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Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of

Primary myelofibrosis (PMF) is an age-related myeloproliferative neoplasm (MPN) characterized by progressive bone marrow fibrosis, increasingly ineffective hematopoiesis, vascular and inflammatory complications, and progression to acute myeloid leukemia (AML) in 20% of cases. PMF is the rarest and the most severe of MPNs, with unfavorable prognosis and challenging treatment. Constitutive activation of the JAK-STAT pathway is an essential contributor to the pathogenesis of PMF. Nonetheless, clinically available JAK inhibitors do not show consistent disease-modifying effects at the molecular and malignant stem cell levels, underscoring the need to identify other targetable pathogenic mechanisms in PMF and related MPNs. We recently described a new signaling cascade involved in the maintenance of the malignant phenotype of stem cells in chronic myeloid leukemia that affects their adhesiveness, interaction with the microenvironment and resistance to imatinib (Chorzalska et al., Leukemia 2014). These phenotypic changes were caused by loss of Abelson interactor-1 (Abi-1) which regulates integrin α4 and the Src family kinases (SFKs) signaling pathways. To further delineate how organ-specific absence of Abi-1 affects hematopoiesis and hematopoietic stem cells, we generated a conditional, inducible bone marrow-specific Abi-1 transgenic mouse knockout model. For this purpose, we crossed conditional Abi-1(fl/fl) knockout mice with transgenic Tg(Mx1-cre+/-) mice (B6.Cg-Tg(Mx1-cre+ 1Cgn/J) that express Cre recombinase under the control of the polyinosinic:polycytidylic acid (poly(I:C)) or interferon-inducible Mx1 promoter. Obtained Abi-1(fl/fl);Tg(Mx1- cre(+)) animals were subjected to bone marrow-specific Abi-1 deletion induced by intraperitoneal administration of poly(I:C), resulting in organ-specific Abi-1 knockout (Abi-1 KO). Abi-1(fl/fl);Tg(Mx1- cre(-)) animals subjected to the same poly(I:C) treatment (Abi-1 WT) were used as controls in all experiments (Figure 1A). We confirmed the absence of Abi-1 protein in the Abi-1 KO bone marrow by immunoblotting (Figure 1B). Abi-1 KO mice developed deranged hematopoiesis with progressive anemia, thrombocythemia, megakaryocytosis, and myeloid hyperplasia (Figure 1C). They also developed progressive splenomegaly with 1.4x, 2.4x, and 4.8x enlargement of the spleen at 4, 28, and 50 weeks after recombination, respectively, and progressive femur pallor (Figure 1D) with bone marrow fibrosis confirmed by trichrome and silver staining (Figure 1D). Using fluorescence-activated cell sorting, we determined that Abi-1 KO resulted in more than two-fold expansion of the most primitive long-term hematopoietic stem cells (Lin-CD34-Sca-1+c-Kit+ CD135-) in the bone marrow and 3.75-fold increase of LSK cells (Lin- Sca-1+c-Kit+) in the peripheral blood compared with controls (Figure 1E). Increased activities of the SFKs and NFκB were observed in Abi-1 KO bone marrow, and confirmed in human CD34+ cells isolated from the marrow of patients with PMF or AML (Figure 1F,G). Furthermore, CD34+ hematopoietic progenitors and granulocytes from PMF patients, as well as CD34+ hematopoietic progenitors from AML patients, showed decreased levels of ABI1 transcript (Figure 1H). Taken together, our results indicate that a targeted deactivation of Abi-1 in the bone marrow causes expansion of hematopoietic stem cells, myeloid hyperplasia with thrombocytosis and overproduction of collagen and reticulin fibers. The Abi-1 KO murine phenotype meets the Mouse Models of Human Cancers Consortium criteria for a myeloproliferative neoplasm with features resembling human PMF (Kogan et al., Blood 2002). These data indicate that loss of Abi-1 is sufficient for initiation of bone marrow fibrosis, and that Abi-1-SFKs-NFκB cross-talk may represent a new molecular target for the treatment of PMF. Bone marrow-specific, conditional Abi-1 KO animals represent a new model of PMF offering a unique opportunity to interrogate the molecular details of the Abi-1-driven mechanism that leads to the development of stem cell-derived MPNs, and to elucidate potential novel therapeutic approaches that directly address their pathogenesis. Disclosures Olszewski: Bristol-Myers Squibb: Consultancy; Genentech: Research Funding; TG Therapeutics: Research Funding. Reagan:Alexion: Honoraria; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

