Oncogenic BRAF Mutations Research Articles

BRAF V600E-Mutant Acute Myeloid Leukemia: A Case Series and Literature Review of a Rare Entity

Background: Although BRAF V600E mutations are common in solid tumors and select hematologic neoplasms, they are reported less frequently in myeloid malignancies. Of the cases of BRAF V600E-mutant acute myeloid leukemia (AML) that have been described, most display monocytic morphology and concurrent KMT2A rearrangement. Strikingly, all cases have been associated with poor survival. Case Presentation: Here, we report two cases of AML, one diagnosed in an elderly male with metastatic lung adenocarcinoma and hepatocellular carcinoma and the other diagnosed in a young boy previously treated for B-cell acute lymphoblastic leukemia. Peripheral blood NGS revealed oncogenic mutations in BRAF p.V600E (VAF = 33%), TET2 p.M508Cfs*25 (VAF = 48%), TET2 p.C211* (VAF = 49%), ZRSR2 p.R295* (VAF = 71%), BRAF p.N581S (VAF = 6%), and EZH2 c.118-2A>G, p.? (VAF = 4%) in case 1 and BRAF p.V600E (VAF = 1%) and KRAS p.G12A (VAF = 28%) in case 2. Cytogenetic workup revealed a complex karyotype in case 1 and an abnormal karyotype with non-clonal aberrations and KMT2A (MLL) rearrangement in case 2. Morphologically, both patients were found to have AML with monocytic features. The post-mortem examination of case 2 also revealed extensive solid organ infiltration, consistent with a monocytic leukemia. Both patients died within days of diagnosis, demonstrating the lethality of this molecular subgroup of AML. Conclusions: Our cases add to the literature, highlighting the poor prognosis of patients diagnosed with BRAF-mutant AML. Although it is uncertain whether the complex karyotype and somatic mutations observed in case 1 and KMT2A rearrangement and variants identified in case 2 may have either independently or cooperatively conferred a poor prognosis, we contend that additional comprehensive studies are needed to further understand the pathophysiology and prognosis of BRAF mutations in AML. We further posit whether patients with BRAF V600E-mutant AML may benefit from the combined use of BRAF inhibitors and/or RAS-pathway-targeting regimens, which are currently FDA-approved for the treatment of BRAF V600-mutant solid tumors and BRAF-mutant histiocytic neoplasms.

Malignant Melanoma of Foot with Brain Metastasis – Case report

Melanoma is a rare type of skin cancer which has a strong correlation between occurrence and exposure to sun ultraviolet radiation. Though being uncommon, it has the highest mortality rate of all skin cancers. The patients with advanced disease may have higher mortality rates. The formation and development of melanoma metastases to brain are not fully understood. The most accepted way is the presence of oncogenic BRAF mutation, which occurs in up to 50% of metastatic melanoma patients. There was a lower survival rate in metastatic melanoma before detailed pathophysiology was understood. There are new research going on immunotherapy and targeted therapy to improve the prognosis of melanoma patients. Hereby, we would like to present a case of melanoma of foot with distant metastasis to brain.

TGFβ-Responsive Stromal Activation Occurs Early in Serrated Colorectal Carcinogenesis.

Activated TGFβ signaling in the tumor microenvironment, which occurs independently of epithelial cancer cells, has emerged as a key driver of tumor progression in late-stage colorectal cancer (CRC). This study aimed to elucidate the contribution of TGFβ-activated stroma to serrated carcinogenesis, representing approximately 25% of CRCs and often characterized by oncogenic BRAF mutations. We used a transcriptional signature developed based on TGFβ-responsive, stroma-specific genes to infer TGFβ-dependent stromal activation and conducted in silico analyses in 3 single-cell RNA-seq datasets from a total of 39 CRC samples and 12 bulk transcriptomic datasets consisting of 2014 CRC and 416 precursor samples, of which 33 were serrated lesions. Single-cell analyses validated that the signature was expressed specifically by stromal cells, effectively excluding transcriptional signals derived from epithelial cells. We found that the signature was upregulated during malignant transformation and cancer progression, and it was particularly enriched in CRCs with mutant BRAF compared to wild-type counterparts. Furthermore, across four independent precursor datasets, serrated lesions exhibited significantly higher levels of TGFβ-responsive stromal activation compared to conventional adenomas. This large-scale analysis suggests that TGFβ-dependent stromal activation occurs early in serrated carcinogenesis. Our study provides novel insights into the molecular mechanisms underlying CRC development via the serrated pathway.

