In malignant B-cell diseases, BCL6 is one of several critical mis-regulated oncogenic factors that are commonly over-expressed in high-risk patient segments in need of safe and tolerable alternatives to immuno-chemotherapeutic standard of care regimens. Furthermore, as a lineage defining factor within the immune microenvironment of follicular diseases (e.g. Teff, Tregs, Tfr and Tfh), targeting BCL6 in this cellular compartment provides an additional layer to the direct anti-tumor mechanism and should lead to therapeutic benefit. Herein, we describe the discovery and preclinical characterization of BMS-986458, a highly selective, orally bioavailable CRL4CRBN E3 ubiquitin ligase-dependent BCL6 ligand directed degrader, as a heterobifunctional molecule that simultaneously co-opts cereblon (CRBN) and the BCL6 N-terminal BTB domain to catalyze proximity induced degradation of BCL6. In vitro, BMS-986458 rapidly degrades BCL6 protein to levels that drive broad anti-tumor effects in 80% of BCL6 expressing NHL cell lines and all ex vivo patient derived xenograft (PDX) models evaluated. Extensive transcriptomic analysis shows the anti-tumor effect of BCL6 degradation is achieved through modulation of a regulon associated with cell-cycle checkpoints, anti-proliferative signaling and interferon response pathways. A novel component of the BCL6 regulon is repression of the highly efficacious therapeutic surface target CD20, whose downregulation is associated with relapsed/refractory (R/R) disease. Using BMS-986458, we demonstrate a broad enhancement of CD20 transcription, surface expression and clustering, increasing up to 20-fold within 72 hrs across multiple Diffuse Large B-cell Lymphoma (DLBCL) cell line models. This enhancement results in potent synergism of BMS-986458 with anti-CD20 agents from both cell intrinsic and ADCC-mediated anti-tumor mechanisms. The cell intrinsic anti-tumor impact of BCL6 degradation was confirmed in vivo using human cell line-derived xenograft (CDX) and PDX models of R/R DLBCL. In these studies, once daily oral dosing of BMS-986458, resulted in deep and sustained degradation of BCL6 leading to CDX tumor regression and significant survival benefit of PDX models. Furthermore, BMS-986458 in combination with anti-CD20, resulted in tumor regression and tumor free animals (<70%), without body weight loss. In additional studies, we demonstrate using both in vitro T-follicular helper cell (Tfh) differentiation assays and evaluation of immunocompetent mice, that BMS-986458 treatment phenotypically modulates lymph-resident Tfh populations without affecting cellular viability, highlighting the potential functional impact of BCL6 degradation on this pro-tumor immune compartment. Non-clinical safety evaluations show that in addition to the absence of impact on normal bone marrow populations in vitro, BMS-986458 is pharmacodynamically active and well tolerated in vivo following 28-day dog toxicity studies. In summary, the robust cell intrinsic mechanism of BMS-986458 across BCL6-expressing NHL tumors together with its stimulation of CD20 surface expression and the immunomodulatory impact on Tfh populations, demonstrate that BCL6 degradation using BMS-986458 has the potential to be highly efficacious and represents a novel mechanism of action for NHL therapy. Collectively, this evidence supports its current clinical investigation as an orally dosed single agent or in combination with an anti-CD20 agent (NCT06090539), providing a potential first-in-class chemo-free therapeutic option for B-cell NHL patients.

