ATM Regulates The Self-Renewal Of Leukemia Stem Cell In MLL-Rearranged Leukemia

Objective:MLL-rearranged leukemia（MLL-r）is a hematological malignancy caused by human p11q23 chromosomal abnormality or MLL gene rearrangement,with poor prognosis,prone to recurrence and high mortality.It is currently believed that the perseveration of leukemia stem cell（LSC）is the main reason for the relapse and refractory of leukemia,because most LSC are in the stationary phase and have strong resistance to conventional radiotherapy and chemotherapy.It plays a significant role in the occurrence,progression and recurrence of leukemia,resulting in failure of treatment of leukemia and high mortality.Therefore,it is urgent to find specific and effective therapeutic targets targeting LSC.In the present study,we mainly explore the related role of Ataxia Telangiectasia Mutated（ATM）in maintaining LSC self-renewal ability in MLL-rearranged leukemia,and provide a novel therapeutic target for the treatment of MLL-rearranged leukemia.Methods:In vitro,we used small-molecule interfering technology（Sh RNA）to reduce the expression of ATM,and examined the cell proliferation,clone formation ability,cell cycle and apoptosis of MLL-r cell lines.In vivo,ATM^+/+and ATM^-/-MLL-AF9 leukemia mouse models were established by bone marrow transplantation,and LSC in the bone marrow cells of MLL-AF9leukemia mice were separated for serial transplantation and limited dilution gradient transplantation.The proliferation ability of the two groups of leukemia cells in vivo,the tumor burden in the liver and spleen of the recipient mice,the survival curve of the leukemia mice,and the frequency of leukemia stem cells were calculated;LSC continuous methylcellulose clones,cell cycle and apoptosis experiments were established in vitro.The LSC from the bone marrow of two groups of recipient mice were sorted,and the differentially expressed genes were analyzed by transcriptome sequencing analysis to enrich the signaling pathways.The expression levels of the differentially expressed genes were verified at the animal and cell line levels,and the molecular mechanism of ATM in LSC was explored.At the molecular mechanism level,in MLL-r cell lines,combined with Q-PCR,western blot,ATM kinase inhibitor experiments,cycloheximide inhibition,co-immunoprecipitation（CO-IP）and ubiquitination experiments to verify the relationship between ATM and target genes.control relationship.Results:1)In vitro studies,we found that ATM knockdown resulted in decreased proliferation and clonogenic ability,cell cycle arrest,and increased apoptosis in MLL-r cell lines（Molm13,MV4-11）.2)In vivo studies showed that in the MLL-AF9 leukemia animal model,ATM deletion caused a sharp decrease in the frequency of LSC in the F1/F2 generation,indicating that ATM deletion caused the depletion of the leukemia stem cell pool in MLL-AF9 leukemia mice,reducing the frequency of leukemia stem cells,thereby reducing the frequency of leukemia stem cells.Affect the occurrence and development of MLL-AF9 leukemia.3)Transcriptome sequencing analysis showed that compared with the ATM^+/+group recipient mice,the ATM^-/-group recipient mice were down-regulated in genes related to maintaining leukemia stem cell stemness,including the DOT1L-regulated transcription factor HOXA9/MEIS1.4)In terms of molecular mechanism,in MLL-r cell line,after ATM knockout,the protein level of DOT1L significantly decreased,but the m RNA level maintain stability.CO-IP experiment proved that there is no direct interaction between ATM and DOT1L,so ATM indirectly regulates the protein level of DOT1L.Conclusion:In the present study,ATM deletion affected the self-renewal capacity of LSC in MLL-AF9-driven leukemia,affected the progression and progression of MLL leukemia,and reduced the frequency of LSC.In mechanistic studies,ATM affects the HOXA9/MEIS1 axis by regulating DOT1L protein stability.This study provides safer and more efficient intervention targets and theoretical basis for the clinical treatment of mixed lineage leukemia.