ALBERT

Description: Background Targeting BCR/ABL by ABL-directed kinase inhibitors (AKIs) induces long lasting remissions in patients with chronic myeloid leukemia (CML), and short remissions in Ph+ acute lymphatic leukemia (ALL). Notably in advanced Ph+ leukemia resistance attributable to either kinase domain mutations in BCR/ABL or non mutational mechanisms remains the major clinical challenge. With the only exception of Ponatinib, a multitargeted kinase inhibitor, all actually approved AKIs are unable to inhibit the „gatekeeper“ mutation T315I. Ponatinib is unable to overcome non mutational resistance in advanced leukemia and has an unfavorable spectrum of undesired side effects, most likely due to the broad spektrum of kinase inhibition. Thus there is the urgent need for further and more selective therapeutical options in treating therapy-resistant advanced Ph+ leukemia. PF114 is an ATP competitor, which was developed focusing on: i.) targeting all known resistance mutations in BCR/ABL but mainly the T315I; ii.) a higher selectivity as compared to Ponatinib in order to reduce undesired side effects; iii.) the ability to overcome also non mutational resistance in advanced Ph+ leukemia; iv.) an activity not only in chronic phase CML but also advanced Ph+ leukemia, Ph+ ALL or blast crisis CML (BC-CML). Methods An toxicity profile of PF-114 as well as its kinase selectivity was investigated. The preclinical evaluation of PF-114 was performed in direct comparison to Ponatinib on golden standard preclinical models of CML and advanced Ph+ leukemia and primary patient-derived long term cultures (PD-LTC) of Ph+ ALL patients. The effects on mutational resistance was investigated i.) on the factor dependence of Ba/F3 cells expressing BCR/ABL or its clinically most relevant resistance mutants (Y253F, E255K, T315I, F317L); ii.) on a PD-LTC of a Ph+ ALL patient harboring the T315I. As models for non mutational resistance we used PD-LTCs from Ph+ ALL patients with different levels of non mutational drug resistance and the SupB15RT, a Ph+ ALL cell line rendered resistant by the exposure to increasing doses of Imatinib and cross-resistant against all approved AKIs. The effects of PF114 in vivo were investigated on the transduction/transplantation model of BCR/ABL- and BCR/ABL-T315I-induced CML-like disease, secondary BCR/ABL-induced murine ALLs as well as on xenografts of PD-LTCs in NSG mice and K562 cells in nude mice. Results PF-114 is an orally available AKI, which is more selective than Dasatinib or Ponatinib (number of kinases inhibited at 100 nM of a drug: Nilotinib - 19; PF-114 - 27; Dasatinib - 48; Ponatinib - 80). It was classified as a class 4 - low-toxic - substance (Hodge/Sterner classification). It efficiently inhibited all tested BCR/ABL mutants in cellular and biochemical assays at dosages of 10-100nM and like Ponatinib it suppressed the up-coming of new resistance mutations in a mutation assay in Ba/F3 cells. It also suppressed growth of Ph+ PD-LTC with non mutational resistance as well as the BCR/ABL-T315I-positve PD-LTC in this dosage range. In all models the effect was independent of the presence of either the p210 or the p185 form of BCR/ABL. No effect of PF114 was seen in PD-LTCs of Ph- ALL. Noteworthy PF-114 (50 mg/Kg) prolonged significantly the survival of mice with both BCR/ABL- and BCR/ABL-T315I-driven CML-like disease as compared to Ponatinib (25 mg/Kg). In all in vivo models of advanced leukemia, PF-114 (50 mg/Kg) significantly inhibited leukemia to a similar extent as Ponatinib (25 mg/Kg). Like Ponatinib, PF-114 was unable to overcome the non mutational resistance in SupB15RT. Conclusions Our work supports clinical evaluation of PF114 as a pan BCR/ABL inhibitor for treatment not only for chronic phase CML, but also for advanced and resistant Ph+ leukemia such as Ph+ ALL or BC-CML. Disclosures: Mian: Fusion Pharma: Research Funding. Zeifman:Fusion Pharma: Employment. Titov:Fusion Pharma: Employment. Stroylov:Fusion Pharma: Employment. Stroganov:Fusion Pharma: Employment. Novikov:Fusion Pharma: Employment. Chilov:Fusion Pharma: Employment. Ruthardt:Fusion Pharma: Research Funding.

