Cooperative Repression of IRF4 and Induction of IRF7 By CDK4/6 Inhibition Sensitizes Mantle Cell Lymphoma Cells to IMiD-Mediated Apoptosis Independent of p16 Deletion or c-Myc Overexpression
Authors: Humos Basel A , Di Liberto Maurizio , Huang Xiangao , Elemento Olivier , Chen-Kiang Selina 
Published: October 2019

Abstract
Lenalidomide (Len) and pomalidomide (Pom) are standard of care therapies for multiple myeloma (MM) and also exhibit clinical efficacy in some lymphomas such as diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). As single agents, each rarely achieves complete remission despite a high overall response rate and durability; however, they potentiate the clinical response to diverse partners including Rituximab in MCL. As immunomodulatory drugs (IMiDs), Len and Pom target multiple cell types in vivo and have been shown to promote the expansion of NK cells in MCL. Len in combination with BTK inhibitors apparently leads to synthetic lethality in the activated B cell-like DLBCL cell lines, but the mechanism that mediates IMiD sensitivity in MCL cells remains largely unknown.
We found that Len markedly enhanced ACP196 (second generation BTK inhibitor) killing of the CCMCL1 MCL cell line, despite c-Myc overexpression and deletion of p16INK4a, but not the JEKO-1 cells, which harbor TP53 deletion. Therefore Len appears to enhance killing by BTK inhibition but does not confer sensitivity to resistant MCL cells. Next, we investigated how inhibition of CDK4 might sensitize MCL cells to Len or Pom killing, given that dysregulation of cyclin D1 and CDK4 (CDK6 is silenced in MCL) underlies uncontrolled proliferation and disease progression, and inhibition of CDK4 is clinically efficacious in MCL. Indeed, priming of CCMCL1 cells by prior induction of G1 arrest with the selective CDK4/6 inhibitor palbociclib markedly sensitized them to Len-induced apoptosis, but only marginally so in JEKO-1 cells. Kinetic studies revealed that Len reduced the IRF4 protein markedly in CCMCL1 cells arrested in G1 by CDK4 inhibition, but marginally so in cycling CCMCL1 cells and not at all in Len-resistant JEKO-1 or Maver-1 cells. Reduction of IRF4 protein by Len in prolonged G1 arrest was concomitant with induction of IRF7 protein and tightly associated with apoptosis, as evidenced by PARP and caspase-3 cleavage. Moreover, inhibition of CDK4 conferred sensitivity to Pom in the Len-resistant JEKO-1 cells, and it was concurrent with reduction of IRF4 and induction of IRF7.
The triple combination of Len, Ibrutinib, and Rituximab in MCL has been shown to be clinically active in relapsed MCL. To explore the underpinning, we found by RNA-seq that CD20 mRNA expression was downregulated in normal primary human peripheral B cells when they were activated into the cell cycle and in primary MCL cells, suggesting an inverse correlation between cell cycling and CD20 mRNA expression. However, surface CD20 expression was not collinear with CD20 mRNA expression and there was no evidence for upregulation of CD20 by BTK inhibition. On this basis, upregulation of the rituximab target CD20 is unlikely to be the primary mechanism for improved clinical response to this triple combination in MCL.
In summary, our data suggest cooperative repression of IRF4 and induction of IRF7 as a central mechanism that underlies enhancement of Len and Pom killing by inhibition of BTK or CDK4, independent of deletion of the physiologic CDK4/6 inhibitor p16INK4a or overexpression of c-Myc. Furthermore, it appears that induction of prolonged early G1 arrest by Palbociclib confers Pom sensitivity independent of p53, implicating a potential to combine Palbociclilb with Pom for treatment of MCL with p53 loss or mutation.

Disclosures
No relevant conflicts of interest to declare.