Interactome of Aiolos/Ikaros in Diffuse Large B-Cell Lymphoma (DLBCL) Reveals Novel Combination of Cereblon Modulators (CELMoD) and Histone Deacetylase (HDAC) Inhibitors
Authors: Hagner Patrick R. , Chiu Hsiling , Chopra Vivek S. , Colombo Martino S. , Patel Nisha , Ortiz Maria , Loos Remco S. , Towfic Fadi , Waldman Michelle , Gandhi Anita K 
Published: February 2020

DLBCL is the most common subtype of non-Hodgkin lymphoma, constituting 30-40% of all new cases. Lenalidomide (Len) and Avadomide (Ava), small molecule cereblon modulators (CELMoD) with clinical activity in DLBCL, bind to cereblon in the CRL4CRBN E3 ligase, leading to ubiquitination and subsequent degradation of transcription factors Aiolos and Ikaros. Aiolos/Ikaros bind to the promoters of interferon stimulated genes (ISGs) and repress transcription in DLBCL cells. Degradation of these substrates by Len and Ava results in an upregulation of ISG expression leading to decreased proliferation and increased apoptosis of DLBCL cells. Ava is directly cytotoxic against ABC and GCB-DLBCL cells while Len has preferential activity in ABC-DLBCL. To date, our understanding of the COO independent activity of Ava is due to increased kinetics and greater depth of degradation of Aiolos/Ikaros compared to Len. We sought to further understand the biology of these substrates and their transcriptionally repressive activities in DLBCL.
Utilizing a comprehensive approach of RNAseq and ChIP-seq of histone modifications (active: H3K27ac, H3K4me3; repressive: H3K27me3 and H3K9me3), we profiled a panel of DLBCL cell lines (2 ABC which are sensitive in proliferation assays to both Len/Ava (TMD8 and U2932), 1 GCB cell line (WSU-DLCL2) which is sensitive only to Ava and an intrinsically resistant GCB cell line (SUDHL-4)). In TMD8 and U2932 (ABC-DLBCL), both Len and Ava treatment resulted in upregulation of ISGs, while only Ava was increased ISG mRNA levels in WSU-DLCL2 (GCB-DLBCL). Neither drug induced ISG expression in the resistant SUDHL-4 (GCB-DLBCL). We found a qualitative association between activity of the drugs and baseline histone modifications of promoters for ISGs with TMD8 and U2932 being relatively enriched for H3K27ac, H3K4me3 and depleted for H3K27me3 and H3K9me3 compared to WSU-DLCL2 and SUDHL-4. SUDHL-4 had the least enrichment for poised and the most enrichment for repressive marks at the transcriptional start sites of ISGs of the cell lines profiled. These data indicate that chromatin modifications, such as decreased active histone marks and increased repressive histone marks at the promoters of ISGs correlate with Len and Ava sensitivity. We hypothesized that Aiolos/Ikaros, known substrates of Len/Ava and repressors of ISGs, were involved in regulation of nearby chromatin landscape through cooperation with transcriptional repressor complexes.
To identify complexes associated with these proteins, we performed TMT labeled proteomics on nuclear lysates immunoprecipitated for Aiolos or Ikaros. We identified a significant enrichment in a number of complexes including the nucleosome remodeling deacetylase (NuRD) complex of which HDAC1 and HDAC2 are integral components. Utilizing a protein ligation assay, the interaction of Aiolos/Ikaros with multiple components of the NuRD complex including CHD4, HDAC1 and HDAC2 was confirmed. Across a panel of 10 cell lines 50-80% of cells were positive for a complex formation of Aiolos/Ikaros with a component of the NuRD complex and a majority of these cells were in the G1 phase of the cell cycle. Given the interaction of Aiolos/Ikaros with the NuRD complex, we hypothesized that a combination treatment of Ava with citarinostat (CC-241), a HDAC 1/2/3/6 inhibitor, would result in enhanced ISG expression and synergistic cell autonomous activity in DLBCL. The combination of Ava and CC-241 led to a significant increase in mRNA expression of ISGs such as IFIT3 by 4.2-14 fold and DDX58 by 6.8-8.2 fold compared to either single agent (IFIT3: Ava 0.5-8 fold; CC-241 1.5-1.6 fold p&lt;0.05-0.001; DDX58: Ava 1.5-3 fold; CC-241 2.2-3 fold p&lt;0.05-0.01). CC-241 demonstrated an inhibition of cell proliferation and increased apoptosis in a dose dependent and COO independent manner (1-4 mM) in a panel of DLBCL cell lines. The combination resulted in a synergistic anti-proliferative activity against 6 of 11 DLBCL cell lines profiled.
These data demonstrate that Aiolos/Ikaros regulate ISG expression in DLBCL in part through directing epigenetic modifications of their promoters. The combination of Ava with CC-241 results in a synergistic inhibition of DLBCL cell proliferation, through enhanced derepression of transcriptional activity at the promoter regions of ISGs. These data highlight the potential of combining avadomide with a HDAC inhibitor such as citarinostat in DLBCL.

Disclosures
Hagner: Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Chiu:Celgene: Employment, Equity Ownership, Patents & Royalties. Chopra:Amgen Inc: Employment; Celgene Corporation: Equity Ownership. Colombo:Celgene: Employment, Equity Ownership. Patel:Celgene: Equity Ownership. Ortiz:Celgene Corporation: Employment, Equity Ownership. Loos:Celgene: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Waldman:Celgene: Employment, Equity Ownership, Patents & Royalties. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.