BRAF-mutant Colorectal Cancer Research Articles

BRAF-Mutated Colorectal Cancer: What Is the Optimal Strategy for Treatment?

The BRAF activating mutation, harbored by approximately 10% of colorectal cancers (CRC), confers dramatic prognosis to advanced diseases. In early-stage setting, the identification of the BRAF mutation does not impact the therapeutic decision. Yet, the BRAF mutation could be considered as a stratification factor in adjuvant trials, because of its prognostic impact after relapse. Moreover, both BRAF mutation and mismatch repair (MMR) statuses should be determined in all CRC to help identify sporadic tumors versus Lynch syndrome-related tumors. Indeed, in patients with MMR-deficient (dMMR) tumors and MLH1 loss of expression, the BRAFV600E mutation indicates a sporadic origin. In advanced BRAF-mutated CRC, the standard of care remains fluoropyrimidine-based cytotoxic regimen in combination with bevacizumab. Although a recent meta-analysis showed that there was insufficient data to justify the exclusion of anti-EGFR monoclonal antibodies, antiangiogenic agents should be preferred in the first-line setting. Despite the lack of a randomized phase 3 study dedicated to BRAF-mutated CRC, chemotherapy intensification combining a quadruple association of 5-fluorouracil, oxaliplatin, irinotecan (FOLFOXIRI), and bevacizumab seems like a valid option. Although first results with BRAF inhibitors as single agents in BRAF-mutated CRC were disappointing, their association with therapies targeting the MAPK pathway seems to overcome the primary resistance to BRAF inhibition. In the field of sporadic CRC, the BRAF mutation is strongly associated with MMR deficiency. Considering breakthrough results of immune checkpoint inhibitors in dMMR repair tumors, determination of the MMR status appears to be mandatory. Given the dramatic prognosis conferred by the BRAF mutation, patients with BRAF-mutated advanced CRC need to be systematically identified and proposed for clinical trial enrolment in order to benefit from innovative therapies.

BRAF +/- MEK inhibition in patients (pts) with BRAF-mutant (mut) metastatic colorectal cancer (mCRC): The Sarah Cannon Research Institute UK’s experience.

645 Background: Activating V600 BRAF mutations are detectable in around 10% of mCRC pts and are associated with poor prognosis. Low response rates have been reported with anti-BRAF monotherapy in this subset of pts while encouraging data exist on enhanced MAPK pathway inhibition with combination therapy (anti-BRAF + anti-MEK and/or anti-EGFR). Methods: We performed a pooled analysis of BRAF-mut mCRC pts enrolled into different phase 1 trials involving the use of anti-BRAF monotherapy alone (Group A) or in combination with a MEK inhibitor (Group B) at therapeutic doses between June 2012 and August 2016. Demographic, baseline, toxicity and efficacy data were retrospectively reviewed. Results: Twenty BRAF-mut mCRC pts were identified in either Group A (n = 13) or Group B (n = 7). All pts had an ECOG performance status ≤ 1. Median age was 54 years (range 33-74). The primary tumor was right-sided in 10 (50%) pts, left-sided in 9 (45%) pts, and unknown in 1 (5%) subject. Median number of previous treatment lines was 1 (range 1-4). The disease control rate was 55%, with 3 (15%) pts experiencing partial response (PR) and 8 (40%) with stable disease. No complete responses were observed, 8 (40%) pts had progressive disease and 1 subject was considered not evaluable. All PRs were achieved in Group B and had duration of 2-4 months (mo). Median PFS was 3.73 mo (95% confidence interval [CI] 3.06-6.79) in the overall population, and showed a trend favoring the combination arm (2.80 mo for Group A vs. 5.03 mo for Group B, hazard ratio 0.71 [CI 0.26-1.95], p = 0.503). Most common toxicities were skin-related side effects (n = 10; 50%), myalgia/arthralgia (n = 5; 25%), nausea (n = 3; 15%), and pyrexia (n = 3; 15%). Grade 3/4 toxicities were uncommon and manageable: neutropenia (n = 1; 5%), hypophosphatemia (n = 1; 5%), pyrexia (n = 1; 5%), and abdominal pain (n = 1; 5%). Conclusions: Our cohort of pts treated with BRAF +/- MEK inhibitors showed a favorable safety profile. An encouraging activity level was found in the combination arm. In line with existing literature data, our findings supporting further development of combined MAPK inhibition in V600-positive mCRC.

