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Targeting Venetoclax-Resistant Acute Myeloid Leukemia Stem Cell
Anagha Inguva Sheth1, Krysta Engel1, Hunter Tolison1, Mark J Althoff1, Anna Krug1, Maria L. Amaya1, Shanshan Pei2, Tracy Young1, Sweta B. Patel1, Mohammad Minhajuddin1, Amanda Winters3, Ian Shelton1, Regan Miller1, Jonathan St-Germain4,5, Tianyi Ling4,5, Courtney Jones4,5, Brian Raught4,5, Austin Gillen1, Monica Ransom1, Sarah Staggs1, Clayton A. Smith1, Daniel A. Pollyea1, Brett M. Stevens1, Craig T. Jordan1*
1Division of Hematology, University of Colorado School of Medicine, Aurora, CO, USA.
2Liangzhu Laboratory, Zhejiang University Medical Center, Bone Marrow Transplantation Center, Hangzhou, China.
3Division of Pediatric Hematology and Oncology, University of Colorado School of Medicine, Aurora, CO, USA.
4Princess Margaret Cancer Centre, University Health Network, Toronto, Canada.
5Department of Medical Biophysics, University of Toronto, Toronto, Canada
Background: Despite the success of venetoclax (ven) based regimens in AML, most patients ultimately relapse on therapy. Consequently, determining mechanisms of ven resistance, and finding novel therapeutic strategies to target resistance have become a priority in the AML field. Notably, ven with azacitidine has been shown to directly target leukemia stem cells (LSCs). We hypothesize this is through OXPHOS inhibition, as OXPHOS activity is a key vulnerability of LSCs that we have shown to be mediated by BCL-2. An unbiased screen of the BCL-2 interactome revealed calcium signaling proteins as a key set of interacting partners. BCL-2 has been shown to regulate intracellular calcium signaling by influencing the activity of calcium channels at the endoplasmic reticulum (ER) and mitochondria. This is crucial for OXPHOS activity as mitochondrial calcium overload or depletion can inhibit TCA cycle and electron transport chain activity. Thus, we hypothesized that BCL-2 inhibition influences OXPHOS in ven sensitive LSCs by perturbing calcium signaling mechanisms. Additionally, we reasoned that drug resistance may arise in LSCs due to lack of calcium perturbation upon ven challenge.
Aims: Our first aim was to elucidate the role of BCL-2 mediated calcium signaling in ven sensitive LSCs and to determine whether calcium is connected to the decreased OXPHOS activity observed upon BCL-2 inhibition. Next, we sought to determine whether calcium biology properties between ven sensitive versus resistant LSCs were different. Lastly, we investigated whether perturbing key calcium signaling proteins in ven resistant LSCs decreases OXPHOS activity and function.
Methods: All studies were conducted in primary human AML LSCs defined by a ROS-low phenotype. Mitochondrial calcium measurements were done using RHOD2-AM via flow cytometry and confirmed with confocal microscopy. Protein-protein interactions were investigated using PLA assays. OXPHOS activity was measured through seahorse assays and TCA cycle enzyme activity assays. All genetic perturbations were done using siRNA via electroporation and confirmed with western blot/qPCR. Bulk RNA sequencing data was analyzed using GSEA. CITE-Seq analysis was performed on 25 AML patient specimens and analyzed using TotalVI 0.18.0. LSC function was assessed by colony formation assays and PDX experiments. PDX experiments were also used to determine efficacy of tumor reduction in ven resistant AML samples.
Results: Venetoclax treatment significantly increased mitochondrial calcium levels in sensitive LSCs, which was phenocopied by genetic inhibition of BCL-2. These findings were evident prior to overt cell death or loss of mitochondrial membrane potential as assessed by DAPI and TMRE staining. Additionally, there was a change in the activity of calcium dependent TCA cycle enzymes upon treatment. Mechanistically, BCL-2 inhibition significantly decreased expression of SERCA, the key calcium influx channel at the ER. The above changes did not occur in ven resistant LSCs. When comparing ven sensitive vs resistant LSCs, there was a striking difference in steady-state mitochondrial calcium levels, with ven resistant specimens showing higher basal levels. Further, genetic analysis revealed resistant LSCs have higher expression of genes involved in calcium transport into the mitochondria (MCU). Pharmacologic (Ru265, MCUi4 and mitoxantrone) and genetic inhibition of MCU led to decreased mitochondrial calcium levels and OXPHOS activity in resistant LSCs. Additionally, pharmacologic inhibition of MCU led to significantly decreased engraftment of venetoclax-resistant AML specimens in NSG-S mice and/or decreased colony formation. Mitoxantrone, the most clinically relevant compound shown to inhibit MCU, led to decreased tumor burden in PDX models and secondary transplantation showed elimination of LSC activity. Importantly, the above findings were observed at drug doses with no discernable effect on normal hematopoietic stem cell activity.
Conclusions: BCL-2 inhibition causes changes in AML LSC metabolism that occur before the onset of overt apoptosis by modulating mitochondrial calcium content and metabolic enzyme activity. This mechanism uniquely occurs in ven sensitive LSCs and indeed represents a distinct feature of their biology. Conversely, ven resistant LSCs have adapted to their unique metabolic demands by increasing basal mitochondrial calcium levels and do not undergo changes in calcium signaling upon ven treatment. Therefore, we propose that these characteristics may allow the use of calcium signaling (or flux) as a prognostic tool to identify patients most likely to respond favorably to ven based therapies. Further, we propose that mitoxantrone is a potent anti-LSC agent specifically in the context of ven-resistant AML due to its recently described activity as an MCU inhibitor. Taken together, our findings have important ramifications for the basic biology of human AML, and the development of improved strategies to target ven resistant LSCs.
Grant information is as follows: AIS is supported by the NCI NRSA F30 grant NCI (F30CA254251). MJA is supported by the NCI F32 award (F32CA275350). MLA is supported by the BL&CS Career Development Award (1IK2BX005603-01A1). AW is supported by American Cancer Society (CSDG-22-018-01-CDP), Morgan Adams Foundation (R6251I), and Swim Across America. CJ is supported by the LLS Special Fellow program. DAP is supported by the Leukemia and Lymphoma Society’s Scholar in Clinical Research Program, the Robert H. Allen MD Chair in Hematology Research and the V Foundation Clinical Scholar Program. BMS is supported by the V Foundation Clinical Scholar Program. CTJ is generously supported by the Nancy Carroll Allen Chair in Hematology Research, a Leukemia and Lymphoma Society SCOR grant (7020-19), NIH R35CA242376, and Veterans Administration merit award BX004768-01.