Expanding the landscape of oncogenic drivers and treatment options in acral and mucosal melanomas by targeted genomic profiling

Abstract

Despite advancements in treating cutaneous melanoma, patients with acral and mucosal (A/M) melanomas still have limited therapeutic options and poor prognoses. We analyzed 156 melanomas (101 cutaneous, 28 acral, and 27 mucosal) using the Foundation One cancer-gene specific clinical testing platform and identified new, potentially targetable genomic alterations (GAs) in specific anatomic sites of A/M melanomas. Using novel pre-clinical models of A/M melanoma, we demonstrate that several GAs and corresponding oncogenic pathways associated with cutaneous melanomas are similarly targetable in A/M melanomas. Other alterations, including MYC and CRKL amplifications, were unique to A/M melanomas and susceptible to indirect targeting using the BRD4 inhibitor JQ1 or Src/ABL inhibitor dasatinib, respectively. We further identified new, actionable A/M-specific alterations, including an inactivating NF2 fusion in a mucosal melanoma responsive to dasatinib in vivo. Our study highlights new molecular differences between cutaneous and A/M melanomas, and across different anatomic sites within A/M, which may change clinical testing and treatment paradigms for these rare melanomas.

Keywords: CRKL; MYC; NF2; acral; dasatinib; fusions; melanoma; mucosal.

Figure 1.. Targeted genomic profiling across melanoma subtypes.

(A) Clinical characteristics and demographics of 156 melanomas included in CGP analysis. (B) Tumor mutational burden (TMB), (C) structural variant frequency, and (D) microsatellite instability were calculated for each tumor and compared between subtypes.

Figure 2.. Genomic alterations (GAs) in common melanoma genes and genes altered in A/M melanomas across melanoma subtypes.

GAs in common melanoma driver genes and genes altered in >5% of A/M melanomas are shown. Genes are grouped by known functional categories, and the percent GA frequency across cutaneous (CUT) and acral/mucosal (A/M) melanomas is indicated on the right. SU, subungual; HN, head and neck; AR, anorectal; VV, vulvovaginal.

Figure 3.. Analysis of common melanoma driver gene GAs across melanoma subtypes.

(A) The frequency of 8 common melanoma driver genes (BRAF, NRAS, HRAS, KRAS, NF1, KIT, GNAQ, and GNA11) is shown for each melanoma subtype. (B, C) The percentage that each specific mutation represents within a given gene is shown for (B) BRAF and (C) RAS. (D) The position of KIT mutated amino acids for each subtype was mapped. Dark gray boxes indicate functional protein domains. (E) Various categories of NF1 GAs are shown as the frequency of each GA type for each subtype of melanoma.

Figure 4.. Efficacy of MAPK inhibition in A/M melanomas in vitro and in vivo.

(A) Cell line GAs in various genes and pathways. (B) Schematic representation of the MAPK signaling pathway. (C) Western blot analysis of proteins involved in MAPK pathway signaling. (D) Cell viability analysis of MAPK inhibitor-treated cell lines. Technical triplicates are shown and error bars represent S.E.M. (E) Clinical timeline of treatments and responses for an acral melanoma patient ultimately undergoing a response to pan-RAF inhibition. (F) Images showing disease burden at baseline and 1.5 years post pan-RAF inhibitor treatment. (G) Cell viability analysis of MEK inhibitor (trametinib) in combination with a pan-RAF inhibitor (LY3009120). Combination Index (CI) values were calculated using CompuSyn software. *CompuSyn cannot accept viability values >100%, therefore CI values for MB 3443 30 nM LY3009120 are higher than actual values.

Figure 5.. Targeting oncogenic pathways and gene amplification events in A/M melanomas

(A, E, I, M) Schematic representation of signaling pathways and inhibitor targets. (B, F, J, N) Western blot analysis of proteins involved in signaling pathways and gene amplification. (C, G, K, O) Cell viability analysis of targeted treatments in A/M melanoma cell lines. Technical triplicates are shown and error bars represent S.E.M. (D, H, L, P) Cell viability analysis of targeted treatment combinations in A/M melanoma cell lines. Technical triplicates are shown and error bars represent S.E.M.

Figure 6.. Potential oncogenic drivers in pan-negative melanomas and responses to targeted therapy.

(A) Potential oncogenic driver GAs were analyzed for 26 pan-negative melanomas. (B) Functional classification of potential oncogenic drivers. (C) Schematic representation of kinase gene fusions. (D) Diagram of splicing events identified in CBL and BRAF genes. (E) MB 1816 melanoma PDX tumors were treated with vehicle or MEK inhibitor (trametinib) or pan-RAF inhibitor (LY3009120) at the indicated doses. Average tumor volumes are shown and error bars represent S.E.M. (F) Schematic representation of the novel NF2-HORMAD2-AS1 gene fusion. (G) qRT-PCR analysis of NF2 gene expression using primers which detect either the 5’ or 3’ portion of the NF2 gene. (H) MB 1816 melanoma PDX tumors were treated with vehicle or multi-kinase inhibitor dasatinib at the indicated dose. Average tumor volumes are shown and error bars represent S.E.M. * p < 0.019.