Fig 1: Genotypic and phenotypic analyses of melanomas derived from patients with germline variant KDR Q472H. (A) Representative images of CD34 expression in primary (bottom) and metastatic (top) melanoma tissue in KDR variant (left) and KDR WT (right). Scale bars are 100 μm and inserts are 50 μm. (B) Dot plot showing log2(CD34) expression of A (p = 0.0045). (C) Nanostring analysis of immunoregulatory gene expression. Differential gene expression analysis (N = 594 genes) illustrates distinct signatures in KDR-Var clusters and KDR-WT clusters in which the variant showed decreased expression (green) of genes associated with chemotactic activity, inflammatory responses, modulation of T cell activity, and antigen presentation (immunosuppression) as well as increased expression (red) of genes associated with cell growth and protein synthesis (proliferation). (D) Metascape pathway analysis of genes that are downregulated in KDR-Var. (E) TCGA dataset showing increased Breslow depth in the KDR-Var melanoma patients (p = 0.0121). (F) Volcano plot of DEGs between KDR-Var and KDR-WT in TCGA cohort analyzed by cBioPortal tool. Red color represents significant DEGs (p < 0.05) (G). Metascape pathway analysis of DEGS upregulated in the KDR-Var TCGA patients showing GO enriched biological processes. (H) Kaplan–Meier curve shows worse progression-free survival in AA (n = 9) homozygous and TA (n = 36) heterozygous KDR-Var patients compared to the KDR-WT TT (n = 64) variant when treated with first-line MAPK targeted therapies (p = 0.022). (I) Kaplan–Meier curve shows worse progression-free survival in KDR-Var patients (TA + AA combined, n = 29) compared to WT (n = 53) when treated with first-line PD-1-based therapy (p = 0.067). p-values were generated using log-rank test.
Fig 2: Analysis of cell line growth, tumor kinetics, and microenvironments in B16 animal models induced with germline variant KDR Q472H. (A–C). Colony assay comparing B16 cell lines induced with KDR WT or KDR Q472H showing increased KDR-Var colony count (p = 0.0242) and colony size (p = 0.0003). (D) Tumor growth curve shows the average tumor volume for mice induced with the KDR variant Q472H at day 29 is greater than the average tumor volume for mice without the KDR variant (p = 0.0027). (E–G). Box blots showing significant reduction in CD45+ tumor-infiltrating lymphocytes in the KDR-Var compared to the KDR-WT (p = 0.0282), while no difference is observed in the peripheral blood. (H) Heat map from RNA-Seq analysis of tumors resected from mice induced with KDR-V B16 cells (n = 4) or KDR WT (n = 5) shows distinct clusters of the two groups comparing DEGs between two groups at p < 0.01. (I) g:Profiler pathway analysis of RNA-seq DEGs comparing KDR-Var to KDR-WT mice tumors showing enriched key pathways and cellular processes like regulation of immune system response, cell proliferation, and signaling by VEGF.
Fig 3: KDR-WT cell lines do not display synergistic cytotoxicity when treated with VEGFR + BRAF inhibitors. Difference between the observed and expected inhibition of cell growth when combining Lenvatinib 10 μM with varying doses of Dabrafenib. Expected inhibition was calculated with Combosyn combination index (n = 4). (A) A375. (B) SKMEL192. (C) SKMEL239. (D) SKMEL28. Yellow, blue and green represent Dabrafenib, Lenvatinib and a combination of both, respectively.
Fig 4: Germline variant KDR Q472H cell lines and murine models respond to Lenvatinib treatment. (A) Growth curves of two KDR homozygous variant and two KDR wild type melanoma cell lines showing decreased proliferation upon the addition of Lenvatinib (VEGFR inhibitor) to Dabrafenib (BRAF inhibitor) and Trametinib (MEK inhibitor) combination in the KDR-Var and not the WT. (B) Representative images of tumors resected from B16 mice post-treatment with control (top) or the anti-VEGFR, Lenvatinib (bottom). (C) Bar graphs of tumor volume at endpoint in KDR WT (left) and KDR variant (right) B16 mouse models post-treatment. (D) Growth curves of tumors with or without Lenvatinib in KDR WT (left) or KDR variant (right).
Fig 5: Germline variant KDR Q472H cell lines display synergistic cytotoxicity when treated with VEGFR + BRAF inhibitors. Difference between the observed and expected inhibition of cell growth when combining Lenvatinib 10 μM with varying doses of Dabrafenib. Expected inhibition was calculated with the Combosyn combination index (n = 4). (A) WM2044 (AA). (B) 501MEL (AA). (C) WM46 (TA). (D) 451LU (TA). Yellow, blue and green represent Dabrafenib, Lenvatinib and a combination of both, respectively.
Supplier Page from Sino Biological, Inc. for Human VEGFR2/KDR/Flk-1/CD309 Gene ORF cDNA clone expression plasmid