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 pRIPK1^Ser166/RIPK1 in both cell lines after one hour treatment (p<0.0001 and p<0.01 respectively). Activation was accompanied by cleavage of RIPK1 after 24 hours in Reh cells only (p<0.05). One hour treatment with PA-PLA₂ was also found to activate pAkt^Ser473/Akt(pan) and pAkt^Thr308/Akt(pan) signalling in Reh cells (p<0.001 and p<0.05 respectively). In contrast, a transient and latent response of Akt activation was observed in SD-1 cells, with pAkt^Ser473/Akt(pan) levels initially decreasing after one hour (p<0.05), before increasing again and resulting in activation at 24 hours (p<0.01). Activation of pAkt^Thr308/Akt(pan) was only observed after 24 hours treatment also (p<0.05).

Examining the signalling axes driving PA-PLA₂'s mechanism of action, phosphoproteomics showed a reduction in pc-Myc^Ser348 expression in both cell lines (p<0.05). Immunoblotting analysis confirmed the c-Myc modifying role of PA-PLA₂, with c-Myc total expression found to be elevated in SD-1 cells after 8 hours treatment (p<0.001).

To 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<0.05), implicating HDAC2 as a possible effector of synergism.

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.

No relevant conflicts of interest to declare.