Abstract IA05: The serrated pathway of colorectal tumorigenesis

Abstract IA05: The serrated pathway of colorectal tumorigenesis

KRAS, NRAS and BRAF mutations in colorectal cancer and melanoma.

Cancers are the group of diseases, which arise because of the uncontrolled behavior of some of the genes in our cells. There are possibilities of gene amplifications, overexpressions, deletions and other anomalies which might lead to the development and spread of cancer. One of the most dangerous ways to the cancers is the mutations of the genes. The mutated genes can start unstoppable proliferation of cells, their uncontrolled motility, protection from apoptosis, the DNA mutation enhancement as well as other anomalies, leading to the cancer. This review focuses on the genes, which are frequently mutated in various cancers and are known to be important in the advance and progression of colorectal cancer and melanoma, namely KRAS, NRAS and BRAF.

MicroRNA Expression in KRAS- and BRAF-mutated Colorectal Cancers.

KRAS and BRAF are two genes commonly mutated in colorectal cancer (CRC). Even though BRAF is a downstream target of KRAS in the MAPK signalling pathway, KRAS- and BRAF-mutated CRCs are found to display several different clinical and histopathological features. We investigated whether a differential expression of microRNAs (miRNAs) could explain the clinicopathological differences seen between KRAS- and BRAF-mutated CRCs. Using a PCR array, we analyzed the expression of 84 different miRNAs in CRC cell lines wild-type in KRAS and BRAF, or mutated in KRAS or BRAF. Ten miRNAs were selected for further analyses in tumor tissue specimens (let-7a, let-7i, miR-10a, miR-10b, miR-31, miR-100, miR-181a, miR-181b, miR-372, and miR-373). BRAF-mutated tumors were found to express significantly higher levels of miR-31 as well as significantly lower levels of miR-373, compared to wild-type tumors. Our results suggest that KRAS- and BRAF-mutated CRCs may have different miRNA signatures compared to CRC tumors wild-type in KRAS and BRAF. However, no difference in expression levels between KRAS- and BRAF-mutated tumors was evident for the miRNAs analyzed in this study.

MAP2K1 Mutation in Colorectal Cancer Patients: Therapeutic Challenge Using Patient-Derived Tumor Cell Lines.

BACKGROUND:The MAP2K1 K57T mutation is known to be a potential mechanism of primary and secondary resistance to EGFR inhibitors in metastatic colorectal cancer (CRC) and has also been reported to promote resistance to BRAF and MEK inhibitors. It is important to overcome therapeutic resistance to EGFR inhibitors to improve the treatment outcomes of metastatic CRC.METHODS: We established patient-derived tumor cells (PDCs) from metastatic lesions that newly appeared during treatment with a BRAF inhibitor (LGX-818) plus an EGFR inhibitor (cetuximab) in a patient with BRAF-mutant CRC. To investigate therapeutic options to overcome acquired resistance due to MAP2K1 mutation in BRAF-mutant CRC, we performed cell viability assays using the PDCs.RESULTS:We tested whether the PDCs were resistant to an EGFR inhibitor (cetuximab) and a BRAF inhibitor (sorafenib) as these cells were established at the time of resistance to the EGFR plus BRAF inhibitors. Moreover, the anti-tumor effect of AZD6244 (MEK inhibitor) was evaluated because PDCs harbored a MAP2K1 mutation at the time of resistance to the EGFR plus BRAF inhibitors. MTT proliferation assays showed that monotherapy with cetuximab, sorafenib, or AZD6244 did not suppress cell viability. We next tested viability of the PDCs to combination treatment with cetuximab plus AZD6244 and sorafenib plus AZD6244. Proliferation of PDCs was significantly inhibited by sorafenib and AZD6244, but not by cetuximab plus AZD6244. Investigation of the combined effect of sorafenib and AZD6244 using the calculated combination index (CI) showed synergistic effects of sorafenib and AZD6244 in combination therapy applied to PDCs with the MAP2K1 K57T mutation.CONCLUSION:Our results suggest that combination treatment with BRAF and MEK inhibitors might be a novel treatment strategy for MAP2K1 K57T-mutant CRC. This finding will be helpful to guide treatment of patients with CRC that is resistant to EGFR inhibitors.

