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Targeting AML MCL-1 re-sensitizes BCL-2 inhibition
driven hematological malignancies9,10 and essential for AML development and AML stem cell self-renewal and survival.11-13 MCL-1 is upregulated in about half of resist- ant/relapsed AML patients and associated with poor prognosis.14 Importantly, high levels of MCL-1 expression are associated with resistance to venetoclax.2,15,16 Furthermore, MCL-1 was reported to increase the gener- ation of reactive oxygen species (ROS) in lung cancer cells,17 and to positively regulate mitochondrial oxidative phosphorylation (OxPhos) in breast cancer, inducing the breast cancer stem cells, and promoting tumor chemore- sistance.18 These studies suggest that, in addition to its anti-apoptotic function, MCL-1 is coupled to tumor cell metabolism to promote cell survival. Indeed, AML cells and stem cells exhibit increased mitochondrial OxPhos,19-21 suggesting that MCL-1 has a role in regulating mitochon- drial OxPhos and altering AML cell metabolism.
Several specific MCL-1 inhibitors have been developed recently. S63845 (Servier/Novartis) was very effective and well tolerated in in vivo models.22 Others such as AZD5991 (AstraZeneca)23 and AMG176/AMG397 (Amgen)24 have entered clinical trials. Additionally, inhibitors of CDK9 that transcriptionally regulates short- lived proteins like MCL-1 are under development as a strategy to target MCL-1, and the CDK9 inhibitor AZD457325 has entered clinical trials.
Rationales supporting BCL-2 and MCL-1 co-targeting have focused on apoptotic mechanisms. Targeting MCL- 1 with a BIM transgene enhanced venetoclax activity against AML.26 We reported a combinatorial regimen of BCL-2 inhibition and MDM2 inhibition-mediated p53- activation that targeted MCL-1 activity and stability. This combination was synthetically lethal to AML cells27 and in early clinical trials elicits 40-50% CR rates among relapsed/refractory AML patients.28 Recent studies in murine models of AML showed that the combined inhi- bition of BCL-2 and MCL-1 has potent antitumor activi- ty, enhances venetoclax activity, and kills venetoclax- resistant disease.23,24,29,30 However, the mechanisms under- lying this synergism, including its effects on AML stem and venetoclax-resistant cells, have not been fully inves- tigated.
Here, we determine the effects of MCL-1 inhibition with the novel MCL-1 inhibitor AZD5991 or the CDK9 inhibitor AZD4573 alone and in combination with vene- toclax on multiple phenotypic AML stem/progenitor cell populations in vitro and in an in vivo patient-derived xenograft (PDX) model. Additionally, our in vitro study was conducted with bone marrow (BM)-derived mes- enchymal stromal cell (MSC) co-culture, which mimics the BM stromal microenvironment.31 In the in vivo study, for the first time, we utilized a PDX model derived from a patient that acquired resistance to venetoclax/ decitabine treatment thus revealing a novel mechanism of synergy involving potential non-apoptotic, metabolic and microenvironmental functions of MCL-1.
Methods
Cells and treatments
AML cell lines (Molm13, MV4-11, and OCI-AML3) and pri- mary AML cells (Online Supplementary Table S1) were obtained as described in the Online Supplementary Appendix. MCL-1-overex- pressing (OE), MCL-1-knockdown (KD), and acquired venetoclax
resistant AML cells were generated as previously described.27 BAX, BAK, or BAX/BAK double KD OCI-AML3 cells using small interfering RNA (siRNA) (control or ON-TARGETplus SMARTpool siRNA for each target, Dharmacon, Chicago, IL) were generated as previously described.32 Human BM-derived MSC were isolated as previously described.33 Cells, cultured under the conditions previously described34 were treated with venetoclax, AZD5991, AZD4573, IACS-10759 (Institute for Applied Cancer Science, MD Anderson, Houston, TX), BL-8040 (BioLineRx Ltd., Israel), and various combinations without or with MSC (AML-to-MSC=4:1). For AZD4573 treatments, cells were exposed to AZD4573 for 6 hours (h), and then the drug was removed.
Cell viability assay
Viable cells and apoptosis were assessed as previously described.34 For leukemia cells co-cultured with MSC, CD45+ cells were counted. Apoptotic cells were defined as annexin V+ and/or 7-aminoactinomycin D+ (AnnV+/7AAD+) CD45+ cells. For patient samples, annexin V positivity was determined in bulk (CD45+), CD34+CD38+/CD38-, and CD34+CD38+/CD38-CD123+ cells.
Western blot analysis and co-immunoprecipitation
Western blot analysis was conducted as described previously.34 Antibodies used are shown in the Online Supplementary Appendix.
Protein determination by flow cytometry
MCL-1 and cell surface CD44 and CXCR4 were also deter- mined by flow cytometry as shown in the Online Supplementary Appendix.
Adhesion and migration assay
Leukemia cells’ migration and adhesion to MSC were assessed as previously described.35
Mitochondrial respiration assay
Mitochondrial respiration was measured using a Seahorse XF extracellular flux analyzer (Agilent Technologies, Inc., Santa Clara, CA) following the manufacturer’s instructions. The oxy- gen consumption rate (OCR) was expressed as pmol/min/1,000 cells or relative to a control.
Reactive oxygen species and glutathione assay
Cellular and mitochondrial ROS were determined by flow cytometry after cells were stained with CellROS deep red or MitoSOX red (ThermoFisher Scientific), for 30 minutes (min) at 37°C and expressed as mean fluorescence intensity shift between stained and unstained live cells (AnnV-/DAPI-).
Total glutathione and glutathione disulfide (GSSG) were deter- mined using a glutathione (GSH) assay kit (Cayman Chemical; Ann Arbor, MI) following the manufacturer’s instructions. Reduced GSH was calculated by subtracting the amount of GSSG from total GSH.
13C -1,2-glucose and 13C -glutamine tracing and ion
5
2
chromatography-mass spectrometry metabolite analysis
Tracing and subsequence metabolite analysis are detailed in the Online Supplementary Appendix.
In vivo experiments
Mouse care and experiments were performed in accordance
with Institution Animal Care and Use Committee approved pro- tocols. See the Online Supplementary Appendix for detailed exper- imental procedures.
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