Gck Inhibition Is a Novel Therapeutic Strategy for RAS Mutated Multiple Myeloma and Overcomes Resistance to IMiDs

Introduction: RAS oncogenes are the most frequently mutated gene family in human cancers. 50% of newly diagnosed multiple myeloma patients carry a RAS/MAPK pathway mutation, with a rising percentage in the relapsed situation¹. Thus, targeting RAS mutations in multiple myeloma will increase therapeutic efficiency and potentially overcome drug-resistance. Unfortunately, RAS mutations have been considered "undruggable" due to a lack of traditional small molecule binding pockets on the proteins. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK), also named mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), is an upstream activator in the MAPK pathway. Indeed, we recently discovered the critical role of GCK in RAS mutated (RAS^mut) MM cell survival and growth. GCK knockdown in RAS^mut MM cells induced MM cell growth inhibition both in vitro and in vivo. However, the detailed mechanism is yet to be defined.

Methods and Results: Our previous data showed that GCK knockdown induces MM cell growth inhibition, associated with the blockage of MKK4/7-JNK phosphorylation and the downregulation of critical transcriptional factors (TFs) including IKZF1/3, BCL-6, and c-MYC proteins. To confirm that GCK knockdown downregulates IKZF1/3 etc at protein level but not mRNA level, we conducted real-time PCR on GCK knockdown MM cells and compared the expression of GCK and TFs to the empty vector (EV) infected MM cells. Results showed that shRNA induced GCK silencing only led to the significantly decreased GCK mRNA, however, did not affect IKZF1 and c-MYC expressions at mRNA level. Consistent with the effects of GCK knockdown, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 proteins, inhibited MM cell proliferation and induced cell apoptosis.

IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Importantly, our data showed that IMiDs-resistant RPMI-8226 MM cells have high expression of GCK. GCK knockdown and inhibition induced IKZF1 downregulation, triggered growth inhibition and cell apoptosis in RPMI-8226 cells, suggesting that GCK regulates IKZF1 degradation via a CRBN-independent mechanism. To confirm this hypothesis, we silenced CRBN in N-Ras^mut H929 MM cells by shRNA lentiviral infection and examined the response to IMiDs and GCK inhibitor. CRBN knockdown was confirmed by western blotting. CRBN silencing in H929 cells resulted in lenalidomide (LEN) resistance, evidenced by the WTS proliferation assay. In contrast, CRBN silencing failed to rescue N-Ras^mut H929 MM cells from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN.

Conclusion: Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RAS^mut MM cells. Importantly, GCK inhibitor downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RAS^mut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance. GCK inhibitors may represent a novel therapy for the treatment of RAS^mut MM patients, especially those who are resistant to IMiDs as well as with refractory or relapsed MM.

References

1. Walker, B.A., et al. Mutational Spectrum, Copy Number Changes, and Outcome: Results of a Sequencing Study of Patients With Newly Diagnosed Myeloma. J Clin Oncol 33, 3911-3920 (2015).