Combined Inhibition of MCL1 By AZD5991 and BCL2/Bclxl By AZD4320 As Promising Strategies to Overcome Acute Leukemia Tumor Burden and Niche Chemoresistance

Abstract

Background. Abnormal regulation of anti-apoptotic proteins belonging to the BCL2 family contributes to therapeutic resistance of many hematological neoplasms. Although Venetoclax, a selective inhibitor of the BCL2 protein, has proven to exert beneficial effects in acute leukemias, the activation of another anti-apoptotic protein, MCL1, a key factor to Venetoclax acquired resistance, frequently reduces its benefits. Additionally, it is known that BCL-xL plays an important pro-survival role in cancer cells by interacting with BCL-2 Hence, we hypothesize that the co-inhibition of MCL1, Bcl-2 and BCL-xL would represent an improved strategy for the treatment of patients with acute leukemia. Thus, in this study, we assessed the efficacy of specific inhibitors of MCL1, BCL2 and BCLxL antiapoptotic proteins. Aims. To investigate the in vitro, ex vivo and in vivo effects of AZD5991 (AstraZeneca), a novel MCL1 inhibitor compound, alone or in combination with AZD4320 (BCL-2/BCL-XL inhibitor compound), venetoclax, cytarabine and doxorubicin. Methods.MCL1 mRNA expression data were obtained from TCGA AML study, performed in mononuclear cells isolated from 163 AML patients (median age 57.5 years [range: 18-88]). Bone marrow (BM) aspirates (25) from healthy donors (HD), Myelodysplastic syndrome (MDS), de novo AML, and acute lymphoblastic leukemia (ALL) patients at diagnosis were submitted to analysis of MCL1 protein levels by flow cytometry and immunofluorescence (IF). A panel of myeloid (OCI-AML3, MOLM13, U937, HL60, KG1a, K562) and lymphoblastic (RS4;11, Jurkat) leukemia cell lines were treated with increasing concentrations of AZD5991 for 48h and the 50% inhibiting concentration and apoptosis (annexin-V stain) rates were determined. Mononuclear and hematopoietic stem (CD34+) cells from HD were submitted to cytotoxic assays. Leukemia cell lines were also tested for reactive oxygen species (ROS) and nitric oxide (NO) production (DAF dye), mitochondrial membrane potential (DilC1 probe) and protein expression (flow cytometry, Western blot) in a time course of 0 to 48h, alone or in combination. Moreover, we tested the AZD5991/AZD4320 action in malignant cells cultured in a 3D-system (in the presence of the mesenchymal cells) and in a monocyte monolayer (2D) in order to evaluate tumor microenvironment (TME) participation. We also developed a xenograft mouse model using U937 leukemia cell line. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests (p<.05). Results. The TCGA study (CBIO Portal) analysis showed high MCL1 gene expression in AML patients was observed (p<.0001). Immunofluorescence and flow cytometry showed that MCL1, BCL-2 and BCL-xL protein levels higher in MDS, AML and ALL BM cells compared to normal cells. All leukemia cell lines were dose-dependent sensitive to AZD5991. Growth inhibiting concentrations were OCI-AML3/0.04μM, U937/4.3μM, RS4;11/3.5μM, Jurkat/0.3μM. Apoptosis was increased in a time and concentration-dependent manner, with greater intensity in OCI-AML3 cells. AZD5991 was able to impair mitochondrial membrane potential with reduced mitochondrial ROS and NO production (p<.01) and decreased MCL1, Bax and Bak but increased Bim protein levels. Coculture in a 3D-system showed that AZD5991 treatment reduced patients’ cell growth, without modulating HD cells. In the 2D-coculture, programmed monocytes intensified apoptosis with synergistic effect between AZD5991 and AZD4320. In vitro experiments demonstrated an additive effect between AZD5991 and AZD4320, venetoclax, cytarabine or doxorubicin. In addition, xenotransplanted mice confirmed the efficacy of AZD5991 compound alone or in combination with AZD4320 due to reduction of tumor weight (fold decrease (FD): 3.8; 2.8) and tumor volume (FD: 6.5; 2.8) compared to vehicle-treated group, respectively. Conclusion. MCL1 overexpression in mononuclear cells from patients with acute leukemia and that present or not resistance to venetoclax treatment, is associated with poor prognosis and relapse disease. Our results demonstrated that co-targeting MCL-1, BCL-2 and BCL-xL, through the AZD5991 and AZD4320 compounds in the treatment of acute leukemias, may enhance therapeutic specificity, overcome chemoresistance and contribute with the cure of these aggressive malignant disorders. Funding: FAPESP grants #2017/21801-2, #2019/25247-5, #2021/05320-0.