Congenital melanocytic nevus (CMN) syndrome represents a mosaic RASopathy, typically caused by postzygotic NRAS codon 61 mutations, which originate in ectodermal precursor cells and result in melanocyte deposits in the skin and central nervous system (CNS). Affected patients are prone to develop uniformly fatal melanomas in the skin and CNS. Here, we report the case of a 2.7-year-old male with CMN syndrome, diffuse leptomeningeal melanosis and CNS melanoma, who underwent experimental therapy with the DNA methyltransferase inhibitor azacitidine in combination with the mitogen-activated protein kinase (MEK) inhibitor trametinib with exceptional clinical and radiological response. Response to combination therapy appeared to be more durable than the treatment response observed in several other severely affected patients treated with trametinib for late-stage disease. Correspondingly, concomitant exposure to trametinib and azacitidine prevented development of trametinib resistance in NRAS-mutated human melanoma cells in vitro. Also, azacitidine was shown to inhibit growth and mitogen-activated protein kinase 1/2 (ERK1/2) phosphoryla-Abbreviations: AKAP9, A-kinase anchoring protein 9; AKT, Ak strain transforming; BRAF, B-raf proto-oncogene, serine/threonine kinase; CMN, congenital melanocytic nevus; CNS, central nervous system; ERK1/2, mitogen-activated protein kinase 1/2; GDP, guanosine diphosphate; GTP, guanosine triphosphate; IC 50 , half-maximal inhibitory concentration; MAPK, microtubule-associated protein kinase; MEK, mitogen-activated protein kinase; mTOR, mechanistic target of rapamycin kinase; NRAS, NRAS proto-oncogene, GTPase; PI3K, phosphatidylinositol 3-kinase; RAS, rat sarcoma virus.

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Functional genomic screening of KRAS-driven mouse sarcomas was previously employed to identify proliferation-relevant genes. Genes identified included Ubiquitin-conjugating enzyme E2 (Ube2c), Centromere Protein E (Cenpe), Hyaluronan Synthase 2 (Has2), and CAMP Responsive Element Binding Protein 3 Like 2 (Creb3l2). This study examines the expression and chemical inhibition of these candidate genes, identifying variable levels of protein expression and significant contributions to rhabdomyosarcoma (RMS) cell proliferation. Chemical treatment of human and murine RMS cell lines with bortezomib, UA62784, latrunculin A and sorafenib inhibited growth with approximate EC50 concentrations of 15-30nM for bortezomib, 25-80nM for UA62784 and 80-220nM for latrunculin A. The multikinase inhibitor sorafenib increased in vitro proliferation of 4 of 6 sarcoma cell lines tested. Latrunculin A was further associated with disruption of the actin cytoskeleton and reduced ERK1/2 phosphorylation. Together, this work advances opportunities for developing therapies to block progression of soft-tissue sarcomas and demonstrates that disruption of the actin cytoskeleton in sarcoma cells by latrunculin A is associated with a reduction in RMS cell growth. (167 words).