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.

Burkitt lymphoma (BL) is characterized by the translocation and dysregulation of the proto-oncogene MYC. Owing to its high aggressiveness, the clinical therapeutics for BL remain unmet. In view of this pathological feature, molecular glue degraders have been widely explored as a targeted therapeutic strategy. This study reports a first-in-class, orally bioavailable dual-target molecular glue degrader that co-degrades GSPT1 and CK1α and demonstrates potent anti-tumor activity in BL. MYC hyperactivity imposes a non-oncogene addiction to GSPT1, its depletion of which collapses protein synthesis and selectively eradicates MYC-addicted tumor cells. Furthermore, down-regulation of CK1α attenuates MYC expression, implying that co-targeting GSPT1 and CK1α may yield superior therapeutic efficacy. To identify the desired molecular glue, we designed a targeted library focused on cereblon (CRBN) and successfully identified the molecular glue degrader, INNO-235. We found that CRBN functions as the requisite E3 ubiquitin ligase for INNO-235, enabling nanomolar-level, proteasome-dependent degradation of both CK1α and GSPT1. Notably, no impact on IKZF1 or IKZF3 was observed. Additionally, INNO-235 demonstrated minimal toxicity in healthy volunteer-derived peripheral blood mononuclear cells, indicating a potentially favorable therapeutic window. In a systematic evaluation, INNO-235 exhibited potent anti-proliferative activity in BL cell lines, including those with TP53 mutations, as shown by nanomolar IC50 values. At the cellular phenotypic level, INNO-235 treatment significantly induced apoptosis and caused cell cycle arrest at the G0/G1 phase. In a Daudi cell-derived xenograft mouse model, orally administered INNO-235 exhibited dose-dependent tumor growth inhibition without observable significant changes in mouse body weight. Notably, compared to INNO-220 (a CK1α single-target molecular glue degrader) and MRT-2359 (a GSPT1 single-target molecular glue degrader), INNO-235 demonstrated superior anti-tumor activity and overall survival. To elucidate INNO-235's mechanism of action, we performed RNA-seq analysis in INNO-235 treated Daudi cells. The results revealed significant enrichment of differentially expressed genes involved in endoplasmic reticulum protein processing, implicating the activation of the integrated stress response (ISR). Subsequent validation confirmed dose-dependent upregulation of key ISR effector (p-eIF2α, ATF4, CHOP) and pro-apoptotic PUMA, concurrent with downregulation of anti-apoptotic BCL-2. These findings indicated that INNO-235 induced p53-independent apoptosis in TP53-mutated BL cells through ISR pathway activation. In conclusion, INNO-235 represents a first-in-class GSPT1/CK1α dual-target molecular glue degrader with significant therapeutic potential for lymphomas. By inducing both p53-dependent and TP53-independent apoptosis, INNO-235 offers a promising therapeutic strategy for TP53-mutated BL and other MYC-driven malignancies with limited treatment options.

