ALBERT

Description: As a result of the t(9;22), more than 95% of CMLs and 20–25% of adult ALLs express the p210(BCR-ABL) or the p185(BCR-ABL) fusion protein respectively. The BCR portion of the fusion protein harbors an N-terminal coiled-coil (CC) domain. The CC contains two alpha helical motifs and assembles to dimers with antiparallel orientation. Two of these dimers associate and lead to the tetramerization of BCR. The second alpha-Helix (Helix-2) contributes the majority of the dimer and tetramer interface. In the case of the BCR/ABL fusion protein, the BCR mediated tetramerization of ABL leads to the constitutive activation of the ABL kinase domain. The subsequent permanent activation of multiple downstream signaling pathways induces the leukemic phenotype. Targeted inhibition of BCR/ABL by the ABL kinase inhibitor Imatinib leads to complete remission in CML and ALL patients. However, a large portion of ALL and CML patients in blast crisis become resistant to Imatinib mainly by the acquisition of point mutations in BCR/ABL. Imatinib binds to the inactive conformation of the ABL-kinase which in case of the BCR/ABL fusion protein is present in monomers. We have previously shown that CC-derived peptides that interfere with BCR/ABL tetramer formation reduce the kinase activity of BCR/ABL in vivo and increase the sensitivity of BCR/ABL expressing cells towards Imatinib. Here we investigated the effects of peptides derived from the CC subdomain Helix-2 on BCR/ABL oligomerization and on the biology of cells expressing Wt BCR/ABL and Imatinib-resistant mutants. First we confirmed the interaction between Helix-2 and BCR/ABL by GST-pull-down and co-immunoprecipitation assays. The influence of Helix 2 peptides on the BCR/ABL oligomerization and its capacity to form high-molecular weight (HMW) complexes in vivo was analyzed by size-exclusion HPLC. The effect of Helix 2 on the leukemogenic potential Helix of BCR/ABL was investigated in retrovirally transduced murine IL-3 dependent Ba/F3 cells and human Philadelphia Chromosome (Ph) negative ALL-cell line Nalm-6 and Ph-positve SupB15 cells. Ba/F3 cells transduced with Imatinib-resistant BCR/ABL point mutations were used to investigate the inhibitory potential of CC specific peptides on mutant BCR/ABL in presence and absence of Imatinib. Here we report that CC-specific Helix-2 peptides interact with Wt and mutant BCR/ABL; decrease the autophosphorylation of BCR/ABL in transduced Ba/F3 cells; disrupt the HMW-complexes of Wt and mutant BCR/ABL; abrogate the growth of human Ph-positive expressing SupB15 cells without affecting Ph-negative Nalm-6 cells; v) increase the Imatinib sensitivity of IL-3 independent Ba/F3 cells expressing BCR/ABL; reduce the Imatinib resistance of Ba/F3-cells expressing BCR/ABL P-loop mutants Y253F and E255K. Taken together these results show that the disruption of BCR/ABL tetramerization has strong inhibitory effects on BCR/ABL transformed cells. Furthermore our results indicate that disruption of BCR/ABL tetramerization is a therapeutic option for the treatment of patients harboring Imatinib-resistant BCR/ABL mutations.