The successful targeting of BCR/ABL by selective ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib alone is unable to eradicate the leukemic clone in Philadelphia chromosome positive (Ph+ ) leukemia. The t(9;22)(q34;q11) is a balanced translocation. Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. The constitutively activated BCR/ABL kinase is responsible for the leukemic transformation through an aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients with chronic myeloid leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. We co-expressed p96ABL/BCRand p185BCR/ ABL from a p2A peptide-linked multi-cistronic retroviral vector, which allows the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The co- expression of p96ABL/BCR enhanced the kinase activity and, as a consequence, the transformation potential of p185BCR/ABL in factor dependent progenitor cells and untransformed fibroblasts. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and primary Ph+ ALL patient-derived long-term cultures (PD-LTCs). Furthermore p96ABL/BCR negatively influenced the response to AKI in these models as shown by an increased response to AKI when p96ABL/BCR was down-regulated. Our in vitro and in vivo stem cell studies on murine fetal liver cells and adult HSCs revealed a functional hierarchy between p96ABL/BCR and p185BCR/ABL. In fact, p96ABL/BCR strongly increased stem cell capacity in replating efficiency and colony forming unit-spleen day 12 (CFU-S12) assays, whereas p185BCR/ABL showed no effect. In contrast co-expression of p96ABL/BCR and p185BCR/ABL increased significantly both serial replating potential and CFU-S12 colony formation as compared to p96ABL/BCR alone. In a syngeneic mouse model co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph + ALL. Disclosures No relevant conflicts of interest to declare.

The t(6,9)(p23,q34) translocation occurs in 1-5% of adult patients with acute myeloid leukemia (AML). It is associated with a poor prognosis and defines a high risk group of AML in the WHO classification. The t(6;9) is in most of the cases the only cytogenetic aberration at diagnosis. The hallmark of t(6;9)-positive AML is the DEK/CAN fusion protein. DEK/CAN is a leukemogenic oncogene, but little is known about the molecular mechanism of DEK/CAN-induced leukemogenesis. The 165 kDa DEK/CAN phosphoprotein is encoded by a single transcript of 5.5Kb. The DEK portion of the DEK/CAN contains all the major functional domains of DEK mediating DNA-binding and multimerization. DEK increases life span of primary cells in culture by inhibiting cellular senescence and apoptosis. Post-translational modifications of DEK, mainly phosphorylation, influence the activity of DEK; unphosphorylated DEK has a higher affinity for DNA than the phosphorylated form, which in turn has a higher ability for multimerization. The main kinases that phosphorylate DEK are Glycogen synthase kinase 3 β (GSK3β) and Casein kinase 2 (CK2). The respective phosphorylation sites are conserved in the DEK portion of DEK/CAN. However, little is known about the role of phosphorylation for the biological functions of DEK/CAN. Therefore we generated several mutants of DEK and DEK/CAN by point-mutating the putative GSK3β-sites (ΔP1) from S to A and by deleting the CK2 sites in addition to these mutations (ΔP2). The reduction of S/T phosphorylation was confirmed by a ProQ staining and affinity chromatography on lysates of 293T cells expressing DEK, DEK/CAN and the respective ΔP1 or ΔP2 mutants. Further biological and biochemical consequences of these mutations for DEK and DEK/CAN were investigated in murine factor dependent 32D progenitor cells and in primary murine Sca1+/lin- hematopoietic stem cells (HSC), retrovirally or lentivirally transduced with DEK, DEK/CAN and/or their phosphorylation mutants ΔP1 or ΔP2, respectively. Here we report that the loss of the GSK3β- and CK2-phosphorylation sites did not interfere with the subnuclear localization of either DEK or DEK/CAN as revealed either by subnuclear fractionation experiments or by co-localization with native DEK/CAN in confocal laser scan microscopy assays on 32D cells co-expressing DEK/CAN and ΔP1-DEK/CAN or ΔP2-DEK/CAN. In contrast, the destruction of GSK3β-phosphorylation sites not only led to a loss of apoptosis inhibition by DEK and DEK/CAN upon factor withdrawal in 32D cells, but also abolished the increased self renewal potential of DEK/CAN-positive HSC. In fact DEK/CAN-positive HSCs significantly increased colony numbers in colony forming units spleen-day 12 (CFU-S12) assays as compared to empty vector controls, whereas ΔP1-DEK/CAN and ΔP2-DEK/CAN did not have any effect. In summary, our results suggest an important role of the GSK3β-phosphorylation for the DEK/CAN-induced leukemogenesis, which establishes the GSK3β-activity as a molecular target for therapeutic intervention in t(6;9)-positive AML. Disclosures No relevant conflicts of interest to declare.

