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DNAs from chronic granulocytic leukaemia (CGL) and chronic myelomonocytic leukaemia (CMML) were assayed for transforming genes by transfection into NIH 3T3 cells. Foci DNA was tagged with a geneticin-resistance cosmid, then followed through a drug selection and tumorigenicity assay. Activated Ha-ras genes, with point mutations at codon 12 (glycine to valine) were subsequently detected. The mutation was detected in the original samples by either MspI/HpaII digestion or polymerase chain reaction (PCR). Although mutations in the ras gene family may occur frequently in leukaemias, these are the first examples of Ha-ras mutations in CGL (blast crisis) and CMML.

A high level of chloroquine resistance was developed in the rodent malaria parasite, Plasmodium chabaudi. This resistance was stable and its inheritance was shown to be multigenic; intermediate levels of resistance were obtained from a cross between highly resistant and sensitive parasites. Chloroquine resistance was shown to segregate independently of pyrimethamine resistance and enzyme markers.

Resistance to cytotoxic agents is a common clinical problem encountered in the treatment of human myelodysplastic syndromes (MDS) and acute myeloblastic leukaemia (AML). Cellular acquisition of the multidrug resistance (MDR) phenotype confers loss of sensitivity to a wide range of structurally dissimilar anti-neoplastic agents. This state can arise through increased expression of the mdrl (P-glycoprotein) gene. We have used the mdrl gene probe to investigate adriamycin resistant (HL60/AR) and vinblastine resistant (CEM/VLB100) human leukaemic cell lines. In addition, peripheral blood or bone marrow cells from 66 patients with MDS and AML have been screened for gene amplification and 40 cases for increased mRNA expression. P-glycoprotein gene amplification was observed only in the (CEM/VLB100) and not in the HL60/AR on any other leukaemic cell line. Gene amplification was not found in any patient's cells. Eighteen out of 40 patients showed an increase (2----20) of mdrl mRNA expression. These results are not only of significance in understanding the biology of human drug resistance but have practical importance in the design of anti-leukaemic therapy.

The human malignant melanoma cell line NK14 contains a novel transforming gene which was identified using DNA transfection into NIH3T3 cells (1). This gene has been assigned to chromosome 19p13.2-q13.2 using human-mouse and human-hamster somatic cell hybrids.

DNA isolated from cell line Mel Swift, a human melanoma cell line, transforms NIH3T3 cells. Southern blot analysis of DNA from secondary foci revealed conserved 8.8- and 7.8-kilobase EcoRI fragments which hybridized with a human repetitive sequence clone, blur 8. The activated transforming gene was identified as N-ras, and the 8.8-kilobase EcoRI fragment from a secondary transformant was cloned. Synthetic 17-mer oligonucleotides which spanned either the normal codon 61 (CAA) or a mutant codon 61 (AAA) were used for hybridization. Cloned N-ras from melanoma cell line Mel Swift hybridized to the mutant (AAA) oligonucleotide. From this we predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift. Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active.

Cellular transforming genes can be detected in human tumours by DNA-mediated transfection into NIH 3T3 mouse fibroblasts1,2. The activated transforming genes have been, in most cases, members of the ras gene family, of which the most frequently found is the c-Ki-ras oncogene3–7 and least frequently the c-Ha-ras gene8–10. An increasing number of studies has identified the presence of activated N-ras (which has no known viral homologue) in human tumour cell lines11–18. Furthermore, other transforming genes, distinct from the ras gene family, have been reported in B-and T-cell lymphomas19,20. The activation of c-Ha-ras and N-ras has been described in some cell lines derived from cases of human malignant melanoma21. Here we describe the presence of transforming activity in the DNA from a human melanoma cell line which shows weak homology with members of the ras oncogene family.