BRAF V600E Mutant Colorectal Cancer Subtypes Based on Gene Expression.

Mutation of BRAF at the valine 600 residue occurs in approximately 10% of colorectal cancers, a group with particularly poor prognosis. The response of BRAF mutant colorectal cancer to recent targeted strategies such as anti-BRAF or combinations with MEK and EGFR inhibitors remains limited and highly heterogeneous within BRAF V600E cohorts. There is clearly an unmet need in understanding the biology of BRAF V600E colorectal cancers and potential subgroups within this population. In the biggest yet reported cohort of 218 BRAF V600E with gene expression data, we performed unsupervised clustering using non-negative matrix factorization to identify gene expression-based subgroups and characterized pathway activation. We found strong support for a split into two distinct groups, called BM1 and BM2. These subtypes are independent of MSI status, PI3K mutation, gender, and sidedness. Pathway analyses revealed that BM1 is characterized by KRAS/AKT pathway activation, mTOR/4EBP deregulation, and EMT whereas BM2 displays important deregulation of the cell cycle. Proteomics data validated these observations as BM1 is characterized by high phosphorylation levels of AKT and 4EBP1, and BM2 patients display high CDK1 and low cyclin D1 levels. We provide a global assessment of gene expression motifs that differentiate BRAF V600E subtypes from other colorectal cancers. We suggest that BRAF mutant patients should not be considered as having a unique biology and provide an in depth characterization of heterogeneous motifs that may be exploited for drug targeting. Clin Cancer Res; 23(1); 104-15. ©2016 AACR.

Triple Therapy Improves Colorectal Cancer Response.

Findings from a phase I/II study indicate a higher response rate among patients with BRAF-mutant metastatic colorectal cancer treated with an EGFR inhibitor alongside dual, as opposed to single-level, MAPK blockade. Panitumumab combined with trametinib and dabrafenib only modestly increased median progression-free survival, however; a short-lived decrease in responders' BRAF V600E mutant allele fraction and the emergence of RAS mutations may have been contributing factors.

BRAF-Mutated Colorectal Cancer Exhibits Distinct Clinicopathological Features from Wild-Type BRAF-Expressing Cancer Independent of the Microsatellite Instability Status.

In patients with colorectal cancer (CRC), the BRAF V600E mutation has been reported to be associated with several clinicopathological features and poor survival. However, the prognostic implications of BRAF V600E mutation and the associated clinicopathological characteristics in CRCs remain controversial. Therefore, we reviewed various clinicopathological features, including BRAF status, in 349 primary CRCs and analyzed the relationship between BRAF status and various clinicopathological factors, including overall survival. Similar to previous studies conducted in Eastern countries, the incidence of the BRAF V600E mutation in the current study was relatively low (5.7%). BRAF-mutated CRC exhibits distinct clinicopathological features from wild-type BRAF-expressing cancer independent of the microsatellite instability (MSI) status. This mutation was significantly associated with a proximal tumor location (P = 0.002); mucinous, signet ring cell, and serrated tumor components (P < 0.001, P = 0.003, and P = 0.008, respectively); lymphovascular invasion (P = 0.004); a peritumoral lymphoid reaction (P = 0.009); tumor budding (P = 0.046); and peritoneal seeding (P = 0.012). In conclusion, the incidence of the BRAF V600E mutation was relatively low in this study. BRAF-mutated CRCs exhibited some clinicopathological features which were also frequently observed in MSI-H CRCs, such as a proximal location; mucinous, signet ring cell, and serrated components; and marked peritumoral lymphoid reactions.

