Acquired BRAF Inhibitor Resistance Research Articles

Single-cell genomics analysis reveals complex genetic interactions in an in vivo model of acquired BRAF inhibitor resistance.

The evolution of therapeutic resistance is a major obstacle to the success of targeted oncology drugs. While both inter- and intratumoral heterogeneity limit our ability to detect resistant subpopulations that pre-exist or emerge during treatment, our ability to analyze tumors with single-cell resolution is limited. Here, we utilized a cell-based transposon mutagenesis method to identify mechanisms of BRAF inhibitor resistance in a model of cutaneous melanoma. This screen identified overexpression of NEDD4L and VGLL3 as significant drivers of BRAF inhibitor resistance in vivo. In addition, we describe a novel single-cell genomics profiling method to genotype thousands of individual cells within tumors driven by transposon mutagenesis. This approach revealed a surprising genetic diversity among xenograft tumors and identified recurrent co-occurring mutations that emerge within distinct tumor subclones. Taken together, these observations reveal an unappreciated genetic complexity that drives BRAF inhibitor resistance.

Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFRhi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma.

Divergent BRAF Inhibitor Resistance Mechanisms Revealed through Epigenetic Mapping

Although tremendous progress has been made in targeted and immune-based treatments for advanced melanoma, there remains a substantial therapeutic failure rate. For patients with BRAF(V600)-mutant melanomas, resistance to BRAF inhibitors remains a significant survival hurdle. Although multiple compensatory mechanisms to bypass BRAF blockade have been discovered, the epigenetic patterns are still poorly characterized. In this report, we generated eight matched pairs of vemurafenib-sensitive/-resistant melanoma lines and subjected these to concurrent RNA-sequencing and H3K27ac chromatin immunoprecipitation sequencing analysis. Globally, we identified two classes of epigenetic profiles that correlate with resistance. Class 1 resistance involves fewer RNA expression alterations accompanied by fewer enhancer mark changes with H3K27ac. Class 2 resistance shows widespread alterations in transcription and enhancer profiles, which converge on epithelial‒mesenchymal transition and hypoxia-related pathways. We also observed significant and dynamic changes in superenhancers that underpin these transcriptomic patterns. We subsequently verified the two-class structure in pre-BRAF inhibitors and postrelapse human melanoma specimens. Our findings reveal a broad and underappreciated spectrum of epigenetic plasticity during acquired BRAF inhibitor resistance.

Clinical Implications of Acquired BRAF Inhibitors Resistance in Melanoma.

Understanding the role of mitogen-activated protein kinase (MAPK) pathway-activating mutations in the development and progression of melanoma and their possible use as therapeutic targets has substantially changed the management of this neoplasm, which, until a few years ago, was burdened by severe mortality. However, the presence of numerous intrinsic and extrinsic mechanisms of resistance to BRAF inhibitors compromises the treatment responses’ effectiveness and durability. The strategy of overcoming these resistances by combination therapy has proved successful, with the additional benefit of reducing side effects derived from paradoxical activation of the MAPK pathway. Furthermore, the use of other highly specific inhibitors, intermittent dosing schedules and the association of combination therapy with immune checkpoint inhibitors are promising new therapeutic strategies. However, numerous issues related to dose, tolerability and administration sequence still need to be clarified, as is to be expected from currently ongoing trials. In this review, we describe the clinical results of using BRAF inhibitors in advanced melanoma, with a keen interest in strategies aimed at overcoming resistance.

Mechanisms of Acquired BRAF Inhibitor Resistance in Melanoma: A Systematic Review.

