Targeting NEK2 Induces Cellular Senescence in B-Cell Malignancies through p53-Independent Signaling Pathways

Background

Multiple Myeloma (MM) is a malignancy of terminally differentiated B-cells homing to and expanding in the bone marrow. We have identified that a chromosomal instability (CIN) gene, the serine-threonine kinase NEK2, is the one mostly significantly upregulated gene in MM cells of patients in the remaining MM cells of patients in complete remission with minimal residual disease (MRD) and also at relapse early after tandem transplants. High expression of NEK2 has been related to poor outcome in MM and many other cancers. We have also shown that pharmacological or genetic inhibition of NEK2 significantly decreases MM cell growth and delays tumor growth in vitro and in vivo. Cellular senescence is defined as a stable cell-cycle arrest which shows a suppression of tumor growth. We investigated whether inhibition of Nek2 induces tumor cell senescence and how Nek2 regulates cellular senescence in B-cell tumors.

Methods

We have generated Nek2 knockout (Nek2^-/-) mice by gene trap and CMV-Cre. These mice develop normally without obvious diseases and were crossed them with EμMyc mice. Flow cytometry and b-galactosidases (b-gal) staining were applied to determine and quantify cellular senescence. RNA sequencing, qRT-PCR, co-immunoprecipitation and western blots were employed to define genes/signaling pathways related to Nek2 cellular senescence using genetic mouse tissues and Nek2-knockout mouse embryonic fibroblast (MEF) cells. MM cell lines ARP1 and OCI-MY5 engineered with NEK2 were used to confirm senescent pathways in MM cells.

Results

Knock-out of Nek2 significantly increased tumor-free survival and overall survival of the EμMyc/Nek2^-/- mice compared to the EμMyc/Nek2^WT mice. The latter spontaneously develop diffuse large B-cell lymphoma (DLBCL) and/or Burkitt's lymphoma. Flow cytometry analyses and histochemistry showed increased b-gal -positive senescent cells derived from EμMyc/Nek2^-/- mouse spleen, lymph notes and bone marrow tissues compared to those derived from EμMyc/Nek2^WTmice. To determine whether cellular senescence played an role in extending Eμ-Myc/ NEK2^-/-mouse survival, Dasatinib (D) and Quercetin (Q), known anti-senescence drugs were used to treat Eμ-Myc/ NEK2^-/-mice. The D + Q treatment prevented the extension of Eμ-Myc/NEK2^-/-mouse survival, indicating senescence plays a key role in survival extension. Mechanistically, we performed RNA-sequencing on splenic premalignant B cells collected from EμMyc/Nek2^-/- and EμMyc/Nek2^WT mice, we found the senescence-related pathways, such as RB1 and p21, were significantly activated in the EμMyc/Nek2^-/- cells. Western blots further confirmed these findings in EμMyc/Nek2^-/- B cells and Nek2^-/- MEF cells. P53 was not increased in Nek2^-/- B cells and MEF cells. Because inhibition of Ezh2 can upregulate p21 expression and trigger the onset of senescence, we explored Ezh2 expression in mouse tissues and MEF cells. Ezh2 was downregulated in EμMyc/Nek2^-/- B cells, Nek2^-/- B cells and MEF cells. To determine whether EZH2 mediates suppression of p21 expression, two EZH2 inhibitors 3-deazaneplanocin A (DZNeP) and EPZ-6438 were used to treat NEK2-overexpressing (NEK2-OE) ARP1 and OCI-MY5 MM cell lines. We observed that NEK2-OE MM cells showed less cellular senescence induced by doxorubicin compared to control MM cells, and the EZH2 inhibitors prevented NEK2 function in antagonizing cellular senescence. Interestingly, down-regulation of p21 by NEK2-OE was also blocked by EZH2 inhibitors.

Conclusion

Our studies demonstrate that high expression of NEK2 inhibits cellular senescence via RB1 and EZH2/p21. This provides a new target for therapy to prevent NEK2-induced drug resistance in MM and other B cell tumors.