Abstract 3078: Role of HuR in metabolic reprogramming of BRAF mutant melanoma treated with dabrafenib

Abstract Introduction: Oncogenic BRAF mutations are present in about 50% of melanoma and are clinically targetable by BRAF inhibitors such as dabrafenib. While BRAF inhibitors show high rates of clinical response, that response is transient and resistance inevitably develops. Previously we have demonstrated that BRAF inhibitors induce a metabolic shift towards the mitochondrial oxidative phosphorylation. We have shown that inhibition of wild type isocitrate dehydrogenase 1 (wtIDH1), a vital anaplerotic and anti-ROS enzyme, demonstrates synergy with dabrafenib in vitro and in vivo. IDH1 expression has been found in pancreatic cancer to be posttranscriptionally regulated by the RNA-binding and stress response protein, HuR (ELAVL1) under nutrient and chemotherapy stress. We hypothesize that HuR functions as a survival mechanism in melanoma under BRAF inhibition, regulating metabolic reprogramming including the expression of IDH1. Methods: The human BRAF V600E mutant melanoma cell line, A375, was used for all experiments. HuR and IDH1 expression was suppressed by siRNA oilgos through lipofectamine transfection, as validated by immunoblot analysis. The bioenergetic assay was conducted using Seahorse XFp mini extracellular analyzer to measure the oxygen consumption rate (OCR) under nutrient stress, both with and without dabrafenib exposure. The impact of HuR on IDH1 expression was further explored using qPCR and Immunoblot. Ivosidenib was used for pharmacologic inhibition of wtIDH as previously described. Results: Mitochondrial respiration (OCR) increased in parental A375 BRAF mutant melanoma cells following nutrient stress with low glucose conditions akin to the tumor microenvironment. OCR was further increased after dabrafenib exposure. Immunoblot analysis revealed that dabrafenib treated cells at 6-hour and 12-hour time points displayed HuR translocation from nucleus to cytoplasm, as detected in cytoplasmic lysates. Transient HuR silencing blocked the adaptive OCR response to both low glucose and BRAF inhibition. This suggests that HuR is involved in the metabolic response to acute BRAF inhibition in BRAF mutant melanoma. HuR cytoplasmic translocation is associated with increased IDH1 expression 24 hours after dabrafenib exposure. However, there is no increase in IDH1 levels under nutrient or treatment stress in HuR knockdown cells, indicating that IDH1 is regulated by HuR. Transient IDH1 knockdown, as well as pharmacologic IDH1 inhibition, blocks the OCR response of melanoma cells to BRAF inhibition and low glucose. This indicates that IDH1 is important in the HuR metabolic response to targeted therapy. Conclusion: We find that BRAF mutant melanoma cells adapt to acute dabrafenib exposure via HuR mediated expression of IDH1 to upregulate mitochondrial metabolism. This demonstrates a targeted therapy induced metabolic vulnerability that can be exploited by wtIDH1 inhibition. Citation Format: Alexander Loftus, Mehrdad Zarei, Hallie Graor, Omid Hajihassani, Christina Boutros, Jordan M. Winter, Luke D. Rothermel. Role of HuR in metabolic reprogramming of BRAF mutant melanoma treated with dabrafenib [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 3078.

Abstract 7268: The SOS1 inhibitor MRTX0902 demonstrates activity across cancer models with mutations in proximal components of the RAS-MAPK pathway

Abstract Genetic alterations in the RAS-MAPK pathway are one of the most frequent causes of human cancers. While targeted therapies are approved for patients harboring mutations in KRAS G12C in advanced or metastatic non-small cell lung cancer, there remains a need for an effective therapy for cancers characterized by additional RAS-MAPK pathway mutations across cancer types. Previous studies have shown that genetic or pharmacological inhibition of Son of Sevenless homolog 1 (SOS1) demonstrates antiproliferative activity in cancer cells addicted to KRAS signaling, including cell lines with oncogenic mutations in KRAS, SOS1, PTPN11, or EGFR or a loss-of-function mutation in NF1. These genetic alterations each function to increase SOS1-mediated GTP-loading of KRAS and increase signaling flux through the MAPK pathway. Inhibition of SOS1 by MRTX0902 is anticipated to shift the tumor cell equilibrium of KRAS towards the inactive GDP-bound KRAS, leading to MAPK pathway inhibition. MRTX0902 is a selective inhibitor of SOS1 under evaluation in clinical trials for the treatment of advanced, unresectable, or metastatic cancers with mutations in the KRAS-MAPK pathway. In the present studies, we have shown that MRTX0902 demonstrates single-agent anti-proliferative activity and modulates ERK1/2 phosphorylation (pERK) across a panel of human tumor cell lines harboring oncogenic mutations in SOS1, PTPN11, BRAF (class III) and NF1. Furthermore, oral administration of MRTX0902 as a monotherapy in preclinical human tumor cell-derived xenograft models harboring these oncogenic mutations results in significant tumor growth inhibition. Additionally, combination with the RAF/MEK clamp avutometinib demonstrated greater antitumor activity versus either monotherapy treatment. These data support the clinical utility of MRTX0902 as a single agent or combination partner of avutometinib for the treatment of human cancers harboring oncogenic mutations implicated in dysregulation of RAS-dependent signaling including SOS1, PTPN11, NF1, and BRAF (class III). Citation Format: Niranjan Sudhakar, Larry Yan, Fadia Qiryaqos, Jade Laguer, David Briere, Allan Hebbert, Andrew Calinisan, Silvia Coma, Jonathan Pachter, James G. Christensen, Peter Olson, Shilpi Khare. The SOS1 inhibitor MRTX0902 demonstrates activity across cancer models with mutations in proximal components of the RAS-MAPK pathway [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 7268.