Simple SummaryPatients with advanced melanoma are often treated with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors. Although these agents prolong life, patients inevitably develop resistance and their cancer progresses. This review examines all of the potential ways that melanoma cells develop resistance to BRAF inhibitors. These mechanisms involve genetic and epigenetic changes that activate different signaling pathways, thereby bypassing the effect of BRAF inhibition, but they also involve a change in cell phenotype and the suppression of anticancer immune responses. Currently, BRAF inhibitor resistance can be partially overcome by combining a BRAF inhibitor with a mitogen-activated protein kinase kinase (MEK) inhibitor, but many other combinations are being tested. Eventually, it may be possible to choose the best combination of drugs based on the genetic profile of an individual’s cancer.This systematic review investigated the literature on acquired v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitor resistance in patients with melanoma. We searched MEDLINE for articles on BRAF inhibitor resistance in patients with melanoma published since January 2010 in the following areas: (1) genetic basis of resistance; (2) epigenetic and transcriptomic mechanisms; (3) influence of the immune system on resistance development; and (4) combination therapy to overcome resistance. Common resistance mutations in melanoma are BRAF splice variants, BRAF amplification, neuroblastoma RAS viral oncogene homolog (NRAS) mutations and mitogen-activated protein kinase kinase 1/2 (MEK1/2) mutations. Genetic and epigenetic changes reactivate previously blocked mitogen-activated protein kinase (MAPK) pathways, activate alternative signaling pathways, and cause epithelial-to-mesenchymal transition. Once BRAF inhibitor resistance develops, the tumor microenvironment reverts to a low immunogenic state secondary to the induction of programmed cell death ligand-1. Combining a BRAF inhibitor with a MEK inhibitor delays resistance development and increases duration of response. Multiple other combinations based on known mechanisms of resistance are being investigated. BRAF inhibitor-resistant cells develop a range of ‘escape routes’, so multiple different treatment targets will probably be required to overcome resistance. In the future, it may be possible to personalize combination therapy towards the specific resistance pathway in individual patients.

Noncanonical EphA2 Signaling Is a Driver of Tumor-Endothelial Cell Interactions and Metastatic Dissemination in BRAF Inhibitor‒Resistant Melanoma

Acquired BRAF/MAPK/extracellular signal‒regulated kinase inhibitor resistance in melanoma results in a new transcriptional state associated with an increased risk of metastasis. In this study, we identified noncanonical ephrin receptor (Eph) EphA2 signaling as a driver of the resistance-associated metastatic state. We used mass spectrometry‒based proteomic and phenotypic assays to demonstrate that the expression of active noncanonical EphA2-S897E in melanoma cells led to a mesenchymal-to-amoeboid transition driven by Cdc42 activation. The induction of mesenchymal-to-amoeboid transition promoted melanoma cell invasion, survival under shear stress, adhesion to endothelial cells under continuous-flow conditions, increased permeability of endothelial cell monolayers, and stimulated melanoma transendothelial cell migration. Invivo, melanoma cells expressing EphA2-S897E or active Cdc42 showed superior lung retention after tail-vain injection. Analysis of BRAF inhibitor‒sensitive and ‒resistant melanoma cells demonstrated resistance to be associated with a mesenchymal-to-amoeboid transition switch, upregulation of Cdc42 activity, increased invasion, and transendothelial migration. The drug-resistant metastatic state was dependent on histone deacetylase 8 activity. Silencing of histone deacetylase 8 led to the inhibition of EphA2 and protein kinase B phosphorylation, reduced invasion, and impaired melanoma cell-endothelial cell interactions. In summary, we have demonstrated that the metastatic state associated with acquired BRAF inhibitor resistance is dependent on noncanonical EphA2 signaling, leading to increased melanoma-endothelial cell interactions and enhanced tumor dissemination.

