MDS Cells Research Articles

MIR9 That Is Aberrantly Expressed in Myelodysplastic Syndrome Blocks Erythroid Differentiation Possibly Through STAT-5-Mediated Signals

Abstract 3842microRNA (miRNA) is a class of 18–24 nucleotides small non-coding RNAs that induce mRNA degradation and negatively modulate protein translation, and plays vital roles in physiological and pathological hematopoiesis. On the other hand, expressional alterations of miRNA are postulated to result in the development of hematological malignancies including myelodysplastic syndrome. RUNX1 belongs to a member of transcription factor that is essential in the development of fetal liver hematopoiesis and is critical in the regulation of adult hematopoiesis. In myelodysplastic syndrome, dysregulated functions of RUNX1 through formation of chimeric genes or point mutations are one of the major causes of their development. We speculate that aberrant expression of miRNA that negatively regulates RUNX1 synthesis could constitute novel mechanism by which RUNX1 is abrogated. To seek for the possibility, we selected 10 microRNAs (MIR27A, MIR27B, MIR9, MIR199A, MIR18A, MIR30A, MIR30B, MIR30C, MIR30D and MIR30E) among RUNX1-related miRNAs whose seed sequences reside within the 3′ UTR of human RUNX1 mRNA as analytical objects. Investigated were 16 MDS patients (RA, 4 cases; RAEB 9, cases; RAEB-t, 2 cases; CMMoL, 1 case), 11 normal controls in our hospital and 86 MDS patients registered to MDS cell bank (RA, 23 cases; RAEB 19, cases; RAEB-t, 18 cases; CMMoL, 3 cases; MDS-derived AML, 23 cases) As a result, as for all RUNX1-related miRNAs, a tendency for higher expression levels of miRNAs in MDS samples compared to control bone marrow samples was observed. Among them, MIR9 was highly expressed in the bone marrow cells of 3 MDS cases including 1 RA case and 2 RAEB cases (approximately 19%), while barely detected in other MDS samples or controls in our institute. MIR9 was also highly expressed in 5 cases of MDS registered in cell bank including 1 RA case, 1 RAEB case, 1 RAEB-t case and 2 MDS-derived AML cases (5.8%). At first, to confirm that MIR9 actually suppresses transcription of RUNX1, we constructed reporter plasmids containing luciferase gene followed by various length of 3′ UTR of RUNX1 mRNA. As a result of reporter assay, overexpression of MIR9 decreased lucifearse activities to approximately 50 % when reporter plasmids containing miR-9 binding site in 3′ UTR of RUNX1 mRNA (2219- and 2260-) were co-transfected in 293 cells. However, when reporter plasmids without MIR9 binding site (2329-, 2411- and 3245-) were co-transfected, overexpression of MIR9 didn’t suppress luciferase activities. Next, we established human hematopoietic cell line, UT-7/GM overexpressing MIR9. Evaluation by counting percentages of benzidine-positive cells, inhibition of hemoglobin synthesis by EPO treatment was observed in MIR9-overexpressing clones. Further, as a result of quantitative real-time PCR and FACS analyses, inhibition of erythroid specific gene (ALAS-E) induction and surface antigen (Glycopholin-A) expression were also observed in those clones. Finally, we analyzed protein expression levels of RUNX1 and several other intracellular signaling molecules that contribute to erythroid differentiation. There is no difference in RUNX1 protein level between mock and MIR9-overexpressing clones. Interestingly, phosphorylation of STAT-5 after EPO treatment in MIR9-overepressing clones was weaker in comparison with mock clones. All these data suggest the possibility that MIR9 functions as a negative regulator of erythroid differentiation possibly through inhibiting STAT-5 phosphorylation and that aberrant expression of MIR9 could at least partly contribute to ineffective erythropoiesis in MDS. Although, we further analyzed protein levels of JAK2 and SH-PTP2 to clarify the molecular basis of inhibition of STAT-5 phosphorylation, there was no obvious difference in expression of JAK2 or SH-PTP2 between mock and MIR9-overexpressing clones. Therefore, MIR9 must influence expression/function of unknown molecule(s) and thus may contribute to pathogenesis of MDS. Disclosures:No relevant conflicts of interest to declare.

