CRL4CRBN E3 Ubiquitin Ligase Research Articles

CRBN modulates synuclein fibrillation via degradation of DNAJB1 in mouse model of Parkinson disease

Cereblon (CRBN) is a substrate recruiter for CRL4CRBN E3 ubiquitin ligase system playing a plethora of pivotal roles for biological systems. Here, we identified DNAJB1 (DJ1) as endogenous substrate of CRBN and report how CRBN influences the aggregation and toxicity of alpha-synuclein (α-SYN) via modulation of DJ1. CRBN interferes with molecular activities of DJ1 in vitro, in cells, and in vivo resulting in a reduced disaggregation of α-SYN fibrils, increased formation of preformed fibrils (PFFs) of α-SYN, and high susceptibility of mice to MPTP and PFF-induced neurotoxicity. Depletion of Crbn improves the behavioral and biochemical responses of mice towards neurotoxic insult. Finally, we designed a peptide inhibitor to inhibit the recruitment of DJ1 to CRBN for ubiquitination, resulting in an enhanced supply of DJ1 to counteract the toxicity of aggregated α-SYN. Our data has important implications for development of CRBN-targeting therapies that could prevent or delay progression of neurodegenerative synucleinopathy.

Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism

Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.

A Lenalidomide-Inducible Suicide Switch for Gene- and Cell-Based Therapy

Cellular immunotherapies, including chimeric antigen receptor (CAR) T cells, have demonstrated promising efficacy against hematologic malignancies and liquid tumors but run the risk of severe life-threatening toxicities. Moreover, further modifications to enhance cellular immunotherapy potency are being tested to overcome treatment failures due to T cell dysfunction or suppression in the tumor microenvironment. Consequently, for novel and/or highly active cellular immunotherapies the inclusion of “suicide switches” may be prudent to suppress treatment-related adverse events by inducing specific depletion of the engineered cells. The clinical deployment of existing cell therapy suicide switches has been limited in part by imperfect performance characteristics, reliance on non-FDA-approved controller drugs, and the potential immunogenicity of non-human sequences. Herein, we report the engineering of a clinically suitable cell therapy suicide switch activated by targeted protein degradation, and the credentialing of this system to rapidly and irreversibly deplete CAR T cells in vitro and in vivo. We and others have previously demonstrated that lenalidomide-inducible degron tags can be linked to proteins of interest to allow for their selective degradation (Jan et al, Science Translational Medicine, 2021; Carbonneau et al, Cell Chemical Biology, 2021; Koduri et al, PNAS, 2019). Lenalidomide acts as a “molecular glue” that recruits neosubstrate proteins to the CRL4CRBN E3 ubiquitin ligase to be ubiquitinated and subsequently degraded by the proteasome. Caspase-activated DNase (CAD) and its inhibitor (ICAD) are broadly expressed pro- and anti-apoptotic proteins, respectively. ICAD serves as a chaperone for CAD folding and sequesters CAD activity by forming an inactive heterodimer with CAD. Upon apoptosis signaling, activated Caspase 3 cleaves ICAD, liberating CAD to form an active homodimer that acts as a pair of “molecular scissors” to create double strand breaks in the genome. We hypothesized that the overexpression of CAD and an ICAD-degron fusion protein at a stoichiometric 1:1 ratio could be well-tolerated, and furthermore, that lenalidomide treatment would deplete the ICAD-degron protein, thereby freeing CAD to cause cell death. After iterative optimization of promoter strength, transgene order, and degron placement, we evaluated an optimized ICAD-degron-CAD lenalidomide suicide switch versus inducible Caspase 9 in primary human T cells. The lenalidomide suicide switch induced more complete cell depletion versus iCasp9, and maximal cell depletion was seen with subtherapeutic nanomolar concentrations of lenalidomide. In multi-day co-culture live cell imaging assays with co-transduced and sorted CAR suicide switch T cells, tumor cell cytolysis and CAR T cell proliferation were comparable with or without expression of the suicide switch. Lenalidomide addition rapidly inhibited tumor cell cytolysis and depleted the suicide switch CAR T cells without subsequent re-expansion. In an in vivo NSG murine xenograft model with JeKo-1 tumor cell engraftment, suicide switch and control CAR T cells demonstrated comparable anti-tumor activity. For the suicide switch CAR T cells, tumor expansion accelerated after pomalidomide treatment, consistent with engineered cell suppression after suicide switch induction. We have also co-delivered the CAR and 2.2 kilobase suicide switch as a single multi-cistronic lentivector. In summary, a lenalidomide-inducible suicide switch composed of the stoichiometric pair of CAD and ICAD-degron proteins enabled rapid, near-complete engineered cell depletion without interfering with CAR T cell growth or effector functions in the models tested. Composed of all-human sequences and a controller available as a generic drug, the lenalidomide-inducible suicide switch is a clinically suitable system that may have broad applications to safeguard the development of highly potent investigational cellular immunotherapies.