Loss of HAT1 expression confers BRAFV600E inhibitor resistance to melanoma cells by activating MAPK signaling via IGF1R

BRAF inhibitors (BRAFi) have been approved for the clinical treatment of BRAF-mutant metastatic melanoma. Although initial responses to BRAFi are generally favorable, acquired BRAFi resistance emerges rapidly, resulting in treatment failure. Only some of the underlying mechanisms responsible for BRAFi resistance are currently understood. Here, we showed that the genetic inhibition of histone acetyltransferase 1 (HAT1) in BRAF-mutant melanoma cells resulted in BRAFi resistance. Using quantitative immunofluorescence analysis of patient sample pairs, consisting of pre-treatment along with matched progressed BRAFi + MEKi-treated melanoma samples, HAT1 downregulation was observed in 7/11 progressed samples (~63%) in comparison with pre-treated samples. Employing NanoString-based nCounter PanCancer Pathway Panel-based gene expression analysis, we identified increased MAPK, Ras, transforming growth factor (TGF)-β, and Wnt pathway activation in HAT1 expression inhibited cells. We further found that MAPK pathway activation following the loss of HAT1 expression was partially driven by increased insulin growth factor 1 receptor (IGF1R) signaling. We showed that both MAPK and IGF1R pathway inhibition, using the ERK inhibitor SCH772984 and the IGF1R inhibitor BMS-754807, respectively, restored BRAFi sensitivity in melanoma cells lacking HAT1. Collectively, we show that the loss of HAT1 expression confers acquired BRAFi resistance by activating the MAPK signaling pathway via IGF1R.

Abstract 4698: Development of a CRISPR-Cas9D10A targetable, high-complexity, single-cell barcoding approach for capture of treatment resistant subclones from heterogeneous cancers

Abstract Cancers are composed of heterogeneous cell populations and the clonal evolution of these cells is one of the key reasons for treatment resistance and tumor recurrence. A fundamental challenge in studying clonal evolution in these tumors is the difficulty in capturing the phenotype-associated (e.g. treatment resistant) sub-populations from the heterogeneous population. We developed an approach to individually barcode and isolate specific cell subpopulations by constructing a Cas9D10A and paired-gRNA targetable unique reporter (CAPTURE) barcoding library with up to 36 million unique barcodes. This approach enabled us to uniquely barcode > 1 million cells, track barcode distribution following treatment and then isolate the resistant subpopulation using the subpopulation-specific barcode. Proof of principle studies showed that specific barcoding cells, even at as low as 0.1%, could be efficiently isolated using barcode targeting and cell sorting. Applied our barcoding approach, we found that A375 cell acquired BRAF inhibitor resistance both from pre-exiting and late-emerging mechanisms. Colony formation experiments showed that the isolated subpopulations were indeed resistant clones. Whole exome, transcriptome and methylome analysis were applied to study the captured subpopulations. Our CAPTURE barcoding approach will enable the identification of the both pre-exiting and late-emerging genetic or epigenetic changes driving treatment resistance. Citation Format: Ze-yan Zhang, Ravesanker Ezhilarasan, Yingwen Ding, Qianghu Wang, Jie Yang, Lihong Long, Roel G. Verhaak, Erik P. Sulman. Development of a CRISPR-Cas9D10A targetable, high-complexity, single-cell barcoding approach for capture of treatment resistant subclones from heterogeneous cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4698.

A targeted genomic alteration analysis predicts survival of melanoma patients under BRAF inhibitors.

Several mechanisms have been described to elucidate the emergence of resistance to MAPK inhibitors in melanoma and there is a crucial need for biomarkers to identify patients who are likely to achieve a better and long-lasting response to BRAF inhibitors therapy. In this study, we developed a targeted approach combining both mRNA and DNA alterations analysis focusing on relevant gene alterations involved in acquired BRAF inhibitor resistance. We collected baseline tumor samples from 64 melanoma patients at BRAF inhibitor treatment initiation and showed that the presence, prior to treatment, of mRNA over-expression of genes’ subset was significantly associated with improved progression free survival and overall survival. The presence of DNA alterations was in favor of better overall survival. The genomic analysis of relapsed-matched tumor samples from 20 patients allowed us to uncover the largest landscape of resistance mechanisms reported to date as at least one resistance mechanism was identified for each patient studied. Alterations in RB1 have been most frequent and hence represent an important additional acquired resistance mechanism. Our targeted genomic analysis emerges as a relevant tool in clinical practice to identify those patients who are more likely to achieve durable response to targeted therapies and to exhaustively describe the spectrum of resistance mechanisms. Our approach can be adapted to new targeted therapies by including newly identified genetic alterations.

