BRAFV600E Mutant Colorectal Cancer Research Articles

Durable Remission After Targeted Therapy in BRAF V600E–Mutant Metastatic Colorectal Cancer: Case Report

ABSTRACT BRAF mutation leads to constitutive activation of the MAPK pathway and is associated with the immune-activating molecular subtype of colorectal cancer. Targeted therapy for BRAF V600E–mutant metastatic colorectal cancer (CRC) has significantly improved outcomes for these patients when combined with anti–epithelial growth factor receptor (EGFR) therapy. However, most patients ultimately develop disease progression. We report a case of a patient with metastatic-deficient mismatch repair, BRAF V600E–mutated CRC, who achieved a durable complete response to vemurafenib plus cetuximab and chemotherapy despite initial high burden of disease, including peritoneal involvement. Recent clinical trials of combined anti-EGFR/anti–BRAF V600E therapy in BRAF V600E-mutant CRCs have found a few instances of robust response. Further study of combined targeted and chemotherapeutic regimens and the immunogenic properties of BRAF mutation may yield promising results.

Epigenome Reprogramming Through H3K27 and H3K4 Trimethylation as a Resistance Mechanism to DNA Methylation Inhibition in BRAFV600E-Mutated Colorectal Cancer.

BRAFV600E-mutated colorectal cancer exhibits a strong correlation with DNA hypermethylation, suggesting that this subgroup of tumors presents unique epigenomic phenotypes. Nonetheless, 5-azacitidine, which inhibits DNA methyltransferase activity, is not efficacious in BRAFV600E colorectal cancer in vivo. We randomized and treated mice implanted with patient-derived tumor xenografts harboring BRAFV600E mutation with control, 5-azacitidine, vemurafenib (BRAF inhibitor), or the combination. Comprehensive epigenomic profiling was conducted on control and 5-azacitidine-treated tumor samples, including DNA methylation, histone modifications, chromatin accessibility, and gene expression. Combinations of epigenetic agents were explored in preclinical BRAFV600E colorectal cancer models. A profound reduction of DNA methylation levels upon 5-azacitidine treatment was confirmed, however, transcriptional repression was not relieved. This study unbiasedly explored the adaptive engagement of other epigenomic modifications upon 5-azacitidine treatment. A loss of histone acetylation and a gain of histone methylations, including H3K27 and H3K4 trimethylation, were observed around these hypomethylated regions, suggesting the involvement of polycomb repressive complex (PRC) activity around the genome with loss of DNA methylation, therefore maintaining the repression of key tumor-suppressor genes. Combined inhibition of PRC activity through EZH2 inhibition with 5-azacitidine treatment additively improved efficacies in BRAFV600E colorectal cancer cells. In conclusion, DNA hypomethylation by 5-azacitidine exhibits a close association with H3K27me3 and PRC activity in BRAFV600E colorectal cancer, and simultaneous blockade of DNA methyltransferase and EZH2 holds promise as a potential therapeutic strategy for patients with BRAFV600E-mutated colorectal cancer.

Peptostreptococcus stomatis promotes colonic tumorigenesis and receptor tyrosine kinase inhibitor resistance by activating ERBB2-MAPK

Peptostreptococcus stomatis (P.stomatis) is enriched in colorectal cancer (CRC), but its causality and translational implications in CRC are unknown. Here, we show that P.stomatis accelerates colonic tumorigenesis in ApcMin/+ and azoxymethane/dextran sodium sulfate (AOM-DSS) models by inducing cell proliferation, suppressing apoptosis, and impairing gut barrier function. P.stomatis adheres to CRC cells through its surface protein fructose-1,6-bisphosphate aldolase (FBA) that binds to the integrin α6/β4 receptor on CRC cells, leading to the activation of ERBB2 and the downstream MEK-ERK-p90 cascade. Blockade of the FBA-integrin α6/β4 abolishes ERBB2-mitogen-activated protein kinase (MAPK) activation and the protumorigenic effect of P.stomatis. P.stomatis-driven ERBB2 activation bypasses receptor tyrosine kinase (RTK) blockade by EGFR inhibitors (cetuximab, erlotinib), leading to drug resistance in xenograft and spontaneous CRC models of KRAS-wild-type CRC. P.stomatis also abrogates BRAF inhibitor (vemurafenib) efficacy in BRAFV600E-mutant CRC xenografts. Thus, we identify P.stomatis as an oncogenic bacterium and a contributory factor for non-responsiveness to RTK inhibitors in CRC.

