Page 113 - Haematologica Vol. 110 - January 2025

ARTICLE - LIPA modulates venetoclax/TKI response in bpCML M. Minhajuddin et al.
Results
Venetoclax and dasatinib target bulk and leukemia stem cell populations in a mouse model of blast phase chronic myeloid leukemia
To assess the therapeutic efficacy of venetoclax and dasati- nib separately and in combination we performed in vitro and in vivo experiments utilizing a mouse model of bpCML. This model employs co-expression of the Bcr-Abl and Nup98- Hoxa9 translocations, independently monitored using the expression of GFP and YFP, respectively (henceforth referred to as GY cells). These translocations are detected in human bpCML and the model has previously been described in detail for studies of leukemia genetics and stem cell biology.30,33,34 To assess the effect of ven/dasa treatment in vitro, the GY cells were treated in cell culture with each drug alone or in combination for 24 h, and viability was assessed with annexin V flow cytometry. Bulk cells treated with the two- drug combination demonstrated a significant loss of viability compared to GY cells treated with either drug alone (Figure 1A). Furthermore, the percentage of phenotypic LSC defined by surface markers (Lin–Sca1+) was also significantly reduced in the ven/dasa-treated group (Figure 1B). To evaluate in vivo activity, syngeneic mice were transplanted with GY cells, (105 cells per mouse through the tail vein) and, at day 5 after transplantation, the mice were treated for 5 days with vehicle, single agents or the ven/dasa combination. The combination of ven/dasa led to a significant reduction in leukemia burden compared to either drug alone (Figure 1C). We also evalu- ated the effect of ven/dasa on the murine LSC population (Lin–Sca1+) and observed a significant decrease in the LSC population compared to that achieved with treatment with the single agents or vehicle control (Figure 1D).
To further investigate the clinical benefit of ven/dasa therapy, mice with established GY leukemic engraftment were treated for 10 days with single agents or the ven/dasa combination and monitored for survival; mice were sacrificed upon ap- pearance of signs of leukemia-related morbidity. No survival benefit was seen with venetoclax alone relative to vehicle, whereas dasatinib alone led to significantly increased survival compared to that of controls. Animals receiving the ven/dasa combination eliminated the leukemic cells and were moni- tored for up to 80 days with no sign of leukemia recurrence as determined by the absence of any GY cells in their bone marrow upon sacrifice (Figure 1E, F).
Kinetics of response to the venetoclax and dasatinib combination in a mouse model of blast phase chronic myeloid leukemia
To better understand the kinetics of drug response, we also performed short-term drug treatment studies. As shown in Figure 2A-C, leukemic mice were treated for 2, 3 and 4 days and effects on leukemia burden as well as on LSC were evaluated. Leukemia burden overall and
LSC percentage were significantly decreased as early as 2 days after treatment initiation. Thus, targeting of primitive populations occurs very rapidly. Notably, analysis of nor- mal progenitor cells, (Lin–Sca1+c-kit+, termed LSK cells), which are co-resident in the same animals, showed no detectable reduction in colony-forming assays implying a leukemia-specific response (Figure 2D).
Due to the known disseminated nature of the GY leuke- mia model, we also assessed the effect of ven/dasa on leukemic infiltration of extramedullary locations such as the spleen. Treatment of mice with ven/dasa resulted in significant inhibition of bulk disease as well as the LSC compartment compared to treatment with either drug alone (Online Supplementary Figure S1A, B). The average spleen size in the combination-treated cohort was reduced to the size of normal spleens (Online Supplementary Figure S1C). Overall, the bpCML mouse model showed the enhanced efficacy of the ven/dasa combination over treatment with the single drugs.
Venetoclax and ponatinib target bulk and leukemia stem cell populations in a mouse model of BCR-ABLT315I mutation
Despite the success of TKI, resistance has been identified in some patients due to point mutations in the BCR-ABL kinase domain. Ponatinib was developed as a TKI that can inhibit all critical kinase domain mutations including T315I.10 Thus, we investigated the addition of venetoclax to ponatinib in the context of T315I-mutant bpCML. We generated a mouse model of leukemia harboring T315I mutant BCR/ABL-GFP and NUP98/HOXA9-YFP (termed GY-315). As expected, in vitro treatment of GY-315 cells showed resistance to dasatinib. Furthermore, GY-315 cells showed no response to ven/dasa (Online Supple- mentary Figure S2A). In contrast, both GY and GY-315 cells responded to ponatinib treatment, confirming that ponatinib is active against both the wild-type as well as the mutant Bcr-Abl oncogene (Online Supplementary Figure S2B).
To assess the effect of the venetoclax and ponatinib combination (ven/pona) in vitro, the GY-315 cells were treated with each drug alone or in combination for 24 h. Treatment with ven/pona resulted in a significant loss of viability of bulk leukemia cells compared to either drug alone (Figure 3A). Furthermore, the percentage of phe- notypic LSC (Lin–Sca1+) was also significantly reduced in the ven/pona-treated group (Figure 3B). To evaluate the effect of these drug combinations in vivo, leukemic mice were generated by transplantation of GY-315 cells into recipient animals followed by treatment with each drug alone or the ven/pona combination. Both single agents resulted in decreased leukemia burden in bone marrow, an effect that was more pronounced in mice treated with ponatinib alone, but the combination of ven/pona led
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