Targeting Unique Synthetic Lethal Interactions between PI3K and MYC in B-ALL

Significance: Activating mutations of PIK3CA and other PI3K subunits are frequent in almost all types of cancer. Likewise, inactivating lesions of PTEN are very common in almost all cancer types. However, activating of PI3KCA and deleterious mutations of PTEN were not found in any of the 987 cases of B-ALL in our study. These findings are in line with recent studies suggesting that rare activating germline mutations in the PI3K pathway predispose to cancer but generally cause B-lymphopenia. Here we studied the underlying reasons for the remarkable sensitivity of normal B-cell precursors and B-ALL and examined strategies to leverage PI3K hyperactivation for therapeutic purposes.

Results: Our study revealed the dysregulated glucose metabolism in PTEN-deficient pre-B ALL cells, which is indicated by increased glucose consumption, lactate production and deprived cellular ATP levels. After closely examining gene expression profile of PTEN KO murine ALL cells, we found glutaminolysis related enzymes and transporters were broadly decreased after PTEN deletion. Considering the observed ATP deficit and decreased glutaminolysis genes expression in PTEN KO ALL cells, we further performed Seahorse analysis and glutamine consumption assay. The results showed significant half-fold reduction of both oxygen consumption rate and glutamine utilization in PTEN KO ALL cells. We noticed most of the downregulated glutaminolysis genes are reported MYC targets and observed significantly decreased Myc protein levels both in PTEN KO and Pik3ca^H1047 overexpressed murine ALL cells. Furthermore, abrogation of PTEN activity by selective PTEN inhibitor SF1670 decreased MYC protein levels in patient-derived pre-B ALL xenografts. Those data indicate MYC as a metabolic activator is negatively regulated by PI3K signaling in pre-B ALL cells.

Considering the hyperactivation of PI3K signaling after PTEN deletion, we performed CRISPR-RNP mediated genetic deletion of key PI3K-adaptor protein genes to compromise PI3K signaling in ALL cells and hopefully mitigate their sensitivity to PTEN deletion. Nevertheless, deletion of one or two of those genes including Cd19, Pik3ap1 and Irs1 did not alleviate PTEN-deletion caused cell death even with slightly decreased basal PI3K signaling level.

Based on the observation of reduced levels of MYC protein and its target genes, we assessed the effect of Myc overexpression in rescuing PTEN-deletion caused cell death. Growth competition assay as well as cell-viability analysis showed profound rescue effects of Myc overexpression in PTEN KO ALL cells. More importantly, Myc overexpression not only increased cellular respiration and glutamine consumption but also recovered ATP levels in those cells. Meanwhile, we found even the overexpressed Myc protein level was reduced upon PTEN deletion, which indicates decreased Myc protein stability after PI3K hyperactivation. By overexpressing the Myc^T58A mutant, we found that proteasomal degradation resistant Myc mutant showed 2-fold enhanced rescue effect than wild type Myc in PTEN KO ALL cells. Our results demonstrate a PI3K-mediated degradation of MYC and its role in regulating glucose and glutamine metabolism in pre-B ALL.

Conclusions: Unlike other cell types, B-cells are under particular metabolic constraints owing to repression of glucose uptake by B-lymphoid transcription factors PAX5 and IKZF1 (Chan et al., Nature 2017; Xiao et al., Cell 2018). As a result of the unique metabolic gatekeeper activity of B-cell-specific PAX5 and IKZF1-function, B-cells survive under conditions of chronic energy deprivation, which makes them particularly susceptible to acute fluctuation of energy expenditure, e.g. by activation of PI3K upon deletion of PTEN. In addition to increasing energy demands by acute PI3K-activation, PTEN-deletion also results in accelerated degradation of MYC, which could otherwise replenish glucose and energy levels. Strikingly, ectopic expression of ubiquitination-resistant T58A-Myc dramatically rescued cell death, which reveals a previously unrecognized synthetic lethality in B-ALL cells. Based on these observation, we propose dual targeting of PTEN with the small molecule phosphatase inhibitor SF1670 (Shojaee et al., Nat Med 2016) with inhibition of glutaminolysis, which erases the rescue effect of Myc on metabolic depletion.