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Supplementary Table 1. Proteomic data

Supplementary Methods, Figures, and Tables

Thalidomide analogues exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2–FGFR1 and ZMYM2–FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment.Significance:We extend the potential clinical scope of thalidomide analogues by the identification of a novel avadomide-dependent CRL4CRBN substrate, ZMYM2. Avadomide induces ubiquitination and degradation of ZMYM2–FGFR1 and ZMYM2–FLT3, two chimeric oncoproteins involved in hematologic malignancies, providing a proof of concept for drug-induced degradation of transcription factor fusion proteins by thalidomide analogues.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1–3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-a-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand–protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology. Binding of the small molecule BI-3802 to the oncogenic transcription factor B cell lymphoma 6 (BCL6) induces polymerization of BCL6, leading to its ubiquitination by SIAH1 and proteasomal degradation.

Molecular glue compounds induce protein–protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines, we identify CR8—a cyclin-dependent kinase (CDK) inhibitor—as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12–cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.

Thalidomide analogues exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2–FGFR1 and ZMYM2–FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment. Significance: We extend the potential clinical scope of thalidomide analogues by the identification of a novel avadomide-dependent CRL4CRBN substrate, ZMYM2. Avadomide induces ubiquitination and degradation of ZMYM2–FGFR1 and ZMYM2–FLT3, two chimeric oncoproteins involved in hematologic malignancies, providing a proof of concept for drug-induced degradation of transcription factor fusion proteins by thalidomide analogues.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins represents a major pharmacologic challenge. The clinical success of thalidomide analogs demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3{Amengual, 2014 #7}{Kronke, 2014 #31}, but a significant subset of proteins are recalcitrant to targeted protein degradation using current approaches4,5. Here we report an alternative mechanism, whereby a small molecule induces highly specific, reversible polymerization, sequestration into cellular foci, and subsequent degradation of a target protein. BI-3802 is a small molecule that binds the BTB domain of the oncogenic transcription factor BCL6 and results in proteasomal degradation6. We used cryo-EM to reveal how the solvent-exposed moiety of a BCL6 inhibitor contributes to a composite ligand/protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to superior pharmacological activity. These findings create new avenues for the development of therapeutics and synthetic biology.

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous “living drugs,” these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4 CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify “super-degron” tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.

Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples., Comprehensive protein ubiquitylation profiling by mass spectrometry typically requires large sample amounts, limiting its applicability to tissue samples. Here, the authors present an optimized proteomics method that enables multiplexed ubiquitylome analysis of cells and tumor tissue samples.

Protein ubiquitylation is involved in a plethora of cellular processes. Defects in the ubiquitin system are at the root of many acquired and hereditary diseases. While antibodies directed at ubiquitin remnants (K-ε-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly-multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here we present a highly-sensitive, rapid and multiplexed protocol for quantifying ∼10,000 ubiquitylation sites from as little as 500 ug peptide per sample from cells or tissue in a TMT10 plex in ca. 5 hr. High-field Asymmetric Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples.

Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.

Lenalidomide mediates the ubiquitination and degradation of Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and casein kinase 1α (CK1α) by facilitating their interaction with cereblon (CRBN), the substrate receptor for the CRL4

To identify novel coding association signals and facilitate characterization of mechanisms influencing glycemic traits and type 2 diabetes risk, we analyzed 109,215 variants derived from exome array genotyping together with an additional 390,225 variants from exome sequence in up to 39,339 normoglycemic individuals from five ancestry groups. We identified a novel association between the coding variant (p.Pro50Thr) in AKT2 and fasting plasma insulin (FI), a gene in which rare fully penetrant mutations are causal for monogenic glycemic disorders. The low-frequency allele is associated with a 12% increase in FI levels. This variant is present at 1.1% frequency in Finns but virtually absent in individuals from other ancestries. Carriers of the FI-increasing allele had increased 2-h insulin values, decreased insulin sensitivity, and increased risk of type 2 diabetes (odds ratio 1.05). In cellular studies, the AKT2-Thr50 protein exhibited a partial loss of function. We extend the allelic spectrum for coding variants in AKT2 associated with disorders of glucose homeostasis and demonstrate bidirectional effects of variants within the pleckstrin homology domain of AKT2., final draft, peerReviewed

Oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI 3-K), occur with high frequency in breast cancer. The protein kinase Akt is considered to be the primary effector of PIK3CA, although mechanisms by which PI 3-K mediates Akt-independent tumorigenic signals remain obscure. We show that serum and glucocorticoid-regulated kinase 3 (SGK3) is amplified in breast cancer and activated downstream of PIK3CA in a manner dependent on the phosphoinositide phosphatase INPP4B. Expression of INPP4B leads to enhanced SGK3 activation and suppression of Akt phosphorylation. Activation of SGK3 downstream of PIK3CA and INPP4B is required for 3D proliferation, invasive migration and tumorigenesis in vivo. We further show that SGK3 targets the metastasis suppressor NDRG1 for degradation by Fbw7. We propose a model in which breast cancers harboring oncogenic PIK3CA activates SGK3 signaling while suppressing Akt, indicative of oncogenic functions for both INPP4B and SGK3 in these tumors.