Therapeutic options for aggressive and high-risk lymphomas, such as double-hit lymphoma (DHL), remain limited. As a novel therapeutic modality, molecular glue degraders represent a promising strategy by harnessing E3 ubiquitin ligases to selectively target oncogenic driver proteins for degradation. Here, we report INNO-235, a novel cereblon (CRBN)-binding molecular glue that concurrently degrades CK1α and GSPT1, effectively inducing apoptosis in DHL cells. Our previous studies demonstrated that selective CK1α molecular glue degraders possess therapeutic potential in certain types of B-cell lymphomas. Building upon this finding and aiming to explore the potential advantages of a multi-target degradation strategy, we rationally designed a unique CRBN-binding molecular glue library. Through library screening efforts, we identified INNO-235 with dual-targeting degrader of GSPT1 and CK1α. INNO-235 demonstrated potentanti-proliferative activity against diffuse large B-cell lymphoma (DLBCL) cell lines, with minimal cytotoxicity in peripheral blood mononuclear cells (PBMCs) from healthy donors. Notably, INNO-235 exhibited single-digit nanomolar antiproliferative activity in all tested DHL cell lines showing a tenfold increase in potency compared to MRT-2359, a GSPT1-selective degrader. Importantly INNO-235 retained robust efficacy in p53-mutant DHL DLBCL cell lines, which are resistant to INNO-220, a selective CK1α degrader. Mechanistic studies confirmed that INNO-235 degrades both GSPT1 and CK1α in a CRBN-dependent manner and maintains strong degradation activity across genetically diverse DHL DLBCL cell lines. Furthermore, we observed that INNO-235 treatment induced apoptosis in DHL DLBCL cell lines irrespective of p53 mutation status and concurrently caused G0/G1 phase arrest in p53 wild-type DHL cell lines. I n vivo evaluation using a Will-2 xenograft model revealed that oral administration of INNO-235 exhibited significant tumor growth inhibition compared to vehicle controls and overperformed either CK1α or GSPT1 single-targeting degrader. Remarkably, in the high-dose INNO-235 group (10mg/kg), half of the treated mice achieved complete tumor regression with no recurrence occurred during the study period. Ex vivo analysis of tumor tissues confirmed CK1α and GSPT1 degradation by INNO-235. To elucidate the mechanism of action of INNO-235, we employed RNA sequencing in Will-2 and found INNO-235 activates both p53 signaling and integrated stress response pathways. INNO-235 exerts its anti-proliferative effects on DHL cells through a dual mechanism. In wild type DHL DLBCL cell lines, INNO-235 triggers CK1α degradation-dependent stabilization and activation of p53 with concomitant p21 upregulation and driving apoptosis; concurrently, GSPT1 degradation induces phosphorylation of eIF2α and upregulates ATF4/CHOP, triggering the terminal integrated stress response. Additionally, we observed concomitant over 60% downregulation of oncogenic c-Myc protein, a key driver of DHL pathogenesis. Notably, CK1α selective degraders failed to inhibit p53-mutant DHL proliferation, whereas INNO-235 maintained nanomolar potency, highlighting the importance of dual-targeting for broad therapeutic efficacy. Collectively, INNO-235 represents a first-in-class, orally bioavailable CRBN-based molecular glue capable of concurrently degrading both GSPT1 and CK1α at nanomolar concentrations. By inducing classical apoptosis in p53 wild-type cells and p53-independent tumor cell apoptosis via the ISR pathway in p53-mutant cells, INNO-235 overcomes the limitations of single-target therapies. This dual targeting GSPT1 and CK1α for degradation presents an attractive therapeutic strategy with a single-agent for DHL across diverse genetic backgrounds. This work provides compelling rationale for clinical translation in genetically diverse high-grade lymphomas.