Description: The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation. The APL is a well characterized subtype of AML, which is related in the 95% of the cases by the presence of the t(15;17) and in 2% by the presence of t(11;17). In several cell models the resulting PML/RAR and PLZF/RAR fusion proteins (X-RAR) recapitulate the leukemic phenotype by inducing a state of refractoriness to various inducers of myeloid differentiation. Accordingly, expression in animal models of both PML/RAR and PLZF/RAR leads to the development of leukemia. The treatment with all trans retinoic acid (t-RA) is able to overcome the block of differentiation of PML/RARα- but not that of PLZF/RAR-positive-blasts. These fusion proteins block differentiation through several mechanisms such as aberrant chromatin modeling by aberrant recruitment of histone deacetylase activity or the deregulation of differentiation-relevant transcription factors such as PU.1, VDR or C/EBPalpha. The deregulated function of these transcription factors can be due to their transcriptional down-regulation or to a sequester by direct interaction. Nothing is known about how the X-RAR block erythroid and megacaryocytic differentiation. Therefore we investigated whether and how the X-RAR interfere with the functionality of the differentiation-relevant transcription factor GATA-1. It has been recently reported that the lack of GATA-1 severely impairs erythroid differentiation and contributes to the accumulation of immature megakaryocytic blasts. He we report thatGATA-1 directly interacts with X-RAR in vivo as revealed by co-immunoprecipitation and mammalian two hybrid assays;GATA-1 expression was not transcriptionally deregulated by the X-RAR;the GATA-1 binding capacity to the H2S beta-globin locus was severely inhibited by the presence of the X-RAR as revealed by ChIP experiments in K562, whereas the transactivation of the GATA-target promotor alpha-IIb was not impaired by the the X-RAR in classical transient promoter studies;treatment with t-RA restored GATA-1 binding to the H2S locus of the beta-globin gene;the overexpression of GATA-1 in the presence of EPO reduced the the colony forming units of PLZF/RAR-positive Sca1+/lin− hematopoietic stem cells (HSC) and diminished the replating efficiency of PML/RAR-positive HSC, but did not increase erythroid differentiation monitored by TER 119 expression. Taken together our data demonstrate that the X-RAR interfere with the functionality of GATA-1 by direct interaction with GATA-1. It remains to definitively clarify whether the X-RAR inhibit the access of GATA-1 to its target promoters or whether they interfere with the accessibility of the GATA-1 for transcriptional co-activators. The fact that the overexpression of GATA-1 did not increase differentiation of HSC and the X-RAR were unable to inhibit the transactivation of a GATA-1 target promoter strongly suggests that the X-RAR interfere with the GATA-1 functionality in the context of the chromatin.

Description: In 95% of chronic myeloid leukemia (CML) and in 25% of acute lymphatic leukemia (ALL) the t(9;22) translocation fuses the bcr gene on chromosome 22 to the abl gene on chromosome 9 and vice versa. On 22+ the different breakpoints leads to the formation of two different major fusion genes: the major breakpoint (M-bcr) related to CML and the minor (m-bcr) related to ALL. The chimaeric fusion gene on 22+ (Philadelphia-chromosome) encodes for the BCR/ABL protein, the p210(BCR/ABL) in CML and the p185(BCR/ABL) in Ph+ALL. The fusion gene on 9+ encodes for the reciprocal ABL/BCR proteins, the p40(ABL/BCR) in CML and the p96(ABL/BCR) in Ph+ALL. The respective ABL/BCR transcripts are detectable in 65% of CML and 100% of Ph+ ALL patients. The ABL/BCRs are BCR mutants and thus N-terminally truncated Rho-guanine-nucleotide exchange factors (Rho-GEF’s). It is known that the N-terminal truncation can confer transformation potential to Rho-GEFs, such as NET-1. In addition, both ABL/BCRs, like wt BCR, contain a C-terminal Rac-GTPase activating protein (GAP)-domain. CML-associated ABL/BCR (p96(ABL/BCR)) differs from the Ph+ ALL-associated p40(ABL/BCR) in that that it misses the ‘dbl homology domain’(DH domains) of potential oncogenic function. Hence it seems that Ph+ALL blasts, in contrast to CML-blasts, express, as a consequence of t(9;22) translocation, two oncogenic fusion proteins, the p185(BCR-ABL) as well as the p40(ABL/BCR) protein. Actually nothing is known about the contribution of the reciprocal t(9;22) translocation products to the CML- and the ALL-phenotype. Thus we studied the phenotype induced by the ABL/BCRs in hemopoietic progenitors. Here we report that both ABL/BCRs i) lost the capacity of wt BCR to suppress the activation of RAC by its Rac-GAP domain, but did not influence the activation status of Rho or cdc42; ii) as a consequence of the deregulation of Rac the cytoskeleton modelling by BCR (Filopodia - cdc42-like phenotype) was altered in p40(ABL/BCR)- and p96(ABL/BCR)-expressing fibroblasts (stress fibers - Rho-like phenotype and “microspikes”, respectively); iii) the increase of migration of BCR-expressing 32D cells into a stroma cell-spheroid model was reverted in p40(ABL/BCR)- and p96(ABL/BCR)-expressing 32D cells; iv) adhesion to TNFalpha activated endothelial cell layer in the “flow chamber” was increased in BCR-positive 32D cells but not in p40(ABL/BCR)- and p96(ABL/BCR)-positive cells. Regarding their leukemogenic potential we showed that i) both ABL/BCRs, in contrast to wt BCR, activated RAS; ii) both ABL/BCRs were unable to transform fibroblasts and to render Ba/F3 cells factor-independent. iii) p96(ABL/BCR) increased the replating efficiency of Sca1+/lin- hemopoietic stem cells (HSC) by selecting a population of immature HSC exclusively expressing c-kit and Sca-1 more strongly than p40(ABL/BCR); iv.) both ABL/BCR blocked the myeloid differentiation of HSC v) the inoculation of p96(ABL/BCR)- or p40(ABL/BCR)-expressing HSC into lethally irradiated recipient mice led in the 40% and 60% of the cases, respectively, to a clinical picture of either acute leukemia or myeloproliferative syndrome within 2–9 months. These data show for the first time that the t(9;22) leads to two leukemogenic fusion proteins - the BCR/ABL and the ABL/BCR - in CML as well as in Ph+ALL, which might represent an additional target for molecular therapy approaches.

