Fig 2: Combinatorial treatment with LDN-193189 and selumetinib synergizes to reduce cellular invasion in MPNST cellsA. Graphical representation of the quantified fluorescence of the cells that invaded through the extracellular matrix (ECM), normalized to invasive activity without any chemoattractant and the vehicle control. LDN-193189 inhibits cellular invasion as compared to the vehicle treated control in LP and HP Nf1(-/-) MPNST cells. Addition of 200 ng/mL of BMP2 promotes invasion in these cells (**P<0.01), which is blocked by the addition of LDN-193189 in all the tested MPNST cells. Cells were stained with CyQuant/GR dye and the number of invaded cells was quantified by a fluorescence plate reader. B. The IC50 graph shows the optimal dose of LDN-193189 needed for half maximal inhibition of invasiveness in MPNSTs. C. Bar graph of the quantified fluorescence of the cells that invaded through the ECM upon treatment with single agents and in combination. The invasive ability of MPNST cells is greatly reduced by LDN-193189 (0.003 µM), whereas selumetinib only affects cellular invasion at 0.1 µM. The combinatorial treatment results in a statistically significant increase of the inhibitory effect of LDN-193189 on invasion. Based on the BI model, the therapeutic interaction of the combination treatment on invasion was synergistic for both the combinations used. Data presented are mean average of three independent experiments ± S.D. with the corresponding P-values (n=3, *P<0.05, **P<0.01, One-way ANOVA followed by Tukey's test for multiple comparisons).

Fig 3: Kaplan-Meier plots of melanoma patients with CAPN1 over expression and NF1 under expression show worse prognosis compared to patients with alterations only in CAPN1 or in NF1(A) Survival plot of patients with CAPN1 over expression or missense mutation in TCGA melanoma (N=355). (B) Survival plot of patients with NF1 under expression or missense mutation in TCGA melanoma (N=355). (C) Survival plot of patients with CAPN1 over expression or missense mutation and NF1 under expression or missense mutation in TCGA melanoma (N=355). A gene in a sample was marked under (over) expressed if its expression level in the sample is below 1/3-quantile (above 2/3-quantile) of its expression level across TCGA melanoma samples. Each association was evaluated using Cox proportional hazard model while controlling for patients’ age, sex, and race, tumors’ genomic instability and tumor stage (see Methods).

Fig 4: Loss of NF1 confers resistance to RAF-targeted therapy. A, Immunoblot of NF1 expression in DBTRG and NMCG1 parental lines (P), and stably infected NF1 knockdown using two different sgRNAs (T1 or T3). B, Immunoblot of DBTRG treated with 30 nmol/L dabrafenib and samples collected at a range of timepoints after treatment. C, Immunoblot of DBTRG cells treated with a range of dabrafenib doses for 24 hours. Growth of NMCG1 (D) or DBTRG (E) parental or NF1 knockdown lines in response to increasing doses of dabrafenib at 72 hours. Data are representative of four experiments. Cytotox Green expression as a marker of apoptosis upon treatment with a range of dabrafenib concentrations in NMCG1 at 72 hours (N = 3; F) and DBTRG at 64 hours (two independent experiments with triplicate at each timepoint; G). H, Growth assay in DBTRG at 48 hours after treatment with RAFi (dabrafenib, 10 nmol/L), MEKi (trametinib, 10 nmol/L), or the combination. Dab, dabrafenib; Tram, trametinib. *, P = 0.05; **, P = 0.01; ***, P = 0.001.

Fig 5: CAPN1 is a novel binding partner of NF1(A) Immunoblots of melanoma cells immunoprecipitated with anti-NF1. Immunoprecipitates were analyzed in parallel by anti-NF1 and anti-CAPN1 immunoblotting. (B) Immunoblots of melanoma cells immunoprecipitated with anti-CAPN1. Immunoprecipitates were analyzed in parallel by anti-NF1 and anti-CAPN1 immunoblotting. WCL – whole cells lysate, RbIgG-IP and MoIgG- Rabbit and mouse IgG isotype control, respectively. (C) Distribution of alterations in NF1, CAPN1, BRAF and NRAS in TCGA melanoma (N=287).