EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib.
ABSTRACT: BRAF mutations occur in 10-15% of colorectal cancers (CRCs) and confer adverse outcome. While RAF inhibitors such as vemurafenib (PLX4032) have proven effective in BRAF mutant melanoma, they are surprisingly ineffective in BRAF mutant CRCs, and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRCs expressed higher levels of phospho-EGFR than BRAF mutant melanomas, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells and markedly improved efficacy in vitro and in vivo. These findings support evaluation of combined RAF and EGFR inhibition in BRAF mutant CRC patients.BRAF valine 600 (V600) mutations occur in 10% to 15% of colorectal cancers, yet these tumors show a surprisingly low clinical response rate (~5%) to selective RAF inhibitors such as vemurafenib, which have produced dramatic response rates (60%–80%) in melanomas harboring the identical BRAF V600 mutation. We found that EGFR-mediated MAPK pathway reactivation leads to resistance to vemurafenib in BRAF-mutant colorectal cancers and that combined RAF and EGFR inhibition can lead to sustained MAPK pathway suppression and improved efficacy in vitro and in tumor xenografts.
Project description:RAF kinase is crucially involved in cell proliferation and survival in colorectal cancer (CRC). Patients with metastatic CRC (mCRC) harboring BRAF mutations (BRAFms) not only experience a poor prognosis but also benefit less from therapeutics targeting ERK signaling. With advances in RAF inhibitors and second-generation inhibitors including encorafenib and vemurafenib, which have been approved for treating BRAF-V600E malignancies, the combinatorial therapeutic strategies of RAF inhibitors elicit remarkable responses in patients with BRAF-V600E mCRC. However, the therapeutic efficacy is restricted by resistance, which might be due to RAF dimerization and reactivation of the MAPK pathway. In addition, the next-generation RAF inhibitors, which are characterized by varying structural and biochemical properties, have achieved preclinical and clinical advances. Herein, we summarize the existing mechanism of RAF kinases in CRC, including MAPK feedback reactivation of resistance to RAF inhibitors. We additionally summarize the development of three generations of RAF inhibitors and different therapeutic strategies including the combination of EGFR, BRAF, and PI3K inhibitors for BRAFm CRC treatment.
Project description:Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in ?2-7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAFV600E-mutant allele, the spectrum of BRAF mutations in LA includes BRAFV600E (?60% of cases) and non-V600E mutant alleles (?40% of cases) such as BRAFG469A and BRAFG466V The presence of BRAFV600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAFV600E-mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAFV600E and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAFV600E that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients.
Project description:Most patients with BRAF(V600)-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF(V600)-mutant metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a "long tail" of new mitogen-activated protein kinase (MAPK) pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAF(V600)-mutant melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies.
Project description:Vemurafenib, a RAF inhibitor, extends survival in patients with BRAF(V600)-mutant melanoma but activates extracellular signal-regulated kinase (ERK) signaling in RAS-mutant cells. In a patient with a BRAF(V600K)-mutant melanoma responding to vemurafenib, we observed accelerated progression of a previously unrecognized NRAS-mutant leukemia. We hypothesized that combining vemurafenib with a MAP-ERK kinase (MEK) inhibitor would inhibit ERK activation in the melanoma and prevent ERK activation by vemurafenib in the leukemia, and thus suppress both malignancies. We demonstrate that intermittent administration of vemurafenib led to a near-complete remission of the melanoma, and the addition of the MEK inhibitor cobimetinib (GDC-0973) caused suppression of vemurafenib-induced leukemic proliferation and ERK activation. Antimelanoma and antileukemia responses have been maintained for nearly 20 months, as documented by serial measurements of tumor-derived DNA in plasma in addition to conventional radiographic and clinical assessments of response. These data support testing of intermittent ERK pathway inhibition in the therapy for both RAS-mutant leukemia and BRAF-mutant melanoma.
Project description:Most melanomas harbor oncogenic BRAF(V600) mutations, which constitutively activate the MAPK pathway. Although MAPK pathway inhibitors show clinical benefit in BRAF(V600)-mutant melanoma, it remains incompletely understood why 10% to 20% of patients fail to respond. Here, we show that RAF inhibitor-sensitive and inhibitor-resistant BRAF(V600)-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-?B signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-?B-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-?B activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-?B activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAF(V600)-mutant melanoma.Although most BRAF(V600)-mutant melanomas are sensitive to RAF and/or MEK inhibitors, a subset fails to respond to such treatment. This study characterizes a transcriptional cell state distinction linked to MITF and NF-?B that may modulate intrinsic sensitivity of melanomas to MAPK pathway inhibitors.