A comprehensive overview of the molecular features and therapeutic targets in BRAFV600E-mutant colorectal cancer.

As one of the most prevalent digestive system tumours, colorectal cancer (CRC) poses a significant threat to global human health. With the emergence of immunotherapy and target therapy, the prognosis for the majority of CRC patients has notably improved. However, the subset of patients with BRAF exon 15 p.V600E mutation (BRAFV600E) has not experienced remarkable benefits from these therapeutic advancements. Hence, researchers have undertaken foundational investigations into the molecular pathology of this specific subtype and clinical effectiveness of diverse therapeutic drug combinations. This review comprehensively summarised the distinctive molecular features and recent clinical research advancements in BRAF-mutant CRC. To explore potential therapeutic targets, this article conducted a systematic review of ongoing clinical trials involving patients with BRAFV600E-mutant CRC.

61P Safety and efficacy of encorafenib, binimetinib, and cetuximab in BRAF V600E-mutated colorectal cancer: A systematic review and single-arm meta-analysis

61P Safety and efficacy of encorafenib, binimetinib, and cetuximab in BRAF V600E-mutated colorectal cancer: A systematic review and single-arm meta-analysis

Abstract LB301: BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation

Abstract Objective: The BRAF V600E mutation drives the generation of an aggressive subtype of colorectal cancer (CRC). Although activation of WNT signalling is a generic hallmark of CRC, mutations in APC are uncommon in BRAF V600E-mutant CRC. Instead, mutations in RNF43 are the suspected WNT signalling driving event in these tumours. Here, we aimed to uncover how the WNT pathway is activated in BRAF V600E-mutant colon cancer. Design: CRISPR-LbCpf1-corrected BRAF (V600E) and RNF43 (P441fs) organoids were analysed for their growth potential in the presence or absence of growth factors, for their ability to form tumours, and by immunoblotting, RNA sequencing, and DNA methylation assays. Results: BRAF E600V organoids regained dependency on epidermal growth factor (EGF) receptor signalling for ERK pathway activation and proliferation, and completely lost tumorigenic potential. Correction of BRAF V600E, but not RNF43 P441fs, caused suppression of WNT target genes, while genes reflecting epithelial differentiation and negative regulators of WNT signalling were upregulated in BRAF E600V organoids. DNA methylation analysis revealed promoter hypermethylation of WNT antagonist genes in BRAF V600E organoids. Gene body hypermethylation, associated with transcriptional upregulation, was observed in key WNT-driven intestinal stem cell marker genes LGR5 and EPHB2, and in the WNT-effector TCF4. Treatment of BRAF V600E organoids with the DNA demethylating agent 5-aza-2′-deoxycytidine resulted in upregulation of WNT antagonist genes and a marked decrease in WNT target gene expression. Conclusions: BRAFV600E, rather than mutant RNF43, is a prominent force behind WNT pathway activation, and is essential for maintaining tumorigenic capacity. BRAFV600E causes widespread dysregulation of DNA methylation leading to activation of WNT signalling. Citation Format: Layla El Bouazzaoui, Jeroen M. Bugter, Emre Küçükköse, André Verheem, Jasmin B. Post, Nicola Fenderico, Inne H. Borel Rinkes, Hugo J. Snippert, Madelon M. Maurice, Onno Kranenburg. BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB301.

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.

