Cotargeting of Bcl-2 and Mcl-1 shows promising antileukemic activity against AML cells including those with acquired cytarabine resistance
2021; Elsevier BV; Volume: 105; Linguagem: Inglês
10.1016/j.exphem.2021.10.006
ISSN1873-2399
AutoresFangbing Liu, Qiushi Zhao, Yongwei Su, Jing Lv, Yuqing Gai, Shuang Liu, Hai Lin, Yue Wang, Guan Wang,
Tópico(s)RNA Interference and Gene Delivery
Resumo•Inhibition of Mcl-1 enhances the antileukemic activity of venetoclax.•Combined Mcl-1 inhibition and venetoclax induce intrinsic apoptosis.•Co-inhibition of Mcl-1 and Bcl-2 has an effect on acquired AraC-resistant cells. Acute myeloid leukemia (AML) remains a clinical challenge. Venetoclax is an effective Bcl-2 selective inhibitor approved by the U.S. Food and Drug Administration (FDA) for treatment of AML in patients who are 75 years and older or who have comorbidities. However, resistance to venetoclax limits its clinical efficacy. Mcl-1 has been identified as one determinant of resistance to venetoclax treatment. In this study, we investigate the Mcl-1 inhibitor S63845 in combination with venetoclax in AML cells. We found that S63845 synergizes with venetoclax in AML cell lines and primary patient samples. Bak/Bax double knockdown and treatment with the pan-caspase inhibitor Z-VAD-FMK revealed that the combination induces intrinsic apoptosis in AML cells. Inhibition of Mcl-1 using another Mcl-1 selective inhibitor, AZD5991, also synergistically enhanced apoptosis induced by venetoclax in a caspase-dependent manner. Importantly, S63845 in combination with venetoclax can effectively combat AML cells with acquired resistance to the standard chemotherapy drug cytarabine. In light of these facts, the combined inhibition of Mcl-1 and Bcl-2 shows promise against AML cells, including relapse/refractory AML. Acute myeloid leukemia (AML) remains a clinical challenge. Venetoclax is an effective Bcl-2 selective inhibitor approved by the U.S. Food and Drug Administration (FDA) for treatment of AML in patients who are 75 years and older or who have comorbidities. However, resistance to venetoclax limits its clinical efficacy. Mcl-1 has been identified as one determinant of resistance to venetoclax treatment. In this study, we investigate the Mcl-1 inhibitor S63845 in combination with venetoclax in AML cells. We found that S63845 synergizes with venetoclax in AML cell lines and primary patient samples. Bak/Bax double knockdown and treatment with the pan-caspase inhibitor Z-VAD-FMK revealed that the combination induces intrinsic apoptosis in AML cells. Inhibition of Mcl-1 using another Mcl-1 selective inhibitor, AZD5991, also synergistically enhanced apoptosis induced by venetoclax in a caspase-dependent manner. Importantly, S63845 in combination with venetoclax can effectively combat AML cells with acquired resistance to the standard chemotherapy drug cytarabine. In light of these facts, the combined inhibition of Mcl-1 and Bcl-2 shows promise against AML cells, including relapse/refractory AML. Acute myeloid leukemia (AML) is a malignant hematologic cancer in which a large number of immature myeloid blood precursor cells proliferate uncontrollably. Standard induction chemotherapy consists of cytarabine (AraC) and an anthracycline, but the majority of patients unfortunately relapse. The 5-year overall survival rate is only about 28% for AML patients and less than 8% in patients more than 60 years old [1Abdallah M Xie Z Ready A Manogna D Mendler JH Loh KP. Management of acute myeloid leukemia (AML) in older patients.Curr Oncol Rep. 2020; 22: 103Crossref PubMed Scopus (3) Google Scholar]. Therefore, new therapies that exhibit activity against AML cells, especially AraC-resistant AML cells, urgently need to be developed. Bcl-2 has been reported to contribute to AML drug resistance and plays a role in its poor prognosis [2Lauria F Raspadori D Rondelli D et al.High bcl-2 expression in acute myeloid leukemia cells correlates with CD34 positivity and complete remission rate.Leukemia. 