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.

Abstract 1384 In acute myeloid leukemia (AML) chimeric genes derived from specific chromosomal translocations, such as t(15;17), t(8;21) or t(6;9) encode aberrant fusion proteins (FPs). These Fps, i.e. PML/RARα (PR), AML-1/ETO (AE) or DEK/CAN (DC), recapitulate the leukemic phenotype in the mouse. In about 50% af AMLs constitutive activation of STAT3 and/or 5 is observed and it is postulated that the aberrant stem cell capacity in leukemia is correlated to the activation of STATs. Furthermore it has been shown in models of the t(15;17)(PR)-positive AML that there is a relationship between STAT-activation and response to arsenic trioxide (ATO). t(6;9)(DC)-positive AML is classified as a separate entity, because of its young age of onset and poor prognosis. The mechanisms by which DC induces leukemia are nearly completely unknown. In order to investigate whether the STAT-activation plays a role for the pathogenesis of t(6;9)-positive AML we investigated the activation of STAT3 and 5 in models of DEK/CAN positive leukemia. As models of DC-induced AML we used either primary Sca1+/lin- murine hematopoietic stem and progenitor cells (mHSPC) retrovirally transduced with DC or as controls with PR. Samples of DC-positive pre-leukemia as well as primary leukemia and 2° leukemia from established DC-induced AML were used for the in vivo studies. Expression and activation of STAT3 and 5 was assessed by either intracellular FACS or Western blotting using antibodies against the phosphorylated (activated) and total STAT3 or 5. The effect of constitutively activated STAT3 (STAT3*) on the stem cell capacity was studied by the retroviral expression of STAT3* in mHSPC which then were subjected to serial replating and colony-forming unit spleen day 12 (CFU-S12) assays. ATO treatment was performed on sublethally irradiated recipients (8 mice/group), from day 5 after inoculation of DC-positive primary leukemic spleen cells for the induction of a 2° leukemia for 14 days. Leukemia development and response to ATO was assessed 30 days post-transplantation by spleen size and determination of the proportion of leukemic cells in the peripheral blood (PB), spleen and bone marrow (BM) in comparison to solvent treated controls. Here we show that i.) both DC and PR strongly activated both STAT3 and 5 in primary blasts from mouse leukemia as as well as in retrovirally transduced primary HSCPs; ii) the expression of constitutively activated STAT3 (STAT*) in HSC led to an increased replating efficiency in the presence of mIL-3, mIL-6 and SCF; iii.) STAT* increased CFU-S12 as a read out for short-term stem cell and early progenitor potential to a similar extent as PR. iv.) the pre-leukemic state of DC-positive AML was characterized by the only activation of STAT3 but not of STAT5; v.) STAT5 activation appeared only in the full blown leukemia; v.) exposure to ATO inhibited phosphorylation of STAT3 and STAT5 in vivo in the PB of the leukemic mice; vi.) the treatment with ATO significantly decreased spleen size and the proportion of leukemic cells in the BM and spleen as compared to solvent treated controls. In summary, our results strongly suggest a direct relationship between the expression of the leukemogenic oncogenes DC or PR and the activation of STATs, which seems to play an important role for the maintenance of the leukemic stem cell. Furthermore ATO seems to represent a novel therapeutic option for the t(6;9)-positive AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3539 The t(9;22)(q34;q11) is a balanced translocation. The cytogenetic correlate of der22 is the so-called Philadelphia chromosome (Ph). Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. BCR/ABL is a constitutively activated kinase which induces the leukemic phenotype by the aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The BCR/ABL kinase activity is efficiently targeted by tyrosin-Kinase inhibitors such Imatinib, Nilotinib, or Dasatinib. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients suffering from chronic myeloid leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute lymphatic leukemia (ALL). In our previous studies we have shown the leukemogenic potential of the ABL/BCR fusion proteins. To further disclose the role of ABL/BCR proteins, mainly p96ABL/BCR, in the transformation process induced by BCR/ABL and the leukemogenesis of Ph+ ALL, we co-expressed p96ABL/BCR and p185BCR/ABL retrovirally in the IL-3 dependent murine Ba/F3 pro-lymphocytic cell line. p96ABL/BCR and p185BCR/ABL were expressed from P2A peptide-linked multicistronic retroviral vectors, which allow the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The effect of p96ABL/BCR on the kinase activity of p185BCR/ABL, was assessed by the rate of autophosphorylation at Y245 and Y412, the BCR/ABL-dependent substrate phosphorylation (CrkL, Bcr) and by the activation of down-stream signaling pathway (Stat, Erk,) determined by Western blotting. Proliferation of the cells was assessed by growth curve and XTT assays upon withdrawal of IL-3. As classical transformation assays we performed focus formation assays (loss of contact inhibition) and colony formation in semi-solid medium (support independent growth) in untransformed Rat-1 fibroblasts. The p96ABL/BCR expression in primary Ph+ ALL patient derived long term cultures (PDLTCs) was targeted by retrovirally transduced shRNA. The efficient targeting of p96ABL/BCR was confirmed by western blotting. Here we report that p96ABL/BCR i.) p96ABL/BCR enhanced not only the autophosphorylation of p185BCR/ABL at Y245, but also the activation of all the downstream signaling pathways; ii.) p96ABL/BCR by itself did not transform Rat-1 cells but impressively increased the number of colonies and foci induced by p185BCR/ABL in Rat-1 cells; iii.) p96ABL/BCR increased the proliferation of p185BCR/ABL-positive Ba/F3 cells; iv.)p96ABL/BCR reduced the responsiveness to TKI in p185BCR/ABL positive Ba/F3 cells; v.) targeting the p96ABL/BCR by shRNA decreased the proliferation of Ph+PDLTCs by the induction of apoptosis and increased their sensitivity towards kinase inhibitors (Imatinib, Nilotinib) and the allosteric inhibition by GNF-2 directed against p185BCR/ABL. Taken together these data suggest that p96ABL/BCR plays an important role in the determination of the leukemic phenotype and the therapy resistance of Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 2964 Poster Board II-940 The t(9;22)-related translocation products are the BCR/ABL fusion proteins. Fusion of BCR to ABL leads to constitutive activation of ABL tyrosine kinase (ATK) activity. c-ABL is finely regulated by a variety of stimuli, whereas constitutively activated ABL induces aberrant proliferation and neoplastic transformation by constitutive activation of RAS, PI3 kinase, and JAK/STAT. Constitutively activated ATK is indispensable for the transformation potential of BCR/ABL. Inhibition of BCR/ABL kinase activity by selective ATP-competitors, such as Imatinib, Dasatinib or Nilotinib, is a valid concept for the causal therapy of Ph+ leukemia. Unfortunately, in advanced Ph+ leukemia, CML-blast crisis, and Ph+ ALL, these compounds select for resistant clones mostly due to the acquisition of point mutations in BCR/ABL that change the affinity for these ATP competitors. Tetramerization of ABL through the N-terminal coiled-coil region (CC) of BCR is essential for aberrant ABL-kinase activation. We recently showed that targeting the CC-domain forces BCR/ABL into a monomeric conformation that abolishes its transformation potential by interfering with its kinase activity. This also increases the sensitivity of the leukemic cells to Imatinib and overcomes the Imatinib resistance of BCR/ABL harboring the Y253F and E255K mutations. Another mechanism to target BCR/ABL consists in the allosteric inhibition. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl binding pocket in a process called “capping”, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, BCR/ABL “escapes” this auto-inhibition. Allosteric inhibition by myristate “mimics”, such as GNF-2, is able to inhibit BCR/ABL. The the “gatekeeper” mutation T315I confers pluri-resistance against ATP competitor, oligomerization as well as allosteric inhibitors Based on data showing that the response to GNF-2 varies with the oligomerization in ABL-chimeras we investigated the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. Therefore we combined GNF-2 with the competitive peptide helix-2, for which we have previously shown the capacity to target BCR/ABL and its Imatinib-resistant