Multiomics Analysis of IMiD/CELMoD Resistant Multiple Myeloma Models Uncovers Novel and Targetable Vulnerabilities in the SREBP Lipid Synthesis Pathway

Background Acquired resistance to immunomodulatory drugs (IMiDs)/cereblon (CRBN) E3 ligase modulators (CELMoDs) is a major challenge in multiple myeloma (MM) treatment. These drugs bind to the CRBN component of the CRL4CRBN E3 ubiquitin ligase and designate a new set of substrates for degradation via the proteasome. Generation of resistance is frequently associated with decreased CRBN expression and this is due to genetic alteration in ~1/3rd of patients. Alternative drivers of the low CRBN state, and other mechanisms of resistance, need to be elucidated. To tackle this complex problem, MM models with acquired IMiD/CELMoD resistance were generated and multiomics analysis performed. Methods IMiD/CELMoD resistant human MM cell lines were generated by treating MM1s and H929 cells with lenalidomide (Len), pomalidomide (Pom) or iberdomide (Iber) at ~10x GI50 concentration for ~12 weeks until resistance was achieved. Resistant cell lines and controls were characterised by whole exome sequencing (WES), RNA-Seq and proteomics. A genome-wide loss-of-function CRISPR screen (Brunello library) was carried out in Iber-resistant MM1s; gene effect scores were calculated with the Chronos model and compared to parental MM1s using data from the Broad Institute DepMap portal. Pathway analysis was performed using g:Profiler. The Membrane Bound Transcription Factor Peptidase, Site 1 (MBTPS1) inhibitor PF-429242 was used to inhibit activation of the Sterol Regulatory-Element Binding Protein (SREBP) pathway and the effect on cell viability measured using CellTiter-Blue®. The Multiple Myeloma Research Foundation CoMMpass database was used to explore the correlation between mRNA expression of SREBP pathway genes in newly diagnosed patients and progression-free survival (PFS). Results All models were resistant to the IMiD/CELMoD with which they were generated (to 20-100x the GI50 concentration) and exhibited cross-resistance to other IMiDs/CELMoDs. Functional assays showed that well-characterised effects of IMiD/CELMoD treatment, e.g. degradation of Ikaros/Aiolos, were abrogated. WES showed Pom-resistant MM1S and Pom- and Iber-resistant H929 cells had acquired mutations in CRBN predicted to have a high impact on function. Len-resistant H929 cells had new copy number loss at the CRBN locus but Len- and Iber-resistant MM1s cells had no genetic changes in CRBN. CRBN protein expression was reduced in all resistant lines compared to control (log2 fold changes (FCs) by proteomics ranging from -0.25 to -1.95, adj p <0.05). Together these models display diverse resistance mechanisms, reflecting the clinical picture. Proteomic analysis of the resistant lines identified key changes in the SREBP pathway. The proteomes of the resistant lines were heterogeneous and the only pathway with common significant enrichment was SREBP/fatty acid metabolism (on analysis performed per cell line of proteins with significantly decreased expression). Stearoyl-CoA Desaturase (SCD), a key effector of the SREBP pathway, was one of only 5 proteins with significantly altered expression in all 6 cell lines compared to control. SCD had log2FCs ranging from -0.37 to -1.50 (adj p <0.05). Other key pathway members are shown in Figure 1. A genome-wide CRISPR screen using Iber-resistant MM1s cells identified potential new dependencies (gene effect score <-1 in resistant cells and >-0.5 in parental cells) in 47 genes including SCD and MBTPS1. MBTPS1 is critical for activation of the SREBP pathway and demonstrated one of the largest changes in gene effect (-1.4 vs -0.3). The activity of PF-429242, an inhibitor of MBTPS1, was explored in the resistant lines. A significant difference in GI50 between Iber-resistant H929 cells and control was found (1uM and >10uM respectively) with greater activity in the resistant cells. The same pattern was observed with the other resistant H929 cell lines, but not the resistant MM1s. Other pathway inhibitors are being explored. The expression of genes encoding SREBP pathway components was explored in the CoMMpass dataset. High SCD or MBTPS1 mRNA expression was associated with significantly worse PFS (logrank p<0.01), providing a further rationale for targeting the pathway. Conclusions Models representing multiple different IMiD/CELMoD resistance mechanisms have unified alterations in the SREBP pathway, highlighting a role in resistance biology and potential novel and targetable vulnerabilities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mechanisms of action of immunomodulatory drugs — from teratogenicity to treatment of multiple myeloma

