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Iswar K. Hariharan - One of the best experts on this subject based on the ideXlab platform.
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Mutation of the Gene Encoding the Ubiquitin Activating Enzyme UBA1 Causes Tissue Overgrowth in Drosophila
Fly, 2007Co-Authors: Cathie M. Pfleger, Kieran F. Harvey, Hua Yan, Iswar K. HariharanAbstract:Protein ubiquitination has been shown to regulate a wide variety of cellular process including cell cycle progression, protein trafficking and apoptosis. Most regulation of ubiquitination occurs at the level of E2 or E3 enzymes and their interactions with specific substrates. In a screen for mutations that cause tissue overgrowth, we recovered multiple mutations in the Drosophila UBA1 gene that encodes the E1 enzyme that is required for the first step of most, if not all, ubiquitination reactions. Previous studies with yeast and mammalian cells have shown that disrupting E1 function results in a cell-cycle arrest. Here we show that in the developing Drosophila eye, clones of cells that are homozygous for partial loss of function alleles of UBA1 show defects in apoptosis. Moreover, clones homozygous for stronger or complete loss of function alleles of UBA1, that are predicted to have a global defect on ubiquitination, survive poorly but are able to stimulate the overgrowth of adjacent wild-type tissue. Experiments with mammalian cells show that reducing the level of RNA of the mammalian UBA1 ortholog, UBE1, also results in increased expression of specific growth factor genes. Our studies show that a reduction in E1 activity can promote tissue growth in a multicellular organism and raise the possibility that changes in E1 activity may occur during normal development or in cancer.
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mutation of the gene encoding the ubiquitin activating enzyme UBA1 causes tissue overgrowth in drosophila
Fly, 2007Co-Authors: Cathie M. Pfleger, Kieran F. Harvey, Hua Yan, Iswar K. HariharanAbstract:Protein ubiquitination has been shown to regulate a wide variety of cellular process including cell cycle progression, protein trafficking and apoptosis. Most regulation of ubiquitination occurs at the level of E2 or E3 enzymes and their interactions with specific substrates. In a screen for mutations that cause tissue overgrowth, we recovered multiple mutations in the Drosophila UBA1 gene that encodes the E1 enzyme that is required for the first step of most, if not all, ubiquitination reactions. Previous studies with yeast and mammalian cells have shown that disrupting E1 function results in a cell-cycle arrest. Here we show that in the developing Drosophila eye, clones of cells that are homozygous for partial loss of function alleles of UBA1 show defects in apoptosis. Moreover, clones homozygous for stronger or complete loss of function alleles of UBA1, that are predicted to have a global defect on ubiquitination, survive poorly but are able to stimulate the overgrowth of adjacent wild-type tissue. Exp...
Samir H. Barghout - One of the best experts on this subject based on the ideXlab platform.
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E1 Enzymes as Therapeutic Targets in Cancer
Pharmacological reviews, 2020Co-Authors: Samir H. Barghout, Aaron D. SchimmerAbstract:Post-translational modifications of cellular substrates with ubiquitin and ubiquitin-like proteins (UBLs), including ubiquitin, SUMOs, and neural precursor cell-expressed developmentally downregulated protein 8, play a central role in regulating many aspects of cell biology. The UBL conjugation cascade is initiated by a family of ATP-dependent enzymes termed E1 activating enzymes and executed by the downstream E2-conjugating enzymes and E3 ligases. Despite their druggability and their key position at the apex of the cascade, pharmacologic modulation of E1s with potent and selective drugs has remained elusive until 2009. Among the eight E1 enzymes identified so far, those initiating ubiquitylation (UBA1), SUMOylation (SAE), and neddylation (NAE) are the most characterized and are implicated in various aspects of cancer biology. To date, over 40 inhibitors have been reported to target UBA1, SAE, and NAE, including the NAE inhibitor pevonedistat, evaluated in more than 30 clinical trials. In this Review, we discuss E1 enzymes, the rationale for their therapeutic targeting in cancer, and their different inhibitors, with emphasis on the pharmacologic properties of adenosine sulfamates and their unique mechanism of action, termed substrate-assisted inhibition. Moreover, we highlight other less-characterized E1s-UBA6, UBA7, UBA4, UBA5, and autophagy-related protein 7-and the opportunities for targeting these enzymes in cancer. SIGNIFICANCE STATEMENT: The clinical successes of proteasome inhibitors in cancer therapy and the emerging resistance to these agents have prompted the exploration of other signaling nodes in the ubiquitin-proteasome system including E1 enzymes. Therefore, it is crucial to understand the biology of different E1 enzymes, their roles in cancer, and how to translate this knowledge into novel therapeutic strategies with potential implications in cancer treatment.
