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International audience; Focal adhesion kinase (FAK) activity contributes to many advanced cancer phenotypes, but little is known about its role in human acute myeloid leukemia (AML). Here, we show that FAK splice variants are abnormally expressed in the primitive leukemic cells of poor prognosis AML patients. In the CD34(+) 38(-) 123(+) long-term culture-initiating cell-enriched leukemic cells of these patients, FAK upregulates expression of Frizzled-4 and phosphorylates Pyk2 to enable the required association of Pyk2 with the Wnt5a/Frizzled-4/LRP5 endocytosis complex and downstream activation of β-catenin, thereby replacing the Wnt3a-controlled canonical pathway used by normal hematopoietic stem cells. Transduction of primitive normal human hematopoietic cells with FAK splice variants induces a marked increase in their clonogenic activity and signaling via the Wnt5a-controlled canonical pathway. Targeting FAK or β-catenin efficiently eradicates primitive leukemic cells in vitro suggesting that FAK could be a useful therapeutic target for improved treatment of poor prognosis AML cases. STEM CELLS2012;30:1597-1610.

Oncogenic pathways underlying in the development of myelodysplastic syndromes (MDS) remain poorly characterized, but mutations of the ten-eleven translocation 2 (TET2) gene are frequently observed. In the present work, we evaluated the prognostic impact of TET2 mutations in MDS. Frameshift, nonsense, missense mutations, or defects in gene structure were identified in 22 (22.9%) of 96 patients (95% confidence interval [CI], 14.5-31.3 patients). Mutated and unmutated patients did not significantly differ in initial clinical or hematologic parameters. The 5-year OS was 76.9% (95% CI, 49.2%-91.3%) in mutated versus 18.3% (95% CI, 4.2%-41.1%) in unmutated patients (P = .005). The 3-year leukemia-free survival was 89.3% (95% CI, 63.1%-97.0%) in mutated versus 63.7% (95% CI, 48.2%-75.4%) in unmutated patients (P = .035). In univariate analysis (Cox proportional hazard model), the absence of TET2 mutation was associated with a 4.1-fold (95% CI, 1.4-12.0-fold) increased risk of death (P = .009). In multivariate analysis adjusted for age, International Prognostic Scoring System, and transfusion requirement, the presence of TET2 mutation remained an independent factor of favorable prognosis (hazard ratio, 5.2; 95% CI, 1.6-16.3; P = .005). These results indicate that TET2 mutations observed in approximately 20% of patients, irrespective of the World Health Organization or French-American-British subtype, represent a molecular marker for good prognosis in MDS.

The metalloprotease 9 (MMP-9), a known mediator of tumour invasion, is secreted as a 92 kDa pro-form but a non-secreted variant of 85 Kda has been described. The importance of this variant pro-form in tumor progression remains poorly defined. We previously showed that the DNA repair protein Ku interacts at the cell surface of leukaemia cell lines with the 85 Kda pro-form of MMP-9 and these Ku/MMP-9 complexes regulates cell invasion, highlighting their importance in haematological malignancies. We demonstrate here that all samples of acute myeloid leukaemia (AML) blasts purified from bone marrow of 16 affected patients express the 85 Kda form of MMP-9. However, only AML that display monocytic lineage markers (AML4/5) express this form at the cell surface with co-expression of the membrane associated form of Ku. Blocking antibodies directed against Ku or MMP-9 specifically inhibited cell invasion of those expressing Ku/MMP-9 on the cell surface. The membrane form of Ku might represent an important factor in the exposition to the cell surface of this specific MMP-9 pro-form in AML with monocytic features. These results might have important functional significance in the occurrence of extra-medullar infiltrates of leukaemia cells that occurs frequently during the onset of monocyte-related AML sub-types.

