Genome-wide CRISPR-Cas9 screen identifies druggable synthetic lethality between LSD1 and MTORC1 in MLL-translocated AML
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ABSTRACT:
PROVIDER: PRJNA522066 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11031281.fastq.gz | Fastqsanger.gz | |||
| SRR11031282.fastq.gz | Fastqsanger.gz | |||
| SRR11031283.fastq.gz | Fastqsanger.gz | |||
| SRR11031284.fastq.gz | Fastqsanger.gz | |||
| SRR11031285.fastq.gz | Fastqsanger.gz |
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Project description:Lysine Specific Demethylase 1 (LSD1 or KDM1A) is one of a number of epigenetic regulators which have recently emerged as candidate therapeutic targets in acute myeloid leukaemia (AML). Pharmacological inhibitors of LSD1 such as the tranylcypromine derivatives have already commenced evaluation in early phase clinical trials; however like all acute leukaemia therapies, it is unlikely that these inhibitors are effective as single agents. Therefore, there is a strong rationale to identify proteins that collaborate with LSD1 to maintain the leukemic phenotype and could be targeted in combination with LSD1 for enhanced therapeutic benefit. Using a genome-wide CRISPR-Cas9 dropout screen in human THP1 cells, we identified multiple regulatory components of the amino acid sensing arm of mTORC1 signalling - RRAGA, MLST8, and LAMTOR2 – as synthetic lethalities with LSD1 inhibition. Genetic and/or pharmacologic inhibition of selected genes in combination with LSD1 inhibition showed significant anti-leukemic activity by inducing a more extensive and wide-ranging myeloid differentiation program and reducing cell proliferation in THP1 and primary human AML cells in vitro and in vivo. In conclusion, we report that inhibition of mTORC1 sensitizes human MLL-translocated AML cells to LSD1 inhibitor-mediated differentiation therefore highlighting a novel combination approach for evaluation in clinical trials.
2019-12-23 | GSE126486 | GEO
Project description:The histone demethylase lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML); tranylcypromine-derivative inhibitors induce loss of clonogenic activity and promote differentiation, in particular in the MLL-translocated molecular subtype of AML. In AML, the use of drugs in combination often delivers superior clinical activity. To identify genes and cellular pathways that collaborate with LSD1 to maintain the leukaemic phenotype, and which could be targeted by combination therapies, we performed a genome-wide CRISPR-Cas9 dropout screen. We identified multiple components of the amino acid sensing arm of mTORC1 signalling-RRAGA, MLST8, WDR24 and LAMTOR2-as cellular sensitizers to LSD1 inhibition. Knockdown of mTORC1 components, or mTORC1 pharmacologic inhibition, in combination with LSD1 inhibition enhanced differentiation in both cell line and primary cell settings, in vitro and in vivo, and substantially reduced the frequency of clonogenic primary human AML cells in a modelled minimal residual disease setting. Synergistic upregulation of a set of transcription factor genes associated with terminal monocytic lineage differentiation was observed. Thus, dual mTORC1 and LSD1 inhibition represents a candidate combination approach for enhanced differentiation in MLL-translocated AML which could be evaluated in early phase clinical trials.
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Project description:In this study, we use the epidermis as a model system to elucidate the cellular effects and signaling feedback sequelae of mTORC1 loss-of-function in epithelial tissue. In mice with conditional epidermal loss of mTORC1 components Rheb or Rptor, we find that mTORC1 loss-of-function unexpectedly results in a profound skin barrier defect with epidermal abrasions, blistering and early postnatal lethality, due to a thinned epidermis with decreased desmosome expression and incomplete biochemical differentiation. Impaired cell-cell adhesion in the context of mTORC1 loss-of-function is caused by constitutive activation of Rho kinase (ROCK) signaling with increased cytoskeletal tension, and inhibition or silencing of ROCK1 is sufficient to rescue keratinocyte adhesion and biochemical differentiation. mTORC1 loss-of-function results in marked feedback up-regulation of upstream TGF-β signaling, triggering ROCK activity and its downstream effects on desmosomal gene expression. These findings elucidate a novel role for mTORC1 in the regulation of epithelial barrier formation, cytoskeletal tension and cell adhesion and underscore the complexity of signaling feedback after mTORC1 inhibition
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Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a M-bM-^@M-^\low/low/high/lowM-bM-^@M-^] or M-bM-^@M-^\LLHLM-bM-^@M-^]. We found that mTORC1 regulated the expression of, among other lysosomal genes, V-ATPases through the transcription factor EB (TFEB, Tcfeb in the mouse). The knockdown of Tfeb resulted in the 'flattening' of the LLHL pattern and allowed the identification of genes regulated by mTORC1 through Tfeb Mouse embryo fibroblasts (MEFs) wild type or deficient in Tsc2 expressing a Tfeb shRNA or scrambled shRNA vector were treated with 25 nM rapamycin or vehicle (methanol) for 24 h under low serum conditions (0.1% FBS)
2011-08-02 | E-GEOD-28021 | biostudies-arrayexpress
Project description:To further explain pathology of mTORC1 -stimulated osteoarthritis, we have employed whole genome microarray expression profiling as a discovery platform to identify miRNAs which involved in development of mTORC1-stimulated osteoarthritis We generated Col2a1-specific deletion of Tsc1 mice. mTORC1 induced miRNAs expression in development of osteoarthritis was measured at eight weeks after birth. Independent experiments were performed using knee joint cartilage from Col2a1Tsc1KO and control mice.