Abstract 5833: Escape form adaptive drug tolerance through OGT and TET1 mediated H3K4me3 remodeling in MAPKi resistant melanoma

Abstract Background Acquired drug resistance in BRAF mutant melanoma is the main cause for disease relapse. We previously described a slow cycling induced drug tolerant state upon continuous BRAF/MEK treatment preceding permanent resistance with generic changes in histone methylation. Rationale Genetic alterations linked to acquired BRAF inhibitor resistance are absent in about 40% of relapsed melanoma patients suggesting the involvement of epigenetic alterations in the development of acquired drug resistance. We investigated epigenetic remodeling in BRAF mutant melanoma upon BRAF/MEK inhibition. Methods An in-vitro model of time lapse dependent transition to acquired drug resistance using mutant BRAF melanoma was used to investigate epigenetic changes following chronic drug exposure. Histone methylation patters were investigated using ChIP-seq, followed by target gene promoter ChIP-PCR and functional verification. Findings were confirmed by gene silencing, combined treatment regimes in vivo, in PDX tumors and clinical data sets. Results A state dependent response to chronic drug treatment was observed. Long term treatment of more than 45 days enables the cells to escape the slow cycling state which results in proliferating cellular clusters (drug-tolerant persister colonies) with stem-like characteristics that regain global H3K4me3. Persister colonies are then giving rise to fast proliferating BRAF/MEK inhibitor resistant cells. H3K4me3 ChIP-seq of colonies compared to parental cells revealed differential marking at promotor regions of several target genes involved in MAPKi resistance, including ARAF, BRAF, and CRAF. H3K4me3 remodeling corresponded to increased gene expression and susceptibility to pan-RAF inhibitors, suggesting an H3K4me3 mediated increase of ARAF and CRAF as a mechanism of BRAF/MEK inhibitor resistance. Two enzymes, OGT and TET1 that are both linked to H3K4me3 regulation in embryonic stem cells are highly upregulated in persister colonies and tumor tissue of PDXs from BRAF mutant melanoma patients under MEK1/2 inhibition. A shift in OGT nuclear localization and O-linked glycosylation patterns was observed in colonies compared to parental cells suggestive of altered transcriptional and protein activity. OGT ChIP-PCR of colonies compared to parental cells confirmed a set of genes with exclusively H3K4me3 marking in colonies. shRNA mediated knockdown of OGT and TET1 blocked H3K4me3 increase in IDTC colonies, prevented colony formation and delayed tumor relapse in a BRAF mutant xenograft mouse model. High TET1 mRNA expression is linked to significantly shorter survival in TGCA data. Conclusion OGT and TET1 mediated epigenetic remodeling through H3K4me3 with upregulation of MAPKi resistant genes is responsible for the emergence of permanent resistance. Both enzymes are promising targets to combat treatment failure and prolong overall survival. Citation Format: Dinoop Ravindran Menon, Heinz Hammerlindl, Abdullah Al Emran, Joachim Torrano, Sabrina Hammerlindl, Gao Zhang, Rajasekharan Somasundaram, Richard A. Sturm, Nikolas K. Haass, Keith Flaherty, Meenhard Herlyn, Helmut Schaider. Escape form adaptive drug tolerance through OGT and TET1 mediated H3K4me3 remodeling in MAPKi resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5833.

