Novel small molecule XPO1/CRM1 inhibitors induce nuclear accumulation of TP53, phosphorylated MAPK and apoptosis in human melanoma cells.

Gregory B. Lesinski,William Senapedis,Jean-Richard Saint-Martin,Sharon Shacham,Krista La Perle,Trinayan Kashyap,Matthew A. Bill,Kari Kendra,Gregory S. Young,Michael Kauffman,Yosef Landesman,Jennifer Yang,Nikolas K. Haass

doi:10.1371/journal.pone.0102983

Gregory B. Lesinski, William Senapedis

Open Access

https://doi.org/10.1371/journal.pone.0102983

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Abstract

XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE). In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI)], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E) cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p<0.0001) and CHL-1 (p = 0.0087) human melanoma cell lines in vivo at well tolerated doses. Inhibition of XPO1 using SINE represents a potential therapeutic approach for melanoma across cells with diverse molecular phenotypes by promoting growth inhibition and apoptosis.

Highlights

Melanoma is the most deadly form of skin cancer, with an estimated 76,100 new cases and 9,710 deaths in the United States alone in 2014 [1]
To determine the potential of XPO1 as a molecular target in melanoma cells, XPO1 protein expression was characterized in a human melanoma tissue microarray, a panel of human metastatic melanoma cell lines (A375, Hs294T, FO-1, Wm1366, CHL-1) or three primary human epidermal melanocyte cell lines (HEM-l, HEMn-MP, HEMn-DP)
XPO1 protein was consistently expressed in a panel of five human metastatic melanoma cell lines, and in three primary human melanocyte cell lines (Fig. 1D)

Summary

Introduction

Melanoma is the most deadly form of skin cancer, with an estimated 76,100 new cases and 9,710 deaths in the United States alone in 2014 [1]. Recent therapeutic approaches including small molecule inhibitors of activated BRAF pathways (vemurafenib, dabrafenib) and immunomodulatory agents represent significant advances in melanoma therapy [3,4]. The genetic and phenotypic heterogeneity of melanoma cells increases the likelihood for the emergence of drugresistant clonal cell populations and eventually disease recurrence [5]. Such resistance mechanisms could be attributed to the fundamental ability of malignant cells to inactivate tumor suppressor pathways and bypass cell cycle checkpoints. This process, termed ‘nuclear export’, is gaining attention as a novel therapeutic target that can be inhibited to promote re-activation of tumor suppressive pathways

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