Description: Abstract 1316 In acute myeloid leukemia (AML), translocations and the resulting fusion proteins (FPs) such as PML/RAR, AML1/ETO and DEK/CAN represent the leukemia initiating event. t(6;9)(DEK/CAN)-positive AML is classified as a separate clinical entity, because of its early onset and poor prognosis. We recently have shown that DEK/CAN is a leukemia-inducing oncogene, which targets a very small subpopulation of primitive hematopoietic stem cells (HSC) for leukemic transformation. Like other FPs, DEK/CAN also interferes with the epigenetic regulation of transcription by modifying key processes of chromatin modeling such as histone acetylation and methylation as well as DNA methylation. In the DEK/CAN fusion protein, all the chromatin binding domains of DEK are conserved and we recently showed that DEK/CAN is associated to chromatin and strongly interferes with chromatin modeling by inhibiting the decondensation of chromatin and accessibility to transcription. As a “Class 1 mutation”, the oncogenic internal tandem duplication (ITD) of the receptor tyrosine kinase Flt3 (Flt3-ITD) is found in 88% of the t(6;9)-positive AML-patients, which otherwise is present in about 30% of other AML cases. The simultaneous presence of Flt3-ITD and DEK/CAN in AML is correlated with a high WBC and significantly lower rates of complete remission. Aim of the study was to determine the effect of Flt3-ITD on the DEK/CAN-induced leukemic phenotype. Therefore we expressed Flt3-ITD and DEK/CAN from a single vector as p2A fusion protein in order to obtain an equimolar expression of the two proteins. We investigated the capacity to mediate factor-independent growth of the single factors and in combination in the myeloid progenitor cell line 32D upon IL-3 withdrawal. The leukemic phenotype was studied in primary Sca1+/Lin- murine hematopoietic stem and progenitor cells (mHSPC) retrovirally transduced with DEK/CAN, FLT3-ITD and FLT3-ITD-p2a-DEK/CAN. These cells were tested for their differentiation potential in liquid culture, for their serial replating capacity in semi-solid medium, their stem cell capacity in colony-forming unit spleen - day12 (CFU-S12) assays, and their potential to induce leukemia in sublethally irradiated recipients. Here we show that I.) Flt3-ITD mediated factor-independent growth alone and in presence of DEK/CAN, but the onset of factor-independent growth was delayed by DEK/CAN; II.) FLT3-ITD did not influence the differentiation potential of DEK/CAN-positive HPSCs; III.) Flt3-ITD increased the colony-number of DEK/CAN-positive, but not the overall serial replating efficiency of DEK/CAN-positive HPSCs; IV.) FLT3-ITD accelerated and increased efficiency of leukemia induction by DEK/CAN in vivo, without modifying either the morphological or the immunological phenotype of DEK/CAN-induced leukemia. Finally we investigated whether FLT3-ITD influences the known capacity of histone-deacetylase (HDAC) inhibitors (HDACi) to revert the leukemogenic potential of DEK/CAN. Therefore we employed a xenograft model based on the patient derived FKH-1 cell line expressing both FLT3-ITD and DEK/CAN. We found that exposure to the HDACi Dacinostat prevented the leukemia-induction in this model. Taken together these findings strongly suggest that DEK/CAN drives the transformation of immature HPSCs which is supported by the presence FLT3-ITD regarding proliferation, without strong effects on the leukemic phenotype induced by DEK/CAN. Disclosures: Bug: Novartis Oncology: Honoraria, Travel grants Other.

