Citron Rho-Interacting Serine/Threonine kinase (CIT) Is a Novel Therapeutic Target in Multiple Myeloma Cells

Introduction: Citron rho-interacting serine/threonine kinase (CIT) is a serine/threonine kinase which is a key component of the midbody and is essential for cytokinesis. CIT localizes to the central spindle and midbody and functions to promote efficient cytokinesis. CIT knockdown may disrupt cytokinesis and therefore cell growth. CIT has been reported to be upregulated and important for growth of several cancers. However, the significance of CIT has not been investigated in the field of multiple myeloma (MM). We therefore dissected the role of CIT in MM growth in vitro and in vivo.

Materials and methods: CIT gene expression in MM cells was compared to normal plasma cells using public-available gene expression profile (GEP) data set (GSE6477). Kaplan-Meier curve for MM patient survival between high and low CIT expressing patients were examined by using the GEP data set (GSE4581). Protein expression of CIT in MM cells was confirmed by proteomic analysis and immunohistochemistry. Knockdown of CIT was performed in MM cell lines MM1s and OPM2 using lentiviral shRNAs. CIT knockdown was confirmed by reduced CIT mRNA in comparison to a scrambled control. Differences in cell proliferation and cell cycle between CIT knockdown cells and scramble control were analyzed by using thymidine uptake and PI staining, respectively. Cytokinesis failure was analyzed by immunofluorescence using alpha-tubulin antibody and DAPI. shCIT OPM2 (n=7) and the scrambled control cells (n=8) were injected subcutaneously into SCID-Bg mice (5x10⁶ cells/mouse) and were followed for tumor development and survival.

Results: CIT expression was significantly higher in MM patients’ plasma cells compared to healthy donors in GEP (p=0.02), proteomic analysis and immunohistochemistry. Also CIT expression was higher in relapsed patients compared to newly diagnosed patients by GEP. MM patients with high CIT expression had significantly worse overall survival compared to low CIT expressing patients (p=0.04). CIT knockdown MM cell lines showed reduced cell proliferation and G2 cell cycle arrest by thymidine uptake and PI staining compared to the scrambled control. Significantly, large amount of multinucleated cells, which indicates cytokinesis failure, were observed in the CIT knockdown cells compared to scrambled control. Reduced tumor growth (p<0.001) and prolonged survival (p<0.001) was observed in CIT knockdown MM cell line injected mice.

Conclusions: shRNA knockdown of CIT in MM cells induces G2 arrest leading to cytokinesis failure in vitro with reduced cell proliferation in vivo. Since MM cells have significantly higher expression of CIT compared to normal plasma cells, CIT represents a novel therapeutic target for MM. Studies are ongoing to develop drugs to target CIT for MM treatment.