Abstract 3241: Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer

Abstract Background: BRAFV600E in colorectal cancer (CRC) defines a unique subgroup of patients with poor prognosis and scarcely benefit from BRAF inhibitor-based treatment. Interestingly, BRAFV600E tumors demonstrated profound CpG island methylator phenotype (CIMP), suggesting extensive epigenetic dysregulation. Thus, we investigated epigenomic regulations in this subset of tumors with treatment-induced DNA demethylation. Methods: We treated six cell lines and two patient-derived xenograft (PDX) models of BRAFV600E CRC with vehicle, 5-azacitidine (DNMTi), vemurafenib (BRAFi), and combination treatment. PDX tumors treated with either vehicle or 5-azacitidine were collected on day 21 and sequenced for DNA methylation (Infinium HumanMethylation 450k BeadChip array), histone marks (ChIP-seq), chromatin accessibility (ATAC-seq), and gene expression (RNA-seq). Comprehensive post-translational modifications (PTMs) in histones with DNMTi or BRAFi treatment were assessed by mass spectrometry (MS). The combinational effect of 5-azacitidine with EZH2 or RNF2 inhibitors (Polycomb repressive complex activity (PRC) inhibition) was assessed through cell viability assays and colony formation assays in BRAFV600E CRC cell lines. Results: Analysis of TCGA COAD/READ tumors demonstrated a higher DNA methylation profile in BRAFV600E tumors than wild-type tumors even among the CIMP population (FDR < 1e-4). The combination of DNMTi with BRAFi showed selective additivity in vitro; however, did not improve efficacies in PDXs. The 5-azacitidine treatment induced profound demethylation in PDXs compared to control samples, predominantly attributed to promoter regions (48%). However, DNA demethylation failed to reactivate classic CIMP markers or tumor suppressor genes in CRC (e.g., CDKN2A, MGMT). Interestingly, demethylated genomic regions gained H3K27 trimethylation, repressive histone mark, in azacitidine-treated samples compared to control (9,431 vs 5,147 peaks respectively). We validated the elevation of H3K27 trimethylation as well as additional histone PTMs following DNMTi in MS-based histone analysis. Finally, combining a demethylating agent with EZH2i or RNF2i, which either deplete H3K27 trimethylation or block the PRC activity respectively, has demonstrated additive growth inhibitory effects in BRAFV600E CRC cell lines (p <0.05). Conclusions: This study unveiled intriguing aspects of demethylation agent treatment, which led to the compensatory engagement of other epigenomic markers, particularly repressive histone marks, resulting in limited efficacies to azacitidine treatment in preclinical BRAFV600E CRC models. This finding suggests the existence of adaptive interactions between epigenetic modifiers and potential clinical strategies in combinational epigenetic therapeutics. Citation Format: Hey Min Lee, Ajay Kumar Saw, Van Karlyle Morris, Anand Kamal Singh, Stefania Napolitano, Alexey Sorokin, Preeti Kanikarla Marie, Oluwadara Coker, Oscar Eduardo Villarreal, Nazanin Esmaeili Anvar, Kunal Rai, Scott Kopetz. Elevated H3K27 trimethylation mediates adaptation to DNA demethylation in BRAFV600E-mutated colorectal cancer [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 3241.

Association of RNF43 Genetic Alterations With BRAFV600E and MSIhigh in Colorectal Cancer.

Recent studies have provided evidence for a predictive value of RNF43 genetic alterations (GAs) as biomarkers for targeted therapies in microsatellite-stable (MSS) colorectal cancer (CRC). These data have the potential to prioritize treatment strategies in patients with BRAFV600E-mutant CRC and help to identify a subgroup that is more likely to derive benefit versus those patients for whom alternative treatment approaches are needed. We were therefore interested in defining the precise frequency of BRAFV600E and RNF43 GAs and their respective overlap in a large cohort of patients with CRC. To address this question, we performed a retrospective analysis that included 52,969 patients diagnosed with CRC from the FoundationCORE database. We observed a striking association of RNF43 GAs with MSI and tumor mutational burden status and BRAFV600E mutations. Overall, 23% of MSS patients with confirmed BRAFV600E mutation harbor an RNF43 GA-which accounts for 1.1% of all patients with CRC and for 15.7% of all CRC BRAFV600E cases. Ongoing phase III clinical trials, such as BREAKWATER, should aim to incorporate broader genetic profiling to further validate the superior sensitivity of patients with RNF43-mutant, MSS BRAFV600E CRC to anti-EGFR-/BRAFi-based therapies.