MET-Driven Resistance to Dual EGFR and BRAF Blockade May Be Overcome by Switching from EGFR to MET Inhibition in BRAF-Mutated Colorectal Cancer.

A patient with metastatic BRAF-mutated colorectal cancer initially responded to combined EGFR and BRAF inhibition with panitumumab plus vemurafenib. Pre-existing cells with increased MET gene copy number in the archival tumor tissue likely underwent clonal expansion during treatment, leading to the emergence of MET amplification in the rebiopsy taken at progression. In BRAF-mutated colorectal cancer cells, ectopic expression of MET conferred resistance to panitumumab and vemurafenib, which was overcome by combining BRAF and MET inhibition. Based on tumor genotyping and functional in vitro data, the patient was treated with the dual ALK-MET inhibitor crizotinib plus vemurafenib, thus switching to dual MET and BRAF blockade, with rapid and marked effectiveness of such strategy. Although acquired resistance is a major limitation to the clinical efficacy of anticancer agents, the identification of molecular targets emerging during the first treatment may afford the opportunity to design the next line of targeted therapies, maximizing patient benefit. MET amplification is here identified-clinically and preclinically-as a new mechanism of resistance to EGFR and BRAF dual/triple block combinations in BRAF-mutated colorectal cancer. Switching from EGFR to MET inhibition, while maintaining BRAF inhibition, resulted in clinical benefit after the occurrence of MET-driven acquired resistance. Cancer Discov; 6(9); 963-71. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 932.

Abstract IA21: Opportunities for integration of PDX models into clinical trials

Abstract Patient-derived xenograft models (PDX) provide opportunities to evaluate efficacy, pharmacodynamics, response durability, and resistance mechanisms in greater details than traditionally possible with sampling of patient tumors. When PDX models are integrated with patients enrolled in clinical trials, or treated with novel agents, there are opportunities to interrogate aspects of cancer biology not traditionally evaluable, while maintaining clinical relevance. This presentation will explore the strengths and limitations of the models, with examples on how these models are being integrated into clinical trials, validation efforts, and novel insights derived from this approach. The concept of a co-clinical trial has been variously defined, including a definition proposed by the NCI as parallel or sequential trials of combination therapy in patients and in mouse and human-in-mouse models of appropriate genotypes to represent the patients. This concept is being extended for PDX models to incorporate tumor models derived from the same patients participating in the clinical trial, both with a goal of improving translational interrogation of the novel agent efficacy as well as emerging efforts to use the PDX models to guide patient therapy. Validation of the PDX models for treatment response/regression will be demonstrated, including recently completed clinical trials utilizing inhibitors of BRAF and MEK with a comparison of the response of the matched PDX models to radiographic measurements from the treated patients. Cohort based validation will be demonstrated through examples with EGFR targeted therapy and comparison to randomized clinical trial results. Limitations of these approaches will be described. PDX models from clinical trials have been demonstrated to model and replicate mechanisms of resistance in some settings, and examples from colorectal and lung cancer will be used to demonstrate this. Resistance of lung cancer to EGFR tyrosine kinase inhibitors is being explored on PDXs obtained from post-progression biopsies. Tumor heterogeneity as a mechanism of resistance has been successfully modeled in BRAF-mutant colorectal cancer, and has been integrated into prospective clinical trials. Finally, efforts to integrate PDX models as predictors of treatment response are described. These include efforts to use matched PDX models to guide treatment decisions in patients as part of prospective clinical trials. Regulatory, logistic, and biologic limitations of such efforts will be discussed. Citation Format: Scott Kopetz. Opportunities for integration of PDX models into clinical trials. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA21.

Molecular Landscape of Acquired Resistance to Targeted Therapy Combinations in BRAF-Mutant Colorectal Cancer.

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. ©2016 AACR.

