The PI3K/AKT/mTOR pathway is a major downstream signaling pathway of the bcr-abl oncogene that is the hallmark of Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (Ph+ ALL) and of CML. Ph+ ALL is a subtype of ALL with a particularly poor prognosis despite the availability of tyrosine kinase inhibitors (TKI) that effectively suppress BCR-ABL kinase activity. Resistance of Ph+ ALL to TKI has been suggested to involve activation of the PI3K signaling pathway, which has also been shown in several other hematologic malignancies to contribute to leukemogenesis and disease progression. Its role in ALL subtypes other than Ph+ ALL has not been clearly established. Moreover, the relative contributions of the individual components of the PI3K/AKT/mTOR signaling pathway to leukemogenesis remain to be resolved. mTOR is a serine/threonine kinase and catalytic subunit of the two biochemically distinct complexes mTORC1 and mTORC2. mTORC1 controls cell growth in response to nutrients and growth factors, whereas mTORC2 is thought to mediate cell proliferation and cell survival. AKT activates mTORC1, which promotes cell growth in part by directly phosphorylating the translational regulators S6K1 and 4E-BP1. Linking mTORC1 regulation to oncogenic PI3K activity provided strong rationale for targeting mTORC1 in cancer, but the effectiveness of targeting mTORC1 is mitigated by strong, mTORC1-dependent negative feedback loops that become inactive on mTORC1 inhibition. mTORC2 directly phosphorylates AKT on a critical regulatory site required for maximal AKT kinase activity. This prompted efforts to develop mTOR inhibitors that target both complexes.
We compared the effects of selective inhibitors of PI3K (NVP-BKM120) and mTORC1 (RAD001) with those of dual PI3K/mTORC1/C2 inhibitors (NVP-BEZ235 & NVP-BGT226) on Ph+ and Ph neg. B-precursor ALL. Long-term serum-free cultures of primary human Ph+ B-ALL (n=6) and Ph- B-ALL (n=6) cells were exposed to increasing concentrations of these inhibitors (NVP-BKM120 (50nM-10uM), RAD001 (5nM-20uM), NVP-BGT226 (1nM-500nM), NVP-BEZ235 (10nM-1uM). All inhibitors were kindly provided by Novartis, Basel, Switzerland. Some of the Ph+ ALL cells are partially resistant to 1st and 2nd generation TKI. Cell proliferation and apoptosis were monitored by XTT-assays and FACS analysis using annexin V/propidium iodide. Phosphorylation of the proteins 4E-BP1 (Thr37/46) & S6 Ribosomal Protein (Ser235/236) downstream of mTOR was assessed by Western Blotting in a time and concentration dependent manner.
In both Ph+ ALL and Ph- ALL, inhibition of PI3K activity by BKM120 suppressed proliferation and induced apoptosis at high nanomolar (IC50≤1μM) and low micromolar (IC50≤5μM) concentrations, respectively. Inhibition of only mTORC1 by RAD001 slightly inhibited proliferation, but failed to induce apoptosis. Combined inhibition of PI3K and both mTOR complexes mTORC1/C2 by NVP-BEZ235 or NVP-BGT226 resulted in a significantly more pronounced suppression of cell growth (BEZ235 (IC50≤200nM), BGT226 (IC50≤20nM) and induction of apoptosis at nanomolar concentrations (BEZ235 (IC50≤250nM), BGT226 (IC50≤25nM)) as compared to both selective inhibitors (NVP-BKM120 and RAD001). The anti-proliferative and pro-apoptotic effects of these inhibitors was independent of bcr-abl status. Comparison of the effect of selective PI3K and mTOR inhibitors on mTOR signaling revealed differential regulation of S6 and 4E-BP1. Whereas selective inhibition of PI3K and mTORC1 by BKM 120 and RAD001, respectively, resulted in dephosphorylation only of the S6 protein, combined inhibition of PI3K and mTORC1 was associated primarily with a decrease of S6 phosphorylation and only minor dephosphorylation of 4E-BP1. On the other hand, exposure to the dual PI3K/mTORC1/C2 inhibitors resulted in nearly complete dephosphorylation of both S6 and 4E-BP1.
These data indicate that in ALL, mTORC2 contributes substantially to regulation of the downstream target 4E-BP1 by mTORC1. Our observation that compounds inhibiting PI3K and both mTOR complexes (mTORC1/mTORC2) have significantly greater growth inhibitory and pro-apoptotic effects than selective inhibition of PI3K and mTORC1 support a functional role of mTORC2 in survival and growth of B-precursor ALL cells. Combined targeting of these complexes may provide a novel therapeutic approach for both Ph+ ALL resistant to ABL TKI and Ph- ALL.
Ottmann:Novartis: Consultancy, Honoraria, Research Funding; BMS: Honoraria, Research Funding.
Asterisk with author names denotes non-ASH members.