1237 Escape form adaptive drug tolerance through OGT and TET1 mediated H3K4me3 remodeling in MAPKi-resistant melanoma

Genetic alterations linked to acquired BRAF inhibitor resistance are absent in about 40% of relapsed melanoma patients suggesting the involvement of epigenetic changes. We investigated epigenetic remodeling in BRAF mutant melanoma upon BRAF/MEK inhibition. Long term treatment of more than 45 days enables the cells to escape the slow cycling state which results in proliferating cellular clusters (drug-tolerant persister colonies) with stem-like characteristics that regain global H3K4me3. H3K4me3 ChIP-seq of colonies compared to parental cells revealed differential marking at promotor regions of several target genes involved in MAPKi resistance, including ARAF, BRAF, and CRAF. H3K4me3 remodeling corresponded to increased gene expression and susceptibility to pan-RAF inhibitors. Two enzymes, OGT and TET1 that are both linked to H3K4me3 regulation are significantly upregulated in persister colonies and tumor tissue of PDXs from BRAF mutant melanoma patients under MEK1/2 inhibition. A shift in OGT nuclear localization and O-linked glycosylation patterns was observed in colonies compared to parental cells and OGT ChIP-PCR confirmed a set of genes with exclusively H3K4me3 marking in colonies. shRNA mediated knockdown of OGT and TET1 blocked H3K4me3 increase in colonies, prevented colony formation and delayed tumor relapse in BRAF mutant xenografts. OGT and TET1 are promising targets to combat treatment failure and prolong overall survivial.

Palbociclib synergizes with BRAF and MEK inhibitors in treatment naïve melanoma but not after the development of BRAF inhibitor resistance.

Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naïve, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug-tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors.

The anti-apoptotic BAG3 protein is involved in BRAF inhibitor resistance in melanoma cells.

BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumour types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. In human melanomas, BAG3 positivity is correlated with the aggressiveness of the tumour cells and can sustain IKK-γ levels, allowing a sustained activation of NF-κB. Furthermore, BAG3 is able to modulate BRAFV600E levels and activity in thyroid carcinomas. BRAFV600E is the most frequent mutation detected in malignant melanomas and is targeted by Vemurafenib, a specific inhibitor found to be effective in the treatment of advanced melanoma. However, patients with BRAF-mutated melanoma may result insensitive ab initio or, mostly, develop acquired resistance to the treatment with this molecule.Here we show that BAG3 down-modulation interferes with BRAF levels in melanoma cells and sensitizes them to Vemurafenib treatment. Furthermore, the down-modulation of BAG3 protein in an in vitro model of acquired resistance to Vemurafenib can induce sensitization to the BRAFV600E specific inhibition by interfering with BRAF pathway through reduction of ERK phosphorylation, but also on parallel survival pathways.Future studies on BAG3 molecular interactions with key proteins responsible of acquired BRAF inhibitor resistance may represent a promising field for novel multi-drugs treatment design.

Acquired BRAF inhibitor resistance: A multicenter meta-analysis of the spectrum and frequencies, clinical behaviour, and phenotypic associations of resistance mechanisms

BackgroundAcquired resistance to BRAF inhibitors (BRAFi) is a near-universal phenomenon caused by numerous genetic and non-genetic alterations. In this study, we evaluated the spectrum, onset, pattern of progression, and subsequent clinical outcomes associated with specific mechanisms of resistance. MethodsWe compiled clinical and genetic data from 100 patients with 132 tissue samples obtained at progression on BRAFi therapy from 3 large, previously published studies of BRAFi resistance. These samples were subjected to whole-exome sequencing and/or polymerase chain reaction-based genetic testing. ResultsAmong 132 samples, putative resistance mechanisms were identified in 58%, including NRAS or KRAS mutations (20%), BRAF splice variants (16%), BRAFV600E/K amplifications (13%), MEK1/2 mutations (7%), and non-mitogen-activated protein kinase pathway alterations (11%). Marked heterogeneity was observed within tumors and patients; 18 of 19 patients (95%) with more than one progression biopsy had distinct/unknown drivers of resistance between samples. NRAS mutations were associated with vemurafenib use (p = 0.045) and intracranial metastases (p = 0.036), and MEK1/2 mutations correlated with hepatic progression (p = 0.011). Progression-free survival and overall survival were similar across resistance mechanisms. The median survival after disease progression was 6.9 months, and responses to subsequent BRAF and MEK inhibition were uncommon (2 of 15; 13%). Post-progression outcomes did not correlate with specific acquired BRAFi-resistance mechanisms. ConclusionsThis is the first study to systematically characterise the clinical implications of particular acquired BRAFi-resistance mechanisms in patients with BRAF-mutant melanoma largest study to compile the landscape of resistance. Despite marked heterogeneity of resistance mechanisms within patients, NRAS mutations correlated with vemurafenib use and intracranial disease involvement.

