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Supplementary Figure 3 - PDF 313K, CRM1 inhibition induces cell cycle arrest and increases cell death when combined with BRAF inhibition

Supplementary Figure 2 - PDF 191K, CRM1 inhibition induces cell cycle arrest and increases cell death when combined with BRAF inhibition

Supplementary Figure 7 - PDF file, TP53 knockdown in A375 reduces the effect of CRM1 inhibition on A375 proliferation but not on caspase-3/7 activity

Supplementary Figure 4 - PDF file 44K, CRM1 inhibition induces a statistically significant increase in caspase-3/7 activity as single therapy or in combination with BRAF inhibition

Supplementary Figure 1 - PDF file 29K, CRM1 and BRAF inhibition decrease melanoma cell proliferation and are synergistic in vitro

Resistance to BRAF inhibitor therapy places priority on developing BRAF inhibitor-based combinations that will overcome de novo resistance and prevent the emergence of acquired mechanisms of resistance. The CRM1 receptor mediates the nuclear export of critical proteins required for melanoma proliferation, survival, and drug resistance. We hypothesize that by inhibiting CRM1-mediated nuclear export, we will alter the function of these proteins resulting in decreased melanoma viability and enhanced BRAF inhibitor antitumoral effects. To test our hypothesis, selective inhibitors of nuclear export (SINE) analogs KPT-185, KPT-251, KPT-276, and KPT-330 were used to induce CRM1 inhibition. Analogs PLX-4720 and PLX-4032 were used as BRAF inhibitors. Compounds were tested in xenograft and in vitro melanoma models. In vitro, we found CRM1 inhibition decreases melanoma cell proliferation independent of BRAF mutation status and synergistically enhances the effects of BRAF inhibition on BRAF-mutant melanoma by promoting cell-cycle arrest and apoptosis. In melanoma xenograft models, CRM1 inhibition reduces tumor growth independent of BRAF or NRAS status and induces complete regression of BRAF V600E tumors when combined with BRAF inhibition. Mechanistic studies show that CRM1 inhibition was associated with p53 stabilization and retinoblastoma protein (pRb) and survivin modulation. Furthermore, we found that BRAF inhibition abrogates extracellular signal–regulated kinase phosphorylation associated with CRM1 inhibition, which may contribute to the synergy of the combination. In conclusion, CRM1 inhibition impairs melanoma survival in both BRAF-mutant and wild-type melanoma. The combination of CRM1 and BRAF inhibition synergizes and induces melanoma regression in BRAF-mutant melanoma. Mol Cancer Ther; 12(7); 1171–9. ©2013 AACR.

Supplementary Figure 5 - PDF file 894K, CRM1 inhibition leads to delayed tumor growth independent of BRAF or NRAS status in melanoma xenograft models

Supplementary Methods and Figure Legends - PDF 133K, Supplementary methods and figure legends

Supplementary Figure 6 - PDF file 57K, CRM1/BRAF inhibition in PTEN null/BRAF mutant melanoma Xenograft model