activation of the HIPPO pathway effector, YAP, upregulation of the YAP target BIRC5 (aka Survivin) and rescued the synthetic lethality due to BET and RAS, RAF or MEK co-inhibition. Phenocopying STK11 loss, constitutively active YAP (YAP5SA), but not transcriptionally inactive YAP (YAPS94A), mitigated the deleterious effects of BET and RAS, RAF or MEK co-suppression in BRAF and KRAS mutant NSCLC cells with potent upregulation of the pro-survival factor BIRC5. Conversely, we found that co-silencing YAP or TEADs with BETs re-sensitized BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 to BET and RAS-MAPK inhibitors combination and caused synthetic lethality. Conclusion: This study uncovered a functional interaction between BET proteins, STK11, HIPPO-YAP and RAS-MAPK signaling pathways in lung cancer. These results will help identify combinatorial strategies for treating aberrant RAS-MAPK pathway driven lung cancer more effectively and forestall drug resistance. Citation Format: Nilanjana Chatterjee, Victor Olivas, Wei Wu, Ben Powell, Trever Bivona. Targeting Hippo-YAP, BRD4 and RAS-MAPK interplay in lung cancer to forestall drug resistance. [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 3878.

Abstract 674: CRISPR and drug screens identify ERK as the mediator of IFNg-induced melanoma growth inhibition

Abstract Interferon-gamma (IFNg) is critical for the success of immune checkpoint blockade therapy. Disruption of IFNg signaling makes tumors resistant to anti-PD-1/L1 treatment primarily by evasion of the growth inhibition (GI) induced by this cytokine. However, the pathways mediating IFNg-induced GI are largely unknown. We used two independent approaches to delineate the IFNg GI pathway, a chemical genomics screen and a whole genome targeting CRISPR/Cas9 screen in an IFNg GI-sensitive patient-derived melanoma (PDM) cell line. Drug screening revealed that treatment with RAF and ERK inhibitors disrupted IFNg GI in PDM cells. For the CRISPR screen, gene-level analysis showed that the magnitude of enrichment for guide RNAs targeting ERK2 in IFNg-treated cells was comparable to proximal IFNg signaling proteins like JAK1, STAT1, IFNGR2, and JAK2 (>26-fold enrichment for ERK2, p = 2.28e-07). Thus, results from both these experiments converged upon ERK as the primary downstream target of IFNg essential for GI induction. We validated the involvement of ERK by detecting a sustained increase in phospho-ERK1/2 levels following IFNg treatment. In live imaging experiments, we found that blocking ERK activity with the ERK inhibitor Ulixertinib blocks the induction of cell death after IFNg treatment in 17 of 23 (~74%) IFNg-sensitive PDM lines covering all the MAPK mutant and triple wildtype molecular subtypes of melanoma. Decreasing and increasing ERK activity levels using shRNA and overexpression approaches also led to a decrease and increase in IFNg-mediated GI, respectively. We have identified NOXA and DR5 as likely mediators of cell death through the IFNg-ERK GI pathway by comparing gene expression profiles of control and IFNg-treated PDM cells. Finally, phorbol 12-myristate 13-acetate (PMA), a known ERK activator, synergizes with IFNg treatment to induce increased GI in PDM cells (Bliss synergy score 16.87, p = 1.83e-123). In summary, the IFNg-ERK signaling axis mediates cell death downstream of IFNg treatment in melanoma cells. This pathway is active in all melanoma subtypes, making it an attractive target to enhance IFNg GI in tumor cells. Our results provide a new understanding of the IFNg GI pathway that will also be crucial to define mechanisms of GI resistance in tumor cells. Citation Format: Ameya S. Champhekar, Rachel Heymans, Cynthia Gonzalez, Guillem Turon Font, Daniel Karin, June Lee, Robert Damoiseaux, Antoni Ribas. CRISPR and drug screens identify ERK as the mediator of IFNg-induced melanoma growth inhibition [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 674.