Abstract 5847: Characterizing mechanisms of BRAF/MEK inhibitor resistance in BRAF V600 mutant colorectal cancer vs. melanoma

Abstract Background: BRAF V600 mutations comprise ~80% of all oncogenic BRAF mutations in metastatic colorectal cancer (mCRC). While most cancers with BRAF V600 mutations can be effectively treated with combined BRAF and MEK inhibition, BRAF V600 mutant mCRC acquire resistance through EGFR-mediated signaling. The profound difference in response rates to BRAF/MEK inhibitors in BRAF V600 mutant mCRC (12%) vs. melanoma (63-68%) is not sufficiently explained by increased EGFR signaling in CRC. Therefore, we sought to characterize the differences that exist between BRAF V600 mutant CRC and melanomas that could explain the differential sensitivity to BRAF/MEK inhibition and identify more effective treatments for BRAF V600 mutant CRC. Methods: We assessed cell survival by clonogenic assay in the presence of small molecule inhibitors. In parallel, we mined TCGA RNAseq data to calculate the MAPK Pathway Activity Score (MPAS). RTK expression was assessed in TCGA data from patients with BRAF V600 mutant CRC vs. melanoma. We developed cells with acquired resistance to BRAF+MEK inhibitors (Encorafenib+Binimetinib; E+B) by culturing cells in increasing concentrations of drug over several months. RNA-sequencing and analysis was performed on parental and resistant cells treated with vehicle control or BRAF+MEK inhibitors. Results: When comparing the efficacy of BRAF+MEK inhibitors across BRAF V600 mutant cells, we found that BRAF V600 mutant CRC cell lines (n=2) were less sensitive than melanoma cell lines (n=2). MPAS scores were significantly lower in BRAF V600 mutant CRC (n=46) vs. melanoma (n=188) tumors. We interrogated protein array data from the DepMap and identified increased activation of several RTKs (EGFR, HER2, HER3) in BRAF V600 CRC (n=8) vs. melanoma (n=39) cell lines. Similarly, we established resistant HT29 (BRAF V600 CRC) cells and observed sustained MAPK pathway activity despite a lack of difference in EGFR activity in the presence of BRAF+MEK inhibitor treatment. Both HT29 parental and resistant cells showed re-establishment of MAPK signaling after 72 hours. Resistant SkMel28 (BRAF V600 melanoma) cells showed increased pERK levels compared to resistant HT29 cells. RNAseq revealed increased expression of multiple RTKs (PDGFRB, ERBB2, ERBB3) and ligands/growth factors for multiple RTKs (FGF3, PDGFA, HB-EGF) in resistant HT29 cells. Shp2 is a protein tyrosine phosphatase that regulates signaling of multiple RTKs to activate the MAPK pathway. Accordingly, we assessed the efficacy of an inhibitor of Shp2 (TNO155) overcoming MAPKi resistance in BRAF V600 CRC cells. We confirmed that E+B+TNO155 more effectively inhibited cell growth and MAPK pathway activity in BRAF V600 CRC cell lines (n=2) in vitro. Conclusion: Together, these data identify key, actionable drivers of MAPK inhibitor resistance in CRC and highlight Shp2 as a potential therapeutic target for BRAF V600 CRC. Citation Format: Jennifer Maxwell, Emmanuelle Rousselle, Lucy An, April Rose. Characterizing mechanisms of BRAF/MEK inhibitor resistance in BRAF V600 mutant colorectal cancer vs. melanoma [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 5847.

