Abstract 4031: CDK4/6 inhibition amplifies IMiD killing of myeloma cells by accelerating CRL4CRBN-dependent degradation of IKZF1 and IKZF3 in opposition to MEIS2
Authors: Huang Xiangao , Jayabalan David , Di Liberto Maurizio , Mark Tomer M. , Chen Zhengming , Rossi Adriana , Schinzel Anna C. , Hahn William C. , Hussein Mohamad A. , Niesvizky Rubin , Chen-Kiang Selina 
Published: August 2015

Abstract
The immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) are effective therapies for hematologic malignancies, especially multiple myeloma (MM). Cereblon (CRBN) is required for IMiD's anti-myeloma activity. Emerging evidence has established that CRBN is a substrate receptor of the CUL4-ROC1-DDB1-CRBN (CRL4CRBN) E3 ligase complex and that the homeobox transcription factor MEIS2 is an endogenous substrate of CRL4CRBN. IMiDs block MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN for ubiquination-proteasome degradation. IKZF1 and IKZF3 appear to be required for MM survival, in part by transcriptional regulation of IRF4.
These findings, obtained in heterologous systems and MM cell lines, suggest that modulation of the CRL4CRBN E3 ligase is a critical early event in IMiD killing of MM cells. We investigated its physiologic relevance in primary bone marrow myeloma cells (BMMCs) in co-culture with HS-5 bone marrow stromal cells. We showed that IMiDs preferentially kill BMMC in G1 arrest and that induction of prolonged early G1 arrest (pG1) by selective inhibition of CDK4/CDK6 with palbociclib (PD 0332991) markedly enhanced Len/Pom killing of BMMCs (14/21), unless the BMMCs were refractory or extremely sensitive to Len (n = 7). Importantly, the IMiD sensitivity in BMMCs ex vivo correlates with subsequent clinical responses to IMiD-based therapies in vivo in 7/7 cases characterized. This novel finding suggests that although IMiDs target multiple cell types, the intrinsic IMiD sensitivity is a major determinant for the clinical response to IMiDs in MM.
Whole transcriptome sequencing and gene set enrichment analysis further revealed that Len induces the interferon response signature genes in BMMCs, and this is enhanced by palbocicib. While CRBN is ubiquitously expressed, IKZF3 and IRF4 (mRNA and protein) and the IKZF1 protein were barely detectable in normal bone marrow plasma cells and prominently increased in BMMCs. Primary myeloma cells thus appear to be addicted to IKZF1/IKZF3-IRF4 signaling for survival, and IMIDs induce CRL4CRBN-mediated degradation of IKZF1 and IKZF3 to antagonize this survival pathway. Accordingly, palbociclib enhancement of Len killing was associated with rapid acceleration of IKZF1/IKZF3 degradation, within one hour of Len treatment in BMMCs. Palbociclib acts in opposition of MEIS2, because MEIS2 knockdown mimicked palbociclib sensitization and MEIS2 reconstitution antagonized palbociclib sensitization.
In summary, we have demonstrated that induction of pG1 by CDK4/6 inhibition sensitizes primary myeloma cells to IMiD killing through rapid acceleration of CRL4CRBN -dependent degradation of IKZF1 and IKZF3 in opposition to MEIS2. These findings are being validated in a clinical trial targeting CDK4/6 with palbociclib in combination with Len in MM.
Note: This abstract was not presented at the meeting.
Citation Format: Xiangao Huang, David Jayabalan, Maurizio Di Liberto, Tomer M. Mark, Zhengming Chen, Adriana Rossi, Anna C. Schinzel, William C. Hahn, Mohamad A. Hussein, Rubin Niesvizky, Selina Chen-Kiang. CDK4/6 inhibition amplifies IMiD killing of myeloma cells by accelerating CRL4CRBN-dependent degradation of IKZF1 and IKZF3 in opposition to MEIS2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4031. doi:10.1158/1538-7445.AM2015-4031