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.

Table S21: Table S21. Drug sensitivity and TP53 mutational status data on squamous cell carcinoma cell lines from COSMIC on Wee1/Chk1 Inhibitor (681460)

Figure S4. Body weight of MK-1775 treated mice

Figure S3. Correlation plots of autologous HNSCC cell line pairs utilizing kinomic profile

Figure S1. Plate layout for both primary validation screen and secondary assay

Figure S2. HFF kinase exclusion plot

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.

Abstract Background Despite significant therapeutic advances in improving lives of multiple myeloma (MM) patients, it remains mostly incurable, with patients ultimately becoming refractory to therapies. MM is a genetically heterogeneous disease and therapeutic resistance is driven by a complex interplay of disease pathobiology and mechanisms of drug resistance. We applied a multi-omics strategy using tumor-derived gene expression, single nucleotide variant, copy number variant, and structural variant profiles to investigate molecular subgroups in 514 newly diagnosed MM (NDMM) samples and identified 12 molecularly defined MM subgroups (MDMS1-12) with distinct genomic and transcriptomic features. Results Our integrative approach let us identify NDMM subgroups with transversal profiles to previously described ones, based on single data types, which shows the impact of this approach for disease stratification. One key novel subgroup is our MDMS8, associated with poor clinical outcome [median overall survival, 38 months (global log-rank p-value < 1 × 10−6)], which uniquely presents a broad genomic loss (> 9% of entire genome, t-test p value < 1e−5) driving dysregulation of various transcriptional programs affecting DNA repair and cell cycle/mitotic processes. This subgroup was validated on multiple independent datasets, and a master regulator analyses identified transcription factors controlling MDMS8 transcriptomic profile, including CKS1B and PRKDC among others, which are regulators of the DNA repair and cell cycle pathways. Conclusion Using multi-omics unsupervised clustering we were able to discover a new high-risk multiple myeloma patient segment. This high-risk group presents diverse previously known genetic markers, but also a new characteristic defined by accumulation of genomic loss which seems to drive transcriptional dysregulation of cell cycle, DNA repair and DNA damage. Finally, our work identified various master regulators, including E2F2 and CKS1B as the genes controlling these key biological pathways.

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 multiplexed cancer cell line screening platform PRISM demonstrated its utility in testing hundreds of cell lines in a single run, possessing the potential to speed up anti-cancer drug discovery, validation and optimization. Here we described the development and implementation of a next-generation PRISM platform combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing, cell line DNA barcoding and next-generation sequencing to enable genetic and/or pharmacological assessment of target addiction in hundreds of cell lines simultaneously. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with a public database.

CC-122 is a next-generation cereblon E3 ligase-modulating agent that has demonstrated promising clinical efficacy in patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Mechanistically, CC-122 induces the degradation of IKZF1/3, leading to T-cell activation and robust cell-autonomous killing in DLBCL. We report a genome-wide CRISPR/Cas9 screening for CC-122 in a DLBCL cell line SU-DHL-4 with follow-up mechanistic characterization in 6 DLBCL cell lines to identify genes regulating the response to CC-122. Top-ranked CC-122 resistance genes encode, not only well-defined members or regulators of the CUL4/DDB1/RBX1/CRBN E3 ubiquitin ligase complex, but also key components of signaling and transcriptional networks that have not been shown to modulate the response to cereblon modulators. Ablation of CYLD, NFKBIA, TRAF2, or TRAF3 induces hyperactivation of the canonical and/or noncanonical NF-κB pathways and subsequently diminishes CC-122-induced apoptosis in 5 of 6 DLBCL cell lines. Depletion of KCTD5, the substrate adaptor of the CUL3/RBX1/KCTD5 ubiquitin ligase complex, promotes the stabilization of its cognate substrate, GNG5, resulting in CC-122 resistance in HT, SU-DHL-4, and WSU-DLCL2. Furthermore, knockout of AMBRA1 renders resistance to CC-122 in SU-DHL-4 and U-2932, whereas knockout of RFX7 leads to resistance specifically in SU-DHL-4. The ubiquitous and cell line-specific mechanisms of CC-122 resistance in DLBCL cell lines revealed in this work pinpoint genetic alternations that are potentially associated with clinical resistance in patients and facilitate the development of biomarker strategies for patient stratification, which may improve clinical outcomes of patients with R/R DLBCL.

