Metastatic cancer remains a clinical challenge; however, patients diagnosed prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion steps within the metastatic cascade, but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome of melanoma cells. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Increased expression of CADM1 resulted in significant inhibition of motility and invasiveness of melanoma cells. In addition, elevated CADM1 elicited caspase-independent cell death in non-adherent conditions. Expression array analysis suggests that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Importantly, tissue microarray analysis and clinical data from TCGA indicate that CADM1 expression is inversely associated with melanoma progression and positively correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be considered a biomarker for favorable prognosis.

MEK-ERK1/2 signaling is elevated in melanomas that are wild-type for both BRAF and NRAS (WT/WT), but patients are insensitive to MEK inhibitors. Stromal-derived growth factors may mediate resistance to targeted inhibitors, and optimizing the use of targeted inhibitors for patients with WT/WT melanoma is a clinical unmet need. Here, we studied adaptive responses to MEK inhibition in WT/WT cutaneous melanoma. The Cancer Genome Atlas data set and tumor microarray studies of WT/WT melanomas showed that high levels of neuregulin-1 (NRG1) were associated with stromal content and ErbB3 signaling. Of growth factors implicated in resistance to targeted inhibitors, NRG1 was effective at mediating resistance to MEK inhibitors in patient-derived WT/WT melanoma cells. Furthermore, ErbB3/ErbB2 signaling was adaptively upregulated following MEK inhibition. Patient-derived cancer-associated fibroblast studies demonstrated that stromal-derived NRG1 activated ErbB3/ErbB2 signaling and enhanced resistance to a MEK inhibitor. ErbB3- and ErbB2-neutralizing antibodies blocked the protective effects of NRG1 and cooperated with the MEK inhibitor to delay tumor growth in both cell line and patient-derived xenograft models. These results highlight tumor microenvironment regulation of targeted inhibitor resistance in WT/WT melanoma and provide a rationale for combining MEK inhibitors with anti-ErbB3/ErbB2 antibodies in patients with WT/WT cutaneous melanoma, for whom there are no effective targeted therapy options. This work suggests a mechanism by which NRG1 regulates the sensitivity of WT NRAS/BRAF melanomas to MEK inhibitors and provides a rationale for combining MEK inhibitors with anti-ErbB2/ErbB3 antibodies in these tumors. .

Background BRAF-mutant melanoma patients respond to BRAF inhibitors and MEK inhibitors (BRAFi/MEKi), but drug-tolerant cells persist, which may seed disease progression. Adaptive activation of receptor tyrosine kinases (RTKs) has been associated with melanoma cell drug tolerance following targeted therapy. While co-targeting individual RTKs can enhance the efficacy of BRAFi/MEKi effects, it remains unclear how to broadly target multiple RTKs to achieve more durable tumour growth inhibition. Methods The blockage of adaptive RTK responses by the new BET inhibitor (BETi), PLX51107, was measured by RPPA and Western blot. Melanoma growth was evaluated in vitro by colony assay and EdU staining, as well as in skin reconstructs, xenografts and PDX models following BRAFi, MEKi and/or PLX51107 treatment. Results Treatment with PLX51107 limited BRAFi/MEKi upregulation of ErbB3 and PDGFR-β expression levels. Similar effects were observed following BRD2/4 depletion. In stage III melanoma patients, expression of BRD2/4 was strongly correlated with ErbB3. PLX51107 enhanced the effects of BRAFi/MEKi on inhibiting melanoma growth in vitro, in human skin reconstructs and in xenografts in vivo. Continuous triple drug combination treatment resulted in significant weight loss in mice, but intermittent BETi combined with continuous BRAFi/MEKi treatment was tolerable and improved durable tumour inhibition outcomes. Conclusions Together, our data suggest that intermittent inhibition of BET proteins may improve the duration of responses following BRAFi/MEKi treatment in BRAF-mutant melanoma.

Metastatic melanoma is the deadliest form of skin cancer; however, patients diagnosed and treated prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion of melanoma cells but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Modulation of CADM1 resulted in significant effects on the migration and invasion of melanoma cells. In addition, elevated CADM1 elicited cell death in non-adherent conditions, an effect that could not be rescued with a pan-caspase inhibitor. Analyses suggest that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Furthermore, clinical data from TCGA indicates that CADM1 expression is correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be a biomarker for improved survival of melanoma patients. Citation Format: Edward J. Hartsough, Michele B. Weiss, Shea A. Heilman, Timothy J. Purwin, Curtis H. Kugel, Sheera R. Rosenbaum, Dan A. Erkes, Manoela Tiago, Inna Chervoneva, Andrew E. Aplin. CADM1 is a TWIST1-regulated suppressor of melanoma invasion and survival [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 105.

