Activity And Mechanism Of Action Of The Novel Bcl-2 Selective Inhibitor ABT-199 In Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a heterogeneous disease characterized by rapid clonal growth of immature myeloid blood cells.AML consists of multiple subtypes,each with different treatment responses and prognoses.It is estimated that 19,520 people will be diagnosed with AML and 10,670 patients will die from this deadly disease in the United States in 2018.Five-year survival rates(25% for adults and 65% for children)remain low despite advances in treatment.Resistance to the front-line chemotherapy(consisting primarily of cytarabine and an anthracycline,such as daunorubicin)remains a major cause of treatment failure,highlighting the urgent need for new and more effective therapies.Most patients with AML become resistant to chemotherapy at some point during the treatment course and succumb to their disease.Therefore,it is critical to prevent chemoresistance or enhance chemosensitivity in a selective fashion to lead to a higher cure rate and a lower toxic burden.The B cell lymphoma 2(BCL-2)gene family encodes more than 20 proteins that regulate the intrinsic apoptosis pathway,and are fundamental to control the balance between cell survival and death.Overexpression of the antiapoptotic Bcl-2 family proteins(Bcl-2,Bcl-x L and Mcl-1,etc.)have been shown to cause resistance to chemotherapy in AML cells and is associated with poor clinical outcome in adult patients with AML.The antiapoptotic Bcl-2 family members,such as Bcl-2,Bcl-x L,and Mcl-1 sequester proapoptotic BH3-only proteins,such as Bim,preventing activation of the proapoptotic proteins Bax and Bak,ultimately preventing mitochondrial outer membrane permeabilization,cytochrome c release,and apoptosis.Thus,inhibition of the antiapoptotic Bcl-2 family members represents a promising approach for the treatment of AML.However,although inhibitors of this family(such as ABT-263,also known as Navitoclax)have been promising,inhibition of Bcl-x L has been associated with platelet death and subsequent thrombocytopenia.Fortunately,the recently developed ABT-199 is specific for Bcl-2,which causes substantially less platelet killing ex vivo and in vivo as compared to navitoclax,making it an attractive potential option for the treatment of AML.In 2017 the US Food and Drug Administration(FDA)approved midostaurin(FLT3 inhibitor),gemtuzumab ozogamicin,CPX-351(liposomal cytarabine and daunorubicin)and enasidenib(IDH2 inhibitor)for the treatment of AML.Although there are many other novel agents that are currently under investigation for the treatment of AML,ABT-199 is of particular interest due to its strong pre-clinical data and promising early clinical results.This study was designed first to identify genetic subgroups,which may respond to ABT-199 favorably,and biomarkers predicting ABT-199 sensitivity in AML.While the AML cell lines tested showed a wide range of sensitivities to ABT-199 monotherapy,one group appeared to be particularly sensitive.Cell lines harboring MLL fusion genes were significantly more sensitive,suggesting that MLL fusion proteins play an important role in Bcl-2 mediated anti-apoptotic effects.Based on the cell line data,patients,especially infants with AML,whose blast cells harbor a MLL gene rearrangement,may benefit from ABT-199 treatment.Importantly,we found that primary AML samples with the APL phenotype are particularly sensitive to ABT-199 monotherapy,suggesting a possible role for this agent in APL patients who do not respond to all-trans retinoic acid and/or arsenic trioxide-based therapies.Further,our data from both AML cell lines and primary patient samples suggest that the ratio of Bcl-2/Mcl-1 transcripts may represent a promising biomarker predicting ABT-199 sensitivity in AML.These results further support the clinical development of ABT-199 for the treatment of AML.ABT-199 has been demonstrated to bind to the BH3 domain binding groove of Bcl-2,disrupting its interactions with pro-apoptotic Bim.Thus,we first investigated the interaction between Bim and Bcl-2,Mcl-1,or Bcl-x L following ABT-199 treatment in AML cell lines and primary patient samples that are sensitive to ABT-199 treatment.ABT-199 treatment resulted in decreased levels of Bcl-2 that co-precipitated with Bim,while total protein levels for Bcl-2,Bcl-x L,and Mcl-1 remained unchanged.This was accompanied by loss of mitochondrial outer membrane potential(MOMP)and PARP cleavage,indicative of apoptosis.Further experiments demonstrated that Bax plays a prominent role in ABT-199-induced cell death in AML cells.Taken together,these results demonstrate that ABT-199 induces apoptosis through the mitochondrial apoptotic pathway in ABT-199-sensitive AML cells.However,even ABT-199 has demonstrated encouraging results in AML,acute lymphoblastic leukemia,chronic lymphocytic leukemia,mantle cell lymphoma,multiple myeloma,and breast cancer,it has limited efficacy in Bcl-x L-and Mcl-1-dependent malignancies.Thus,intrinsic drug resistance remains a concern.Understanding the molecular mechanisms of resistance to ABT-199 will allow for rationally designed combination regimens to increase its antileukemic efficacy.In this study,we identified Bim sequestration by Mcl-1 as a mechanism of resistance to ABT-199 in AML cells.Binding to Bim likely contributed to increased Mcl-1 protein stability and presumably decreased levels of free Bim,preventing its induction of apoptosis.To overcome this resistance,AML cells were treated with ABT-199 in combination with daunorubicin or cytarabine,which increased DNA damage and decreased Mcl-1 protein levels,resulting in synergistic induction of cell death in both ABT-199-resistant and ABT-199-sensitive cell lines.Additionally,we demonstrated that these combinations are synergistic in AML cell lines and primary patient samples independent of their sensitivities to ABT-199,thus providing evidence that screening for ABT-199 sensitivity is not necessary,therefore supporting clinical testing of these combinations in AML patients regardless of their sensitivities to ABT-199.In addition,our studies using primary patient samples further support the clinical development of combined ABT-199 and daunorubicin or cytarabine treatment as well as provide guidance for designing other ABT-199 combination therapies for the treatment of AML.