Transactivation of Met signaling by oncogenic Gnaq drives the evolution of melanoma in Hgf-Cdk4 mice

Recent pan-cancer genomic analyses have identified numerous oncogenic driver mutations that occur in a cell-type and tissue-specific distribution. For example, oncogenic mutations in Braf and Nras genes arise predominantly in melanocytic neoplasms of the epidermis, while oncogenic mutations in Gnaq/11 genes arise mostly in melanocytic lesions of the dermis or the uvea. The mechanisms promoting cell-type and tissue-specific oncogenic events currently remain poorly understood. Here, we report that Gnaq/11 hotspot mutations occur as early oncogenic drivers during the evolution of primary melanomas in Hgf-Cdk4 mice. Additional single base substitutions in the Trp53 gene and structural chromosomal aberrations favoring amplifications of the chromosomal region containing the Met receptor gene accumulate during serial tumor transplantation and in cell lines established in vitro. Mechanistically, we found that the GnaqQ209L mutation transactivates the Met receptor. Overexpression of oncogenic GnaqQ209L in the immortalized melanocyte cell line promoted in vivo growth that was enhanced by transgenic Hgf expression in the tumor microenvironment. This cross-signaling mechanism explains the selection of oncogenic Gnaq/11 in primary Hgf-Cdk4 melanomas and provides an example of how oncogenic driver mutations, intracellular signaling cascades, and microenvironmental cues cooperate to drive cancer development in a tissue-specific fashion.

Identification of MAPK and mTOR pathway alterations in HER2-amplified colorectal cancer.

185 Background: Human epidermal growth factor receptor 2 (HER2) amplification is now a highly actionable alteration for patients with colorectal cancer (CRC) with distinct combination therapies. Recent studies have shown that oncogenic KRAS, BRAF, and PIK3CA mutations, may serve as biomarkers of response to HER-directed treatments such as Trastuzumab and Tucatinib/Pertuzumab. In this study, we investigated the incidence of MAP kinase and mTOR pathway alternations in patients with HER2-amplified CRC. Methods: The cBioPortal for Cancer Genomics was utilized to obtain data and graphics on HER2 amplification in CRC. cBioPortal was queried to select 21 published studies that contain CRC specimens (assessed April 2023). Clinical, specimen, copy number alteration (CNA), and somatic mutation data of each study were downloaded and aggregated across all studies, followed by filtering, standardization, and harmonization to generate ~30 analysis-ready fields encompassing demographic variables, HER2 amplification, and KRAS/NRAS/PIK3CA/BRAF mutations. Results: Among a pool of 4822 CRC patients with known gender, 44.4% (2139) were female and 55.6% (2683) were male. The incidence of HER2 amplification in the overall cohort was 2.6% (123/4822) and was noted to be higher in males (3.1%, 82/2683) than in females (1.9%, 41/2139). The incidence of HER2 amplification was highest in Asian CRC patients (3.9%, 9/228), followed by Black CRC patients (3.0%, 9/298), and then Caucasian CRC patients (2.7%, 57/2118). Rates of HER2 amplification were similar in the colon and the rectum at 2.6% (61/2360 and 26/1015, respectively). After analyzing rates of concurrent MAPK and mTOR pathway mutations in patients with HER2-amplified CRC, KRAS mutations were noted to be the most common mutation in HER2-amplified colon cancer (27.9%, 17/61), but were less common in HER2-amplified rectal cancer (7.7%, 2/26). PIK3CA mutations were also common, with an incidence of 13.1% (8/61) in HER2-amplified colon cancer and 7.7% (2/26) in HER2-amplified rectal cancer. NRAS mutations occurred at comparable, low rates in HER2-amplified colon and rectal cancer, at 3.3% (2/61) and 3.8% (1/26) respectively. No BRAF mutations were reported in either disease group. Conclusions: In this study, we identified that MAPK and mTOR pathway alterations are relatively common among patients with HER2-amplified CRC. These somatic events appear to be more common in HER2-amplified colon cancer than rectal cancer. Next generation based molecular profiling is recommended to identify potential resistance mechanisms before initiation of HER2-directed therapy.