1997; 11: 2075-2078Crossref PubMed Scopus (109) Google Scholar, 3Schimmer AD O'Brien S Kantarjian H et al.A phase I study of the pan bcl-2 family inhibitor obatoclax mesylate in patients with advanced hematologic malignancies.Clin Cancer Res. 2008; 14: 8295-8301Crossref PubMed Scopus (159) Google Scholar, 4Davids MS Letai A. Targeting the B-cell lymphoma/leukemia 2 family in cancer.J Clin Oncol. 2012; 30: 3127-3135Crossref PubMed Scopus (208) Google Scholar, 5Saxena A Viswanathan S Moshynska O Tandon P Sankaran K Sheridan DP. Mcl-1 and Bcl-2/Bax ratio are associated with treatment response but not with Rai stage in B-cell chronic lymphocytic leukemia.Am J Hematol. 2004; 75: 22-33Crossref PubMed Scopus (101) Google Scholar]. As a result, the Bcl-2 selective inhibitor venetoclax was developed. It has since shown such promise that it was approved by the U.S. Food and Drug Administration (FDA) for use in combination with low-dose cytarabine, azacytidine, or decitabine in newly diagnosed AML patients who are ineligible for intensive chemotherapy. Thus far, the combinations show promise, though the median overall survival is 14.2–16.4 months [6Wei AH Strickland Jr, SA Hou JZ et al.Venetoclax combined with low-dose cytarabine for previously untreated patients with acute myeloid leukemia: Results from a phase Ib/II study.J Clin Oncol. 2019; 37: 1277-1284Crossref PubMed Scopus (306) Google Scholar, 7DiNardo CD Pratz KW Letai A et al.Safety and preliminary efficacy of venetoclax with decitabine or azacitidine in elderly patients with previously untreated acute myeloid leukaemia: a non-randomised, open-label, phase 1b study.Lancet Oncol. 2018; 19: 216-228Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 8Pollyea DA Pratz K Letai A Jonas BA. Venetoclax with azacitidine or decitabine in patients with newly diagnosed acute myeloid leukemia: long term follow-up from a phase 1b study.Am J Hematol. 2021; 96: 208-217Crossref PubMed Scopus (23) Google Scholar]. Previously, it was reported that A1210477, an Mcl-1 selective inhibitor, enhances the activity of venetoclax against AML cells [9Luedtke DA Niu X Pan Y et al.Inhibition of Mcl-1 enhances cell death induced by the Bcl-2-selective inhibitor ABT-199 in acute myeloid leukemia cells.Signal Transduct Target Ther. 2017; 2: 17012Crossref PubMed Scopus (68) Google Scholar]. Encouraging as the results were, A1210477 is not being pursued in the clinic, and the antileukemic activity of the combination against AraC resistant cells was left unanswered. As others have also investigated combined Bcl-2 and Mcl-1 inhibition [10Bhatt S Pioso MS Olesinski EA et al.Reduced mitochondrial apoptotic priming drives resistance to BH3 mimetics in acute myeloid leukemia.Cancer Cell. 2020; 38 (e876): 872-890Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 11Hormi M Birsen R Belhadj M et al.Pairing MCL-1 inhibition with venetoclax improves therapeutic efficiency of BH3-mimetics in AML.Eur J Haematol. 2020; 105: 588-596Crossref PubMed Scopus (11) Google Scholar, 12Moujalled DM Pomilio G Ghiurau C et al.Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in pre-clinical models of acute myeloid leukemia.Leukemia. 2019; 33: 905-917Crossref PubMed Scopus (82) Google Scholar, 13Ramsey HE Fischer MA Lee T et al.A novel MCL1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemia.Cancer Discov. 2018; 8: 1566-1581Crossref PubMed Scopus (163) Google Scholar, 14Tron AE Belmonte MA Adam A et al.Discovery of Mcl-1-specific inhibitor AZD5991 and preclinical activity in multiple myeloma and acute myeloid leukemia.Nat Commun. 2018; 9: 5341Crossref PubMed Scopus (217) Google Scholar], their antileukemic activity against relapsed/refractory AML remains to be elucidated. In this study, the antileukemic activity of venetoclax in combination with Mcl-1 inhibition by second-generation selective inhibitors was investigated. Combination treatment exhibited synergistic antileukemic activity in both AML cell lines and primary AML patient samples. Most importantly, we found that the combination treatment has potent antileukemic activity against AML cell lines with acquired AraC resistance, suggesting that the combination may show promise in AML after standard induction therapy has failed. Venetoclax, S63845, and AZD5991 were