Germinal Center Kinase As a Therapeutic Target in Multiple Myeloma

Introduction: Despite many new drug approvals, Multiple Myeloma remains an incurable disease due to development of drug resistance. This process is driven by clonal evolution in which kinases play a major role. Germinal center kinase (GCK) is the founding member of the GCKs, a group of Ser/Thr kinases homologous to the saccharomyces cerevisiae Ste20p, and participates in B cell differentiation. Among human tissues, GCK is highly expressed in transformed lymphocytes, spleen and whole blood. However, the expression and function of GCK in human myeloma is totally unknown.

Methods and Results: First we analyzed the expression level of GCK in human MM cells and found that GCK is abundantly expressed in all the MM cell lines we tested. To determine whether GCK is required for MM cell growth, we introduced inducible shRNA to knock down GCK expression in MM cells by lentiviral mediated transduction. MM cell lines MM.1S and RPMI-8266 were stably transduced with shRNA lentivirus generated from a robust inducible knockdown vector pLKO-tet-On. Doxycycline-induced GCK-tet-on-shRNA expression resulted in GCK protein knockdown and significant inhibition of cell growth by 75% in WST-1 proliferation assay. In accordance, cell cycle analysis revealed that the knockdown of GCK decreased cells in S-phase with concomitant increase in G0/G1 growth arrest (55% vs 67%). Importantly, GCK knockdown resulted in significant increase (3.21% vs 60.8%) of apoptotic cells suggesting that inhibition of GCK is a potential therapeutic target in MM cells. This was further confirmed by treating MM cells lines (MM.1S, RPMI-8266 and H929) with GCK inhibitors TL4-12, NG25 and Bay 61-3606. We found that these drugs dose dependently and significantly inhibit multiple myeloma cell growth, with IC₅₀ 7, 1.6 and 0.5 µM, respectively in H929 cells by WST-1 proliferation assay. Mechanistic studies showed that Bay 61-3606 dose-dependently decreases the protein levels of IKZF1/IKZF3 and c-Myc, increases p53 level and induces PARP cleavage resulting in cell cycle G1 arrest (60.8 vs. 69.8 with 1 µM) and apoptosis (9.16 vs. 29.4 at 1 µM) in MM.1S cells. To confirm the critical role of GCK in in vivo growth of MM cells, we generated subcutaneous MM xenografts in SCID Beige (CB17.Cg-PrkdcscidLystbg-J/Crl) mice using doxycycline-inducible GCK shRNA MM.1S cells. Doxycycline or vehicle treatment was started after the tumor was established on day 16. In contrast to vehicle-treated MM.1S-tet-on-shGCK or doxycycline-treated MM.1S-tet-on-sh-control tumors, doxycycline treated animals bearing MM.1S-tet-on-shGCK xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 30 days. Immunohistochemistry staining of tumors confirmed the decrease of GCK and IKZF1 expression in doxycycline-treated mice bearing MM.1S-tet-on-shGCK tumors compared with tumors of vehicle- or non-doxycycline treated mice.

Conclusion: Our findings show that GCK is a potential target for multiple myeloma therapy. GCK inhibition results in significant anti-MM effects by inducing cell cycle arrest and cell apoptosis accompanied by IKZF1/3 down regulation. Furthermore, the inhibition of in vivo tumor growth correlates with the doxycycline-induced GCK knockdown, further confirming the critical role of GCK in MM tumorigenesis.