Introduction. Immunomodulatory drugs (IMiDs) are a class of chemical derivatives of thalidomide with numerous immunomodulatory, antiangiogenic, anti-inflammatory, and cytostatic effects in multiple myeloma (MM).Aim — to highlight the history of the discovery of IMiDs and discuss the molecular mechanisms of their therapeutic activity.Basic information. In 2010, more than half a century after the German company Chemie Grünenthal began the clinical use of thalidomide, the first understanding of the molecular mechanism of thalidomide and its structural derivatives appeared. Hiroshi Handa and colleagues from the Tokyo Medical University discovered that the drug thalidomide binds to the protein Cereblon (CRBN), a substrate receptor of the CRL4CRBN E3 ubiquitin ligase. Subsequent generations of immunomodulatory drugs (IMiDs) — lenalidomide and pomalidomide, are structurally like thalidomide. The glutarimide ring of IMiDs is inserted into the receptor pocket of the CRBN. In this case, the variable phthalimide part of the drug protrudes from the binding domain, changing the configuration of the CRBN in such a way that it allows it to interact with proteins (neosubstrates) with which it does not react under physiological conditions. It was later found that ubiquitin-mediated degradation of two transcription factors (Ikaros and Aiolos) underlies the antitumor and immunomodulatory activity of IMiDs, which have shown unique clinical efficacy in the treatment of multiple myeloma. A natural continuation of the success of IMiDs was the creation of a series of therapeutic molecules (Iberdomide, etc.) belonging to a new class of drugs called CELMoDs (Cereblon E3 Ligase Modulating Drugs). The presented literature review is devoted to the history of the discovery of IMiDs and a discussion of the molecular mechanisms of their therapeutic activity.

The role of E3 ubiquitin ligase in multiple myeloma: potential for cereblon E3 ligase modulators in the treatment of relapsed/refractory disease

Introduction Insights into the mechanisms of protein homeostasis and proteasomal degradation have led to new strategies of redirecting the ubiquitin–proteasome system (UPS) to reduce or eliminate proteins or survival factors key to malignant pathobiology, multiple myeloma (MM) in particular. These strategies have enabled researchers to target proteins that were previously considered difficult to modulate by pharmacological means. Areas covered This review provides a brief overview of UPS biology, particularly the role of the CRL4CRBN E3 ubiquitin ligase complex, and summarizes current strategies for co-opting the UPS, including CELMoD compounds, SNIPERs, PROTACs, and degronimids. A detailed discussion is provided on lead CELMoD compounds iberdomide and mezigdomide, which are currently being evaluated in clinical trials in patients with MM. Expert opinion Since a high proportion of patients develop drug resistance, it is vital to have novel therapeutic agents for treating relapsed patients with MM more effectively. It is encouraging that the expanding pathophysiological insight into cellular signaling pathways in MM increasingly translates into the development of novel therapeutic agents such as targeted protein degraders. This holds promise for improving outcomes in MM and beyond.

High-resolution structures of the bound effectors avadomide (CC-122) and iberdomide (CC-220) highlight advantages and limitations of the MsCI4 soaking system

Cereblon (CRBN) is the substrate receptor of the CRL4CRBN E3 ubiquitin ligase and is a central player in targeted protein degradation. It is the target of the thalidomide-derived immunomodulatory drugs (IMiDs) and is one of the most widely employed receptors for proteolysis-targeting chimeras (PROTACs), both of which induce the ubiquitination and subsequent proteasomal degradation of target proteins. Structural studies of ligand binding to CRBN are crucial to elucidate the mechanisms of action and for mediation of side effects, ultimately aiding the development of next-generation IMiDs and PROTACs. With this aim, a crystal-soaking system based on the single-domain bacterial homologue MsCI4 has previously been established and used to delineate the binding modes of several classes of small molecules, including FDA-approved drugs, at the molecular level. Here, this system was used to characterize the binding of the next-generation IMiDs avadomide (CC-122) and iberdomide (CC-220) at high resolution, highlighting the advantages and limitations of the MsCI4 system and its implications for the development of future cereblon effectors.