purchased from Selleck Chemicals (Houston, TX, USA). Z-VAD-FMK was purchased from Sigma-Aldrich (St. Louis, MO, USA). MV4-11, U937, and THP-1 were purchased from the American Type Culture Collection (Manassas, VA, USA). MOLM-13 was purchased from AddexBio (San Diego, CA, USA). The cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) and 2 mmol/L L-glutamine, plus 100 U/mL penicillin and 100 μg/L streptomycin, in a 37°C humidified atmosphere containing 5% CO2/95% air, as previously described [15Li X Su Y Madlambayan G et al.Antileukemic activity and mechanism of action of the novel PI3K and histone deacetylase dual inhibitor CUDC-907 in acute myeloid leukemia.Haematologica. 2019; 104: 2225-2240Crossref PubMed Scopus (34) Google Scholar]. The cell lines were tested for the presence of mycoplasma using polymerase chain reaction (PCR) on a monthly basis. Diagnostic blast samples were obtained from the First Hospital of Jilin University, Changchun, China. Written informed consent was provided according to the Declaration of Helsinki. This study was approved by the Human Ethics Committee of The First Hospital of Jilin University. All clinical samples were screened for FLT3-ITD, NPM1, C-kit, CEBPA, IDH1, IDH2, and DNMT3A gene mutations by PCR amplification and automated DNA sequencing and for fusion genes by real-time (RT)-PCR, as described previously [16Niu X Wang G Wang Y et al.Acute myeloid leukemia cells harboring MLL fusion genes or with the acute promyelocytic leukemia phenotype are sensitive to the Bcl-2-selective inhibitor ABT-199.Leukemia. 2014; 28: 1557-1560Crossref PubMed Scopus (71) Google Scholar,17Qi W Xie C Li C et al.CHK1 plays a critical role in the anti-leukemic activity of the wee1 inhibitor MK-1775 in acute myeloid leukemia cells.J Hematol Oncol. 2014; 7: 53Crossref PubMed Scopus (39) Google Scholar]. Patient characteristics are summarized in Supplementary Table E1 (online only, available at www.exphem.org). Following drug treatment, AML cells were stained with annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Beckman Coulter, Brea, CA, USA) and then subjected to flow cytometry analysis, as previously described [18Wang G Edwards H Caldwell JT et al.Panobinostat synergistically enhances the cytotoxic effects of cisplatin, doxorubicin or etoposide on high-risk neuroblastoma cells.PLoS One. 2013; 8: e76662Crossref PubMed Scopus (31) Google Scholar] Apoptotic events are expressed as the mean percentage of annexin V+/PI– (early apoptotic) and annexin V+/PI+ (late apoptotic and/or dead) cells ± the standard error from one representative experiment. Combination index (CI) values were calculated using CompuSyn software (ComboSyn Inc. Paramus, NJ, USA). CI 1 indicate synergistic, additive, and antagonistic effects, respectively. Western blotting was performed as previously described [18Wang G Edwards H Caldwell JT et al.Panobinostat synergistically enhances the cytotoxic effects of cisplatin, doxorubicin or etoposide on high-risk neuroblastoma cells.PLoS One. 2013; 8: e76662Crossref PubMed Scopus (31) Google Scholar] Antibodies directed toward β-actin, Bcl-2, Mcl-1, Bax, and PARP (Proteintech, Chicago, IL, USA); cleaved (cf) caspase 3 and Bim (Cell Signaling Technologies, Danvers, MA, USA); and Bak and Bcl-xL (Bimake, Houston, TX, USA) were used for Western blot analysis. AML cell lines were treated for 4 hours with either S63845 or venetoclax, alone or in combination, then lysed with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 5 mmol/L MgCl2, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 20 mmol/L Tris, and 0.05% Tween-20 in the presence of protease inhibitors. Co-immunoprecipitation of Bim was carried out as described previously [19Xie C Edwards H Xu X et al.Mechanisms of synergistic antileukemic interactions between valproic acid and cytarabine in pediatric acute myeloid leukemia.Clin Cancer Res. 2010; 16: 5499-5510Crossref PubMed Scopus (65) Google Scholar,20Niu X Zhao J Ma J et al.Binding of released Bim to Mcl-1 is a mechanism of intrinsic resistance to ABT-199 which can be overcome by combination with daunorubicin or cytarabine in AML cells.Clin Cancer