Fibroblast-derived neuregulin 1 promotes compensatory ErbB3 receptor signaling in mutant BRAF melanoma.

Rapidly accelerated fibrosarcoma (RAF) inhibitors are first-line treatments for patients harboring V600E/K mutant BRAF melanoma. Although RAF inhibitors produce high response rates, the degree of tumor regression is heterogeneous. Compensatory/adaptive responses to targeted inhibitors are frequently initiated by the activation of growth factor receptor tyrosine kinases, including ErbB3, and factors from the tumor microenvironment may play an important role. We have shown previously that mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF) melanoma cells have enhanced activation of ErbB3 following RAF inhibition. However, the source of neuregulin 1 (NRG1), the ligand for ErbB3, is unknown. In this study, we demonstrate that NRG1 is highly expressed by dermal fibroblasts and cancer-associated fibroblasts (CAFs) isolated from mutant BRAF melanomas. Conditioned medium from fibroblasts and CAFs enhanced ErbB3 pathway activation and limited RAF inhibitor cytotoxicity in V600 mutant BRAF-harboring melanomas. Targeting the ErbB3/ErbB2 pathway partially reversed the protective effects of fibroblast/CAF-derived NRG1 on cell growth properties of RAF inhibitor-treated melanoma cells. These findings support the idea that NRG1, acting in a paracrine manner, promotes resistance to RAF inhibitors and emphasize that targeting the ErbB3/ErbB2 pathway will likely improve the efficacy of RAF inhibitors for mutant BRAF melanoma patients.

SOX10 Loss Sensitizes Melanoma Cells to Cytokine-Mediated Inflammatory Cell Death.

The transcription factor, SOX10, plays an important role in the differentiation of neural crest precursors to the melanocytic lineage. Malignant transformation of melanocytes leads to the development of melanoma, and SOX10 promotes melanoma cell proliferation and tumor formation. SOX10 expression in melanomas is heterogeneous, and loss of SOX10 causes a phenotypic switch toward an invasive, mesenchymal-like cell state and therapy resistance; hence, strategies to target SOX10-deficient cells are an active area of investigation. The impact of cell state and SOX10 expression on antitumor immunity is not well understood but will likely have important implications for immunotherapeutic interventions. To this end, we tested whether SOX10 status affects the response to CD8+ T cell-mediated killing and T cell-secreted cytokines, TNFα and IFNγ, which are critical effectors in the cytotoxic killing of cancer cells. We observed that genetic ablation of SOX10 rendered melanoma cells more sensitive to CD8+ T cell-mediated killing and cell death induction by either TNFα or IFNγ. Cytokine-mediated cell death in SOX10-deficient cells was associated with features of caspase-dependent pyroptosis, an inflammatory form of cell death that has the potential to increase immune responses. IMPLICATIONS: These data support a role for SOX10 expression altering the response to T cell-mediated cell death and contribute to a broader understanding of the interaction between immune cells and melanoma cells.

EYA2 tyrosine phosphatase inhibition reduces MYC and prevents medulloblastoma progression.

BACKGROUND: Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. METHODS: Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. RESULTS: EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. CONCLUSIONS: Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.

Targeting SOX10-deficient cells to reduce the dormant-invasive phenotype state in melanoma.

Cellular plasticity contributes to intra-tumoral heterogeneity and phenotype switching, which enable adaptation to metastatic microenvironments and resistance to therapies. Mechanisms underlying tumor cell plasticity remain poorly understood. SOX10, a neural crest lineage transcription factor, is heterogeneously expressed in melanomas. Loss of SOX10 reduces proliferation, leads to invasive properties, including the expression of mesenchymal genes and extracellular matrix, and promotes tolerance to BRAF and/or MEK inhibitors. We identify the class of cellular inhibitor of apoptosis protein-1/2 (cIAP1/2) inhibitors as inducing cell death selectively in SOX10-deficient cells. Targeted therapy selects for SOX10 knockout cells underscoring their drug tolerant properties. Combining cIAP1/2 inhibitor with BRAF/MEK inhibitors delays the onset of acquired resistance in melanomas in vivo. These data suggest that SOX10 mediates phenotypic switching in cutaneous melanoma to produce a targeted inhibitor tolerant state that is likely a prelude to the acquisition of resistance. Furthermore, we provide a therapeutic strategy to selectively eliminate SOX10-deficient cells.

