CDK4/6 Inhibition Reverses MEIS2 Inhibition of CRL4CRBN By Both Disrupting MEIS2-CRBN Association and Opposing Regulation of MEIS2 and CRBN Synthesis That Enhances IMiD Therapy in Multiple Myeloma

The immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) are standard of care for multiple myeloma (MM). Cereblon (CRBN), a component of the CRL4^CRBN E3 ligase, is required for IMiD’s anti-myeloma activity. Binding of IMiD to CRBN promotes the recruitment of transcription factors IKZF1 and IKZF3 to CRL4^CRBN for ubiquitination and degradation, leading to loss of IRF4, an IKZF1/3 target essential for survival of myeloma cells. MEIS2 was identified by protein array as an endogenous substrate that competes with IMiD for binding to the same domain on CRBN. However, whether MEIS2 functions as an inhibitor of CRBN and its relevance in IMiD therapy has not been investigated.

To address this, first we discovered that high MEIS2 RNA expression in myeloma cells before therapy was tightly associated with poorer overall survival (OS) (p-value = 0.0001) in the CoMMpass Bor-Lenalidomide-Dex clinical trial (N=272). Conversely, high CRBN RNA expression correlated with improved OS (p-value =0.0077). Next, we demonstrated that MEIS2 protein was differentially expressed in primary MM cells but absent in normal plasma cells (PC)s, B cells and lymphoma cells. In contrast, the expression of CRBN protein varied in PCs, and was significantly greater in treatment-naïve MM cells than in IMiD refractory MM cells. This resulted in a ~3-fold increase in the MEIS2 to CRBN ratio in IMiD refractory patients, implicating a role for MEIS2 in inhibiting CRBN and promoting clinical resistance to IMiD therapy.

In addition, high expression of core genes that promote cell cycle progression through early G1 (CDK4 and CDK6) and late G1 (E2F1, CDK2, CCNA2) also tightly correlated with poorer OS. This is in line with dysregulation of G1-S progression in relapsed-refractory MM we previously reported (Ely et al, 2005) and suggests that inhibition of CDK4/6 may reprogram MEIS2 for enhanced IMiD killing. Indeed, induction of prolonged early G1 arrest by CDK4/6 inhibition with palbociclib 1) enhanced Len and Pom killing in model MM cell lines; 2) reduced the MEIS2 protein by transcriptional repression and increased the CRBN protein by post-transcriptional regulation in cooperation with Len; 3) this led a > 5 fold reduction in the MEIS2/CRBN protein ratio, exacerbated loss of IKZF1/3 and IRF4, and enhanced apoptosis (caspase- cleavage). Importantly. these findings were validated in freshly isolated primary bone marrow myeloma cells (BMMCs) (n=27) in a stromal co-culture, and the IMiD response ex vivo mimicked the clinical response to the first post-biopsy Len or Pom therapy in patients independent of prior therapy. Thus, induction of early G1 arrest by CDK4/6 inhibition reprograms primary MM cells for IMiD killing by reducing the MEIS2 to CRBN protein ratio through opposing regulation of MEIS2 and CRBN.

Loss and gain of function studies demonstrated that MEIS2 inhibits IMiD killing by attenuating the induction of interferon downstream of the IKZF1/3-IRF4 axis, more prominently in early G1 arrest induced by CDK4/6 inhibition in MM cell lines as well as primary BMMCs. Further biochemical analyses revealed that CDK4/6 inhibition rapidly accelerated the displacement of MEIS2 from CRBN within one hour of Len treatment, concurrent with enhanced ubiquitination and degradation of IKZF1 and IKZF3 in both MM cell lines and primary BMMCs.

In summary, we have demonstrated for the first time that MEIS2, an endogenous CRBN substrate, is a negative regulator of IMiD therapy in MM: 1) High expression of MEIS2 is a prognostic indicator for poor OS in IMiD therapy, in opposition to improved OS with high CRBN expression; 2) MEIS2 inhibits IMiD killing of MM cells by attenuating the induction of interferon; 3) inhibition of CDK4/6 enhances IMiD killing by rapidly displacing MEIS2 from CRBN through enhanced Len recruitment of IKZF1/3 and by time-dependent opposing regulation of MEIS2 and CRBN synthesis. Selective expression of MEIS2 in MM cells but not in normal PCs, B cells or lymphoma cells further suggest MEIS2 as a druggable target in MM. In this study, we provide the first evidence that CDK4/6 inhibition reverses MEIS2 inhibition of CRBN that has a potential to enhance the clinical response to IMiD. The prospect that through cell cycle control CDK4/6 inhibition may also enhance the anti-MM activity of CELMoDs, the newly developed IMiDs such as iberdomide, in relapsed-refractory setting is exciting and warrants future investigation.