Abstract 1096: microRNA-193a-3p acts as a tumor suppressor in BRAF-mutated colorectal cancer

Abstract The aim of this study was to identify microRNAs (miRNAs) specifically dysregulated in BRAF-mutant colorectal cancer, a malignant subtype of colorectal cancer. The genome-wide miRNA expression analysis in a screening set (n = 30) identified 22 miRNAs specifically dysregulated in BRAF-mutant cancers. Among the top 5 dysregulated miRNAs, the validation analysis using another set of patients (n = 34) confirmed miR-31 and miR-135b as significantly up-regulated miRNAs and miR-193a-3p as a significantly down-regulated one. Moreover, functional assays supported its tumor-suppressive role in colorectal cancer. Finally, the expression status of miR-193a-3p correlated with clinical outcome of patients treated with anti-EGFR antibodies. Taken together, these results show a novel functional role of miR-193a-3p as a tumor suppressive miRNA in colorectal cancer, particularly with BRAF mutations. Further studies are needed to clarify the precise molecular mechanisms as to how this miRNA is involved in colorectal tumorigenesis, which could help further develop a therapeutic strategy for colorectal cancer. Citation Format: Hidekazu Takahashi, Masanobu Takahashi, Shin Takahashi, Hideki Shimodaira, Chikashi Ishioka. microRNA-193a-3p acts as a tumor suppressor in BRAF-mutated colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1096.

Abstract 2115: MEK/ERK and MCL-1 inhibition synergistically reverse apoptosis resistance in colon cancer cells with BRAF(V600E)-mediated MCL-1-upregulation

Abstract Background. Oncogenic BRAFV600E mutations in colorectal cancer (CRC) are associated with poor prognosis and treatment resistance. BRAF mutations activate MAP kinase signaling and may confer apoptosis resistance by upregulating anti-apoptotic BCL-2/BCL-XL/MCL-1 proteins. Results. Ectopic expression of mutant BRAFV600E was shown to potently activate MEK/ ERK that mediated the phosphorylation (T163) and upregulation of MCL-1, but not BCL-2 or BCL-XL. MEK/ERK-mediated phosphorylation of MCL-1 (T163) increased its protein stability, shown by an MCL-1 phospho-mimic mutant. In human colon cancers, mutant vs wild-type BRAF was associated with increased MCL-1 expression by IHC. MEK/ERK inhibition by cobimetinib induced pro-apoptotic BIM in BRAF mutant CRC cell lines (HT-29, RKO), and suppressed phospho-MCL-1/MCL-1 proteins as did ERK siRNA. Suppression of MCL-1 by shRNA or using a small molecule MCL-1 inhibitor (A-1210477) synergistically enhanced cobimetinib-induced apoptosis (Table) that was associated with BAK/BAX activation. A-1210477 antagonized MCL-1 by disrupting its interaction with BAK and BIM proteins, shown by immunoprecipitation. Conclusion. BRAF/MEK/ERK induces upregulation of MCL-1 via phosphorylation/stabilization to confer apoptosis resistance that can be overcome by combined ERK and MCL-1 inhibition, suggesting a novel therapeutic strategy against BRAF mutant CRCs. Drug-induced apoptosis (annexin V) in CRC cell linesDrugsHT-29 % apoptosisRKO % apoptosisDMSO16.842.11A-1210477 (2.5 μM)29.5222.88Cobimetinib(5 μM)28.0421.05Combination53.2250.93 Citation Format: Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. MEK/ERK and MCL-1 inhibition synergistically reverse apoptosis resistance in colon cancer cells with BRAF(V600E)-mediated MCL-1-upregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2115.

