The t(4;14) chromosomal translocation drives overexpression of the histone methyltransferase NSD2 and defines a high-risk segment of multiple myeloma (MM) patients. Herein, we report the discovery of NSD2-LDD, a cereblon-recruiting and PWWP1-mediated ligand directed degrader (LDD) that selectively and potently eliminates full length and PWWP1 domain containing NSD2 protein isoforms. NSD2-LDD treatment induces global loss of H3K36me2 leading to promoter-proximal spreading of H3K27me3 and re-wiring of cis-regulatory interactions that reverse t(4;14) transcriptional programs. These effects suppress MM disease-associated phenotypes including stromal adhesion, three-dimensional colony growth and paracrine signaling. By integrating patient single cell profiles with model 3D epigenomic and spatial transcriptomics, we delineate t(4;14) disease state together with the tumor-intrinsic reprogramming and resultant remodeling of the bone marrow microenvironment upon NSD2 degradation. In cell line derived xenografts and genetically engineered mouse models of t(4;14), NSD2-LDD extends median survival accompanied by tumoral H3K36me2 loss and niche re-modelling. Although the NSD2-LDD response is restricted to PWWP1-containining models, collectively this work validates NSD2 as a tractable dependency and supports clinical development of NSD2 degradation as a novel, targeted therapeutic strategy in high-risk MM.

B-cell lymphoma 6 protein (BCL6) is an oncogenic driver dysregulated and overexpressed in subtypes of high-risk non-Hodgkin lymphoma (NHL). Development of agents that induce the targeted degradation of BCL6 would offer a promising novel therapeutic approach. For this purpose, we employed ligand-directed degraders, heterobifunctional molecules linking a BCL6-binding ligand to a cereblon recruiter, enabling cereblon-mediated BCL6 degradation. Through a focused optimization effort, we identified highly potent BCL6 degraders, culminating in the selection of BMS-986458 for clinical development. BMS-986458 induces rapid cereblon-dependent BCL6 degradation while sparing known CRBN neosubstrates such as CK1α, GSPT1, Aiolos, Ikaros, or SALL4. Oral administration of BMS-986458 results in dose-dependent pharmacokinetics, pharmacodynamics, and significant antitumor efficacy in mouse models of lymphoma. A potential first-in-class agent, BMS-986458, is currently being evaluated in a phase 1/2 clinical trial (NCT06090539) for patients with relapsed/refractory NHL.

BMS-986365 overcomes resistance to ARPIs due to an adaptative mechanism in vitro.

Supplementary Fig. 1. Golcadomide induced transition from CRBNopen to CRBNclosed for efficient recruitment and degradaion. Supplementary Fig. 2. Antiproliferative effect of golcadomide is mediated via CRBN-based IKZF1/3 degradation. Supplementary Fig. 3. Golcadomide is broadly active with potent cell autonomous antiproliferative activity. Supplementary Fig. 4. Antiproliferative effect of golcadomide is mediated via CRBN based IKZF1/3 degradation. Supplementary Fig. 5. Golcadomide demonstrated minimal broad cytotoxicity and reversible effects on neutrophil maturation. Supplementary Fig. 6. Golcadomide stimulated immune activation, reversed T cell exhaustion, and exhibited potent antitumor synergy with rituximab. Supplementary Fig. 7. Overview of different treatment conditions and technical replicates in the CRISPR screen. Supplementary Fig. 8. CRISPR screen identified genes and pathways that exacerbated or attenuated antiproliferative effects of golcadomide in SU-DHL-4 upon inactivation. Supplementary Fig. 9. KO of PRC2 complex components enhanced cell antiproliferation by golcadomide. Supplementary Fig. 10. Apoptosis induction is linked to the antiproliferative effects of golcadomide. Supplementary Fig. 11. NF-κB hyperactivation reduced golcadomide-induced DLBCL cell apoptosis. Supplementary Fig. 12. XPO1 inhibitor enhances antiproliferative effects of golcadomide. Supplementary Fig. 13. AMBRA1 knockout renders resistance to golcadomide via cyclin D3/CDK4/6/Rb axis in DLBCL. Supplementary Fig. 14. KCTD5 ameliorated the antiproliferative effects of golcadomide via GNG5. Supplementary Fig. 15. Chemical synthesis of golcadomide. Supplementary Fig. 16. Representative gating strategies for flow cytometry-based assays.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous disease with limited treatment options and a poor prognosis, especially for patients refractory to standard therapies. We report the discovery of golcadomide (CC-99282), an oral cereblon-modulating CELMoD agent designed using target-specific knowledge and optimized pharmacologic properties for the treatment of DLBCL. Golcadomide exhibited rapid, deep, and sustained degradation of transcription factors IKZF1 and IKZF3, surpassing the antitumor activity of the IMiD agent lenalidomide in preclinical models. In human lymphoma cell lines, golcadomide downregulated MYC, activated IFN-stimulated genes, and promoted antiproliferation, apoptosis, and immunogenic cell death. In mouse xenografts, golcadomide preferentially distributed to tissues known to be affected by lymphoma, resulting in enhanced tumor regression and tumor-free outcomes. Pharmacologic and CRISPR screening further revealed genes and pathways underlying golcadomide's antitumor efficacy. These findings supported golcadomide as a promising drug candidate for DLBCL, providing a strong rationale for future golcadomide-based regimens.Significance: Golcadomide is an oral cereblon-modulating agent for the treatment of DLBCL. It exhibited rapid, deep, and sustained degradation of IKZF1 and IKZF3, preferentially accumulated in lymphoma residence tissues, and delivered robust antitumor activity. These results provide a strong rationale for continued clinical investigation of golcadomide for patients with DLBCL.