Abstract A090: Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations

Abstract Alterations in the RAS-MAPK pathway, such as mutations in NF1, RAS, and BRAF, often lead to oncogenic signaling and result in aberrant cell proliferation and tumor growth. Oncogenic BRAF mutations may be activated as either monomers or dimers, while oncogenic RAS mutations or loss-of-function mutations in NF1, a negative regulator of RAS, drive activation of RAF dimers to transduce MAPK signaling. Currently approved BRAF inhibitors are selective against monomeric BRAF mutations and largely inactive against dimeric RAF mutants. Additionally, these agents may also promote deleterious paradoxical activation of RAF in select contexts. Moreover, FDA-approved KRAS G12C mutant-selective inhibitors are ineffective for cancer patients who harbor other (non-G12C) KRAS, NRAS, and NF1 mutations which all lead to active RAF dimer. There remains a high unmet clinical need for a highly CNS penetrant RAF inhibitor that targets a broad spectrum of oncogenic RAF conformations in the context of RAF, RAS, and other upstream driver mutations. BDTX-4933 is a next generation MasterKey RAF inhibitor designed to address the shortcomings of previous generation RAF inhibitors. BDTX-4933 is an orthosteric inhibitor that potently targets the oncogenic RAF conformation shared by groups of RAF driver mutations and induces an inactive conformation of RAF. As a MasterKey inhibitor, BDTX-4933 potently inhibits a broad spectrum of BRAF mutations and active RAF dimers promoted by upstream oncogenic MAPK pathway alterations, such as KRAS, NRAS, and NF1 mutations. In vivo, once daily oral dosing of BDTX-4933 resulted in robust and sustained target engagement, inhibiting ERK phosphorylation without inducing paradoxical activation. In a mouse breadth of efficacy study with patient derived xenografts (PDX), BDTX-4933 demonstrated tumor growth inhibition and regression as a single agent across models expressing a variety of targeted BRAF or MAPK pathway oncogenic mutations. Additionally, BDTX-4933 as a single agent and in combination with a MEK inhibitor, demonstrates anti-tumor efficacy in KRAS mutant lung PDX models. In a PDX model of BRAF V600E positive tumor that progressed following BRAF and MEK combination therapy, BDTX-4933 achieved robust tumor growth regression. 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 RAF inhibitor profile to treat cancer patients harboring BRAF mutations or RAF dimer-promoting upstream genetic alterations. BDTX-4933 is currently in a Phase I clinical study in patients with solid tumor harboring sensitive BRAF and RAS alterations (NCT05786924). For more information on this active Phase 1, please contact: ClinicalTrials@bdtx.com. Citation Format: Yoon-Chi Han, Pui Yee Ng, Luisa Shin Ogawa, Shao Ning Yang, Miao Chen, Darlene Romashko, Tai-An Lin, Elizabeth Buck. Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations [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 A090.

Current Treatment of Melanoma Brain Metastases.

Melanoma is a type of skin cancer in which there is a strong correlation between its occurrence and exposure to ultraviolet radiation. Although it is not the most common skin cancer, it has the highest mortality rate of all skin cancers. The prognosis of patients is significantly worsened by melanoma metastasis to the brain, which often occurs in patients with advanced disease. The formation and development of melanoma metastases to the brain involve a very complex process, and their mechanisms are not fully understood. One of the ways for metastatic melanoma cells to survive and develop cancer in the brain environment is the presence of oncogenic BRAF mutation, which occurs in up to 50% of metastatic melanoma cases. Before discovering new methods of treating metastases, the overall survival of patients with this disease was 6 months. Currently, research is being conducted on new drugs using immunotherapy (immune checkpoint inhibitors: anti-PD-1, anti-CTLA-4) and targeted therapy (BRAF and MEK inhibitors) to improve the prognosis of patients. In this article, we summarize the current state of knowledge about the results of treating brain metastases with new systemic therapies.

BRAF Mutations in Melanoma: Biological Aspects, Therapeutic Implications, and Circulating Biomarkers.

Melanoma is an aggressive form of skin cancer resulting from the malignant transformation of melanocytes. Recent therapeutic approaches, including targeted therapy and immunotherapy, have improved the prognosis and outcome of melanoma patients. BRAF is one of the most frequently mutated oncogenes recognised in melanoma. The most frequent oncogenic BRAF mutations consist of a single point mutation at codon 600 (mostly V600E) that leads to constitutive activation of the BRAF/MEK/ERK (MAPK) signalling pathway. Therefore, mutated BRAF has become a useful target for molecular therapy and the use of BRAF kinase inhibitors has shown promising results. However, several resistance mechanisms invariably develop leading to therapeutic failure. The aim of this manuscript is to review the role of BRAF mutational status in the pathogenesis of melanoma and its impact on differentiation and inflammation. Moreover, this review focuses on the mechanisms responsible for resistance to targeted therapies in BRAF-mutated melanoma and provides an overview of circulating biomarkers including circulating tumour cells, circulating tumour DNA, and non-coding RNAs.