Description: Abstract 598 The t(9;22) translocation (Ph+) leads to the formation of the chimeric bcr/abl fusion gene, which encodes the BCR/ABL fusion protein. Depending on the minor and major breakpoint (m-BCR or M-BCR, respectively) on chromosome 22, either the p185-BCR/ABL (p185) or the p210-BCR/ABL (p210) fusion protein is expressed. In contrast to p210, p185 lacks the putatively oncogenic dbl- and pleckstrin homology domains. In the majority of the cases Ph+ ALL patients harbor the p185, but with increasing age the number Ph+ ALL patients with the p210 increases up to an 40%. Only few functional and biological differences between p185 and p210 are known. Both exhibit a constitutively activated kinase activity responsible for the induction of the leukemic phenotype in contrast to their physiological counterpart, c-ABL, whose kinase activity is finely regulated. One reason is that BCR/ABL “escapes” auto-inhibition mechanisms of c-ABL, such as the allosteric inhibition by “capping”. “Capping” is the process by which myristoylation of the N-terminal cap region of c-ABL leads to an auto-inhibited conformation through its binding to the hydrophobic myristoyl binding pocket (MBP) in the kinase domain. The cap region of c-ABL is encoded by the exon I, which is replaced by the N-terminus of BCR in the fusion protein. Myristate “mimicks” and MBP binders, such as GNF-2, aim to restore the allosteric inhibition through the binding to the MBP in BCR/ABL. The aim of our study was to further develop allosteric inhibition in Ph+ ALL. Therefore we investigated the anti-proliferative activity of GNF-2 in different Ph+ leukemia models such as patient derived Ph+ lymphatic cell lines, long term cultures of Ph+ ALL patient derived bone marrow cells (LTC) and factor dependent lymphatic Ba/F3 cells retrovirally transduced with either p185 or p210. Here we report that i.) the IC50 of GNF-2 for p210-positive ALL cells were much lower (cell lines =125nM; LTC from two different patients 100–300nM) than that for p185-positive ALL cells (cell lines=500 nM; LTC from two different patients 500–1000nM); ii.) factor independent growth of Ba/F3 cells expressing p185 was inhibited with an IC50 of 750nM whereas that of Ba/F3 cells expressing p210 was abolished already at 125nM; iii.) 1000nM GNF-2 was able to abolish colony formation in soft agar of p210-positive but not of p185-positive LTC; iv.) increasing concentrations of GNF-2 abolished in p210-positive ALL cells the BCR/ABL kinase activity, whereas in p185-positive cells it only reduced it as shown by the effects of GNF-2 on the autophosphorylation (Y245) and substrate phosphorylation (Crkl) activity as well as on down stream signaling (Stat5) of BCR/ABL. Our here presented data provide first evidence of a differential response of p185- and p210-positive Ph+ ALL cells to the allosteric inhibition by GNF-2, as a expression of functional and biological differences between the two fusion proteins within the same disease entity. It remains to clarify not only the mechanisms of this differential response, such as three dimensional conformation, different kinase activity and related signal transdcution, but also its prognostic significance for patients with p210-positive Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.

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 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: 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.

Description: Acute myeloid leukemias (AML) are characterized by recurrent genomic alterations, often in transcriptional regulators, which form the basis on which current prognostication and therapeutic intervention is overlaid. Three subtypes of AML carrying specific translocations, namely t(15;17), t(11;17) and t(6;9), are notable for being associated with a smaller number of co-existing driver mutations than e.g. AML with normal karyotype. This strongly suggests that the function of their aberrant gene products, PML/RAR and DEK/CAN, respectively, may subsume the functions of other driver mutations. Thus we hypothesized that these functions, while as yet elusive, not necessarily require sequential acquisition of secondary genomic alterations. We elected to study AML with the t(6;9), defined as a distinct entity by the WHO classification, because of its particular biological and high risk clinical features and unmet clinical needs. Most t(6;9)-AML patients are young, with a median age of 23-40 years, complete remission rates do not exceed 50% and median survival after diagnosis is only about 1 year. We used a novel "subtractive interaction proteomics" (SIP) approach to understand the mechanisms by which the t(6;9)-DEK/CAN nuclear oncogene induces this highly resistant leukemic phenotype. Based on Tandem