Cereblon E3 Ligase Modulator Research Articles

All-oral triplet iberdomide, ixazomib, and dexamethasone in elderly patients with multiple myeloma at first relapse: Results of the IFM phase 2 study I2D.

7507 Background: The triplet combination daratumumab, lenalidomide and dexamethasone (DRd) and bortezomib, lenalidomide and dexamethasone (VRd) are to date the standard of care for patients with transplant ineligible (TI) newly diagnosed multiple myeloma (MM). Most TI patients therefore present with MM refractory to lenalidomide and daratumumab at first relapse and represent a difficult-to-treat population. Iberdomide is a novel oral cereblon E3 ligase modulator (CELMoD) that demonstrated promising activity in MM patients refractory to lenalidomide/pomalidomide. Here, we report efficacy and safety results of the oral triplet iberdomide, ixazomib and dexamethasone in elderly patients with MM at first relapse. Methods: The Intergroupe Francophone du Myélome (IFM) prospective, multicenter, phase 2 study I2D enrolled MM patients aged over 70 years at first relapse (NCT04998786).Patients received oral iberdomide (1.6 mg on day 1 to 21), ixazomib (3 mg on day 1,8,15) and dexamethasone (20 mg on day 1,8,15,22 on cycle 1-2 and 10 mg on day 1,8,15,22 on cycle 3-6) (28-day cycle) until disease progression or unacceptable toxicity. The primary endpoint was very good partial response (VGPR) rate. Results: Seventy patients were included from Dec 2021 to May 2023 in 19 IFM centers. Median age was 76. The International Myeloma Working Group (IMWG) frailty score was >2 in 35 (50%) patients. In evaluable patients (54/70), cytogenetic analysis revealed del(17p) in 10 patients (18.5%) and t(4;14) in 8 (15%) patients. Based on inclusion criteria, all patients received 1 prior line of treatment, including lenalidomide in 87% (refractory, 74%) and daratumumab in 40% (refractory, 37%) of patients. Median time from MM diagnosis to study enrolment was 28 months. At data cut-off, 36 (51%) patients discontinued the study due to disease progression (n=30), adverse event (n=4) or death (n=2). After a median follow-up of 12 months, the overall response rate was 64%, including 33% VGPR. The median progression-free survival (PFS) was 13 months and the 12-month overall survival (OS) was 85% (77-95% 95% CI). In patients with MM refractory to both lenalidomide and daratumumab (n=26), the median PFS was 10 months. Overall, the triplet I2D was well tolerated. Most common non-hematologic adverse events (AE) were infection (30% of patients), peripheral neuropathy (20%), diarrhea (19%), and were mostly grade 1 or 2. Most common grade 3-4 treatment related AEs (>5% of patients) were neutropenia (46%), thrombocytopenia (9%) and infection (8%). Four patients discontinued treatment due to a severe AE (cytopenia (n=3), skin rash (n=1). Conclusions: The oral tripletiberdomide, ixazomib and dexamethasone demonstrated a favorable efficacy / safety profile in elderly MM patients at first relapse, including in patients with lenalidomide and daratumumab refractory disease. Clinical trial information: NCT04998786 .

Iberdomide increases innate and adaptive immune cell subsets in the bone marrow of patients with relapsed/refractory multiple myeloma

Iberdomide is a potent cereblon E3 ligase modulator (CELMoD agent) with promising efficacy and safety as a monotherapy or in combination with other therapies in patients with relapsed/refractory multiple myeloma (RRMM). Using a custom mass cytometry panel designed for large-scale immunophenotyping of the bone marrow tumor microenvironment (TME), we demonstrate significant increases of effector T and natural killer (NK) cells in a cohort of 93 patients with multiple myeloma (MM) treated with iberdomide, correlating findings to disease characteristics, prior therapy, and a peripheral blood immune phenotype. Notably, changes are dose dependent, associated with objective response, and independent of prior refractoriness to MM therapies. This suggests that iberdomide broadly induces innate and adaptive immune activation in the TME, contributing to its antitumor efficacy. Our approach establishes a strategy to study treatment-induced changes in the TME of patients with MM and, more broadly, patients with cancer and establishes rational combination strategies for iberdomide with immune-enhancing therapies to treat MM.

Ex vivo expansion and activation of Vγ9Vδ2 T cells by CELMoDs in combination with zoledronic acid.

As multiple myeloma (MM) progresses, immune effector cells decrease in number and function and become exhausted. This remains an insurmountable clinical issue that must be addressed by development of novel modalities to revitalize anti-MM immunity. Human Vγ9Vδ2 T (Vδ2+ γδ T) cells serve as the first line of defense against pathogens as well as tumors and can be expanded ex vivo from peripheral blood mononuclear cells (PBMCs) upon treatment with amino-bisphosphonates in combination with IL-2. Here, we demonstrated that next-generation immunomodulators called cereblon E3 ligase modulators (CELMoDs), as well as lenalidomide and pomalidomide, expanded Th1-like Vδ2+ γδ T cells from PBMCs in the presence of zoledronic acid (ZA). However, the expansion of Th1-like Vδ2+ γδ T cells by these immunomodulatory drugs was abolished under IL-2 blockade, although IL-2 production was induced in PBMCs. BTN3A1 triggers phosphoantigen presentation to γδ T-cell receptors and is required for γδ T-cell expansion and activation. ZA but not these immunomodulatory drugs upregulated BTN3A1 in monocytes. These results suggest that immunomodulatory drugs and ZA have cooperative roles in expansion of Th1-like Vδ2+ γδ T cells, and provide the important knowledge for clinical application of human Vδ2+ γδ T cells as effector cells.

Abstract 3305: Golcadomide (CC-99282) is a novel CELMoD® agent with antiproliferative activity and combinatorial potential in disease models of chronic lymphocytic leukemia

Abstract Chronic Lymphocytic Leukemia (CLL) is an adult leukemia characterized by the accumulation of incompetent B lymphocytes expressing CD19, CD20, CD23, and CD5. Current therapies include chemotherapy and combination with targeted therapies such as ibrutinib (Bruton’s tyrosine kinase inhibitor), venetoclax (BCL2 inhibitor), and obinutuzumab (anti-CD20). The tumor microenvironment promotes CLL development and drug resistance. Golcadomide (GOLCA, CC-99282) is an oral cereblon E3 ligase modulator (CELMoD®) agent with immunomodulatory and tumor cell-autonomous activities under clinical investigation for R/R non-Hodgkin lymphomas and CLL/SLL. Here we investigated the effect of GOLCA on inhibiting proliferation and inducing apoptosis of CLL preclinical models by degrading Ikaros and Aiolos. We also studied its combination with other anti-CLL agents. A panel of 10 CLL cell lines and 15 primary CLL patient samples were used to assess efficacy of GOLCA alone or in combination with ibrutinib (Ibru), venetoclax (Ven), and obinutuzumab (Obi). Primary CLL cells were grown ex vivo in a co-culture system with CD40L expressing fibroblasts designed to mimic the CLL lymph node microenvironment. Substrate degradation, viability, cell cycle and immunophenotyping were assessed by flow cytometry. GOLCA showed potent in vitro antiproliferative activity on 6 out of 10 CLL cell lines, including those with high-risk features, with IC50 of 1-20 nM. It also inhibited CLL stimulated cell proliferation and induced apoptosis in all evaluated patient samples in the co-culture system, with low to sub-nanomolar IC50, independent of IGHV mutation status and other chromosomal characteristics. Cell-cycle analysis confirmed inhibition of proliferation in the 5 patient samples evaluated after 3 days of treatment with GOLCA, with a dose-dependent decrease in the fraction of cells in the S phase. GOLCA also demonstrated degradation of the proximal substrates Ikaros and Aiolos in tumor and T cells from CLL patient samples. In combination with Obi, Ven, and Ibru, GOLCA led to synergistic or additive tumor cell toxicity in most patient samples evaluated, suggesting potential clinical benefit of these targeted agent combinations. Additive tumor toxicity was observed in two out of four patient samples when GOLCA was combined with Ibru. We have previously demonstrated that GOLCA induces immune activation in T cells from healthy donor PBMCs. In co-cultures of CLL PBMCs and CD40L-expressing fibroblasts, GOLCA increased T cell numbers and activation characteristics. Ven inhibited this immune activation, while Obi or Ibru maintained the immune activation. Collectively, these findings suggest that GOLCA, alone or in combination with targeted agents, may produce clinical benefit in CLL patients. Citation Format: Antonia Lopez-Girona, Maria Dolores Jimenez-Nunez, Diego Sobradillo, Soraya Carrancio, Preethi Janardhanan, Gauri Deb, Lynda Groocock, Daniel Pierce, Neil Bence, Mark Rolfe. Golcadomide (CC-99282) is a novel CELMoD® agent with antiproliferative activity and combinatorial potential in disease models of chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3305.