CC-90009 is a novel cereblon E3 ligase modulator (CELMoD) currently under investigation in a phase I clinical study in relapsed or refractory acute myeloid leukemia (R/R AML) (CC-90009-AML-001; NCT02848001). CC-90009 coopts the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex to target the translation termination factor G1 to S phase transition 1 (GSPT1) for ubiquitination and proteasomal degradation, resulting in rapid induction of apoptosis and growth inhibition in AML cell lines and primary patient blasts. To further elucidate the mechanism of action of CC-90009 in AML, we performed a genome-wide CRISPR/Cas9 screen to identify gene(s) whose knockout abrogate(s) the response to CC-90009 in a sensitive AML cell line. In addition to well-established key regulatory proteins required for the activity of all known cereblon modulators, which include components of the CRL4CRBN complex, E2 ubiquitin conjugating enzymes UBE2G1 and UBE2D3, and members of the neddylation and deneddylation machinery, interestingly, the screen identified the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreased the production of full-length CRBN transcript via modulating alternative splicing of CRBN mRNA, leading to significant downregulation of cereblon expression and hence diminished response to CC-90009. The screen also revealed that mTOR signaling and the integrated stress response (ISR) specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Since CC-90009 inhibits protein translation, it is reasonable to expect interactions with regulators of this pathway. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 , at least in part 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. Loss of GCN2 significantly attenuated the growth inhibitory effect of CC-90009, and this effect can be rescued with GCN2 wild-type but not enzymatically-dead mutants. Collectively, the antitumor activity of CC-90009, a first-in-class GSPT1 degrader, in AML cell lines is mediated by multiple layers of signaling networks and machinery, the elucidation of which reveals the underlying mechanism by which CC-90009 exerts its anti-AML activity and informs on the pathways for further study of CC-90009's clinical utility. Disclosures Lu: Celgene Corporation: Employment, Equity Ownership. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Jang:Celgene: Employment, Equity Ownership. Wang:Celgene: Employment, Equity Ownership. Rolfe:Celgene: Employment, Equity Ownership.

The effectiveness of most cancer targeted therapies is short-lived. Tumors often develop resistance that might be overcome with drug combinations. However, the number of possible combinations is vast, necessitating data-driven approaches to find optimal patient-specific treatments. Here we report AstraZeneca’s large drug combination dataset, consisting of 11,576 experiments from 910 combinations across 85 molecularly characterized cancer cell lines, and results of a DREAM Challenge to evaluate computational strategies for predicting synergistic drug pairs and biomarkers. 160 teams participated to provide a comprehensive methodological development and benchmarking. Winning methods incorporate prior knowledge of drug-target interactions. Synergy is predicted with an accuracy matching biological replicates for >60% of combinations. However, 20% of drug combinations are poorly predicted by all methods. Genomic rationale for synergy predictions are identified, including ADAM17 inhibitor antagonism when combined with PIK3CB/D inhibition contrasting to synergy when combined with other PI3K-pathway inhibitors in PIK3CA mutant cells.

AbstractPurpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with high mortality and a lack of targeted therapies. To identify and prioritize druggable targets, we performed genome analysis together with genome-scale siRNA and oncology drug profiling using low-passage tumor cells derived from a patient with treatment-resistant HPV-negative HNSCC.Experimental Design: A tumor cell culture was established and subjected to whole-exome sequencing, RNA sequencing, comparative genome hybridization, and high-throughput phenotyping with a siRNA library covering the druggable genome and an oncology drug library. Secondary screens of candidate target genes were performed on the primary tumor cells and two nontumorigenic keratinocyte cell cultures for validation and to assess cancer specificity. siRNA screens of the kinome on two isogenic pairs of p53-mutated HNSCC cell lines were used to determine generalizability. Clinical utility was addressed by performing drug screens on two additional HNSCC cell cultures derived from patients enrolled in a clinical trial.Results: Many of the identified copy number aberrations and somatic mutations in the primary tumor were typical of HPV(−) HNSCC, but none pointed to obvious therapeutic choices. In contrast, siRNA profiling identified 391 candidate target genes, 35 of which were preferentially lethal to cancer cells, most of which were not genomically altered. Chemotherapies and targeted agents with strong tumor-specific activities corroborated the siRNA profiling results and included drugs that targeted the mitotic spindle, the proteasome, and G2–M kinases WEE1 and CHK1. We also show the feasibility of ex vivo drug profiling for patients enrolled in a clinical trial.Conclusions: High-throughput phenotyping with siRNA and drug libraries using patient-derived tumor cells prioritizes mutated driver genes and identifies novel drug targets not revealed by genomic profiling. Functional profiling is a promising adjunct to DNA sequencing for precision oncology. Clin Cancer Res; 24(12); 2828–43. ©2018 AACR.

The effectiveness of most cancer targeted therapies is short lived since tumors evolve and develop resistance. Combinations of drugs offer the potential to overcome resistance, however the number of possible combinations is vast necessitating data-driven approaches to find optimal treatments tailored to a patient’s tumor. AstraZeneca carried out 11,576 experiments on 910 drug combinations across 85 cancer cell lines, recapitulating in vivo response profiles. These data, the largest openly available screen, were hosted by DREAM alongside deep molecular characterization from the Sanger Institute for a Challenge to computationally predict synergistic drug pairs and associated biomarkers. 160 teams participated to provide the most comprehensive methodological development and subsequent benchmarking to date. Winning methods incorporated prior knowledge of putative drug target interactions. For >60% of drug combinations synergy was reproducibly predicted with an accuracy matching biological replicate experiments, however 20% of drug combinations were poorly predicted by all methods. Genomic rationale for synergy predictions were identified, including antagonism unique to combined PIK3CB/D inhibition with the ADAM17 inhibitor where synergy is seen with other PI3K pathway inhibitors. All data, methods and code are freely available as a resource to the community.

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.

There is currently no standard approach to classify high-risk newly diagnosed Multiple Myeloma (ndMM). Various efforts have yielded approaches based on single molecular data types, including gene expression (GE), mutation (SNV), copy number alteration (CNA), and structural variation (SV) profiling. A comprehensive classification integrating heterogeneous molecular information may improve prognosis and treatment of high-risk ndMM patients.