PTEN Is Essential for Normal Cytokine Signaling and Oncogenic Transformation of Pre-B Cells

Background: PTEN is a negative regulator of PI3K-AKT signaling and a potent tumor suppressor throughout all main types of human cancer. Recent work demonstrated that loss of PTEN in T cell acute lymphoblastic leukemia (ALL) confers glucocorticoid resistance. To develop a model for glucocorticoid resistance in pre-B ALL, we studied Cre-mediated deletion of Pten.

Results: Surprisingly, loss of one or both alleles of Pten caused rapid cell death in pre-B ALL cells and was sufficient to cure transplant recipient mice from leukemia. Furthermore, deletion of Pten in pre-B cells disabled oncogenic transformation by BCR-ABL1 and NRAS^G12D oncogenes. A reanalysis of genetic lesions of PTEN in human cancer revealed a high frequency across all main subtypes of mature B cell lymphoma, myeloid leukemias and T cell lineage leukemia/lymphoma (11.3% in 1,697 samples). Strikingly, however, lesions of PTEN were not detected in any of 670 pre-B ALL patient samples. Whereas high expression levels of PTEN predict favorable outcomes for patients with T-ALL, pre-B ALL patients with high PTEN expression levels at the time of diagnosis have particularly poor clinical outcomes (P=0.01). Studying the unexpected role of PTEN in B cell lineage ALL, we developed a genetic model for Cre-mediated deletion of Pten in mouse pre-B ALL cells carrying BCR-ABL1 or NRAS^G12D oncogenes. Deletion of one or both alleles of Pten reduced the colony forming ability (p < 10E-06) and proliferation rate (p < 10E-04) of pre-B ALL cells while strongly increasing cellular senescence (P < 10E-03). Since immediate effects of Pten deletion include hyperactivation of PI3K-AKT signaling, we hypothesized that small molecule inhibitors that limit PI3K-AKT pathway activity may rescue the pre-B ALL cells from cell death when Pten is inducibly deleted. Therefore, we tested the effect of AKT and PI3K inhibitors AZD5363 and BKM120 on Pten deleted cells and confirmed that chemical inhibition of PI3K-AKT pathway indeed rescued cell death of pre-B ALL cells upon Pten deletion. Also, global tyrosine phosphoprofiling by quantitative mass spectrometry revealed that Cre-mediated deletion of Pten strongly induced activation of CBL, a ubiquitin ligase that induces degradation of the IL7 receptor in pre-B cells and oncogenic tyrosine kinases in leukemia. Genetic experiments confirmed that deletion of Pten and hyperactivation of AKT engaged a novel, CBL-dependent pathway of negative feedback inhibition of STAT5 and tyrosine kinase signaling. Consistent with these finding, Reverse Phase Protein Array (RPPA) measurements for 155 newly diagnosed cases of pre-B ALL revealed a strong negative correlation between STAT5 protein levels and phospho-ATK^T308 (P=0.0021) and phospho-AKT^S473 (P=0.0005).

Unlike pre-B ALL, deletion of Pten in CML like cells showed no toxicity. To further study the lineage specifity of Pten dependency we used a doxycycline-inducible CEBPa construct to differentiate pre-B cell ALL into myeloid lineage leukemia cells. Similar to our finding in CML like cells, unlike the pre-B ALL, myeloid lineage Leukemia showed no dependency on Pten and the deletion of Ptencaused no significant change in the cells viability. Studying the basal level of PTEN, pre-B cells showed ~40-fold higher expression levels than myeloid lineage cells. Consistently, genetic knockdown of PTEN using two different shRNA was only toxic in patient-derived pre-B but not myeloid leukemia cells.

Clinical relevance: To find out the clinical potential of our finding, we tested the effects of SF1670 a small molecule inhibitor of PTEN on a panel of pre-B ALL and CML. Interestingly, the pre-B ALL showed more sensitivity to PTEN inhibition compared to CML cells.

Conclusion: Our analysis uncovered an unexpected vulnerability of human Pre-B ALL cells toward PTEN inhibition and hyperactivation of the PI3K-AKT pathway, which provides an explanation why activating mutations of this pathway are negatively selected in human pre-B ALL. Since short (few hours) and profound inhibition of Pten is sufficient to commit human pre-B ALL cells to cell death, transient inhibition of this pathway or transient hyperactivation of PI3K-AKT may be a promising strategy to target multi-drug resistant human ALL.