Abstract
Abstract 513
Background:
The phosphatase and tensin homolog (PTEN) tumor suppressor is a negative regulator of PI3K/AKT signaling and frequently deleted in solid tumors and in T cell lineage acute lymphoblastic leukemia (ALL). Recent work by our group demonstrated that Pten-deletion cooperates with WNT/β-catenin signaling in self-renewal signaling of leukemia stem cells in T cell lineage ALL (Guo et al., Nature 2008). Although PTEN deletions are uncommon in B cell lineage ALL and chronic myeloid leukemia, recent work identified Pten as a tumor suppressor in B cell lineage ALL and chronic myeloid leukemia (CML; Peng et al., Blood 2010). This study showed that Pten-deletion accelerated leukemia in B cell lineage ALL and CML and increased self-renewal capacity of leukemia stem cells.
Approach:
To investigate the mechanism of Pten-mediated negative regulation of leukemia proliferation and stem cell self-renewal in B cell lineage ALL and CML, we established a conditional mouse model for inducible ablation of Pten in BCR-ABL1-driven B cell lineage ALL and CML. To this end, B cell precursors or Lin− Sca-1+ c-Kit+ (LSK) cells from the bone marrow of Pten-fl/fl mice were transduced with retroviral BCR-ABL1 under B lymphoid or myeloid conditions. When growth-factor-independent B cell lineage or CML-like leukemia formed, Pten-fl/fl leukemia cells were transduced with tamoxifen (4-OHT)-inducible Cre-ERT2 or an ERT2 empty vector control.
Results:
Deletion of Pten was induced by 4-OHT treatment and near-complete deletion of Pten was observed on day 2 after Cre-ERT2 induction as determined by genomic PCR and Western blot. As expected, deletion of Pten resulted in a strong increase of phospho-AKT, as determined by Western blot. Surprisingly, deletion of Pten in B cell lineage leukemia and CML-like disease did not accelerate leukemia cell growth and had the opposite effect. After three days of Cre-induction, the vast majority of both B cell lineage and CML-like leukemia cells underwent cellular senescence, as measured by staining for senescence-associated β-galactosidase activity (>100-fold increase; p=0.0008). In addition, Pten-deletion induced cell cycle arrest in both G0/G1 and G2/M phase of the cell cycle, which is consistent with cellular senescence. While growth kinetics and viability of Pten-fl/fl leukemia cells carrying the ERT2 empty vector control remained unchanged, Pten-deletion caused a reduction of viability by 80% within 6 days. Compared to B cell lineage leukemia and CML-like leukemia, Pten protein levels were very low in normal B cell precursors and myeloid progenitor cells and the consequences of Pten-deletion were less drastic in normal as compared to leukemia cells. Consistent with cellular senescence, deletion of Pten resulted in dramatic upregulation of p53 and p21 but not p27 cell cycle inhibitors. We recently identified BCL6 as a FoxO-dependent suppressor of p53 in BCR-ABL1-driven leukemias (Duy et al., J Exp Med 2010). Since Pten functions as a positive regulator of FoxO1, FoxO3A and FoxO4, we tested whether excessive upregulation of p53 and cellular senescence were a consequence of loss of BCL6/FoxO function downstream of Pten-deletion. Treatment of BCR-ABL1 B cell lineage and CML-like leukemia cells with Imatinib results in strong upregulation of BCL6 expression downstream of FoxO factors. Upon deletion of Pten, however, Imatinib-treatment failed to upregulate BCL6. In the absence of Pten, BCL6 protein expression was undetectable and p53 protein levels were excessively increased.
Conclusion:
Pten has been extensively studied as a tumor suppressor in a broad range of malignancies. It is frequently deleted in solid tumors and also in T cell lineage ALL, where Pten-deletion accelerates leukemia cell growth by increased PI3K/AKT survival signaling. In B cell lineage and CML-like leukemia, Pten deletion also increases PI3K/AKT signaling. Unlike T-ALL, however, B cell lineage leukemia and CML cells undergo cellular senescence and cell cycle arrest. In B cell lineage ALL and CML, the BCL6 transcriptional repressor is required to overcome p53-dependent senescence. Here we unexpectedly identify the PTEN phosphatase as a central requirement for FoxO-dependent upregulation of BCL6. Hence, PTEN signaling via FoxO/BCL6 is required for the ability of the BCR-ABL1 ALL and CML cells to evade p53-mediated cellular senescence.
Disclosures:
No relevant conflicts of interest to declare.
Temporally distinct roles for tumor suppressor pathways in cell cycle arrest and cellular senescence in Cyclin D1-driven tumor