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Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

ABSTRACT: Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.

SUBMITTER: Shirasaki R

PROVIDER: S-EPMC8485877 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Functional genomics identify distinct and overlapping genes mediating resistance to different classes of heterobifunctional degraders of oncoproteins

Project description:Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR/Cas9-based gene-editing studies. We observed that myeloma cell resistance to "degraders" of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; involves loss-of-function for the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for CRBN- vs. VHL-based degraders explains mechanistically the lack of cross-resistance for degraders targeting the same protein via different E3 ligase/CRLs.

2021-02-12 | GSE162205 | GEO

Functional genomics identify distinct and overlapping genes mediating resistance to different classes of heterobifunctional degraders of oncoproteins

Project description:Functional genomics identify distinct and overlapping genes mediating resistance to different classes of heterobifunctional degraders of oncoproteins

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DNA cytosine hydroxymethylation levels are distinct among non-overlapping classes of peripheral blood leukocytes.

Project description:BackgroundPeripheral blood leukocytes are the most commonly used surrogates to study epigenome-induced risk and epigenomic response to disease-related stress. We considered the hypothesis that the various classes of peripheral leukocytes differentially regulate the synthesis of 5-methylcytosine (5mCG) and its removal via Ten-Eleven Translocation (TET) dioxygenase catalyzed hydroxymethylation to 5-hydroxymethylcytosine (5hmCG), reflecting their responsiveness to environment. Although it is known that reductions in TET1 and/or TET2 activity lead to the over-proliferation of various leukocyte precursors in bone marrow and in development of chronic myelomonocytic leukemia and myeloproliferative neoplasms, the role of 5mCG hydroxymethylation in peripheral blood is less well studied.ResultsWe developed simplified protocols to rapidly and reiteratively isolate non-overlapping leukocyte populations from a single small sample of fresh or frozen whole blood. Among peripheral leukocyte types we found extreme variation in the levels of transcripts encoding proteins involved in cytosine methylation (DNMT1, 3A, 3B), the turnover of 5mC by demethylation (TET1, 2, 3), and DNA repair (GADD45A, B, G) and in the global and gene-region-specific levels of DNA 5hmCG (CD4+ T cells≫CD14+ monocytes>CD16+ neutrophils>CD19+ B cells>CD56+ NK cells>Siglec8+ eosinophils>CD8+ T cells).ConclusionsOur data taken together suggest a potential hierarchy of responsiveness among classes of leukocytes with CD4+, CD8+ T cells and CD14+ monocytes being the most distinctly poised for a rapid methylome response to physiological stress and disease.

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Potent and Selective Mitogen-Activated Protein Kinase Kinase 1/2 (MEK1/2) Heterobifunctional Small-molecule Degraders.

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Functional characterization of Prickle2 and BBS7 identify overlapping phenotypes yet distinct mechanisms.

Project description:Ciliopathies are genetic disorders that are caused by dysfunctional cilia and affect multiple organs. One type of ciliopathy, Bardet-Biedl syndrome, is a rare disorder characterized by obesity, retinitis pigmentosa, polydactyly, mental retardation and susceptibility to cardiovascular diseases. The Wnt/Planar cell polarity (PCP) has been associated with cilia function and ciliogenesis in directing the orientation of cilia and basal bodies. Yet the exact relationship between PCP and ciliopathy is not well understood. Here, we examine interactions between a core PCP component, Prickle2 (Pk2), and a central BBS gene, Bbs7, using gene knockdown in the zebrafish. pk2 and bbs7 knockdown both disrupt the formation of a ciliated organ, the Kupffer?s vesicle (KV), but do not display a synergistic interaction. By measuring cell polarity in the neural tube, we find that bbs7 activity is not required for Pk asymmetric localization. Moreover, BBS protein complex formation is preserved in the Pk2-deficient (Pk2(-/-)) mouse. Previously we reported an intracellular melanosome transport delay as a cardinal feature of reduced bbs gene activity. We find that pk2 knockdown suppresses bbs7-related retrograde transport delay. Similarly, knockdown of ift22, an anterograde intraflagellar transport component, also suppresses the bbs7-related retrograde delay. Notably, we find that pk2 knockdown larvae show a delay in anterograde transport. These data suggest a novel role for Pk2 in directional intracellular transport and our analyses show that PCP and BBS function independently, yet result in overlapping phenotypes when knocked down in zebrafish.

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Project description:Chemically induced proximity between certain endogenous enzymes and a protein of interest (POI) inside cells may cause post-translational modifications to the POI with biological consequences and potential therapeutic effects. Heterobifunctional (HBF) molecules that bind with one functional part to a target POI and with the other to an E3 ligase induce the formation of a target-HBF-E3 ternary complex, which can lead to ubiquitination and proteasomal degradation of the POI. Targeted protein degradation (TPD) by HBFs offers a promising approach to modulate disease-associated proteins, especially those that are intractable using other therapeutic approaches, such as enzymatic inhibition. The three-way interactions among the HBF, the target POI, and the ligase-including the protein-protein interaction between the POI and the ligase-contribute to the stability of the ternary complex, manifested as positive or negative binding cooperativity in its formation. How such cooperativity affects HBF-mediated degradation is an open question. In this work, we develop a pharmacodynamic model that describes the kinetics of the key reactions in the TPD process, and we use this model to investigate the role of cooperativity in the ternary complex formation and in the target POI degradation. Our model establishes the quantitative connection between the ternary complex stability and the degradation efficiency through the former's effect on the rate of catalytic turnover. We also develop a statistical inference model for determining cooperativity in intracellular ternary complex formation from cellular assay data and demonstrate it by quantifying the change in cooperativity due to site-directed mutagenesis at the POI-ligase interface of the SMARCA2-ACBI1-VHL ternary complex. Our pharmacodynamic model provides a quantitative framework to dissect the complex HBF-mediated TPD process and may inform the rational design of effective HBF degraders.

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