Project description:We reported the expression profiling between mTORC1 high and low mouse AML cells with MLL-AF9 utilizing a novel fluorescent probe named mVenus-TOSI (TOr-Signal-Indicator). For this purpose, 3 mTOR high (mVenus low) and 4 mTOR low (mVenus high) Mouse AML cells were harvested from 4 mice. The transcriptomic profiles were distinctive and gene ontology analysis revealed that mTORC1 activity was associated with differentially expressed immune response and metabolism related genes. Further, gene signature enrichment analysis (GSEA) validated that the mTORC1 signal signature correlated with mTORC1 activity and that leukemia stem cell (LSC), Myc, and cell cycle related signatures were enriched in mTORC1 high cells.
Project description:There are two possible mechanisms for the shift of gene expression to a high mTORC1 activity signature after chemotherapy; one is the selection of mTORC1 high cells and the second is the induction of mTORC1 activity following chemotherapy. We assessed these two possible scenarios by introducing genetic barcodes using the ClonTracer library that allowed for clonal tracking of ~0.2 million AML cells. We found that a significant proportion of unique barcodes are represented in both the mTORC1 high and low cell populations. Since barcodes only infect a single cell which has to exist in a high or low state, a significant proportion of barcodes that are represented by cells in both states suggests that induction occurs. Otherwise the proportion should be close to an extreme (0 or 1) meaning most of the populations exist only in mTORC1 high or mTORC1 low states. Additionally, clonal abundance based on barcodes revealed more similarity with respect to the cumulative proportion of represented barcodes between mTORC1 high and low cells in treated cohorts compared with non-treated cohorts. These data suggest a more similar distribution of mTORC1 high and low cells in these populations which would arise primarily due to induction rather than selection.
Project description:To obtain the biologic insight of TET1 expression,we analyzed microRNAs expression profiles from 12 AML patients with high TET1 expression and 12 cases with low TET1 expression.
Project description:We reported the expression profiling between mouse AML cells with or without chemotherapy. For this purpose, mouse AML cells were harvested from mice treated with or without chemotherapy drugs on day 7. Gene expression analysis of AML cells with or without chemotherapy identified clearly distinguishable transcriptomic profiles. GO analysis revealed that gene alterations after chemotherapy were related to immune response, RNA biology and several metabolomic processes. mTORC1 signal-related GSEA signatures were also enriched in AML cells after chemotherapy including those of stemness and impaired differentiation.
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.
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
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)