ALBERT

Description: Abstract 3236 Bcr-Abl is a leukemogenic fusion gene that by itself is sufficient for cellular transformation (Daley et al.) and is the hallmark of chronic myeloid leukemia and Philadelphia chromosome positive (Ph+) ALL. The Bcr-Abl fusion protein is a constitutively active tyrosine kinase (TK) which disrupts multiple cellular signalling pathways controlling apoptosis, cell cycle, proliferation and DNA repair. In Ph+ ALL, a subtype of ALL with a particularly poor prognosis, targeted inhibition of Bcr-Abl activity by Abl kinase inhibitors such as imatinib has improved treatment outcome but has not abrogated the frequent development of clinical resistance. In addition to mutations in the Bcr-Abl tyrosine kinase domain (TKD), it has become apparent that other resistance mechanisms contribute to disease progression. The activity of proteins involved in the above-mentioned signalling pathways and possibly resistance to TK inhibitors (TKI) is controlled at least partially by posttranslational modifications such as phosphorylation, which is regulated by the balance between kinases and protein tyrosine phosphatases (PTP). We previously showed that PTP1B is a negative regulator of Bcr-Abl-mediated transformation and modulates sensitivity to the TKI imatinib (Koyama et al). We hypothesized that other phosphatases for which Bcr-Abl is a substrate may also contribute to resistance, one candidate being Suppressor of T-cell receptor Signalling 1 (STS-1), which negatively regulates the endocytosis of receptor TK involved in a variety of hematologic malignancies. It was the aim of this study to determine whether: i) Bcr-Abl is a substrate of STS-1 ii) STS-1 is able to dephosphorylate Bcr-Abl iii) expression of STS-1 reduces the proliferation of Bcr-Abl expressing cells by inhibiting Bcr-Abl kinase activity iv) the level of STS-1 expression modulates the sensitivity of Bcr-Abl positive cells to TKI In order to answer these questions, we used 293T cells, a human primary embryonal kidney cell line, and the IL3-dependent murine pro B cell line Ba/F3. Both cell lines were modified with constructs encoding both forms of Bcr-Abl (p185/p210) and Sts-1. For experiments with endogenous Bcr-Abl (p185) and Sts-1 we used Sup B15 cells, a human B cell precursor leukemia, and its TKI-resistant subline (Sup B15 RT), which was generated in our lab and is highly resistant not only to imatinib but also to 2nd generation TKIs (Nilotinib & Dasatinib), with no evidence of TKD mutations or transcriptional up-regulation of Bcr-Abl. In all above described cell lines the interaction between Bcr-Abl and Sts-1 could be shown in an overexpressed system (293T & Ba/F3) and on an endogenous level (Sup B15 & Sup B15 RT) by using co-IPs followed by SDS-PAGE and Western blotting. The functional relevance was examined by testing the ability of Sts-1 to dephosphorylate Bcr-Abl. Complete dephosphorylation of Bcr-Abl was shown for p185bcr-abl and p210bcr-abl in 293T cells. To verify that the functional activity was also present in hematopoietic cells, we analyzed the ability of Sts-1 to dephosphorylate Bcr-Abl in Ba/F3 and Sup B15 cells. Dephosphorylation was observed in both cell lines but was less pronounced than in 293T cells. We therefore more closely examined the most important tyrosine (Tyr) residues of Bcr-Abl and identified Tyr245 and Tyr412 as the major targets of Sts-1. Phosphorylation of Tyr245 and Tyr412 was decreased by ∼60% in Ba/F3 cells and ∼39% in Sup B15 cells. These two residues are known to be important for regulating cell proliferation, survival and cell motility. In a competitive proliferation assay in the absence of IL3, the proliferation rate of BA/F3 cells infected with Bcr-Abl and Sts – 1 was reduced compared to a Bcr-Abl infected control population. When treated with imatinib the Sts-1 expressing cells showed an approximately 5-fold reduced proliferation rate compared to cells lacking Sts-1, or to imatinib-resistant cells harbouring the Bcr-Abl “gatekeeper mutation” T315I. The expression level of Sts-1 was found to be approximately 3-fold lower in the Sup B15 RT compared to the WT cell line. Regulation appeared to occur at the transcriptional level as shown by quantitive RT-PCR. These results show that Bcr-Abl is a substrate of Sts-1, that this phosphatase modulates Bcr-Abl kinase activity and may abrogate the response to TKI. This suggests that phosphatases may contribute to the development of clinical resistance of Ph+ leukemias to TKIs. Disclosures: Ottmann: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

Description: Abstract 1032 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. Disclosures: Ottmann: Novartis: Consultancy, Honoraria, Research Funding; BMS: Honoraria, Research Funding.