Introduction: Compounds inducing cereblon-mediated degradation of Ikaros and Aiolos (I/A), like lenalidomide (LEN) and avadomide, have demonstrated antitumor activity in non-Hodgkin lymphoma (NHL), with significant but limited cell-autonomous activity in diffuse large B-cell lymphoma (DLBCL) cell lines. Their pharmacodynamic activities also include T-cell activation, increased macrophage tracking, and increased regulatory T lymphocytes (T-regs) in peripheral blood (PB) [Carpio C et al Blood 2020]. High immune infiltration in tumors at baseline (BL) was correlated with improved outcome (Risueño A et al Blood 2020). Golcadomide (GOLCA) is a potential first-in-class cereblon E3 ligase modulatory drug (CELMoD™) that, compared with immunomodulatory (IMiD) agents like LEN, has shown more profound cell autonomous activity and immune stimulation in preclinical DLBCL models (Lopez-Girona A et al Hematological Oncology, 2021). Its potent I/A degradation, prolonged exposure, and high tissue penetration predicted superior GOLCA activity vs IMiDs, including in tumors with low immune infiltration. In the CC-99282-NHL-001 study, GOLCA ± rituximab showed a manageable, predictable safety profile characterized by an on-target toxicity of neutropenia and promising efficacy in relapsed/refractory NHL in heavily pretreated pts (Chavez JC et al Blood 2023). Here, we describe pt immune profiles, their modulation by GOLCA and association with objective clinical response in the CC-99282-NHL-001 DLBCL biomarker cohort. Methods: Cell intrinsic and immune-mediated activity of GOLCA in evaluable pts with DLBCL enrolled in the study were assessed by RNA-Seq, whole genome sequencing, immune-fluorescence and flow cytometry (FC) in screening and on-treatment biopsies, and by FC in PB samples. Results: I/A degradation was necessary but not always sufficient for GOLCA activity. We observed deep I/A degradation in biopsies and PB samples from all evaluable pts independent of response, suggesting additional tumor-related features influence response to GOLCA. In contrast to IMiDs, efficacy of GOLCA (best overall response and progression-free survival) was independent of BL tumor microenvironment (TME) content. Despite this, BL CD4 naive T cells, T follicular helper and CD8 T cell levels were higher in responders while mono/macrophages were lower. GOLCA sensitivity analysis was run separately in immune infiltration (TME+) vs high B-cell content (TME−) tumors to explore immune mediated and tumor intrinsic mechanisms. Low leukocyte migration and low chemokine signaling were enriched in TME− responders. TME+ tumors showed distinct expression levels of genes involved in immune activation and trafficking (↑AIRE, ↑CLDN12, ↑IQGAP3, ↓MADD, ↓UBE7). GOLCA responders showed a significant increase in pathways of chemokine and cytokine receptor binding and chemokine signaling. Interferon (IFN)-stimulating genes and other proteins involved in immunoregulatory/inflammatory processes (IFI6, OAS3, CCL3L etc) were upregulated in responders, with a modest increase in pathways like tumor necrosis factor alpha and IFNα/β. GOLCA-induced I/A degradation led to increased natural killer- (NK) and T-cell activation with dose/schedule-dependent T-reg increase and decrease in naive T cells in PB. Response was associated with increased PD-1, potentially due to increased T-cell activation. GOLCA responders showed a significant decrease in B-cell content and increase in NK and dendritic cells, macrophage and T-cell subsets. In these responders, there was positive correlation of T-regs between PB at Cycle 1 Day 15 and on-treatment biopsies. Other T-cell populations like naive and memory T cells showed anti-correlation, suggesting peripheral changes may be due to trafficking. In non-responders, these changes were less pronounced. Conclusion: These results show GOLCA fulfills its design intent, causing deep and prolonged I/A degradation in NHL lesions resulting in BL TME-independent activity, compared with IMiDs like LEN which are TME-dependent. BL immune profiles and their modulation on treatment link to treatment effect and imply an immune contribution to GOLCA efficacy. These data support broader activity of GOLCA than IMiDs in NHL, with high potential for combination with anti-lymphoma agents acting on tumor B cells and/or TME.

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.