Cell-based therapies are emerging as potent agents against cancer and other diseases, but are uniquely uncontrolled "living drugs". For example, chimeric antigen receptor (CAR) T cells can effectively target hematologic malignancies yet pose a risk for toxic hyperactivation. Future cell-based therapies, including CAR T cells, could be improved by incorporating specific and reversible control systems. However, clinically suitable ON- and OFF-switches engineered from non-immunogenic human polypeptide sequences and regulated by non-immunosuppressive FDA-approved drugs are needed. Here we report the engineering of a robust lenalidomide-responsive degron tag, which we then used to construct a degradable CAR affording reversible OFF-switch functional control at clinically relevant lenalidomide doses. Thalidomide, lenalidomide, and pomalidomide are effective and clinically approved therapies for multiple myeloma, subtypes of non-Hodgkin lymphoma, and myelodysplastic syndrome with chromosome 5q deletion. These drugs exert therapeutic properties by acting as molecular glue, bridging interactions between the CRL4CRBN ubiquitin ligase and disease-relevant proteins that are subsequently ubiquitinated and degraded by the proteasome. A set of Cys2-His2 (C2H2) zinc fingers have emerged as degron motifs mediating drug-dependent interactions with the CRL4CRBN ubiquitin ligase. We hypothesized that these small, modular, human polypeptide domains could be further engineered and repurposed as tags to induce drug-dependent depletion of engineered proteins. By systematically shuffling the subdomains of all known zinc finger degrons and functionally screening this hybrid zinc finger library, we engineered "super-degron" tags that are more efficiently degraded at lower drug concentrations than any C2H2 zinc finger in the human proteome. We then incorporated one of these super-degrons (SD01) into a second-generation CAR targeting CD19 (FMC63-4-1BB-CD3z), thereby constructing a degradable CAR (Figure 1A). In a Jurkat T cell model, addition of lenalidomide induced rapid and near-complete depletion of the degradable CAR (Figure 1B/C). When co-cultured with target cells expressing CD19, therapeutically relevant lenalidomide concentrations robustly inhibited T cell activation (Figure 1D). Indeed, the IC50 for inhibition of IL2 secretion and CD69 expression were 2 and 22 nM lenalidomide, respectively, whereas in myeloma patients the plasma concentration of lenalidomide following one 25 mg oral dose decays from ~1000 to 10 nM over the course of 24 hours (Connarn et al, CPDD, 2017). In primary T cells, 100 nM lenalidomide suppressed degradable CAR effector functions including tumor cell killing and cytokine release (Figure 1E). Using pomalidomide, which has a longer in vivo half-life, we demonstrated robust and reversible depletion of the degradable CAR in T cells engrafted in NSG mice. Together, these findings demonstrate reversible OFF-switch control of degradable CARs at clinically relevant lenalidomide concentrations. Experiments are underway to determine whether, in the context of tumor clearance in NSG mice, degradable CAR T cell effector functions can be paused and subsequently released with short-term administration of lenalidomide. Whereas the current management of CAR T cell hyperactivation syndromes consists of supportive care, tocilizumab, and/or high-dose corticosteroids, we propose that cytokine release and CAR-related encephalopathy syndromes may be more easily diagnosed and managed with degradable CARs. The super-degron tags presented here are generalizable and clinically suitable tools to achieve chemical genetic control of diverse genetically engineered cell therapies. Disclosures Jan: Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.. Sievers:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.. Maus:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.. Ebert:Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute.; Deerfield: Research Funding; Celgene: Research Funding.