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Preclinical evaluation of the selective small-molecule UBA1 inhibitor, TAK-243, in acute myeloid leukemia
Leukemia, 2019Co-Authors: Samir H. Barghout, Parasvi S. Patel, Xiaoming Wang, Simon Kavanagh, Ondrej Halgas, Sara F. Zarabi, Marcela Gronda, Rose Hurren, Danny V. Jeyaraju, Neil MacleanAbstract:Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.
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a genome wide crispr cas9 knockout screen identifies bend3 as a determinant of sensitivity to UBA1 inhibition in acute myeloid leukemia
Blood, 2018Co-Authors: Samir H. Barghout, Rose Hurren, Neil Maclean, Aaron D. Schimmer, Geethu Thomas, Zachary BlatmanAbstract:UBA1 is the major ubiquitin-activating enzyme that initiates the ubiquitylation cascade whereby proteins are tagged with mono- or polyubiquitin to mark them for proteasomal degradation or modify their functions. Despite having equal levels of UBA1 protein, AML cell lines and primary AML cells are more dependent on UBA1 activity compared to normal hematopoietic cells, rendering them more vulnerable to UBA1 inhibition. Recently, we demonstrated that inhibiting UBA1 with the small-molecule inhibitor TAK-243 was selectively cytotoxic to a subset of AML cells and stem cells in vitro and in vivo through a mechanism at least partly dependent on inducing ER stress (Leukemia, 2018). To identify potential determinants of sensitivity/resistance to TAK-243 (Millennium Pharmaceuticals, Takeda) in AML, we conducted a genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with cytotoxic TAK-243 concentrations corresponding to the IC90 and IC99. By next-generation sequencing and enrichment analysis, we then identified genes whose knockout renders AML cells resistant to TAK-243. We identified 34 hits in the IC90 and 11 hits in the IC99 arms of the screen (cut off FDR To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells using the 4 top performing gRNAs in the screen. We confirmed target knockout by immunoblotting. BEND3 knockout did not alter the basal proliferation rate of the cells. However, knockout of BEND3 rendered OCI-AML2 cells resistant to TAK-243 with up to a 4-fold increase in the IC50 by the MTS assay. Resistance to TAK-243 was confirmed by Annexin V/PI staining, PARP cleavage, and colony-forming assays. Cells were cross resistant to the NEDD8-activating enzyme inhibitor pevonedistat (2-fold IC50 increase), but not bortezomib or daunorubicin. As assessed by immunoblotting, BEND3 knockout did not change expression of UBA1, or the related enzymes UBA2, UBA3, or UBA6. BEND3 knockout was associated with reduced induction of ER stress as assessed by levels of CHOP and ATF4 after TAK-243 treatment. Conclusions Through a genome-wide CRISPR screen, we identified BEND3 as a determinant of sensitivity to TAK-243 in AML. Mechanistically, lack of BEND3 expression dampens the ER stress response to UBA1 inhibition. Thus, these results may highlight a new mechanism of sensitivity to TAK-243. Disclosures Schimmer:Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity9s Board of Directors or advisory committees.