Introduction: Treatment of AML (Acute Myeloid Leukemia) has been clinically challenging with modest success and poor patient outcomes. The limited efficacy of single-agent treatments has encouraged the investigation of rationally designed combinations to increase drug activity and achieve deeper and prolonged remission avoiding the development of resistance. Azacitidine (AZA) and BCL2 inhibitor venetoclax (VEN)-based combinations became the new standard of care for the treatment of newly diagnosed AML patients who are ineligible for intensive chemotherapy upon its approval in 2018 1-3. Despite this recent approval, reduced response rates in mature phenotypes (AML M4 and higher), tolerability, and duration of response remain suboptimal. Therefore, innovative combination approaches are still a need for AML. A first-in-class, potent, and selective CK1α-targeting oral CELMoD, BMS-986397, is being investigated for the treatment of R/R AML and HR-MDS (High-Risk Myelodysplastic Syndrome) harboring functional TP53 (Tumor protein 53) in a clinical study (NCT04951778). Owing to its mechanism, combination with other agents that target apoptosis pathways such as VEN or hypomethylating agents, could improve efficacy in AML. In this regard, this study seeks to investigate a convenient strategy for BMS-986397 combinations that would circumvent resistance and inform future decisions in the FIH trial to improve the treatment of R/R leukemia, including specific AML subtypes. Methods: Viability studies in a panel of 16 AML cell lines were performed to test the combinatorial potential of BMS-986397 with VEN and/or AZA. These studies were expanded with ex vivo clonogenic assays in bone marrow mononuclear cells (BMNCs) from 15 TP53 WT (Wild Type) AML patients as a single agent and, in double or triple combinations with VEN and AZA. The impact on normal hematopoietic stem (HSC) and progenitor cells was evaluated by assessing the effect on the growth of granulocyte/monocyte (CFU-GM) and burst forming unit of erythrocytes (BFU-E) progenitor cells in human bone marrow (BM) CD34+ cells from 4 healthy volunteers (HV). Leukemic blasts were phenotypically and molecularly characterized, including different FAB subtypes. A drug combination analysis was conducted to determine synergy (Bliss) and additivity (HSA) scores. In vivo studies in 2 AML-PDXs (Patient Derived Xenograft) from patients with different AML subtypes were explored in monotherapy and in VEN and/or AZA combinations followed by an immunophenotyping assessment of the blood, spleen, and BM and PD (pharmacodynamics) of human primary leukemic cells. Results: BMS-986397 shows additivity/synergy in 7/16 AML cell lines in combination with VEN and/or AZA. Ex vivo testing in normal hematopoietic cells reveals that double combinations of BMS-986397 with VEN or AZA do not induce a significant impact on CFU-GM and BFU-E when compared with their single agent activity. However, triple combination of BMS-986397/VEN/AZA exhibits an increase on myelotoxic effect on these healthy progenitors (median IC50 291.6nM in doublets vs 88.1nM in triplets). In AML patient samples, BMS-986397/VEN or BM-986397/AZA combinations exhibit similar activity inhibiting the growth of AML leukemic progenitor cells compared with the triplet combination and, in most cases, similar or superior activity to the VEN/AZA combination. In 12 out 15 AML samples, additive effect mediated by triple BMS-986397/VEN/AZA combination is recapitulated for at least one of the double combinations, overcoming the potential toxicity provoked by triplets in normal hematopoietic progenitors. Interestingly, acute monoblastic/monocytic leukemia (AML M5) samples, described as resistant to VEN/AZA treatment 4, have shown benefit after including BMS-986397 in the combination backbone (6.6 fold-change excess additivity score). In vivo pharmacology studies have also evidenced an improvement in mice survival to >80 days (vs 60 days in VEN/AZA groups) with no signs of leukemia after BMS-986397 and/or VEN/AZA combinations, even in M5 AML-PDX models. Conclusions: BMS-986397 combination with VEN and/or AZA unveiled preclinical efficacy improvement with tolerable safety in primary AML. These data support the use of combination-based therapies targeting different mechanism of action to achieve expanded clinical success transforming the therapeutic landscape for patients diagnosed with AML.

