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Supplementary Data from Interactome of Aiolos/Ikaros Reveals Combination Rationale of Cereblon Modulators with HDAC Inhibitors in DLBCL

Supplementary Table S1. Summary of avadomide plasma pharmacokinetic parameters by day and dose level Supplementary Figure S1. Representative flow cytometry analysis of Aiolos degradation in peripheral B cells (A) and T cells (B). Supplementary Figure S2. IL-2 expression correlates with Aiolos degradation in treated primary human T cells in vitro.

Purpose:Cereblon (CRBN), a substrate receptor of the E3 ubiquitin ligase complex CRL4CRBN, is the target of the small molecules lenalidomide and avadomide. Upon binding of the drugs, Aiolos and Ikaros are recruited to the E3 ligase, ubiquitylated, and subsequently degraded. In diffuse large B-cell lymphoma (DLBCL) cells, Aiolos and Ikaros are direct transcriptional repressors of interferon-stimulated genes (ISG) and degradation of these substrates results in increased ISG protein levels resulting in decreased proliferation and apoptosis. Herein, we aimed to uncover the mechanism(s) Aiolos and Ikaros use to repress ISG transcription and provide a mechanistic rationale for a combination strategy to enhance cell autonomous activities of CRBN modulators (CELMoD).Experimental Design:We conducted paired RNA sequencing with histone modification and Aiolos/Ikaros chromatin immunoprecipitation sequencing to identify genes regulated by these transcription factors and to elucidate correlations to drug sensitivity. We confirmed Aiolos/Ikaros mediated transcriptional complex formation in DLBCL patient samples including those treated with avadomide.Results:In DLBCL, the repression of ISG transcription is accomplished in part through recruitment of large transcriptional complexes such as the nucleosome remodeling and deacetylase, which modify the chromatin landscape of these promoters. A rational combination approach of avadomide with a specific histone deacetylase inhibitor leads to a significant increase in ISG transcription compared with either single agent, and synergistic antiproliferative activity in DLBCL cell lines.Conclusions:Our results provide a novel role for lineage factors Aiolos and Ikaros in DLBCL as well as further insight into the mechanism(s) of Aiolos and Ikaros–mediated transcriptional repression and unique therapeutic combination strategies.

Supplementary Figure from Interactome of Aiolos/Ikaros Reveals Combination Rationale of Cereblon Modulators with HDAC Inhibitors in DLBCL

Supplementary Table from Interactome of Aiolos/Ikaros Reveals Combination Rationale of Cereblon Modulators with HDAC Inhibitors in DLBCL

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Purpose: Cereblon (CRBN), a substrate receptor of the E3 ubiquitin ligase complex CRL4CRBN, is the target of the small molecules lenalidomide and avadomide. Upon binding of the drugs, Aiolos and Ikaros are recruited to the E3 ligase, ubiquitylated, and subsequently degraded. In diffuse large B-cell lymphoma (DLBCL) cells, Aiolos and Ikaros are direct transcriptional repressors of interferon-stimulated genes (ISG) and degradation of these substrates results in increased ISG protein levels resulting in decreased proliferation and apoptosis. Herein, we aimed to uncover the mechanism(s) Aiolos and Ikaros use to repress ISG transcription and provide a mechanistic rationale for a combination strategy to enhance cell autonomous activities of CRBN modulators (CELMoD). Experimental Design: We conducted paired RNA sequencing with histone modification and Aiolos/Ikaros chromatin immunoprecipitation sequencing to identify genes regulated by these transcription factors and to elucidate correlations to drug sensitivity. We confirmed Aiolos/Ikaros mediated transcriptional complex formation in DLBCL patient samples including those treated with avadomide. Results: In DLBCL, the repression of ISG transcription is accomplished in part through recruitment of large transcriptional complexes such as the nucleosome remodeling and deacetylase, which modify the chromatin landscape of these promoters. A rational combination approach of avadomide with a specific histone deacetylase inhibitor leads to a significant increase in ISG transcription compared with either single agent, and synergistic antiproliferative activity in DLBCL cell lines. Conclusions: Our results provide a novel role for lineage factors Aiolos and Ikaros in DLBCL as well as further insight into the mechanism(s) of Aiolos and Ikaros–mediated transcriptional repression and unique therapeutic combination strategies.