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A Genome-Wide CRISPR/Cas9 Knockout Screen Identifies BEND3 As a Determinant of Sensitivity to UBA1 Inhibition in Acute Myeloid Leukemia
Blood, 2018Co-Authors: Samir H. Barghout, Rose Hurren, Neil Maclean, Zachary Blatman, Geethu E. Thomas, Aaron D. SchimmerAbstract:Abstract UBA1 is the major ubiquitin-activating enzyme that initiates the ubiquitylation cascade whereby proteins are tagged with mono- or polyubiquitin to mark them for proteasomal degradation or modify their functions. Despite having equal levels of UBA1 protein, AML cell lines and primary AML cells are more dependent on UBA1 activity compared to normal hematopoietic cells, rendering them more vulnerable to UBA1 inhibition. Recently, we demonstrated that inhibiting UBA1 with the small-molecule inhibitor TAK-243 was selectively cytotoxic to a subset of AML cells and stem cells in vitro and in vivo through a mechanism at least partly dependent on inducing ER stress (Leukemia, 2018). To identify potential determinants of sensitivity/resistance to TAK-243 (Millennium Pharmaceuticals, Takeda) in AML, we conducted a genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with cytotoxic TAK-243 concentrations corresponding to the IC90 and IC99. By next-generation sequencing and enrichment analysis, we then identified genes whose knockout renders AML cells resistant to TAK-243. We identified 34 hits in the IC90 and 11 hits in the IC99 arms of the screen (cut off FDR < 0.2). These hits are involved in signaling pathways including transcriptional regulation, histone methylation, ubiquitin conjugation, cell cycle progression, mTOR and NF-κB signaling pathways, consistent with the broad range of pathways regulated by UBA1-mediated ubiquitylation. We focused our investigation on BEN domain-containing protein 3 (BEND3) that ranked as a top hit in both arms (FDR = 0.0012). Compared to control, all 6 BEND3-targeting gRNAs were enriched up to 10,000 times. BEND3 is a transcriptional repressor that regulates heterochromatin organization. To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells using the 4 top performing gRNAs in the screen. We confirmed target knockout by immunoblotting. BEND3 knockout did not alter the basal proliferation rate of the cells. However, knockout of BEND3 rendered OCI-AML2 cells resistant to TAK-243 with up to a 4-fold increase in the IC50 by the MTS assay. Resistance to TAK-243 was confirmed by Annexin V/PI staining, PARP cleavage, and colony-forming assays. Cells were cross resistant to the NEDD8-activating enzyme inhibitor pevonedistat (2-fold IC50 increase), but not bortezomib or daunorubicin. As assessed by immunoblotting, BEND3 knockout did not change expression of UBA1, or the related enzymes UBA2, UBA3, or UBA6. BEND3 knockout was associated with reduced induction of ER stress as assessed by levels of CHOP and ATF4 after TAK-243 treatment. Conclusions Through a genome-wide CRISPR screen, we identified BEND3 as a determinant of sensitivity to TAK-243 in AML. Mechanistically, lack of BEND3 expression dampens the ER stress response to UBA1 inhibition. Thus, these results may highlight a new mechanism of sensitivity to TAK-243. Disclosures Schimmer: Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.