Pharmacologic agents that induce protein degradation represent an emerging and important class of therapeutic agents. Thalidomide and thalidomide derivatives (TDs) function by bringing target proteins and the E3 ubiquitin ligase substrate adaptor CRBN into proximity leading to ubiquitination and proteasomal degradation of critical oncoproteins. While highly effective, most patients eventually develop resistance to these agents, though the mechanisms by which this occurs and why some patients respond to different drugs within the class remains largely unknown. One major impediment to monitoring these drugs' activity is the need to quantitatively measure multiple protein targets simultaneously. To overcome this issue, we have developed a novel targeted mass spectrometry (MS) assay to measure the levels of CRBN, 8 substrate proteins that are degraded in the presence of TDs (IKZF1, IKZF3, CK1α, ZFP91, RNF166, ZNF692, GSPT1 and GSPT2), and 2 housekeeping control proteins (β-actin and GAPDH). This assay exhibits several advantages over currently used methodologies in that it is quantitatively precise and multiplexed for high throughput, allowing for sensitive detection of a panel of proteins from a single sample. The precision of quantitation of our MS assay enabled us to investigate the relationship between substrate level and drug-mediated degradation by measuring all known substrates in dense time course experiments following treatment of MM1S cells with lenalidomide. These experiments revealed that substrates are degraded in an ordered fashion with IKZF1 and IKZF3 being degraded before CK1α and ZFP91, suggesting that substrates may compete for access to CRBN. To test this directly we overexpressed either RNF166 or ZNF692, two recently identified substrates, in MM1S cells, which led to lenalidomide resistance. This resistance was not mediated by any direct cellular dependence on these proteins but instead was due to their competition for binding to CRBN leading to diminished degradation of other essential lenalidomide targets, IKZF1, IKZF3, and CK1α. Thus, increased expression of proteins capable of interacting with CRBN represents a novel potential mechanism of resistance to this class of drugs. CRBN is essential for lenalidomide-mediated degradation of substrates and sensitivity to drug. Quantitative detection of CRBN in our MS assay enabled us to study the effect of changes in CRBN levels on the activity of TDs. Loss of CRBN led to decreased degradation of all substrates and resistance, whereas overexpression of CRBN augmented degradation of all substrates leading to increased sensitivity to lenalidomide in MM1S cells. In a panel of ten MM cell lines CRBN level correlated with increased substrate degradation and was a significant determinant of differential sensitivity to lenalidomide. These findings demonstrate that CRBN is a limiting factor for both degradation of substrates and sensitivity to TDs. Finally, we took advantage of the high throughput nature of our MS assay and generated dense dose response and kinetic curves for 3 additional TDs (pomalidomide, avadomide and CC-885) in MM1S cells. By combining this with in vivo binding data we have created a detailed map of substrate binding, degradation and order of interaction for each TD. Among other findings, these data demonstrate that lenalidomide differs from pomalidomide and avadomide in that it is a superior degrader of CK1α but inferior at degrading IKZF3 and ZFP91, features that likely underlie some of the differential clinical activity and disease specificity of these agents. In sum, we have developed a highly sensitive, multiplexed quantitative MS assay allowing us to characterize the biochemical activity of TDs in unprecedented detail. Using this assay we show that substrates compete for access to a limiting pool of CRBN and identify a novel mechanism of resistance. The modularity of our assay allows for the detection of new substrates as they are discovered or reconfiguration of the assay to study other degraders. Future translation of this assay to clinical samples will facilitate accurate monitoring of pharmacodynamic activity and elucidation of mechanisms of resistance in vivo. Disclosures Munshi: OncoPep: Other: Board of director.

Lenalidomide exerts its therapeutic effects in the malignancy multiple myeloma by facilitating the degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by the CRL4-CRBN E3 ubiquitin ligase. In the following study we utilized a positive selection, genome-scale CRISPR-Cas9 screen in the lenalidomide-sensitive myeloma cell line, MM1S, to further our understanding of the molecular machinery which regulates and is required for lenalidomide-mediated modulation of CRL4-CRBN. The gRNAs demonstrating the greatest enrichment following selection with lenalidomide belong to cereblon, the molecular target of lenalidomide and substrate receptor for the CRL4-CRBN ubiquitin ligase. Additionally, 20 of the top 30 genes highlighted by the screen are implicated in both the positive and negative regulation of cullin-ring ligases (CRLs), emphasizing both the importance of cereblon's E3 ligase function in mediating cell death as well as the equilibrium of forces required for proper function of CRLs. Among these genes were the ubiquitin-donor E2 enzymes UBE2D3 and UBE2G1; despite the relevance of E2s to E3 ligase biology it has been difficult to determine via traditional methods which of the ~35 E2 enzymes are utilized by a given E3 ligase. Here we demonstrate that G1 and D3 fulfill distinct functional roles and cooperate with the CRL4CRBN E3 ligase to ubiquitinate an aiolos reprorter with lysine-48 linkages. In aggregate these findings confirm the power of CRISPR-Cas9 positive selection screening to reveal drug mechanism-of-action and provides a paradigm for the identification and functional characterization of E2-E3 pairings. Disclosures Fischer: Novartis: Consultancy.