Fueling the Fire: Inflammatory Forms of Cell Death and Implications for Cancer Immunotherapy.

Unleashing the immune system with immune checkpoint inhibitors (ICI) has significantly improved overall survival for subsets of patients with stage III/IV cancer. However, many tumors are nonresponsive to ICIs, in part due to a lack of tumor-infiltrating lymphocytes (TIL). Converting these immune "cold" tumors to "hot" tumors that are thus more likely to respond to ICIs is a major obstacle for cancer treatment. Triggering inflammatory forms of cell death, such as necroptosis and pyroptosis, may alter the tumor immune microenvironment and the influx of TILs. We present an emerging view that promoting tumor-localized necroptosis and pyroptosis may ultimately enhance responses to ICI. SIGNIFICANCE: Many tumor types respond poorly to ICIs or respond but subsequently acquire resistance. Effective therapies for ICI-nonresponsive tumors are lacking and should be guided by evidence from preclinical studies. Promoting inflammatory cell death mechanisms within the tumor may alter the local immune microenvironment toward an ICI-responsive state.

SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects.

Developmental factors may regulate the expression of immune modulatory proteins in cancer, linking embryonic development and cancer cell immune evasion. This is particularly relevant in melanoma because immune checkpoint inhibitors are commonly used in the clinic. SRY-box transcription factor 10 (SOX10) mediates neural crest development and is required for melanoma cell growth. In this study, we investigate immune-related targets of SOX10 and observe positive regulation of herpesvirus entry mediator (HVEM) and carcinoembryonic-antigen cell-adhesion molecule 1 (CEACAM1). Sox10 knockout reduces tumor growth in vivo, and this effect is exacerbated in immune-competent models. Modulation of CEACAM1 expression but not HVEM elicits modest effects on tumor growth. Importantly, Sox10 knockout effects on tumor growth are dependent, in part, on CD8+ T cells. Extending this analysis to samples from patients with cutaneous melanoma, we observe a negative correlation with SOX10 and immune-related pathways. These data demonstrate a role for SOX10 in regulating immune checkpoint protein expression and anti-tumor immunity in melanoma.

PLX3397 inhibits the accumulation of intra-tumoral macrophages and improves bromodomain and extra-terminal inhibitor efficacy in melanoma.

Bromodomain and extra-terminal inhibitors (BETi) delay tumor growth, in part, through tumor cell intrinsic alterations and initiation of anti-tumor CD8+ T-cell responses. By contrast, BETi effects on pro-tumoral immune responses remain unclear. Here, we show that the next-generation BETi, PLX51107, delayed tumor growth to differing degrees in Braf V600E melanoma syngeneic mouse models. These differential responses were associated with the influx of tumor-associated macrophages during BETi treatment. Tumors that were poorly responsive to PLX51107 showed increased influx of colony-stimulating factor-1 receptor (CSF-1R)-positive tumor-associated macrophages. We depleted CSF-1R+ tumor-associated macrophages with the CSF-1R inhibitor, PLX3397, in combination with PLX51107. Treatment with PLX3397 enhanced the efficacy of PLX51107 in poorly responsive Braf V600E syngeneic melanomas in vivo. These findings suggest that tumor-associated macrophage accumulation limits BETi efficacy and that co-treatment with PLX3397 can improve response to PLX51107, offering a potential novel combination therapy for metastatic melanoma patients.

FOXD3 Regulates VISTA Expression in Melanoma.

Immune checkpoint inhibitors have improved patient survival in melanoma, but the innate resistance of many patients necessitates the investigation of alternative immune targets. Many immune checkpoint proteins lack proper characterization, including V-domain Ig suppressor of T cell activation (VISTA). VISTA expression on immune cells can suppress T cell activity; however, few studies have investigated its expression and regulation in cancer cells. In this study, we observe that VISTA is expressed in melanoma patient samples and cell lines. Tumor cell-specific expression of VISTA promotes tumor onset in vivo, associated with increased intratumoral T regulatory cells, and enhanced PDL-1 expression on tumor-infiltrating macrophages. VISTA transcript levels are regulated by the stemness factor Forkhead box D3 (FOXD3). BRAF inhibition upregulates FOXD3 and reduces VISTA expression. Overall, this study demonstrates melanoma cell expression of VISTA and its regulation by FOXD3, contributing to the rationale for therapeutic strategies that combine targeted inhibitors with immune checkpoint blockade.