The ras gene family, H-ras, K-ras and N-ras, code for 21kD proteins that have GTPase activity and have been implicated in the control of cell proliferation (McKay et al. 1986; Trahey et al. 1987). Mutations in these genes give rise to abnormal protein products that have the capacity to transform certain cells to a malignant phenotype. Ras mutations have been found in a wide range of human malignancies and N-ras has been particularly implicated in AML, chronic myeloid leukaemia (CML), and acute lymphoblastic leukaemia (ALL) (Janssen et al.1985; Hirai et al. 1985). Activation of these genes has been associated with mutations in codons 12/13 or codon 61 and lesions of this type have recently been described in MDS. Hirai et al. (1987) described three MDS patients with codon 13 mutations in N-ras and Liu et al. (1987) two patients with a similar mutation in K-ras. We have assessed the mutational status of members of the ras gene family by polymerase chain reaction and hybridisation with synthetic oligonucleotide probes in 50 patients with MDS together with the use of a nude mouse tumorigenicity assay in some cases (Padua et al. 1988). We have also examined material from 70 haematologically normal patients in complete remission following cytotoxic therapy for lymphoma, but in whom a higher than normal incidence of MDS and leukaemia might be expected in subsequent years.

Activated ras genes have been implicated in a wide variety of neoplasms [2]. N-ras in particular has been shown to be involved in acute myelogenous leukaemia (AML) with activating mutations around codons 12/13 and 61 [3, 7]. The myelodysplastic syndromes (MDS) are a group of preleukaemias, a proportion of which will develop AML. Hirai et al. [9] described three MDS patients with N-ras mutations in codon 13 and Liu et al. [12] showed K-ras activations in two MDS patients. In this study we have screened DNA from peripheral blood or bone marrow of 50 MDS patients for ras mutations around codons 12/13 and 61 of H, K and N-ras and around codon 117 of N-ras. A mutation in position 117 of H-ras has been reported to be an activating mutation in vivo in chemically induced murine liver tumours [15]. Mutations in codons 116–119 of H-ras have been shown to reduce the ability of the H-ras p21 protein to bind and hydrolyse guanosine triphosphate (GTP) and some of these mutations are capable of activating the transforming potential of the normal gene [5, 20].

Summary X-chromosome DNA probes for the phosphoglycerate kinase (PGK) and hypoxanthine phosphoribosyl transferase (HPRT) genes were used to study clonality in haemopoietic cells from 63 women with myeloproliferative disease, idiopathic erythrocytosis. secondary erythrocytosis or normal red cell volumes. A total of 25 women (39%) were heterozygous for one of the polymorphisms associated with these genes. Clonality was demonstrated in five out of six patients with polycythaemia vera (PV) and in three other patients with myeloproliferative disease. In all cases of PV, including the patient in whom clonality was not demonstrated, cultures of peripheral blood showed growth of endogenous erythroid colonies.

We have found a new allele at the structural locus for glucosephosphate isomerase (called Gpi-1c) in a population of wild mice. The Gpi-1c allele codes for an enzyme of greater cathodal electrophoretic mobility than either the Gpi-1a or Gpi-1b alleles found in the wild and in the SM/J and C57BL/6J inbred strains. Mice homozygous for Gpi-1c have erythrocyte enzyme activity reduced to 33% of normal levels, altered pH profile, lowered heat stability, and normal Km's when compared with SM/J and C57BL/6J mice. The activity of the enzyme in brain, liver, and kidney is not so markedly lowered, although the electrophoretic mobility, pH profile, and heat stability are altered in these tissues. Deficiencies of erythrocyte glucosephosphate isomerase in man, to this level, can cause severe hemolytic anemia. Homozygotes for Gpi-1c show only mild hematological symptoms. The frequency of Gpi-1c in wild populations of mice is discussed and the occurrence of a further rare allele Gpi-1d is reported.