BRAF - a tumour-agnostic drug target with lineage-specific dependencies.

In June 2022, the FDA granted Accelerated Approval to the BRAF inhibitor dabrafenib in combination with the MEK inhibitor trametinib for the treatment of adult and paediatric patients (≥6 years of age) with unresectable or metastatic BRAFV600E-mutant solid tumours, except for BRAFV600E-mutant colorectal cancers. The histology-agnostic approval of dabrafenib plus trametinib marks the culmination of two decades of research into the landscape of BRAF mutations in human cancers, the biochemical mechanisms underlying BRAF-mediated tumorigenesis, and the clinical development of selective RAF and MEK inhibitors. Although the majority of patients with BRAFV600E-mutant tumours derive clinical benefit from BRAF inhibitor-based combinations, resistance to treatment develops in most. In this Review, we describe the biochemical basis for oncogenic BRAF-induced activation of MAPK signalling and pan-cancer and lineage-specific mechanisms of intrinsic, adaptive and acquired resistance to BRAF inhibitors. We also discuss novel RAF inhibitors and drug combinations designed to delay the emergence of treatment resistance and/or expand the population of patients with BRAF-mutant cancers who benefit from molecularly targeted therapies.

Development and validation of a clinical prognostic model for BRAF V600E-mutated colorectal cancer patients based on pathological stage, microsatellite status, and primary tumor site.

To develop and validate a prognostic model for patients with BRAF V600E-mutated colorectal cancer. The clinical and pathological information of 206 patients with BRAF V600E-mutated colorectal cancer diagnosed in Cancer Hospital, Chinese Academy of Medical Sciences, and Peking Union Medical College from 2014 to 2021 was retrospectively collected. Least absolute shrinkage and selection operator (LASSO) regression, Cox regression, and nomograms were used to develop clinical prognostic models. The differentiation was measured using C-statistic, and the predicted variability was evaluated using the calibration curve. The prognostic model was externally validated using validation set data from 164 patients pooled from five studies. Our clinical prognostic model included three variables: pathological stage, microsatellite status, and primary tumor site. In internal validation, the model had a concordant index of 0.785 (95% CI [0.732-0.839]) and a concordant index of 0.754 (95% CI [0.698-0.810]) using pathological staging. External validation confirmed the robustness of the model with a consistency index of 0.670 (95% CI [0.617-0.724]) and a consistency index of 0.584 (95% CI [0.546-0.622]) using pathological staging. Likelihood ratio test results show that our model is better (internal validation, p = 5.141e-03; external validation, p = 2.728e-05). The calibration graph drawn based on the prediction and the actual situation is close to the 45° diagonal. By adding microsatellite status and primary tumor site on the basis of pathological stage, we improved the discriminability and prediction accuracy of the model and successfully established a prognosis model for patients with BRAF V600E mutation of colorectal cancer.

BRAFV600E mutation and DSS treatment synergize to induce cecal tumor formation in mice

BRAF mutation is a driver mutation in colorectal cancer (CRC), and BRAFV600E mutation is found in 10–15 % of all CRCs. BRAF mutant CRCs in patients are primarily localized in the right colon, including the cecum. However, in the Vill-Cre;BRAFV600E/+ mice, adenomas mainly developed in the small intestines of the mice, and no tumor formed in the cecum. The mice model of BRAFV600E-mutant CRC with tumors in the cecum is lacking. Dextran Sulfate Sodium (DSS) treatment induces colitis in mice. Acute DSS treatment does not lead to tumor formation. We show that DSS treatment and BRAFV600E mutation synergistically induced cecal tumorigenesis, and cecal tumors formed within three months after five-day DSS treatment. The location of the adenomas supports the patient relevance of the model. Our BRAFV600E/DSS model provides a valuable in vivo model for future identification and validation of novel therapeutic approaches for treating BRAF-mutant CRC. Our results are consistent with the notion that BRAFV600E mutation is an oncogenic event that can shift controlled regeneration to unrestrained oncogenesis.