Project description:In melanoma, dysregulation of the MAPK pathway, usually via BRAF(V600) or NRAS(Q61) somatic mutations, leads to constitutive ERK signaling. While BRAF inhibitors are initially effective for BRAF-mutant melanoma, no FDA-approved targeted therapies exist for BRAF-inhibitor-resistant BRAF(V600), NRAS mutant, or wild-type melanoma.The 50% inhibitory concentration (IC50) of SCH772984, a novel inhibitor of ERK1/2, was determined in a panel of 50 melanoma cell lines. Effects on MAPK and AKT signaling by western blotting and cell cycle by flow cytometry were determined.Sensitivity fell into three groups: sensitive, 50% inhibitory concentration (IC50) < 1 ?M; intermediately sensitive, IC50 1-2 ?M; and resistant, >2 ?M. Fifteen of 21 (71%) BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH772984. All three (100%) BRAF/NRAS double mutants, 11 of 14 (78%) NRAS mutants and 5 of 7 (71%) wild-type melanomas were sensitive. Among BRAF(V600) mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH772984. SCH772984 caused G1 arrest and induced apoptosis.Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines and significantly delayed the onset of acquired resistance in long term in vitro assays. Therefore, SCH772984 may be clinically applicable as a treatment for non-BRAF mutant melanoma or in BRAF-mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors.
Project description:The use of targeted therapeutics directed against BRAF(V600)-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAF(V600)-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor-resistant alleles were sensitive to RAF/MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents.
Project description:The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N = 571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted in vitro studies using both wild-type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with a concomitant decrease in trypsin-1 and -2 secretion. Notably, no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in the therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.
Project description:Vemurafenib, a selective inhibitor of BRAF(V600), has shown significant activity in BRAF(V600) melanoma but not in less than 10% of metastatic BRAF(V600) colorectal cancers (CRC), suggesting that studies of the unique hypermethylated phenotype and concurrent oncogenic activation of BRAF(mut) CRC may provide combinatorial strategies.We conducted comparative proteomic analysis of BRAF(V600E) melanoma and CRC cell lines, followed by correlation of phosphoinositide 3-kinase (PI3K) pathway activation and sensitivity to the vemurafenib analogue PLX4720. Pharmacologic inhibitors and siRNA were used in combination with PLX4720 to inhibit PI3K and methyltransferase in cell lines and murine models.Compared with melanoma, CRC lines show higher levels of PI3K/AKT pathway activation. CRC cell lines with mutations in PTEN or PIK3CA were less sensitive to growth inhibition by PLX4720 (P = 0.03), and knockdown of PTEN expression in sensitive CRC cells reduced growth inhibition by the drug. Combined treatment of PLX4720 with PI3K inhibitors caused synergistic growth inhibition in BRAF-mutant CRC cells with both primary and secondary resistance. In addition, methyltransferase inhibition was synergistic with PLX4720 and decreased AKT activation. In vivo, PLX4720 combined with either inhibitors of AKT or methyltransferase showed greater tumor growth inhibition than PLX4720 alone. Clones with acquired resistance to PLX4720 in vitro showed PI3K/AKT activation with EGF receptor (EGFR) or KRAS amplification.We show that activation of the PI3K/AKT pathway is a mechanism of both innate and acquired resistance to BRAF inhibitors in BRAF(V600E) CRC and suggest combinatorial approaches to improve outcomes in this poor prognosis subset of patients.
Project description:RAF inhibitors such as vemurafenib and dabrafenib block BRAF-mediated cell proliferation and achieve meaningful clinical benefit in the vast majority of patients with BRAF(V600E)-mutant melanoma. However, some patients do not respond to this regimen, and nearly all progress to therapeutic resistance. We used a pooled RNA interference screen targeting more than 16,500 genes to discover loss-of-function events that could drive resistance to RAF inhibition. The highest ranking gene was NF1, which encodes neurofibromin, a tumor suppressor that inhibits RAS activity. NF1 loss mediates resistance to RAF and mitogen-activated protein kinase (MAPK) kinase kinase (MEK) inhibitors through sustained MAPK pathway activation. However, cells lacking NF1 retained sensitivity to the irreversible RAF inhibitor AZ628 and an ERK inhibitor. NF1 mutations were observed in BRAF-mutant tumor cells that are intrinsically resistant to RAF inhibition and in melanoma tumors obtained from patients exhibiting resistance to vemurafenib, thus showing the clinical potential for NF1-driven resistance to RAF/MEK-targeted therapies.