Abstract 4803: BMAL2 is a KRAS-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma

Abstract To identify novel drivers of PDA malignancy, we employed a regulatory network analysis, which accurately infers the activity of transcription factors and other regulatory proteins based on the integrated expression of their positive and negative target genes. This highly validated approach enables the identification of the most hyper-activated and hyper-repressed regulatory proteins (i.e. the “master regulators (MRs)”) that drive phenotypic distinctions. We applied this technique to a set of 200 laser capture microdissected human PDA samples as well as 45 low-grade precursors for which we had matched histopathological, clinical, and epidemiological annotation. We identified the MRs associated with four malignancy phenotypes: precursors vs. PDA (initiation), low-grade vs. high grade histopathology (progression), survival post resection, and association with KRAS activity. Integrating across these phenotypes, the top MR of PDA malignancy was found to be BMAL2, a member of the PAS family of bHLH transcription factors. Although the canonical function of BMAL2 is linked to the circadian rhythm protein CLOCK, gene set enrichment analysis highlighted a potential role in hypoxia response. We previously demonstrated that PDA in humans and in the genetically engineered “KPC” mouse model is hypovascularized, hypoperfused, and profoundly hypoxic, with a partial oxygen pressure <1mmHg. Given the close homology of BMAL2 to HIF1B (ARNT) and its potential to heterodimerize with HIF1A, we investigated whether BMAL2 plays a role in the hypoxic response of PDA. Indeed, BMAL2 activity was induced in response to hypoxia and inhibited following treatment with multiple RAF, MEK, and ERK inhibitors, validating its computationally-inferred association with RAS activity. Strikingly, knockout or knockdown of BMAL2 in human PDAC cells led to defects in viability and invasion in the setting of hypoxia. BMAL2 knockout cells lost the ability to induce glycolysis upon exposure to severe hypoxia and this was associated with a loss of expression of the glycolysis enzyme LDHA. A large-scale CRISPR screen found that LDHA was the single most critical gene necessary for viability of PDA cells in the setting of hypoxia. Moreover, the set of inferred transcriptional targets of BMAL2 were highly enriched in hypoxia-responsive proteins, including GLUT1, the second top hit in our hypoxia survival CRISPR screen. Strikingly, knockout of BMAL2 led to a complete loss of HIF1A stabilization in response to hypoxia, consistent with the stabilizing role of HIF1A heterodimerization partners such as HIF1B. By contrast, HIF2A was further upregulated under hypoxia in the setting BMAL2 loss. We conclude that BMAL2 is a key master regulator of hypoxia responses in PDA that serves as a molecular switch between the disparate metabolic roles of HIF1A- and HIF2A-dependent hypoxia responses. This will be further validated in ongoing metabolomic studies Citation Format: Alvaro Curiel Garcia, Carlo H. Maurer, Kate Hollinshead, Pasquale Laise, Anthony Andren, Stephen A. Sastra, Carmine F. Palermo, Irina R. Sagalovskiy, Li Zhang, Sam Holmstrom, Kristen Johnson, Gulam A. Manji, Alina Luga, Costas A. Lyssiotis, Andrea Califano, Alec Kimmelman, Kenneth P. Olive. BMAL2 is a KRAS-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma. [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 4803.

Abstract 2: Vertical MAPK pathway targeting in novel genetically engineered mouse and cell line models of NF1-altered melanoma: the mSK-Mel murine cohort