Abstract 1078: Micro RNAs as promising therapeutic targets for anti-metastatic therapy in colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the third most common cancer and is a leading cause of cancer related death worldwide and arises by accumulation of genetic and epigenetic alterations. Recently, it has been demonstrated that microRNAs (miRNAs) play critical roles in tumor progression in various cancers. In this study, we tried to find miRNAs associated with distant metastasis and poor outcome and evaluate those miRNAs as therapeutic target for advanced CRC. Methods: MiRNA array was done on CRC specimens derived from tumors with various gene status (KRAS/BRAF/microsatellite instability [MSI] status) with reference of their normal mucosal specimens. The array analysis revealed that a set of miRNAs was specifically down-regulated in CRC with BRAF V600E mutation without MSI, considered to be the poorer outcome. To examine whether the set of miRNAs causes distant metastasis or not, we investigated malignant potential of cell lines transfected and knock-downed the set of miRNAs by siRNA. Expression status of ZEB2 and Epithelial-Mesenchymal Transition (EMT) markers, E-cadherin, were evaluated. In addition, we analyzed expression level of the set of miRNAs in a cohort of 67 stage IV CRCs (TNM staging system by UICC 7th) by quantitative reverse transcription PCR using a comparative Ct method and examined association of target-miRNA fold change (tumor/normal tissue) and their clinocopathilogical findings. Patient survival analysis were performed by Cox proportional hazard model and Kaplan-Meier analysis. Results: In vitro, cell lines transfected the set of miRNAs significantly reduced their malignant potentials. In contrast cell lines knock-downed the set of miRNAs by siRNA obviously increased their malignant potentials. Finally, to confirm our results obtained from in vitro assays, we analyzed expression level of the set of miRNAs in a cohort of stage IV CRCs. Of 67 patients with stage IV CRCs, 15 patients showed KRAS mutation, 4 patients showed BRAF mutation. CRCs with the lower expression level of the set of miRNAs showed poor outcome compared with those with the higher expression level by Cox proportional hazard model and Kaplan-Meier analysis. Conclusions: Our data indicate that miRNAs associated with BRAF mutant CRCs is promising prognostic biomarkers and therapeutic targets for anti-metastatic therapy in CRC. Citation Format: Takashi Kawai, Takeshi Nagasaka, Yoshiko Mori, Tomokazu Fuji, Fumitaka Taniguchi, Keisuke Kimura, Toshiaki Toshima, Kazuya Yasui, Yuzo Umeda, Hiroshi Tazawa, Ajay Goel, Toshiyoshi Fujiwara. Micro RNAs as promising therapeutic targets for anti-metastatic therapy in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1078.

Abstract 3394: Bevacizumab enhances the antitumor effect of BRAFV600E inhibition in colorectal and melanoma xenograft models

Abstract BRAF inhibitors (BRAFi) have shown improved response rate and overall survival (OS) compared to standard chemotherapy in metastatic melanoma. Combinations of BRAF and MEK targeted agents further improve progression-free survival (PFS) and OS when compared with single-agent BRAFi, but the development of resistance remains a major obstacle to long-term disease control. Thus, additional strategies must be exploited to increase the efficacy of BRAF-MEK blockade in metastatic melanoma and circumvent or delay the onset of resistance. We have previously demonstrated in murine xenograft models of BRAF mutant melanomas and colorectal cancers (CRC) that the vemurafenib analog, PLX4720, has cytostatic activity and induces vascular normalization by changing the pro-angiogenic program triggered by the oncogenic mutation (Bottos A et al., 2012;109(6):E353-9). We hypothesized that BRAF-targeted inhibitors could cooperate with anti-angiogenic regimens in the treatment of BRAF-mutant melanoma and colorectal tumors. Here, we demonstrated that when associated with the VEGFA neutralizing antibody bevacizumab (COMBO), PLX4720 shows enhanced anti-cancer effects and induces a de novo transcriptional signature in BRAF mutant melanoma and colorectal xenografts. Interestingly, in the COMBO, bevacizumab does not alter the improvement in blood perfusion and tumor vessel normalization observed with PLX4720 alone. Although targeted inhibition of either BRAF or VEGFA initially suppresses the tumor growth, only combined inhibition of both pathways results in an evident apoptotic effect. Furthermore, bevacizumab delays the onset of resistance to PLX4720 by recruiting macrophages with a M1-like phenotype and inducing a remodeling of the extracellular matrix. The latter is characterized by a reduction in collagen deposition and of cancer-associated fibroblasts (CAFs), known to sustain cancer progression. The effect of bevacizumab on acquired resistance to PLX4720 is transient and depends on the presence of M1 macrophages. Indeed, 3/6 xenografts treated with COMBO start re-growing after 6 weeks with a dramatic reduction of infiltrating macrophages and an increase of CAFs. Collectively, our findings offer a new perspective for the management of clinical resistance to BRAF inhibitors and offer a rationale to combine BRAFV600E inhibitors with anti-angiogenic regimens. Citation Format: Valentina Comunanza, Davide Corà, Emanuele Middonti, Federica Di Nicolantonio, Enzo Medico, Dario Sangiolo, Federico Bussolino. Bevacizumab enhances the antitumor effect of BRAFV600E inhibition in colorectal and melanoma xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3394.