CC-99282 (BMS-986369) is a novel, oral CELMoD® agent under investigation in a phase 1/2 clinical study in patients (pts) with relapsed or refractory (R/R) NHL (NCT03930953), where early CC-99282 monotherapy data show a manageable safety profile and promising efficacy in heavily pretreated pts. Mechanistically, CC-99282 interacts with CRBN, a CRL4CRBN E3 ubiquitin ligase substrate receptor, to induce recruitment and ubiquitin-mediated proteasomal degradation of the transcription factors Ikaros and Aiolos. Compared with immunomodulatory agents such as lenalidomide that also degrade Ikaros/Aiolos, CC-99282 demonstrates a faster, deeper, and more sustained degradation of these transcription factors. This leads to derepression of cyclin-dependent kinase (CDK) inhibitors and interferon-stimulated genes, such as IRF7, and reduction of the critical oncogenic factor, c-Myc, resulting in potent, autonomous cell killing and apoptosis in a large panel of genetically heterogeneous NHL cell lines. CC-99282 also demonstrated in vivo activity in NHL xenograft models, leading to tumor regression and tumor-free animals (Lopez-Girona et al. Hematol Oncol 2021). Antitumor effects of CC-99282 were independent of cell origin (activated B-cell, germinal center B-cell, or primary mediastinal B-cell lymphoma subtypes of diffuse large B-cell lymphoma) or the presence of high-risk chromosomal translocations (MYC, BCL2, and/or BCL6) (Carrancio et al. Blood 2021). To further elucidate the mechanism of action of CC-99282 and identify additional genes and pathways that modulate CC-99282-mediated antiproliferative effects, a genome-wide CRISPR/Cas9 pooled screen was performed in SU-DHL-4, an NHL cell line with moderate sensitivity to CC-99282. Top-ranked CC-99282-sensitizing or resistance genes identified in the screen were then characterized in a panel of NHL cell lines, using genetic tools and orthogonal confirmatory methods. As expected, genetic knockout (KO) of the CC-99282 targets Ikaros or Aiolos sensitized NHL cells to CC-99282-mediated antiproliferation and attenuated cell survival. KO of select epigenetic modifiers, cell-cycle activators, nucleoplasmic transport factors, and anti-apoptotic genes also enhanced the antiproliferative effect of CC-99282, with the anti-apoptotic gene BCL2 among the top-ranked hits. Venetoclax (VEN), a BCL2 inhibitor approved for the treatment of pts with chronic lymphocytic leukemia and acute myeloid leukemia, was tested in aggressive NHL cell lines alone or in combination with CC-99282. Sensitivity to VEN largely correlated with BCL2 expression, and a synergistic effect with CC-99282 was observed in 58.8% of the aggressive NHL cell lines tested. With respect to genes that reduced sensitivity to CC-99282, top-ranked candidates included previously identified components/regulators of the CUL4-DDB1-RBX1-CRBN E3 ubiquitin ligase complex, such as CRBN and UBE2G1, neddylation and deneddylation machinery, and members of key signaling pathways, such as repressors of NF-κB signaling and cell-cycle regulators. KO of NF-κB suppressor genes induced hyperactivation of the canonical and/or noncanonical NF-κB pathway and reduced CC-99282-induced tumor cell apoptosis. Resistance to CC-99282 was also conferred by KO of autophagy and beclin 1 regulator 1 (AMBRA1), an E3 ligase adaptor that regulates the stability of cyclin D, and low AMBRA1 level was reported as a poor prognosis marker for pts with diffuse large B-cell lymphoma (Simoneschi et al. Nature 2021). AMBRA1 KO led to CC-99282 resistance by promoting stabilization of cyclin D3, activation of CDKs, phosphorylation of retinoblastoma, and subsequent cell-cycle progression, thus reducing CC-99282-induced antiproliferation. Lastly, combination of CC-99282 with CDK4/6 inhibitors palbociclib/abemaciclib, which prevent cell-cycle progression, showed strong synergistic effects in NHL cell lines without RB1 loss or CCND3 mutations. CC-99282 is a novel CELMoD agent that demonstrates potent antiproliferative and pro-apoptotic effects in NHL cells. This study revealed genetic alterations that may be associated with clinical response to CC-99282 in pts with NHL. These in vitro data highlight potential biomarkers that could facilitate patient stratification in future clinical trials and provide rationale for combination therapies that may improve clinical outcomes for pts with R/R NHL.

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).