Introduction: The Casein Kinase 1α (CK1α) gene resides on chromosome 5q and has haploid status in del5q MDS (Myelodysplastic Syndrome). Lenalidomide is a weak, but significant degrader of CK1α, and has clinical efficacy in del5q MDS relative to non-del5q MDS. Therefore, development of a strong degrader of CK1α that may have activity in myeloid malignancies irrespective of 5q status is warranted. We identified BMS-986397 as a potent, specific, oral CELMoD molecular glue degrader of CK1α. Here, we present preclinical evidence of p53-dependent efficacy of BMS-986397 in AML (Acute Myeloid Leukemia) and HR-MDS (High-Risk Myelodysplastic Syndrome). Methods: To assess the anti-proliferative activity and molecular mechanism of BMS-986397, AML parental and genetically engineered cell lines with CRISPR/Cas9-mediated knockout of Cereblon (CRBN) or TP53 or overexpressing a non-degradable CK1α mutant were evaluated by proliferative assays, flow cytometry, and immunoblotting. As BMS-986397 is pharmacology active in different species, informative in vivo studies in rodent models and in ex vivo models using primary samples from healthy volunteers and AML patients were conducted to assess pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity. Additionally, single- and repeat-dose toxicity studies in rats, monkeys and primary human samples were executed to determine the CK1α on-target toxicity and the toxicologic profile of BMS-986397. All preclinical data described above were leveraged and integrated into a PK/PD model to establish key safety, efficacy, and PD relationships informing the starting dose/schedules for the FIH (Firs-in-Human) clinical study. Results: BMS-986397 exhibits a strong antiproliferative effect in TP53 WT (Wild-Type) AML cancer cell lines through potent and selective degradation of CK1α regardless of FAB subtype or any common oncogenic-driver mutations, with the exception of TP53. Degradation of CK1α leads to p53 stabilization and the consequent induction of p53 transcriptional targets including p21, PUMA, and BAX, thereby inducing cell cycle arrest and acute apoptosis of AML cells. This anti-AML activity is dependent on upon CRBN, CK1α, and p53. The growth inhibitory effect of BMS-986397 is also observed in hematopoietic progenitors and leukemic cells from multiple AML patient samples harboring functional p53, while sparing normal T-lymphocytes. Normal hematopoietic progenitors are less responsive to BMS-986397 treatment in a time and dose-dependent fashion. Pharmacodynamic (PD) studies indicate that sustained CK1α degradation for a minimum of 48 hours is required to stabilize p53 and, therefore, achieve antileukemic efficacy compromising AML blasts viability. A faster recovery of normal marrow progenitors and stem cells is observed compared to AML blasts, further supporting a reasonable therapeutic index for the treatment of AML and HR-MDS. Similarly, in vivo pharmacology studies in cell line-derived AML xenograft models determined a dose- and schedule-dependent PK/PD relationship, a significant CK1α degradation and consequent activation of downstream pathways led to marked tumor burden reduction and prolonged mice survival. Integrative modeling of PK/PD, efficacy, and toxicity data established key safety, efficacy, and target engagement relationships which suggested that short and intense schedules followed by prolonged off-treatment period instead of more frequent intermittent schedules may maximize cytotoxicity on AML blasts while allowing for extended recovery period of hematopoietic progenitors. Conclusions: BMS-986397 is a novel CELMoD agent with a first in class mechanism targeting CK1α degradation exhibiting strong anti-leukemic activity as a single agent in models of TP53 WT AML and HR-MDS. These data support the clinical investigation of BMS-986397 in patients with R/R AML and HR-MDS patients (NCT04951778).