Description: Histone deacetylase inhibitors (HDI) have attracted considerable attention because of their ability to overcome the differentiation block in leukemic blasts either alone, or in combination with differentiating agents such as all-trans retinoic acid (ATRA). We have previously reported favorable effects of the potent HDI valproic acid (VPA) in combination with ATRA in a small subset of patients with advanced acute myeloid leukemia (AML) leading to blast cell reduction and improvement of hemoglobin. This effect was accompanied by hypergranulocytosis most likely due to an enhancement of non-leukemic myelopoiesis and suppression of malignant hematopoiesis rather than enforced differentiation of leukemic cells. These data prompted us to investigate the impact of VPA on normal hematopoietic stem cells (HSC). Differentiation of cord blood-derived, purified CD34+ cells was assessed by FACS analysis after a 7-days suspension culture in presence of early acting cytokines and 30–150μg/mL VPA. VPA prevented differentation of CD34+ cells in a dose-dependent manner: concomitant with an increase of CD34+ cells from 17 to 47%, the proportion of monocytic CD14+ cells decreased from 27 to 3% (n=3). In addition, VPA induced a 30-fold amplification of CD34+ bone marrow (BM) cells within 10 days as determined by colony assays (n=3). To evaluate the functional capacity of VPA-treated HSC, murine Sca1+/lin−s cells were harvested from colony assays and replated. VPA treatment allowed up to four cycles of replating in contrast to VPA-naïve control cells. Further analysis demonstrated that the stimulatory effect of VPA on the in vitro growth and colony formation capacity of HSC was mainly due to accelerated cell cycle progression. VPA strongly increased the proportion of cells in S phase compared to untreated controls (38 vs. 17%, resp.), as detected by propidium iodid staining and BRDU incorporation as well as reduced expression of the CDK-inhibitor p21cip-1/waf using murine HSC after 7 days of culture. Downregulation of p21cip-1/waf was confirmed in CD34+ BM cells showing maximum inhibition after 48 hours of VPA treatment and no recovery thereafter. Recent results indicate that VPA exerts inhibitory activity on GSK3beta by phosphorylation on Ser-9 and stimulates Akt in human neuroblastoma cells. GSK3beta is an effector of the Wnt-signaling pathway located upstream of beta-catenin. Wnt-signaling can directly stimulate the proliferation of HSC, expand the HSC pool and lead to upregulation of HoxB4. Here we show that VPA increased the inhibition-associated phosphorylation of GSK3beta on Ser-9 in human CD34+ BM cells after 48 hours as well as in murine Sca1+/lin− cells after 7 days. Exposure to VPA enhanced beta-catenin and Akt activity not only in CD34+ HSC but also in KG-1 and TF-1 cells with maximum activation after 48 hours of VPA stimulation. Moreover, VPA lead to an 8-fold increase of the HoxB4 level in CD34+ BM cells as determined by real time PCR at 48 hours. In conclusion, we show that VPA i.) expands HSC as assesed by phenotype and function; ii.) accelerates cell cycle progression of HSC accompanied by the down-regulation of p21cip-1waf; iii.) activates the GSK3beta depending beta-catenin pathway and Akt and iv.) up-regulates HoxB4. Our data strongly suggest that VPA is able to influence some of the signaling pathway considered relevant for proliferation and self-renewal which might request reconsideration of their employment for the treatment of AML.