Introduction: CC-99282 is an Ikaros- and Aiolos-targeting oral CELMoD agent in development for the treatment of relapsed/refractory non-Hodgkin lymphomas (R/R NHL). While patients with R/R NHL tend to have poor prognoses, agents mediating Ikaros/Aiolos degradation, such as lenalidomide (LEN) and avadomide (AVA), have shown promise in the R/R NHL setting. Methods: The effects of CC-99282 were studied in lymphoma and nontumorigenic human cell lines, primary cells, and lymphoma xenograft models. Shotgun proteomics, immunoblotting, enzyme fragment complementation assays, and flow cytometry studies assessed substrate degradation selectivity of CC-99282. CRISPR/Cas9 gene editing was performed in lymphoma cell lines and a T-cell restimulation assay evaluated immune-stimulatory effects. Results: CC-99282 demonstrated potent autonomous cell killing and apoptosis in DLBCL cells, independent of the origin subtype, presence of high-risk chromosomal translocations, or acquired resistance to doxorubicin. Of 23 cell lines tested, 20 showed sensitivity to CC-99282 with IC50 of 1–500 nM; similar low IC50 values were achieved only in 3 cell lines treated with AVA and none with LEN. No general cytotoxicity was observed in nontumorigenic cells in culture treated with ≤10 µM of CC-99282. While genetic knockout of either IKZF1/Ikaros or IKZF3/Aiolos sensitized DLBCL cells, the expression of degradation-resistant mutants of Ikaros/Aiolos protected them from the antiproliferative effects of CC-99282. The deep and sustained degradation of Ikaros/Aiolos in DLBCL cells treated with CC-99282 correlated with: the induction of interferon (IFN)-inducible genes (IRF7, IFIT3, and DDX58); a reduction of the highly critical oncogenic factors c-Myc and IRF4; and the induction of cleaved caspases and PARP. Several DLBCL xenograft models treated with CC-99282 (1–30 mg/kg) at different dosing schedules showed significant (P < 0.0001) tumor regression and tumor-free status (Figure). The strong antitumor activity was accompanied with robust distribution of CC-99282 across multiple tissues, including effectively crossing the blood-brain barrier in the intracranial xenograft model of central nervous system lymphoma. Degradation of Ikaros/Aiolos by CC-99282 in T cells correlated with increased secretion of IL-2, a hallmark of immune activation. CC-99282 was able to reverse T-cell exhaustion and induce secretion of the effector cytokines/chemokines GM-CSF, IFNγ, and TNFα, at concentrations (0.1–100 nM) that induced strong antitumor effects in vitro. The research was funded by: Bristol Myers Squibb Keywords: Molecular Targeted Therapies Conflicts of interests pertinent to the abstract A. Lopez-Girona Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb L. Groocock Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb Z. Mo Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb R. K. Narla Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb P. Janardhanan Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb S. Wood Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb D. Mendy Employment or leadership position: Bristol Myers Squibb L. Barnes Employment or leadership position: Bristol Myers Squibb S. Peng Employment or leadership position: Bristol Myers Squibb D. Jankeel Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb C. Fontanillo Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb S. Carrancio Employment or leadership position: Bristol Myers Squibb Stock ownership: Bristol Myers Squibb J. Hansen Employment or leadership position: Bristol Myers Squibb

Many patients with multiple myeloma (MM) initially respond to treatment with modern combination regimens including immunomodulatory agents (lenalidomide and pomalidomide) and proteasome inhibitors. However, some patients lack an initial response to therapy (i.e., are refractory), and although the mean survival of MM patients has more than doubled in recent years, most patients will eventually relapse. To address this need, we explored the potential of novel cereblon E3 ligase modulators (CELMoDs) for the treatment of patients with relapsed or refractory multiple myeloma (RRMM). We found that optimization beyond potency of degradation, including degradation efficiency and kinetics, could provide efficacy in a lenalidomide-resistant setting. Guided by both phenotypic and protein degradation data, we describe a series of CELMoDs for the treatment of RRMM, culminating in the discovery of CC-92480, a novel protein degrader and the first CELMoD to enter clinical development that was specifically designed for efficient and rapid protein degradation kinetics.