Clinical outcomes with frontline immunotherapy (IO) in BRAF-mutant NSCLC.

e21089 Background: Oncogenic BRAF mutations are present in approximately 4% of non-small cell lung cancers (NSCLC). They are categorized as class I, II, or III according to their effect on the MAPK pathway. Little is known about how oncogenic BRAF mutations influence tumor PD-L1 expression and response to IO. We evaluated the clinical outcomes of frontline IO among BRAF classes. Methods: We performed a retrospective analysis of patients with BR AF-mutant NSCLC from January 1, 2012 through March 30, 2020. BRAF mutations were detected using next-generation sequencing or genotyping methods. PD-L1 expression was assessed using 22C3 or SP263 assays. Patient demographics were collected through electronic medical records review. In those with advanced/metastatic disease, we captured response to anticancer therapies using standardized real-world (rw) criteria and calculated rwOS using Kaplan-Meier method with log-rank test. Results: Of all 9,004 lung cancer patients (all histologies) with genotyping testing, 79 had NSCLC and an oncogenic BRAF mutation. 52% were females, 95% had adenocarcinoma, 81% were active/former smokers, and 76% had PD-L1 ≥1%. 39 patients had class I, 24 had class II, and 16 had class III mutations. Of these, 43 patients had advanced/metastatic disease evaluable for response. Patients with class I mutations who received first-line IO trended towards improved rwOS (42.6 months) vs. frontline anti-BRAF therapy (22.7 months; p= 0.51) and chemotherapy (19.6 months; p= 0.63). Patients with non-class I mutations, also trended towards improved rwOS with frontline IO (18.8 months) compared to frontline chemotherapy (9.9 months; p= 0.25). Outcomes were better in all patients with PD-L1 levels ≥1% and ≥50% who received IO. Patients with non-class I variants tended to have shorter rwOS with IO or chemotherapy compared to those with class I mutations (Table 1). Despite an overall trend towards improved outcomes with first-line IO, results did not reach significance likely due to the limited number of evaluable patients. Conclusions: The use of frontline IO may be associated with better outcomes than frontline chemotherapy or targeted therapies in patients with BRAF-mutant NSCLC. Patients with class I mutations and/or higher tumor PD-L1 expression seem to derive the most benefit. [Table: see text]

Oncogenic BRAF and p53 Interplay in Melanoma Cells and the Effects of the HDAC Inhibitor ITF2357 (Givinostat).

Oncogenic BRAF mutations have been widely described in melanomas and promote tumour progression and chemoresistance. We previously provided evidence that the HDAC inhibitor ITF2357 (Givinostat) targets oncogenic BRAF in SK-MEL-28 and A375 melanoma cells. Here, we show that oncogenic BRAF localises to the nucleus of these cells, and the compound decreases BRAF levels in both the nuclear and cytosolic compartments. Although mutations in the tumour suppressor p53 gene are not equally frequent in melanomas compared to BRAF, the functional impairment of the p53 pathway may also contribute to melanoma development and aggressiveness. To understand whether oncogenic BRAF and p53 may cooperate, a possible interplay was considered in the two cell lines displaying a different p53 status, being p53 mutated into an oncogenic form in SK-MEL-28 and wild-type in A375 cells. Immunoprecipitation revealed that BRAF seems to preferentially interact with oncogenic p53. Interestingly, ITF2357 not only reduced BRAF levels but also oncogenic p53 levels in SK-MEL-28 cells. ITF2357 also targeted BRAF in A375 cells but not wild-type p53, which increased, most likely favouring apoptosis. Silencing experiments confirmed that the response to ITF2357 in BRAF-mutated cells depends on p53 status, thus providing a rationale for melanoma-targeted therapy.