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Cereblon E3 ligase modulator (CELMoD®) agents, including classic immunomodulatory (IMiD®) agents, have shown antitumor activity in MM. ALNUC is an asymmetric 2-arm, humanized IgG TCE that binds to BCMA and CD3ε in a 2 + 1 format. ALNUC mediates myeloma tumor cell killing by recruiting T cells to BCMA-expressing target cells, leading to immune synapse formation and T cell cytotoxic activity. Combining CELMoD agents with ALNUC may enhance antitumor activity. Aims: Evaluate the anti-MM potential of ALNUC+pomalidomide (POM) and novel CELMoD agents mezigdomide (MEZI) and iberdomide (IBER) in preclinical models. Methods: To evaluate effects of IMiD and CELMoD agents on ALNUC-mediated T cell anti-MM activity, healthy donor (HD) T cells and/or BCMA-expressing MM cell lines were pretreated with POM, MEZI, IBER, or DMSO for 16 h (T cells) or 72 h (MM cells) prior to co-culture; ALNUC was then added for 72 h and T cell activation and MM target cell depletion were measured. To evaluate IMiD and CELMoD agent effects on artificially exhausted T cells, HD CD3+ T cells were pretreated with anti-CD3/CD28 beads and POM, MEZI, IBER, or DMSO for 7 d prior to co-culture with MM cells and ALNUC. To determine if CELMoD treatment could reverse T cell dysfunction in patients (pts) with R/R MM, pretreatment PBMCs from pts with MM who had response (n=2) or nonresponse (n=3) to ALNUC in a phase 1 trial (NCT03486067) were co-cultured with H929 cells. Co-cultures were pretreated with MEZI 1 nM for 24 h prior to adding ALNUC. Flow cytometry was used to evaluate T cell proliferation and activation and MM cell apoptosis in all co-culture models. The effects of concurrent and sequential MEZI and ALNUC were studied in a humanized mouse H929 MM xenograft model. Results: In co-cultures with HD T cells, pretreatment of MM cells with MEZI, IBER, and POM increased ALNUC-induced tumor-cell killing potency and efficacy, resulting in IC50 decreases and greater reductions of MM cells across all cell lines vs ALNUC alone. ALNUC antitumor activity against the OPM-2 cell line was also enhanced by pre-treating T cells with MEZI and IBER, but not POM. Overall, MEZI showed the greatest enhancement of ALNUC-mediated activity in this in vitro model. In artificially exhausted HD T cells, enhanced antitumor activity was observed with MEZI and IBER, but not POM, vs DMSO. Based on the strong activity of MEZI in vitro, its ability to enhance ALNUC antitumor activity was tested in a humanized mouse MM xenograft model. Priming or concurrent treatment with MEZI enhanced T cell activation, promoted T cell infiltration of tumor tissue, and increased ALNUC-induced tumor clearance. The ability of MEZI to enhance ALNUC-mediated T-cell function was also examined in a co-culture model using MM pt-derived PBMCs harvested before ALNUC treatment. Pretreatment with MEZI increased ALNUC-mediated antitumor activity vs control in samples from both responder and nonresponder pts. Summary/Conclusion: The combination of ALNUC with novel CELMoD agents showed enhanced T cell mediated antitumor activity in both in vitro and ex vivo MM models. MEZI showed the greatest ability to enhance TCE-mediated antitumor activity while reversing artificial T cell dysfunction/exhaustion in vitro. Priming and concurrent treatment with MEZI enhanced ALNUC-induced T cell infiltration and activation in a MM xenograft model. The ability of MEZI to enhance ALNUC T cell antitumor function was confirmed using PBMCs from ALNUC monotherapy-treated pts with MM. These results provide a strong biological rationale for combining MEZI or IBER with ALNUC in the clinic to enhance responses in pts with MM. Keywords: Bispecific, Multiple myeloma, B-cell maturation antigen, Imids

T-Cell Tumor Infiltration in Tissue-Matched Baseline (screening) and On-Treatment Metastatic Tumors (arm B parts 1 and 2)

Table S1. Baseline Characteristics in the Stromal SPARC-Evaluable Population

Figure S1 . Representative SPARC staining. (A) Representative SPARC staining in stroma. (B) Representative SPARC staining in tumor epithelia.

Percentage lymphocyte subset Ki67+. All patient visits are binned by treatment cycle. Data at baseline and treatment cycle 3 were compared using the Wilcoxon signed rank test. All available data are illustrated; however, statistical comparisons were performed only for data available at both baseline and treatment cycle 3 (n=29).

Table S2. Baseline Characteristics in the Tumor SPARC-Evaluable Population

Investigator-assessed PFS and OS in patients with PD-L1 expression cutoff of 1% (A, B) or 5% (C, D). HR, hazard ratio; mo, months; OS, overall survival; PD-L1, programmed cell death ligand-1; PFS, progression-free survival.

Concentrations of CXCL10 (A) and sIL2Rα (B) at baseline and peak on-treatment values. Investigator-assessed PFS in patients with high or low serum cytokines CXCL10 (C) and sIL2Rα (D). CXCL10, interferon gamma-induced protein 10; mo, months; NE, not estimable; NR, nonresponder; PFS, progression-free survival; R, responder; sIL2Rα, soluble form of interleukin 2 receptor alpha; Tx, treatment.

Baseline (screening) vs treatment cycle 2 (P=.06; paired t test). For patient 5, baseline data were not available.

Avadomide is a cereblon E3 ligase modulator and a potent antitumor and immunomodulatory agent. Avadomide trials are challenged by neutropenia as a major adverse event and a dose-limiting toxicity. Intermittent dosing schedules supported by preclinical data provide a strategy to reduce frequency and severity of neutropenia; however, the identification of optimal dosing schedules remains a clinical challenge. Quantitative systems pharmacology (QSP) modeling offers opportunities for virtual screening of efficacy and toxicity levels produced by alternative dose and schedule regimens, thereby supporting decision-making in translational drug development. We formulated a QSP model to capture the mechanism of avadomide-induced neutropenia, which involves cereblon-mediated degradation of transcription factor Ikaros, resulting in a maturation block of the neutrophil lineage. The neutropenia model was integrated with avadomide-specific pharmacokinetic and pharmacodynamic models to capture dose-dependent effects. Additionally, we generated a disease-specific virtual patient population to represent the variability in patient characteristics and response to treatment observed for a diffuse large B-cell lymphoma trial cohort. Model utility was demonstrated by simulating the avadomide effect in the virtual population for various dosing schedules and determining the incidence of high-grade neutropenia, its duration, and the probability of recovery to low-grade neutropenia.