Upon differentiation, hematopoietic stem cells (HSC) give rise to multipotent progenitors (MPP) that retain the ability to produce all blood lineages but have lost their self-renewal capacity. MPP are then orientated towards either the lymphoid or myeloid lineages, developing into common myeloid progenitors (CMP) or lymphoid-primed multipotent progenitors which can still produce certain myeloid cell types. CMP can differentiate into either granulocyte-macrophage progenitors (GMP) or megakaryocyte-erythroid progenitors. GMP finally differentiate into granulocyte or monocyte progenitors (GP/MP). This study aimed to investigate whether acute myeloid leukemia (AML) samples can be sub-classified based on the stage of arrest in differentiation (Stage of Leukemia Arrest, SLA). Understanding which critical differentiation program is specifically altered could be of special interest to design new therapeutic strategies aimed at re-inducing the differentiation process in AML. Based on CD34/CD117/CD13/CD33/MPO expression, we defined phenotypic signatures that identify all normal hematopoietic stem and progenitor cells (HSPC). Applied to leukemic hematopoiesis, these signatures allowed us to sub-classify 932 AML patients from Toulouse University Hospital (test cohort) and 142 AML patients from The Cancer Genome Atlas Network, published in NEJM in 2013 (control cohort). Globally, AML with an HSC phenotype (henceforth termed HSC-L) represented 1% of the cases, while MPP-L, CMP-L, GMP-L and GP/MP-L accounted for 15%, 30%, 22% and 30% of the cases, respectively. To validate our phenotypic signature and SLA, we generated a transcriptomic signature of normal hematopoietic differentiation from three databases including normal HSPC assessment (GSE21973, GSE42414 and E-TABM-978). Unsupervised clustering and gene set enrichment analysis (GSEA) of a test cohort (n=40) and a control cohort (n=142) confirmed that AML shared a common transcriptomic signature with their SLA. To strengthen the relationship between SLA and HSPC, we then compared their energetic metabolism. Using metformin as an enhancer of glycolytic (GLY) metabolism (e.g. Pasteur effect), we constructed transcriptomic signatures for AML cell lines with either a GLY or with an oxidative (OX) metabolism. We showed that, as expected normal HSC and MPP were enriched in GLY genes, whereas CMP and GMP were enriched in OX genes set. Similarly, HSC-L and MPP-L highly expressed genes related to GLY metabolism; CMP-L samples could be subdivided between GLY AML and OX AML; and GMP-L highly expressed genes related to OX metabolism. Each AML subgroup defined accordingly to the SLA had a particular clinical presentation (Table). Interestingly, the least differentiated AML (HSC-L, MPP-L and CMP-L) were enriched in HSC and LSC genes sets. These AML samples blocked at early stage of hematopoiesis gave higher engraftment levels in NSG mice compared to GMP-L and GP/MP-L (21%, vs 5%, panel of 59 AML tested in 376 mice, p We then sought to investigate the distribution of AML mutations according to SLA classification. We took advantage of the fully annotated TCGA database and found that 80% of HSC-L and 25% of MPP-L had RUNX1 mutations, 80% of GMP-L had either CEBPA mutation or RUNXL1-RUNX1/CBFB-MYH11 AML, and 80% of GP/MP-L had NPM1 mutation. The subgroups of CMP-L showed a greater molecular heterogeneity. AML and normal HSPC shared phenotypic, transcriptomic and metabolic signatures. This is of special interested for differentiation therapy as specific critical dysregulated transcriptional factors (e.g. CEBPA) could be related to specific stage of arrest. Identifying each of them could allow to propose targeted differentiating therapy and maximize therapeutic responses. Table. HSC-L MPP-L CMP-L GMP-L GP/MP-L frequency 1% 15% 30% 22% 30% Median WBC at diagnosis (G/L) 4 25 20 35 54 organ infiltration NA spleen varied lymph nodes gengiva or lymph nodes CFU-L (% of blasts) 0.7 8.1 9.6 8.7 6.9 metabolism GLY GLY GLY/OX OX NA oncogenic event RUNX1m RUNX1m/TP53m unknown CEBPAm/CBF NPM1c BAALC/ERG/MN1 expression high high mid mid low stem cell signature high high high low low Median DFS (months) 5 22.9 13.7 41.3 >120 Median OS (months) 6.6 26.6 16.3 >120 83.3 Disclosures Huguet: Novartis: Consultancy, Research Funding; BMS: Consultancy, Speakers Bureau; ARIAD: Consultancy, Speakers Bureau; PFIZER: Consultancy, Speakers Bureau.

