Mutated and enucleated TIM3 regulates function of Acute Myeloid Leukemia stem cells through c-MYB Free

Drug resistance and recurrence are important causes of poor prognosis in acute myeloid leukemia (AML), and leukemia stem cells (LSCS) are the key factor for drug resistance and recurrence. However, the relevant mechanisms by which different mutations affect the function of LSCS remain unclear. Clarifying the specific mechanisms by which mutations influence chemotherapy resistance in LSCS and identifying the corresponding targets are urgent scientific issues to be addressed. Previously, we observed in the clinical cohort that the incidence of TIM3 mutations was approximately 5.30% in AML, the TIM3 p.Y82C mutation was an incidence rate of 4.08% significantly. The 3-year cumulative recurrence rates in the TIM3^wt group and the TIM3^mut group were 22.9% and 37.5% respectively (P = 0.013), the overall survival rate (OS) was 70.8% and 50% respectively (P = 0.02), and the event-free survival rate (EFS) was 56.3% and 33.3% respectively (P = 0.006), it was suggested that patients with TIM3 mutations have a higher proportion of drug resistance and a poorer clinical prognosis. In order to explore the impact and mechanism of Tim-3 mutation on AML, TIM3-Y82C mutant mice were constructed by CRISPR/CAS9，then MLL-AF9 and Hoxa9 AML mouse models were constructed. Through the incidence rate of mice, colony-forming unit (CFU), extreme dilution test, IC50 of chemotherapy drugs and survival statistics, it was found that the TIM3-Y82C mutation could promote the onset of AML, enhance the stemness of LSC and chemotherapy resistance. Through immunofluorescence and nuclear-cytoplasmic separation experiments of mouse bone marrow and patient bone marrow cells, it was proved that the TIM3-Y82C mutation affects LSC function by entering the nucleus. By constructing nuclear and nucleated AML cell lines, it was found that the TIM3-Y82C mutation enhances chemotherapy resistance by entering the nucleus. To further clarify the mechanism, we used protein-protein interaction analysis found that the TIM3-Y82C mutation was related to the interaction of hematopoietic transcription factor C-MYB. In vivo, MLL-AF9 and Hoxa9 AML mice were constructed, it was furtherly confirmed that mutated and enucleated TIM3 interacted with C-MYB regulates stemness and drug resistance of LSC through C-MYB expression knockout, chemotherapy drug IC50 and survival statistics, thereby affecting AML survival. Finally, through high-throughput screening of 1,140 drugs in the small molecule compound drug library, we found tyrosine kinase/adaptors have unique efficacy in TIM3 mutated AML mice. In summary，our study firstly discovered that TIM3 mutation is an important target for the poor prognosis of AML. Targeting C-MYB or combined therapy with tyrosine kinase/adaptors may be of great significance for improving the efficacy and prognosis of TIM3-mutated AML.