Abstract 703: A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression

Abstract BACKGROUND: BRAF/MEK inhibitors have shown promising levels of response in melanomas harboring the BRAF V600E mutation, however responses tend to be short-lived and resistance is a major clinical problem. In the current study, we evaluated the pharmacological activity of BI847325, a dual inhibitor of MEK and Aurora kinases against multiple melanoma cell line models. METHODS: The cytotoxic effect of BI847325 was evaluated in vitro in a panel of selected BRAF-mutant vemurafenib resistant cell lines by Alamar blue and Annexin V binding assay. 3D spheroid model systems and colony formation assays demonstrated the long-term growth inhibitory effect of BI847325. Western blot analysis and qRT-PCR studies were carried out to evaluate the mechanism underlying BI5-mediated cytotoxicity. In vivo studies in Balb SCID mice were performed to assess the suppression of BRAF-mutant xenografts on treatment with BI847325. RESULTS: BI847325 potently reduced the growth and survival of BRAF-mutant melanoma cell lines with acquired and intrinsic BRAF inhibitor resistance (NRAS mutations, BRAF splice forms, Cyclin D1 amplification, RTK upregulation, PTEN loss, COT amplification). We confirmed that BI847325 induced apoptosis through decrease in Mcl-1 mRNA and protein expression and increase in BIM expression. The effects of BI847325 upon BIM and Mcl-1 expression could not be mimicked by the combination of other MEK and aurora kinase inhibitors. For the first time we demonstrated that BI847325 reverses the acquired vemurafenib resistance by inhibiting MEK expression at mRNA and protein level. A strong suppression of MEK expression was observed without recovery following 72 h of washout. In vivo studies revealed complete tumor suppression with no recurrence over a period of 65 days of treatment with 70mg/kg/week dose of BI847325. In contrast, treatment with vemurafenib analog PLX4720 in the same mouse model was associated with tumor relapse after 30 days of treatment. BI847325 also successfully suppressed the long-term growth of xenografts with acquired vemurafenib resistance. Analysis of tumor samples complied with in vitro results demonstrating inhibition of phospho-ERK, phospho-Histone3, Mcl-1 and total MEK. CONCLUSION: In conclusion we report for the first time that BI847325, a novel ATP-competitive MEK/ Aurora kinase inhibitor effectively inhibits BRAF-mutant melanoma and overcomes vemurafenib-resistance by decreasing expression of MEK and Mcl-1; in vitro and in vivo. Further preclinical and clinical investigations towards this would open new avenues in treatment of melanoma. Citation Format: Manali S. Phadke, Patrizia Sini, Keiran Smalley. A novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 reverses acquired BRAF inhibitor resistance through suppression of Mcl-1 and inhibition of MEK expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2015-703

The Novel ATP-Competitive MEK/Aurora Kinase Inhibitor BI-847325 Overcomes Acquired BRAF Inhibitor Resistance through Suppression of Mcl-1 and MEK Expression