Aberrant expression of anaplastic lymphoma kinase (ALK) is the oncogenic driver of 3-7% of non-small cell lung cancer (NSCLC) cases. Six ATP-competitive tyrosine kinase inhibitors (TKIs) of ALK have been approved by the FDA to treat patients with ALK-positive NSCLC, but mutations conferring drug resistance often occur in the ATP-binding pocket of ALK signifying a need for alternative modalities to inhibit ALK function. Here we report that ALK-driven tumor growth can be suppressed by novel molecular glue cereblon E3 ligase modulatory drugs (CELMoDs) that trigger ubiquitylation and subsequent proteasomal degradation of ALK. A cryo-EM structure of the ALK-CELMoD-cereblon ternary complex revealed that ALK interacts with cereblon and drug through a site distal to its ATP-binding pocket and features a surprising degron structure distinct from the canonical G-loop degron established for other cereblon neosubstrates. Subsequent lead optimization identified a series of ALK CELMoDs with improved brain penetration. Our in vitro and in vivo studies support the hypothesis that a brain penetrant ALK CELMoD would be an effective therapy for ALK-positive NSCLC with similar or better efficacy and safety profile compared to TKIs. Furthermore, combination treatment with TKIs might enhance ALK CELMoD efficacy due to distinct binding sites and mechanisms of action. Finally, the novel degron suggests that the degradome of CELMoDs goes beyond G-loop-containing proteins, thus emphasizing the importance and potential of this modality to target a wide spectrum of previously un-druggable proteins in human diseases. Zheng Wang, Massimo Ammirante, Martina Malatesta, Alex Cortez, Young Chen, Jinyi Zhu, Dahlia Weiss, Vijaya Lakshmi Dommeti, Geraldine Hernandez, Joshua M. Baughman, Adwait Sathe, Matthew Groza, Maria Donoso, Roxxana Beltran, Regina Paramitha, Ryan Davison, Atefeh Garzan, Barbra Pagarigan, Michelle Slade, Jacobo Fuentes, Jennifer Buenviaje, Shan Yu, Xinde Zheng, Andres H de la Peña, Mariko Riley, Gabe Mintier, Surendra Nayak, Shuichan Xu, Rama Narla, Ellen Filvaroff, Deborah Mortensen, Lihong Shi, Celia Fontanillo Fontanillo, Christoph Zapf, Neil Bence, Mark Rolfe. Brain-penetrant molecular glue degraders targeting ALK via a novel degron: a potential therapeutic approach for ALK-positive NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6378.

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.