Background: Modalities of monitoring and clinical impact of post transfusional iron overload in MDS patients remain controversial. Labile Plasma Iron (LPI) is a component of Non-Transferrin Bound Iron (NTBI) that is thought to contribute to oxidative stress in MDS. However, little data support this hypothesis and most studies on LPI have only addressed its ability to produce free radicals in vitro. The aim of this work was to assess LPI and to evaluate the correlation between LPI and plasma and blood markers of peroxidation in MDS patients Design and Methods: In 67 non-chelated MDS patients, we evaluated: hemoglobin (Hb), indices of iron overload, peroxidation status at inclusion and/or during follow up. The peroxidation status was evaluated by measuring blood Glutathione (GSH) and plasma Advanced Oxidation Protein Products (AOPP). LPI was measured using the Afferix kit. NTBI was measured by a flow cytometry- based assay (FeFlobead) in a subset of patients with high transferrin saturation. Results: Median age of the 67 patients was 79 years (range 61-93). 38 patients were included at diagnosis of MDS and 29 were included during the disease course (after a median of 50 months, range 3-168, for the latter group). WHO sub-type was: 14 refractory anemia, 22 refractory anemia with ring sideroblasts, 7 refractory cytopenia with mutlilineage dysplasia, 4 5q minus syndrome, 16 refractory anemia with excess of blasts ( RAEB) (type I n=6, Type II n=5, 5 RAEB-t/AML , 3 chronic myelo monocytic leukemia type I, one unclassifiable case. Prognostic score according IPSS was low in 43 patients, Int-1 in 13, Int-2 in 6 and high in 5. 36/67 patients had been transfused and had received a median of 22 packed RBCs (range 2-102). Transfused patients (N=36) were more heavily iron overloaded than non-transfused patients (N=31), as indicated by significantly higher median transferrin (Tf ) saturation (42% versus 62%) (P 50 %. However, the mean value of LPI was very similar in transfused and non-transfused group (0.34 microM versus 0.38). 8/67pts had LPI levels above limit upper value (≥ 0.4 µM) and their SF was significantly higher than that of other patients(p=0.02). However no significant difference was found between patients with LPI < 0.4 µM and ≥ 0.4 µM, for Tf saturation, blood GSH or AOPP median values. No correlation between LPI and NTBI was seen. GSH and AOPP levels did not statistically differ according (IPSS) groups. 14 patients were studied during follow up, 7 of them after 3 months of iron chelation, and 7 after treatment with EPO. While chelation slightly improved markers of oxidative stress (MAO) (p= NS), EPO induced a very highly significant improvement of MAO-(p= 0.005). Conclusion: We found no evidence to support LPI pro-oxidant role in vivo. A few patients had an elevated LPI level in this large, moderately transfused, MDS cohort. Utility of LPI in monitoring of post transfusional iron overload remain to be demonstrated. Results obtained following EPO treatment suggest that ineffective erythropoiesis rather than iron overload may be responsible for RBC oxidative stress Disclosures Rose: novartis: Honoraria, Research Funding; celgene: Honoraria. Fenaux:Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding. Beaumont:novartis: Research Funding.

The mammalian target of rapamycin (mTOR) is a key regulator of growth and survival in many cell types. Its constitutive activation has been involved in the pathogenesis of various cancers. In this study, we show that mTOR inhibition by rapamycin strongly inhibits the growth of the most immature acute myeloid leukemia (AML) cell lines through blockade in G0/G1 phase of the cell cycle. Accordingly, 2 downstream effectors of mTOR, 4E-BP1 and p70S6K, are phosphorylated in a rapamycin-sensitive manner in a series of 23 AML cases. Interestingly, the mTOR inhibitor markedly impairs the clonogenic properties of fresh AML cells while sparing normal hematopoietic progenitors. Moreover, rapamycin induces significant clinical responses in 4 of 9 patients with either refractory/relapsed de novo AML or secondary AML. Overall, our data strongly suggest that mTOR is aberrantly regulated in most AML cells and that rapamycin and analogs, by targeting the clonogenic compartment of the leukemic clone, may be used as new compounds in AML therapy.

