Improving Pediatric Melanoma Outcomes in a Longitudinally Evaluated Cohort

Division Of Basic Sciences - Nci

Investigators

Abstract

We have made progress on each of three specific aims for P-MOLE. Specific Aim 1: Determine germline variants and phenotypes associated with PM. We have completed whole genome sequencing on germline samples from 75+ patients with PM and are in the process of analyzing these data. In the course of this analysis, we have identified rare germline variants of the LZTR1 gene in children with spitzoid melanoma and US melanoma-prone families. We have established a pipeline to functionally characterize these variants of unknown significance. Specifically, we co-transfect FLAG-tagged wild type or variant LZTR1 and HA-tagged RIT1 into HEK293T cells. Steady state protein expression of the over-expressed RIT1 and LZTR1 are assessed, as well as MEK and ERK phosphorylation. Variants of LZTR1 that are known to induce MAPK pathway activation (R170Q, Y193H, and G248R) are used as positive controls in the assay. This assay has been used to validate the function of other LZTR1 VUS (Dr. Pau Castel, personal communication). In follow up experiments, we assess the stability of the LZTR1 variants using cycloheximide chase assays and assess whether the LZTR1 variant remains able to interact with RIT1 using co-immunoprecipitation experiments. In addition, model organism experiments are pivotal for understanding the developmental impact of variants of unknown significance. The LCDS zebrafish facility, led by Dr. Christine Kettenhofen (Insinna), has established a phenotyping pipeline to rank the severity of the defects associated with impaired RAS signaling in zebrafish. These assays allow effective quantification of the phenotypes observed at different developmental stages including embryo elongation during early convergence and extension (CE) at the gastrulation stage (11 hours post-fertilization, hpf), increased heart size and heart jogging laterality randomization (20-24 hpf), shortening of the body size (3 days post-fertilization, dpf), and widening of the head (5 dpf). To adapt these experiments for the functional characterization of novel LZTR1 variants, we will first depleted lztr1 expression in zebrafish embryos by injecting an ATG morpholino at the 1 cell stage. A CE defect was observed in lztr1 morphants. We are currently attempting to rescue this phenotype by co-injecting human LZTR1 wildtype mRNA and compare the function of novel LZTR1 variants. We will classify variants as likely pathogenic if they are unable to rescue the morpholino-induced CE defect and submit this data to ClinVar. Specific Aim 2: Characterize genomic and transcriptomic alterations in PM and descriptively correlate with outcomes. We have completed RNAseq for 75 pediatric melanoma cases and are currently analyzing these data. In addition, we have completed single cell RNAseq and spatial transcriptomic profiling on 25 cases of pediatric melanoma, and are analyzing the data. Specific Aim 3: Establish humanized mouse models of PM and employ these models to study mechanisms of PM resistance to immune checkpoint and MAPK pathway inhibition. Working with Dr. Simone Difilippantonio and Dr. Cindy Chao, we have established a humanized model of melanoma (A375 cell line xenograft) that is responsive to immune checkpoint inhibition. In our next experiment, we will test the efficacy of the immune checkpoint inhibitor nivolumab in a humanized PDX model of PM.