Lenalidomide- and pomalidomide-based therapies are effective drugs in the treatment of patients with multiple myeloma (MM), however most patients with MM eventually relapse or become resistant. CC-92480, a novel cereblon (CRBN) E3 ligase modulator (CELMoD) with multiple activities including potent immunomodulation and single-agent antiproliferative effects, is being investigated in a phase 1 clinical trial (CC-92480-MM-001; NCT03374085) for patients with relapsed/refractory MM (RRMM). The present study investigates the preclinical data and mechanism of action of CC-92480 in MM models. CELMoD agents bound to CRBN confer differentiated substrate-degradation specificity on the CRL4CRBN E3 ubiquitin ligase. CRBN-modulator agents mediate destruction of Ikaros and Aiolos, transcription factors that contribute to myeloma cell survival. CC-92480 was found to produce rapid, deep, and sustained degradation of Ikaros and Aiolos, with superior antimyeloma activity. Accordingly, in a CRBN protein competitive binding assay, CC-92480 displaced a Cy-5-labeled CELMoD analog from CRBN with a 50% inhibitory concentration (IC50) value of 0.03 μM, whereas lenalidomide competed with an IC50 value of 1.27 μM in the same assay, demonstrating a higher binding affinity of CC-92480 for CRBN. Additionally, CC-92480 promoted the recruitment of Ikaros to the CRBN E3 ligase complex more effectively than pomalidomide in 2 orthogonal CRBN/Ikaros binding assays; it also triggered a more extensive cellular ubiquitination of Ikaros, and a faster, more efficient depletion of cellular Ikaros and Aiolos than pomalidomide. In various MM cell lines, including those with acquired resistance to lenalidomide or pomalidomide and low levels of CRBN, CC-92480 produced robust degradation of Ikaros and Aiolos followed by strong reduction of 2 additional and highly critical transcription factors, c-Myc and interferon regulatory factor 4, which are linked to the induction of apoptosis as measured by cleaved caspase-3. The tumoricidal activity of CC-92480 was shown to be CRBN dependent, since the effect was prevented by complete loss of CRBN or by the stabilization of Ikaros and Aiolos. CC-92480 displayed broad and potent antiproliferative activity across a panel of 20 MM cell lines that are either sensitive, have acquired resistance, or are refractory to lenalidomide or pomalidomide; the cell lines also contained diverse chromosomal translocations and oncogenic drivers typically found in MM patients. Approximately half of the MM cell lines evaluated were highly sensitive to CC-92480, with IC50 values for antiproliferative activity ranging from 0.04 to 5 nM; only 2 cell lines had IC50 values > 100 nM. CC-92480 inhibits cell proliferation and induces apoptosis in MM cell lines that are not sensitive to lenalidomide or pomalidomide. This panel of cell lines includes both refractory cell lines and resistant cell lines generated through continuous exposure to lenalidomide and pomalidomide that acquired low levels of CRBN protein or mutations in the CRBN gene. CC-92480 also induced deep destruction of Ikaros and Aiolos in cultures of peripheral blood mononuclear cells (PBMCs), which led to the activation of T cells and increased production of the cytokines interleukin-2 and interferon gamma. These responses occurred at the range of CC-92480 concentrations that show potent tumoricidal effect against MM cells. The T cell activation and enhanced cytokine production by CC-92480 led to the potent and effective immune-mediated killing of MM cells in co-cultures with PBMCs. CC-92480 is a potent antiproliferative and proapoptotic novel CELMoD with enhanced autonomous cell-killing activity in MM cells that are either sensitive, resistant, or have acquired resistance to lenalidomide and pomalidomide. CC-92480 has a unique and rapid degradation profile stemming from the enhanced efficiency to drive the formation of a protein-protein interaction between Ikaros and Aiolos and CRBN, inducing cytotoxic effects in a CRL4CRBN-dependent fashion that leads ultimately to the induction of apoptosis, even in the context of low or mutated CRBN protein. Additionally, similar to lenalidomide, CC-92480 conserves immunomodulatory activity against MM cells. These data support the clinical investigation of CC-92480 in patients with RRMM. Disclosures Lopez-Girona: Celgene Corporation: Employment. Havens:Pfizer: Employment, Equity Ownership; Celgene: Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Gaffney:Celgene: Employment. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Matyskiela:Celgene corporation: Employment. Khambatta:Celgene: Employment. Wong:Celgene Corporation: Employment, Equity Ownership. Hansen:Celgene Corporation: Employment. Pierce:Celgene Corporation: Employment, Equity Ownership. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Carmichael:Celgene plc: Employment, Equity Ownership.