CC-99282 is a novel CELMoD® agent that was optimized for activity in non-Hodgkin lymphoma (NHL). CC-99282 potently degrades Ikaros and Aiolos, resulting in enhanced antiproliferative, apoptotic, and immune-stimulatory activity in diffuse large B-cell lymphoma (DLBCL) models, including those with acquired chemoresistance (Lopez-Girona A, et al. Hematol Oncol. 2021). In lymphocytes, Ikaros negatively regulates gene expression via histone modifications, including polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) (Oravecz A, et al. Nat Commun. 2015); in NHL, these epigenetic mechanisms are unclear. Using DLBCL models, we explored the relationship between CC-99282 activity and epigenetic status, and the mechanism of synergy of CC-99282 treatment and inhibition of EZH2, a PRC2 component. Baseline characteristics of human DLBCL lines and effects of CC-99282 ± tazemetostat (TAZ), a representative EZH2i, were evaluated in vitro and in vivo by chromatin immunoprecipitation sequencing, gene expression, flow cytometry, immunoblotting, enzyme fragment complementation assays, and/or CRISPR/Cas9 gene editing. Analysis of Ikaros/Aiolos degradation in > 20 DLBCL cell lines showed their loss is necessary, but not sufficient for CC-99282 efficacy. Evaluation of baseline histone marks showed that the sensitive cell lines exhibit aberrant, higher H3K27me3 coverage at promoter regions of expressed genes. This suggests a direct correlation between H3K27me3 status at these regions and CC-99282 sensitivity. In T cells, Oravecz et al. found that loss of Ikaros reduces H3K27me3 at specific chromatin sites due to its interaction with PRC2. We confirmed that these affected regions are enriched in genes upregulated upon CC-99282 treatment in DLBCL cell lines, suggesting a similar role for Ikaros in DLBCL. Pathway analysis following CC-99282 or TAZ treatment demonstrated high overlap between pathways altered by both agents. CC-99282 + TAZ combined demonstrated additive and/or synergistic antiproliferative and apoptotic effects in DLBCL cell lines. This combination did not alter Ikaros degradation or overall H3K27me3 status but increased downstream effects. Results were confirmed by CRISPR/Cas9 knockout competition assays with EZH2 and other PRC2 components. This effect was independent of cell of origin, EZH2 mutational status, or degree of CC-99282 sensitivity. Synergy was confirmed using the SU-DHL6 and DB xenograft models that are intrinsically resistant to EZH2is and CC-99282, respectively. Combination treatment yielded durable responses and tumor-free animals. Collectively, these data suggest that Ikaros could act as an epigenetic modulator through PRC2 recruitment and support the combination of CC-99282 with EZH2is in NHL (NCT03930953) to favor broad and durable clinical responses. Citation Format: Soraya Carrancio, Celia Fontanillo, Ryan Galasso, Martino Colombo, Scott Wood, Carla Guarinos, Alejandro Panjkovich, Diana Jankeel, Adam Blattler, Preethi Janardhanan, Matthew Groza, Jim Leisten, Rama Krishna Narla, Antonia Lopez-Girona, Daniel W. Pierce. Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3932.