Mezigdomide-A Novel Cereblon E3 Ligase Modulator under Investigation in Relapsed/Refractory Multiple Myeloma.

Mezigomide is an oral cereblon E3 ligase modulator (CELMoD) that is under clinical investigation in patients with relapsed/refractory (RR) multiple myeloma (MM). Like other CELMoD compounds, mezigdomide acts by altering the conformation of cereblon within the cullin 4A ring ligase-cereblon (CRL4CRBN) E3 ubiquitin ligase complex, thereby recruiting novel protein substrates for selective proteasomal degradation. These include two critical lymphoid transcription factors, Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), also known as Ikaros and Aiolos, which have important roles in the development and differentiation of hematopoietic cells, in MM pathobiology, and in suppressing the expression of interferon-stimulating genes and T-cell stimulation. Among the CELMoDs, mezigdomide has the greatest cereblon-binding potency, plus the greatest potency for the degradation of Ikaros and Aiolos and subsequent downstream antimyeloma effects. Preclinical studies of mezigdomide have demonstrated its anti-proliferative and apoptotic effects in MM, along with its immune-stimulatory effects and its synergistic activity with other antimyeloma agents, including in lenalidomide-/pomalidomide-resistant MM cell lines and mouse xenograft models. Early-phase clinical trial data indicate notable activity in heavily pretreated patients with RRMM, including those with triple-class-refractory disease, together with a tolerable and manageable safety profile. This review summarizes current preclinical and clinical findings with mezigdomide and its potential future roles in the treatment of MM.

Management of Adverse Events Associated with Pomalidomide-Based Combinations in Patients with Relapsed/Refractory Multiple Myeloma.

Multi-agent regimens incorporating immunomodulatory (IMiD®) agents such as thalidomide, lenalidomide, and pomalidomide have become the preferred standard of care for the treatment of patients with multiple myeloma (MM), resulting in improved survival outcomes. Currently, there are three IMiD agents approved for the treatment of MM: thalidomide, lenalidomide, and pomalidomide. Lenalidomide is commonly used to treat patients with newly diagnosed MM and as maintenance therapy following stem cell transplant or after disease relapse. Pomalidomide, the focus of this review, is approved in patients with relapsed/refractory MM (RRMM). Despite survival benefits, IMiD agents each have different safety profiles requiring consideration both prior to starting therapy and during treatment. Adverse event (AE) management is essential, not only to ensure treatment adherence and thus ensure optimal efficacy but also to maintain patient quality of life. Here, we discuss AEs associated with pomalidomide and present five clinically relevant hypothetical case studies in patients with RRMM to provide scenario-driven guidance regarding treatment selection and AE prevention and management in the clinical setting. Lastly, as new treatment approaches continue to be explored in MM, we also discuss novel cereblon E3 ligase modulator (CELMoD™) agents including iberdomide (CC-220) and mezigdomide (CC-92480).

Investigation of the Synergistic Combination of a Novel Celmod (CC-99282) and BET Inhibitors in Preclinical DLBCL

Background: Golcadomide (GOLCA; CC-99282) is a novel cereblon E3 ligase modulator (CELMoD), which binds to cereblon (CRBN), a substrate receptor of CRL4 CRBN E3 ubiquitin ligase, and induces proteasome-dependent degradation of Ikaros and Aiolos, transcription factors essential for B cell malignancies. GOLCA was shown to exhibit promising cell of origin independent activity in pre-clinical DLBCL models and is currently being investigated in early clinical trials (NCT03930953) for relapsed/refractory non-Hodgkin lymphomas (R/R NHL). Ikaros and Aiolos have previously been shown to have occupancy on the promoter of the proto-oncogene MYC, which is subsequently downregulated by CELMoDs in DLBCL models. Given MYC is a well-studied downstream target of BET inhibitors and a driver of lymphoma disease, we investigated the mechanistic synergy of anti-proliferative and pro-apoptotic effects of GOLCA in combination with BMS-986158, a potent BET inhibitor. Results: Dose titration and analysis of the anti-proliferative effects of DLBCL cell lines (SU-DHL-4, WSU-DLCL2 and CARNAVAL) treated with either GOLCA or BMS-986158 suggests significant single agent activity of either agent. Bliss synergy score was determined and strong synergism of the two drugs was observed on day 5, with maximal bliss score (14.3-29.8) centered at low doses (1.25 and 2.5 nM) of BMS-986158 and sub-lethal doses of GOLCA (5-50 nM). Similar synergistic effects were found when combining GOLCA with JQ1, another tool compound BET inhibitor, confirming BET inhibition potentiates the anti-DLBCL effects of the CELMoD agent. Incucyte-based live cell image confirmed the enhanced anti-proliferative capacity of the combination compared to single agents. EdU (5-ethynyl-2′-deoxyuridine)-based cell cycle analysis revealed an increased proportion of G1-phase cells in combination treatment (67%) compared to GOLCA (38%), BMS-986158 (43%) and DMSO (35.4%) in the GOLCA resistant SU-DHL-4 cells. To identify key pathways responsible for the observed synergistic combination effect, we performed transcriptomic analyses of SU-DHL-4 and WSU-DLCL2 cells treated with GOLCA/BMS-986158 for 72 hrs. Compared to either single agents, the combination treatment downregulated significantly more genes (~1000 and 500 genes in SU-DHL-4 and WSU-DLCL2, respectively), which were enriched with cell cycle pathways. Gene set enrichment analysis (GSEA) showed the top downregulated gene sets in combination treatment were E2F targets such as CHEK1 and G2M checkpoint molecules including AURKA and CDC45. Given the critical role of CDKN1A (gene encodes p21) in cell cycle arrest, we examined its expression by real-time PCR. We found treatment of GOLCA induced CDKN1A mRNA expression (16.4 and 25.1-fold in SU-DHL-4 and WSU-DLCL2, respectively); remarkably, this induction was further enhanced by the combination with BMS-986158 (40.1 and 128.5-fold in SU-DHL-4 and WSU-DLCL2, respectively). Western blot confirmed the synergistic induction of p21 in combination compared to single agents, with a concomitant reduction of phosphorylated Rb, E2F target genes (Chk1, SKP2 and E2F1) and MYC. Since the main targets of GOLCA and BMS-986158 are transcription factors (Ikaros and Aiolos) and transcriptional regulator (Brd4), respectively, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) with antibodies targeting these three proteins along with various histone marks including H3K27ac and H3K4me3. Interestingly, the chromatin occupancies of Ikaros/Aiolos/Brd4 were highly overlapping in genes that are upregulated ( CDKN1A) and downregulated (e.g. CHEK1 and MYC) by the combination treatment. Further characterization of the interplay between Ikaros/Aiolos/Brd4 and their loci-specific interactomes may help elucidate the synergism at epigenetic levels. Conclusions: BET inhibition potentiates anti-proliferative effects of GOLCA in DLBCL cells through synergistic induction of p21 and inhibition of E2F signaling. The synergetic effects of BMS-986158 and GOLCA suggest potential clinical benefits of combining lower doses of single agents to achieve better efficacy with reduced risk of adverse effects.

