Antileukemic Activity And The Underlying Molecular Mechanisms Of Contargeting PI3K,HDAC,and Bcl-2 In Preclinical Models Of Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is an aggressive myeloid malignancy characterized by the clonal expansion,differentiation arrest,and deregulated proliferation of myeloid blasts.Standard induction therapy(7 + 3 regimen)for AML patients has largely unchanged over the past 40 years,and 5-year overall survival rates(approximately 25% for adults and 65% for children)remain unacceptably low.The majority of patients over the age of 60 cannot tolerate induction chemotherapy,resulting in dismal prognosis.This highlights the urgent need of more effective therapies for patients with AML.Dyregulation of apoptosis has been recognized as a key mechanism of tumorigenesis and resistance to cancer treatment.The Bcl-2 family members are central regulators of mitochondrial apoptosis and their interaction controls cell fate.Overexpression of anti-apoptotic Bcl-2 family proteins(Bcl-2,Bcl-xL and Mcl-1)is a prognostic factor and is associated with chemotherapy resistance in AML,making anti-apoptotic Bcl-2 family proteins promising therapeutic targets for the treatment of AML.Venetoclax is an oral,selective Bcl-2 inhibitor,which has been approved by the US FDA in 2018 for the treatment of AML.Although Venetoclax shows promising clinical activity against AML,its antileukemic activity is limited by both intrinsic and acquired resistance.Our previous studies have demonstrated that Venetoclax treatment releases Bim from Bcl-2,however Mcl-1 sequesters the released Bim,preventing apoptosis to occur,demonstrating Mcl-1 a key factor involved in the resistance to Venetoclax in AML.Further,we have previously shown that Venetoclax also enhances DNA damage induced by DNA damaging agents in AML cells.Therefore,we hypothesized that simultaneously downregulating Mcl-1,upregulating Bim,and inducing DNA damage can maximally enhance venetoclax-induced cell death.The PI3K/AKT and Ras/Raf/MEK/ERK pathways play important roles in cellular proliferation,differentiation,and survival.It has been reported that 50-80% of AML patients have constitutively active PI3K/AKT pathway,which correlates with poor prognosis.Further,it has been reported that simultaneous inhibition of the PI3K/AKT and Ras/Raf/MEK/ERK pathways results in down-regulation of Mcl-1 and upregulation of Bim and cell apoptosis.It has been previously reported that histone deacetylase(HDAC)inhibitors synergize with PI3 K inhibitors through inactivation of both the PI3K/AKT and Ras/Raf/MEK/ERK pathways in preclinical models of AML,indicating that PI3 K and HDAC dual inhibitors should have the ability to simultaneously down-regulate Mcl-1 and up-regulate Bim.CUDC-907 is a novel PI3 K and HDAC dual inhibitor which can directly inhibit the PI3K/AKT pathway and can also inactivate the Ras/Raf/MEK/ERK pathway through inhibition of HDAC,indicating that CUDC-907 is able to down-regulate Mcl-1 and up-regulate Bim.Since HDAC inhibitors have been reported possessing the ability to down-regulate DNA repair related proteins in cancer cells leading to the induction of DNA damage,we hypothesized that combining CUDC-907 with Venetoclax will result in synergistic antileukemic activity against AML.To begin to test our hypothesis,we first tested the antileukemic activity and underlying molecular mechanism of CUDC-907 in preclinical models of AML.We demonstrated that CUDC-907 induced apoptosis in AML cell lines and primary patient samples and showed in vivo efficacy in an AML cell line-derived xenograft mouse model.CUDC-907-induced apoptosis was partially dependent on Mcl-1,Bim,and c-Myc.CUDC-907 induced DNA damage in AML cells while sparing normal hematopoietic cells.Down-regulation of CHK1,Wee1,and RRM1,and induction of DNA damage also contributed to CUDC-907-induced apoptosis of AML cells.In addition,CUDC-907 treatment decreased leukemia progenitor cells in primary acute myeloid leukemia samples ex vivo,while also sparing normal hematopoietic progenitor cells.Next,we investigated the combination of CUDC-907 and Venetoclax in preclinical models of AML both in vitro and in vivo.We demonstrated that CUDC-907 and Venetoclax synergistically induced apoptosis in AML cell lines and primary AML patient samples ex vivo.CUDC-907 down-regulated CHK1,Wee1,RRM1,and c-Myc,which were found to play an important role in Venetoclax-induced apoptosis.We also found that Venetoclax treatment impairs repair of CUDC-907-induced DNA damage.In an AML cell line derived xenograft mouse model,CUDC-907 significantly enhanced Venetoclax efficacy,supporting the development of CUDC-907 in combination with Venetoclax for the treatment of AML.In summary,combined CUDC-907 and Venetoclax treatment shows great synergistic antileukemic activity against preclinical models of AML.Our results form a solid foundation for the clinical development of this promising combination therapy for the treatment of AML.