623P VIC regimen (vemurafenib/irinotecan/cetuximab) versus bevacizumab plus chemotherapy as first-line treatment for BRAF V600E-mutated advanced colorectal cancer

623P VIC regimen (vemurafenib/irinotecan/cetuximab) versus bevacizumab plus chemotherapy as first-line treatment for BRAF V600E-mutated advanced colorectal cancer

Clinical research progress on BRAF V600E-mutant advanced colorectal cancer.

Colorectal cancer is one of the malignant tumors that pose a serious threat to human health. A particularly bad prognosis might be expected for colorectal tumors with the unique molecular subtype BRAF V600E mutation. With the development of precision therapy, the advent of molecularly targeted therapies and immune checkpoint inhibitors has improved the outcome of intermediate to advanced colorectal cancer. However, the duration of drug benefit is usually short, and overall survival and progression-free survival remain suboptimal. Therefore, investigators are exploring more rational, safe, and effective drug combination regimens through clinical trials to provide longer survival for patients with such genetic mutations with metastatic colorectal cancer (mCRC). This article reviews the progress of clinical research on molecularly targeted drugs, immune checkpoint inhibitors, first-line chemotherapeutic agents, and different combination therapy regimens (including different targeted drug combinations, immune combination targeting, and chemotherapy combination targeting) for colorectal cancer patients with BRAF V600E mutation, which provides a reference for further in-depth clinical exploration of the treatment of colorectal cancer patients with BRAF V600E mutation.

A randomized trial of consolidation-targeted adjuvant therapy with encorafenib and cetuximab versus usual care for patients with stage II/III BRAF V600E colon cancer: Alliance for Clinical Trials in Oncology A022004.

TPS3641 Background: Patients with mismatch repair proficient (pMMR) BRAF V600E mutant high-risk stage II (T4) or stage III colon cancer have a substantial risk of recurrence despite standard adjuvant therapy. In patients with metastatic BRAF V600E mutant colorectal cancer, the combination of the RAF inhibitor encorafenib and the EGFR inhibitor cetuximab has been shown to improve overall and progression-free survival compared to standard therapy after at least one prior line of therapy. This study will evaluate if this combination improves disease-free survival (DFS) in patients with resected BRAF V600E mutant pMMR/microsatellite stable (MSS) high-risk stage II (T4) or stage III colon cancer after standard adjuvant therapy. Methods: The study has a seamless phase II/III design, enrolling a maximum of 394 patients. BRAF mutation status can be determined locally or submitted for central testing in trial pre-screening. Eligible patients will be randomized 1:1 to usual surveillance versus 6 months of treatment with encorafenib (300mg oral daily) plus cetuximab (500mg/m2 intravenous every 14 days). Stratification factors are adjuvant chemotherapy regimen (FOLFOX versus CAPOX), duration of adjuvant therapy (3 months versus > 3 months), tumor stage (T4N0 versus T1-3N1 versus T4N1/TanyN2), and ctDNA marker status (detectable versus undetectable) at randomization. Adjuvant therapy must be completed at most 8 weeks prior to registration. For the phase II component, in the ctDNA detectable cohort, the primary endpoint will be ctDNA clearance rate at 6 months, and, in the ctDNA undetectable cohort, the primary endpoint will be 6-month ctDNA recurrence-free survival, where events consist of death, recurrence, or ctDNA positivity. The primary endpoint of the phase III component is DFS, and secondary endpoints are overall survival, adverse events, and alternative DFS calculated from the date of primary resection, rather than date of study randomization. Patient reported symptoms (PRO-CTCAE) will be collected and analyzed as an exploratory endpoint, and serial blood banking will support future correlative studies. The trial will proceed to the phase III portion if either of the two endpoints in the phase II cohorts is statistically superior in patients receiving encorafenib plus cetuximab. The phase III study sample size will provide 80% power at a one-sided α of 0.05 to detect an approximately 10% improvement in the three-year DFS rate, assuming 68% in the control arm. Two interim analyses will be performed for DFS endpoint. Support: U10CA180821, U10CA180882; https://acknowledgments.alliancefound.org . U10CA180820 (ECOG-ACRIN); U10CA180868 (NRG); U10CA180888 (SWOG); Pfizer, Eli Lilly; Clinicaltrials.gov ID: NCT05710406. Clinical trial information: NCT05710406 .