Abstract A036: Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation

Abstract The RAS-RAF-MEK-ERK signaling axis is upregulated in many human cancers due to its central role in cell growth, differentiation and survival. Oncogenic transformation frequently arises from RAS, BRAF and MEK mutations that activate the pathway. In the clinic, similar transformations also recur as resistance mutations to KRAS.G12C inhibitor treatment. BRAF mutations at V600 (Class I) activate BRAF by releasing the kinase’s activation segment and rendering BRAF activity dimerization-independent. Yet, it remains unclear how a diverse set of dimerization-dependent BRAF mutations (Class II/III) activate the pathway. MEK mutations are assumed to bypass the RAF node entirely. We previously reported a 2.9-Å-resolution crystal structure of human BRAF kinase domain (BRAFKD) in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that stabilize an inactive, pre-signaling monomeric conformation of BRAFKD and explain how ATP breaks RAF dimers in solution. Surprisingly, the β3-αC loop of MEK provides additional stabilizing interactions to the BRAF-bound AMP-PCP molecule. Utilizing an in vitro RAF dimerization assay, we find that MEK not only modulates RAF-RAF dimerization, it also enhances both the dimer-breaking effect ATP and the dimer-forming effect of paradoxical activator molecules, such as GDC-0879. Structural analysis reveals that all common oncogenic BRAF mutations alter key interactions with ATP that stabilize the inactive monomer conformation. We find ATP is unable to break RAF dimers containing Class I, II or III mutations, confirming these mutants counteract the inhibitory effects of ATP binding by lowering the threshold for RAF dimerization and thus pathway activation. We further find that oncogenic MEK mutants, in particular β3-αC loop deletions, favor RAF-RAF dimerization, even in the presence of ATP. Our study establishes a framework for rationalizing oncogenic BRAF mutations, and suggests that oncogenic MEK mutants can act, at least in part, by promoting RAF dimerization. This model provides a better understanding of activation mechanisms in the context of constantly evolving resistance mutations where oncogenesis continues to depend on the MAPK pathway. Citation Format: Timothy J. Wendorff, Jennifer Kung, Jawahar Sudhamsu. Oncogenic mutations in BRAF and MEK weaken the ATP-stabilized inactive conformation of RAF to promote RAF dimerization and MAPK pathway activation [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 A036.

Acral BRAF‐mutated tubular adenoma should be distinguished from HPV42‐related digital papillary adenocarcinoma

Acral <i>BRAF</i>‐mutated tubular adenoma should be distinguished from <scp>HPV42</scp>‐related digital papillary adenocarcinoma

Abstract 940: Comprehensive characterization of MET exon 14 skipping mutations in non-small cell lung cancer

Abstract Background: MET exon 14 skipping mutation (METΔex14) is a key driver event in non-small cell lung cancer (NSCLC), and can be acquired following drug resistance to MET, EGFR or ALK inhibitors. Clinical and genomic features of METΔex14 in NSCLC remain to be further characterized. Methods: A total of 556 baseline samples, 54 MET inhibitor-resistant samples and 16 EGFR/ALK inhibitor-resistant samples from 586 METΔex14 NSCLC patients were included in the analysis. Tissue and/or plasma samples were subjected to targeted next-generation sequencing (NGS) using Geneseeq’s 139/425 cancer-relevant gene panels. Results: The overall frequency of METΔex14 was 1.1%, with a total of 266 distinct variants detected. Of these, 40.2% were base substitutions in the intronic non-coding region immediately adjacent to the splice acceptor site, followed by indels (25.6%) and base substitutions (4.5%) in the splice donor site, and indels (21.4%) and base substitutions (2.3%) in the splice acceptor site, etc. No associations with clinical features were found among different variants. At baseline, METΔex14 commonly co-occurred with TP53 mutations (40.8%), and CDK4 (15.8%) and MET (7.9%) amplifications. In 42 (7.6%) patients, concurrent oncogenic mutations in EGFR, KRAS, ALK, RET, BRAF, and ERBB2 were also detected. Such patients displayed a different spectrum of METΔex14 variants compared with those without concurrent oncogenic mutations (P=0.08). In patients treated with MET inhibitors, no difference in survival was seen among different METΔex14 variants (P=0.38). Notably, activation of EGFR or MET (kinase domain mutations/amplification) conferred primary resistance to savolitinib and crizotinib. In addition, patients with high chromosomal instability or low intratumoral heterogeneity had unfavorable PFS. Secondary resistance to MET-TKI included MET activation (D1228N/H, Y1230C/S, amplification), EGFR activating mutations/amplification, KRAS G12V, as well as FGFR2/4 amplification. Surprisingly, we identified a FGFR3-TACC3 fusion as a previously unreported secondary resistance mechanism to savolitinib in the second line setting. Finally, we found that MET exon 14 skipping mutation might be a rare secondary resistance mechanism to EGFR/ALK inhibitors. Conclusion: In this study, we characterized METΔex14 in a large cohort of NSCLC patients and explored potential primary and secondary resistance mechanisms to savolitinib and crizotinib, which may facilitate drug development and help inform clinical actions. Citation Format: Xue Wu, Yaru Zhang, Jiaohui Pang, Li Lin, Jiani C. Yin, Haimeng Tang, Yang Shao. Comprehensive characterization of MET exon 14 skipping mutations in non-small cell lung 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 940.