Resistance to BRAF inhibitors is a major clinical problem. Here, we evaluate BI-847325, an ATP-competitive inhibitor of MEK and Aurora kinases, in treatment-naïve and drug-resistant BRAF-mutant melanoma models. BI-847325 potently inhibited growth and survival of melanoma cell lines that were both BRAF inhibitor naïve and resistant in 2D culture, 3D cell culture conditions, and in colony formation assays. Western blot studies showed BI-847325 to reduce expression of phospho-ERK and phospho-histone 3 in multiple models of vemurafenib resistance. Mechanistically, BI-847325 decreased the expression of MEK and Mcl-1 while increasing the expression of the proapoptotic protein BIM. Strong suppression of MEK expression was observed after 48 hours of treatment, with no recovery following >72 hours of washout. siRNA-mediated knockdown of Mcl-1 enhanced the effects of BI-847325, whereas Mcl-1 overexpression reversed this in both 2D cell culture and 3D spheroid melanoma models. In vivo, once weekly BI-847325 (70 mg/kg) led to durable regression of BRAF-inhibitor naïve xenografts with no regrowth seen (>65 days of treatment). In contrast, treatment with the vemurafenib analog PLX4720 was associated with tumor relapse at >30 days. BI-847325 also suppressed the long-term growth of xenografts with acquired PLX4720 resistance. Analysis of tumor samples revealed BI-847325 to induce apoptosis associated with suppression of phospho-ERK, total MEK, phospho-Histone3, and Mcl-1 expression. Our studies indicate that BI-847325 is effective in overcoming BRAF inhibitor resistance and has long-term inhibitory effects upon BRAF-mutant melanoma in vivo, through a mechanism associated with the decreased expression of both MEK and Mcl-1.

MiR-200c/Bmi1 axis and epithelial-mesenchymal transition contribute to acquired resistance to BRAF inhibitor treatment.

Resistance to BRAF inhibitors (BRAFi) is one of the major challenges for targeted therapies for BRAF-mutant melanomas. However, little is known about the role of microRNAs in conferring BRAFi resistance. Herein, we demonstrate that miR-200c expression is significantly reduced whereas miR-200c target genes including Bmi1, Zeb2, Tubb3, ABCG5, and MDR1 are significantly increased in melanomas that acquired BRAFi resistance compared to pretreatment tumor biopsies. Similar changes were observed in BRAFi-resistant melanoma cell lines. Overexpression of miR-200c or knock-down of Bmi1 in resistant melanoma cells restores their sensitivities to BRAFi, leading to deactivation of the PI3K/AKT and MAPK signaling cascades, and acquisition of epithelial-mesenchymal transition-like phenotypes, including upregulation of E-cadherin, downregulation of N-cadherin, and ABCG5 and MDR1 expression. Conversely, knock-down of miR-200c or overexpression of Bmi1 in BRAFi-sensitive melanoma cells activates the PI3K/AKT and MAPK pathways, upregulates N-cadherin, ABCG5, and MDR1 expression, and downregulates E-cadherin expression, leading to BRAFi resistance. Together, our data identify miR-200c as a critical signaling node in BRAFi-resistant melanomas impacting the MAPK and PI3K/AKT pathways, suggesting miR-200c as a potential therapeutic target for overcoming acquired BRAFi resistance.

Overcoming Acquired BRAF Inhibitor Resistance in Melanoma via Targeted Inhibition of Hsp90 with Ganetespib

Activating BRAF kinase mutations serve as oncogenic drivers in over half of all melanomas, a feature that has been exploited in the development of new molecularly targeted approaches to treat this disease. Selective BRAF(V600E) inhibitors, such as vemurafenib, typically induce initial, profound tumor regressions within this group of patients; however, durable responses have been hampered by the emergence of drug resistance. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90, in melanoma lines harboring the BRAF(V600E) mutation. Ganetespib exposure resulted in the loss of mutant BRAF expression and depletion of mitogen-activated protein kinase and AKT signaling, resulting in greater in vitro potency and antitumor efficacy compared with targeted BRAF and MAP-ERK kinase (MEK) inhibitors. Dual targeting of Hsp90 and BRAF(V600E) provided combinatorial benefit in vemurafenib-sensitive melanoma cells in vitro and in vivo. Importantly, ganetespib overcame mechanisms of intrinsic and acquired resistance to vemurafenib, the latter of which was characterized by reactivation of extracellular signal-regulated kinase (ERK) signaling. Continued suppression of BRAF(V600E) by vemurafenib potentiated sensitivity to MEK inhibitors after acquired resistance had been established. Ganetespib treatment reduced, but not abolished, elevations in steady-state ERK activity. Profiling studies revealed that the addition of a MEK inhibitor could completely abrogate ERK reactivation in the resistant phenotype, with ganetespib displaying superior combinatorial activity over vemurafenib. Moreover, ganetespib plus the MEK inhibitor TAK-733 induced tumor regressions in vemurafenib-resistant xenografts. Overall these data highlight the potential of ganetespib as a single-agent or combination treatment in BRAF(V600E)-driven melanoma, particularly as a strategy to overcome acquired resistance to selective BRAF inhibitors.

