Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma

Melanoma harboring BRAF mutations frequently develop resistance to BRAF inhibitors, limiting the impact of treatment. Here, we establish a mechanism of resistance and subsequently identified a suitable drug combination to overcome the resistance. Single treatment of BRAF mutant melanoma cell lines with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in overexpression of Mcl-1. Overexpression of Mcl-1 in A375 and SK-MEL-28 by transfection completely blocked BRAF and MEK1/2 inhibitor-mediated inhibition of cell survival and apoptosis. Melanoma cells resistant to BRAF inhibitors showed massive expression of Mcl-1 as compared to respective sensitive cell lines. Silencing of Mcl-1 using siRNA completely sensitized resistant melanoma cells to growth suppression and induction of apoptosis by BRAF inhibitors. In vivo, vemurafenib resistant A375 xenografts implanted in athymic nude mice showed substantial tumor growth inhibition when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA. Immunohistochemistry and western blot analyses demonstrated enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was diminished in the tumors of mice treated with either of the combination. Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1. These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors

A melanocyte lineage program confers resistance to MAP kinase pathway inhibition

BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1. RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4. Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations. Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors. These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB). Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors. Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF. Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance. Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics

BRAF inhibition is associated with increased clonality in tumor-infiltrating lymphocytes

There have been significant advances with regard to BRAF-targeted therapies against metastatic melanoma. However, the majority of patients receiving BRAF inhibitors (BRAFi) manifest disease progression within a year. We have recently shown that melanoma patients treated with BRAFi exhibit an increase in melanoma-associated antigens and in CD8+ tumor-infiltrating lymphocytes in response to therapy. To characterize such a T-cell infiltrate, we analyzed the complementarity-determining region 3 (CDR3) of rearranged T-cell receptor (TCR) β chain-coding genes in tumor biopsies obtained before the initiation of BRAFi and 10–14 d later. We observed an increase in the clonality of tumor-infiltrating lymphocytes in 7 of 8 patients receiving BRAFi, with a statistically significant 21% aggregate increase in clonality. Over 80% of individual T-cell clones detected after initiation of BRAFi treatment were new clones. Interestingly, the comparison of tumor infiltrates with clinical responses revealed that patients who had a high proportion of pre-existing dominant clones after the administration of BRAFi responded better to therapy than patients who had a low proportion of such pre-existing dominant clones following BRAFi. These data suggest that although the inhibition of BRAF in melanoma patients results in tumor infiltration by new lymphocytes, the response to treatment appears to be related to the presence of a pre-existing population of tumor-infiltrating T-cell clones

Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion.

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance

Proteasomal down-regulation of the proapoptotic MST2 pathway contributes to BRAF inhibitor resistance in melanoma

The RAS-RAF-MEK-ERK pathway is hyperactivated in most malignant melanomas, and mutations in BRAF or NRAS account for most of these cases. BRAF inhibitors (BRAFi) are highly efficient for treating patients with BRAFV600E mutations, but tumours frequently acquire resistance within a few months. Multiple resistance mechanisms have been identified, due to mutations or network adaptations that revive ERK signalling. We have previously shown that RAF proteins inhibit the MST2 proapoptotic pathway in a kinase-independent fashion. Here, we have investigated the role of the MST2 pathway in mediating resistance to BRAFi. We show that the BRAFV600E mutant protein, but not the wild-type BRAF protein, binds to MST2 inhibiting its proapoptotic signalling. Down-regulation of MST2 reduces BRAFi-induced apoptosis. In BRAFi-resistant cell lines, MST2 pathway proteins are down-regulated by ubiquitination and subsequent proteasomal degradation rendering cells refractory to MST2 pathway–induced apoptosis. Restoration of apoptosis can be achieved by increasing MST2 pathway protein expression using proteasome inhibitors. In summary, we show that the MST2 pathway plays a role in the acquisition of BRAFi resistance in melanoma.Science Foundation IrelandCDAUCD’s School of Medicine Ad Astra Fellowshi

PDGFRα up-regulation mediated by sonic hedgehog pathway activation leads to BRAF inhibitor resistance in melanoma cells with BRAF mutation