Pharmacodynamic Biomarkers and CtDNA Support the Mechanism of Action and Clinical Efficacy of Golcadomide (CC-99282) Combined with R-CHOP in Previously Untreated Aggressive B-Cell Lymphoma

Introduction Golcadomide (GOLCA, CC-99282) is an orally available cereblon E3 ligase modulator (CELMoD ®) that potently degrades target proteins Ikaros and Aiolos, enhancing antiproliferative, apoptotic, and immunomodulatory activity and preferentially distributing to lymphoid organs. In a phase 1 study, GOLCA monotherapy previously demonstrated a manageable safety profile with promising clinical activity in relapsed/refractory non-Hodgkin lymphoma (Michot Blood 2021). In the ongoing CC-220-DLBCL-001 study (NCT04884035), GOLCA dose finding is underway in patients with previously untreated aggressive B-cell lymphoma (BCL), in combination with R-CHOP in 21-day (D) cycles (C), at a variety of GOLCA dose levels (DLs) and schedules: DL-1 = 0.2 mg D1-7, DL1 = 0.4 mg D1-7, and DL2 = 0.4 mg D1-10. Here, we report Ikaros degradation, immune modulation, circulating tumor DNA (ctDNA) assessment, and baseline tumor genomics. Methods Peripheral blood was collected to analyze Ikaros and Aiolos levels in T cells by flow cytometry (C1D1, C1D7, and C2D1). Immune phenotyping of T and natural killer (NK) cells was performed by flow cytometry (C1D1, C1D15, C2D1, and C3D1). ctDNA levels were analyzed by the PhasED-Seq assay (timepoints C1D1, C2D1, C3D1, and end of treatment [EOT]; Kurtz Nat Biotechnol 2021); somatic variants were filtered by performing PhasED-seq on buffy coats. Pretreatment biopsies were analyzed by whole genome sequencing and whole transcriptome sequencing. DL1 (0.4 mg D1-7) and DL2 (0.4 mg D1-10) data were pooled. Results Ikaros levels in T cells at 5 hours after the first dose had a 1.1% median increase with 0.2 mg and 44% median decrease with 0.4 mg; by C1D7 pretreatment, Ikaros substantially decreased with both DLs (median: 0.2 mg, 87% decrease; 0.4 mg, 83% decrease). Ikaros differences between 0.2 mg and 0.4 mg were not substantial. By C2D1, Ikaros levels recovered to baseline. Based on preclinical benchmarks, steady-state Ikaros degradation in T cells suggested that direct tumor cell killing by GOLCA was achieved. Changes in T and NK cell phenotypes in peripheral blood included activation, increased proliferation, increased T effector memory and regulatory cell levels, and decrease in naive T cells. Changes were seen by C1D15 and maintained at C2D1 and C3D1. Overall, immune changes between 0.2 mg and 0.4 mg dose levels were not substantially different. Phased variant data from ctDNA and buffy coats were evaluable for 34 patients (0.2 mg, n =15; 0.4 mg, n = 19). Baseline ctDNA levels were similar between the 0.2 mg and 0.4 mg cohorts and, consistent with prior studies, correlated with International Prognostic Index and cell of origin. Previously reported landmarks such as early molecular response (EMR; ≥ 2 log decrease) at C2D1 and major molecular response (MMR; ≥ 2.5 log decrease) at C3D1 were assessed (Kurtz J Clin Oncol 2018) . A high fraction of patients designated high risk by pretreatment ctDNA (≥ 2.5 log haploid genome equivalents [HGE]/mL; Kurtz J Clin Oncol 2018) were clinical responders. Patients who achieved minimal residual disease (MRD) negativity on treatment generally remained MRD negative at subsequent timepoints. At C2D1, the EMR and MRD negativity rates for 0.2 mg were 73% (11/15) and 20% (3/15), respectively; for 0.4 mg these rates were 83% (15/18) and 44% (8/18). At C3D1, the MMR and MRD negativity rates for 0.2 mg were 83% (10/12) and 42% (5/12), respectively; for 0.4 mg these were 83% (10/12) and 58% (7/12). Although ctDNA differences between 0.2 mg and 0.4 mg were not substantial, numerically greater ctDNA changes were seen with 0.4 mg GOLCA + R-CHOP vs 0.2 mg GOLCA + R-CHOP and vs R-CHOP and R-CHOP-like regimens from other cohorts ( Figure). Insufficient samples were available to draw conclusions about EOT, but additional ctDNA data will be presented. Whole genome and transcriptome sequencing data from GOLCA + R-CHOP-treated patients will be shown. Conclusions Pharmacodynamic biomarkers and ctDNA levels indicate that both tumor-intrinsic and immune-modulating mechanisms of action of GOLCA remain relevant in combination with R-CHOP in previously untreated aggressive BCL. ctDNA data during treatment are consistent with the efficacy noted on clinical restaging. Translational data demonstrate the potency of GOLCA and will support future frontline clinical trial designs in patients with untreated aggressive BCL. Study support: Celgene, a Bristol-Myers Squibb Company

Efficacy and Safety of Golcadomide, a Novel Cereblon E3 Ligase Modulator (CELMoD) Agent, Combined with Rituximab in a Phase 1/2 Open-Label Study of Patients with Relapsed/Refractory Non-Hodgkin Lymphoma

Introduction: Golcadomide is a novel, oral, small-molecule CELMoD ® agent that co-opts cereblon to induce targeted degradation of the transcription factors Ikaros/Aiolos, which are crucial to B-cell malignancy development. We previously reported promising efficacy with a predictable and manageable safety profile of golcadomide in patients with relapsed/refractory (R/R) non-Hodgkin lymphoma (Michot et al. ICML 2023, presentation #90). Methods: CC-99282-NHL-001 (NCT03930953) is a 2-part, multicenter, first-in-human study with dose escalation (Part A) of golcadomide monotherapy and expansion (Part B) ± combination partners. Patients with R/R diffuse large B-cell lymphoma (DLBCL) or R/R follicular lymphoma and disease progression after ≥ 2 lines of therapy or transplant-ineligible patients with R/R DLBCL after ≥ 1 line of therapy were included. Following dose exploration in Part A, golcadomide was administered at 0.2 or 0.4 mg alone or with rituximab on 2 intermittent schedules in Part B. Total duration of treatment is up to 2 years. Here, we report efficacy and safety results for golcadomide + rituximab in patients with DLBCL treated in Part B expansion on a 14 days on/14 days off schedule. The efficacy-evaluable population consisted of patients completing ≥ 1 cycle of golcadomide (taking ≥ 75% of assigned doses) and having baseline and ≥ 1 post-baseline tumor assessment. Results: As of May 10, 2023, 35 patients with DLBCL were enrolled and 34 received ≥ 1 dose of golcadomide or rituximab (safety population); 17 (49%) patients were ongoing and 17 (49%) had discontinued treatment, mostly due to progressive disease (n = 10, 29%). The median age was 60 years (range, 35-80). The median number of prior anticancer therapies was 4.5 (range, 1-11); 68% (n/N = 23/34) of patients had prior CAR T-cell therapy and 49% (n/N = 17/35) were refractory to their last regimen. Golcadomide median treatment duration was 8 weeks (range, 4-48) and median follow up was 15 weeks (range, 2-55). In the efficacy-evaluable population (n = 16), overall response rate (complete response [CR] + partial response) was 50% (n = 8), with CR occurring in 13% (n = 2) of patients. Median duration of response was 17.4 weeks (range, 7.4-45.9). Response data in the 0.2-mg and 0.4-mg golcadomide-treated groups are shown in the table. In the safety population (n = 34), neutropenia was the most common any-grade treatment-emergent adverse event (TEAE), occurring in 15 (44%) patients, all of which were grade 3/4. No febrile neutropenia occurred. The most common treatment-related adverse event (TRAE) for golcadomide was neutropenia (all grade 3/4), occurring in 14 (41%) patients, comprising 5/19 (26%) patients treated at the 0.2-mg and 9/15 (60%) patients treated at the 0.4-mg dose level. Granulocyte colony-stimulating factors were used in 14 (41%) patients. Six serious TRAEs related to golcadomide were reported (n = 1 each), all in the 0.4-mg group. One grade 5 TEAE occurred (tubulo-interstitial nephritis in the 0.2-mg group), which was reported to be due to progressive disease and considered unrelated to study treatment. TEAEs led to golcadomide discontinuation in 2 (6%) patients (1 at each dose level) and rituximab discontinuation in 5 (15%) patients. Similar pharmacokinetic (PK) exposures of golcadomide were observed when golcadomide was dosed alone and in combination with rituximab, indicating no apparent impact of rituximab on golcadomide PK. Analysis of circulating tumor DNA data is underway and will be presented. Conclusions: Golcadomide oral therapy combined with rituximab showed promising efficacy in heavily pretreated patients with R/R DLBCL, including 1 durable response over 300 days. Golcadomide can be safely combined with rituximab, and the combination showed a safety profile similar to that previously reported for golcadomide monotherapy. This study is ongoing, with continued enrolment in the monotherapy and golcadomide + rituximab combination expansion cohorts.