Abstract 2614 We previously demonstrated that although retinoic acid (RA) has targeted efficacy in Acute Promyelocytic Leukemia (APL), heterogeneity exists leading to the appearance of un-targeted clones at the time of relapse. Characterization of these clones is not yet fully unraveled though we and others have previously highlighted the roles of RARα mutations, pharmacogenomics or APL miRNome. We recently identified that the ERK1/2 pathway synergized with RA to restore the transcriptional activity of RA in resistant APL cells, thus restoring RA induced differentiation (Cassinat et al. Mol Cell Biol 2011). These results suggest that targeting interconnected signaling pathways could optimize differentiation therapy efficacy. To this effect, we studied known signaling pathway activators or inhibitors that could potentiate with RA and identified Lithium chloride (LiCl). Treatment of the ATRA sensitive-APL NB4 cell line with LiCl (25mM) decreases proliferation and increases apoptosis (25% and 40% of Annexin V-positive cells at day 1 and 2 respectively) with evidence of caspase 3 cleavage at day 2. Because NB4 cells fully differentiated with RA alone we were unable to observe any synergy when combined with LiCl. Treatment of the RA-resistant APL UF-1 cell line with RA or LiCl alone does not induce differentiation. Combination of RA+LiCl restores differentiation after 3 days of culture (65% CD11b positive and 55% NBT test positive cells). Similar results were obtained with different GSK3 inhibitors, suggesting that the LiCL effects were in part linked to its well characterized GSK3 inhibitory activity. Interestingly, we noted that LiCl treatment induces rapid phosphorylation of ERK1/2 and pretreatment with the MEK/ERK1/2 inhibitor UO126 fully abolished the differentiation induced by the RA+LiCl combination. The combination restores in UF-1 the expression of RA target genes (such as RARα2) to the same levels obtained in NB4 cells treated by RA alone. The level of luciferase activity of an RA responsive element reporter gene was increased with the RA+LiCl combination compared to RA alone. Both target gene expression and luciferase activiy were abolished after inhibition of the MEK/ERK1/2 pathway. Thus, increase in differentiation of UF-1 cells by RA+LiCl is linked to increased RA transcriptional activation. Similar studies in fresh APL patient cells confirmed both the increase in differentiation and level of RA target gene expression and their inhibition by UO126. Finally, to translate these findings in vivo, we used the APL-transplantable mouse model. Plasma lithium levels in treated mice were measured between 0.6 and 1.05 mmol/l, levels reached in humans. When LiCl was combined with RA we repeatedly observed a pronounced survival advantage compared to mice treated by RA alone as evaluated by Kaplan Meier analysis. In this work we demonstrate that LiCl, a well tolerated agent in humans, has the potential, when combined with RA, to restore RA induced transcriptional activation and differentiation in RA resistant APL cells. Furthermore, this combination also increases RA efficacy in an in vivo APL mouse model. Disclosures: Off Label Use: Lithium is a mood modulator administered for bipolar disorders.

Background In spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease. Methods We performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively. Results Analysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples. Conclusions These two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0235-8) contains supplementary material, which is available to authorized users.

