Fig 1: CAPN1 inhibition stabilizes NF1 levels, affecting RAS signaling and cell proliferation(A) NF1 wild type cells (A375, 74T) were treated with increasing concentrations of Calpain inhibitor I (µM) for 6 hours or with DMSO as control, and NF1 levels were tested by immunoblot. (B) NF1 mutant cells (108T, 76T) were treated with increasing concentrations of Calpain inhibitor I (µM) for 6 hours or with DMSO as control, and NF1 levels were tested by immunoblot. Ratios of NF1 levels to GAPDH were generated using Image lab (BioRad) and Microsoft Excel analysis. (C) 74T and A375 cells were treated with 3 µM or 5 µM of Calpain inhibitor I for 16 hours, respectively. Cell lysates were analyzed by western blot with the indicated antibodies. RAS-GTP levels were assessed by RAS pulldown assay after treatment with 50 µM of Calpain inhibitor I, respectively for 6 hours. (D) A375 and 74T cells were seeded in the 96 well plates with 10% FBS and cells were treatedwith 2.5 or 5 µM of Calpain inhibitor I, respectively. DMSO was used as a control for this experiment. The average cell number was measured by assessing DNA content using SYBR green I in two independent experiments with six replicates each. Error bars, s.e.m.
Fig 2: 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 3: CAPN1 is responsible for NF1 degradation in vitroCell lysates of A375, 74T and 293T stably over expressing NF1 (293T-NF1) were incubated with the indicated units of purified CAPN1 with 10 mM CaCl2 for 1 hour, resolved by SDS-PAGE and blotted with anti-NF1 (polyclonal antibody) and anti-GAPDH. Anti-Tubulin served as a positive control for the assay. Molecular weights are given in kilodaltons on the left. The arrows indicate the detection of full length NF1 and the black boxes indicate an approximately 40 kDa proteolytic fragment of NF1. Addition of 45 µM of Calpain inhibitor I blocked this degradation (last lane on each blot).
Fig 4: 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).
Fig 5: NF1 mutant and wild-type cell lines show enhanced sensitivity to combined CAPN1 and MEKi inhibition(A) Representative dose response curves were generated using NF1 mutant cell lines (108T and 76T) treated with constant concentration of Calpain inhibitor I (6 µM) and increasing concentrations of Trametinib (1 pM - 10 µM) for 72 hours before assessing viability by Cell Titer-Glo Luminescent Cell viability assay (n=3). The relative cell number after cells were treated with Calpain inhibitor I and Trametinib is plotted as percent survival, as compared to Trametinib-treated control, versus log Trametinib concentration in pM. (B) Representative dose response curves were generated using NF1 wild-type cell lines (A375 and 74T) treated with constant concentration of Calpain inhibitor I (6 µM and 4 µM, respectively) and increasing concentrations of Trametinib (1 pM - 10 µM) for 72 hours before assessing viability by Cell Titer-Glo Luminescent Cell viability assay (n=3). The relative cell number after cells were treated with Calpain inhibitor I and Trametinib is plotted as percent survival, as compared to Trametinib-treated control, versus log Trametinib concentration in pM. **P<0.01 and ***P<0.0001 Trametinib versus Trametinib and Calpain inhibitor I (Student t test). (C) EC50 values for inhibition of cell growth by 72 hours treatment with Trametinib or with the combination of Trametinib and Calpain inhibitor I.
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