Development of PDE6D and CK1α Degraders through Chemical Derivatization of FPFT-2216.

Immunomodulatory drugs are a class of drugs approved for the treatment of multiple myeloma. These compounds exert their clinical effects by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif, resulting in degradation of degron-containing targets. However, although many cellular proteins feature the degron motif, only a subset of those are degradable via this strategy. Here, we demonstrated that FPFT-2216, a previously reported "molecular glue" compound, degrades PDE6D, in addition to IKZF1, IKZF3, and CK1α. We used FPFT-2216 as a starting point for a focused medicinal chemistry campaign and developed TMX-4100 and TMX-4116, which exhibit greater selectivity for degrading PDE6D and CK1α, respectively. We also showed that the region in PDE6D that interacts with the FPFT-2216 derivatives is not the previously pursued prenyl-binding pocket. Moreover, we found that PDE6D depletion by FPFT-2216 does not impede the growth of KRASG12C-dependent MIA PaCa-2 cells, highlighting the challenges of drugging PDE6D-KRAS. Taken together, the approach we described here represents a general scheme to rapidly develop selective degraders by reprogramming E3 ubiquitin ligase substrate specificity.

Overcoming IMiD resistance in T-cell lymphomas through potent degradation of ZFP91 and IKZF1

AbstractImmunomodulatory (IMiD) agents like lenalidomide and pomalidomide induce the recruitment of IKZF1 and other targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and degradation. These agents are highly active in B-cell lymphomas and a subset of myeloid diseases but have compromised effects in T-cell lymphomas (TCLs). Here, we show that 2 factors determine resistance to IMiDs among TCLs. First, limited CRBN expression reduces IMiD activity in TCLs but can be overcome by newer-generation degrader CC-92480. Using mass spectrometry, we show that CC-92480 selectively degrades IKZF1 and ZFP91 in TCL cells with greater potency than pomalidomide. As a result, CC-92480 is highly active against multiple TCL subtypes and showed greater efficacy than pomalidomide across 4 in vivo TCL models. Second, we demonstrate that ZFP91 functions as a bona fide transcription factor that coregulates cell survival with IKZF1 in IMiD-resistant TCLs. By activating keynote genes from WNT, NF-kB, and MAP kinase signaling, ZFP91 directly promotes resistance to IKZF1 loss. Moreover, lenalidomide-sensitive TCLs can acquire stable resistance via ZFP91 rewiring, which involves casein kinase 2–mediated c-Jun inactivation. Overall, these findings identify a critical transcription factor network within TCLs and provide clinical proof of concept for the novel therapy using next-generation degraders.

Ligands for cereblon: 2017–2021 patent overview

ABSTRACT Introduction Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase has been extensively studied due to its involvement in many biological processes. It has also been identified as the target for immunomodulatory drugs (IMiDs). CRBN ligands are also important components of proteolysis-targeting chimeras (PROTACs), special bifunctional constructs capable of targeted degradation of aberrantly acting proteins using the cell’s ubiquitin-proteasome machinery. Areas covered Due to upsurge of the PROTAC technology, the patenting activity of new CRBN ligands has been on the rise in the last 5 years. The present review covers two broadly defined areas of CRBN ligand design. One covers ‘thalidomide-like’ molecules representing modifications of various parts of classical IMiDs. The other areas – non-thalidomide-like compounds – are compounds that are structurally distinct from the classical IMiDs. Efforts toward creating new CRBN ligands reflected in non-patent literature are briefly discussed with emphasis on the rational, crystallography-driven approaches. Expert opinion The chemical space of CRBN ligands which is related to the classical IMiDs (thalidomide/lenalidomide/pomalidomide) is comprehensively covered by the current patent literature. The promising area of research is in the identification of non-thalidomide-like chemotypes capable of binding to CRBN. Rational, crystallography-driven approaches currently exploited in academia will significantly aid in this endeavor.

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the β-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.