Res. 2016; 22: 4440-4451Crossref PubMed Scopus (108) Google Scholar]. Two micrograms of anti-Bim antibody (Cell Signaling Technology), 1 mg protein lysate, and protein A agarose beads (Roche Diagnostics, Indianapolis, IN, USA) were used. Proteins were eluted with 50 mmol/L glycine at pH 2.0 and then analyzed via Western blotting as described. The lentiCRISPRv2 plasmid was a gift from Feng Zhang at the Broad Institute of MIT and Harvard (Addgene plasmid 52961). Guide RNAs were designed using the CRISPR design tool (http://crispr.mit.edu). The nontarget control (NTC) and Mcl-1 vectors were generated using Zhang's protocol, which is available on Addgene's website (www.addgene.org). Lentivirus production and transduction were carried out as described earlier, except that psPAX2 (gift from Didier Trono at the Swiss Institute of Technology, Addgene Plasmid No. 12260) was used instead of delta 8.2. Lentivirus production and transduction were carried out as previously described [20Niu X Zhao J Ma J et al.Binding of released Bim to Mcl-1 is a mechanism of intrinsic resistance to ABT-199 which can be overcome by combination with daunorubicin or cytarabine in AML cells.Clin Cancer Res. 2016; 22: 4440-4451Crossref PubMed Scopus (108) Google Scholar,21Xie C Drenberg C Edwards H et al.Panobinostat enhances cytarabine and daunorubicin sensitivities in AML cells through suppressing the expression of BRCA1, CHK1, and Rad51.PLoS One. 2013; 8: e79106Crossref PubMed Scopus (69) Google Scholar]. The pMD-VSV-G and delta 8.2 plasmids were gifts from Dr. Dong at Tulane University. Non-template-negative control (NTC)-, Bax-, Bak-, and Bim-shRNA lentiviral constructs were purchased from Sigma-Aldrich. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT, Sigma-Aldrich) assays were performed as previously described [19Xie C Edwards H Xu X et al.Mechanisms of synergistic antileukemic interactions between valproic acid and cytarabine in pediatric acute myeloid leukemia.Clin Cancer Res. 2010; 16: 5499-5510Crossref PubMed Scopus (65) Google Scholar,20Niu X Zhao J Ma J et al.Binding of released Bim to Mcl-1 is a mechanism of intrinsic resistance to ABT-199 which can be overcome by combination with daunorubicin or cytarabine in AML cells.Clin Cancer Res. 2016; 22: 4440-4451Crossref PubMed Scopus (108) Google Scholar]. Differences in cellular apoptosis (comparison of the sum of annexin V+/PI– and Annexin V+/PI+ cells) between treatment groups and/or untreated cells were compared using a pairwise two-sample t test. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad, San Diego, CA). Error bars represent ±SEM. The significance level was set at p < 0.05. AML cell lines were treated with various concentrations of venetoclax and S63845 both alone and in combination for only 4 hours. The combination synergistically induced apoptosis in AML cell lines regardless of their inherent venetoclax sensitivity (intrinsic venetoclax-resistant cell lines OCI-AML3, THP-1, and U937; intrinsic venetoclax-sensitive cell lines MV4-11 and MOLM-13; Figure 1A). The combination treatment substantially increased cleavage of caspase 3 and PARP compared with single-drug treatment (Figure 1B). Furthermore, synergistic induction of apoptosis was confirmed in six primary AML patient samples ex vivo (Figure 1C). Increased cleavage of caspase 3 and PARP following combination treatment was confirmed in two primary AML patient samples ex vivo (Figure 1D).Figure 1Venetoclax and S63845 synergistically induce apoptosis in acute myeloid leukemia (AML) cells. (A) OCI-AML3, THP-1, U937, MOLM-13, and MV4-11 cells were treated with variable concentrations of venetoclax and S63845, both alone and in combination, for 4 hours. The cells were stained with annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) and analyzed by flow cytometry. (B) OCI-AML3, THP-1, U937, MOLM-13, and MV4-11 cells were treated with venetoclax and S63845, alone and in combination, for 4 hours. Whole-cell lysates were subjected to Western blot analysis. (C) Primary AML patient samples were treated with variable concentrations of venetoclax and S63845, both alone and in combination, for 4 hours. The cells were stained with annexin V–FITC/PI and analyzed by flow cytometry. CompuSyn software was used to calculate the CI values. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively. **p < 0.01 and ***p < 0.001 compared with vehicle control and individual drug treatment. (D) Primary AML patient samples AML Nos. 206 and 208 cells were treated with venetoclax and S63845, alone and in combination, for 4 hours. Whole-cell lysates were subjected to Western blot analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine if the combination treatment induces caspase-dependent apoptosis, THP-1, U937, and MV4-11 cells were treated with the pan-caspase inhibitor Z-VAD-FMK alone or in combination with combined S63845 and venetoclax. Z-VAD-FMK treatment completely abolished apoptosis induced by combined S63845 and venetoclax (Figure 2A), which was accompanied by complete attenuation of caspase 3 cleavage (Figure 2B). Consistent with these results, Bak/Bax double knockdown in both MV4-11 and THP-1 cells abolished drug-induced apoptosis (Figure 2C,D). Taken together, these results indicate that venetoclax and S63845 synergistically induce intrinsic apoptosis. To begin to elucidate the mechanism of action, we began by determining the changes in Bcl-2 family protein levels. Consistent with our previous findings [9Luedtke DA Niu X Pan Y et al.Inhibition of Mcl-1 enhances cell death induced by the Bcl-2-selective inhibitor ABT-199 in acute myeloid leukemia cells.Signal Transduct Target Ther. 2017; 2: 17012Crossref PubMed Scopus (68) Google Scholar], venetoclax treatment resulted in increased Mcl-1 protein levels in both THP-1 and U937 cells, and inhibition of Mcl-1 resulted in a substantial increase in Mcl-1 protein in all three cell lines tested as well as one primary patient sample (Figure 3). The combination treatment reduced Mcl-1 protein levels in THP-1, U937, MV4-11, and primary AML patient sample AML No. 206, at least to baseline levels. Single-drug and combination treatment had little to no effect on other Bcl-2 family proteins. Next, we investigated if the drug combination affects the interaction between Bim and Mcl-1 and Bcl-2. THP-1 cells were treated with vehicle control, venetoclax, and S63845, alone or in combination, for 4 hours, and the Bim was immunoprecipitated (Figure 4A). Venetoclax treatment reduced the amount of Bcl-2 bound to Bim with concomitant upregulation of Mcl-1. S63845 treatment decreased the amount of Mcl-1 bound to Bim while increasing the amount of Bcl-2 bound to Bim. Combination treatment resulted in downregulation of Bcl-2 and Mcl-1 bound to Bim. However, combination treatment substantially reduced the amount of Bim immunoprecipitated. To determine if Bim plays a role in the antileukemic activity of the combination, Bim shRNA knockdown was performed in THP-1 cells (Figure 4B). Knockdown of Bim almost completely blocked apoptosis induced by venetoclax or S63845. Although knockdown of Bim significantly reduced apoptosis induced by the combination of venetoclax and S63845, the magnitude was minimal (Figure 4C). Surprisingly, Bim knockdown in MV4-11 cells had little to no effect on drug-induced apoptosis (Figure 4D,E). In contrast, in U937 cells, Bim knockdown significantly and substantially hindered S63845-induced apoptosis and apoptosis induced by the combination of venetoclax and S63845 (Figure 4F,G). These results suggest that Bim can be dispensable in the apoptosis induced by the combination of venetocalx and S63845 in AML cells. To determine whether Mcl-1 downregulation has an effect similar to that of Mcl-1 inhibition by S63845 on apoptosis induced by venetoclax in AML cells, CRISPR knockdown of Mcl-1 was performed in THP-1 cells (Figure 5A). Knockdown of Mcl-1 (80% compared with NTC) significantly enhanced apoptosis induced by venetoclax. However, the level of apoptosis induced by venetoclax in the Mcl-1 knockdown cells was significantly lower than that induced by the combination of venetoclax and S63845 in the NTC cells (Figure 5B). To determine if the significantly stronger enhancement by S63845 than Mcl-1 knockdown on apoptosis induced