2016-07-30 | E-GEOD-71484 | biostudies-arrayexpress
Project description:To screen miRNAs specifically regulated by mTORC1 or mTORC2, a global miRNA expression profile in MCF-7 cells treated with rapamycin or PP242 (mTORC1/2 kinase inhibitor) was developed using microarray. control, rapamycin or PP242 treated human MCF-7 cells were harvested 48h post-treatment and subjected to total RNA extraction.
2014-09-11 | E-GEOD-61289 | biostudies-arrayexpress
Project description:The mechanistic target of rapamycin mTORC1 is a key regulator of cell metabolism and autophagy. Despite widespread clinical use of mTOR inhibitors, the role of mTORC1 in renal tubular function and kidney homeostasis remains elusive. By utilizing constitutive and inducible deletion of conditional Raptor alleles in renal tubular epithelial cells, we discovered that mTORC1 deficiency caused a marked concentrating defect, loss of tubular cells and slowly progressive renal fibrosis. Transcriptional profiling revealed that mTORC1 maintains renal tubular homeostasis by controlling mitochondrial metabolism and biogenesis as well as transcellular transport processes involved in counter-current multiplication and urine concentration. Although mTORC2 partially compensated the loss of mTORC1, exposure to ischemia and reperfusion injury exaggerated the tubular damage in mTORC1-deficient mice, and caused pronounced apoptosis, diminished proliferation rates and delayed recovery. These findings identify mTORC1 as an essential regulator of tubular energy metabolism and as a crucial component of ischemic stress responses. Pharmacological inhibition of mTORC1 likely affects tubular homeostasis, and may be particularly deleterious if the kidney is exposed to acute injury. Furthermore, the combined inhibition of mTORC1 and mTORC2 may increase the susceptibility to renal damage. Raptor fl/fl*KspCre and Raptor fl/fl animals were sacrificed at P14 before the development of an overt functional phenotype. Kidneys were split in half and immediately snap frozen in liquid nitrogen.
2014-06-09 | E-GEOD-54417 | biostudies-arrayexpress
Project description:The gut microbiome of translocated populations of the Seychelles warbler
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Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a M-^Slow/low/high/lowM-^T or M-^SLLHLM-^T, which allowed the identification of genes regulated by mTORC1 by performing the appropriate comparisons
2011-08-02 | E-GEOD-27982 | biostudies-arrayexpress
Project description:Distinct molecular subgroups and subtypes have been identified for medulloblastoma (MB), one of the most malignant brain tumors of childhood, with the sonic hedgehog (SHH)-activated subgroup of patients with mutations in p53 having a very severe outcome, associated with unfavorable histological large cell/anaplastic (LC/A) features. Aim of this study was to determine whether hyperactivation of mechanistic Target Of Rapamycin Complex 1 (mTORC1) pathway, whose role in molecular subtypes of MB is currently undefined, underlies the acquisition of LC/A histology in this subtype. MBs developing in p53-deficient Ptch+/- SHH mice show LC/A features that correlate with mTORC1 hyperactivation, which, mechanistically, is mediated by a decrease in the p53-dependent expression of the negative mTORC1 regulator Tsc2. Ectopic mTORC1 activation in mouse MB cancer stem cells (CSCs) promotes the acquisition of the LC/A phenotype. Furthermore, mTORC1 inhibition in both autochthonous p53-mutant SHH MBs and CSC-derived LC/A MBs results in reduced tumor burden and enhanced survival. Most remarkably, mTORC1 hyperactivation in tumor cells is detected only in human p53-mutant SHH MB samples and treatment with rapamycin of a human preclinical model phenocopying this subgroup decreases tumor growth and malignancy. As such, mTORC1 may act as a new and specific druggable target for this subset of SHH MB, thus resulting in the implementation of a more stringent MB risk stratification and in the potentially rapid translation of this precision medicine approach into the clinical setting.
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