Ruth Halaban and colleagues report exome sequences of 147 melanoma tumors. They identified a recurrent somatic activating alteration in the Rho GTPase RAC1 in sun-exposed melanomas. We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

Combination checkpoint blockade (CCB) targeting inhibitory CTLA4 and PD1 receptors holds promise for cancer therapy. Immune-related adverse events (IRAEs) remain a major obstacle for the optimal application of CCB in cancer. Here, we analyzed B cell changes in patients with melanoma following treatment with either anti-CTLA4 or anti-PD1, or in combination. CCB therapy led to changes in circulating B cells that were detectable after the first cycle of therapy and characterized by a decline in circulating B cells and an increase in CD21^lo B cells and plasmablasts. PD1 expression was higher in the CD21^lo B cells, and B cell receptor sequencing of these cells demonstrated greater clonality and a higher frequency of clones compared with CD21^hi cells. CCB induced proliferation in the CD21^lo compartment, and single-cell RNA sequencing identified B cell activation in cells with genomic profiles of CD21^lo B cells in vivo. Increased clonality of circulating B cells following CCB occurred in some patients. Treatment-induced changes in B cells preceded and correlated with both the frequency and timing of IRAEs. Patients with early B cell changes experienced higher rates of grade 3 or higher IRAEs 6 months after CCB. Thus, early changes in B cells following CCB may identify patients who are at increased risk of IRAEs, and preemptive strategies targeting B cells may reduce toxicities in these patients.

BRAF^V600E/K is a frequent mutationally active tumor‐specific kinase in melanomas that is currently targeted for therapy by the specific inhibitor PLX4032. Our studies with melanoma tumor cells that are BRAF^V600E/K and BRAF^WT showed that, paradoxically, while PLX4032 inhibited ERK1/2 in the highly sensitive BRAF^V600E/K, it activated the pathway in the resistant BRAF^WT cells, via RAF1 activation, regardless of the status of mutations in NRAS or PTEN. The persistently active ERK1/2 triggered downstream effectors in BRAF^WT melanoma cells and induced changes in the expression of a wide‐spectrum of genes associated with cell cycle control. Furthermore, PLX4032 increased the rate of proliferation of growth factor‐dependent NRAS Q61L mutant primary melanoma cells, reduced cell adherence and increased mobility of cells from advanced lesions. The results suggest that the drug can confer an advantage to BRAF^WT primary and metastatic tumor cells in vivo and provide markers for monitoring clinical responses.

Activating mutations in BRAF kinase are common in melanomas. Clinical trials with PLX4032, the mutant-BRAF inhibitor, show promising preliminary results in patients selected for the presence of V600E mutation. However, activating V600K mutation is the other most common mutation, yet patients with this variant are currently excluded from the PLX4032 trials. Here we present evidence that a patient bearing the BRAF V600K mutation responded remarkably to PLX4032, suggesting that clinical trials should include all patients with activating BRAF V600E/K mutations.