Abstract 3782: Development of a dual inhibitor of pan-RAF and VEGFR2 for the treatment of metastatic colorectal cancer with mutant K-RAS

Abstract Signal transduction in the RAS-RAF-MEK-ERK (MAPK) pathway plays a key role in cell survival, growth and proliferation. The pathway is controlled by extracellular signals through receptor tyrosine kinases (RTK) and is activated by oncogenic mutations in many types of cancer. K-RAS mutation has been reported in 40% of colorectal cancer patients. These patients have acquired resistance to EGFR tyrosine kinase inhibitors. Therefore, there is a need for the development of new therapeutic approaches for the treatment of these cancers. The B-RAF selective inhibitors have been approved for the treatment of human melanoma with a B-RAF V600E mutation. However, B-RAF specific inhibitor activates C-RAF in RAS mutant cells by inducing RAF dimer formation and paradoxical activation of C-RAF. These observations require the development of pan-RAF inhibitors without inducing paradoxical activation. In addition, as VEGFR2 represents one crucial promoter of tumor angiogenesis in colorectal cancer, the inhibition of VEGFR2 has been expected to be synergistic for the treatment of colorectal cancer. Therefore, we tried to find dual inhibitors of pan-RAF and VEGFR2 using structure based molecular modeling and identified a selective inhibitor, C1005, which binds to the DFG-out inactive conformation of B-RAF. In addition, C1005 potentially inhibited B-RAF, C-RAF, B-RAF V600E and VEGFR2 with IC50 value of 2.7 nM, 0.7 nM, 5.2 nM and 11nM, respectively. C1005 completely inhibited the phosphorylation of MEK-ERK without paradoxical activation in K-RAS mutant cells compared with Vemurafenib and Regorafenib. We examined the effects of C1005 on cell proliferation of colorectal cancer cells with K-RAS or B-RAF mutation. C1005 displayed potent anti-proliferative activities not only in K-RAS mutant colorectal cancer cells but also in B-RAF mutant colorectal cancer cells. C1005 effectively blocked cell proliferation of HCT-116, LS-513 and WiDr at EC50 of 145 nM, 79 nM and 636 nM, respectively. In addition, growth inhibition of VEGF-stimulated HUVEC indicates that C1005 is a potent inhibitor of the angiogenesis. To study in vivo effect, we tested C1005 in colorectal cancer xenografts with mutant K-RAS. Oral treatment of animals bearing xenograft tumors by 30 and 60 mg/kg, twice daily (BID) of C1005, induced a dose-dependent tumor growth inhibition. We observed 77% and 89% reduction of tumor volume, respectively. In this study, we discovered an orally active dual inhibitor of pan-RAF and VEGFR2. C1005 has been shown to suppress mutant K-RAS colorectal cancer cells without activating the MAPK pathway. Our findings suggest that C1005 could be an excellent preclinical candidate to treat mutant K-RAS driven colorectal cancer. Citation Format: Sungpyo Hong, Young-Il Choi, Jihye Choi, Wonyoung Kim, Ho-Seok Kwon, Yong Bin Park, Min-Hyo Ki, Hee Jong Shin, Michael Lee, Soon Kil Ahn. Development of a dual inhibitor of pan-RAF and VEGFR2 for the treatment of metastatic colorectal cancer with mutant K-RAS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3782.