Downregulation of IRS2 in myelodysplastic syndrome: A possible role in impaired hematopoietic cell differentiation

Insulin receptor substrate 2 (IRS2) is an adaptor protein that associates with the receptor of erythropoietin, insulin-like growth factor 1 and thrombopoietin; however, its role is not known in myelodysplasia. We, herein, report a significantly lower IRS2 expression in MDS cells, compared to normal cells. IRS2 expression was reduced in high-risk, compared to low-risk disease, and positively correlated with neutrophil and platelet counts. IRS2 was upregulated during erythroid differentiation of CD34+ cells from normal donors and low-risk MDS patients and also during erythroid, granulocytic and megakaryocytic differentiation in cell lines. These results suggest that defective IRS2 expression plays a role in the impaired hematopoietic cell differentiation in MDS.

Effect of Lenalidomide Treatment on Lipid Signalling Pathways in Low-Risk MDS Patients

Abstract 4635Nuclear inositide signalling pathways are involved in the MDS progression to AML. Indeed, in the last few years our group demonstrated not only that MDS cells can show alterations on PI-PLCbeta1 and Akt pathways, but also that Akt is inversely correlated with PI-PLCbeta1, therefore affecting MDS cell survival and differentiation. Lenalidomide has proven effectiveness in 70–80% low-risk MDS cases with del(5q), resulting in transfusion-independence with a rise in hemoglobin levels, suppression of the 5q clone, and improvement of bone marrow morphologic features. In particular, in del(5q) MDS, Lenalidomide probably acts by directly suppressing the dysplastic clone, while in non-del(5q) it might enhance an effective erythropoiesis, possibly via activation of the EPO signalling, which in turn is associated with PI-PLCgamma1 pathways. However, the exact molecular mechanisms underlying the effect of Lenalidomide in MDS cells are still not completely clarified. Interestingly, Lenalidomide might inhibit the phosphatase PP2A, whose gene is located in the common deleted region and which usually targets Akt. Indeed, Akt-dependent pathways are critical in low-risk MDS cells, which display a marked apoptosis and a low proliferation rate. In this study we examined four MDS patients treated with Lenalidomide, and compared them with four low-risk MDS patients (IPSS: Low or Int-1) who only received best supportive care. In our study, all of the patients treated with Lenalidomide were affected by del(5q) low-risk MDS (IPSS: Low or Int-1), with transfusion-dependent anemia, and had only received supportive care before undergoing Lenalidomide treatment. Clinically, all of the patients responded to Lenalidomide: one patient reached Complete Remission, whilst the other three patients showed erythroid Hematologic Improvement. In contrast, all of the patients who were treated only with best supportive care maintained a Stable Disease. As for the molecular effects of Lenalidomide on lipid signalling pathways, we analyzed the expression of critical molecules involved in both cell proliferation and differentiation, that is PI-PLCbeta1 and its downstream target Cyclin D3, as well as PI-PLCgamma1, which is linked with EPO signalling. Ongoing analyses are also trying to examine the effect of Lenalidomide on Akt phosphorylation and Globin genes, which are specifically associated with erythropoiesis. So far, our results indicate that, in our responder patients, both PI-PLCbeta1 and Cyclin D3 are not significantly affected by Lenalidomide, whereas PI-PLCgamma1 is specifically induced, as compared with both healthy subjects and low-risk MDS patients treated with supportive care. Overall, these findings hint at a specific activation of PI-PLCgamma1 signalling following Lenalidomide treatment, and possibly pave the way to larger investigations aiming to better understand the role of these pathways in the mechanism of action of Lenalidomide in del(5q) MDS. Disclosures:No relevant conflicts of interest to declare.

A Phase II Clinical Study of Erlotinib for Treatment of Myelodysplastic Syndromes