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. The t(4;14) translocation activates nuclear receptor binding SET domain protein 2 (NSD2) to define a molecularly distinct subset of multiple myeloma patients with especially unfavorable prognosis. Despite the clear delineation of t(4;14) as a segment with NSD2 activation as the driver, an incomplete understanding of the mechanistic cascade towards myelomagenesis in these patients has been a major hurdle. We thus developed a tool CRL4CRBN E3 ubiquitin ligase-dependent ligand-directed degrader (LDD) that rapidly catalyzes proximity-induced degradation of NSD2 to facilitate the multi-omics characterization of NSD2's role in myeloma, uncovering direct impacts on both the local activity and long-range organization of cis-regulatory elements governing critical programs. Methods. t(4;14) and non-t(4;14) multiple myeloma preclinical models, at baseline and with NSD2-LDD treatment, were profiled using: CUT&RUN/ChIP-Seq (CTCF, H3K27ac/me3, H3K36me2/3, H3K4me1/3), Hi-C, ATAC-seq, RNA-seq, histone mass spectrometry. Soft agar colony formation, fibronectin cell adhesion, and FBS chemotaxis migration assays were used to validate the phenotypic consequences in vitro, whereas cell-line derived xenograft models were used to assess tumor control in vivo. Additionally, a co-culture system of myeloma cells with bone marrow stromal cells was used to evaluate both paracrine signaling and cell adhesion-mediated drug resistance. Results. NSD2 degradation-dependent therapeutic benefit was demonstrated across multiple in vivo models: up to 73% tumor volume reduction in mice bearing subcutaneous KMS34 tumors as well as intrafemoral engraftment of luciferase-labelled KMS34 cells that not only saw significant survival benefit (p = 0.034) but also absence of extra-nodal growth - contrasting CNS-resident and contra-lateral metastases in the vehicle group. Seeing that the in vivo efficacy of NSD2 degradation coincided with deep NSD2 degradation (-94%) as well as loss of its direct catalytic product H3K36me2 (-74%), we turned to in vitro models to elucidate NSD2's trans-omics mechanistic cascade. Consistent with H3K36me2 loss (9x) and H3K27me3 gain (3x) being the most significant changes among the 40 histone post-translational modifications detected by mass spectrometry, cells treated with NSD2-LDD exhibit global restoral of transcriptional control across multiple modalities with: (1) 3x excess number of down-regulated genes based on RNA-seq, (2) 6x more sites with reduced accessibility based on ATAC-seq, (3) 2x surplus of genomic regions with significantly more heterochromatic compartment score based on Hi-C. Besides changes to local activity, comparison of genome-wide Hi-C contact probability further revealed a specific gain of long-range interactions around the scale of 2-megabase upon NSD2 degradation; accordingly, we observed an 20% increase in the strength of heterochromatic long-range interaction. We then integrated 7 different epigenetic signals to categorize 110k candidate cis-regulatory regions into 6 baseline chromatin states such as boundaries, promoters, enhancers, polycomb regions, etc. to determine the predictors of genes responsive to NSD2 modulation. Ultimately, we pinpointed H3K36me2 at enhancers coincident with H3K27me3 repressive looping at promoters to primarily underlie transcriptional down- and up-regulation upon treatment, respectively influencing tumor-extrinsic (e.g., cell-cell junction: CD44, JAM2, TJP1) and intrinsic (e.g., B cell markers: LAIR1, POU2AF1, IL12RB1) pathways. These effects were subsequently reflected in not only dose-dependent reduction in growth, adhesion, and migration, but also elevated sensitivity to dexamethasone (monoculture) and bortezomib (HS-5 co-culture) following NSD2-LDD pre-treatment. Conclusions. We leverage targeted NSD2 degradation to clarify the epigenetic signaling circuitry driving t(4;14) multiple myeloma, implicating repressive polycomb regions and activating enhancers in balancing normal programming versus disease-associated processes. With evidence across in vitro and in vivo models, we affirm the effectiveness of targeted NSD2 degradation against a t(4;14)-specific chromatin-based vulnerability not addressed by existing approaches and rationalizes the combination of NSD2 degraders with standard of care regimens for increased efficacy.