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TAK-243 Is a Selective UBA1 Inhibitor That Displays Preclinical Activity in Acute Myeloid Leukemia (AML)
Blood, 2017Co-Authors: Samir H. Barghout, Parasvi S. Patel, Xiaoming Wang, Simon Kavanagh, Marcela Gronda, Rose Hurren, Danny V. Jeyaraju, Neil Maclean, Marc L. HyerAbstract:Introduction: Ubiquitin-like Modifier Activating Enzyme 1 (UBA1; UAE) is the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Compared to normal hematopoietic cells, AML cell lines and primary AML cells have equal levels of UBA1 protein, but increased requirement for this enzyme. TAK-243 is a potent and selective inhibitor of UBA1 and we determined the preclinical activity, biological effects and mechanisms of resistance to the drug in AML. Results: TAK-243 reduced growth and viability of human AML cell lines (OCI-AML2, TEX, U937 and NB4) in a concentration- and time-dependent manner with IC509s ranging from 15-40 nM after treatment for 48 hours. In primary AML samples, most (n=18/21) were sensitive to TAK-243 with an IC50 Binding of TAK-243 to UBA1 and related E1 enzymes was measured in intact AML cells using the cellular thermal shift assay (CETSA). In AML cell lines and primary AML samples, TAK-243 bound UBA1 at concentrations associated with cell death, but bound other E1 enzymes UBA3 and UBA6 only at much higher concentrations. Next, we evaluated the biological effects of UBA1 inhibition by TAK-243. At concentrations associated with cell death, TAK-243 decreased the abundance of poly- and mono-ubiquitylated proteins in OCI-AML2 cells and primary AML samples. In addition, TAK-243 treatment increased PERK phosphorylation, CHOP, XBP1s and ATF4 which are markers of proteotoxic stress and unfolded protein response. TAK-243 inhibited DNA double strand break (DSB) repair as evidenced by reduced recruitment of 53BP1 to DSBs and sustained γH2AX foci after 3 Gy of irradiation. We assessed the preclinical efficacy and toxicity of TAK-243 in mouse models of AML. OCI-AML2 cells were injected subcutaneously (sc) into SCID mice, and when tumors were palpable, mice were treated with TAK-243 (20 mg/kg sc twice weekly). TAK-243 significantly delayed tumor growth in mice (T/C=0.02) with no toxicity as evidenced by no changes in mouse body weight, serum chemistry, or organ histology. In tumors and organs isolated from the above treated mice, TAK-243 preferentially reduced levels of mono- and poly-ubiquitylated proteins in tumors over normal tissues. As an additional model, primary AML cells from 2 patients were injected into the femurs of NOD-SCID mice. Two weeks after injection, mice were treated with TAK-243 (20 mg/kg sc twice weekly). After 3 weeks of treatment, mice were sacrificed, and AML engraftment in the non-injected femur was measured by flow cytometry. TAK-243 reduced primary AML tumor burden in both tested samples without toxicity. Using secondary transplantations, we demonstrated that TAK-243 had targeted the leukemic stem cells. To understand mechanisms of resistance to TAK-243, we selected a population of TAK-243-resistant OCI-AML2 by culturing cells with increasing concentrations of the drug. Persisting cells were 33-fold more resistant to TAK-243 compared to wild-type cells (IC50 757 vs 23 nM), but had a normal rate of proliferation and remained equally sensitive to bortezomib, daunorubicin, mitoxantrone and the NEDD8-activating enzyme inhibitor pevonedistat. Using CETSA, we showed reduced binding of TAK-243 to UBA1 in the resistant cells. We sequenced UBA1 exons 12-16 and 23-24 that span the adenylation domain. Resistant cells had a missense mutation in exon 16 resulting in substitution of tyrosine with cysteine at codon 583 (Y583C). Y583 in human UBA1 corresponds to Y551 in yeast UBA1, which makes a favorable interaction with TAK-243 in its UBA1 binding site. Therefore, Y583C substitution is predicted to interfere with TAK-243 binding to UBA1. Conclusions: TAK-243 is a potent and selective UBA1 inhibitor that displays preferential activity towards AML cells over normal hematopoietic cells. Acquired mutations affect drug binding and may be a clinically relevant mechanism of resistance. These data support conducting a clinical trial of TAK-243 in patients with AML. Disclosures Hyer: Takeda Pharmaceuticals International Co.: Employment. Berger: Takeda Pharmaceuticals International Co.: Employment. Traore: Takeda Pharmaceuticals International Co.: Employment. Sintchak: Takeda Pharmaceuticals International Co.: Employment. Milhollen: Takeda Pharmaceuticals International Co.: Employment. Schimmer: Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.
Cathie M. Pfleger - One of the best experts on this subject based on the ideXlab platform.
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Mutation of the Gene Encoding the Ubiquitin Activating Enzyme UBA1 Causes Tissue Overgrowth in Drosophila
Fly, 2007Co-Authors: Cathie M. Pfleger, Kieran F. Harvey, Hua Yan, Iswar K. HariharanAbstract:Protein ubiquitination has been shown to regulate a wide variety of cellular process including cell cycle progression, protein trafficking and apoptosis. Most regulation of ubiquitination occurs at the level of E2 or E3 enzymes and their interactions with specific substrates. In a screen for mutations that cause tissue overgrowth, we recovered multiple mutations in the Drosophila UBA1 gene that encodes the E1 enzyme that is required for the first step of most, if not all, ubiquitination reactions. Previous studies with yeast and mammalian cells have shown that disrupting E1 function results in a cell-cycle arrest. Here we show that in the developing Drosophila eye, clones of cells that are homozygous for partial loss of function alleles of UBA1 show defects in apoptosis. Moreover, clones homozygous for stronger or complete loss of function alleles of UBA1, that are predicted to have a global defect on ubiquitination, survive poorly but are able to stimulate the overgrowth of adjacent wild-type tissue. Experiments with mammalian cells show that reducing the level of RNA of the mammalian UBA1 ortholog, UBE1, also results in increased expression of specific growth factor genes. Our studies show that a reduction in E1 activity can promote tissue growth in a multicellular organism and raise the possibility that changes in E1 activity may occur during normal development or in cancer.
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mutation of the gene encoding the ubiquitin activating enzyme UBA1 causes tissue overgrowth in drosophila
Fly, 2007Co-Authors: Cathie M. Pfleger, Kieran F. Harvey, Hua Yan, Iswar K. HariharanAbstract:Protein ubiquitination has been shown to regulate a wide variety of cellular process including cell cycle progression, protein trafficking and apoptosis. Most regulation of ubiquitination occurs at the level of E2 or E3 enzymes and their interactions with specific substrates. In a screen for mutations that cause tissue overgrowth, we recovered multiple mutations in the Drosophila UBA1 gene that encodes the E1 enzyme that is required for the first step of most, if not all, ubiquitination reactions. Previous studies with yeast and mammalian cells have shown that disrupting E1 function results in a cell-cycle arrest. Here we show that in the developing Drosophila eye, clones of cells that are homozygous for partial loss of function alleles of UBA1 show defects in apoptosis. Moreover, clones homozygous for stronger or complete loss of function alleles of UBA1, that are predicted to have a global defect on ubiquitination, survive poorly but are able to stimulate the overgrowth of adjacent wild-type tissue. Exp...
Aaron D. Schimmer - One of the best experts on this subject based on the ideXlab platform.
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E1 Enzymes as Therapeutic Targets in Cancer
Pharmacological reviews, 2020Co-Authors: Samir H. Barghout, Aaron D. SchimmerAbstract:Post-translational modifications of cellular substrates with ubiquitin and ubiquitin-like proteins (UBLs), including ubiquitin, SUMOs, and neural precursor cell-expressed developmentally downregulated protein 8, play a central role in regulating many aspects of cell biology. The UBL conjugation cascade is initiated by a family of ATP-dependent enzymes termed E1 activating enzymes and executed by the downstream E2-conjugating enzymes and E3 ligases. Despite their druggability and their key position at the apex of the cascade, pharmacologic modulation of E1s with potent and selective drugs has remained elusive until 2009. Among the eight E1 enzymes identified so far, those initiating ubiquitylation (UBA1), SUMOylation (SAE), and neddylation (NAE) are the most characterized and are implicated in various aspects of cancer biology. To date, over 40 inhibitors have been reported to target UBA1, SAE, and NAE, including the NAE inhibitor pevonedistat, evaluated in more than 30 clinical trials. In this Review, we discuss E1 enzymes, the rationale for their therapeutic targeting in cancer, and their different inhibitors, with emphasis on the pharmacologic properties of adenosine sulfamates and their unique mechanism of action, termed substrate-assisted inhibition. Moreover, we highlight other less-characterized E1s-UBA6, UBA7, UBA4, UBA5, and autophagy-related protein 7-and the opportunities for targeting these enzymes in cancer. SIGNIFICANCE STATEMENT: The clinical successes of proteasome inhibitors in cancer therapy and the emerging resistance to these agents have prompted the exploration of other signaling nodes in the ubiquitin-proteasome system including E1 enzymes. Therefore, it is crucial to understand the biology of different E1 enzymes, their roles in cancer, and how to translate this knowledge into novel therapeutic strategies with potential implications in cancer treatment.