Abstract 1714 BACKGOUNDErlotinib is an oral small molecule tyrosine kinase inhibitor that inhibits intracellular EGFR tyrosine kinase. Preclinical data suggest that erlotinib has in vivo and in vitro efficacy in MDS and AML (Boehrer et al., Blood, 2008), inducing apoptosis in MDS and AML cell lines and primary myeoblasts, and promoting myeloid differentiation. The antitumor effect of erlotinib was demonstrated in AML xenograft mouse model. Anecdotal case reports documented the haematological activity of erlotinib in patients who had lung cancer and concomitant MDS or AML. The precise non-EGFR kinase targets responsible for erlotinib activity in myeloid malignancies are not clear, but may involve aberrant Lyn, Syk and mTOR-mediated signaling. We report results of a phase II trial examining the activity of erlotinib in the treatment of poor prognostic group of myelodysplastic syndromes patients. METHODSThis is a single-institution two-stage phase 2 study. Eligibility includes confirmed int-2 or high risk MDS, low or int-1 risk patients with symptomatic anemia/ transfusion dependent anemia or patients with platelet counts < 50 × 109/L or a significant clinical hemorrhage requiring platelet transfusions, ANC < 1 × 109/L, and RAEB-t by FAB classification are also. All patients signed written infomred consent. Prior intensive induction chemotherapy, malignancy in the past 2 years, known history of HIV infection, ECOG PS 3–4 were key exclusions. Erlotinib was given as an oral 150 mg daily dose for 16 weeks. The dose was adjusted for diarrhea, rash and pulmonary toxicty. Study assessments included baseline and weekly complete blood counts and repeat bone marrow aspirate and biopsy at weeks 8 and 16; non-responders were removed from study after 16 weeks. Stable or responding patients continued study treatment until evidence of disease progression or relapse. The primary endpoint was the overall response rate ORR (CR, PR, marrow CR, or HI) as defined by the IWG 2006 criteria. Secondary endpoints inluced overall survival (OS), prgoression free survival (PFS), and leukemia free survival (LFS). The study was approved by scientific review committee and IRB. RESULTSBetween September 2009 and January 2011, 39 patients signed consent at Moffitt Cancer Center (4 patients were found to be ineligible after signing informed consent, one patient had CML blast crisis transformation upon review but received < 1 week erlotinib). For the 35 patients treated on protocol, the mean age was 73 years, 80% were male, and majority 92% were Caucasian. According to the WHO classification cases included RCMD 2, RAEB-I 8, RAEB-II 9, CMML 6, AML 8 (RAEB-t by FAB), MDS/MPN-U 1. The IPSS risk group was low in 2 patients (4.2%), int-1 in 6 (17.14%), int-2 in 13 (37.14%) and high risk in 13 (37.14%) when applicable. The median number of prior treatments was 2, All patients had received a hypomethylating agent (azacitidine or decitabine). The median duration of follow up was 17 month.The best responses for all eligible subjects on study by IWG criteria were 3 marrow CR, 2 HI, and 11 stable disease, for a combined ORR of 5/34 (14.7%). 4 death occurred on study (sepsis, intracranial hemorrhage, sudden death, AML). Nine patients were not evaluable for response (withdrew consent or off study due to adverse event before first evaluation). The overall best response for evaluable patients (n=26) was 5/26 (19%) (3 marrow CR and 2 HI). The most common observed grade 3/4 toxicity according to CTCAE v3 were diarrhea (17.1%), rash (17.1) %, fatigue, thrombocytopenia, and anorexia (5.7%) for each.The median OS survival was 6.8 month (95%CI 4.9–13.2), the median PFS was 3.6 mo (95%CI 2–4.8), and LFS was 5 mo (95%CI 3.4–7.3). The median OS was 16.5 mo (95%CI 2.9–30) for patients who had HI or mCR, 7.1 mo (95% CI 0–12.5) for patients who had stable disease, and 5 month (95%CI 1–9) for patients who had progressive disease or were not evaluable. (p=0.06). ConclusionErlotinib has acceptable toxicity profile in MDS patients and anti-leukemia activity in poor risk MDS patients after azanucleosides failure. Preclinical data suggest potential synergism in combination with azanucleosides and our future direction is to test erlotinib in combination with azacitidine for higher risk MDS patients. Disclosures:Komrokji:Genentech: Research Funding. Off Label Use: erlotinib for treatment of MDS.

