Synergistic Growth Inhibition of NPM1 Mutant AML PDX By Combined Therapy with BCL-2 Inhibitor Venetoclax (ABT-199) and Menin Inhibitor DS-1594b In Vivo

Introduction: Mixed-lineage-leukemia (MLL) translocations are prevalent in hematologic malignancies, resulting in aggressive leukemias resistant to standard therapies and low 5-year survival rates. These translocations lead to the production of MLL fusion proteins that promote leukemia development by enhancing proliferation, inhibiting hematopoietic differentiation, and driving leukemogenesis. Acute myeloid leukemia (AML), an aggressive blood cancer with a grim prognosis, particularly in elderly patients, frequently involves mutations in the nucleophosmin (NPM1) gene. Importantly, the interaction between Menin, MLL, and MLL fusion proteins play a crucial role in the pathogenesis of AML, both with MLL rearrangements (MLLr) and in leukemias carrying NPM1 mutations. Inhibition of the Menin-MLL interaction using novel Menin inhibitors has demonstrated efficacy in pre-clinical and clinical studies for both AML subsets. In this study, we investigate the effectiveness of the novel Menin-MLL inhibitor DS-1594b, developed by Daiichi Sankyo Co. Ltd., in combination with the BCL-2 antagonist venetoclax (ABT-199), in a patient-derived xenograft (PDX) model of NPM1-mutated acute leukemia. We have previously reported synergistic lethality and differentiation-inducing effects of this drug combination in both AML cell lines and primary leukemia samples in vitro (Ciaurro et al., Blood 2022). Our findings shed light on the potential of this therapeutic combination for the targeted treatment of AML with NPM1 mutations.

Methods: NSG mice (female, 8-10 weeks old) were purchased from Jackson Laboratory. Mice were inoculated via the tail vein with NPM1m PDX/luc/GFP (3×10 ⁶ cells). Once leukemia engraftment was confirmed by bioluminescent imaging mice were randomized in 4 groups: vehicle, ABT199, DS-1594b alone, and combination. After 12 days drugs were administered as follows: DS1594b at a dosage of 50 mg/kg by oral gavage for 4 weeks; ABT-199 at a dosage of 50 mg/kg by oral gavage for 2 weeks and 100mg/kg for the other 2 weeks. NSG mice were euthanized by CO ₂ inhalation at the endpoint of the study in accordance with IACUC protocols. BM and spleens were harvested in a sterile environment and flushed in HBSS with 1% FBS. For CyTOF data spleen cells were initially barcoded and stained with metal-conjugated antibodies according to Fluidigm's protocols and samples were analyzed by CyTOF (Helios) Mass Cytometer.

Results: New in vitro data confirmed synergistic lethality on additional AML cell lines and demonstrated upregulation of PARP-C and Caspase3-C apoptosis pathways using western blot analysis. Further, additional 3 primary AML patient samples showed additive or synergistic growth inhibition when treated with combination of drugs. In the in vivo experiment with NPM1m PDX/luc/GFP AML PDX model, we found that co-treatment with DS-1594b and venetoclax for four weeks resulted in a greater reduction in AML burden compared to treatment with either agent alone or a vehicle control as shown by Bioluminescence imaging. Moreover, weight of mice in the combo group was not significantly affected, while mice lost weight in vehicle and in single agent cohort due to rapid disease progression. The survival curve clearly demonstrated the benefits of the combination treatment, with the DS-1594b group having modest anti-tumor efficacy, and the combination group showing the greatest efficacy (Fig. 1A). To study the effects on cell differentiation, we used CyTOF technique, which allowed us to analyze a complete panel of 30 markers. We found two populations of cells, which we named populations (pop) 3 and pop 4, that were enriched in the group treated with the combination of drugs compared to the control group (Fig 1B). These populations of cells had higher levels of expression of CD33 and CD38, which are well-known markers of cellular differentiation.

Conclusions: In this study, we present the in vivo effectiveness of DS-1594b in combination with venetoclax in a PDX model of NPM1-mutated AML. Moreover, the combination treatment exhibits differentiation-inducing effects, which contribute to its therapeutic effectiveness, and was safe. Overall, our findings strongly indicate that the combination of DS-1594b and venetoclax exhibits promising anti-leukemic activity in vivo and holds potential as a therapeutic strategy for AML patients with mtNPM1 mutations.