CADM1 is a TWIST1-regulated suppressor of invasion and survival.

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.

MIG6 Is MEK Regulated and Affects EGF-Induced Migration in Mutant NRAS Melanoma.

Activating mutations in neuroblastoma RAS viral oncogene homolog (NRAS) are frequent driver events in cutaneous melanoma. NRAS is a guanosine triphosphate-binding protein whose most well-characterized downstream effector is RAF, leading to activation of mitogen-activated protein kinase (MEK)-extracellular signal-regulated protein kinase 1/2 signaling. Although there are no Food and Drug Administration-approved targeted therapies for melanoma patients with a primary mutation in NRAS, one form of targeted therapy that has been explored is MEK inhibition. In clinical trials, MEK inhibitors have shown disappointing efficacy in mutant NRAS patients, the reasons for which are unclear. To explore the effects of MEK inhibitors in mutant NRAS melanoma, we used a high-throughput reverse-phase protein array platform to identify signaling alterations. Reverse-phase protein array analysis of phospho-proteomic changes in mutant NRAS melanoma in response to trametinib indicated a compensatory increase in v-akt murine thymoma viral oncogene homolog signaling and decreased expression of mitogen-inducible gene 6 (MIG6), a negative regulator of epidermal growth factor receptor/v-erb-b2 erythroblastic leukemia viral oncogene homolog receptors. MIG6 expression did not alter the growth or survival properties of mutant NRAS melanoma cells. Rather, we identified a role for MIG6 as a negative regulator of epidermal growth factor-induced signaling and cell migration and invasion. In MEK-inhibited cells, further depletion of MIG6 increased migration and invasion, whereas MIG6 expression decreased these properties. Therefore, a decrease in MIG6 may promote the migration and invasiveness of MEK-inhibited mutant NRAS melanoma, especially in response to epidermal growth factor stimulation.

RAC1 P29S regulates PD-L1 expression in melanoma.

Whole exome sequencing of cutaneous melanoma has led to the detection of P29 mutations in RAC1 in 5-9% of samples, but the role of RAC1 P29 mutations in melanoma biology remains unclear. Using reverse phase protein array analysis to examine the changes in protein/phospho-protein expression, we identified cyclin B1, PD-L1, Ets-1, and Syk as being selectively upregulated with RAC1 P29S expression and downregulated with RAC1 P29S depletion. Using the melanoma patient samples in TCGA, we found PD-L1 expression to be significantly increased in RAC1 P29S patients compared to RAC1 WT as well as other RAC1 mutants. The finding that PD-L1 is upregulated suggests that oncogenic RAC1 P29S may promote suppression of the antitumor immune response. This is a new insight into the biological function of RAC1 P29S mutations with potential clinical implications as PD-L1 is a candidate biomarker for increased benefit from treatment with anti-PD1 or anti-PD-L1 antibodies.

ErbB3-ErbB2 Complexes as a Therapeutic Target in a Subset of Wild-type BRAF/NRAS Cutaneous Melanomas.

The treatment options remain limited for patients with melanoma who are wild-type for both BRAF and NRAS (WT/WT). We demonstrate that a subgroup of WT/WT melanomas display high basal phosphorylation of ErbB3 that is associated with autocrine production of the ErbB3 ligand neuregulin-1 (NRG1). In WT/WT melanoma cells displaying high levels of phospho-ErbB3, knockdown of NRG1 reduced cell viability and was associated with decreased phosphorylation of ErbB3, its coreceptor ErbB2, and its downstream target, AKT. Similar effects were observed by targeting ErbB3 with either siRNAs or the neutralizing ErbB3 monoclonal antibodies huHER3-8 and NG33. In addition, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro, and xenograft growth in vivo. Pertuzumab also potentiated the effects of MEK inhibitor on WT/WT melanoma growth in vitro and in vivo. These findings demonstrate that targeting ErbB3-ErbB2 signaling in a cohort of WT/WT melanomas leads to tumor growth reduction. Together, these studies support the rationale to target the NRG1-ErbB3-ErbB2 axis as a novel treatment strategy in a subset of cutaneous melanomas.