New mechanistic and therapeutic insights into purified phospholipase A2 from Pseudechis australis snake venom - a novel anticancer agent for the treatment of precursor B-cell acute lymphoblastic leukaemia

Introduction
Precursor B-cell acute lymphoblastic leukaemia (pre-B ALL) is the most common childhood malignancy and the leading cause of cancer-related death in children and young adults. Standard treatment options for pre-B ALL typically involve multiple regimens of DNA-damaging chemotherapy agents, which despite their high curative rates, are often tolerated poorly by patients.
Previously our group has purified, and characterised a novel anti-leukaemia compound, termed PA-PLA2 from Pseudechis australis snake venom. Comprised of three phospholipase A2 (PLA2) enzymes, PA-PLA2 was shown to induce necrotic cell death in pre-B ALL cells, with RNA-sequencing analysis revealing enrichment of PI3K/Akt signalling as well as inflammation and immune response genes. Importantly, PA-PLA2 has also been shown to be non-cytotoxic towards normal stem cells from donor peripheral blood and bone marrow, raising the prospect of its use as a novel and selective treatment.
In this study, we further explore the mechanism of action and therapeutic potential of PA-PLA2 in relevant models of pre-B ALL.

Methods
PA-PLA2 was purified and characterised previously using, reverse-phase high-performance liquid chromatography and mass spectrometry (Boncan et al., 2021). Reh and SD-1 pre-B ALL in vitro cell lines, were used to study PA-PLA2's anti-leukaemia action. Protein immunoblotting was used to validate previously performed RNA-sequencing, proteomics and phosphoproteomics analysis. Densitometry analysis of immunoblots was completed using ImageJ software.
To assess the therapeutic potential of PA-PLA2 in combination with standard of care therapies, we performed a drug screen, comprised of a custom panel of 48 standard of care and clinical agent compounds. CellTox™ Green cytotoxicity assay was used as a readout of cell death, with effective drug combinations validated using CellTiter® Glo viability assay, and combination indices (CI) calculated using CompuSyn software.

Results
Time course protein immunoblotting analyses of PA-PLA2-sensitive cell lines, Reh and SD-1 treated with IC50 doses of PA-PLA2 (Reh; 1µg/mL, SD-1; 25µg/mL) revealed activation of pRIPK1Ser166/RIPK1 in both cell lines after one hour treatment (pExamining the signalling axes driving PA-PLA2's mechanism of action, phosphoproteomics showed a reduction in pc-MycSer348 expression in both cell lines (pTo evaluate the therapeutic benefit of including PA-PLA2 in standard of care regimens, we performed a drug screen that identified combinations of PA-PLA2 with Cytarabine, Idarubicin-HCl or Vorinostat (SAHA) as being synergistic in Reh cells. SAHA displayed the best synergism profile, with CI values of < 1 being obtained for all combinations of PA-PLA2 (0.1, 0.5, 1, 2.5 and 5µg/mL) with SAHA (1,10 and 20µM). Importantly, proteomics analysis revealed increased expression of HDAC2 in Reh cells (p
Conclusion
This study has identified RIPK1 and Akt as effectors of the mechanism of action of PA-PLA2. PA-PLA2 treatment had a modifying role on the expression of the multifunctional transcription factor c-Myc. Importantly PA-PLA2 was shown to act synergistically with Cytarabine as well as SAHA and Idarubicin-HCl, with HDAC2 implicated as a possible effector of synergism with SAHA.
Overall, this study has further elucidated the mechanism of action of PA-PLA2, with the identified synergy of PA-PLA2 with standard of care agents possibly yielding a novel, treatment option for patients with pre-B ALL.