Synergistic Loss of IRF4 and Induction of IRF7 Sensitizes Primary Myeloma Cells to IMiD Killing by IFNβ in Prolonged Early G1 Arrest Induced by CDK4/CDK6 Inhibition
Authors: Huang Xiangao , Jayabalan David , Liberto Maurizio Di , Harvey Jackson D , Schinzel Anna C. , Mark Tomer M , Hussein Mohamad , Randolph Sophia , Hahn William C. , Niesvizky Ruben , Chen-Kiang Selina 
Published: October 2019

Abstract
Abstract 572
The immunomodulatory drugs (IMiD) lenalidomide (Len) and pomalidomide (Pom) are effective therapies for multiple myeloma (MM), improving both disease-free and overall survival in relapsed or refractory MM with a favorable toxicity profile. However, MM remains incurable due to the eventual development of drug resistance, and the mechanism of IMiD action is not well understood. Developing novel mechanism-based combination therapies and defining the mechanism of IMiD action are thus timely and necessary.
By inducing prolonged early G1 arrest (pG1) through inhibition of CDK4/CDK6 with a highly specific, potent and reversible inhibitor, PD 0332991, we have now developed a novel sequential combination therapy that both inhibits proliferation of MM cells and sensitizes them to IMiD killing. Our rationales are as follows: 1) cell cycle dysregulation underlies unrestrained proliferation of MM cells in relapse, as in other cancers; 2) dysregulation of CDK4 or CDK6, which drives cell cycle progression through early G1, precedes the increase in proliferation in MM progression; 3) inhibition of CDK4/CDK6 by PD 0332991 arrests the cell cycle in early G1 in all Rb-positive primary bone marrow myeloma cells (BMMM)s, ex vivo and in MM patients in a phase I/II clinical trial; 4) pG1 sensitizes MM cells to killing by diverse clinically relevant agents in pG1 and in subsequent synchronous S phase entry after the release of early G1 block.
Our replication kinetics data show that Len induces a dose-dependent late G1 arrest by 48 hours in MM cell lines, but apoptosis and reduction of viable cells is not evident until 72 hours, and appears independent of late G1 arrest. However, killing by Len or Pom is markedly accelerated and enhanced in pG1 induced by PD 0332991 for 24 hours (twice the time needed to induce G1 arrest in MM cells). Importantly, acceleration of early G1 arrest by PD 0332991 sensitizes BMMMs to killing by Len (16/20 cases) and by Pom (3/4 cases) despite protection by bone marrow stromal cells. Thus, IMiDs preferentially target MM cells arrested in early G1, in contrast to most cytotoxic agents, which primarily target tumor cells in S phase, thereby providing a strong rationale for improving IMiD therapy by prior induction of pG1.
Whole transcriptome sequencing (WTS, RNA-Seq) and q-PCR analyses of BMMMs further revealed that correlating with Len killing, genes of the interferon (IFN) signaling pathway are coordinately and prominently induced by Len, and by Len and pG1 in synergy, but not by pG1 alone. These data provide the first direct evidence for induction of IFN by IMiD and enhancement by pG1 in BMMMs, suggesting a pivotal role for IFN in mediating IMiD killing in synergy with pG1 in MM.
pG1 halts scheduled gene expression in early G1 and prevents the expression of genes programmed for other cell cycle phases, as we have demonstrated by WTS in conjunction with q-PCR and immunoblot analyses. Synergistic induction of IFN may stem from the imbalance in gene expression in pG1 and its interplay with IMiD signaling. Indeed, pG1 activates the synthesis of IRF4, an essential survival factor of MM cells, but markedly amplifies the loss of IRF4 protein induced by Len or Pom through transcriptional and posttranscriptional mechanisms. This leads to induction of IRF7, a primary and direct target of IRF4 repression, and IFNb, which is activated by IRF7. The essential roles of IRF4 and IRF7 in mediating IMiD killing and pG1 sensitization by IFNb signaling have been further confirmed by shRNA silencing in representative MM cell lines that have been characterized by WTS and shown to recapitulate pG1 sensitization of Len and Pom killing.
In summary, we have developed a novel sequential combination therapy that both inhibits proliferation and enhances IMiD killing of MM cells by induction of pG1 through selective CDK4/CDK6 inhibition. This therapy combines oral compounds with excellent toxicity profiles and acts in pG1; thus, it may serve as a maintenance therapy to both control tumor expansion and prevent self-renewal. This study presents the first WTS-validated therapeutic strategy in MM, and demonstrates, for the first time, that the IRF4-IRF7-IFNb pathway mediates IMiD killing and pG1 amplifies it. Further investigation may uncover novel molecular therapeutic targets and biomarkers for genome-based patient stratification for cell cycle-based IMiD combination therapies.

Disclosures:
Huang: Celgene, Corp: Research Funding. Off Label Use: PD 0332991 is a CDK4/CDK6 selective inhibitor Lenalidomide is an Immunomodulatory drug. Mark:Millenium Inc.: Speakers Bureau; Celgene Corp: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hussein:Celgene, Corp: Employment, Equity Ownership. Randolph:Pfizer, Inc.: Employment, Equity Ownership. Niesvizky:Onyx, Millemium, Celgene. Speakers bureau: Millenium and Celgene: Consultancy, Research Funding. Chen-Kiang:Bristol-Myers Squibb: Consultancy; Pfizer, Inc.: Research Funding.