Introduction: Mezigdomide (BMS-986348) is a novel cereblon (CRBN) E3 ligase modulator (CELMoD®) that induces potent degradation of IKZF1 (Ikaros) and IKZF3 (Aiolos), transcription factors that contribute to multiple myeloma (MM) survival. The direct tumoricidal effect of mezigdomide is mediated by rapid, deep, and sustained degradation of Ikaros and Aiolos and induction of apoptosis in MM cells. In parallel, mezigdomide induces degradation of Ikaros and Aiolos in peripheral blood mononuclear cells (PBMCs), leading to the activation of T cells. This dual mechanism of cell-autonomous tumor inhibition and immune activation has been invoked to explain the clinical observations highlighting the impact of patient immune status on treatment outcomes. Here, we provide data indicating that these mechanisms may be one and the same: the primary site of action of mezigdomide is in the tumor cell, and results in both cell-autonomous viability loss and sensitization to immune-mediated killing. Methods: To assess the impact of mezigdomide on interactions with T cells, we combined mezigdomide and alnuctamab (BMS-986349; CC-93269), an asymmetric 2-arm, humanized immunoglobulin G1-based T-cell engager that promotes T-cell-mediated killing of target cells by recruiting T cells to BCMA-expressing target cells, leading to immune-synapse formation and T-cell cytotoxic activity. Donor T cells (n = 3) or target MM cells (n = 3 cell lines with various BCMA-expression levels tested: H929 > OPM2 > L363) were pretreated with mezigdomide for 16 or 72 hours, respectively, and after mezigdomide wash off, T cells and MM cells were combined in a co-culture system followed by alnuctamab treatment for 72 hours. BCMA was measured before and after mezigdomide pretreatment. Target-cell viability, T-cell proliferation, and cytokine release were measured at the end of the co-culture period. Primity BioScreen, a flow-cytometry-based assessment of membrane-associated proteins, was performed on target MM cells (n = 5 different cell lines) and PBMCs following mezigdomide pretreatment to profile potential changes in surface proteins important for immune-synapse formation. Results: Pretreatment of target MM cells with mezigdomide for 72 hours led to enhanced cytotoxic activity of alnuctamab. These results were reproducible across 3 healthy donors and various MM cell lines, including lenalidomide- and pomalidomide-resistant derivates, but not in CRBN-deficient cell lines. Profiling of secreted factors associated with T-cell cytotoxic function revealed increased release of interleukin-2 after mezigdomide pretreatment of either T cells only or T cells and MM cells; further experiments utilizing conditioned media showed no effect of mezigdomide-induced secreted factors on T-cell-mediated killing, suggesting a primary role for T-cell-to-target-cell interaction. Primity BioScreen revealed that the profile of adhesion molecules important for T-cell-mediated killing had been shifted both in MM cells and in PBMCs after mezigdomide pretreatment, revealing several candidates potentially mediating the enhanced cytotoxicity of alnuctamab following mezigdomide pretreatment. Conclusions: Collectively, our data demonstrate that pretreatment with mezigdomide induces enhanced immune sensitization in MM cells via upregulation of membrane-associated cell-adhesion molecules leading to increased cytotoxic T-cell activity. These results support the clinical investigation of mezigdomide in combination with T-cell-engaging therapeutic modalities in patients with MM.