IKZF1/3 and CRL4CRBN E3 Ubiquitin Ligase Mutations Associate with IMiD Resistance in Relapsed Multiple Myeloma

Immunomodulatory drugs (IMiDs) provide the backbone for various Multiple Myeloma (MM) treatment regimens. Cereblon (CRBN) is the known key modulator of the IMiD anti-tumor effects as it is the intermediate protein within the CRL4CRBN E3 ubiquitin ligase (CRL4) complex that targets the degradation of the two transcription factors, Ikaros (IKZF1) and Aiolos (IKZF3). We hypothesize that the formation of thiscomplex (CRBN-DDB1-CUL4B-ROC1) is essential for the IMiD's action and that mutations that occur in involved genes affect its assemblage, thus induce IMiD resistance.Targeted sequencing was performed using the M3P (v2.0 or v3.0) gene selection. Average sequencing depth was 700X. We have combined data from different cohorts analyzed with the M3P for a total of 501 patients, 337 newly diagnosed and 164 pretreated. We also included data from the CoMMpas trial (804 newly diagnosed MM analyzed by WES with 100X average depth).We analyzed a total of 1.305 MM cases and identified 53 mutations in 42 patients within CRBN, IKZF1, IKZF3 and CUL4B . Mutation frequency increased significantly after treatment (Z-score: 6.9, p<0.001), with 1.9% in untreated (22/1141) and 12.2% in (20 /164) pretreated cases. Of note, this analysis does not include either DDB1, one of the binding partners of CRBN in the complex, nor ROC1, in the pretreated cohort, as it was not part of the M3P gene selection. In untreated patients, however, mutation incidence was low with 4 out of 804 (0.5%) for DDB1 and no mutation was identified in ROC1 within patients included into the CoMMpass trial.CRBN was the most commonly mutated gene within the investigated CRL4 complex genes (21 mutations /15 MM patients), followed by CUL4B (15/13). Of note, in IKZF3 (11/11) and IKZF1 (6/6) we identified two new hotspots (IKZF3 p.G159R/A and IKZF1 p.A152T, two patient each). Furthermore, five of the patients harbored mutations in IKZF1/3 within 30 amino acids previously described to be essential for IMiD sensitivity in vitro (Krönke et al., Science, 2014). 91% (20/22) of the CRBN and CUL4B mutations found in treated patients were nonsense or were located within close proximity to the binding sites of the proteins (Figure), causing loss/weakening of lenalidomide (LEN)-CRBN, CRBN-DDB1, CUL4B-DDB1 or CUL4B-ROC1 interactions. This includes a patient having progressed on Thalidomide and Pomalidomide therapy with parallel evolution of 7 subclonal mutations in CRBN, 3 located in the LEN binding and 4 in the DDB1 binding site. Response data were available for 19 out of 20 treated patients with mutations in the CLR4 complex and, strikingly, 18 (95%) of them were clinically unresponsive to IMiD treatment.In summary, CLR4 mutations are predominantly selected by therapy and correlate with IMiD resistance. In our cohort of pretreated patients the incidence of CLR4 mutations was 12%, thus we provide evidence of a significant role of single point mutations as an IMiD drug resistance mechanism in MM.Figure: Mutations in CRL4CRBN E3 ubiquitin ligase clustered in the binding sides of complex unitsA) Mutations close to the binding side of Lenalidomide. B) Mutations in the interface CRBN-DDB1 and C) DDB1-CUL4B. D) Mutations in the binding side of ROC1. The structure of the CRL4 complex was produced by the alignments LEN-CRBN-DDB1 (PDB: 4CI2) and DDB1-CUL4B-ROC1 (PDB 4A0C). CRBN CTD (LEN binding) domain is shown in gray and HBD (DDB1 binding) in plun, LEN in orange, DDB1 in sky blue, CUL4B in pink and ROC1 in yellow. [Display omitted] DisclosuresMai:Mundipharma: Other: Travel grants; Janssen: Honoraria, Other: Travel grants; Onyx: Other: Travel grants; Celgene: Other: Travel grants; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Raab:Heidelberg Pharma GMBH: Research Funding. Sonneveld:Celgene Corporation, Amgen, Janssen, Karyopharm, PharmaMar, SkylineDx: Honoraria; Celgene Corporation, Amgen, Janssen, Karyopharm, SkylineDx, PharmaMar: Consultancy; Celgene, Amgen, Janssen, Karyopharm, Takeda: Consultancy, Honoraria, Research Funding. Stewart:Bristol-Myers Squibb: Consultancy; Roche: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy.