Efficacy of vemurafenib based regimens in colorectal cancer: A systematic review of clinical trials.

e15566 Background: Colorectal cancer (CRC) is the 3rd most common cancer worldwide, and BRAF gene mutation is present in almost 10% of these cancers. It results in constitutive activation of the mitogen-activated protein kinase pathway (MAPKP). Abnormal activation of MAPKP leads to uncontrolled mitotic activity and inhibition of apoptosis. In this systematic review, we will assess the efficacy of vemurafenib (BRAF inhibitor) based regimens in CRC. Methods: We followed PRISMA guidelines to conduct this systematic review. A literature search was performed on PubMed, Clinicaltirals.gov, and Embase with mesh terms, ‘’Colorectal Neoplasms’’ and “Proto-Oncogene Proteins B-raf” from the inception of data till 01/05/2023. We screened 4572 articles and included 1 randomized clinical trial (RCT, N = 100), and 4 non-randomized trials (nRCTs, N = 85), measuring the efficacy of vemurafenib (V) based regimens in BRAF V600E-mutated CRC. Results: In 5 clinical trials (N = 185), 90 patients were treated with triple therapy, 45 with dual therapy, and 50 were in the control arm. In triple therapy, 69 patients were treated with V plus irinotecan (I) and cetuximab (C), overall response rate (ORR) was 17% and 35%, and progression-free survival (PFS) was 4.2 and 7.7 months in the randomized and the non-randomized study, respectively. PFS was improved significantly with a hazard ratio (HR) of 0.50 (95% CI, 0.32 to 0.76, P = .001) in favor of V+I+C compared to C+I. Among combinations of V+C, adding 5-fluorouracil/leucovorin/irinotecan (FOLFIRI) as third combination showed the highest ORR of 81% and overall survival (OS) and PFS of 15.4 and 9.7 months, respectively. In two drug regimens, V was combined with Cobimetinib (Co) and Panitumumab (P) in two non-randomized studies, and partial response (PR) was 26% and 13%, respectively. (Table 1). Conclusions: BRAF + EGFR inhibitor combination is showing promising antitumor effects, and the addition of the FOLFIRI regimen was more effective than adding irinotecan alone while comparing treatment regimens in BRAF V600E-mutated CRC patients with metastasis. V+Co showed antitumor effects in CRC BRAF mutated patients who were heavily pre-treated. More randomized trials needed. [Table: see text]

Antitumor Efficacy of Dual Blockade with Encorafenib + Cetuximab in Combination with Chemotherapy in Human BRAFV600E-Mutant Colorectal Cancer.

Encorafenib + cetuximab (E+C) is an effective therapeutic option in chemorefractory BRAFV600E metastatic colorectal cancer (mCRC). However, there is a need to improve the efficacy of this molecular-targeted therapy and evaluate regimens suitable for untreated BRAFV600E in patients with mCRC. We performed a series of in vivo studies using BRAFV600E mCRC tumor xenografts. Mice were randomized to receive 5-fluoruracil (5-FU), irinotecan, or oxaliplatin regimens (FOLFIRI or FOLFOX), (E+C) or the combination. Patients received long-term treatment until disease progression, with deescalation strategies used to mimic maintenance therapy. Transcriptomic changes after progression on cytotoxic chemotherapy or targeted therapy were assessed. Antitumor activity of either FOLFIRI or E+C was better as first-line treatment as compared with second-line, with partial cross-resistance seen between a cytotoxic regimen and targeted therapy with an average 62% loss of efficacy for FOLFIRI after E+C and a 45% loss of efficacy of E+C after FOLFIRI (P < 0.001 for both). FOLFIRI-treated models had upregulation of epithelial-mesenchymal transition (EMT) and MAPK pathway activation, where E+C treated models had suppressed MAPK signaling. In contrast, with chemotherapy with E+C, EMT and MAPK signaling remained suppressed. FOLFOX or FOLFIRI, each in combination with E+C, were the most active first-line treatments as compared with E+C or to chemotherapy alone. Furthermore, FOLFOX in combination with E+C as first-line induction therapy, followed by E+C ± 5-FU as maintenance therapy, was the most effective strategy for long-term disease control. These results support the combination of cytotoxic chemotherapy and molecular-targeted therapy as a promising therapeutic approach in the first-line treatment of BRAFV600E mCRC.