The cereblon E3 ligase modulator (CELMoD) CC-885 co-opts the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex to trigger the ubiquitination and proteasomal degradation of the translation termination factor G1 to S phase transition 1 (GSPT1), resulting in robust growth inhibition in AML cell lines and primary patient blasts (Matyskiela ME, et al. Nature. 2016;535:252-7). However, CC-885 also triggers off-target degradation of other cereblon (CRBN) neosubstrates, potentially leading to toxicities. Here, we present the identification of a novel GSPT1-selective CELMoD, CC-90009, and the mechanistic rationale for its clinical development in relapsed or refractory (R/R) AML (CC-90009-AML-001; NCT02848001). To identify new CELMoD agents, we performed cell-based phenotypic screening using a panel of 11 human AML cell lines harboring common oncogenic mutations. The screening identified CC-90009, which demonstrated potent antiproliferative and proapoptotic activity with 50% inhibitory concentration (IC50) values ranging from 3 to 75 nM in 10 out of 11 cell lines. Maximal apoptosis was reached between 16 and 48 hours post exposure to CC-90009 in 5 AML cell lines with cells being committed to apoptosis within 8-16 hours of treatment. CC-90009 was then tested in a panel of samples obtained from 30 patients with newly diagnosed or R/R AML. Bone marrow aspirates obtained during patient diagnosis were plated without separation of constituent cells and tested for sensitivity to CC-90009 using the PharmaFlow PM test which was reported to predict clinical response to standard-of-care chemotherapy with 81% accuracy in AML (Martínez-Cuadrón D, et al. Leuk Res. 2019;76:1-10). The efficacy of CC-90009 was concentration dependent in 26 of 30 patient samples, with an average half-maximal response concentration (EC50) of 21 nM. Leukemic cell killing was rapid and highly efficient: > 82% of leukemic cells were eliminated within 24 hours and nearly all were eliminated within 96 hours. In contrast, CC-90009 showed only modest activity against normal lymphocytes in the same patient samples. Knockout of CRBN via CRISPR/Cas9-mediated gene editing completely abrogated the activity of CC-90009 in sensitive AML cell lines, suggesting that the anti-AML activity of CC-90009 is mediated by the degradation of GSPT1. Tandem mass tag quantitative mass spectrometry analysis of AML cells treated with CC-90009 revealed that CC-90009 selectively reduced the abundance of GSPT1 with little to no effect on the rest of the proteome. The degradation of GSPT1 by CC-90009 was blocked by proteasomal inhibition or inactivation of the CRL4CRBN E3 ubiquitinligase complex. Overexpression of a GSPT1-degradation-resistant mutant, G575N, conferred complete resistance to CC-90009, while RNA interference (RNAi)-mediated partial knockdown of GSPT1 gene expression enhanced the response to CC-90009. Mechanistically, GSPT1 degradation induced by CC-90009 triggers the activation of the integrated stress response pathway, which is associated with the induction of apoptosis and inhibition of proliferation. CC-90009 is a CELMoD and first-in-class GSPT1 degrader entering clinical development. The degradation of GSPT1 was confirmed to be essential for CC-90009-induced apoptosis and antiproliferative activity. The profound antiproliferative activity of CC-90009 in > 80% of human AML cell lines and patient blasts strongly supports the ongoing phase 1 study in R/R AML. Disclosures Lopez-Girona: Celgene Corporation: Employment. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Lu:Celgene Corporation: Employment, Equity Ownership. Rappley:Celgene Corporation: Employment. Fontanillo:Celgene Corporation: Employment. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Daniel:Celgene Corporation: Employment. Hansen:Celgene Corporation: Employment.

The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the K48-linked polyubiquitination of CRL4CRBN neomorphic substrates via a sequential ubiquitination mechanism. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, it will be of fundamental interest to explore if loss of UBE2G1 activity is linked to clinical resistance to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins.

SummaryPomalidomide is an IMiD® immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, ; San Miguel et al, 2013; Richardson et al, 2014; Scott, ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti‐proliferative and pro‐apoptotic activity in both lenalidomide‐sensitive and lenalidomide‐resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single‐agent treatment in xenografts of lenalidomide‐resistant H929 R10‐1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide‐sensitive H929 MM cell lines, were also observed in PomDex‐treated lenalidomide‐resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide‐sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex‐treated samples, highlighted by the modulation of pro‐apoptotic pathways in lenalidomide‐resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide‐resistant setting.