Abstract 5486: Combining bromodomain and extraterminal (BET) protein inhibitor treatment with standard MAPK inhibitors improved MAPK signaling inhibition in colorectal cancer

Abstract Background: Overexpression and activation of the MAPK signaling pathway is known to play a key role in colorectal cancer (CRC) progression. Substantial efforts have been made to target MAPK signaling pathways in CRC, particularly blocking oncogenic mutations in KRAS or BRAF. However, standard treatments targeting MAPK signaling have demonstrated limited activities in CRC patients and are in strong need of improved efficacies. Thus, we investigated how the combination of BET inhibitor treatment could affect MAPK signaling activation. Methods: We performed a wide-scale drug combination study testing 15 drug combinations using CRC patient-derived xenograft (PDX) models. PDX models were treated for 21 days, and tumor volume and body weight changes were measured for assessment of treatment responses and toxicities. For the BET inhibitor (iBET-151) plus MEK inhibitor (binimetinib or trametinib) combination, RNA-seq and RPPA was performed on PDX tumor tissues collected after 7 days of treatment for pharmacodynamics study (n=3 per treatment condition). Tumors collected on day 21 were sequenced for H3K27Ac ChIP-seq and IHCs for understanding epigenomic changes in active enhancers and target validations. Results: Our preliminary screening of 15 drug combinations in CRC PDX models have shown the most promising and active responses in a combination of BETi with MEKi (5 out of 20 PDXs obtained tumor regression). Preclinical H3K27ac ChIP-seq data has shown lost enhancer peaks after BET inhibition were significantly associated with RAS signaling pathways (q&amp;lt;0.05). Gene set enrichment analysis on preclinical transcriptomic data also demonstrated downregulation of KRAS-signaling in BETi plus MEKi combination treatment compared to MEKi as monotherapy (p&amp;lt;0.05). MAPK activation scores (MPAS) were also significantly downregulated in transcriptomic and enhancer levels under BETi plus MEKi combinations compared to MEKi. Downstream transcription factors of MAPK signaling pathways, such as MYC and FOSL1, were synergistically downregulated in combination with BETi. Also, we found downregulation of EREG, a member of the epidermal growth factor (EGF) family, under BET inhibition which may play a role in MAPK downregulation together with standard therapy. Conclusions: Some studies have shown MAPK signaling activation as a resistance mechanism of BET inhibition in multiple cancer types. However, the understanding of the crosstalk between the activity of BET inhibition in a combination with MAPK signaling inhibition is limited in CRC. We found BET inhibition induces deeper inhibition of MAPK pathway activation together with standard targeted therapies. Our data demonstrated transcriptomic and epigenetic levels of the mechanism of BETi activity together with MAPK inhibition in CRC. Citation Format: Hey Min Lee, Alexey Sorokin, Preeti Kanikarla Marie, Saikat Chowdhury, Anand Singh, Amanda Anderson, Jumanah Yousef Alshenaifi, Funda Meric-Bernstam, Kunal Rai, Scott Kopetz. Combining bromodomain and extraterminal (BET) protein inhibitor treatment with standard MAPK inhibitors improved MAPK signaling inhibition in colorectal 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 5486.

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.

Age-related next-generation sequencing mutational analysis in 1196 melanomas.

Melanoma mutational burden is high and approximately 50% have oncogenic mutations in BRAF. We sought to evaluate age-related mutational differences in melanoma. We analyzed melanoma samples in the Genomics Evidence Neoplasia Information Exchange database. Targetable mutations were identified using the Precision Oncology Knowledge Base (OncoKB). We found 1194 patients with a common set of 30 genes. The top mutated genes in patients <40 years old (y/o) (n = 98) were BRAF (59%), TP53 (31%), NRAS (17%), and PTEN (14%); in 40-59 y/o (n = 354) were BRAF (51%), NRAS (30%), TP53 (26%), and APC (13%); and in ≥60 y/o (n = 742) were BRAF (38%), NRAS (33%), TP53 (26%), and KDR (19%). BRAF mutations were almost mutually exclusive from NRAS mutations in <40 y/o (58/59). Mutational burden increased with age, with means of 2.39, 2.92, and 3.67 mutations per sample in patients <40, 40-59, and ≥60 y/o, respectively (p < 0.0001). There were 10 targetable mutations meeting OncoKB criteria for melanoma: BRAF (level 1), RET (level 1), KIT (level 2), NRAS (level 3A), TP53 (level 3A), and FGFR2, MET, PTEN, PIK3CA, and KRAS (level 4). Mutations in melanoma have age-related differences and demonstrates potential targetable mutations for personalized therapies.

MAPK inhibitors dynamically affect melanoma release of immune NKG2D-ligands, as soluble protein and extracellular vesicle-associated.

Metastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi in vitro, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi in vitro, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients.