Today, I would like to share with you an article published by the British Cancer Institute of the University of Manchester in the Nature journal Leukemia, titled "Pre-clinical activity of combined LSD1 and mtorc1 inhibitionin mll-translocated acute myeloid leukaemia". The team identified mTorC1 signaling as a cellular sensitizer for LSD1 inhibition using genome-wide CRISPR-Cas9 screening. Multiple components of the amino acid-sensitive arm of mtorc1 signaling RRAGA, MLS8, WDR24 and LAMTOR 2 were identified as sensitizers for cell inhibition of LSD1, which provides a new research direction for acute myeloid leukemia.
Background:Lysine-specific demethylase 1 (LSD 1, also known as KDM 1A) is a potential target for acute myeloid leukemia (AML) as well as certain solid malignancies. Preclinical studies of AML have shown that LSD 1 knockdown or LSD 1 pharmacological inhibition promotes the differentiation of AML cells with chromosomal translocation of MLL. Currently, the most effective method of AML is a combination of medications. To address this issue, we used a genome-wide CRISPR-Cas9 screening approach to find the most effective targets for AML. Research processA genome-wide CRISPR screen library (123,411 sgRNAs targeting 19,050 protein-coding genes and 1864 microRNA precursor genes) was constructed and transfected into a THP1 AML cell line for genome-wide CRISPR screening (by mock and OG-86 (LSD1 inhibitor: Oryzon Genomics, Compound 86) Then, the surviving cells were collected and sequenced with MISEQ to analyze genes with important cellular functions, and the synergistic activation of myeloid cell differentiation with combined pharmacological inhibition of LSD1 and mTorc1 was verified by RNA sequencing in vitro and in mice. Findings:1.Screening and identification of LSD1 inhibitory genes and allergens in human THP1 AML cellsTHP 1 AML cells exhibit MLL gene rearrangements and are similar to MLL cells with MLL translocation in primary patients in terms of inhibition of LSD 1 response. OG-86 is a potent and specific trans-cytosine derivative LSD 1 inhibitor. In this article, human THP1 AML cells were screened for loss-of-function CRISPR-Cas9 genome-wide in the presence and absence of OG-86, and Mageck analysis identified 7 candidate genes with high scores, in which targets encoding genes for multiple positive regulators of mTorc1 signaling were depleted, including MLS8, Rraga, LaMtor2, WDR24, and AKT1. THP 1 AML cells were treated with OG-86 250 nM (red line) or DMSO (blue line) in combination with different concentrations of MK 2206, PP 242, or RAD 001, and RAD001 was finally selected5um and OG-86 250 nm were combined.
Figure 1 Identification of genetic sensitizers for LSD1 inhibition & combined pharmacological inhibition of LSD 1 and mTorc 1 in human THP1 AML cells2.In vitro analysis of combined pharmacological inhibition of LSD 1 and MT0RC 1
THP 1 AML cells were treated with DMSO, OG-86 (250 nM), RAD 001 (40 nM), or a combination of both for 24 h. Then, transcriptomic sequencing was performed to detect the differentially expressed genes in the four samples, and it was found that the cells treated with OG-86 RAD 001 were significantly different from other groups, group A genes were down-regulated and related to active metabolism, while group B genes were up-regulated, which were related to immune function, and their survival rate and surface markers were detected, and OG-86 RAD 001 was found Flow cytometry showed that the activity of non-leukemia stem cell surface markers in the OG86 treatment group was significantly up-regulated by rapid induction of myeloid differentiation program (marked by cell surface proteins CD11B and CD86), which further indicated that the combination of drugs could promote the differentiation of AML cells more than single treatment. 
Fig.2 Combinatorial pharmacological synergy by inhibition of LSD 1 and mTorc 1.
Activates bone marrow differentiation in vitro.
3.In vivo analysis of combined pharmacological inhibition of LSD 1 and MT0RC 1
To determine whether in vitro observation can also be observed in vivo, we co-cultured primary MLL-transtranslocated acute myeloid leukemia cells from 7 patients with DMSO, OG-86 (250 nM), RAD001 (2 M), and a combination of DMSO for 7 days, then injected into mice, and 14 weeks later by oral gavage with control (H2O), RAD 001 (5 mg kg), OG-98 (3 mg kg), and RAD 001 (5 mg). kg) and OG-98 (3 mg kg) for 5 days, human CD45+ cells from murine bone marrow were screened by immunomagnetic strains, and then transcriptomic sequencing was performed to detect differentially expressed genes in four samples. By combining inhibition of LSD1 and mTorc1 to synergistically activate myeloid differentiation programs in vivo, the data showed that the treated OG-98 RAD 001 cells were significantly different from other groups, and the down-regulation of group A genes and the up-regulation of group B genes were obtained by reclustering, and the expression of the up-regulated gene set in the treated OG-98 RAD 001 cells was significantly higher than that of other groups. There was significant overlap between the group B genome upregulated by THP 1 AML cells and primary patient AML cell xenograft combined with **, and these genes included transcription factors that play an important role in monocyte macrophage differentiation. In vitro and in vivo analyses of ML translocated cell lines and primary samples, the combination of LSD 1 and mTorc 1 inhibits the synergistic upregulation of induced monocyte macrophage differentiation procedures. 
Fig.3 Combinatorial pharmacological synergy by inhibition of LSD 1 and mTorc 1.
Activates bone marrow differentiation in vivo.
4.Combined inhibition of LSD1 and mTorc1 impairs the in vivo clonal activity of primary AML cells
In order to validate a potential clinical protocol for the combination of LSD1 and mTorC1 inhibition: the feasibility of promoting the differentiation of residual clonal AML cells after minimal residual disease, the primary AML cells with less burden were transferred to NSG mice, and after 3 weeks with the drug** mice were found to be well-tolerated with the RAD001 og-98 combination after 4 weeks, **There was no significant difference in weight at the end. The functional potential of transplanted human AML cells was evaluated by ** cells and cloning assays, and it was found that OG-98 reduced the number of transplanted AML cells, while RAD001 reduced the clonal activity of transplanted cells. Bone marrow transplant clonal AML cells were significantly reduced in the OG-98 RAD001 combination treatment group compared to all other groups. Thus, inhibition of concomitant pharmacology-induced monocyte macrophage differentiation procedures for LSD 1 and MTORC 1 is inhibited in in vitro and in vivo analysis of ML translocated cell lines and primary samples. OG-86 alone has little overall effect on cell growth, while RAD 001 shows moderate antiproliferative activity, and this combination inhibits cell growth again significantly and significantly.
Fig.4 Combined pharmacological inhibition of LSD 1 and mTorc 1 impairs the clonogenic activity of primary AML cells in vivoConclusions
The authors identified multiple components of the amino acid-sensitive arm of the mTorC1 signaling RRAGA, MLS8, WDR24, and LAMtor2 as sensitizers for cellular inhibition of LSD1. Downregulation of mtorc1 components or pharmacological inhibition of mtorc1 in combination with LSD1 inhibition enhances the differentiation of cell lines and primary cells in vitro and in vivo. Therefore, mTorC1 and LSD1 inhibition are candidate combination methods for MLL translocation and AML-enhanced differentiation that can be evaluated in early clinical trials. 
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