The simultaneous occurrence of Philadelphia positive chronic myeloid leukemia (Ph+ CML) and B-cell chronic lymphocytic leukemia (B-CLL) is a rare event which raises the possibility that the two malignant clones derive from a common, or distinct, malignant stem cells. In this study, we used combined CD19-based cell-sorting and fluorescence in situ hybridisation (FISH) to investigate whether or not the BCR-ABL fusion gene was present in the malignant B-cells of a patient who presented a Ph+ CML/B-CLL association. The CD19+ cells lacked the BCR-ABL rearrangement whereas all CD19-cells exhibited the fusion gene. This result demonstrates that B-cell transformation occurred in a Ph-B-cell subset.

In light of the emerging concept of a protective function of the mitogen-activated protein kinase (MAPK) pathway under stress conditions, we investigated the influence of the anthracycline daunorubicin (DNR) on MAPK signaling and its possible contribution to DNR-induced cytotoxicity. We show that DNR increased phosphorylation of extracellular-regulated kinases (ERKs) and stimulated activities of both Raf-1 and extracellular-regulated kinase 1 (ERK1) within 10 to 30 minutes in U937 cells. ERK1 stimulation was completely blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor PD98059 or the Raf-1 inhibitor 8-bromo-cAMP (cyclic adenosine monophosphate). However, only partial inhibition of Raf-1 and ERK1 stimulation was observed with the antioxidant N-acetylcysteine (N-Ac). Moreover, the xanthogenate compound D609 that inhibits DNR-induced phosphatidylcholine (PC) hydrolysis and subsequent diacylglycerol (DAG) production, as well as wortmannin that blocks phosphoinositide-3 kinase (PI3K) stimulation, only partially inhibited Raf-1 and ERK1 stimulation. We also observed that DNR stimulated protein kinase C zeta (PKCzeta), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNR-triggered PKCzeta activation. Finally, we found that the expression of PKCzeta kinase-defective mutant resulted in the abrogation of DNR-induced ERK phosphorylation. Altogether, these results demonstrate that DNR activates the classical Raf-1/MEK/ERK pathway and that Raf-1 activation is mediated through complex signaling pathways that involve at least 2 contributors: PC-derived DAG and PI3K products that converge toward PKCzeta. Moreover, we show that both Raf-1 and MEK inhibitors, as well as PKCzeta inhibition, sensitized cells to DNR-induced cytotoxicity.

Previous studies have established that protein kinase C-zeta (PKC-zeta) is critical for neuronal cell differentiation. However, the role of PKC-zeta in haematopoietic cell differentiation is less clear. In this study, we have investigated the influence of PKC-zeta overexpression on the phenotype of the human monocytic U937 leukaemic cells. In two PKC-zeta-overexpressing clones (U937 zetaJ and U937 zetaB), PKC-zeta expression levels and activity were three to fourfold higher, and the enzyme accumulated both in the cytoplasm and in the nucleus compared with U937 control cells. PKC-zeta-overexpressing U937 cells exhibited an erythroid phenotype characterized by high levels of glycophorin A, cell haemoglobinization, increased GATA-1 transcripts and protein expression, compared with controls. Immunoprecipitation studies revealed that GATA-1 protein was constitutively phosphorylated in PKC-zeta-overexpressing cells. Moreover, GATA-1 did not interact with PKC-zeta but interacted with ERK1, which was constitutively activated and accumulated in the nucleus of U937 zetaJ. However, ERK1 phosphorylation inhibition by PD098059 did not influence either GATA-1 phosphorylation or GATA-1/ERK1 interaction. Collectively, these results suggest a model in which PKC-zeta induces MEK-dependent ERK1 activation, ERK1 translocation to the nucleus, GATA-1/ERK1 interaction and ERK1-independent GATA-1 phosphorylation resulting in GATA-1 accumulation. To conclude, this study provides evidence for the role of PKC-zeta in erythroid gene regulation.