Abstract 2818: An unbiased tumor cell panel profiling method to identify drug-drug interactions reveals synergy between the CDK4 and CDK 6 inhibitor abemaciclib and the Raf dimer and pan-Raf inhibitor LY3009120 in Ras mutant cancers

Abstract Drug sensitivity profiling across genomically characterized panels of tumor cells can identify the molecular determinants of drug response. By testing compound combinations in an unbiased format, the same methodology can be used to identify the genomic context of drug-drug synergy. Based on this principle, we developed an unbiased combination screening protocol to identify synergistic interactions with LY3009120, a novel Raf dimer inhibitor that inhibits all three Raf isoforms (Peng et al. 2015, Cancer Cell 28:384-98). Inhibitors of the Ras-MAPK pathway have proven very effective in the treatment of BRAF-mutant melanoma but are, in general, only partially effective in the treatment of BRAF-mutant colorectal cancer and Ras mutant cancers. LY3009120 combined with various compounds was screened across panels of genomically characterized tumor cells. These screens identified a strong synergy with abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6). Statistical analysis of effects in over 500 cancer cell lines showed that mutations in BRAF or Ras family genes were strongly associated with sensitivity to this combination. Strong synergy was observed in skin, colorectal, lung and pancreatic cancers with Ras/Raf mutations, but was also observed in various cancer cells wild type for Ras pathway genes. This included tumor types sensitive to single agent abemaciclib, such as mantle cell lymphoma, ER+ breast cancers, certain leukemias, squamous non-small cell lung cancer, and/or lung cancer with receptor tyrosine kinase activation. In vitro and in vivo analyses of the effects of the combination treatment on signaling pathways in KRAS mutant cancers led to potential mechanistic explanations for the differing efficacy of the combination, which manifests as regression of tumor xenografts in rodent models. Citation Format: Xueqian Gong, Wenjuan Wu, Li-Chun Chio, Susan Pratt, Constance King, Yue Webster, Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Philip Iversen, Christoph Reinhard, Shih-Hsun Chen, Richard Bechmann, Sheng-Bin Peng, Sean Buchanan. An unbiased tumor cell panel profiling method to identify drug-drug interactions reveals synergy between the CDK4 and CDK 6 inhibitor abemaciclib and the Raf dimer and pan-Raf inhibitor LY3009120 in Ras mutant cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2818.

Evaluation of [18F]Fluorothymidine as a Biomarker for Early Therapy Response in a Mouse Model of Colorectal Cancer.

In oncology, positron emission tomography imaging using dedicated tracers as biomarkers may assist in early evaluation of therapy efficacy. Using 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), we investigated the early effects of chemotherapeutic treatment on cancer cell proliferation in a BRAF-mutated colorectal cancer xenograft model. Colo205 subcutaneously inoculated animals underwent 90-min dynamic imaging before and 24h after treatment with vehicle (control), cetuximab (resistant) or irinotecan (sensitive). Total distribution volume was quantified from dynamic data, and standardized uptake values as well as tumor-to-blood ratios were calculated from static images averaged over the last 20min. In vivo imaging data was correlated with ex vivo proliferation and thymidine metabolism proteins. All imaging parameters showed a significant post-treatment decrease from [18F]FLT baseline uptake for the irinotecan group (p ≤ 0.001) as compared with the cetuximab and vehicle group and correlated strongly with each other (p ≤ 0.0001). In vivo data were in agreement with Ki67 staining, showing a significantly lower percentage of Ki67-positive cells in the irinotecan group as compared with other groups (p ≤ 0.0001). Tumor expression of thymidine kinase 1 phosphorylated on serine 13, thymidylate synthase, and thymidine phosphorylase remained unaffected, while thymidine kinase 1 expression was, surprisingly, significantly higher in irinotecan-treated animals (p ≤ 0.01). In contrast, tumor ATP levels were lowest in this group. [18F]FLT positron emission tomography was found to be a suitable biomarker of early tumor response to anti-proliferative treatment, with static imaging not being inferior to full compartmental analysis in our xenograft model. The dynamics of thymidine kinase 1 protein expression and protein activity in low ATP environments merits further investigation.

O-026 Combination of encorafenib and cetuximab with or without alpelisib in patients with advanced BRAF-mutant colorectal cancer (BRAFm CRC): phase 2 results

O-026 Combination of encorafenib and cetuximab with or without alpelisib in patients with advanced BRAF-mutant colorectal cancer (BRAFm CRC): phase 2 results