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

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

Over expression of the PI3 kinase/mTOR/AKT pathway has been well documented in MM patient biopsies and human MM cell lines, suggesting this pathway plays a key role in the survival and proliferation of malignant plasma cells. Rapamycin and the rapalogs are allosteric inhibitors of the mTORC1 complex (consisting of mTOR, raptor, mLST8 and PRAS40), inducing mainly cytostatic effects but not cell death. Inhibition of mTORC1 prevents a negative feedback loop to the mTORC2 complex (consisting of mTOR, Rictor, mLST8 and Sin 1) leading to the phosphorylation of AKT. Phosphorylated AKT is a key inducer of anti-apoptosis mechanisms and cell cycle progression, which may explain the limited results of the rapalogs in the clinic. Recently developed mTOR kinase inhibitors (i.e., CC-223) target both mTORC1 and mTORC2 complexes in order to inhibit tumor growth and importantly, induce cell death. Here we evaluate the effects of CC-223 on a panel of MM cell lines, in combination with current standard of care agents in MM (the corticosteroid, dexamethasone [DEX] and the IMiD® immunomodulatory drugs, lenalidomide [LEN] and pomalidomide [POM]), as well as in the context of LEN resistance.

Abstract Abstract 2963 Background: The immunomodulatory agents thalidomide (THAL), lenalidomide (LEN), and pomalidomide (POM) have significant activity in a wide range of hematologic cancers. THAL is primarily a potent anti-angiogenic agent with minimal immunomodulatory activity. LEN and POM both demonstrate significant immunomodulatory activity. Additionally, POM displays anti-myeloma activity in patients with LEN-resistant multiple myeloma (MM). Recently, modulation of cereblon (CRBN)-bound E3 ubiquitin ligase complexes has been implicated in the mechanisms of action of THAL, LEN, and POM. This has enabled rational development of a next generation of compounds. CC-122 is a non-phthalimide analog of the immunomodulatory drugs and a first in class PPM™ pleiotropic pathway modulator that binds the CRBN-DDB1-Cul4-Roc1 E3 ubiquitin ligase complex. This study investigated the anti-proliferative, immunomodulatory, and anti-angiogenic activity of CC-122 in MM and lymphoma cells. Results: CC-122 inhibited proliferation of H929 MM cells in a CRBN-dependent and dose-dependent manner (IC50 = 43 nM). CC-122 induced cell cycle arrest at G0/G1 stage, which was associated with reduced retinoblastoma protein phosphorylation, and increased CDK inhibitor p27 protein expression. CC-122 also inhibited the growth of LEN-resistant H929 cells, although the proliferation IC50 for CC-122 was relatively higher in LEN-resistant cells vs H929 control cells (Table). CC-122 has significant anti-myeloma activity, and has greater activity in LEN-resistant H929 cells vs LEN and POM. Compared with LEN, CC-122 had a greater anti-proliferative effect in diffuse-large B-cell lymphoma (DLBCL). Furthermore, CC-122 had greater anti-proliferative effects in ABC- and PBML-subtypes compared with GCB subtype lines. In ABC-subtype U2932 and OCI-Ly10-DLBCL cell lines, 10 μM CC-122 treatment significantly inhibited DNA-binding of NF-κB p65 (P < .001), p50 subunits (P < .05), and IRF4 in a CRBN-dependent manner. In vivo anticancer activity of CC-122 was demonstrated in xenograft models of human lymphoma and MM. CC-122 exhibits potent immunomodulatory activity in whole blood, T, and natural killer (NK) cells. Additionally, CC-122 enhanced the anti-CD3 mAb-stimulated T-cell production of IL-5, IL-13, GM-CSF, IFN-γ, RANTES, and TNF-α. The immunomodulatory activity of CC-122 was 10-fold more potent vs LEN. We investigated the anti-angiogenic properties of CC-122. In a human umbilical artery sprout outgrowth assay, CC-122 inhibited new vessel growth as well as endothelial cell migration and invasion. It also inhibited endothelial cell sprout formation and migration in a growth factor-induced endothelial cell migration and invasion assay. CC-122 has significantly greater anti-angiogenic activity compared with the LEN and POM in human angiogenesis assays (Table). In contrast, it has less of an anti-platelet effect as measured by megakaryocyte proliferation vs LEN and POM. CRBN binding competition assays were conducted with THAL-binding beads. As demonstrated by the higher IC50 concentration, CC-122 has less potency with regard to CRBN binding compared with LEN or POM. Conclusion: Together, these data demonstrate that the first-in-class PPM™ CC-122 has anti-proliferative, immunomodulatory, and anti-angiogenic properties that may have clinical significance in the treatment of advanced refractory lymphoproliferative disorders and is currently in Phase I studies. Furthermore the data suggest that the potency of binding to CRBN per se does not explain the broad pleiotropic activity of CC-122. Disclosures: Gandhi: Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Parton:Celgene Corp: Employment, Equity Ownership. Wu:Celgene Corp: Employment, Equity Ownership. Kosek:Celgene Corp: Employment, Equity Ownership. Zhang:Celgene Corp: Employment, Equity Ownership. Capone:Celgene Corp: Employment, Equity Ownership. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.