Abstract Large scale clinical genomic sequencing efforts have revealed inactivating mutations in the RAS-GTPase Neurofibromin 1 (NF1) in a significant subset of melanomas. To date, immunotherapy and MAPK pathway-directed targeted therapies have been largely inactive in this molecularly defined cohort and immunogenic models that reflect the distinct co-mutation patterns found in NF1-mutant melanoma patients are lacking. Leveraging a population-scale tumor genomic profiling initiative, we identified TP53 as a gene significantly co-altered with NF1 in melanoma. We thus generated and molecularly characterized a cohort of genetically engineered mice with targeted deletion of NF1 in melanocytes. Melanocyte-specific, homozygous knockout of NF1 induced hyperpigmentation yet was insufficient for tumorigenesis. Addition of TP53 knockout and/or conditional activating mutation in BRAF (BRAFVE), resulted in melanoma formation with variable and high penetrance, respectively, along with histologic features consistent with human melanomas. Tumor latency and overall survival in NF1/TP53 double knockout mice was similar to NF1/BRAFVE double mutants. NF1 knockout did not shorten the latency to tumor formation in the setting of BRAFVE/TP53 mutation but did intensify melanocytic hyperpigmentation in all genetic backgrounds tested. To facilitate preclinical and functional studies, we derived 22 congenic cell lines from harvested mouse tumors from NF1 knockout mice, with and without BRAFVE mutation, and tested their sensitivity to targeted agents. As expected, loss of NF1 conditioned the response to BRAF inhibition, while NF1-mutant cells retained sensitivity to MEK inhibition. To abrogate the effects of adaptive RAS reactivation after MEK inhibitor therapy, combined MEK/SHP inhibition in NF1/TP53 knockout cells and BRAF/SHP inhibition in NF1/TP53/BRAFVE mutant cells strongly blunted ERK phosphorylation and cell proliferation better than single agent therapy. However, this response to the addition of SHP inhibition was transient and ERK rebound was driven by continued MEK activation and dependance. In syngeneic xenograft models of NF1/TP53/BRAFVE mutation, MEK inhibition alone, or in combination with RAF and/or SHP inhibition, induced tumor regression and delayed the onset of resistance and progression as compared to doublet RAF/SHP inhibitor therapy. Overall, we demonstrated the efficacy and feasibility of vertical MAPK pathway targeting in a novel cohort of genetically relevant mouse and cell line models of NF1-mutant melanoma and provide justification for future studies of vertical MAPK pathway targeting to achieve maximal ERK pathway inhibition in this molecularly defined patient cohort. Citation Format: Alexis M. Jones, Aphrothiti J. Hanrahan, Moriah H. Nissan, Sebastien Monette, Ziyu Chen, Wenhuo Hu, Sandra Misale, Isabell Schulze, Naresh Vasani, Cailian Liu, Xia Yang, Mohsen Abu-Akeel, Elisa de Stanchina, Nikolaus Schultz, Michael F. Berger, Neal Rosen, Taha Merghoub, David B. Solit. Vertical MAPK pathway targeting in novel genetically engineered mouse and cell line models of NF1-altered melanoma: the mSK-Mel murine cohort [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 2.

Abstract 570: Dual acting bone defender agents inhibit prostate cancer-mediated bone destruction

Abstract Introduction: Bone metastasis is clinically observed in approximately 90% of patients with advanced prostate cancer (PCa). Metastatic bone destruction is driven by a self-reinforcing process coupling osteoclast (OC)-mediated bone degradation to PCa growth, and where cell movement acts to keep these two cooperating cell types in close proximity and to move them across the bone surface. We sought to create a new class of therapeutics that would be highly effective at inhibiting PCa-mediated bone destruction by targeting both OC function and cell motility. Methods: We synthesized new chemical entities, termed Dual Acting Bone Defender agents (DABDs), that couple the small molecule anti-motility agent, KBU2046, with bisphosphonate (bis), through a chemical linker. Assays for in vitro efficacy included: cell migration and invasion, binding to bone mineral, inhibiting osteoclast (OC)-mediated bone destruction, inhibition of Raf1 phosphorylation and inhibition of protein prenylation. In vivo assays included intra-cardiac (IC) injection of human PCa cells into mice, followed by real time monitoring of tumor growth and bone destruction by IVIS and CT imaging, respectively. Findings: We successfully created a novel route of chemical synthesis allowing us to access and probe unique chemical space, providing for single small molecules with bis functional groups linked to a KBU2046 core through a linker moiety. DABDs retain the cellular and molecular characteristics of KBU2046 and bis. Like KBU2046, they inhibit PCa cell motility and phosphorylation of Raf1 on its activation motif. They also inhibit the motility of breast and lung cancer cells. Like bis, they inhibit OC-mediated bone mineral destruction, prenylation of Ras small GTPase proteins, and chemically bind hydroxyapatite. Administered prior to IC injection, DABDs are so effective that they prolong animal survival and inhibit bone destruction in a dose-dependent manner. In mice with established bone metastasis to the femur, after IC injection, DABDs inhibit the growth of tumors. Doses 3,000 fold higher than that required for efficacy had no clinical or critical organ toxicity. Conclusions: DABDs represent a new multifunctional class of therapeutic agents that inhibit PCa-mediated bone destruction and have a high potential for advancing clinical efficacy in PCa patients with bone metastasis. Citation Format: Fangfang Qiao, Ryan Gordon, Abhinandan Pattanayak, Katelyn O’Neill, Nina Chaika, Weining Chen, Rachel Rhatigan, Raymond Bergan. Dual acting bone defender agents inhibit prostate cancer-mediated bone destruction [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 570.

