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.
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).
The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4
) 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 CRL4
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 CRL4
to eliminate disease-driving proteins.
Summary
Overexpression of the transcription factor interferon regulatory factor‐4 (IRF4), which is common in multiple myeloma (MM), is associated with poor prognosis. Patients with higher IRF4 ...expression have significantly poorer overall survival than those with low IRF4 expression. Lenalidomide is an IMiD® immunomodulatory compound that has both tumouricidal and immunomodulatory activity in MM. This study showed that lenalidomide downregulated IRF4 levels in MM cell lines and bone marrow samples within 8 h of drug exposure. This was associated with a decrease in MYC levels, as well as an initial G1 cell cycle arrest, decreased cell proliferation, and cell death by day 5 of treatment. In eight MM cell lines, high IRF4 levels correlated with increased lenalidomide sensitivity. The clinical significance of this observation was investigated in 154 patients with MM. Among MM patients with high levels of IRF4 expression, treatment with lenalidomide led to a significantly longer overall survival than other therapies in a retrospective analysis. These data confirm the central role of IRF4 in MM pathogenesis; indicate that this is an important mechanism by which lenalidomide exerts its antitumour effects; and may provide a mechanistic biomarker to predict response to lenalidomide.
Summary
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.
Decreased p27Kip1 levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCFSkp2 E3 ligase function, resulting in enhanced p27 ubiquitination and ...proteasome-mediated degradation. While proteasome inhibitors stabilize p27Kip1, agents inhibiting SCFSkp2 may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCFSkp2 ligase function in vitro, and induced specific accumulation of p21 and other SCFSkp2 substrates in cells without activating a heat-shock protein response. CpdA prevented incorporation of Skp2 into the SCFSkp2 ligase, and induced G1/S cell-cycle arrest as well as SCFSkp2- and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCFSkp2 inhibitors as a novel class of antitumor agents.
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.
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.
Summary
Pomalidomide 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.
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.
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.
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