Affinity Precipitation (TAP) for the enrichment of proteins complexes associated with SILAC-technology followed by LC-MS/MS we developed SIP as a comparison between the interactome of an oncogene and those of its functionally inactive mutants in order to obtain eventually only relevant interaction partners (exclusive binders) in the same genetic background. This is achieved by the subtraction of binders that are common to four functionally inactive mutants classifying them as not relevant. Bioinformatic network analysis of the 9 exclusive binders of DEK/CAN revealed by SIP (RAB1A, RAB6A, S100A7, PCBD1, Clusterin, RPS14 and 19, IDH3A, SerpinB3) using BioGrid, IntAct and String together with Ingenuity© Pathway Analysis (IPA), indicated a functional relationship with ABL1-, AKT/mTOR-, MYC- and SRC family kinases-dependent signaling. Interestingly, we found all these signaling pathways strongly activated in an autonomous manner in four DEK/CAN-positive leukemia models, DEK/CAN expressing U937 cells, t(6;9)-positive FKH-1 cells, primary syngeneic murine DEK/CAN-driven leukemias, and t(6;9)-positive patient samples. Bioinformatic analysis of the phopshoproteomic profile of FKH1 cells upon molecular targeting of single pathways (imatinib for ABL1, PP2 for SFKs, dasatinib for ABL1/SFK and Torin1 or NVP-BEZ-235 for mTOR/AKT) revealed that these signaling pathways were organized in clusters creating a network with nodes that are credible candidates for combinatorial therapeutic interventions. On the other hand inhibition of individual outputs had the potential to activate interconnected pathways in a detrimental manner with consequential clinical impact e.g. the activation of STAT5 by the inhibition of mTOR/AKT in these cells. Treatment of mice injected with primary syngeneic DEK/CAN-induced leukemic cells with dasatinib (10mg/kg) and NVP-BEZ-235 (45mg/kg) alone and in combination for 14 days led to a strong reduction of leukemia burden in all cohorts (each cohort n=7). In fact, as compared to untreated controls (146.6 +/- 36mg), mice treated with NVP-BEZ 235 alone and in combination (61.7 +/-4.7mg and 65.3+/- 4.6mg, respectively) showed a statistically significant reduction of spleen size whereas those treated with dasatinib alone (77.5 8 +/- 5.4mg) did not reach statistical significance. Taken together the here presented results reveal specific interdependencies between a nuclear oncogene and kinase driven cancer signaling pathways providing a foundation for the design of therapeutic strategies to better address the complexity of cancer signaling. In addition, it provides evidence for the need of a more in depth analysis of indirect effects of molecular targeting strategies in a preclinical setting not only in AML but in all cancer types. Disclosures Ottmann: Novartis: Consultancy; Pfizer: Consultancy; Fusion Pharma: Consultancy, Research Funding; Amgen: Consultancy; Celgene: Consultancy, Research Funding; Takeda: Consultancy; Incyte: Consultancy, Research Funding.

Description: Abstract 2424 Acute myeloid leukemia (AML) is characterized by an abnormal accumulation of hematopoietic progenitors in the bone marrow (BM). The AML phenotype is induced and maintained by specific chromosomal translocations, such as t(8;21), t(15;17), t(6;9). These leukemia initiating events lead to an accelerated proliferation due to a differentiation block that prevents progenitors from reaching the post-proliferative stage of blast cells. This is supported by the aberrant stem cell capacity of poorly defined leukemic stem cells (LSC). The related AML associated fusion proteins (FPs) such as PML/RARα-t(15;17) (P/R), AML-1/ETO-t(8;21) or DEK/CAN-t(6;9) (D/C) recapitulate the leukemic phenotype in vitro and in vivo. Deregulated activation of the Wnt-signaling by FPs lead to aberrant self renewal of LSC in AML and is fundamental for maintenance of the LSC population. Wnt-signaling can be inhibited by non steroidal anti inflammatory drugs (NSAID, i.e. Sulindac, Indomethacin), mainly dual COX 1/2 inhibitors that in high concentrations, which are relevant for the Wnt-signaling inhibition also target the 5-Lipoxygenase (5-LO). Both COX1/2 and 5-LO are the key enzymes in the arachidonic acid metabolism. COX1/2 are the key enzymes for the synthesis of prostaglandines whereas 5-LO is responsible for the biosynthesis of leukotrienes, a group of pro-inflammatory lipid mediators. Reportedly the loss of 5-LO abolished the LSC of BCR/ABL induced CML-like disease. Therefore we targeted 5-LO by selective inhibitors (CJ 13,610 or Zileuton) and have recently shown that the inhibition of the 5-LO enzymatic activity interferes with the stem cell capacity of both PML/RARα and DEK/CAN positive LSC. In order to definitively show that the effects of the 5-LO