Abstract 3308 Background: Animal models enable us to understand disease progression and provide us with reagents to test various therapeutic strategies. We have previously developed a mouse model of myelodysplasia/acute myelogenous leukemia (MDS/AML) progression using mutant NRASD12 and overexpression of human hBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). Expanded leukemic stem cells (LSC) were identified as Lin-/Sca1+/KIT+ (LSK) populations, with increased myeloid colony growth and were transplantable. Increased hBCL-2 and RAS-GTP complex were observed in both MDS/AML diseases. The MDS-like disease had increased apoptosis, whilst the AML-like mice had liver apoptosis patterns similar to wild type. The single NRASD12 line also had increased apoptosis. In this present study using a BCL-2 homology domain 3 (BH3) mimetic ABT-737 (Abbott), we have evaluated the effects of targeting BCL-2 in our preclinical models. Methods & Results: Treatment with the inhibitor shows a reduction of LSK cells, reduced progenitor numbers in colony assays and clearance of the liver infiltrations in both MDS and AML models. Gene expression profiling of the MDS mice shows regulation of 399 genes upon treatment including 58 genes expressed by the single mutant RAS mice and not expressed in the untreated AML mice. 78 genes were shared between single NRASD12 and diseased mice and not the treated mice. These studies potentially identify the contribution of NRASD12 genes to disease progression. By confocal microscopy we observed that in the MDS mice the majority of the RAS and BCL-2 co-localized to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localized in the mitochondria, where BCL-2 is normally found (Omidvar et al 2007). After treatment with the inhibitor the AML co-localization of RAS and BCL-2 shifted to the plasma membrane where single NRASD12 is normally localized. Furthermore, increased RAS-GTP levels was detected in both Sca1+ and Mac1+ enriched spleen cells and interestingly an increase in BCL-2 expression was observed in peripheral blood and in spleen cells after treatment; this increase in BCL-2 was associated with a decrease in the phosphorylation of serine 70 and an increase in phosphorylation of threonine 56 of BCL-2. ABT-737 treatment led to increased phosphorylated ERK resembling RAS and reduced MEK and AKT phosphorylation, changes detected by western blots and the nanoimmunoassay (NIA, NanoPro, Cell Biosciences) that might account for the increased apoptosis, measured by TUNEL and In vivo imaging by single-photon emission computed tomography (SPECT) using Tc-99m-labelled AnnexinV (SPECT). In contrast, although treated MDS mice had increased apoptosis they did not have an increase in overall expression of BCL-2 or in RAS-GTP levels. Treatment of both MDS and AML models with this inhibitor significantly extended lifespan from diagnosis with mean survival of 28 days untreated vs 80 days treated (p=0.0003) and mean survival from birth of 39 untreated vs 85 days treated (p Conclusions: Genomics, proteomics and imaging have been employed in the MDS/AML models to characterize disease progression and follow response to treatment to the BH3 mimetic ABT-737 in order to gain molecular insights in the evaluation of the efficacy. ABT-737 appears to target LSCs, induce apoptosis, regulating RAS and BCL-2 signalling pathways, which translated into significantly increased survival. Disclosures: Padua: Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Auboeuf:GenoSplice technology: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. de la Grange:GenoSplice technology: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Fenaux:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding. Tu:Cell Biosciences Inc;: Employment. Yang:Cell Biosciences Inc;: Employment. Weissman:Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Felsher:Cell Bioscience:. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Background The combination of ATRA and ATO is very effective in treating APL, both in mouse models (Lallemand JEM 1999) and in patients (Lo Coco NEJM 2013). Having previously shown that the combination of ATRA and a specific DNA vaccine (pCDNA3PML-RARAFrC) rescues APL mice from relapse and enhances specific immune responses (Padua Nat Med 2003; Robin Blood 2006; Furugaki Blood 2010; Pokorna Mol Cell Probes 2013), we applied this approach using a novel non-specific DNA, pVAX14, as add-on therapy to ATRA +/- ATO. Methods APL transplanted mice (APL cells injected in syngeneic 6-week old mice, Brown PNAS 1997) were treated with various combinations of ATRA +/- ATO and DNA (pVAX1 (control DNA), pCDNA3PML-RARAFrC (specific DNA vaccine) or pVAX14. pVAX14 is a novel construct containing GC-rich sequences and coding for unique peptides, 3 of which we have shown to be immunogenic. Mice were monitored for survival, MRD (by RQ-PCR of PML-RARA) on days 19, 37, 57 and 67 in peripheral blood (PB), and as a measure of LICs by secondary transplantation of bone marrow (BM) or spleen cells from long term (more than 120 days) survivors (LTS) into syngeneic lethally irradiated mice. Immune responses were assessed on CD4 Memory T-cells (Tmem), IFNg production (ELISPOT) and anti-RARa (ELISA) antibody production. Functional studies including challenge experiments of LTS with leukemic cells assessed the protective effects of immunotherapy. Results 1) Survival . In a first set of experiments, ATRA+pVAX14 significantly increased survival compared to placebo, ATRA alone or ATRA+pVAX1 (control DNA) (p ATRA+ATO > ATRA (p ATRA+pVAX14 ≈ ATRA+ATO+pVAX1 (control DNA) ≈ ATRA+ATO > ATRA+pVAX1 (Control DNA) > ATRA (p Conclusion The combination of the non-specific vaccine (pVAX14) with ATRA+ATO further improved survival over ATRA+ATO alone and elicited specific humoral and T-cell mediated responses targeting LICs. This vaccine is also effective in a mouse model of high risk MDS (Submitted to ASH 2103). SP, LG and CLP contributed equally to this work. Disclosures: Patel: CEFIPRA: Research Funding. Fenaux:Celgene: Honoraria, Research Funding. Chomienne:Vivavacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. , I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees. Padua:Vivavacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. I, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. I Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees.

Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.

Background: MDS lack therapeutic approaches in many instances and very few animal models of MDS available for preclinical testing of new drugs have been reported. We have created transgenic animal models of HR-MDS and acute myelogenous leukemia(AML) post-MDS using mutant NRAS and BCL-2 overexpression (Omidvar Cancer Res.2007;67:1657; Rassool Cancer Res. 2007;67:8762) and reported the efficacy of azacitidine in the HR-MDS model (Gorombei Blood 2013;122:1514) and of the BCL2 inhibitor, ABT-737, on the AML post-MDS model (Beurlet Blood 2013;122:2864). The objective of this study was to determine the effects of this inhibitor on our mouse model of HR-MDS. Methods: The mice were followed for disease and once the platelets fell ( Disclosures de la Grange: Genosplice: Employment. Fenaux:Celgene, Janssenm, Novartis: Research Funding.

Abstract 2938 Background: Myelodysplastic syndromes (MDS) are clonal stem cell hematological disorders characterized by ineffective hematopoiesis leading to cytopenia, which evolve to acute myeloid leukemia (AML). The ability of the DNA vaccination to induce effective immune responses has been demonstrated in different preclinical models of diseases. Having previously shown that DNA vaccination with a PMLRARaFrC plasmid in combination with all-trans retinoic acid (ATRA) induced long term remissions with appropriate immune responses in a mouse model of acute promyelocytic leukemia (APL), our aim was to use this strategy in a mouse model of MDS we have created using transgenic mice bearing a mutant NRAS and overexpression of human BCL-2. Methods: We used our MDS-like triple transgenic mice bearing NRASD12/MMTVLTRtTA/TetoBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). We cloned part of the kanamycin resistance gene and adjacent pVax1 plasmid sequences flipped so the antisense sequences were inserted into the pVax1 vector (named Flipper). These sequences code for 5 polypeptides. We treated the MDS mice with either DNA alone, ATRA alone (10mg daily release for 21 days) or a combination of ATRA+DNA or as add-on therapy with 5-azacytidine (5-aza). 5-aza (5mg/kg) was administered 3 times a week and continued until death. Treatment with ATRA and/or DNA was initiated after 12 courses of 5-aza injections. Results and Conclusions: Sequencing of the Flipper insert predicted 5 peptides, which were synthesized and shown not to affect growth in vitro of myeloid leukemic NB4, K562 and p39 cell lines. In vivo injections of healthy mice with these peptides had no effect on mortality. The inserted sequences did not appear to have detrimental effects. The studies on our MDS mouse model show that DNA vaccination, alone or in combination with ATRA induces a long-term survival of the treated mice compared to untreated controls (p Disclosures: Fenaux: Novartis, Janssen, Cilag, Roche, Amgem, GSK, Merck and Cephalon: Honoraria, Research Funding. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Padua:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The hallmark (8:14) translocation of Burkitt’s lymphoma causes MYC overexpression, a primary event in the development of Burkitt’s lymphoma. While Burkitt’s lymphoma comprises nearly one third of childhood malignancies, it is rare in adults. Among many explanations is the possibility that the capacity for a lymphocyte to undergo malignant transformation by MYC activation is influenced by factors related to the developmental state of the host. To address this possibility, we used transgenic mice in which the MYC proto-oncogene is conditionally regulated via the Tetracycline Regulatory System (Tet system). When MYC was activated in cohorts of mice less than 3 weeks of age, mice universally succumbed to lymphoma within 23 weeks. However, when MYC was activated in adult mice (over 7 weeks of age) the mice failed to develop lymphoma within 50 weeks. We were not able to detect differences in the effects of MYC on the proliferation or apoptosis in lymphocytes from adult versus neonatal hosts. We next explored whether the concomitant activation of MYC with a second oncogene could induce lymphomagenesis in adult mice. When we conditionally induced both MYC and BCL2 or MYC and RAS in adult hosts, mice succumbed to lymphoma with a mean latency of 24–27 weeks. Our results suggest that in adult hosts, concurrent activation of at least two oncogenes is required for MYC to initiate tumorigenesis.

