Immunomodulatory Drugs Are Efficacious Against Primary Effusion Lymphoma By Targeting IKZF1, IRF4 and MYC in a CRBN-Dependent Manner and Are Synergistic with BRD4 Inhibitors
Authors: Gopalakrishnan Ramakrishnan , Matta Hittu , Tolani Bhairavi , Triche Timothy Junius , Chaudhary Preet 
Published: October 2019

Abstract
Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma localized predominantly in body cavities. Kaposi’s sarcoma-associated herpes virus is the causative agent of PEL. PEL is an incurable malignancy and has extremely poor prognosis when treated with conventional chemotherapy. PEL cells universally express interferon regulatory factor 4 (IRF4). We found that shRNA-mediated knockdown of IRF4 is toxic to PEL. Further, we observed that FDA-approved immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show selective cytotoxicity towards PEL in a panel of cell lines comprising 11 hematological malignancies. The cytotoxic effect of IMiDs towards PEL was observed within their physiologically achievable concentrations and involved the downregulation of IRF4 and MYC. Genome-wide microarray examination (illumina) of PEL cells treated with lenalidomide followed by Gene Set Enrichment Analysis (http://www.broadinstitute.org/gsea) revealed activation of interferon (IFN) signaling. Further, recombinant IFNs α, β and γ are very effective and selective against PEL in a panel of cell lines tested. However, blocking the IFN pathway using specific blocking antibodies did not block the anti-proliferative potential of IMiDs towards PEL suggesting that activation of IFN pathway is not responsible for the cytotoxicity of IMiDs against PEL. Ikaros family proteins IKZF1 and IKZF3 are transcription factors which play crucial roles in immunity and cell-fate decisions. Recently, it has been shown that IMiDs selectively degrade these transcription factors (Kronke et al., and Lu et al., Science, 2014). In PEL, IKZF1 is strongly downregulated upon treatment with IMiDs. Additionally, shRNA-mediated knockdown of IKZF1 is toxic to PEL. Cereblon (CRBN) is a direct cellular binding target of IMiDs. IMiDs have no significant effect on the expression of CRBN in PEL and shRNA-mediated knockdown of CRBN (shCRBN) is not toxic to PEL. However, all the anti-PEL effects of IMiDs were blunted in cells expressing shCRBN. CRBN is a component and substrate receptor for Culling-RING ubiquitin ligase complex (CRL) (Ito et al., Science, 2010) and CRL activity depends on NEDDylation (Soucy et al., Nature, 2009). MLN4924, small molecule NEDD8-activating enzyme inhibitor phenocopied the effects of shCRBN on the anti-PEL activities of IMiDs suggesting that CRBN and its CRL activity is essential for the anti-proliferative activity of IMiDs in PEL. MYC is a downstream target of IMiDs in PEL but the role of MYC on the activity of IMiDs is not known. While investigating the role of MYC in IMiDs activity against PEL we found that shRNA-mediated knockdown of MYC enhanced the cytotoxicity of IMiDs suggesting a potential synergism between IMiDs and MYC inhibition. BRD4 inhibitors have been shown to block MYC expression (Delmore et al., Cell, 2011; Mertz et al., PNAS, 2011). We observed that lenalidomide is synergistic with all the BRD4 inhibitors (JQ-1, IBET151 and PFI-1) tested in PEL. Combined treatment of IMiDs with JQ-1 at low doses resulted in comprehensive downregulation of MYC and significant increase in the number of cells undergoing apoptosis as observed by AnnexinV staining and PARP cleavage. Knocking down BRD4 using specific shRNA also enhanced the cytotoxicity of IMiDs towards PEL suggesting that the synergism observed between IMiDs and BRD4 inhibition may not be limited only to the BRD4 inhibitors used in our study. Furthermore, combined administration of lenalidomide and JQ-1 significantly increased the survival of PEL bearing NOD.SCID mice in an orthotopic xenograft model. These results provide the basis for clinical testing of IMiDs alone and in combination with BRD4 inhibitors for PEL.

Disclosures
Gopalakrishnan: University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties. Chaudhary:University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties.