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A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity.

ABSTRACT: TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.

SUBMITTER: Barghout SH

PROVIDER: S-EPMC8021101 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity

Project description:TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 (UBA1) that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase 1 clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243

2021-01-13 | GSE164639 | GEO

A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity

Project description:A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity

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Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth.

Project description:Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.

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A genome-scale CRISPR-Cas9 screening in myeloma cells identifies regulators of immunomodulatory drug sensitivity.

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A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia.

Project description:Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as DOT1L, BCL2, and MEN1, and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose KAT2A as a candidate for downstream study. KAT2A inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies.

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A CRISPR/Cas9-mediated screen identifies regulators of early plasma cell differentiation

Project description:The differentiation of B cells into antibody-secreting cells depends on cell division-coupled, epigenetic and other cellular processes that are incompletely understood. We have developed a CRISPR/Cas9-based screen that models an early stage of T cell-dependent plasma cell differentiation and measures B cell survival or proliferation versus the formation of CD138+ plasmablasts. Here, we refined and extended this screen to more than 500 candidate genes that are highly expressed in plasma cells. Among known genes whose deletion preferentially affected plasmablast formation were the transcriptional regulators Prdm1, Irf4 and Pou2af1, and the Ern1 gene encoding the ER stress sensor IRE1a. Moreover, we newly identified several genes involved in NF-kB or p38 signaling (Pim2, Nfkbia, Mapk14), vesicle trafficking (Arf4, Preb) and epigenetic regulators that form part of the NuRD complexes (Hdac1, Mta2, Mbd2). Defective plasmablast formation caused by Ern1 deletion could not be rescued by spliced XBP1 (XBP1s), a transcription factor dependent on and downstream of IRE1a, suggesting that in early plasma cell differentiation IRE1a acts independently of XBP1s. The deletion of ARF4, a small GTPase required for ER-to-Golgi transport, impaired plasmablast formation by blocking antibody secretion. Plasmablast formation after Hdac1 deletion was consistently reduced by about 50%, while deletion of the closely related Hdac2 gene had no effect. Hdac1 knock-out led to strongly perturbed protein expression of antagonistic transcription factors that specify plasma cell versus B cell identity (by decreasing IRF4 and BLIMP1 and increasing BACH2 and PAX5). Taken together, our results highlight specific and non-redundant roles for Ern1, Arf4 and Hdac1 in the early steps of plasma cell differentiation.

2022-12-22 | PXD038201 | Pride

A CRISPR/Cas9 Functional Screen Identifies Rare Tumor Suppressors.

Project description:An enormous amount of tumor sequencing data has been generated through large scale sequencing efforts. The functional consequences of the majority of mutations identified by such projects remain an open, unexplored question. This problem is particularly complicated in the case of rare mutations where frequency of occurrence alone or prediction of functional consequences are insufficient to distinguish driver from passenger or bystander mutations. We combine genome editing technology with a powerful mouse cancer model to uncover previously unsuspected rare oncogenic mutations in Burkitt's lymphoma. We identify two candidate tumor suppressors whose loss cooperate with MYC over-expression to accelerate lymphomagenesis. Our results highlight the utility of in vivo CRISPR/Cas9 screens combined with powerful mouse models to identify and validate rare oncogenic modifier events from tumor mutational data.

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In vivo CRISPR/Cas9 screening identifies Pbrm1 as a regulator of myeloid leukemia development in mice.

Project description:CRISPR/Cas9 screening approaches are powerful tool for identifying in vivo cancer dependencies. Hematopoietic malignancies are genetically complex disorders in which the sequential acquisition of somatic mutations generates clonal diversity. Over time, additional cooperating mutations may drive disease progression. Using an in vivo pooled gene editing screen of epigenetic factors in primary murine hematopoietic stem and progenitor cells (HSPCs), we sought to uncover unrecognized genes that contribute to leukemia progression. We, first, modeled myeloid leukemia in mice by functionally abrogating both Tet2 and Tet3 in HSPCs, followed by transplantation. We, then, performed pooled CRISPR/Cas9 editing of genes encoding epigenetic factors and identified Pbrm1/Baf180, a subunit of the polybromo BRG1/BRM-associated factor SWItch/Sucrose Non-Fermenting chromatin-remodeling complex, as a negative driver of disease progression. We found that Pbrm1 loss promoted leukemogenesis with a significantly shortened latency. Pbrm1-deficient leukemia cells were less immunogenic and were characterized by attenuated interferon signaling and reduced major histocompatibility complex class II (MHC II) expression. We explored the potential relevance to human leukemia by assessing the involvement of PBRM1 in the control of interferon pathway components and found that PBRM1 binds to the promoters of a subset of these genes, most notably IRF1, which in turn regulates MHC II expression. Our findings revealed a novel role for Pbrm1 in leukemia progression. More generally, CRISPR/Cas9 screening coupled with phenotypic readouts in vivo has helped identify a pathway by which transcriptional control of interferon signaling influences leukemia cell interactions with the immune system.

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Project description:To identify novel targets for acute myeloid leukemia (AML) therapy, we performed genome-wide CRISPR-Cas9 screening using AML cell lines, followed by a second screen in vivo. Here, we show that the mRNA decapping enzyme scavenger (DCPS) gene is essential for AML cell survival. The DCPS enzyme interacted with components of pre-mRNA metabolic pathways, including spliceosomes, as revealed by mass spectrometry. RG3039, a DCPS inhibitor originally developed to treat spinal muscular atrophy, exhibited anti-leukemic activity via inducing pre-mRNA mis-splicing. Humans harboring germline biallelic DCPS loss-of-function mutations do not exhibit aberrant hematologic phenotypes, indicating that DCPS is dispensable for human hematopoiesis. Our findings shed light on a pre-mRNA metabolic pathway and identify DCPS as a target for AML therapy.

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