Antileukemic Activity And Mechanism Of Action Of Targeting HDAC,PI3K,and FLT3 In FLT3-mutated Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a hematologic malignancy caused by abnormal proliferation and differentiation disorders.AML is the most common acute leukemia in adults.For fit patients,the standard of care is the "7+3" conventional chemotherapy along with hematopoietic stem cell transplantation,while for unfit patients the recently FDA approved Venetoclax in combination with hypomethylating agents or low-dose cytarabine therapies are becoming the standard of care.Although the majority of patients achieve complete remission,relapses and drug resistance are common which result in the 5-year overall survival rate of about 30%.Therefore,there is an urgent need for new treatments with high efficacy and low toxicity to improve the treatment outcome of AML.FMS-like tyrosine kinase 3(FLT3)mutations occur in approximately 30% of AML patients,about 25% are internal tandem duplication(ITD)mutations in the Juxtamembrane domain,and 7-10% are point mutations in the tyrosine kinase domain(TKD).FLT3 mutations lead to constitutive activation of the receptor tyrosine kinase and its downstream signaling pathways,PI3K/AKT/m TOR,RAS/RAF/MEK/ERK,and JAK2/STAT5,promoting cell proliferation and survival.Hence,FLT3 mutations often indicate a very poor prognosis.On November 28,2018,the US Food and Drug Administration(FDA)approved gilteritinib for the treatment of adult patients with relapsed or refractory FLT3-mutated AML.However,it only shows limited clinical efficacy.Therefore,there is an urgent need of agents to enhance the antileukemic activity of gilteritinib to break through the current treatment bottleneck of FLT3-mutated AML.CUDC-907 is a dual inhibitor of PI3 K and HDAC with potent antitumor activity against preclinical models of a variety of tumors.Our previous studies have shown that CUDC-907 can downregulate FLT3,prompting us to hypothesize that downregulation of FLT3 by CUDC-907 enhances the anti-leukemia activity of gilteritinib.In addition,our previous studies have found that activation of ERK is closely related to gliteritinib resistance and CUDC-907 can effectively inactivate ERK.Based on these findings,we overall hypothesis of this study was that CUDC-907 can synergistically enhance the antileukemic activity of gilteritinib by suppressing FLT3 and its downstream signaling pathways and overcoming gilteritinib resistance in FLT3-mutated AML.To test this hypothesis,we first verified that CUDC-907 downregulates FLT3 through protein degradation mediated by the proteasome pathway.Next,we determined the antileukemic activities of CUDC-907 and gilteritinib,alone or in combination,against FLT3-mutated AML.CUDC-907 and gilteritinib synergistically induced apoptosis in FLT3-mutated AML cell lines and primary patient samples,but spared healthy human peripheral blood mononuclear cells,indicating that the combination has a good safety profile.We also found that the combination can significantly kill AML progenitor cells,but has little effect on normal hematopoietic progenitor cells,which further proves the excellent tumor selectivity of this combination.Finally,in a FLT3-ITD AML cell line MV4-11-derived xenograft NSGS mouse model,the combination was well tolerated and showed excellent in vivo antileukemic efficacy.Next,we thoroughly investigated the mechanism responsible for the synergistic antileukemic activity of CUDC-907 and gilteritinib against FLT3-mutated AML.The combination of CUDC-907 and gilteritinib resulted in suppression/inhibition of FLT3 protein,leading to inactivation of the downstream PI3K/AKT/m TOR,RAS/RAF/MEK/ERK,and JAK2/STAT5 signaling pathways.This was accompanied by downregulation of anti-apoptotic protein Mcl-1 and oncoprotein c-Myc,and upregulation of pro-apoptotic protein Bim.Bak and Bax dual silencing resulted in partial rescue of the cells from CUDC-907 and gilteritinib,alone or in combination.In addition,we found that gilteritinib treatment resulted in activation of ERK,while CUDC-907 treatment resulted in activation of STAT5,which in turn leads to drug resistance.Interestingly,the combination of both agents reciprocally overcame these mechanisms of drug resistance.In addition,through metabolomics analysis,we found that the combination of CUDC-907 and gilteritinib significantly downregulated the levels of cellular metabolites and had persitent antileukemic effects.In summary,our results demonstrate that the combination of CUDC-907 and gilteritinib has synergistic antileukemic activity against FLT3-mutated AML both in vitro and in vivo.Through downregulation and inhibition of FLT3,the combination inactivates downstream MAPK/ERK and JAK2/STAT5 signaling pathways,accompanied by induction of Bim and downregulation of Mcl-1,c-Myc,and cellular metabolism,resulting in persistent antileuemic effects.The results of this study provide a solid theoretical and experimental basis for the clinical evaluation of this promising combination therapy for the treatment of FLT3-mutated AML.