The immune modulatory drug lenalidomide has shown promising anti-tumor activity in a clinical trial of patients with advanced hepatocellular carcinoma (HCC). The present study explored whether lenalidomide can enhance the anti-tumor activity of sorafenib, the standard molecular targeted therapy for HCC.The anti-tumor efficacy of single-agent or combination treatment was measured by change in tumor volume and animal survival using an orthotopic liver cancer model. Distribution of T-cell subpopulations in tumor-infiltrating lymphocytes (TILs) and splenocytes derived from tumor-implanted mice was measured by flow cytometry. Depletion of relevant T-cell subpopulations or cytokines was done by co-administration of relevant antibodies with study drug treatment. Tumor cell apoptosis and tumor angiogenesis were measured by transferase deoxytidyl uridine end labeling assay and immunohistochemical study, respectively.Combination of sorafenib and lenalidomide produced significant synergistic anti-tumor efficacy in terms of tumor growth delay and animal survival. This synergistic effect was associated with a significant increase in interferon-γ expressing CD8(+) lymphocytes in TILs and a significantly higher number of granzyme- or perforin-expressing CD8(+) T cells, compared with vehicle- or single-agent treatment groups. Combination treatment significantly increased apoptotic tumor cells and vascular normalization in tumor tissue. The synergistic anti-tumor effect was abolished after CD8 depletion.Lenalidomide can enhance the anti-tumor effects of sorafenib in HCC through its immune modulatory effects, and CD8(+) TILs play an important role in the anti-tumor synergism.

Cereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4(CRBN) . T cell co-stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4(CRBN) substrates responsible for T cell co-stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4(CRBN) is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4(CRBN) with enhanced ubiquitination leading to cereblon-dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression. The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

Background CRBN, a target of thalidomide and IMiDs® immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM), is a component of the E3 ubiquitin cullin 4 ring ligase (CRL4) complex that also includes DDB1, Roc1, and Cul4. Two CRBN mutations have been reported in multiple myeloma (MM) patients: truncating mutation (Q99) and point mutation (R283K). One copy of the CRBN gene was shown to be deleted in the MM1S and MM1S.R cell lines. No DDB1 mutation has been described previously. Results We investigated the incidence of CRBN and DDB1 mutations by next-generation sequencing in 20 MM cell lines and MM subjects. Of 90 MM patients, 24 were newly diagnosed and 66 were relapsed and refractory of which 36 patients were LEN resistant. Out of the cell lines tested, 1 heterozygous CRBN mutation (D249Y) was found in the LEN-resistant ANBL6R cells, which is located in the putative DDB1 binding domain, and 2 single silent mutations were identified in the KMS-12-BM (rs17027638) and OPM-2 cells. One DDB1 heterozygous mutation (E303D) was identified in ANBL6 cells. In the cohort of patients assessed, no CRBN mutation was detected; however, 5 single nucleotide variations (SNV) were identified. Three of the 5 SNVs were at position 735 (Y245Y) and 1 each at position 219 (H73H) and 939 (C313C), respectively. The first 2 SNVs (rs17027638 and rs1045309) are described but not the last. We found a single SNV (P51P; rs2230356) in DDB1 gene the patient samples. Conclusion Mutations within the coding sequences of CRBN and DDB1 are rare in MM patients and cell lines. Most intrinsically LEN-resistant cells and cell lines made resistant to LEN or POM do not have CRBN or DDB1 mutations, suggesting the potential role of other sources, such as genetic or epigenetic pathways in developing resistance to IMiD drug–based therapy. Disclosures: Thakurta: Celgene: Employment, Equity Ownership. Gandhi:Celgene: Employment, Equity Ownership. Waldman:Celgene: Employment, Equity Ownership. Bjorklund:Celgene: Employment, Equity Ownership. Lentzsch:Celgene: Research Funding. Schey:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; NAPP: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees. Madan:Covance Genomics Lab: Employment. Ning:Celgene: Employment, Equity Ownership. Mendy:Celgene: Employment, Equity Ownership. Lopez-Girona:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Avet-Loiseau:Celgene: Research Funding. Chopra:Celgene: Employment, Equity Ownership.