Safety and Efficacy of a Novel Mammalian-Derived Asparaginase without Glutaminase Activity in Combination with Venetoclax for Acute Myeloid Leukemia

Background

Acute Myeloid Leukemia (AML) with specific vulnerabilities, such as low asparagine synthetase (ASNS) expression, may be susceptible to asparagine depletion. Currently available asparaginase therapies can cause hypersensitivity reactions due to their bacterial origin and other side effects associated with their glutaminase activity. Enzyme by Design developed a mammalian-derived, glutaminase-free asparaginase (EBD) designed to address these issues, and we studied its efficacy and safety both alone and in combination with Venetoclax (VEN), in AML cell lines, primary patient samples, and preclinical models.

Methods

Western Blot was used to quantify ASNS protein levels in AML cell lines. Cell Titer Glo (CTG) assay was performed to evaluate the efficacy of EBD, VEN, and the combination of VEN-EBD in AML cell lines with various ASNS protein levels. Colony-forming unit (CFU) assays assessed the impact of EBD and VEN-EBD on CD34+ cells in primary AML patient samples. Additionally, 3 PDX models were leveraged for in vivo testing, including PDX-1 (deletion 7q and FLT3-ITD, DNMT3A, IDH1, KIT, NPM1 mutation), PDX-2 (diploid cytogenetics and IDH1 mutation), and PDX-3 (deletion 7 and TP53 mutation). NSG mice were injected with PDX cells following sublethal irradiation. The mice were then randomized into four groups (n=8) to receive vehicle, EBD (750-1500 IU/kg, i.p., twice or three times a week for 3 weeks), VEN (100 mg/kg, p.o., 5 days a week for 2 weeks), or a combination of VEN and EBD. Tumor burden and drug tolerance were monitored weekly via hCD45% in peripheral blood. Three mice were sacrificed at week-3 to assess leukemia burden in multiple organs, while the remaining mice were observed for survival. Asparaginase activity of EBD was measured using a continuous enzyme-coupled spectrophotometric assay. The lack of glutaminase activity of EBD was confirmed using both nuclear magnetic resonance and a continuous enzyme-coupled spectrophotometric assay.

Results

EBD had higher efficacy in AML cell lines with lower ASNS levels. Specifically, OCI-AML6 cells (-7/7q, low ASNS level) had an IC₅₀ of 1.1e-4 IU/mL, while MOLM-13 (high ASNS level) had an IC₅₀ of 0.751 IU/mL, indicating a negative correlation between EBD efficacy and ASNS levels. VEN-EBD co-exposure decreased VEN IC₅₀ from 757 to 674 nM in OCI-AML6 and from 24 to 12.8 nM in MOLM-13. CFU assays using CD34⁺ cells from three primary AML patients showed that EBD (0.5 IU/mL) treatment reduced colony count by 52.8% (range 33.3-56.8%, p<0.01) compared to DMSO. VEN (100 nM) led to a 19.1% reduction (range 4.2-15.9%, p=0.58), and the combination of VEN-EBD yielded a 75.9% reduction (range 61.4-95.5%, p<0.005), indicating potentially synergistic VEN-EBD efficacy in eliminating leukemic stem cells (LSCs). In PDX in vivo testing, EBD and VEN-EBD reduced leukemic burden compared to the vehicle group, with VEN-EBD achieving the highest reduction after 3 weeks of treatment (average circulating hCD45%: 19.3%, 14.3%, 5.1%, and 2.3% for vehicle, VEN, EBD, and combination, respectively). Additionally, EBD (p=0.002) and VEN-EBD (p=0.0003) significantly reduced leukemia burden in bone marrow (BM), liver, and spleen, with reductions of 36.2% (EBD) and 25.0% (VEN-EBD) in BM, 15.3% (EBD) and 7.5% (VEN-EBD) in spleen, and 22.4% (EBD) and 11.0% (VEN-EBD) in liver. In comparison, leukemia infiltration in vehicle and VEN groups ranged from 60.0-74.4% in BM, 12.0-22.6% in spleen, and 54.0% in liver in PDX-2. Mice in the combination treatment cohort survived longer (p=0.0038), with a median overall survival of 47 days compared to 44, 42.5, and 45.5 days for vehicle, VEN, and EBD, respectively, in PDX-1.

Conclusion

The novel asparaginase EBD exhibited significant efficacy in AML cell lines with low ASNS expression. Additionally, VEN-EBD has superior efficacy across multiple AML cell lines in comparison to monotherapy. AML characterized by 7/7q deletion is particularly sensitive to EBD due to low ASNS expression resulting from the loss of one allele of the ASNS gene located on 7q21.3. EBD and VEN-EBD reduce LSCs, as indicated by the decreased CFU in primary AML patient samples. Furthermore, EBD and VEN-EBD markedly reduce leukemia burden and improve survival in AML PDX models compared to VEN monotherapy and vehicle groups, suggesting that EBD is a potential anti-AML drug in patients with low ASNS. Analysis of VEN-EBD synergy and potential biomarkers of low ASNS are ongoing.