Epigenetic agents such as bromodomain and extra terminal domain inhibitors (BETi) slow tumor growth via tumor intrinsic alterations; however, their effects on antitumor immunity remain unclear. A recent advance is the development of next-generation BETi that are potent and display a favorable half-life. Here, we tested the next-generation BETi, PLX51107, for immune-based effects on tumor growth in BRAF V600E melanoma syngeneic models. PLX51107 delayed melanoma tumor growth to differing degrees and altered the immunogenicity by lowering PD-L1 and FasL and increasing MHC-I in highly responsive melanomas. Moreover, PLX51107 treatment increased activated, proliferating and functional CD8+ T cells in tumors, leading to CD8+ T-mediated tumor growth delay. PLX51107 led to increased CD8-promoting, antitumor dendritic cells by acting as a Cox2 inhibitor. The accumulation of CSF-1R+ tumor-associated macrophages in poorly responsive melanoma limited PLX51107 efficacy. Importantly, PLX51107 delayed the growth of tumors that progressed on anti-PD-1 therapy, a response associated with decreased Cox2 levels, decreased PD-L1 expression and increased MHC-I expression on nonhematopoietic cells and increased intratumoral CD8+ T cells. Thus, next-generation BETi represent a potential first-line and secondary treatment strategy for metastatic melanoma by eliciting effects, at least in part, on antitumor immunity. Citation Format: Dan A. Erkes, Conroy O. Field, Claudia Capparelli, Manoela Tiago, Timothy J. Purwin, Shea A. Heilman, Inna Chervoneva, Adam C. Berger, Sheera R. Rosenbaum, Edward J. Hartsough, Jessie Villanueva, Andrew E. Aplin. The next-generation BET inhibitor, PLX51107, delays melanoma growth, altering the tumor immune microenvironment via Cox2 inhibition [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A33.

<div>Abstract<p>MEK–ERK1/2 signaling is elevated in melanomas that are wild-type for both BRAF and NRAS (WT/WT), but patients are insensitive to MEK inhibitors. Stromal-derived growth factors may mediate resistance to targeted inhibitors, and optimizing the use of targeted inhibitors for patients with WT/WT melanoma is a clinical unmet need. Here, we studied adaptive responses to MEK inhibition in WT/WT cutaneous melanoma. The Cancer Genome Atlas data set and tumor microarray studies of WT/WT melanomas showed that high levels of neuregulin-1 (NRG1) were associated with stromal content and ErbB3 signaling. Of growth factors implicated in resistance to targeted inhibitors, NRG1 was effective at mediating resistance to MEK inhibitors in patient-derived WT/WT melanoma cells. Furthermore, ErbB3/ErbB2 signaling was adaptively upregulated following MEK inhibition. Patient-derived cancer-associated fibroblast studies demonstrated that stromal-derived NRG1 activated ErbB3/ErbB2 signaling and enhanced resistance to a MEK inhibitor. ErbB3- and ErbB2-neutralizing antibodies blocked the protective effects of NRG1 <i>in vitro</i> and cooperated with the MEK inhibitor to delay tumor growth in both cell line and patient-derived xenograft models. These results highlight tumor microenvironment regulation of targeted inhibitor resistance in WT/WT melanoma and provide a rationale for combining MEK inhibitors with anti-ErbB3/ErbB2 antibodies in patients with WT/WT cutaneous melanoma, for whom there are no effective targeted therapy options.</p><p><b>Significance:</b> This work suggests a mechanism by which NRG1 regulates the sensitivity of WT NRAS/BRAF melanomas to MEK inhibitors and provides a rationale for combining MEK inhibitors with anti-ErbB2/ErbB3 antibodies in these tumors. <i>Cancer Res; 78(19); 5680–93. ©2018 AACR</i>.</p></div>

<p>Supplemental Fig. S1. ErbB3 and ErbB2 are phosphorylated in the majority of WT/WT melanoma patient. Supplemental Fig. S2. Melanoma cells utilized in this study do not express NRG1. Supplemental Fig. S3. NRG1 rescues the inhibitory effects of MEK inhibitor on melanoma cell growth. Supplemental Fig. S4. Trametinib induces adaptive activation of ErbB2/ErbB3 dimer following NRG1 treatment. Supplemental Fig. S5. ERK1/2 phosphorylation modulates ErbB2 activation. Supplemental Fig. S7. LJM716 and pertuzumab reverse the protective effects provided by CAF-derived NRG1 in MEK inhibited melanoma cells and increase the efficacy of MEK inhibitors in vivo.</p>

<p>Article File and Figures</p>