Preclinical and Translational Biomarker Analyses to Inform Clinical Development of Mezigdomide (CC-92480) in Combination with Dexamethasone and Daratumumab in Multiple Myeloma

Introduction: Mezigdomide (MEZI) is a novel, oral cereblon (CRBN) E3 ligase modulator (CELMoD) that induces rapid and potent degradation of lkaros and Aiolos. Reduction of these transcription factors results in direct tumoricidal and immunomodulatory effects in multiple myeloma (MM). MEZI showed promising efficacy and safety in combination with dexamethasone in the phase 1/2 CC-92480-MM-001 trial (NCT03374085) in Relapsed/Refractory MM. MEZI is currently being investigated in combination with daratumumab and dexamethasone (MEZI + DARA + DEX or MEZI-Dd) in the phase 1/2 CC-92480-MM-002 trial (NCT03989414). In MEZI-Dd cohort, MEZI is administered at three different schedules (B1 for 21/28 days; B2 for 14/21 days; B3 for 7/14 days), and at two different doses (0.3mg; 0.6mg). Here we report preclinical data and pharmacodynamic (PD) biomarker analyses from blood and bone marrow samples collected in CC-92480-MM-002 to support dose and schedule optimization of MEZI-Dd. Methods:Preclinical studies on the pro-apoptotic effects of MEZI on daratumumab-mediated complement-dependent cytotoxicity (CDC) and peripheral blood mononuclear cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) were performed in MM cell lines treated with MEZI-D in comparison to single-agent MEZI or DARA. Clinical PD biomarker analyses included peripheral blood samples collected on treatment Cycle (C)1 Day (D)1 and mid C1-C3 for the expression of Aiolos in T-cells and immunomodulation by flow cytometry. Bone marrow samples were collected for immunohistochemistry at screening and mid C2. Serum free light chain (sFLC) and soluble B-cell maturation antigen (BCMA) were analyzed as biomarkers for tumor burden from C1-C6. Results: Preclinically, combinations of MEZI-D induced synergistic anti-tumor activity in MOLP-8 cells. The MEZI-D combination showed greater anti-MM activities across the dosing gradient than either single-agent DARA (~2-4.5 fold) or MEZI (~1.7-2.4 fold) in a CDC assay. MEZI-D also induced significantly more cell apoptosis than POM-D (p<0.05) in an ADCC assay. In the clinic, MEZI-Dd was sufficient to induce rapid Aiolos degradation in the peripheral blood T-cells for doses as low as 0.3mg MEZI, but 0.6mg MEZI was necessary to result in >50% substrate degradation, suggesting 0.6mg MEZI induced deeper substrate degradation. While 0.3mg MEZI-Dd decreased total CD56+/CD16+ NK-cell counts (Median -83.8%) by mid C1 via immunophenotyping analyses, the combination increased %Ki67+ proliferative NK-cell (Median 251.9%). 0.3mg MEZI-Dd also showed consistent trends of schedule-dependent PD immune effects by mid C3, including increased %Ki67+ proliferative CD3+ T-cell (Median B1: 113.1%, B2: 365.1%, B3: 37.4%), decreased CD45RA+CCR7+ naïve CD4+ T-cell (Median B1: -82.0%, B2: -62.3%, B3: -43.9%), increased CD45RO+CCR7- effector memory CD4+ T-cell (Median B1: 142.3%, B2: 82.9%, B3: 13.9%), and increased HLA-DR expressing CD4+ T-cell activation (Median B1: 149.3%, B2: 107.9%, B3: 17.1%). Thus, greater PD immune effects were observed with schedules of 21/28 and 14/21 days, compared to the 7/14 schedule. At 0.6mg MEZI-Dd, greater median changes of PD immune effects were induced with the 7/14 schedule, compared to 0.3mg MEZI-Dd. Conversely, at continuous dosing schedules longer than 7 days, MEZI-Dd PD immune effects of 0.6mg MEZI were similar to 0.3mg MEZI. In addition, greater reduction of tumor burden was observed at 0.6mg MEZI-Dd, compared to 0.3mg MEZI-Dd across different schedules, as assessed by sFLC and soluble BCMA analyses. Conclusions: Our data show dose- and schedule-dependent PD effects of MEZI-Dd. PD analyses at 0.3mg MEZI suggest at least two weeks of continuous dosing may be needed for greater PD activities in combination with daratumumab, compared to one week of dosing. For continuous dosing of one week, a higher dose at 0.6mg MEZI may be needed to achieve greater PD activities. Further investigation of doses at 0.6mg and 1mg MEZI is underway to determine the optimal dose. Moreover, MEZI induction of NK-cell proliferation and/or activation may provide mechanistic rationale for possible MEZI-D synergy in the clinic.

Synthetic Lethal Interactions with IRAK4 Inhibition in Myeloid Malignancies

Overexpression of immune-related genes is widely reported in acute myeloid leukemia (AML). Research from our lab and others has demonstrated that AML leukemic stem and progenitor cells (LSPC) rely on innate immune signaling through IRAK4 (reviewed in Bennett et al., Blood 2023). Moreover, AML LSPCs express a hypermorphic isoform of IRAK4 (IRAK4-L), due to U2AF1- and SF3B1-dependent mis-splicing. Building on this preclinical evidence, several IRAK4 inhibitors are now undergoing clinical trials in AML patients. In Phase-1 findings with a selective IRAK4 inhibitor (CA-4948; Curis Therapeutics), it was found that MDS and AML patients with splicing factor mutations responded best to monotherapy IRAK4 inhibition, although the overall response rate with monotherapy was modest. These findings suggest that IRAK4 is a relevant target in myeloid malignancies, but the identification of synthetic lethal dependencies may be crucial in revealing effective combination therapies. In this study, we sought to identify synthetic lethal interactions upon IRAK4 inhibition in AML. For this, we first generated isogenic AML cells that are proficient (WT THP1) or deficient for IRAK4 (IRAK4 KO THP1) using site-directed mutagenesis with CRISPR-Cas9. Deletion of IRAK4 resulted in a modest (~50%) reduction in leukemic colony formation and negligible differences in growth potential. We used the isogenic WT and IRAK4 KO AML cells to screen for synthetic lethal interactions by performing a 2400+ drug in vitro screen. Hits were prioritized for potency in IRAK4 KO cells vs WT AML cells, degree of fitness, and overall effect. Among these hits, the Cereblon E3 ligase modulator (CELMoD) CC-885 emerged as the top target. Independent validation confirmed that CC-885 significantly decreased proliferation and viability of isogenic IRAK4 KO MDS (MDSL) and AML (THP1) cells by approximately 3-4-fold compared to WT MDS or AML cells. Colony formation in methylcellulose after treatment with CC-885 was also significantly reduced in IRAK4 KO compared to WT MDS and AML cells. Based on Caspase-3 activation and AnnexinV staining, CC-885 induced significantly more apoptosis of IRAK4 KO cells as compared to WT AML cells. We extended our synthetic lethal analysis to a clinical-stage IRAK4 inhibitor (CA-4948). MDS and AML cells were treated with CA-4948 concurrently with CC-885 or pretreated for 7 days prior to treatment with CC-885. Unexpectedly, we found that only prior exposure of the AML cells to the IRAK4 inhibitors resulted in sensitivity to CC-885. These findings indicate that IRAK4 inhibition reprograms AML cells so that they acquire a synthetic lethal interaction with CC-885. CC-885 is known to target multiple substrates for degradation by the ubiquitin proteasome, including IKZF1, IKZF3, CK1a, and GSPT1. We compared the effect of CC-885 to CC-90009, a CELMoD with GSPT1 specific activity in the isogenic IRAK4 KO and WT AML cell lines. CC-90009 did not result in complete cell death up to concentrations of 10mM in both WT and IRAK4 KO, suggesting that the selective sensitivity of IRAK4 KO AML cells to CC-885 is not due to inhibition of GSPT1. In support of these interpretations, IRAK4 deficient AML cells exhibit increased protein expression of IKZF2 and IKZF3, but not GSPT1 or CK1a. Given the unique responses of IRAK4 KO cells to CELMoDs, we posited that IRAK4 inhibition alters the neosubstrate profile of CELMoDs in AML cells. Total proteome characterization of CC-885 treated WT and IRAK4 KO THP1 cells by mass spectrometry identified a significant decrease in 90 protein substrates in IRAK4 KO cells treated with CC-885 as compared to WT cells treated with CC-885. These findings suggest that IRAK4 inhibition alters the pool of neosubstrates in AML cells for certain CELMoDs. Overall, our study demonstrates that IRAK4 is a therapeutic target in AML, but that combination therapies, such as with certain CELMoDs, will be necessary to achieve better clinical responses.