Control of BRAF(V600E) metastatic melanoma by BRAF inhibitor (BRAF-I) is limited by intrinsic and acquired resistance. Growth factor receptor up-regulation is among the mechanisms underlying BRAF-I resistance of melanoma cells. Here we demonstrate for the first time that PDGFRα up-regulation causes BRAF-I resistance. PDGFRα inhibition by PDGFRα-specific short hairpin (sh)RNA and by PDGFRα inhibitors restores and increases melanoma cells' sensitivity to BRAF-I in vitro and in vivo. This effect reflects the inhibition of ERK and AKT activation which is associated with BRAF-I resistance of melanoma cells. PDGFRα up-regulation is mediated by Sonic Hedgehog Homolog (Shh) pathway activation which is induced by BRAF-I treatment. Similarly to PDGFRα inhibition, Shh inhibition by LDE225 restores and increases melanoma cells' sensitivity to BRAF-I. These effects are mediated by PDGFRα down-regulation and by ERK and AKT inhibition. The clinical relevance of these data is indicated by the association of PDGFRα up-regulation in melanoma matched biopsies of BRAF-I +/- MEK inhibitor treated patients with shorter time to disease progression and less tumor regression. These findings suggest that monitoring patients for early PDGFRα up-regulation will facilitate the identification of those who may benefit from the treatment with BRAF-I in combination with clinically approved PDGFRα or Shh inhibitors

Clinical Profiling of BCL-2 Family Members in the Setting of BRAF Inhibition Offers a Rationale for Targeting De Novo Resistance Using BH3 Mimetics

While response rates to BRAF inhibitiors (BRAFi) are high, disease progression emerges quickly. One strategy to delay the onset of resistance is to target anti-apoptotic proteins such as BCL-2, known to be associated with a poor prognosis. We analyzed BCL-2 family member expression levels of 34 samples from 17 patients collected before and 10 to 14 days after treatment initiation with either vemurafenib or dabrafenib/trametinib combination. The observed changes in mRNA and protein levels with BRAFi treatment led us to hypothesize that combining BRAFi with a BCL-2 inhibitor (the BH3-mimetic navitoclax) would improve outcome. We tested this hypothesis in cell lines and in mice. Pretreatment mRNA levels of BCL-2 negatively correlated with maximal tumor regression. Early increases in mRNA levels were seen in BIM, BCL-XL, BID and BCL2-W, as were decreases in MCL-1 and BCL2A. No significant changes were observed with BCL-2. Using reverse phase protein array (RPPA), significant increases in protein levels were found in BIM and BID. No changes in mRNA or protein correlated with response. Concurrent BRAF (PLX4720) and BCL2 (navitoclax) inhibition synergistically reduced viability in BRAF mutant cell lines and correlated with down-modulation of MCL-1 and BIM induction after PLX4720 treatment. In xenograft models, navitoclax enhanced the efficacy of PLX4720. The combination of a selective BRAF inhibitor with a BH3-mimetic promises to be an important therapeutic strategy capable of enhancing the clinical efficacy of BRAF inhibition in many patients that might otherwise succumb quickly to de novo resistance. Trial Registrations: ClinicalTrials.gov NCT01006980; ClinicalTrials.gov NCT01107418; ClinicalTrials.gov NCT01264380; ClinicalTrials.gov NCT01248936; ClinicalTrials.gov NCT00949702; ClinicalTrials.gov NCT0107217

Tumor microenvironment induces innate RAF-inhibitor resistance through HGF secretion

Drug resistance remains a vexing problem in the treatment of cancer patients. While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anti-cancer drugs. We found that stroma-mediated resistance is surprisingly common – particularly to targeted agents. We further characterized the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most of these patients exhibit some degree of innate resistance1-4. Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition. Immunohistochemistry confirmed stromal HGF expression in patients with BRAF-mutant melanoma and a statistically significant correlation between stromal HGF expression and innate resistance to treatment. Dual inhibition of RAF and MET resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance

Targeting endothelin receptor signalling overcomes heterogeneity driven therapy failure

Approaches to prolong responses to BRAF targeting drugs in melanoma patients are challenged by phenotype heterogeneity. Melanomas of a “MITF‐high” phenotype usually respond well to BRAF inhibitor therapy, but these melanomas also contain subpopulations of the de novo resistance “AXL‐high” phenotype. > 50% of melanomas progress with enriched “AXL‐high” populations, and because AXL is linked to de‐differentiation and invasiveness avoiding an “AXL‐high relapse” is desirable. We discovered that phenotype heterogeneity is supported during the response phase of BRAF inhibitor therapy due to MITF‐induced expression of endothelin 1 (EDN1). EDN1 expression is enhanced in tumours of patients on treatment and confers drug resistance through ERK re‐activation in a paracrine manner. Most importantly, EDN1 not only supports MITF‐high populations through the endothelin receptor B (EDNRB), but also AXL‐high populations through EDNRA, making it a master regulator of phenotype heterogeneity. Endothelin receptor antagonists suppress AXL‐high‐expressing cells and sensitize to BRAF inhibition, suggesting that targeting EDN1 signalling could improve BRAF inhibitor responses without selecting for AXL‐high cells