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a genome wide crispr cas9 knockout screen identifies bend3 as a determinant of sensitivity to UBA1 inhibition in acute myeloid leukemia
Blood, 2018Co-Authors: Samir H. Barghout, Rose Hurren, Neil Maclean, Aaron D. Schimmer, Geethu Thomas, Zachary BlatmanAbstract:UBA1 is the major ubiquitin-activating enzyme that initiates the ubiquitylation cascade whereby proteins are tagged with mono- or polyubiquitin to mark them for proteasomal degradation or modify their functions. Despite having equal levels of UBA1 protein, AML cell lines and primary AML cells are more dependent on UBA1 activity compared to normal hematopoietic cells, rendering them more vulnerable to UBA1 inhibition. Recently, we demonstrated that inhibiting UBA1 with the small-molecule inhibitor TAK-243 was selectively cytotoxic to a subset of AML cells and stem cells in vitro and in vivo through a mechanism at least partly dependent on inducing ER stress (Leukemia, 2018). To identify potential determinants of sensitivity/resistance to TAK-243 (Millennium Pharmaceuticals, Takeda) in AML, we conducted a genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with cytotoxic TAK-243 concentrations corresponding to the IC90 and IC99. By next-generation sequencing and enrichment analysis, we then identified genes whose knockout renders AML cells resistant to TAK-243. We identified 34 hits in the IC90 and 11 hits in the IC99 arms of the screen (cut off FDR To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells using the 4 top performing gRNAs in the screen. We confirmed target knockout by immunoblotting. BEND3 knockout did not alter the basal proliferation rate of the cells. However, knockout of BEND3 rendered OCI-AML2 cells resistant to TAK-243 with up to a 4-fold increase in the IC50 by the MTS assay. Resistance to TAK-243 was confirmed by Annexin V/PI staining, PARP cleavage, and colony-forming assays. Cells were cross resistant to the NEDD8-activating enzyme inhibitor pevonedistat (2-fold IC50 increase), but not bortezomib or daunorubicin. As assessed by immunoblotting, BEND3 knockout did not change expression of UBA1, or the related enzymes UBA2, UBA3, or UBA6. BEND3 knockout was associated with reduced induction of ER stress as assessed by levels of CHOP and ATF4 after TAK-243 treatment. Conclusions Through a genome-wide CRISPR screen, we identified BEND3 as a determinant of sensitivity to TAK-243 in AML. Mechanistically, lack of BEND3 expression dampens the ER stress response to UBA1 inhibition. Thus, these results may highlight a new mechanism of sensitivity to TAK-243. Disclosures Schimmer:Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity9s Board of Directors or advisory committees.
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A Genome-Wide CRISPR/Cas9 Knockout Screen Identifies BEND3 As a Determinant of Sensitivity to UBA1 Inhibition in Acute Myeloid Leukemia
Blood, 2018Co-Authors: Samir H. Barghout, Rose Hurren, Neil Maclean, Zachary Blatman, Geethu E. Thomas, Aaron D. SchimmerAbstract:Abstract UBA1 is the major ubiquitin-activating enzyme that initiates the ubiquitylation cascade whereby proteins are tagged with mono- or polyubiquitin to mark them for proteasomal degradation or modify their functions. Despite having equal levels of UBA1 protein, AML cell lines and primary AML cells are more dependent on UBA1 activity compared to normal hematopoietic cells, rendering them more vulnerable to UBA1 inhibition. Recently, we demonstrated that inhibiting UBA1 with the small-molecule inhibitor TAK-243 was selectively cytotoxic to a subset of AML cells and stem cells in vitro and in vivo through a mechanism at least partly dependent on inducing ER stress (Leukemia, 2018). To identify potential determinants of sensitivity/resistance to TAK-243 (Millennium Pharmaceuticals, Takeda) in AML, we conducted a genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with cytotoxic TAK-243 concentrations corresponding to the IC90 and IC99. By next-generation sequencing and enrichment analysis, we then identified genes whose knockout renders AML cells resistant to TAK-243. We identified 34 hits in the IC90 and 11 hits in the IC99 arms of the screen (cut off FDR < 0.2). These hits are involved in signaling pathways including transcriptional regulation, histone methylation, ubiquitin conjugation, cell cycle progression, mTOR and NF-κB signaling