Mezigdomide Treatment in Relapsed-Refractory Myeloma Patients Shifts Bone Marrow NK and T Cell Populations from Exhaustion to Activation

Background The effect of immunomodulatory drugs (IMiDs) and novel cereblon E3 ligase modulators (CELMoDs) on the tumour microenviroment (TME) of multiple myeloma (MM) patients remains poorly understood. A recent multi-center phase 1/2 trial (CC-92480-MM-001) of CELMoD Mezigdomide (MEZI) demonstrated clinical response in triple-class refractory, heavily pre-treated MM patients (Richardson et al, EHA abstracts 2020). Here, we report results from high dimensional mass cytometry immune profiling of baseline and treatment samples from relapsed-refractory MM (RRMM) patients to understand the effect of MEZI on the bone marrow (BM) TME. In addition, we analyze baseline samples to evaluate BM immune phenotypes that may correlate with response to MEZI. Methods We identified 45 RRMM patients with paired bone marrow aspirate samples pre- (screening) and post- (cycle 2 onwards) MEZI treatment, and 32 additional patients with isolated screening samples. Of the 45 patients, 39 received a dose of 0.8-1.0mg daily for 14 or 21 days of a 28-day cycle, 6 received 0.2-0.6mg on varying schedules as part of the trial design. CD138-negative BM mononuclear cells were isolated from these patients and profiled at single-cell resolution using a previously described 39-marker TME CyTOF-panel (van Oekelen, ASH 2021). Both manual hierarchical gating and unsupervised computational workflows were used for CyTOF data analysis. Around 5 million total cells were analyzed and over 100 immune populations. Samples with <2500 total events were excluded for quality-control. Population frequencies used for comparison are reported as median percentage of the parent population. Statistical comparisons between groups were assessed using the Wilcoxon test. Results Upon MEZI treatment, both adaptive and innate BM immune compartments demonstrated a significant increase in activated populations and marked reduction in exhausted/senescent populations (Figure 1A). Total NK and NKT populations expanded on MEZI (p<0.01 for both). Both CD4 and CD8 T cell populations shifted towards an effector phenotype with significant increases in effector memory (CCR7-CD45RA-) populations (p<0.001 for both) and concurrent reductions of and naïve (p<0.001), central memory (p<0.01 for CD4, NS for CD8) and TEMRA populations (p<0.001). HLADR-expressing CD4, CD8 and NKT populations significantly increased on MEZI (p<0.001 for all) as did ICOS-expressing CD4 (p<0.01), NK (p<0.001) and NKT cells (p<0.05). Senescent NK, NKT, CD4 and CD8 populations expressing KLRG1 decreased on MEZI treatment (p<0.01 for all) and CD57 positive CD8 and NKT cell populations also reduced (p<0.05). CD8, NK and NKT cells expressing the inhibitory checkpoint molecule TIGIT, showed a near 3-fold reduction of on treatment (p<0.001 for all). Although PD1-positive populations did not change on MEZI, PD1/TIGIT double-positive CD4 and CD8 populations showed significant reduction (p<0.05 for both). A dose-dependent reduction of TIGIT-expressing CD8 and NKT populations was seen at higher doses of MEZI (p<0.05 for both), suggesting a direct effect of MEZI on the BM TME. We further correlated depth-of-response with baseline immune composition in 77 patients (Figure 1B). Patients with progression of disease (n=9) on MEZI, when compared to all other patients (n=68), were found to have higher frequencies of PD1 (p<0.05) and TIGIT (p<0.001) positive, as well as dual PD1 and TIGIT positive (p<0.001), CD8 and NKT populations. Number of prior lines (median = 6) and type of MM therapy prior to MEZI did not significantly affect baseline PD1 or TIGIT positive populations. Conclusion MEZI treatment leads to significant activation of adaptive and innate BM immune populations with concurrent reduction of exhausted and senescent populations. These findings are concordant with prior observations of IMiD-mediated immune-stimulation. Patients progressing through MEZI had higher levels of immune checkpoint expression at baseline, which may be helpful for patient selection for this novel CELMoD. These results also suggest that MEZI may be useful in combination with other agents for increasing immune activation and reducing immune exhaustion. Further analyses comparing the effect of different CELMoDs on the BM TME are ongoing.

Iberdomide Enhances Dara Mediated Cytotoxicity through Upregulation of CDC Activity and Elevated NK Cell Mediated ADCC

Background: Multiple Myeloma (MM) is an incurable hematological malignancy with current treatments comprising combination regimens, typically featuring immunomodulatory agents (IMiDs) due to their potent anti-tumor activity and immunomodulatory effects. Clinical studies utilizing the combination of IMiD agents and daratumumab (DARA), an anti-CD38 monoclonal antibody, have shown improved patient outcomes for MM. However, patients ultimately relapse demonstrating the need for novel therapies. Iberdomide (IBER) is a novel cereblon E3 ligase modulator. It promotes faster and deeper degradation of the substrate targets compared to IMiDs, resulting in enhanced anti-proliferative, pro-apoptotic and immune stimulatory activity. In early clinical studies, IBER has been shown to overcome resistance to IMiDs and in the ongoing Phase 3 EXCALIBER study (NCT04975997) is being explored for treatment of newly diagnosed (ND) and relapsed/refractory (RR) MM in combination with DARA and dexamethasone. Here, we investigate the potential mechanisms of synergy between IBER and DARA in preclinical models. Methods: Complement dependent cytotoxicity (CDC) of DARA was assessed with 15% human serum for human complement components and assays were performed by titrating either single agent or combinations in a tritiated thymidine assay. Antibody dependent cellular cytotoxicity (ADCC) was performed by co-culturing MM cells with varying expression levels of CD38 and effector cells and assessing target cell viability with annexin-v/topro-3 flow. A humanized PBMC adoptive transfer mouse model, utilizing human PBMC engraftment 14 days prior to implantation, was used to assess in vivo activity of IBER, DARA and IBER + DARA. Results: In CDC assays, we observed variable sensitivity of MM cells to complement-specific CDC in the presence of DARA (0.1-1 µg/ml), regardless of cell surface CD38 expression. Notably, IBER treatment resulted in upregulation of CD38 surface expression in 60% of MM cell lines tested. LP1 and MOLP8 displayed susceptibility to DARA-mediated CDC. DARA showed a dose-dependent increase in CDC activity in both cell lines. Notably, IBER+DARA also showed ~35% more synergistic CDC activity in both cell lines, than the single agents. In ADCC assays, we observed pretreatment of PBMC with IBER (vs. DMSO) significantly increased DARA mediated ADCC in H929 and LP-1 cells. To confirm that this effect was directed by NK cells, we cultured NK cells for 24 hr in supernatants from stimulated PBMC in presence of IBER prior to co-culture with MM cells. We noted that IBER-treated PBMC supernatant significantly enhanced the NK ADCC activity of DARA suggesting that IBER enhancement of cytokine secretion from PBMC increases NK cell mediated ADCC. Orthogonally, we pre-treated MM cells with IBER or DMSO prior to co-culture in ADCC assay. Notably, IBER treatment of MM cells, without treating immune cells, also increased DARA-mediated ADCC, indicating that IBER directly increases sensitivity to DARA-directed ADCC. To investigate the MOA of IBER-DARA synergy in ADCC, we analyzed gene expression and cell surface changes of a panel of NK-specific receptor ligands on MM cells following treatment with IBER. We observed IBER-induced upregulation of MICA mRNA levels (~2x) and surface expression (~40%) in MM target cells. We used RNAi to knock down Ikaros, an IBER substrate, which resulted in increased MICA expression (~3x) in MM cells. Furthermore, we observed that RNAi knock down of MICA in MM cells resulted in a significant decrease (60%, p<0.0001) in MM cell killing by NK cells, suggesting IBER-enhanced sensitivity of MM cells to NK-cell killing was in part, due to the upregulation of MICA through degradation of Ikaros. Lastly, we examined the anti-MM effects of IBER in a humanized xenograft model with a lenalidomide resistant cell line. Longitudinal analysis identified persistence of human PBMC engraftment and activation of immune subsets (NK cells and T-cells) that were increased upon IBER treatments, consistent with clinical observations. The combination of IBER/DARA resulted in ~22% tumor growth inhibition (TGI) in comparison to single agents, DARA and IBER and ~37% TGI in comparison to vehicle. Conclusion: Taken together, these data demonstrate that IBER and DARA work synergistically in preclinical models primarily through enhancement of both ADCC and CDC. This combination has the potential to improve outcomes in MM.