Patients with myelodysplastic syndromes (MDS) have high frequencies of cytogenetic abnormalities and evidence is accumulating of associations between exposure history and primary MDS. The objective of this article is to examine the relationship between histories of occupational or environmental exposure and presence of cytogenetic abnormalities. A case control study of MDS patients estimated lifetime exposure to more than 90 potential hazards in 400 age, sex, and area of residence matched patient and control pairs. A parallel cytogenetics study undertaken at time of diagnosis, independently of any knowledge of exposure history, identified 75 cytogenetically abnormal and 139 normal (186 not studied). Odds ratios of MDS patients and their matched controls were compared for 3 groups: cytogenetically abnormal, normal, and not known. The odds ratios for all exposures combined were possibly higher among cytogenetically abnormal 2.0 (95% confidence interval 0.8-5.9) than among normal 1.0 (0.6-1.8). This pattern was observed for exposure to semimetals, abnormal 4.0 (0.4-195.1) and normal 0.5 (0.1-1.0) and inorganic dusts, 1.6 (0. 6-3.8) and 0.4 (0.1-1.4) respectively. The pattern was principally in abnormalities in chromosomes 5 and 7. For organic chemicals and radiation, the odds ratios for both cytogenetically abnormal and normal were marginally raised: organic 1.8 (0.6-6.0) and 1.3 (0.6-2.9), respectively, and radiation 1.7 (0.5-5.6) and 1.3 (0.4-4.7) respectively. For radiation, abnormalities were mostly in chromosome 8. This study of association between exposures and cytogenetics in primary MDS complements those previously reported in secondary MDS and may provide some insight into pathogenetic mechanisms that lead to development of MDS.