Abstract The androgen receptor (AR) is a key driver of the cellular processes that contribute to the pathology of prostate cancers at most stages, and drugs that inhibit the activity of this receptor or interfere with androgen biosynthesis have become the cornerstone of treatments for prostate cancer. While both types of therapy have had a significant positive impact on disease progression and overall survival, de novo and acquired resistance remains an impediment to a durable clinical response in patients with metastatic castration-resistant prostate cancer (mCRPC). The mechanisms underlying drug resistance are complex and multifactorial, being attributable to AR overexpression, point mutations in the receptor that alter drug pharmacology, and to the upregulation of receptor-associated coregulators, which render AR less dependent on an activating ligand. Regardless, absolute inhibition of AR signaling remains the objective of contemporary approaches to treat prostate cancer. Recently, there has been specific interest in the development of approaches to eliminate AR proteins using drugs that enable targeted degradation of AR in cancer cells.This puts in context the significance of our discovery of BMS-986365 (also known as CC-94676), a heterobifunctional ligand-directed degrader (LDD) that enables the CRL4CRBN E3 ligase-dependent ubiquitination and degradation of AR (DC50 10 to 40 nM, Ymin 7 to 19%). BMS-986365 is a highly potent and selective AR degrader that induces rapid and deep degradation of both wildtype and mutant forms of the receptor residing in either the cytoplasmic or nuclear compartments of the cell. The drug is ~100-fold more potent than enzalutamide (ENZ) at inhibiting androgen-stimulated transcription of AR target genes, and 10 to 120-fold more potent than ENZ at inhibiting AR-dependent proliferation of multiple prostate cancer cell lines in vitro. In animal models of advanced prostate cancer, BMS-986365 demonstrates on-target activity, degrading AR, suppressing AR signaling, and inhibiting tumor growth. Indeed, tumor volume reductions of 63-92% were achieved by BMS-986365 in validated models of advanced CRPC and therapy resistant patient-derived xenografts, including those with acquired resistance to ENZ. Collectively our preclinical data suggest that the AR degrader BMS-986365 is superior to standard-of-care AR antagonists, such as ENZ, in both preclinical and disease-relevant animal models, and support its clinical development for treatment of prostate cancer. BMS-986365 has advanced into clinical studies where it has demonstrated encouraging clinical activity in patients with mCRPC. Citation Format: Shuichan Xu, Surendra Nayak, John D. Norris, Massimo Ammirante, Emyly Rychak, Suzanne E. Wardell, Toshiya Tsuji, Ken Liu, Joseph Meiring, Joseph R. Piccotti, Deepak Dalvie, Debbie Liao, Raju Kandimalla, Nadia Guerrero, Lisa Sapinoso, Jennifer G. Baker, Yeeun Bae, Joshua Baughman, Brandon Toyama, Celia Fontanillo Fontanillo, Stephen Norris, Evan J. Horn, Veronique Plantevin-Krenitsky, Deborah Mortensen, Brian Cathers, Marie Hong Nguyen, Joshua D. Hensen, Lawrence G. Hamann, Donald P. McDonnell, Rama Krishna Narla, Mark Rolfe. Discovery of BMS-986365, a ligand-directed androgen receptor degrader (AR LDD) with a dual mechanism-of-action and best-in-class potential, for the treatment of advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND02.

