New Strategies Of Killing Both Bulk Cells And Stem Cells In Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a clonal disease characterized by uncontrolled proliferation and accumulation of myeloid progenitor cells in the bone marrow and affecting the differentiation of normal blood cells.The incidence of AML increases with age,with a median age of disgnosis between 67-70 years.The "7 + 3" induction chemotherapy,widely accepted for the treatment of AML,results in complete remission in 75% of patients under the age of 60.However,most of these patients relapse and die from the disease.The overall five-year survival rate is only approximately 30%.The treatment outcome is even worse in AML patients with the age of 60 years or older with the median overall survival(OS)less than 1 year.Studies have shown that AML stem cells are the main cause of therapy resistance and relapse of the disease.Thus,eradication of AML stem cells is critical for a cure.This highlights the urgent need of novel therapies which can target both AML bulk cells and stem cells to improve the cure rate of AML.The first part of my dissertation focuses on the antileukemic activity and underlying molecular mechanisms of targeting the PI3/K/mTOR,ERK,and Bcl-2signaling network in AML.The PI3K/mTOR signaling pathway is essential for the growth and survival of AML cells.More than 60% of AML patients have constitutively active PI3K/mTOR signaling.Therefore,targeting PI3K/mTOR signaling pathway represents a good approach for treating AML.Unfortunately,although promising activity were observed with single PI3 K inhibitors or mTOR inhibitors,patients eventually die due to the negative feedback mechanisms,which reactivate these signaling pathways resulting in drug resistance.Further,many studies have revealed that inhibition of the PI3 K / mTOR signaling pathway causes compensatory activation of the ERK signaling,resulting resistance to apoptosis.In addition,it has been well documented that the antiapoptotic Bcl-2 family proteins are overexpressed in AML,protecting them from cell death.Basedon resports that the PI3K/mTOR and ERK signaling pathways promote the antiapoptotic function of Bcl-2,we hypothesized that simultaneous targeting of the PI3 K,mTOR,ERK,and Bcl-2 signaling network with the novel selective PI3K/mTOR dual inhibitor VS-5584,ERK inhibitor SCH772984,and the Bcl-2 selective inhibitor ABT-199,would result in superior antileukemic activity against AML.To test this hypothesis,we first measured the antileukemic activity of VS-5584 against AML.The results showed that VS-5584 induced caspase-independent cell death.Consistent with other reports,we found that VS-5584 treatment resulted in upregulation of p-ERK,which could be abolished by the ERK selective inhibitor SCH772984.Moreover,the combination of VS-5584 and SCH772984 resulted in synergistic induction of cell death in both AML cell lines and primary patient samples.However,the levels of cell death induced by this combination were modest in some AML cell lines and patient samples.Mechanistic studies revealed that the combination of VS-5584 and SCH772984 downregulated Mcl-1 and upregulated Bim,however,the upregulated Bim failed to induce apoptosis due to its binding to Bcl-2.The addition of ABT-199,a selective inhibitor of Bcl-2,completely abolished the binding of Bim to Bcl-2,resulting in significantly increased AML cell death.Finally,results from colony forming assays showed that simultaneous inhibition of PI3K/mTOR and ERK,followed by inhibition of Bcl-2 significantly reduced the number of AML-CFUs but had no effect on the CFUs in human CD34+ cord blood.These results suggest that the three drug combination is able to suppress AML progenitor cells but spares normal human hematopoietic progenitors.These studies provide a novel stretagy for the treatment of AML bulk cells.The second part of my disseration focused on the antileukemic activity and molecular mechanisms of ONC213,a new member of the imipridone family of anticancer agents.By using AML cell lines and primary patient samples,we demonstrated that ONC213 stongly inhibited cell growth and induced cell death at least partially through the intrinsic apoptotic pathway.After establishing the antileukemic activity of ONC213,we began investigating the underlying molecular mechanism.ONC201,the first generation of ONC213,was shown to inactivate AKT and ERK and induce the expression of ATF4,CHOP,DR5,and TRAIL,resulting in extrinsic apoptosis.Unlike ONC201,ONC213 did not reduce phosphorylated AKT and ERK,nor did it induce TRAIL and DR5 prior to induction of cell death in AML cells.It has been reported that the antileukemic activity of ONC212,another analog of ONC201,is correlated with the expression of GPR132 mRNA in AML cell lines.Our study with both AML cell lines and primary patient samples demonstrated that the antileukemic activity of ONC213 is not related to GPR132.Further studies revealed that ONC213 treatment resulted in significant reductions in basal oxygen comsuption rate(OCR),maximal respiration,and ATP production,implying that ONC213 inhibits mitochondrial oxidative phosphorylation(OXPHOS).Switching glucose to galactose in the cell culture media,which forces cells to use OXPHOS to produce ATP,significantly enhanced the sensitivity of AML cells to ONC213.In addition,ONC213 treatment downregulated NDUFB8 and SDHB,which are critical for the stability of OXPHOS complex I and II,respectively.Electron microscopy imaging revealed that ONC213 treatment increased the number of mitochondria,and was accompanied by altered mitochondrial morphology.In addition,we also demonstrated that ONC213 downregulated Mcl-1.Further,overexpression of Mcl-1 significantly attenuated the suppression of basal and maximal respiration and ATP production by ONC213 treatment and partially rescued the cells from ONC213 treatment.Consistent with these findings,AML cells with acquired ONC213 resistance showed significantly decreased OXPHOS and increased Mcl-1.ONC213 treatment failed to decrease Mcl-1 and induce cell death in the ONC213 resistance cells even in galactose medium.Finally,ex vivo treatment of primary AML patient samples with ONC213 almost completely suppressed in vivo engraftment in immunconrpomised mice,suggesting that ONC213 can effectively kill AML stem cells.Treatment with ONC213 significantly prolonged survival of NSGS mice bearing MV4-11 leukemia,demonstrating its potent antileukemic activity in vivo.Taken together,these studies demonstrate that ONC213 effectively targets AML bulk cells and stem cells both in vitro and in vivo via suppresion of mitochondrial respiraiton and Mcl-1.In summary,this dissertation provides strong evidence that targeting PI3K/ mTOR,ERK,and Bcl-2 signaling network can effectively kill AML bulk cells and targeting mitochondrial respiration may selectively eradicate AML stem cell.Combination of these novel strategies may represent an effective therapy to achieve a cure of AML.