Regulation of AMPK Activity by CRBN Is Independent of the Thalidomide-CRL4CRBN Protein Degradation Axis.

Cereblon (CRBN), a primary target of immune-modulatory imide drugs (IMiDs), functions as a substrate receptor in the CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN) E3 ubiquitin ligase complex. Binding of IMiDs to CRBN redirects the CRL4CRBN E3 ubiquitin ligase to recruit or displace its substrates. Interaction between CRBN and the AMPK α subunit leads to CRL4CRBN-dependent degradation of the γ subunit and inhibits AMPK activity. However, the effect of thalidomide on the function of CRBN as a negative regulator of AMPK through interaction with the α subunit remains unclear. Here, we show that thalidomide does not affect AMPK activation or the binding affinity between CRBN and the AMPK α subunit. Thalidomide had no effect on AMPK activity independent of CRBN expression. The N-terminal region and C-terminal tail of CRBN, which is distinct from the IMiD binding site, were critical for interaction with the AMPK α subunit. The present results suggest that CRL4CRBN negatively regulates AMPK through a pathway independent from the CRBN-IMiD binding region.

Avadomide induces degradation of ZMYM2 fusion oncoproteins in hematologic malignancies.

Thalidomide analogs 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.

Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide.

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 CRL4CRBN 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.

Leveraging gene expression subgroups to classify DLBCL patients and select for clinical benefit from a novel agent

AbstractDiffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, commonly described by cell-of-origin (COO) molecular subtypes. We sought to identify novel patient subgroups through an unsupervised analysis of a large public dataset of gene expression profiles from newly diagnosed de novo DLBCL patients, yielding 2 biologically distinct subgroups characterized by differences in the tumor microenvironment. Pathway analysis and immune deconvolution algorithms identified higher B-cell content and a strong proliferative signal in subgroup A and enriched T-cell, macrophage, and immune/inflammatory signals in subgroup B, reflecting similar biology to published DLBCL stratification research. A gene expression classifier, featuring 26 gene expression scores, was derived from the public dataset to discriminate subgroup A (classifier-negative, immune-low) and subgroup B (classifier-positive, immune-high) patients. Subsequent application to an independent series of diagnostic biopsies replicated the subgroups, with immune cell composition confirmed via immunohistochemistry. Avadomide, a CRL4CRBN E3 ubiquitin ligase modulator, demonstrated clinical activity in relapsed/refractory DLBCL patients, independent of COO subtypes. Given the immunomodulatory activity of avadomide and the need for a patient-selection strategy, we applied the gene expression classifier to pretreatment biopsies from relapsed/refractory DLBCL patients receiving avadomide (NCT01421524). Classifier-positive patients exhibited an enrichment in response rate and progression-free survival of 44% and 6.2 months vs 19% and 1.6 months for classifier-negative patients (hazard ratio, 0.49; 95% confidence interval, 0.280-0.86; P = .0096). The classifier was not prognostic for rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone or salvage immunochemotherapy. The classifier described here discriminates DLBCL tumors based on tumor and nontumor composition and has potential utility to enrich for clinical response to immunomodulatory agents, including avadomide.

Using an in-Vivo Degron-Based Approach to Interrogate Dependencies of Serially Acquired Mutations - Including DNMT3a-R882 and NPM1c - in Acute Myeloid Leukemia