Introduction Compounds inducing CRBN-mediated degradation of Ikaros/Aiolos have demonstrated antitumor activity in pts with NHL. The pharmacodynamic (PD) effects of these agents include T-cell activation, increased trafficking of macrophages, and increased number of regulatory T cells (Tregs) in peripheral blood (PB). Furthermore, high baseline immune infiltration in tumors has been correlated with improved outcomes. CC-99282 (BMS-986369) is a novel CELMoD® agent that has enhanced antiproliferative, apoptotic, and immune-stimulatory effects compared with other immunomodulatory agents in various preclinical NHL models. Part A of the CC-99282-NHL-001 study (NCT03930953) showed CC-99282 monotherapy has a predictable and manageable safety profile, dominated by the class effect of neutropenia, and promising efficacy (objective response rate 42%) in heavily pretreated pts with R/R NHL. Methods CC-99282-mediated immune effects and the dependency of response on immune changes were evaluated using biomarker data from part A of CC-99282-NHL-001, including longitudinal substrate degradation, circulating tumor DNA (ctDNA), ex vivo cytokine secretion, immunophenotyping, and baseline tumor RNA-seq data. Results At steady state, CC-99282 induced deep (> 90%) and sustained degradation of Ikaros/Aiolos in monocytes, B cells, and T cells that was independent of response. PD effects were also confirmed in tumor cells obtained at screening and on treatment. PB T cells from pts dosed with CC-99282 showed increased secretion of IL-2, TNFα, INFγ, and GM-CSF on ex vivo stimulation with anti-CD3. Tumor and PB biomarkers were analyzed for correlation to response. ctDNA data available for 39/50 pts treated in part A showed high tumor burden (> 2.5 log10 of tumor-specific mutant sequence [Kurtz, et al. J Clin Oncol 2018]) in 28/39 pts (72%) at study entry, and yet 13/28 pts (43%) still achieved a partial or complete response. B-cell content estimation in screening biopsies, calculated from bulk RNA-seq data using cell-type-specific markers (Bindea, et al. Immunity 2013), confirmed similar B-cell content in responders and non-responders prior to CC-99282 treatment. In contrast to treatment with other CELMoD agents, this analysis showed that durable responses to CC-99282 were observed in pts with R/R NHL despite their high tumor burden and low immune infiltration. Baseline PB immunophenotype was not significantly associated with response. However, unsupervised clustering of changes in immune cell subsets in pts between baseline and cycle (C) 1 day (D) 15, revealed 5 clusters (Figure 1). Cluster 5, enriched in diffuse large B-cell lymphoma (DLBCL) non-responders, showed upregulation of peripheral Tregs without changes in T-cell activation. Clusters 1 and 4 also showed upregulation of Tregs, with cluster 1 showing additional immune activation. Cluster 2, enriched for follicular lymphoma (FL), only showed T-cell activation. Cluster 3 was enriched in responders without changes in immune subsets upon CC-99282 treatment, suggesting a stronger direct effect on tumor cells in the early stages of treatment. Baseline tumor biopsies from pts in all clusters had similar Treg levels, independent of their immune changes upon treatment. Further, longitudinal analysis of ctDNA during treatment showed that all clusters of responders had a similar decrease in ctDNA that was maintained until the end of treatment despite increases in Tregs. Immunophenotyping at C2D1 and C3D1 showed that immune activation was maintained in responders and increases in Tregs progressively extended to all pts independently of response. The implications of these differences remain unclear. The only previous systematic analyses of the prognostic role of Tregs in lymphoma concluded that increase in Tregs correlated with longer overall survival for pts with DLBCL, but found no significant association in pts with FL (Glowala-Kosinska, et al. Eur J Haematol 2013; Peng, et al. J Cancer Res Clin Oncol 2020). Conclusion Taken together, these results indicate a link between patterns of immune activation and response to CC-99282, but whether this link is causative, or correlative, remains to be elucidated. Responder subsets were identified based on effects on Tregs and immune activation; however, all cases led to durable responses. Further study and validation of these data could provide rationale for pt selection and combination strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction: CC-90009 is a novel cereblon E3 ligase modulator (CELMoD ®) agent that is a first-in-class degrader of translation termination factor G1 to S phase transition 1 (GSPT1). CC-90009 has demonstrated antileukemic activity as a single agent and is currently under investigation in patients with acute myeloid leukemia (AML; NCT02848001). Treatment with CC-90009 led to rapid reductions in peripheral and bone marrow blasts, and demonstrated preliminary promising efficacy, including several complete remissions, in patients with relapsed or refractory AML. Here, we describe the identification and preclinical activity of select anti-AML agents as potential combination partners of CC-90009 to further improve its efficacy and therapeutic index. Based on these results, the combination activity of CC-90009 with venetoclax (VEN)/azacitidine (AZA) is being evaluated in a phase 1/2 trial in patients with AML (NCT04336982).

CC-99282 is a novel, oral CELMoD ® agent currently under investigation in phase 1 clinical studies in patients with relapsed or refractory (R/R) non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Mechanistically, CC-99282 interacts with the CRL4 CRBN E3 ubiquitin ligase substrate receptor CRBN to induce recruitment and ubiquitin-mediated proteasomal degradation of transcription factors Ikaros and Aiolos. The design intent for CC-99282 included efficient absorption, deep tissue distribution, and prolonged exposure to optimize activity in bulky lymphoma lesions. Recently, we reported that CC-99282 shows potent antitumor activity in different preclinical models of diffuse large B cell lymphoma (DLBCL; Lopez-Girona, et al. Hematol Oncol. 2021). Here, we provide an expanded analysis of CC-99282 activity as a monotherapy, as well as examine its synergistic activity with anti-CD20 antibody treatment, in preclinical models of NHL including DLBCL and follicular lymphoma (FL).