Novel prognostication biomarker adipophilin reveals a metabolic shift in uveal melanoma and new therapeutic opportunities.

Metastatic uveal melanoma remains incurable at present. We previously demonstrated that loss of BAP1 gene expression in tumour cells triggers molecular mechanisms of immunosuppression in the tumour microenvironment (TME) of metastatic uveal melanoma. Adipophilin is a structural protein of lipid droplets involved in fat storage within mammalian cells, and its expression has been identified in uveal melanoma. We comprehensively evaluated adipophilin expression at the RNA (PLIN2) and protein levels of 80 patients of the GDC-TCGA-UM study and in a local cohort of 43 primary uveal melanoma samples respectively. PLIN2 expression is a survival prognosticator biomarker in uveal melanoma. Loss of adipophilin expression is significantly associated with monosomy 3 status and nuclear BAP1 losses in uveal melanoma tumours. Integrative transcriptomic and secretome studies show a relationship between transient loss of adipophilin expression and increased levels of tumour-associated macrophages and hypoxia genes, suggesting PLIN2-dependent changes in oxygen and lipid metabolism in the TME of low and high-metastatic risk uveal melanoma. We designed four adipophilin-based multigene signatures for uveal melanoma prognostication using a transcriptomic and secretome survival-functional network approach. Adipophilin-based multigene signatures were validated in BAP1-positive and BAP1-negative uveal melanoma cell lines using a next-generation RNA sequencing approach. We identified existing small molecules, mostly adrenergic, retinoid, and glucocorticoid receptor agonists, MEK, and RAF inhibitors, with the potential to reverse this multigene signature expression in uveal melanoma. Some of these molecules were able to impact tumour cell viability, and carvedilol, an adrenergic receptor antagonist, restored PLIN2 levels, mimicking the expression of normoxia/lipid storage signatures and reversing the expression of hypoxia/lipolysis signatures in co-cultures of uveal melanoma cells with human macrophages. These findings open up a new research line for understanding the lipid metabolic regulation of immune responses, with implications for therapeutic innovation in uveal melanoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Structure and RAF family kinase isoform selectivity of type II RAF inhibitors tovorafenib and naporafenib

Upon activation by RAS, RAF family kinases initiate signaling through the MAP kinase cascade to control cell growth, proliferation, and differentiation. Among RAF isoforms (ARAF, BRAF, and CRAF), oncogenic mutations are by far most frequent in BRAF. The BRAFV600E mutation drives more than half of all malignant melanoma and is also found in many other cancers. Selective inhibitors of BRAFV600E (vemurafenib, dabrafenib, encorafenib) are used clinically for these indications, but they are not effective inhibitors in the context of oncogenic RAS, which drives dimerization and activation of RAF, nor for malignancies driven by aberrantly dimerized truncation/fusion variants of BRAF. By contrast, a number of "type II" RAF inhibitors have been developed as potent inhibitors of RAF dimers. Here, we compare potency of type II inhibitors tovorafenib (TAK-580) and naporafenib (LHX254) in biochemical assays against the three RAF isoforms and describe crystal structures of both compounds in complex with BRAF. We find that tovorafenib and naporafenib are most potent against CRAF but markedly less potent against ARAF. Crystal structures of both compounds with BRAFV600E or WT BRAF reveal the details of their molecular interactions, including the expected type II-binding mode, with full occupancy of both subunits of the BRAF dimer. Our findings have important clinical ramifications. Type II RAF inhibitors are generally regarded as pan-RAF inhibitors, but our studies of these two agents, together with recent work with type II inhibitors belvarafenib and naporafenib, indicate that relative sparing of ARAF may be a property of multiple drugs of this class.

Prognosis and Characterization of Microenvironment in Cervical Cancer Influenced by Fatty Acid Metabolism-Related Genes.