Investigating the Functional Impact of CRBN Mutations on Response to IMiD/Celmod Agents in Myeloma

Introduction Immunomodulatory drugs (IMiDs) have revolutionized the treatment of myeloma (MM), with lenalidomide (Len) and pomalidomide (Pom) approved and novel cereblon E3 ligase modulators (CELMoDs, e.g. iberdomide, Iber and mezigdomide, Mezi) in clinical trials. IMiDs and CELMoDs bind to cereblon (CRBN), which is part of the CRL4 CRBN Ubiquitin E3 ligase complex, leading to the ubiquitination and subsequent degradation of neo-substrates including the transcription factors Ikaros and Aiolos which are critical for MM cell survival. Previous data suggests almost one third of myeloma patients acquire CRBN mutations by the time they are Pom refractory. Some events, including stop codons/frameshift mutations and copy loss, have clearly explicable effects on cereblon function. However, many are point mutations with some occurring outside of the thalidomide binding domain and at variable cancer clonal fractions. We sought to understand which mutations were deleterious to cereblon function and whether any mutations were associated with a differential response between IMiDs and CELMoDs. Methods From the 36 CRBN mutations previously reported in patient datasets, 12 missense mutations were selected for functional investigation (fig.1a). All mutations were mapped on to the publicly available crystallographic 3D structures of CRBN bound to IMiDs and CELMoDs to aid in predicting the potential functional effect of each mutation. CRBN knock out (KO) MM1.s cell lines were generated using CRISPR-Cas9 and confirmed to have homozygous loss at the first few exons of the CRBN sequence. Once validated, two independent MM1.s CRBNKO cell line clones were transduced with plasmids expressing either CRBN wild type control (CRBN WT) or mutated CRBN (generated using site directed mutagenesis) leading to stable expression. All cell lines were assessed for response to IMiDs and CELMoDs using cell viability assays (CellTitre Blue, 5 days, Len max. concentration 20uM, Pom 8uM, Iber 2uM and Mezi 1uM) and their ability to degrade neo-substrates assessed using Western blotting for Aiolos and downstream effects using RT-qPCR for IRF4, at 24 hrs. Results Knock out of CRBN in MM1.s cells led to complete resistance to IMiDs/CELMoDs. The re-expression of wild type CRBN re-sensitised MM1.s CRBNKO cells to IMiDs/CELMoDs with degradation of Aiolos, downregulation of IRF4 and a similar effect on cell viability to that seen in parental MM1.s. In contrast, the “positive control” mutation p.W386A located within the tri-tryptophan IMiD/CELMoD binding pocket appeared to completely inactivate CRBN with no change in Aiolos/IRF4 expression levels and no effect on cell viability after incubation with all IMiDs/CELMoDs. A second mutation, also in very close proximity to the tri-tryptophan binding pocket and within the neo-substrate binding area (p.H397Y), had the same effect suggesting mutations here entirely prevent all IMiD and CELMoD activity (fig.1b). In contrast, some CRBN mutations appeared to have no deleterious effect on CRBN function with response to all IMiDs and CELMoDs regained on re-expression at the same magnitude as seen with wild type CRBN re-expression. These mutations included all those located in the Lon protease-like domain (p.D50H, p.A143V, p.L190F, p.R283K) and two within the thalidomide binding domain but not close to the tri-tryptophan binding pocket nor neo-substrate binding area (p.A347V and p.W415G)(fig.1b). The remainder of the mutations studied appeared to abrogate the activity of the IMiDs lenalidomide and pomalidomide but retained some response (both degradation and viability) to iberdomide (p.C326G), and to a greater extent mezigdomide (p.C326G, p.P352S, p.C366Y and p.F381S), suggesting these compounds were able to partially compensate the effect of the mutation (fig.1b). This may be due to the more potent CELMoD compounds being able to overcome structural changes induced by some mutations but not others. Discussion These data highlight key differences in the functional impact of different CRBN missense mutations that have been previously identified in patients. These results may have important implications for the interpretation of CRBN sequencing results from patients for future therapy decisions, particularly differentiating those who may, despite relapsing on IMiDs with CRBN mutations, have the potential to still benefit from the use of CELMoD agents.

Mezigdomide Reverses T-Cell Exhaustion through Degradation of Aiolos/Ikaros and Reinvigoration of Cytokine Production Pathways

Introduction: T cell exhaustion (Tex) is characterized by progressive decline in activation and proliferation and has been implicated as a major resistance mechanism of T cell immunotherapies such as BCMA directed bispecifics approved for the treatment of multiple myeloma (MM). Understanding and overcoming Tex is key for relapsed patient populations facing drug resistance. Here, by using the next generation cereblon E3 ligase modulator (CELMoD) Mezigdomide (Mezi), a specific and potent Aiolos/Ikaros degrader to date, we revealed for the first time through comprehensive genomic/epigenomic and functional analyses that Aiolos/Ikaros are two major transcription factors that contribute to the regulation of the Tex phenotype and their degradation reverses the negative impact on activation, proliferation and tumor cell killing. Methods: Tex were generated ex vivo from normal donors through multiple rounds of anti-CD3/CD28 dynabead stimulations over the course of 14 days. Unstimulated (Tn) or transiently stimulated T cells (Tact) from the same donors were used for differential comparisons. Comprehensive genomic analyses were conducted to compare the three groups of T cells using RNAseq, ATACseq, whole genome bisulfite sequencing and ChIPseq for Aiolos/Ikaros and histone modifications (H3K27ac, H3K27me3, H3K4me3 H3K4me1), with and without Mezi treatment for 72 hrs. Cytokine/chemokines were measured by Luminex multiplex cytokine assays. Exhaustion markers were determined by flow cytometry. Mezi effects on Tex mediating tumor cell killing in combination with anti-CD3/BCMA bispecific antibody Alnuctamab (Alnuc) was also assessed via both incucyte and flow cytometry. Results: After multiple rounds of stimulation, the viability of Tex remained over 90%, with proliferation decreased from 3-6-fold in initial stimulation to 1-1.4-fold in the last stimulation, as well as 98% reduction in IL-2 secretion. RNAseq analysis showed more proinflammatory cytokine expression from Tex was significantly down-regulated compared to Tact, such IL-1/6/9/17/21/23 and TNF. Exhaustion markers CTLA4, PD1, TIM3, LAG3, TOX, PDL1, and ENTPD1 (CD39) were analyzed via flow cytometry and confirmed by RNAseq to be significantly increased in expression after initial activation, then maintained and/or increased further with subsequent re-stimulations. Treatment of Mezi significantly down regulated PD1, TIM3, TIGIT, and up-regulated proinflammatory cytokines/chemokines such as IL-1/23/3/6/7, CCL1/2/17/20, CXCL1/3/11/12 and TNF, as well as cell surface interaction related integrins, RAS signaling and proliferation related pathways, as revealed by both Luminex multiplex cytokine analysis and RNAseq (Fig.A). Using ATAC-seq, we found that the peaks gained or lost in Tex at promoters and distal sites were significantly correlated with gene expression changes from the RNAseq analysis, with the strongest effect seen at promoters. We identified that canonical RUNX1/2/3 binding motifs were significantly enriched in the gained peaks in Tex compared to Tact, with RUNX2/3 themselves also showing increased mRNA expression. After treatment of Mezi, Tex gained peaks were enriched with NFATC2 and RUNX3 binding sites whose gene expression levels were also increased (Fig. B). Enrichment analysis of the cis-regulated genes of the gained peaks in Tex after treatment highlights biological processes related to cell-cell adhesion, cytokine production and other T cell activation processes. Notably, TF analysis also showed that, after treatment, Ikaros motifs were enriched in the Tex gained peaks. Flow cytometry analysis revealed that Tex cells have the highest expression of Ikaros, especially in PD1 + TIM3 + CD4 subsets. To investigate the effect of Aiolos/Ikaros in Tex functionally, we set up co-culture assays using Tex cells with BCMA expressing tumor cells in the presence of Alnuc. We found that Mezi treatment to Tex significantly enhanced killing of tumor cells and reduced the EC 50 of Alnuc. 5-10 fold compared to DMSO controls. Conclusion: We demonstrated here that Aiolos/Ikaros regulate and maintain T cell exhaustion. Mezi mediated degradation of both transcription factors at the same time resulted in enhanced proinflammatory cytokine expression and reduction of exhaustion associated markers, as well as enhanced Tex killing of BCMA expressing tumor cells in combination with Alnuctamab.

