Page 112 - Haematologica Vol. 110 - January 2025

ARTICLE - LIPA modulates venetoclax/TKI response in bpCML M. Minhajuddin et al.
TKI.7-9 Ponatinib and asciminib are next-generation TKI that can overcome resistance to T315I mutations.10,11 However, both have the potential for considerable toxicity12 and do not always allow for durable responses in bpCML. More ef- fective and safer therapeutic interventions are necessary to improve the cure rate in cpCML.13
The anti-apoptotic protein BCL-2 has recently been validated as a target in acute leukemia. The BCL-2 family of proteins are key regulators of mitochondrial-mediated apoptosis.14 Work from several groups has demonstrated that inhibition of BCL-2 is cytotoxic to acute myeloid leukemia (AML) cells.15 In addition, we have previously shown that BCL-2 is overex- pressed in AML LSC and is important for their survival.16 The expression of BCL-2 has been reported to be higher in CML than in normal hematopoietic stem cells and is further in- creased in bpCML.17 CML cell survival is in part also mediated by upregulation of other BCL-2 family proteins including BCL-XL and MCL-1.18,19 We have previously demonstrated that BCL-2 inhibition with venetoclax can eradicate LSC populations in the context of AML via a mechanism involving perturbation of energy metabolism20 suggesting that similar activities may be relevant to bpCML. Exciting preclinical studies by Carter et al. demonstrated in vivo targeting of LSC in a mouse model of cpCML, as well as in vitro targeting of primitive human bpCML cells, using a combination of venetoclax and the TKI nilotinib.21 Subsequent clinical testing of venetoclax with TKI in heavily pretreated bpCML patients has shown promising results.22 Metabolic aberrancies appear to underlie much of veneto- clax resistance in AML. One potential culprit could be the enzyme lysosomal acid lipase (LAL), encoded by the lipase A (LIPA) gene, the only known intracellular lipase active at an acidic pH that hydrolyzes cholesteryl ester and triglyceride in the lysosome.23 LIPA-mediated lipid catabolism releases fatty acids for use as an energy source. Recent work has re- vealed that fatty acids derived from LAL-mediated lipolysis have important functional impacts on macrophage alterna- tive activation,24 metabolic reprogramming of CD8+ memory T cells,25 and lipid mediator synthesis.26 It has also been demonstrated that increased fatty acid metabolism may be a universal mechanism for therapeutic resistance in AML, relevant to conventional chemotherapy27 and venetoclax.28,29 Similarly, metabolic shifts may be important in the develop- ment of therapy resistance in bpCML. In the present study, we evaluated the activity of the combination of venetoclax and dasatinib (ven/dasa) in bpCML mouse models and primary bpCML patients’ samples. Studies were designed to identify pathways that could potentially enhance the relative efficacy of the drug combination in functionally defined LSC.
Methods
Cell culture
Base medium of Iscove modified Dulbecco medium with 10% fetal bovine serum and 1% penicillin/streptomycin was
used, supplemented with 10 nM of human cytokines, stem cell factor, interleukin-3, and FLT3.
Human primary blast phase chronic myeloid leukemia samples
Primary bpCML samples were obtained from patients after informed consent for sample procurement. All specimen acquisition was approved by the University of Colorado In- stitutional Review Board.
Mouse strains and husbandry
Wild-type C57BL/6J mice, breeders of B6 Cd45.1, Pep Boy mice were purchased from Jackson Laboratory. All mice were housed at the University of Colorado Anschutz Medical Campus Animal Facility in a specific pathogen-free facility with individually ventilated cages. Mice were provided ad libitum access to rodent chow diet. All animal experiments were approved by the Office of Laboratory Animal Resources at the University of Colorado Anschutz Medical Campus.
Mouse studies
The genetically induced “GY” mouse model of bpCML, driven by the dual translocations Bcr-Abl (positive for green fluo- rescent protein, GFP+) and Nup98-Hoxa9 (positive for yel- low fluorescent protein, YFP+), was developed as previously described.30 A T315I version of this syngeneic mouse model was also generated for this study, using the same protocol as described previously,30 except using the T315I mutant version of the Bcr-Abl gene. In parallel, patient-derived xenograft studies were performed as previously described.31 Briefly, NSG-S mice were first engrafted with a human primary bp- CML sample through tail vain injection. After 4 weeks, they were treated by oral gavage with either venetoclax alone (100 mg/kg/day) or dasatinib (20 mg/kg/day) or the combination.
RNA sequencing
Bcr-Ablwt “GY” syngeneic mice were treated with ven/dasa or vehicle for 4 h on day 11 after transplant of the leukemia cells. Mice were sacked after the treatment, bone marrow was isolated and sorted for LSC (Lin–Sca1+), and total RNA from LSC was isolated using the RNeasy Plus kit (Qiagen) with the manufacturer’s protocol. Library construction and sequencing were performed according to a previously de- scribed protocol.29 Single-end reads of 100 nucleotides were generated for each sample on the Illumina HiSeq2500 plat- form. Methods for RNA-sequencing analysis can be found in the Online Supplementary Materials.
Metabolomics
Murine LSC (GY+ Lin–Sca1+) were isolated using the BD ARIA II cell sorter, treated for 4 h with ven/dasa or vehicle, and subjected to metabolomic analyses (0.2x106 cells/sample) via ultrahigh pressure liquid chromatography - mass spec- trometry (Vanquish and Q Exactive, Thermo Fisher) as pre- viously described.32
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