Description: Abstract 2953 Poster Board II-929 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. t(6;9)-positive AML is classified as a separate entity, because of its young age of onset and poor prognosis. The t(6;9) associated fusion protein is DEK/CAN. Assuming that in AML the genetic aberration, here the t(6;9) and the expression of DEK/CAN, represents the initiation event of the leukemogenic process we wanted i.) to disclose its effects on the biology of primitive hematopoietic stem cells (HSC) and its leukemogenic potential and ii.) to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that i.) DEK/CAN induced leukemia from the Sca1+/lin− HSC with a frequency of 20% and a long latency of 8-12 months. ii.) DEK/CAN did not efficiently block the differentiation of committed progenitors; iii.) DEK/CAN increased number of colony forming cells in Sca1+/lin− HSC which did not exhibit increased replating efficiency as compared to controls; iv.) DEK/CAN augmented ST-HSC potential but not LT-HSC of murine Sca1+/lin− HSCs, most likely due to its incapacity to up-regulate p21Cip1/Waf1 expression. Based on the hypothesis that DEK/CAN exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare DEK/CAN-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed DEK/CAN in Sca1+/lin− cells and enriched this population for LT- (lin−/Sca1+/c-Kit+/Flk2−) and ST-HSC (lin−/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. We here report that i.) after 4 to 42 weeks, 6/6 mice developed AML without signs of differentiation in the group transplanted with the lin−/Sca1+/c-Kit+/Flk2− population but not from that transplanted with lin−/Sca1+/c-Kit+/Flk2+ cells; ii.) the DEK/CAN-induced AML was efficiently transplanted into secondary recipients exhibiting a very aggressive clinical picture; iii.) the leukemic cell population gave origin to four different clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, all able - after sorting - to give leukemia in sublethally irradiated recipients: lin−/Sca1+/c-Kit+/CD34− (LT) lin−/Sca1+/c-Kit+/CD34+ (ST), Sca1−/c-Kit+/Mac1+/Gr1+, Sca1−/c-Kit+/Mac1−/Gr1−. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine DEK/CAN leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

Description: Central nervous system (CNS) relapse accompanying the prolonged administration of imatinib mesylate has recently become apparent as an impediment to the therapy of Philadelphia chromosome–positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib mesylate into the cerebrospinal fluid because of the presence of P-glycoprotein at the blood-brain barrier. To overcome imatinib mesylate–resistance mechanisms such as bcr-abl amplification, mutations within the ABL kinase domain, and activation of Lyn, we developed a dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25 to 55 times more potent than imatinib mesylate in vitro and at least 10 times more potent in vivo. The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. We found that INNO-406, like imatinib mesylate, is a substrate for P-glycoprotein. The concentrations of INNO-406 in the CNS were about 10% of those in the plasma. However, this residual concentration was enough to inhibit the growth of Ph+ leukemic cells which expressed not only wild-type but also mutated BCR-ABL in the murine CNS. Furthermore, cyclosporine A, a P-glycoprotein inhibitor, augmented the in vivo activity of INNO-406 against CNS Ph+ leukemia. These findings indicate that INNO-406 is a promising agent for the treatment of CNS Ph+ leukemia.

Description: Leukemia-specific translocations such as t(15;17), t(11;17), or t(8;21) lead to the expression of aberrant fusion proteins (FP) such as PML/RAR, PLZF/RAR and AML-1/ETO. These FP induce and maintain the leukemic phenotype by blocking terminal differentiation of early hematopoietic progenitors and by interfering with the biology of the leukemic counterpart of the hematopoietic stem cells. The t(6;9) with its DEK/CAN FP is of particular interest because it is more frequent in young patients and associated with a poor prognosis. The t(6;9)-DEK/CAN fusion occurs with an incidence of 1–5% in adult patients with AML and in most of the cases t(6;9)-positive AML is classified as FAB-M2 or M4. In contrast to other FP the overexpression of DEK/CAN does not block VitD3-induced differentiation in U937 cells. These findings called in question the leukemogenic potential of DEK/CAN. To further disclose the role of DEK/CAN in leukemogenesis we investigated its effect on the biology of early hematopoietic stem cells (HSC) in comparison to PML/RAR. Therefore we retrovirally transduced Sca1+/linmurine HSC (SL cells) with DEK/CAN and PML/RAR and performed both differentiation and stem cells assays (morphology, surface marker expression, replating efficiency, colony forming unit - spleen - CFU-S). Here we show that i.) DEK/CAN in contrast to PML/RAR was apparently unable to block terminal differentiation of SL cells as revealed by morphology and the expression of differentiation-specific surface markers; ii.) DEK/CAN slightly increased the replating efficiency of SL cells, but did not reach the level of PML/RAR with a reduced number of either colony forming units as plating rounds with respect to PML/RAR; iii.) the increased replating efficiency was related to an increased stem cell capacity as revealed by the fact that in contrast to mock infected control cells DEK/CAN-positive cells gave origin to a positive CFU-S assay even after one or two plating rounds in semi-solid medium similar to PML/RAR. Apparently DEK/CAN seems to share with other FP the capacity to increase the self renewal of HSC, but not that to block terminal differentiation. These findings strongly suggest that the effect of DEK/CAN is limited to a very small subset of cells within the stem cell compartment which might represent the origin of a DEK/CAN-positive leukemia. Actually the capacity of DEK/CAN to give origin to leukemia in vivo is under investigation.