Increasing evidence suggests that diverse activation patterns of metabolic signalling pathways may lead to molecular diversity of cervical cancer (CC). But rare research focuses on the alternation of fatty acid metabolism (FAM) in CC. Therefore, we constructed and compared models based on the expression of FAM-related genes from the Cancer Genome Atlas by different machine learning algorithms. The most reliable model was built with 14 significant genes by LASSO-Cox regression, and the CC cohort was divided into low-/high-risk groups by the median of risk score. Then, a feasible nomogram was established and validated by C-index, calibration curve, net benefit, and decision curve analysis. Furthermore, the hub genes among differential expression genes were identified and the post-transcriptional and translational regulation networks were characterized. Moreover, the somatic mutation and copy number variation landscapes were depicted. Importantly, the specific mutation drivers and signatures of the FAM phenotypes were excavated. As a result, the high-risk samples were featured by activated de novo fatty acid synthesis, epithelial to mesenchymal transition, angiogenesis, and chronic inflammation response, which might be caused by mutations of oncogenic driver genes in RTK/RAS, PI3K, and NOTCH signalling pathways. Besides the hyperactivity of cytidine deaminase and deficiency of mismatch repair, the mutations of POLE might be partially responsible for the mutations in the high-risk group. Next, the antigenome including the neoantigen and cancer germline antigens was estimated. The decreasing expression of a series of cancer germline antigens was identified to be related to reduction of CD8 T cell infiltration in the high-risk group. Then, the comprehensive evaluation of connotations between the tumour microenvironment and FAM phenotypes demonstrated that the increasing risk score was related to the suppressive immune microenvironment. Finally, the prediction of therapy targets revealed that the patients with high risk might be sensitive to the RAF inhibitor AZ628. Our findings provide a novel insight for personalized treatment in CC.

Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance.

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

Mechanistic model of MAPK signaling reveals how allostery and rewiring contribute to drug resistance

BRAF is prototypical of oncogenes that can be targeted therapeutically and the treatment of BRAFV600E melanomas with RAF and MEK inhibitors results in rapid tumor regression. However, drug‐induced rewiring generates a drug adapted state thought to be involved in acquired resistance and disease recurrence. In this article, we study mechanisms of adaptive rewiring in BRAFV600E melanoma cells using an energy‐based implementation of ordinary differential equation (ODE) modeling in combination with proteomic, transcriptomic and imaging data. We develop a method for causal tracing of ODE models and identify two parallel MAPK reaction channels that are differentially sensitive to RAF and MEK inhibitors due to differences in protein oligomerization and drug binding. We describe how these channels, and timescale separation between immediate‐early signaling and transcriptional feedback, create a state in which the RAS‐regulated MAPK channel can be activated by growth factors under conditions in which the BRAFV600E‐driven channel is fully inhibited. Further development of the approaches in this article is expected to yield a unified model of adaptive drug resistance in melanoma.

MAPK Pathway Inhibitors in Thyroid Cancer: Preclinical and Clinical Data.

Thyroid cancer is the most common endocrine cancer, with a good prognosis in most cases. However, some cancers of follicular origin are metastatic or recurrent and eventually become radioiodine refractory thyroid cancers (RAIR-TC). These more aggressive cancers are a clinical concern for which the therapeutic arsenal remains limited. Molecular biology of these tumors has highlighted a hyper-activation of the Mitogen-Activated Protein Kinases (MAPK) pathway (RAS-RAF-MEK-ERK), mostly secondary to the BRAFV600E hotspot mutation occurring in about 60% of papillary cancers and 45% of anaplastic cancers. Therapies targeting the different protagonists of this signaling pathway have been tested in preclinical and clinical models: first and second generation RAF inhibitors and MEK inhibitors. In clinical practice, dual therapies with a BRAF inhibitor and a MEK inhibitor are being recommended in anaplastic cancers with the BRAFV600E mutation. Concerning RAIR-TC, these inhibitors can be used as anti-proliferative drugs, but their efficacy is inconsistent due to primary or secondary resistance. A specific therapeutic approach in thyroid cancers consists of performing a short-term treatment with these MAPK pathway inhibitors to evaluate their capacity to redifferentiate a refractory tumor, with the aim of retreating the patients by radioactive iodine therapy in case of re-expression of the sodium-iodide symporter (NIS). In this work, we report data from recent preclinical and clinical studies on the efficacy of MAPK pathway inhibitors and their resistance mechanisms. We will also report the different preclinical and clinical studies that have investigated the redifferentiation with these therapies.