Iberdomide Maintenance after Autologous Stem-Cell Transplantation in Newly Diagnosed MM: First Results of the Phase 2 EMN26 Study

Background. Iberdomide is a novel oral cereblon E3 ligase modulator (CELMoD TM) with enhanced direct antitumor effects and immune stimulatory effects, compared to lenalidomide or pomalidomide. In the ongoing phase 1/2 CC-220-MM-001 study, iberdomide plus dexamethasone had a favorable safety profile and demonstrated clinically meaningful activity in triple-class refractory multiple myeloma (MM) patients, including those refractory to lenalidomide and pomalidomide (IMiDs ®). Based on these data, we aimed to evaluate the safety and clinical activity of 3 different doses of iberdomide as a novel maintenance treatment post-transplant in newly diagnosed MM patients. Here we report results from the first interim analysis for patients who have been treated with at least 6 treatment cycles, or discontinued treatment earlier. Methods. The EMN26 study is a multicohort, phase 2 study conducted in 4 European countries. Patients aged 18 years or older with MM, who had achieved at least a partial response (PR) after induction therapy containing a proteasome inhibitor (PI) plus IMiD followed by single or double autologous stem-cell transplantation (ASCT) +/- consolidation, were enrolled into one of 3 different cohorts (iberdomide 0.75, 1.0, or 1.3 mg on days 1-21 of each 28-day cycle; treatment continued until progression or unacceptable toxicity; 40 patients in each cohort). The primary outcome is improvement in response, and secondary outcomes include safety and progression-free survival (PFS). Response was evaluated at screening and after every cycle (bone marrow analysis was done at screening, at 6 and 12 months after treatment initiation, and to confirm (s)CR). This trial is ongoing and is registered with ClinicalTrials.gov (NCT04564703). Results. At data cut-off (May 31, 2023) 31 patients were enrolled in the 0.75 mg cohort, and 40 patients each in the 1.0 and 1.3 mg cohorts (total of 111). A total of 69 patients had received ≥6 cycles of iberdomide treatment or discontinued earlier (n=34 in 1.0 mg cohort; n=35 in 1.3 mg cohort; n=0 in 0.75 mg cohort [this cohort was added later]). Median age of these 69 patients was 59 years, and 57% were male. At diagnosis, 37% of patients presented with International Staging System (ISS) stage 1 disease, 35% with ISS stage 2, and 28% with stage 3. High-risk disease (del(17p), t(4;14), and/or t(14;16)) was present in 14% of patients. All patients received a PI/IMiD-containing induction regimen which also included daratumumab in 41% of patients. Double ASCT was administered to 19% and post-ASCT consolidation to 7%. Best response at the time of enrollment in the study was PR in 15%, very good (VG)PR in 59%, complete response (CR) in 12%, stringent (s)CR in 15% [≥CR: 26%] in the 1.0 mg cohort, and PR in 3%, VGPR in 69%, CR in 11%, sCR in 17% [≥CR: 29%] in the 1.3 mg cohort. After 6 treatment cycles, there was comparable deepening of response in both cohorts (1.0 mg cohort: PR 6%, VGPR 44%, CR 3%, sCR 47% [≥CR: 50%]; 1.3 mg cohort: PR 3%, VGPR 37%, CR 9%, sCR 51% [≥CR: 60%]). Improvement of response was reported in 48% (90% CI 32-65%) of patients treated with 1.0 mg iberdomide and 45% (90% CI 29-62%) in the 1.3 mg iberdomide cohort ( Figure), which are significantly higher than the null hypothesis of ≤20% response improvement within 6 months. The most common grade 3 or worse adverse events (AEs) during cycles 1-6 were neutropenia (21% in 1.0 mg cohort and 46% in 1.3 mg cohort), infections (3% and 14%), fatigue/asthenia (12% and 14%). There were no events of ≥grade 3 thrombocytopenia, anemia, diarrhea, VTE, or neuropathy. Dose reductions were used to manage AEs in 18% of patients in the 1.0 mg cohort and 31% in the 1.3 mg cohort. Treatment discontinuation occurred in 3 patients in 1.0 mg cohort (1 due to AE, 2 PD), and 4 patients in 1.3 mg cohort (2 due to AE, 1 PD, and 1 death [unknown cause]). PFS at 6 months was 97% and 94% in the 1.0 and 1.3 mg cohorts. With longer follow-up, results from the 0.75 mg cohort and MRD conversion data will be presented at the meeting. Conclusions. Iberdomide represents a novel effective post-ASCT maintenance strategy with a favorable safety profile and superior response improvement at 6 months than what has been observed with lenalidomide maintenance (26% at 6 months in the EMN02 study). Additional follow-up is needed to define the recommended maintenance dose that will be used in the randomized phase 3 EXCALIBER maintenance study, which will evaluate iberdomide vs. lenalidomide maintenance post-ASCT.

Celmod Induced Disruption of Granulopoiesis Continuum Identified Using Humanized Cereblon Mice

Introduction: CRBN mediated degradation of Ikaros by Pomalidomide (Pom) and Mezigdomide (Mezi) results in robust antiproliferative activity against myeloma cells, with concomitant activation of immune cells such as T and NK cells. However, degradation of Ikaros has been shown to mediate maturation arrest of neutrophils leading to neutropenia. Unlike chemotherapy drugs, CELMoD driven neutropenia is an on-target adverse effect. Although the next generation cereblon E3 ligase modulators are potent and highly selective, neutrophil biased adverse effects remain persistent. Here, we sought to define the impact of Ikaros degradation through a longitudinal assessment of granulopoiesis in the bone marrow of humanized CRBN mice treated with Pom and Mezi. Methods: We have performed multi-parametric studies leveraging a humanized CRBN (hCRBN) C57BL/6 mouse model. Driven by the endogenous mouse CRBN promoter, human CRBN was inserted into the mouse CRBN gene, preventing expression of mCRBN. These hCRBN mice are capable of specific degradation of CELMoD mediated substrates, thus enabling an in vivo model to understand pathophysiological mechanisms. Homozygous hCRBN mice were treated daily with Pom and Mezi via oral gavage until days 7, 14 and 21 when mice were euthanized. Peripheral blood and bone marrow were collected for flow cytometric and molecular analyses. Critical population classes such as multipotent progenitors, lineage committed progenitors, pre-myelocytes, immature neutrophils and mature neutrophils were defined by immunophenotypes, and pharmacodynamic parameters such as Aiolos/Ikaros degradation were evaluated. All data is represented as fold change over median of vehicle control. Results: The hCRBN mouse model was able to recapitulate Pom and Mezi driven clinical neutropenia. Peripheral absolute neutrophil count gradually decreased with increased dosing (QD7, QD14 and QD21) in mice treated with Pom (26%, 45% and 74%) and Mezi (35%, 57% and 70%). Treatment with Pom and Mezi resulted in disruption of early hematopoietic multi-progenitor populations leading to a widespread effect at multiple stages of haematopoiesis. Intriguingly, in bone marrow these effects were both temporally differential for multi-potent progenitors, and across various stages of neutrophil maturation. Myeloid biased, MPP3 population increased to ~55% and ~103% at QD7, followed by a steady decline, renormalizing to baseline at QD14 and QD21 for Pom and Mezi respectively. The first myeloid lineage committed progenitors, CMP population, reduced to 21% (Pom) and 42% (Mezi) at QD7 stabilizing at ~17-21% below baseline by QD21. In contrast GMP population, descendants from CMP, increased to ~50% (Pom) and ~117% (Mezi) at QD7. For Pom treatment (but not with Mezi), GMP proportions renormalized to baseline at QD21. Pre-myelocytes, one of the earliest neutrophil maturation stages, shrunk to ~24-30% at QD21 without any differences between Pom and Mezi. On the other hand, immature neutrophils steadily diminished to 51% (Pom) and 70% (Mezi) at QD21. This effect was more pronounced in mice treated with Mezi. Mature neutrophils in bone marrow, part of a highly dynamic reservoir that extravasates into blood, increased to 10-18% at all the time points for both Pom and Mezi. Overall, impairment of granulopoiesis is more profound in hCRBN mice treated with Mezi than Pom, owing to the more potent Ikaros degradation. Mechanistically, degradation of neo-substrates such as Ikaros affect the myelopoiesis and neutrophil maturation but are likely not the sole drivers. Addition of corticosteroids such as dexamethasone have been shown to mitigate IMiD/CELMoD mediated neutropenia in the clinical setting. Mezi treated animals who were co-administered dexamethasone demonstrated partially enhanced early progenitor populations compared to single agent Mezi. To investigate patterns of Ikaros transcriptional control, specific populations were flow sorted in parallel, for focused molecular analyses such as RNAseq using the SMARTseq platform and will be included in the presentation. Conclusion: These studies delineate previously unreported effects of CELMoDs on a multifaceted continuum of myelopoiesis. These insights will help rationally design dose and schedule considerations for CELMoD treatment regimens alone, or in combination with other therapeutic agents to alleviate CELMoD induced neutropenia.