Description: Abstract 2579 Chronic myelogenous leukemia (CML) and Philadephia chromosome-positive acute lymphatic leukemia (Ph+ ALL) is caused by the t(9;22) which fuses the BCR to ABL resulting in a deregulated tyrosine kinase activity. ABL Kinase inhibitiors (AKI) such as imatinib, are effective in the early stage CML, but in advanced stages patients relapse as a result of point mutations within the BCR/ABL. However a certain group of resistant patients do not have point mutations, which can explain the resistance. The mechanisms of resistance remain often unknown. CD133 (PROMININ-1 or PROM-1; in the mouse: Prom-1) is a novel hematopoietic stem/progenitor cell (HSPC) marker, but its biological function is nearly unknown. Its expression is found on various leukemic cells. It has been shown that PROM-1-negative colon cancer-initiating cells were more aggressive than PROM-1-positive cells, suggesting that cancer-initiating cells are in fact PROM-1-negative. We examined the role of PROM-1 in the normal and BCR/ABL-induced malignant haematopoiesis in Prom-1−/− in comparison to Prom+/+ murine HSPCs. Our results suggest that PROM-1 plays an important role in the induction of the BCR/ABL related leukemic phenotype. BCR/ABL induced a significantly higher colony number in Prom−/− as compared to Prom+/+ HSPCs in factor independent CFU-Assays, which did not respond to 1μM Imatinib. In fact in comparison to Prom +/+ HSPCs Prom −/− HSPCs exhibited in presence as well as in absence of BCR/ABL a different response to Imatinib characterized by a significantly increase of immature c-Kit and Sca-1-positive cells. Furthermore in a transduction/transplantation model, BCR/ABL induced a significantly higher rate of ALLs (50%) in the Prom−/− than in Prom+/+ background, where only CML-like diseases were seen. Based on these results we studied the role of PROM-1 for the non-mutational resistance in Ph+ ALL. We investigated the expression of PROM-1 in different non mutational resistance models of Ph+ ALL among the patient derived cell lines (SupB15 and Tom-1) as well as 7 primary long term cultures derived from patient with Ph+ ALL (PDLTCs). These PDLTCs exhibit several grades of resistance against established ABL-kinase inhibitors (AKI). Furthermore there is a cross resistance against other molecular therapy approaches, such as allosteric inhibition by GNF-2 or oligomerization inhibition by competitive peptides. In these cells there was a direct relationship between PROM-1 expression and response to AKIs. In fact sensitivity to AKIs increased with the expression of PROM-1. In these PDLTCs targeting PROM-1 by siRNA reduced and the over expression of PROM-1 increased the response to AKIs. Interestingly, also the only PDLTC harboring the BCR/ABL-T315I was PROM-1 negative. Furthermore the induction of resistance by increasing concentrations of either Imatinib or Nilotinib in the Ph+ ALL line SupB15 was accompanied by a progressive reduction of PROM-1 expression on the surface (CD133). In summary, our data show for the first time the importance of PROM-1/CD133 for the determination of the leukemic phenotype and as a potential marker for resistance to AKIs in Ph+ ALL, which is actually under examination in cohorts of Ph+ ALL patients with non mutational resistance. Disclosures: No relevant conflicts of interest to declare.

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.