SPECIFIC AIMSMany studies have demonstrated that 1-β-D-arabinofuranosylcytosine (Ara-C) triggers a wide spectrum of intracellular signals, which may contribute downstream of drug-DNA incorporation to modulate Ara-C cytotoxicity. Therefore, in this study we sought to determine the respective roles of reactive oxygen species (ROS) and p53/p56 Lyn kinase in the regulation of sphingomyelinase (SMase) activation in Ara-C-triggered JNK activation and apoptosis in leukemic myeloid cells.PRINCIPAL FINDINGSU937 and HL-60 cells were treated in kinetic experiments with 40 μM Ara-C and analyzed for cell viability and DNA fragmentation using the [3H]thymidine release assay, poly (ADP-ribose) polymerase (PARP) cleavage, and DAPI staining. Ara-C induced a significant loss of cell viability with DNA fragmentation of 9.7% as soon as 6 h and 45.9% at 24 h. We also observed typical morphological features of apoptosis by DAPI staining and PARP cleavage. The enumeration of morphologically apoptotic cells was estimated at 31% ...

Anthracyclines such as daunorubicin (DNR) generate radical oxygen species (ROS), which account, at least in part, for their cytotoxic effect. We observed that early ceramide generation (within 6-10 min) through neutral sphingomyelinase stimulation was inhibitable by the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate, which led to a decrease in apoptosis (>95% decrease in DNA fragmentation after 6 h). Furthermore, we observed that DNR triggers the c-Jun N-terminal kinase (JNK) and the transcription factor activated protein-1 through an antioxidant-inhibitable mechanism. Treatment of U937 cells with cell-permeant ceramides induced both an increase in ROS generation and JNK activation, and apoptosis, all of which were antioxidant-sensitive. In conclusion, DNR-triggered apoptosis implicates a ceramide-mediated, ROS-dependent JNK and activated protein-1 activation.