Correction to: Leukemia; doi:10.1038/leu.2012.119; advance online publication, 3 May 2012 Since the publication of this article, the authors have noticed an error in Figure 4a, specifically that the structures of the methyl-pomalidomide enantiomers were missing a carbonyl group. The error has now been rectified, and the correct article (with the correct figure 4) appears in this issue.

Abstract Abstract 4043 Background: Cereblon (CRBN), a component of the DDB1-CUL4A-Roc1 ubiquitin ligase complex, has been identified as a target of the immunomodulatory agents thalidomide, lenalidomide, and pomalidomide (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011; Ito et al. Science. 2010.). CRBN binding by these agents mediates their anti-proliferative effects in multiple myeloma (MM) cells (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011). However, the role of CRBN quantification as a marker for disease responsiveness or resistance to these drugs remains to be fully defined. Furthermore, it is unclear whether measuring mRNA or protein expression is the best approach for development of a quantitative CRBN expression assay. In order to define the optimal assay approach, we have studied CRBN mRNA and protein expression in MM cell lines (n=20) and MM patient samples. Methods: CRBN isoform mapping was undertaken using a nested PCR approach and Sanger sequencing. Commercially available and newly generated rabbit anti-CRBN antibodies were characterized with recombinant human CRBN protein and MM cell line extracts via western blot analysis. Results: Our data show that in addition to the transcript for full length protein (GenBank Accession NM_016302.3), in MM cells there are at least 6 alternatively spliced isoforms of CRBN as depicted in Figure 1. Five of the 6 CRBN isoforms (CRBN-003, -004, -005, -006, and -007) contain novel splice junctions not previously described. In addition, 3 of the identified transcripts (CRBN-002, -003, and -005) contain in-frame ORFs, suggesting they encode variants of CRBN protein. Of note, exon 10, which contains a portion of the IMiD-binding domain, is not present in CRBN-002. The functional consequence of CRBN-002 remains to be elucidated, but may be a marker of drug resistance. In order to measure CRBN protein levels, we developed and characterized three rabbit monoclonal antibodies to CRBN including antibody CRBN65, which has the potential to discriminate between the different CRBN protein products, including CRBN-002 by western blot analysis. Additionally, we compared 8 commercially available CRBN antibodies. Western blot analysis of cell lines with commercial and newly developed antibodies identified full length protein at 51 kD. Most commercial antibodies also identified multiple bands of other sizes which may represent CRBN protein variants; however, many are likely non-specific bands as they are larger than full-length CRBN. Conclusion: We have identified novel splice variants of CRBN from MM cell lines and primary tumor samples. The structure of the isoforms and their potential ability to be translated into several protein variants of CRBN reflect the complex regulation of the CRBN gene. These data suggest that accurate quantification of CRBN mRNA level in clinical studies may require measurement of both full-length CRBN mRNA as well as other mRNA isoforms. Currently available primers and gene expression arrays are not capable of identifying and/or resolving the complex set of CRBN isoforms present in cells. These data also demonstrate that CRBN65 is a highly specific and sensitive antibody that could be used for detection of CRBN and its key variants. Taken together, our data emphasize the importance for developing standardized reagents and assays for both mRNA and protein level measurement of CRBN before using them as markers for clinical response or resistance. Disclosures: Gandhi: Celgene Corp: Employment, Equity Ownership. Waldman:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Aukerman:Celgene Corp: Employment, Equity Ownership. Chen:Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Rychak:Celgene Corp: Employment, Equity Ownership. Miller:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Gonzales:Celgene Corp: Employment, Equity Ownership. Cathers:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.