Abstract 1377: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with encorafenib and cetuximab in BRAF V600E mutant colorectal cancer models

Abstract Background: Cancer cells activate autophagy through ULK1/2 kinases as an adaptive stress response (ASR) mechanism to therapies targeting the RTK/RAS/MAPK/PI3K pathways, limiting antitumor response. BRAF signals through the MAPK pathway while EGFR signals upstream through both the MAPK and PI3K pathways, suppressing ULK1/2 kinases and autophagy. Inhibition of mutant BRAF and EGFR reverses this suppression, activating the autophagy ASR1-4. The BRAF V600E mutation occurs in ~10% of colorectal cancer (CRC) patients. Approved treatments for these patients include the BRAF V600E inhibitor encorafenib in combination with the EGFR antibody, cetuximab. Treatment with encorafenib + cetuximab (E+C) is initially successful, but drug resistance develops either through RTK/MAPK resistant mutations or ASR pathways including autophagy. DCC-3116 is a selective, investigational, potent, first-in-class inhibitor of the ULK1/2 kinases in clinical development in combination with targeted therapies that activate the autophagic ASR pathway. Herein we demonstrate ULK1/2 kinases and autophagy are activated upon treatment with E+C. A combination of E+C with DCC-3116 leads to inhibition of ULK1/2-mediated ATG13 phosphorylation (pATG13) and autophagy in vitro. In a PK/PD model, DCC-3116 inhibits E+C induced pATG13 and leads to synergistic tumor growth inhibition (TGI) in a preclinical model of BRAF V600E mutated CRC. Methods: ULK1/2 activity was measured in cells using an ELISA for the ULK substrate phospho-ATG13 (pATG13). Autophagic flux was measured by monitoring mCherry/GFP tagged LC3 in HT-29 and Colo-205 cells. Xenograft models were performed at a CRO. Results: E+C treatment led to the activation of ULK1/2 2-3-fold in BRAF V600E mutated CRC cell lines. DCC-3116 inhibited both E+C -induced and basal pATG13 with IC50 values of 80 nM and 234 nM in HT-29 and Colo-205 cells, respectively. E+C induced autophagic flux ~3-fold which was potently inhibited by DCC-3116 with IC50 values of 65 nM in HT-29 and 40 nM in Colo-205 cells. In a PK/PD model, DCC-3116 inhibited ULK1/2-mediated pATG13 induced by E+C. The combination of DCC-3116 with E+C resulted in greater TGI compared to single agent or the combination of E+C in BRAF V600E models. Conclusions: These preclinical data demonstrate that BRAF inhibitors in combination with EGFR blockade such as E+C activate ULK-mediated autophagy as an ASR resistance mechanism which can be inhibited by DCC-3116, providing the rationale to study the combination of DCC-3116 with E+C in BRAF V600E mutated CRC patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors. (NCT04892017).

BAYONET trial: A multicenter phase II trial of staged combination with encorafenib + binimetinib + cetuximab following encorafenib + cetuximab in patients with BRAF V600E-mutant metastatic colorectal cancer.