by venetoclax was due to an off-target effect, we employed another Mcl-1 selective inhibitor, AZD5991, which has a different chemical structure compared with S63845. Inhibition of Mcl-1 using AZD5991 also synergistically enhanced apoptosis induced by venetoclax (Figure 5C). The pan-caspase inhibitor Z-VAD-FMK almost completely abolished apoptosis induced by combined venetoclax and AZD5991 treatment, indicating that the combination induces caspase-dependent apoptosis (Fig 5D). Bak/Bax double knockdown also resulted in near-complete rescue of the cells from combined venetoclax and AZD5991 treatment (Figure 5E). Consistent with results illustrated in Figure 5B, the magnitude of enhancement on apoptosis induced by venetoclax by Mcl-1 knockdown was significantly lower than that of the combination of venetoclax and AZD5991 in NTC cells (Figure 5F). These results suggest that Mcl-1 inhibition is a more robust approach than Mcl-1 downregulation in enhancing apoptosis induced by venetoclax in AML cells. Resistance to chemotherapy remains a challenge in the treatment of AML. To determine if the combination of venetoclax and S63845 can combat AML cells resistant to chemotherapy, AraC-resistant MV4-11 and U937 cells were generated by culturing the cells in stepwise increasing concentrations of AraC. To ensure that downstream experimental results were due to the effects of the investigation drug treatments without the interference of AraC, the AraC-resistant cells were cultured with or without AraC for 5 days and then treated with variable concentrations of AraC. MTT assays were performed, and half-maximal inhibitory concentrations (IC50) were calculated. Although there were differences between the AraC-resistant cells cultured with and without AraC, they were at least 10 times more resistant to AraC, based on their AraC IC50 values (Figure 6A,B). These results illustrate that the AraC resistance is maintained when AraC is removed from the culture medium for 5 days. S63845 treatment induced significantly more apoptosis in AraC-resistant MV4-11 and U937 cells (Figure 6C). Combination treatment induced significantly less apoptosis in MV4-11 AraC-resistant cells, though the magnitude was minimal. Venetoclax in combination with S63845 induced significantly more apoptosis in U937 AraC resistant cells than parental U937 cells. Taken together, these results suggest that acquired AraC resistance does not confer cross-resistance to combined venetoclax and S63845 treatment. Combined inhibition of Bcl-2 and Mcl-1 has shown promise in AML cell lines and ex vivo primary AML patient samples [9Luedtke DA Niu X Pan Y et al.Inhibition of Mcl-1 enhances cell death induced by the Bcl-2-selective inhibitor ABT-199 in acute myeloid leukemia cells.Signal Transduct Target Ther. 2017; 2: 17012Crossref PubMed Scopus (68) Google Scholar,11Hormi M Birsen R Belhadj M et al.Pairing MCL-1 inhibition with venetoclax improves therapeutic efficiency of BH3-mimetics in AML.Eur J Haematol. 2020; 105: 588-596Crossref PubMed Scopus (11) Google Scholar, 12Moujalled DM Pomilio G Ghiurau C et al.Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in pre-clinical models of acute myeloid leukemia.Leukemia. 2019; 33: 905-917Crossref PubMed Scopus (82) Google Scholar, 13Ramsey HE Fischer MA Lee T et al.A novel MCL1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemia.Cancer Discov. 2018; 8: 1566-1581Crossref PubMed Scopus (163) Google Scholar]. Recently, Hormi et al. [11Hormi M Birsen R Belhadj M et al.Pairing MCL-1 inhibition with venetoclax improves therapeutic efficiency of BH3-mimetics in AML.Eur J Haematol. 2020; 105: 588-596Crossref PubMed Scopus (11) Google Scholar] investigated venetoclax and S63845 in AML cells. They found that AML cells resistant to venetoclax were highly sensitive to S63845 treatment. Consistent with their findings, we also found that S63845 strongly synergizes with venetoclax in inducing apoptosis in AML cells (Figure 1A,C). Additionally, we found that Mcl-1 selective inhibitor AZD5991 also significantly and synergistically enhanced the antileukemic activity of venetoclax against AML cells (Figure 5). Moujalled et al. [12Moujalled DM Pomilio G Ghiurau C et al.Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in pre-clinical models of acute myeloid leukemia.Leukemia. 2019; 33: 905-917Crossref PubMed Scopus (82) Google Scholar] found that chemotherapy relapsed/refractory AML patient samples were sensitive to the combination of the Bcl-2 inhibitor S55746 and S63845, both in vitro and in vivo. Consistent with their findings, the combination of S63845 and venetoclax exhibited potent antileukemic activity in AML cell lines with acquired AraC resistance (Figure 6), suggesting that the combination may have activity against relapse/refractory AML. Ramsey et al. [13Ramsey HE Fischer MA Lee T et al.A novel MCL1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemia.Cancer Discov. 2018; 8: 1566-1581Crossref PubMed Scopus (163) Google Scholar] found that samples taken from AML patients who had become resistant to the combination of venetoclax and low-dose AraC respond ex vivo to combined Bcl-2 and Mcl-1 inhibition. Mcl-1 has been reported to be a mechanism of intrinsic resistance to venetoclax, and treatment with venetoclax can lead to Mcl-1-driven resistance [10Bhatt S Pioso MS Olesinski EA et al.Reduced mitochondrial apoptotic priming drives resistance to BH3 mimetics in acute myeloid leukemia.Cancer Cell. 2020; 38 (e876): 872-890Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar,13Ramsey HE Fischer MA Lee T et al.A novel MCL1 inhibitor combined with venetoclax rescues venetoclax-resistant acute myelogenous leukemia.Cancer Discov. 2018; 8: 1566-1581Crossref PubMed Scopus (163) Google Scholar,20Niu X Zhao J Ma J et al.Binding of released Bim to Mcl-1 is a mechanism of intrinsic resistance to ABT-199 which can be overcome by combination with daunorubicin or cytarabine in AML cells.Clin Cancer Res. 2016; 22: 4440-4451Crossref PubMed Scopus (108) Google Scholar]. Bhatt et al. [10Bhatt S Pioso MS Olesinski EA et al.Reduced mitochondrial apoptotic priming drives resistance to BH3 mimetics in acute myeloid leukemia.Cancer Cell. 2020; 38 (e876): 872-890Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar] reported that Mcl-1 is a mechanism of resistance to venetoclax in patient-derived xenograft AML models and that the combination of venetoclax and S63845 reduced leukemia burden compared with single-agent treatment. Interestingly, the amount of apoptosis induced by venetoclax in the Mcl-1 knockdown cells was significantly lower than that induced by the combination of venetoclax and S63845 or AZD5991 in the NTC cells (Figure 5). This finding suggests that Mcl-1 inhibition is potentially more robust than Mcl-1 downregulation in enhancing the antileukemic activity of venetoclax. Our findings using Bim knockdown models suggest that the role Bim plays is partial at best in combined Bcl-2 and Mcl-1 inhibition. As our data indicate that induction of apoptosis by the combination requires Bak/Bax, other BH3 proteins may play a role in antileukemic activity. Alternatively, Greaves et al. [22Greaves G Milani M Butterworth M et al.BH3-only proteins are dispensable for apoptosis induced by pharmacological inhibition of both MCL-1 and BCL-XL.Cell Death Differ. 2019; 26: 1037-1047Crossref PubMed Scopus (38) Google Scholar] found that the BH3-only proteins were dispensable for combined inhibition of Bcl-xL and Mcl-1 in colorectal cancer cells, which could play a role in AML cells. Importantly, our study indicates that Mcl-1 inhibition in combination with venetoclax exhibits synergistic antileukemic activity against AML cells, especially AraC-resistant AML cell lines. In this study, we used S63845, the clinical derivative of which, S64315, is being used in clinical studies for AML (NCT02979366, NCT03672695, and NCT04629443), as well as AZD5991, which is also being tested in clinical studies (NCT03218683). Both drugs are being tested not only alone, but also in combination with venetoclax. Our findings here further support clinical investigation of combined Bcl-2 and Mcl-1 inhibition in patients with AML, especially those with refractory/relapsed disease.
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