Pharmacological induction of translational readthrough of nonsense mutations in the retinoblastoma (RB1) gene.

The retinoblastoma protein (Rb) is encoded by the RB1 tumor suppressor gene. Inactivation of RB1 by inherited or somatic mutation occurs in retinoblastoma and various other types of tumors. A significant fraction (25.9%) of somatic RB1 mutations are nonsense substitutions leading to a premature termination codon (PTC) in the RB1 coding sequence and expression of truncated inactive Rb protein. Here we show that aminoglycoside G418, a known translational readthrough inducer, can induce full-length Rb protein in SW1783 astrocytoma cells with endogenous R579X nonsense mutant RB1 as well as in MDA-MB-436 breast carcinoma cells transiently transfected with R251X, R320X, R579X or Q702X nonsense mutant RB1 cDNA. Readthrough was associated with increased RB1 mRNA levels in nonsense mutant RB1 cells. Induction of full-length Rb protein was potentiated by the cereblon E3 ligase modulator CC-90009. These results suggest that pharmacological induction of translational readthrough could be a feasible strategy for therapeutic targeting of tumors with nonsense mutant RB1.

Mezigdomide (MEZI) Plus Dexamethasone (DEX) and Daratumumab (DARA) or Elotuzumab (ELO) in Patients (pts) with Relapsed/Refractory Multiple Myeloma (RRMM): Results from the CC-92480-MM-002 Trial

Introduction : MEZI is a novel oral cereblon E3 ligase modulator (CELMoD ™) with enhanced tumoricidal and immune-modulatory effects compared with immunomodulatory drugs (IMiDs ®). In preclinical studies, MEZI showed synergy with anti-myeloma drugs including DEX, proteasome inhibitors (PIs), and anti-CD38 monoclonal antibodies (mAbs), and is being assessed in the phase 1/2 CC-92480-MM-002 (NCT03989414) trial with different treatment combinations in RRMM. Here we report the preliminary efficacy and safety of MEZI, DARA, and DEX (MeziDd) over 3 dosing schedules, as well as MEZI, ELO and DEX (MeziEd), in pts with RRMM. Methods : Eligible pts had: RRMM, received 2-4 prior lines of therapy, a minimal response or better to ≥ 1 prior regimen, and disease progression during or after their last therapy. In the MeziDd cohort, oral MEZI was given at escalating doses on days (D) 1-21 per 28-D cycle (C) (subcohort B1); on D1-14 per 21-D cycle from C1-8 and D1-21 per 28-D cycle ≥ C9 (subcohort B2); or on D1-7 and D15-21 per 28-D cycle from C1-6 and D1-21 per 28-D cycle ≥ C7 (subcohort B3). Intravenous (IV; 16 mg/kg) or subcutaneous (1800 mg) DARA was given weekly (C1-2), then biweekly (C3-6), and monthly (≥ C7) for subcohorts B1 and B3, and weekly (C1-3) then on D1 of each 21-D (C4-8) or 28-D (≥ C9) cycle for subcohort B2; with weekly oral/IV DEX (40 mg; 20 mg > 75 y or body mass index < 18.5 kg/m 2). In the MeziEd cohort, oral MEZI was given at escalating doses on D1-21 per 28-D cycle, with weekly (C1-2; 10 mg/kg) then monthly (≥ C3; 20 mg/kg) IV ELO, and oral/IV DEX on ELO dosing days (36 mg; 16 mg > 75 y) plus on D8, 15, and 22 of ≥ C3 (40 mg; 20 mg > 75 y). Thromboembolism (TE) prophylaxis was given within 48 hours of C1D1 and until 48 hours after last MEZI dose. Primary objectives were to determine the recommended dose and regimen and to assess safety and preliminary efficacy as overall response rate (ORR). Results : As of the May 25, 2023 data cut, 57 pts were enrolled and had received MeziDd (B1: n = 23; B2: n = 16; B3: n = 18) at the 0.3 mg or 0.6 mg dose. Median age was 67 (range, 45-83) y, median time since initial diagnosis was 8.2 (1.0-15.8) y, and median number of prior regimens was 2 (2-5). Prior therapies included stem cell transplantation (15.8%), IMiD agents (100%), PIs (98.2%), and anti-CD38 mAbs (8.8%); 82.5% of pts were refractory to IMiD agents and 61.4% to PIs. Extramedullary plasmacytomas were present in 4 (7.0%) pts. Forty-five of 57 (78.9%) pts continued treatment; the main reason for discontinuation was progressive disease (5 pts; 8.8%). In the safety population (n = 56), ORR was 75.0% overall, with 2 (3.6%) stringent complete responses, 8 (14.3%) complete responses, 16 (28.6%) very good partial responses, and 16 (28.6%) partial responses (Figure). Duration of response and progression-free survival data were not yet mature. Grade (Gr) 3/4 treatment-emergent adverse events (TEAEs) occurred in 43/56 (76.8%) pts and were mostly hematologic (Table). Gr 3/4 neutropenia occurred in 30 (53.6%) pts; Gr 3/4 thrombocytopenia occurred in 4 (7.1%) pts (3/4 pts were in subcohort B1-0.6 mg) and was not associated with bleeding. Gr 3/4 infections occurred in 11 (19.6%) pts and proved manageable in all cases (Table). Non-hematologic Gr 3/4 TEAEs were low. No Gr 3/4 TE or peripheral neuropathy was observed. MEZI dose reductions were needed for 7 (30.4%) pts in subcohort B1 (1 at 0.3 mg, 6 at 0.6 mg) and 1 (5.6%) pt in subcohort B3 (at 0.6 mg). In the evaluable population (n = 35), 5 (14.3%) pts had ≥ 1 dose-limiting toxicity. The maximum tolerated dose was not reached. MeziDd had pharmacodynamically active immune stimulation in T and NK cells in all 3 schedules and both doses. Twenty pts received MeziEd at 0.3 mg (n = 11) or 0.6 mg (n = 9); median prior lines of therapy was 3 (2-4) and 3 (2-5), and most pts (72.7% and 100%) had prior anti-CD38 mAb exposure. ORRs were 36% and 56%, respectively. The safety profile was consistent with known TEAEs and was generally well tolerated, although 2 Gr 3 pulmonary embolisms occurred. Conclusions : MeziDd showed promising efficacy and a manageable safety profile in pts with RRMM and 2-4 prior lines of therapy, as did MeziEd in pts with prior anti-CD38 mAb therapy. The immune activity of MEZI was consistent with previous preclinical reports. Improved safety and efficacy may be achieved by schedule and dose adjustments. These results support further evaluation of MEZI plus anti-myeloma mAbs in phase 1/2 and phase 3 studies. Updated results will be presented at the meeting.

Model based assessment of food and acid reducing agent effects on oral absorption of mezigdomide (CC-92480), a novel cereblon E3 ligase modulator.

Mezigdomide is a novel cereblon E3 ligase modulator (CELMoD) agent with enhanced autonomous cell-killing activity in multiple myeloma (MM) cells, and promising immunomodulatory and antitumor activity in patients with MM. We developed a population pharmacokinetics (PKs) model for mezigdomide in healthy subjects (HSs), and quantified effects of high-fat meal and proton pump inhibitor (PPI) on human disposition parameters. Plasma concentrations from 64 HS in two phase I clinical studies (NCT03803644 and NCT04211545) were used to develop a population PK model. The HSs received single oral doses of 0.4-3.2 mg mezigdomide with full PK profiles collected. A two-compartment linear PK model with first-order absorption and lag time best described mezigdomide PK profiles in HSs. The population PK parameters of absorption rate constant, lag time, central volume of distribution, clearance, peripheral volume of distribution, and intercompartmental clearance were estimated to be 1.18 h-1 (interoccasion variability [IOV]: 65%), 0.423 h (IOV: 31%), 440 L (interindividual variability [IIV]: 63%), 35.1 L/h (IIV: 40%), 243 L (IIV: 26%), and 36.8 L/h (IIV: 26%), respectively. High-fat meal increased oral bioavailability by ~30% and PPI co-administration decreased oral bioavailability by ~64%. Mezigdomide demonstrated a linear dose-exposure relationship in HSs. The PK model suggests a modest effect of high-fat meal, and a substantial effect of PPIs on mezigdomide oral bioavailability. This population PK model enables data integration across studies to identify important covariate effects and is being used to guide dose selection in clinical study designs for mezigdomide in patients with MM.