A Novel AKT1 Mutant Amplifies an Adaptive Melanoma Response to BRAF Inhibition

BRAF inhibitor (BRAFi) therapy leads to remarkable anti melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants, causing late, acquired resistance. We show here that BRAFi (or BRAFi + MEKi) therapy in patients frequently led to rebound phosphorylated AKT (p-AKT) levels in their melanomas early on-treatment. In cell lines, BRAFi treatment led to rebound levels of receptor tyrosine kinases (RTK; including PDGFRβ), phosphatidyl (3,4,5)-triphosphate (PIP3), pleckstrin homology domain recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by the introduction of a mutant AKT1 (Q79K) known to confer acquired BRAFi resistance. Functionally, AKT1(Q79K) conferred BRAFi resistance via amplification of BRAFi-elicited PI3K-AKT signaling. In addition, mitogen-activated protein kinase pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy.

Abstract 5237: Systems level modeling following BRAF inhibition reveals an essential role for Ephrin A2 receptor in therapeutic escape.

Abstract Although initially striking, the therapeutic efficacy of BRAF inhibition is eventually undermined by adaptive cellular signaling that leads to progressive disease. To date, several mechanisms of BRAF inhibitor resistance have been reported focusing primarily on single alterations. To address how global rewiring of melanoma signaling promotes resistance, we utilized mass spectrometry based phosphoproteomic and network analyses to identify signaling changes in tyrosine, threonine and serine that occur prior to and following acquired BRAF inhibitor resistance. This comprehensive bioinformatics approach uncovered a “resistance interactome” consisting of ∼150 nodes that was marked by significant alterations and the emergence of signaling nodes such as ITGB1, EphA2, EphB4, FAK1, STAT3 and PXN. GeneGo pathway enrichment analysis revealed enhanced cytoskeletal rearrangement, adhesion, integrin signaling, cell migration and extracellular matrix remodeling. Consistent with our bioinformatic predictions, melanoma cell lines with acquired vemurafenib resistance were considerably more invasive than their drug naïve counterparts and showed increased trans-endothelial cell migration. Confirmatory Western blot studies showed increased expression and phosphorylation of EphA2 receptor at Ser897, FAK1 at Ser397 and paxillin at Y118. Knockdown of EphA2 in the resistant cultures inhibited ERK signaling and completely prevented cell invasion and trans-endothelial cell migration. Functionally, EphA2 expression appeared to be regulated by an epigenetic mechanism involving promoter methylation and HDAC1. The clinical relevance of these findings was demonstrated in matched pairs of biopsies from patients receiving either BRAF or BRAF+MEK inhibitors, with significant Ephrin A2 receptor expression being observed in a subset of those on therapy. In summary, chronic MAPK pathway inhibition leads to major remodeling of the melanoma signaling network with EphA2 emerging as a key driver of the resistance phenotype. Citation Format: Kim H. T. Paraiso, Bin Fang, Dale Han, Inna Fedorenko, Jobin John, John Koomen, Jennifer Wargo, Keith Flaherty, Friedegund Meier, Kerian S. M. Smalley. Systems level modeling following BRAF inhibition reveals an essential role for Ephrin A2 receptor in therapeutic escape. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5237. doi:10.1158/1538-7445.AM2013-5237