TPS271 Background: While the triplet combination of encorafenib (ENCO) + binimetinib (BINI) + cetuximab (CET) indicated higher response rate compared to the doublet combination of ENCO + CET, no significant survival benefits of the triplet combination were observed in patients with BRAF V600E-mutant metastatic colorectal cancer (mCRC) according to BEACON CRC study. Although ENCO + CET is the standard 2nd-line therapy in the United States and European Union, poor prognoses are expected after disease progression. As resistant mechanisms of BRAF + EGFR blockage, several MAPK pathway alternations, including RAS and RAF mutations were reported, which suggests the additional blockade of MAPK signaling may be an effective strategy for ENCO + CET refractory mCRC. In addition, the preclinical study suggested BRAF inhibitor + anti-EGFR antibody resistant BRAF V600E-mutant colorectal cancer cell-lines were sensitive to the combination of BRAF inhibitor + MEK inhibitor + anti-EGFR antibody (Oddo D, et al. Cancer Res. 2016). Methods: BAYONET is a single-arm multicenter phase II trial to evaluate the efficacy and safety of the staged combination with ENCO + BINI + CET for patients with BRAF V600E-mutant mCRC who were refractory to ENCO + CET. The main eligibility criteria are as follows; RAS wild-type/ BRAF V600E-mutant mCRC; age ≥ 20; ECOG PS 0 or 1; within 4 weeks from the last administration of previous ENCO or CET; no administration of other systemic therapy after refractoriness to ENCO + CET; complete response, partial response, or ≥4 months of stable disease were observed in the previous ENCO + CET. Included patients receive the combination treatment of ENCO (300mg once a day) + BINI (45mg twice a day) + CET (400mg/m2 initial dose and then 250mg/m2 once a week) in a 28 day-cycle as a study treatment. The primary endpoint of this trial is 12-week progression-free survival (PFS) rate. The secondary endpoints include PFS, overall survival, objective response rate, disease control rate, time to treatment failure, and the incidence of adverse events. The targeted sample size was calculated to be 30 on the basis of a power of 80%, a significant level of 10% (one-sided), the threshold 12-week PFS rate of 20%, and the expected 12-week PFS rate of 40%. As a translational analysis, circulating tumor DNA for next-generation sequencing using Guardant360 is collected at twice time points (before and after study treatment) to investigate the resistance mechanisms. Enrollment started from Jan 2022 and is ongoing at 25 facilities in Japan. As of Sep 20, 2022, 9 patients were enrolled. Clinical trial information: jRCTs031210510 .

Metastatic pattern is a prognostic factor in BRAFV600E mutant colorectal cancer

BackgroundDespite recent advancements in the treatment of metastatic BRAFV600E colorectal cancer (CRC), prognosis remains poor. However, a some patients with BRAFV600E disease have superior outcomes compared to the overall cohort and the prognostic factors associated with this improved survival are not well understood. MethodsWe conducted a single center retrospective review of patients with metastatic CRC and available next generation sequencing data. Patients with confirmed BRAFV600E disease were selected for the final analysis. We collected baseline demographic characteristics, concurrent mutations, and metastatic pattern. The primary endpoint was overall survival (OS). Univariate and multivariable logistic regression was used to examine the association between baseline concurrent somatic mutations and sites of metastatic disease with survival. ResultsOf 466 patients with metastatic CRC, 50 harbored BRAFV600E disease and 42 were included in the final analysis. The median OS in this cohort was 18.7 months (95% CI: 5.55–31.8). There was no association between baseline concurrent somatic mutations and OS. On univariate analysis, patients with lymph node only disease at the time of metastatic disease were more likely to have longer OS (hazard ratio [HR] = 0.30, 95% CI: 0.09–0.98, p = 0.047) and patients with peritoneal disease were more likely to have shorter OS (HR = 2.78, 95% CI: 1.12–6.88, p = 0.03). However, these associations did not retain statistical significance on multivariable analysis. ConclusionsThe pattern of metastatic disease in BRAFV600E CRC may be a prognostic factor and future studies are needed to better understand the underlying mechanisms and potentially change clinical practice for a select patient population. MicroAbstractSelect patients with metastatic BRAFV600E colorectal cancer may have better than expected survival but are not well characterized. We conducted a retrospective review of 42 patients with metastatic BRAFV600E colorectal cancer and showed that lymph node only disease at the time of metastatic disease was associated with superior survival.