mutants. Here we demonstrate that i.) the helix-2 strongly increased the inhibitory effects of 2μM GNF-2 on the factor independent growth of BCR/ABL expressing Ba/F3 progenitors; ii.) both helix-2 and GNF-2 were able to decrease the transformation potential of BCR/ABL in Rat-l fibroblasts; iii.) the combination of helix-2 and GNF-2 completely abolished the colony formation of BCR/ABL-positive Rat-1 cells in semi-solid medium; iv.) in combination helix-2 and GNF-2 were able to abrogate factor independent growth of Ba/F3 cells expressing BCR/ABL-T315I, whereas no effect was seen by the single agents; v.) the helix-2/GNF-2 combination nearly completely abolished the transformation potential of BCR/ABL-T315I in Rat-1 cells; vi.) the inhibitory effects of helix-2/GNF-2 were due to an inhibition of the BCR/ABL dependent signaling as shown by a reduced activation of STAT5 and a block of substrate phosphorylation (Crkl). The fact that the helix-2 is able to overcome the resistance of BCR/ABL-T315I against GNF-2 strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 3980 Poster Board III-916 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. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted 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 PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-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 PML/RAR 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. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia 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. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. 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 PML/RAR 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.

Acute myeloid leukemia (AML)-associated fusion proteins (AAFP), such as PML/RAR, PLZF/RAR or AML1/ETO have leukemogenic potential in vivo which is related to their capacity to induce a differentiation block and aberrant self renewal in early hematopoietic progenitors. The potential of the AAFP to induce the leukemic phenotype is strictly related to their capacity to oligomerize and to form high molecular weight complexes (HMW). Furthermore we recently showed that the deregulation of the Wnt-signaling seems to be a key event for the leukemogenesis by the above mentioned AAFP because it is indispensable for the aberrant self renewal of leukemic stem cells (LSC). A „tandem affinity purification” (TAP) screen of KG1 cells expressing PLZF/RAR, PML/RAR or AML-1/ETO for elucidating the composition of the related HMW revealed the „adenomatous polyposis coli” protein (APC) as an interaction partner of PLZF/RAR. APC is a key inhibitory regulator of the Wnt-signaling and is frequently inactivated by mutations in colon carcinomas. Therefore the role of the interaction between APC and PLZF/RAR for the leukemogenesis was further investigated. Here we report that i.) only PLZF/RAR strongly interacted with APC as confirmed by co-immunoprecipitation experiments; ii.) the overexpression of APC reverted the aberrant activation of the Wnt-signaling by PLZF/RAR as revealed by a reduction of the TCF/LEF mediated transcriptional activity; iii.) the overexpression of APC also reduced the self renewal potential of PLZF/RAR-positive HSC as revealed by a reduced replating efficiency of these cells in semi solid medium, as well as by a reduction of the colony number in colony forming units spleen (CFU-S) assays; iv.) APC was able to revert the leukemogenic potential of PLZF/RAR-positive LSC as revealed by the retroviral overexpression of APC in bone marrow isolated from mice with PLZF/RAR-induced AML which were inoculated into secondary recipients. In fact until now the presence of APC significantly increased the survival of these secondary recipients with respect to mock-infected controls. Taken together these data strongly suggest that the direct interaction with PLZF/RAR is an important mechanism for the functional inactivation of APC which contributes to the aberrant activation of the Wnt-signaling in PLZF/RAR-positive cells. Furthermore our data provide further evidence that the aberrantly activated Wnt-signaling pathway represents a valid target for molecular therapy approaches.

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.

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.