The reactivation of fetal hemoglobin (HbF, α2γ2) is a recognized disease modifier for sickle cell disease (SCD) where treatments are limited and there remains a significant unmet need. Achieving a concentration of 30% total HbF, distributed homogeneously in at least 70% of HbF-expressing (F) red blood cells (equating to 10 pg per F cell), has been shown to inhibit the polymerization of sickle hemoglobin (HbS, α2βS2) under deoxygenated conditions and significantly ameliorate SCD-related symptoms (Steinberg MH et al., Blood, 2014). Several transcriptional repressors of HbF have been identified, including B-cell lymphoma/leukemia 11A (BCL11A), zinc finger and BTB domain containing 7A (ZBTB7A) and widely interspaced zinc finger protein (WIZ), which until now, were considered undruggable. Here, we describe the development of BMS-986470, a novel, orally bioavailable HbF-activating cereblon (CRBN) E3 ligase modulator (CELMoD™) agent that targets ZBTB7A and WIZ for degradation and is currently under clinical development for SCD (NCT06481306). To identify HbF-activating CELMoD compounds, a phenotypic screen was conducted using primary erythroblasts derived from healthy CD34+ cells. Compounds with dual degrading activity targeting ZBTB7A and WIZ emerged as the most effective γ-globin inducers, resulting in the highest levels of HbF. BMS-986470 was subsequently designed following an extensive structure-activity-relationship optimization campaign to define the optimal degradation of ZBTB7A and WIZ for maximal γ-globin induction. Global proteomic profiling of BMS-986470 demonstrated that ZBTB7A and WIZ were the predominant proteins regulated in the proteome, and predictive modeling confirmed that they were the major substrates contributing to HbF induction. Degrons in both proteins were identified, and ternary complex structures of ZBTB7A:BMS-986470:CRBN/DDB1 and WIZ:BMS-986470:CRBN/DDB1 were resolved at high resolution, explaining the selectivity and potency of BMS-986470 against both substrates. Genetically engineered HUDEP-2 cells were established to validate the advantage of ZBTB7A and WIZ dual degradation over the degradation of WIZ alone at inducing HbF. Knockout of either ZBTB7A or WIZ alone significantly induced γ-globin expression and the dual silencing of both transcription factors synergistically enhanced the proportion of F-cells to >95% and total HbF tetramer to >80%. Introducing single point mutations into the degrons of either ZBTB7A or WIZ protein alone reduced BMS-986470-mediated γ-globin induction, and expression of double point mutations in both ZBTB7A and WIZ significantly abrogated γ-globin induction. BMS-986470 was also a potent degrader of ZBTB7A and WIZ in primary erythroblasts derived from healthy donor and SCD patient samples, achieving levels of >90 % F-cells and >40 % total HbF, without affecting erythroblast viability or erythroid differentiation. In these preclinical models, HbF induction by BMS-986470 was significantly higher than the standard of care, hydroxyurea. Utilizing a murine model of human erythropoiesis, BMS-986470 did not affect human erythroblast differentiation and demonstrated a significant dose-dependent decrease in hZBTB7A and hWIZ protein, along with an increase in F-cells and γ-globin expression. Notably, mice treated at the minimal efficacious dose achieved up to a 3-fold increase in total HbF levels as compared to the vehicle control. BMS-986470 also significantly induced F-cells and γ-globin protein in the peripheral blood of Townes mice expressing human CRBN, and reduced sickling under hypoxic conditions, ex vivo. In naïve healthy cynomolgus monkeys treated daily with BMS-986470 for 16 days, with a 2-week observation period, significant, dose dependent degradation of WIZ and ZBTB7A was evident, along with an increase in circulating immature erythrocytes, and increased HBG1/2 transcript, γ-globin protein and erythroid progenitor markers in the peripheral blood and bone marrow. BMS-986470 was well-tolerated at all doses tested. In summary, we disclose the discovery and preclinical characterization of BMS-986470, a potential first-in-class, dual degrader of ZBTB7A and WIZ, with robust γ -globin induction activity leading to HbF levels predicted to significantly ameliorate SCD pathology. BMS-986470 is currently under clinical investigation (NCT06481306) for patients with SCD.

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).

Supplementary Figure S1:Effect of CB-5083 on cell growth and survival in multiple myeloma and solid tumor cell lines; Supplementary Figure S2:CB-5083 activates UPR;Supplementary Figure S3:CB-5083 transcriptional response is unique in comparison to proteasome inhibitors;Supplementary Figure S4:CB-5083 enhances the antitumor activity of proteasome inhibitors; Supplementary Figure S5: Nrf1 upregulation induced by bortezomib is inhibited by CB-5083; Supplementary Figure S6:CB-5083 demonstrates a broad activity in multiple myeloma relevant in vivo models; Supplementary Table S1: List of antibodies used in the manuscript.

Supplementary Fig. S1 from Comparison of biochemical and biological effects of ML858 (salinosporamide A) and bortezomib

Supplementary Table 1. Proteomic data

Supplementary Data, Figure Legends 1-3 from Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

Supplementary Figure 1 from Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

Supplementary Figure 3 from Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer

Supplementary Figure 2 from Evaluation of the Proteasome Inhibitor MLN9708 in Preclinical Models of Human Cancer