inhibitors are related to the inhibition of 5-LO, we extended our studies on P/R- and D/C-positive LSC in a 5-LO−/−background. Here we show that loss of 5-LO expression not only failed to inhibit the stem cell activity of P/R or D/C expressing LSC, but significantly increased the colony number and the replating efficiency of D/C-positive LSC as compared to 5-LO+/+ controls. No significant differences were seen between short-term (ST) or long-term (LT) stem cell capacity of P/R or D/C expressing HSC in 5-LO−/− as compared to a 5-LO+/+ background. These data led us to the conclusion that the inhibition, but not the absence of 5-LO is important for the effect of 5-LO inhibitors on the Wnt-signaling. In order to prove the hypothesis that the inactive 5-LO is the inhibitor of the Wnt-signaling, we expressed 5-LO in U937 cells which are 5-LO-negative. We used U937 P/R9 cells, in which the inducible expression of PML/RAR strongly activates the Wnt-signaling as revealed by the Topflash/Fopflash transactivation system. The expression of 5-LO in absence of leukotriene stimulation reduced the Wnt-signaling activation by P/R to the levels seen in controls. These effects of 5-LO on Wnt-signaling can be explained by the direct interaction between 5-LO and β-catenin as revealed by co-immunoprecipitations in U937 cells. Taken together our data strongly suggest 1.) an important role of 5-LO in the regulation of the Wnt-signaling pathway and thereby for the maintenance of LSC and 2.) that the inhibitory effect of 5-LO inhibitors are not due to the interruption of the 5-LO mediated lipid signaling but to the presence of an inactivated 5-LO protein, which acquires novel functions that are disconnected from the lipid signaling. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1846 Chromosomal translocations such as t(15;17), t(8,21) or t(6;9) lead to the formation of chimeric genes encoding the PML/RAR, AML-1/ETO or DEK/CAN fusion proteins (FP). These FP are able to induce and to maintain acute myeloid leukemia (AML) by both blocking terminal differentiation of early hematopoietic progenitors and increasing the self renewal potential of the leukemic stem cells (LSC). LSCs are potential therapeutic targets and it is of great importance to elucidate which signaling pathways control their development and maintenance. Recently it has been shown that the presence of the 5-Lipoxygenase activity (5-LO) is indispensable for the induction and the maintenance of the BCR/ABL induced CML-like disease in mice. Its depletion or inhibition impairs the LSCs in the CML-like disease. 5-LO is the key enzyme in the biosynthesis pathway of leukotrienes, a group of proinflammatory lipid mediators derived from arachidonic acid. Furthermore we have shown that Sulindac sulfide, a dual Cycloxygenase/5 –LO inhibitor, was able, at 5-LO inhibitory concentrations, to interfere with the stem cell capacity of PML/RAR-positive LSC. It also overcame the differentiation block in PML/RAR-positive HSC. To disclose whether a “leukemic stem cell therapy” in AML is feasible if based on selectively targeting the 5-LO, we used two different selective 5-LO inhibitors, Zileuton and CJ-13,610, in a PML/RAR- and DEK/CAN-positive leukemia model. Zileuton, an anti-asthmatic drug, is a reversible inhibitor of 5-LO activity which leads to the inhibition of leukotrienes (LTB4, LTC4, LTD4, and LTE4) formation. CJ-13,610 is novel non redox, non iron chelating 5-LO inhibitor. As stem cell models we used Sca-1+/lin-murine HSC retrovirally transduced either with PML/RAR or DEK/CAN. Here we report that both Zileuton and CJ -13,610 at clinically feasible concentrations of 0.3 – 3μM interfered with the aberrant replating efficiency of PML/RAR and DEK/CAN expressing HSCs; ii.) inhibited the short-term stem cell (ST-HSC) capacity of PMR/RAR- and DEK/CAN-positive HSCs as assessed by colony forming unit-spleen day 12 assays in lethally irradiated recipient mice; and iii.) reduced the frequency of long-term HSC in a long term competitive repopulation stem cell assays. The effects of both compounds were not due related to the induction of apoptosis. Interestingly, on normal control HSC both Zileuton and CJ-13,610 exhibited a “paradox” effect by increasing ST-HSC as well as LT-HSC capacity. Our here presented data establish the inhibition of 5-LO by selective inhibitors as a feasible approach of molecular stem cell therapy in AML. Furthermore it strongly suggest an important role of leukotrienes for the maintenance of leukemic stem cells. The exact mechanisms by which the inhibition of 5-LO interferes with the LSC have still to be disclosed. Disclosures: Off Label Use: The use of anti-inflammatory drugs such as Zileuton and CJ-13610 as novel approach for stem cell treatment in AML is discussed.