Paraoxonase 2 Enables Initiation of B-ALL By Subverting Metabolic Gatekeeper Functions

Background and significance: The B-lymphoid transcription factors IKZF1 and PAX5 are essential for early B-cell development but also function as metabolic gatekeepers by restricting glucose uptake across the cell membrane (Chan et al., Nature 2017; Xiao et al., Cell 2018). Paraoxonase 2 (PON2) is used as a diagnostic marker and included in a 15 gene diagnostic LDA panel (NCT02883049) for the identification of Ph+ and Ph-like ALL, a B-ALL subgroup with poor outcome and frequent deletion of IKZF1. Pon2 is a member of detoxifying enzymes that are located to the mitochondrial membrane, and hydrolyze lactone metabolites. While PON2 is typically not expressed in normal B-cell precursors, we studied the functional role of high expression levels of Pon2 in B-ALL.

Results: Indeed, high Pon2 mRNA levels at diagnosis predicted poor clinical outcomes for both children (COG P9906; n=207; P=1.09e-05) and adults (ECOG; n=215; P=0.003). While PON2 mRNA and protein levels are barely detectable in normal B-cell precursors we found >10-fold increased mRNA and protein levels in multiple B-ALL. Pon2-deficient mice have a number of subtle defects, including propensity to neurogenerative disease and atherosclerosis. However, deletion of Pon2 had profound effects in two models for B-ALL (BCR-ABL1- and NRAS^G12D). Compared to Pon2 wildtype, Pon2^-/- B-ALL cells failed to form colonies in semisolid agar, were prone to cell cycle arrest in G₀/G₁ phase. Consistent with these observations, Pon2^-/- B-ALL cells had a substantially reduced proliferation rate and expressed Arf and p21 checkpoint proteins at substantially increased levels compared to Pon2^+/+ ALL cells. These in vitro findings suggest an important role of PON2 in B-lymphoid leukemogenesis, which was confirmed in mouse transplant experiments for BCR-ABL1 and NRAS^G12D B-ALL models. Pon2-deficiency substantially prolonged survival of recipient mice of BCR-ABL1 B-ALL cells (P=0.0001). Strikingly, NSG mice transplanted with Pon2-deficient NRAS^G12D B-ALL cells did not develop any leukemia and survived for indefinite periods of time. Based on engineered expression of Cas9 and PON2-specific cRNAs, genetic deletion of PON2 replicated these findings in patient-derived B-ALL xenografts (PDX).

Mechanism: Reflecting PAX5 and IKZF1 function, B-lymphoid cells exhibit transcriptional repression of glucose transport (e.g. repression of INSR, GLUT1). Interestingly, deletion of PON2 decreased glucose uptake even further. Intracellular ATP levels in Ph⁺ ALL cells were drastically reduced and activated the AMPK energy-stress sensor in both murine BCR-ABL1 and NRAS^G12D B-ALL models and human Ph+ B-ALL cells. Reconstitution of PON2 restored ATP production as early as 3 hours after PON2 induction. Mechanistically, PON2 was previously identified as an inhibitor of STOM and GLUT1 interactions. By disrupting STOM: GLUT1 interactions, PON2 can restore glucose transport. STOM-knockdown in PON2^-/- ALL cells replenished cellular ATP levels as did PON2 reconstitution. We conclude that PON2 serves a critical role in B-ALL by subverting the metabolic gatekeeper function of IKZF1 and PAX5 through interference with STOM:GLUT1 interactions.

Therapeutic targeting of PON2: While PON2 activity typically results in detoxification of lactone-metabolites, lactone-hydrolysis of the N-(3-oxododecanoyl)-homoserine lactone (3OC12) prodrug results in cytotoxic byproducts. Hence, we tested the concept of leveraging PON2 lactonase activity in a prodrug-approach as a synthetic lethal in Pon2+ B-ALL cells. 3OC12 had profound cytotoxic effects in wildtype but not Pon2^-/- B-ALL cells while reconstitution or overexpression of PON2 restored cytotoxic responses to 3OC12.

Conclusion: We describe the previously unrecognized function of the detoxifying PON2 lactonase as a central factor in glucose and energy supply in B-ALL, hence subverting metabolic gatekeeper functions of PAX5 and IKZF1. In addition to identifying PON2 as the biomarker for for Ph⁺ and Ph-like ALL and outcome predictor for patients with B-ALL, our results indicate PON2 facilitate glucose uptake and ATP production via its interaction with STOM. PON2 protects B-ALL cells against glucose depletion and enables malignant growth. On the other hand, we demonstrate a targeted approach that leverages PON2-lactonase as a synthetic lethal to the lactone-prodrug 3OC12.