Studies On Antitumor Activity And Mechanism Of Combined Inhibition Of PI3Kδ And FLT3 Or BCL-2 In Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is the most common malignant myeloid disorder in adults.Treatment of AML has limited efficacy,producing a low cure rate for the past 40 years.Many patients with AML have genetic abnormalities,which are consistently associated with poor outcomes.Thus,individualized molecular-targeting approaches are needed for the treatment of AML.Class I phosphatidylinositol 3-kinase(PI3K)and its downstream target protein kinase B(AKT)are frequently constitutively activated in AML cells.In this study,we investigated the expression levels of the four different catalytic isoforms of class I PI3 Ks in AML cell lines.And we found that in contrast to the other class I isoforms,which exhibited more widely varied expression levels,PI3Kδ was consistently expressed at a high level in AML cells.Thus,PI3Kδ might be a potential therapeutic target in AML.CAL101 is a potent,first-in-class selective inhibitor of PI3Kδ that was approved by the US Food and Drug Administration for the treatment of relapsed chronic lymphocytic leukemia,relapsed follicular lymphoma,and relapsed small lymphocytic lymphoma.However,CAL101 showed weaker inhibitory activity toward AML compared with that toward chronic lymphocytic leukemia.Thus,combination therapies are needed to be explored for the treatment of AML.FMS-like tyrosine kinase 3(FLT3)mutations that result in constitutive activation of FLT3 kinase are the most frequently identified genetic alterations in about 30% of adult AML patients.FLT3 inhibitors induce responses in AML patients harboring FLT3 mutations,but responses are not durable,and early clinical studies have revealed several resistant mechanisms of FLT3 inhibitors.Given the limited and transient efficacy of FLT3 inhibitors,combination therapies are being explored,such as combination treatments with FLT3 inhibitors and chemotherapeutic agents or signaling pathway inhibitors.In this study,we demonstrate that combined inhibition of PI3Kδ and FLT3 exerts synergistic antitumor activity in FLT3-activated AML.Synergistic antiproliferative effects were observed in FLT3-activated MV-4-11 and EOL-1 AML cell lines,but not in FLT3-independent RS4;11 and HEL cells,as demonstrated by both pharmacological inhibition and silencing of PI3Kδ/FLT3.Combined treatment with PI3Kδ and FLT3 inhibitors more effectively inhibited AKT and extracellular signal regulated kinase(ERK)phosphorylation,and induced apoptosis more efficiently than either agent alone.This synergistic effect was confirmed in hematopoietic 32 D cells transfected with an FLT3 internal tandem duplication(ITD)mutant,but not FLT3 wild type.In in vivo FLT3-activated AML xenografts,a PI3Kδ inhibitor CAL101 combined with FLT3 inhibitor led to significantly enhanced antitumor activity compared with either agent alone,in association with simultaneous inhibition of AKT and ERK.In early clinical studies,patients often acquire tyrosine kinase domain(TKD)point mutations of FLT3 and result in resistance to FLT3 inhibitors after prolonged monotreatment with FLT3 inhibitors.We generated an MV-4-11/su cell line resistant to FLT3 inhibitors by culturing cells in the continuous presence of escalating doses of sunitinib.And next-generation sequencing of FLT3 revealed a D835 H substitution within that TKD in the MV-4-11/su cell line.CAL101 combined with FLT3 inhibitors overcame acquired drug resistance in MV-4-11/su cells.The effects of overcoming acquired drug resistance were futher confirmed in 32 D cells transfected with FLT3-ITD mutant and D835 Y or D835 H point mutations.Thus,PI3Kδ inhibitor CAL101 combined with FLT3 inhibitors overcame acquired drug resistance in FLT3-ITD AML cells.Taken together,combined inhibition of PI3Kδ and FLT3 may be a promising strategy in FLT3-activated AML,particularly for patients with FLT3-inhibitor-resistant mutations.In addition,AML cells often up-regulate B cell lymphoma-2(BCL-2),to avoid apoptosis.And over-expression of myeloid cell leukemia-1(MCL-1)in cells can cause resistance to BCL-2 inhibitors.Thus,we investigated the antitumor activity of combined inhibition of PI3Kδ and BCL-2 in AML cells.We found that combined inhibition of PI3Kδ and BCL-2 exerted synergistic antiproliferative effects and induced apoptosis more efficiently in AML cells with BCL-2 and MCL-1 expression,and it could overcome the resistance to BCL-2 inhibitors due to MCL-1 over-expression.However,no synergistic effects were seen in AML cells almost lack MCL-1 expression.Combined treatment with PI3Kδ and BCL-2 inhibitors inhibited AKT phosphorylation and decreased the expression of MCL-1 more effectively.In the end,combined inhibition of MCL-1 and BCL-2 also exerted synergistic antiproliferative effects in AML cells.And these results futher demonstrated that MCL-1 was important in the combination therapy of PI3Kδ and BCL-2 inhibition.Thus,combined inhibition of PI3Kδ and BCL-2 may be a promising strategy in AML.In conclusion,we found that combined inhibition of PI3Kδ and FLT3 yielded synergistic antitumor effects in FLT3-activated AML,and combined inhibition of PI3Kδ and BCL-2 exerted synergistic antitumor effects in AML with BCL-2 and MCL-1 expression for the first time.These findings are important and may provide some guidance for clinical combination scheme in AML.