While a number of targeted therapies have recently become available in the treatment of acute myeloid leukemia (AML), it remains to be determined whether early acquired mutations are essential for ongoing leukemia maintenance in the face of sequential acquisition of additional mutations. In some cases, our ability to answer this question is hampered by the lack of targeted therapies to a number of these mutations. Of all AML mutations, those involving DNMT3a and NPM1c are some of the most frequent - often acquired before the final transforming event. Here we report the development of an in-vivo degron-based approach to interrogate the requirement for DNMT3a-R882 and NPM1c after leukemia development. Using an IKZF-based degron system offers us the opportunity to address the ongoing requirement of particular oncogenes for AML maintenance - by way of a conditional system that can be rapidly modulated. Thalidomide and its related analogues act by recruiting various substrates to the CRL4CRBN E3-ubiquitin ligase complex mediating subsequent ubiquitination and proteasomal degradation. In a majority of cases - the basis of this degradation has been determined to be the result of recognition and binding to a degron motif within C2H2 zinc fingers. Using more detailed analysis with single amino-acid resolution, we have engineered 'degrons' sensitive or resistant to degradation by thalidomide and its related analogues. In-vivo degradation using pomalidomide revealed a dependence of GMP cells on the presence of NPM1c but not DNMT3a R882. Using our cereblon (CRBN) knock-in mice engineered to mediate sensitivity to these compounds, we generated triple-mutant acute-myeloid leukemia - sequentially introducing DNMT3a-R882, NPM1c and N-RAS G12D mutations whilst altering the site of the degron tag in each case. Degradation-resistant degron constructs were used as a control in each case. Findings were subsequently correlated and validated with in-vitro treatments of triple mutant cells. RNA-seq was also performed after both in-vitro and in-vivo treatments to further delineate acute transcriptional changes in the absence of NPM1c - highlighting a number of changes including those within the Hox family of genes Together, these findings suggest triple mutant leukemia remains dependent on the leukemia-inducing mutation of NPM1c. In contrast, whilst epigenetic alterations induced by DNMT3a mutations are essential for leukemia development - these do not appear to be essential for ongoing leukemia maintenance. Further work is currently underway to determine how modulating NPM1c impacts long-term survival from leukemia and whether the epigenetic changes associated with DNMT3a R882 mutations are indeed reversible in the context of leukemia. This work highlights an approach to interrogate a protein of interest in-vivo using a conditional system that is reversible, rapidly acting and that uses a series of FDA-approved compounds with known kinetics. We envisage this will be generalisable as a tool to be used in other similar contexts. Disclosures No relevant conflicts of interest to declare.

Mutations in CRBN and Other Cereblon Pathway Genes Are Only Associated with Acquired Resistance to Immunomodulatory Drugs in a Subset of Patients and Cell Line Models

Mutations in CRBN and Other Cereblon Pathway Genes Are Only Associated with Acquired Resistance to Immunomodulatory Drugs in a Subset of Patients and Cell Line Models

Crystal structure of the SALL4-pomalidomide-cereblon-DDB1 complex.

Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4CRBN E3 ubiquitin ligase is a plausible major driver of thalidomide teratogenicity. The structure of the second zinc finger of SALL4 in complex with pomalidomide, cereblon and DDB1 reveals the molecular details of recruitment. Sequence differences and a shifted binding position relative to Ikaros offer a path to the rational design of cereblon-binding drugs with reduced teratogenic risk.

Cyclosporine Broadens the Therapeutic Potential of Lenalidomide in Myeloid Malignancies.

The immunomodulatory drug lenalidomide is used for the treatment of certain hematologic malignancies, including myelodysplastic syndromes (MDS). Lenalidomide interacts with cereblon (CRBN), a component of the CRL4CRBN E3 ubiquitin ligase complex, leading to ubiquitination and subsequent degradation of substrates, such as transcription factor Ikaros (Ikaros family zinc finger 1, IKZF1). With a genome loss of function screen, we recently identified two novel pathways mediated by lenalidomide in MDS. In this review, we summarized the major findings of these two pathways and their clinical implications. Depletion of G protein-coupled receptor 68 (GPR68) or an endogenous calcineurin (CaN) inhibitor, regulator of calcineurin 1 (RCAN1), reversed the inhibitory effect of lenalidomide on MDSL cells, an MDS cell line. Intriguingly, both GPR68 and RCAN1 expression levels were upregulated in MDSL cells after treatment with lenalidomide that was dependent on diminishment of IKZF1, indicating that IKZF1 functioned as a transcription repressor for GPR68 and RCAN1. Mechanistic studies revealed that upregulation or activation of GPR68 induced a Ca2+/calpain pro-apoptotic pathway, while upregulation of RCAN1 inhibited the CaN pro-survival pathway in MDSL cells. Notably, the pharmacological CaN inhibitor, cyclosporine, enhanced the sensitivity to lenalidomide in MDS as well as acute myeloid leukemia (AML). Surprisingly, pretreatment with lenalidomide reversed the immunosuppressive effects of cyclosporine on T lymphocytes. Our studies suggest that lenalidomide mediates degradation of IKZF1, leading to derepression of GPR68 and RCAN1 that activates the Ca2+/calpain pro- apoptotic pathway and inhibits the CaN pro-survival pathway, respectively. Our studies implicate that cyclosporine extends the therapeutic potential of lenalidomide to myeloid malignancies without compromising immune function.