Abstract 1385 The JAK2V617F mutation is present in the majority of polycythemia vera (PV) patients and half of those with essential thrombocythemia and primary myelofibrosis. JAK2V617F is a gain-of-function mutation resulting in constitutive JAK2 signalling involved in the pathogenesis of these diseases. JAK2V617F has been shown to promote S phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell cycle transition, is constitutively over-expressed in JAK2V617F -positive cell lines, JAK2-mutated patient CD36+ progenitors and in vitro differentiated proerythroblasts. Accordingly, CDC25A is also overexpressed in bone marrow and spleen of Jak2V617F knock-in mice compared to wild-type (WT) littermates. Using murine FDC-P1-EPOR and human HEL and SET-2 cell lines, we found that JAK2V617F-induced CDC25A up-regulation was not caused by increased transcription or CDC25A stability or the involvement of its known upstream regulators AKT and MAPK. Instead, our results suggest that CDC25A is originaly regulated at the translational level through the implication of the transcription factor STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2V617F-expressing erythroid progenitors, while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2V617F patients, making of this protein an attracting potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Abstract 3556 IDH1/2, TET2 and DNMT3A mutations have been reported in myeloid malignancies including de novo AML. In this study, we have analyzed the frequency and prognostic impact of these mutations in a large retrospective cohort of patients (pts) with secondary AML (SA) which encompass myelodysplasia-related changes (MRC) AML and therapy-related (TR) AML according to the WHO classification. Bone marrow samples were collected from 247 pts at diagnosis with SA and the mutational status of IDH1/2, TET2 and DNMT3A genes together with other genes frequently mutated in AML (NPM1, FLT-3, N and K-RAS, WT1) was determined by Sanger sequencing or high resolution melting analysis. The cohort of 247 pts consisted in 201 MRC AML and in 46 TR AML, 39.5% of which with a normal karyotype (NK). The frequency of IDH1/2, TET2 and DNMT3A mutations was 12.6, 19.8 and 4.5%, respectively. Two pts had both TET2 and IDH1/2 mutations, 2 pts had TET2 and DNMT3A mutations and 5 pts had both IDH1/2 and DNMT3A mutations showing that these mutations were not mutually exclusive in SA. IDH1/2 and TET2 mutations were significantly more frequent in MRC AML (14.1 and 22.3%) than in TR AML (6.4 and 8.7%) (P =0.04 and P =0.03) while the frequency of DNMT3A mutations was identical in the two subgroups. The SA pts harbouring at least one IDH1/2 or TET2 or DNMT3A mutation were significantly older (P Complete remission rate and overall survival were evaluated in a group of 158 pts which had received intensive chemotherapy at diagnosis, and were identical in the IDH1/2 or TET2 or DNMT3A mutated and unmutated groups. These mutations did significantly influence survival neither in the subgroup of pts with normal karyotype, nor in the subgroup of MRC-AML, or TR-AML which were of very poor prognosis. These data show that IDH1/2, TET2 or DNMT3A mutations could modify the clinical presentation without impact on prognosis. Disclosures: No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) are heterogeneous hematopoietic disorders characterized by an accumulation of immature leukemic cells and by a block in cell differentiation. In spite of recent therapeutical advances and frequent complete remission, all patients shortly relapse leading to an urgent need for more specific targeted treatments. Beside the development of epigenetic and differentiation therapies for AML, therapeutic strategies targeting protein tyrosine kinases are also attractive and promising. Here we report a novel oncogenic activity of the cMet related receptor tyrosine kinase Ron (Recepteur d'Origine Nantais) in AML. Ron is expressed in most epithelial tissues as well as in normal hematopoietic CD34+ cells and macrophages. Its oncogenic properties have been documented in solid tumors and a truncated form of Ron (short form of Ron, sf-Ron) lacking most of the Ron receptor extracellular domain but retaining the whole transmembrane and intracellular domains has been detected in several tissues and in KG1 AML cell line. We found that sf-Ron was expressed in 44% of AML patients cells (n=87), whereas 38% of patient cells did not express neither fl-Ron nor sf-Ron. Interestingly only fl-Ron is expressed in CD34+ normal cells suggesting that sf-Ron could be specific of leukemic cells. Accordingly, sf-Ron was found to be active in AML samples unlike the fl-Ron. Inhibition of sf-Ron activity by either the dual c-Met/Ron inhibitor SU-11274 or a specific siRNA approach inhibited clonogenic properties and induced apoptosis only in sf-RON positive AML cells. Moreover, the leukemic compartment CD34+CD38-CD123+ often associated with the chemoresistance and clinical relapse, was also sensitive to this treatment. Additionally, we observed an original link between sfRon and the tyrosine kinase Lyn previously described to be strongly deregulated in AML cells. Indeed, we showed that sf-Ron is associated with Lyn and these two tyrosine kinases activate each other. Finally, a recent study described an epigenetic mechanism of expression regulation of Ron and sf-Ron, showing the existence of two distinct CpG islands. Indeed, 5-Azacytidine treatment of AML cells led to a marked down-regulation of sf-Ron in AML cells. This effect was accompanied by a strong reduction of Lyn activity. In conclusion, our study suggests that targeting sf-Ron activity might be of therapeutic value in AML and uncovers novel mechanisms by which hypomethylating agents would act on AML and myelodysplastic syndromes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3130. doi:10.1158/1538-7445.AM2011-3130

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes which are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is responsible for a strong up-regulation of miR-125b (6 to 90 fold). In vitro experiments revealed that miR-125b was able to block monocytic and granulocytic differentiation of leukemic cells and primary CD34+ human blasts. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation and myeloid neoplasms carrying the t(2;11) translocation define a new clinico-pathological entity.

While in RARS-T, JAK2V617F mutation is common and associated with good prognosis, the clinical and prognostic impact of this mutation in other MDS is unknown. We collected data from 132 non-RARS-T MDS with known JAK2V617F mutation status. JAK2V617F mutation was significantly correlated with lower progression to AML (p