Background: Melanoma is a heterogeneous malignancy that presents an immense challenge in therapeutic development. Recent approaches targeting the oncogenic MAP kinase pathways have shown tremendous improvement in the overall survival of patients with advanced melanoma. However, there is still an urgent need for identification of new strategies to overcome drug resistances and to improve therapeutic efficacy. Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) belongs to a selected group of mitotic kinases and is required for normal mitosis progression. In contrast to inhibitors of other mitotic kinases, anti-tumor potential of haspin inhibitors has not been well explored. Herein, we aim to examine effects of CHR-6494, a small molecule inhibitor of haspin, in melanoma cells.Methods: Anti-tumor activities of the haspin inhibitor CHR-6494 were tested in a number of melanoma cell lines either as a single agent or in combination with the MEK inhibitor Trametinib (GSK1120212). Experiments are based on: 1) Cell viability determined by the crystal violet staining assay; 2) apoptotic responses measured by the caspase 3/7 activity assay and western blot analysis for the level of cleaved PARP (Poly ADP-Ribose Polymerase); 3) cell cycle analysis conducted using flow cytometry; and 4) cell migratory ability assessed by the scratch assay and the transwell migration assay.Results: We have found that CHR-6494 alone elicits a dose dependent inhibitory effect on the viability of several melanoma cell lines. This growth inhibition is accompanied by an increase in apoptotic responses. More importantly, CHR-6494 appears to synergize with the MEK inhibitor Trametinib in suppressing cell growth and enhancing apoptosis in both wild type and BRAFV600E mutant melanoma cell lines. Administering of these two small molecules as a combination is also capable of suppressing cell migration to a greater extent than the individual agent.Conclusion: These results suggest that haspin can be considered as a viable anti-melanoma target, and that concomitant inhibition of haspin and MEK activities with small molecules could represent a novel therapeutic strategy with improved efficacy for treatment of melanoma.

Our recent studies have shown that haspin, a protein kinase imperative for mitosis, is engaged in the interphase progression of HeLa and U2OS cancer cells. In this investigation, we employed the Fucci reporter system and time-lapse imaging to examine the impact of haspin gene silencing on cell cycle progressions at a single-cell level. We found that the loss of haspin induced multiple cell cycle defects. Specifically, the S/G2 duration was greatly prolonged by haspin gene depletion or inhibition in synchronous HeLa cells. Haspin gene depletion in asynchronous HeLa and U2OS cells led to a similarly protracted S/G2 phase, followed by mitotic cell death or postmitotic G1 arrest. In addition, haspin deficiency resulted in robust induction of the p21 CIP1/WAF1 checkpoint protein, a target of the p53 activation. Also, co-depleting haspin with either p21 or p53 could rescue U2OS cells from postmitotic G1 arrest and partially restore their proliferation.These results substantiate the haspin's capacity to regulate interphase and mitotic progression, offering a broader antiproliferative potential of haspin loss in cancer cells.