pathways, consistent with the broad range of pathways regulated by UBA1-mediated ubiquitylation. We focused our investigation on BEN domain-containing protein 3 (BEND3) that ranked as a top hit in both arms (FDR = 0.0012). Compared to control, all 6 BEND3-targeting gRNAs were enriched up to 10,000 times. BEND3 is a transcriptional repressor that regulates heterochromatin organization. To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells using the 4 top performing gRNAs in the screen. We confirmed target knockout by immunoblotting. BEND3 knockout did not alter the basal proliferation rate of the cells. However, knockout of BEND3 rendered OCI-AML2 cells resistant to TAK-243 with up to a 4-fold increase in the IC50 by the MTS assay. Resistance to TAK-243 was confirmed by Annexin V/PI staining, PARP cleavage, and colony-forming assays. Cells were cross resistant to the NEDD8-activating enzyme inhibitor pevonedistat (2-fold IC50 increase), but not bortezomib or daunorubicin. As assessed by immunoblotting, BEND3 knockout did not change expression of UBA1, or the related enzymes UBA2, UBA3, or UBA6. BEND3 knockout was associated with reduced induction of ER stress as assessed by levels of CHOP and ATF4 after TAK-243 treatment. Conclusions Through a genome-wide CRISPR screen, we identified BEND3 as a determinant of sensitivity to TAK-243 in AML. Mechanistically, lack of BEND3 expression dampens the ER stress response to UBA1 inhibition. Thus, these results may highlight a new mechanism of sensitivity to TAK-243. Disclosures Schimmer: Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.
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Targeting the Ubiquitin E1 as a Novel Anti-Cancer Strategy
Current pharmaceutical design, 2013Co-Authors: Julie L. Lukkarila, Stacey-lynn Paiva, Sara R. Da Silva, Patrick T. Gunning, Aaron D. SchimmerAbstract:The proteasomal pathway of protein degradation involves two discrete steps: ubiquitination and degradation. Blocking protein degradation by inhibiting the proteasome has well described biologic effects and proteasome inhibitors are approved for the treatment of multiple myeloma and mantle cell lymphoma. In contrast, the biological effects and potential therapeutic utility of inhibiting the ubiquitination cascade and the initiating enzyme UBA1 are less well understood. UBA1 is the initial enzyme in the ubiquitination cascade and initiates the transfer of ubiquitin molecules to target proteins where they are degraded by the proteasome. Here, we review the biological effects of UBA1 inhibition and discuss UBA1 inhibitors as potential anti-cancer agents. Similar to proteasome inhibition, blocking UBA1 elicits an unfolded protein response and induces cell death in malignant cells over normal cells. Chemical UBA1 inhibitors have been developed that target different regions of the enzyme and inhibit its function through different mechanisms. These molecules are useful tools to understand the biology of UBA1 and highlight the potential of inhibiting this target for the treatment of malignancy.
Isabelle Guénal - One of the best experts on this subject based on the ideXlab platform.
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The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the β-TrCP homologue slimb
Apoptosis, 2014Co-Authors: Jessie Colin, Julie Garibal, Amandine Clavier, Aurore Rincheval-arnold, Sébastien Gaumer, Bernard Mignotte, Isabelle GuénalAbstract:The ubiquitin–proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila , the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax -induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme UBA1 as a suppressor of bax -induced cell death. We then demonstrated that UBA1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase—an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of β-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.
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The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the b-TrCP homologue slimb
Apoptosis, 2014Co-Authors: Jessie Colin, Julie Garibal, Amandine Clavier, Aurore Rincheval-arnold, Sébastien Gaumer, Bernard Mignotte, Isabelle GuénalAbstract:The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme UBA1 as a suppressor of bax-induced cell death. We then demonstrated that UBA1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of b-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.
