Person:

Lawrence, Donald

An effective treatment for metastatic melanoma remains one of the most elusive goals in all of oncology. Several generations of therapeutic trials have yet to yield any agents that can significantly prolong survival for widespread disease. Despite this disheartening history, our understanding of the biology and molecular genetics of melanoma hold the promise of a new era of molecular targets. One pathway that appears to be universally activated in and critically needed for melanoma growth is the Ras/mitogen activated protein (MAP) kinase signaling cascade. Since the enzymatic functions of the signaling partners are well characterized, this pathway offers many potential “druggable” candidates including Braf, Mek and Ras itself. In this review, we describe this pathway in the context of melanoma tumorigenesis and discuss some of the current relevant pharmacologic treatments and clinical trials.

There have been significant advances with regard to BRAF-targeted therapies against metastatic melanoma. However, the majority of patients receiving BRAF inhibitors (BRAFi) manifest disease progression within a year. We have recently shown that melanoma patients treated with BRAFi exhibit an increase in melanoma-associated antigens and in CD8+ tumor-infiltrating lymphocytes in response to therapy. To characterize such a T-cell infiltrate, we analyzed the complementarity-determining region 3 (CDR3) of rearranged T-cell receptor (TCR) β chain-coding genes in tumor biopsies obtained before the initiation of BRAFi and 10–14 d later. We observed an increase in the clonality of tumor-infiltrating lymphocytes in 7 of 8 patients receiving BRAFi, with a statistically significant 21% aggregate increase in clonality. Over 80% of individual T-cell clones detected after initiation of BRAFi treatment were new clones. Interestingly, the comparison of tumor infiltrates with clinical responses revealed that patients who had a high proportion of pre-existing dominant clones after the administration of BRAFi responded better to therapy than patients who had a low proportion of such pre-existing dominant clones following BRAFi. These data suggest that although the inhibition of BRAF in melanoma patients results in tumor infiltration by new lymphocytes, the response to treatment appears to be related to the presence of a pre-existing population of tumor-infiltrating T-cell clones.

A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1) transporter inhibition. We identified drugs sensitizing cell lines that are BRAFV600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR) and platelet derived growth factor receptor (PDGFR) family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis in vitro and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs), demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK) kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.

Abstract To establish the maximum tolerated dose (MTD), dose‐limiting toxicities (DLT), safety profile, and anti‐tumor efficacy of RAF265. We conducted a multicenter, open‐label, phase‐I, dose‐escalation trial of RAF265, an orally available RAF kinase/VEGFR‐2 inhibitor, in patients with advanced or metastatic melanoma. Pharmacokinetic (PK) analysis, pharmacodynamics (PD) and tumor response assessment were conducted. We evaluated metabolic tumor response by 18[F]‐fluorodeoxyglucose‐positron‐emission tomography (FDG‐PET), tissue biomarkers using immunohistochemistry (IHC), and modulators of angiogenesis. RAF265 has a serum half‐life of approximately 200 h. The MTD was 48 mg once daily given continuously. Among 77 patients, most common treatment‐related adverse effects were fatigue (52%), diarrhea (34%), weight loss (31%) and vitreous floaters (27%). Eight of 66 evaluable patients (12.1%) had an objective response, including seven partial and one complete response. Responses occurred in BRAF‐mutant and BRAF wild‐type (WT) patients. Twelve of 58 (20.7%) evaluable patients had a partial metabolic response. On‐treatment versus pretreatment IHC staining in 23 patients showed dose‐dependent p‐ERK inhibition. We observed a significant temporal increase in placental growth factor levels and decrease in soluble vascular endothelial growth factor receptor 2 (sVEGFR‐2) levels in all dose levels. RAF265 is an oral RAF/VEGFR‐2 inhibitor that produced antitumor responses, metabolic responses, and modulated angiogenic growth factor levels. Antitumor activity occurred in patients with BRAF‐mutant and BRAF‐WT disease. Despite low activity at tolerable doses, this study provides a framework for the development of pan‐RAF inhibitors and modulators of angiogenesis for the treatment of melanoma.

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies.

Despite initial responses, most melanoma patients develop resistance to immune checkpoint blockade (ICB). To understand the evolution of resistance, we studied 37 tumor samples over 9 years from a metastatic melanoma patient with exceptional response followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of 7 lineages with multiple convergent, but independent resistance-associated alterations (RAAs). All recurrent tumors emerged from a lineage characterized by loss of chromosome 15q, with post-treatment clones acquiring additional genomic driver events. Deconvolution of bulk RNAseq and highly-multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition amongst different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFR-High tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated 2 distinct NGFR spatial patterns with high polarity and proximity to immune cells in subcutaneous tumors versus a diffuse spatial pattern in lung tumors, suggesting different roles of this neural crest-like program in different tumor microenvironments. Broadly, this study establishes a high-resolution map of the evolutionary dynamics of resistance to ICB, characterizes a de-differentiated, neural crest tumor population in melanoma immunotherapy resistance, and describes site specific differences in tumor-immune interactions via longitudinal analysis of a melanoma patient with an unusual clinical course.