Background We have established animal models of MDS and acute myelogenous leukemia (AML) using NRASD12 and overexpression of BCL-2 (Omidvar Cancer Res 2007). These models have identified a novel MDS biomarker, the RAS:BCL-2 complex (Le Pogam, Leuk Res 2013) and a BH3 mimetic inhibitor, ABT-737, was shown to target leukemic cells and increase life span in both models (Beurlet, Blood 2013, Gorombei, EHA 2013). Here we have studied the benefit of an immunotherapeutic approach in HR-MDS by taking advantage of the reported immunomodulating effect of azacitidine (AZA) (Goodyear, Blood 2010) and of a non-specific DNA vaccine (pVAX14) we have designed. Methods pVAX14 is a non-specific DNA plasmid which, in an APL mouse model , gives similar survival to the specific PML-RARA DNA we previously designed (Padua, Nat Med 2003) (results submitted to ASH 2013). pVAX14 is a novel construct containing GC-rich sequences and coding for unique peptides, 3 of which we have shown to be immunogenic; ATRA was combined for its immunomodulatory properties. Survival efficacy was measured in mice treated with AZA alone (1mg/kg intraperitoneally 3 times per week until death), pVAX14 (6 weekly injections of 50 mg DNA injected intradermally, ATRA (10 mg 21-day release pellets) or their combinations, and untreated controls. MDS was monitored with biomarkers previously validated for this model, (Beurlet Blood 2013): peripheral blood (PB) counts, PB blasts (Mac1hi/Gr1lo) and spleen cell AKT, MEK1 and ERK1/2 levels by the Nanoimmunoassay (NIA) (Fan, Nat Med 2009). Immunomonitoring was based on lymphocyte subpopulations including Memory T-cells (CD4+/CD44hi/CD62Llo), IFNg production (ELISPOT) and Toll-like receptor-9 (TLR-9) activation measured on MYD88 transcripts by RQ-PCR. Results 1) Survival benefit: in a first cohort of HR-MDS mice, pVAX14 treatment significantly prolonged survival (median survival 100 days in treated versus 10 days in untreated mice) (Kaplan Meier p 2) Hematological parameters: survival advantage of mice treated with pVAX14 alone, AZA alone and AZA+pVAX14+ATRA was corroborated with lack of leukemic progression assessed on days 13, 32 and 55, as shown by stable platelet counts and peripheral blasts (Mac-1hi/GR-1lo population) and downregulation of RAS signaling proteins with dephosphorylation of AKT, MEK1 and ERK1/2. 3) Enhanced immune responses: survival advantage and absence of leukemic progression were correlated with enhanced immune responses: increased IFNg production (p Conclusions 1) AZA increases survival in this HR-MDS model. Immune mechanisms seem to be implicated but we are currently analyzing other potential mechanisms of action, including DNA methylation 2) pVAX14+ATRA as add-on therapy to AZA further improves survival, and potentiates the immune responses initiated by AZA. This adjuvant DNA immunotherapy may thus be a promising approach for MDS treatment. PG and CLP contributed equally to this work. Disclosures: Pierre: Celgene: Honoraria, Research Funding. Christine:VivaVacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study., I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees. Padua:Vivavacs: Equity Ownership, I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study., I have patents pending through INSERM/Paris-Diderot related to technology employed in this present study. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees.

MYC overexpression is thought to induce tumorigenesis through a variety of different mechanisms including the induction of proliferation, inhibition of differentiation and disruption of genomic stability. Conventional transgenic systems that have been used to study the role of oncogenes in tumorigenesis continuously overexpress transgenes and hence preclude the investigation of the initial and age specific consequences of oncogene activation. To investigate the developmental specific consequences of MYC overexpression in the pathogenesis of lymphoma in vivo, we used transgenic mice in which the MYC proto-oncogene is conditionally regulated via the Tetracycline Regulatory System (Tet system). The ability of MYC to induce lymphomagenesis was found to be inversely correlated with the age of the host at the time of MYC activation. When MYC was activated constitutively, the mean time until tumor development was 13 weeks. When MYC was activated at increasing developmental ages of 3 and 5 weeks, identical tumors arose, but with an increased mean tumor latency. When MYC was activated at 7 weeks, an age equivalent to an adult mouse, no tumors developed even after 40 weeks of observation. However, we found that in adult mice, if both MYC and BCL2 are overexpressed at 7 weeks of age, mice succumbed to lymphoma with a mean latency of 27 weeks. Surprisingly, we could not find evidence that MYC overexpression induces apoptosis or that BCL2 overexpression reduced apoptosis. We conclude that the ability of MYC to induce lymphomagenesis is highly dependent on the developmental context. MYC and BCL2 cooperate to permit tumorigenesis in adult mice. Since lymphoma occurs generally as a disease in adults, this suggests that previous reports may have greatly overestimated the ability of MYC activation to induce tumorigenesis and underestimated the potential cooperation between MYC and BCL2 oncogenes. Our results also suggest that BCL2 may cooperate with MYC to induce lymphomagenesis through additional mechanisms other than preventing MYC from inducing apoptosis.