Cooperative Supression of MEIS2 Mediates Dexamethasone Enhancement of Lenalidomide Killing in Myeloma Cells

Dexamethasone (Dex) enhances the clinical response to most therapies in multiple myeloma, the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide in particular. As a steroid, Dex may modulate the therapeutic response by acting on multiple cell types in vivo. Whether and how Dex enhances the tumor intrinsic clinical response to IMiDs is not well understood. To address this important question, we focus on the role of Dex in modulating the proximal events in IMiD signaling that lead to the killing of myeloma cells.

Cereblon (CRBN), a substrate receptor of the CRL4^CRBN E3 ligase, is required for IMiD's anti-myeloma activity. MEIS2 is a homeobox transcription factor of the mammalian myeloid ecotropic insertion site (MEIS) family, which plays a pivotal role in development and leukemogenesis. Recent crystal structure studies in conjunction with biochemical screen have identified MEIS2 as an endogenous substrate of CRBN. IMiDs apparently bind CRBN and block MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4^CRBN for degradation. This then leads to loss of IRF4, a target of IKZF1/3, necessary for myeloma survival. This model posits that MEIS2 inhibits the function of CRBN in myeloma cells. However, as all the studies of MEIS2 were performed heterologous cell lines, evidence that supports a function of MEIS2 in myeloma cells is lacking.

We now showed by RNA-seq and protein analysis that MEIS2 was absent in normal bone marrow plasma cells (BMPC), but highly expressed in freshly isolated primary bone marrow myeloma cells (BMMC)s at both RNA and protein levels. We further showed that MEIS2 was regulated by the cell cycle: reduced in G1 arrest and restored in S phase in human myeloma cell lines (HMCL)s as well as primary BMMCs. Depletion of MEIS2 markedly enhanced lenalidomide killing, and overexpression of MEI2 attenuated lenalidomide killing of HMCL MM1.S. These results provide the first evidence that MEIS2 is aberrantly expressed in primary myeloma cells and acts within myeloma cells to inhibit IMiD killing.

To investigate if Dex enhances the myeloma intrinsic response to IMiDs, we first determined that lenalidomide and pomolidomide induced late G1 arrest within 24 hours, as evidenced by the repression of cyclin A, the increases in p21 and p27 proteins, and the reduction of myeloma cells in S phase by BrdU labeling. Kinetic studies further indicate that induction of G1 arrest precedes apoptosis, detectable at 72 hours in MM1.S and CAG cells. These data suggest that IMiDs preferentially kill myeloma cells arrested in G1. Dex killed CAG and MM1.S cells marginally at 10 nM and killing was not greater at 100 nM. However, concurrent addition of Dex enhanced lenalidomide killing, more prominently in CAG cells then in MM1.S cells. Addition of Dex at these concentrations to CAG cells after 48 hours of exposure to lenalidomide leads to synergistic and greater killing in 24 hours in a dose-dependent manner. These data indicate that Dex enhances lenalidomide killing, preferentially in G1 arrest.

To investigate the mechanism by which Dex enhances lenalidomide killing, we found that Dex did not regulate MEIS2 or CRBN protein expression in CAG cells, in line with its inability to significantly kill myeloma cells. Sequential addition of lenalidomide and Dex led to a > 2-fold reduction in the MEIS2 protein, which corresponds to a > 2-fold reduction in the MEIS2/CRBN ratio and synergistic killing. Concurrent addition of Dex and lenalidomide also resulted in a 2-fold reduction of the MEIS2 protein and the MEIS2/CRBN ratio, but it required 72 hours of the presence of Dex instead of 24 hours.

In summary, our study demonstrates, for the first time, that MEIS2 is expressed in primary myeloma cells and inhibits IMiDs killing. Dex enhances IMiD killing of myeloma cells through cooperative suppression of MEIS2 at proximal IMiD signaling. These findings suggest a novel mechanism by which Dex enhances the anti-myeloma activity of IMiD, which has important implication for myeloma therapy.