Azacitidine Is Feasible and Safe in Patients with MDS and Co-Existing Plasma Cell Dysplasias (PCD) - Report on 9 Patients From the Austrian Azacitidine Registry (AAR)

Abstract 5056Azacitidine (AZA) has been approved for the treatment of higher-risk MDS and therefore marks a cornerstone in the development of hypomethylating agents. Recent clinical trials raise questions regarding optimal sequencing and also about combining hypomethylating agents with other classes of drugs targeting epigenetic processes. It has been postulated that unspecific demethylation of promotors may lead to the activation of oncogenes, and therefore result in an elevated rate of secondary malignancies.We report on 9 patients from the Austrian Azacitidine Registry (AAR) with co-excisting plasma cell dyscrasias (PCD) receiving treatment with AZA for MDS. Six male and 3 female patients had a median age of 73 years at time of diagnosis of MDS and 73 years at time of diagnosis of PCD. They were diagnosed with Waldenstrom macroglobulinemia (WMG) (n=3), multiple myeloma (n=2) and MGUS (IgG (n=2), IgM (n=1), IgA (n=1)). All PCD were diagnosed either before or at least at the same time as MDS. None occurred after diagnosis of MDS or after treatment initiation with AZA. However, one patient with multiple myeloma likely had treatment–related MDS due to prior treatment with MPT (melphalan, prednisone, thalidomide).At the time of writing, the mean number of AZA cycles given was 9 (range 1–27). Two patients were concomitantly treated with Rituximab (R) whilst on AZA treatment. Importantly, no additional toxicities occurred and there seemed to be no negative interaction between Rituximab and AZA. In all patients treated with AZA for MDS, M-gradient remained stable, and in some cases significant improvement was noted. One patient even achieved complete remission of WMG following AZA/R treatment (data will be shown; Weiss L et al, J Clin Oncol. 2011 Jul 25 [Epub ahead of print]). Importantly, no progression of PCD was seen in any of the 9 patients treated with AZA, and PCD did not contribute to death in any of these patients.In conclusion, AZA treatment of MDS in patients with co-existing PCD seems safe, combination with Rituximab seems feasible, no aggravation of PCD was seen and remarkably, one patient with WMG achieved CR following AZA/R. Disclosures:Pleyer:Celgene: Research Funding. Egle:AOP Orphan Pharmaceuticals AG: Research Funding. Greil:AOP Orphan Pharmaceuticals AG: Research Funding.

Final Results from an International, Multi-Center, Single-Arm Study Evaluating the Safety and Efficacy of Romiplostim in Adults with Primary Immune Thrombocytopenia (ITP),

Abstract 3279▪▪This icon denotes a clinically relevant abstract Background:ITP is an autoimmune disorder characterized by increased platelet destruction and suboptimal platelet production. Romiplostim stimulates platelet production via the TPO-receptor, and is recommended for second- and third-line treatment of chronic ITP in adults. We report final data from a large prospective study of romiplostim in adults with ITP of varying duration and severity. Methods: Eligibility criteria were broad: patients ≥18 years of age, who had received prior ITP therapies (final protocol amendment: ≥1, previous amendments: ≥3), with low platelet counts (final amendment: ≤ 30 × 109/L, previous amendments: ≤ 10, ≤ 20 × 109/L) or experiencing uncontrolled bleeding. The only excluded comorbidities were: hematological malignancy, myeloproliferative neoplasms, MDS and bone marrow stem cell disorder. Romiplostim was initiated at 1 (final amendment) or 3 (previous amendments) μg/kg/week, with dose adjustments allowed to maintain platelet counts ≥50 × 109/L. Patients could continue on study until they had access to commercially available romiplostim. Rescue medications were allowed at any time; concurrent ITP therapies could be reduced when platelet counts were > 50 × 109/L. Primary endpoint was incidence of adverse events (AEs) and antibody formation. Secondary endpoint was platelet response, defined as either (1) doubling of baseline count and ≥ 50 × 109/L or (2) ≥20 × 109/L increase from baseline. Results: A total of 407 patients received romiplostim, 60% of whom were female. Median (Q1, Q3) time since ITP diagnosis was 4.25 (1.20, 11.40) years (maximum 57.1 years), with 51% of patients splenectomised and 39% receiving baseline concurrent ITP therapies. Seventy-one percent of patients completed the study, with requirement for alternative therapy and withdrawn consent the most common reasons for discontinuation (5% each). Median (Q1, Q3) on-study treatment duration was 44.29 (20.43, 65.86) weeks (maximum 201 weeks), with a total of 20,201 subject-weeks on study. Incidence and type of AEs were consistent with previous studies. The most common serious treatment-related AEs were cerebrovascular accident, headache, bone marrow reticulin fibrosis (with no evidence of positive trichrome staining for collagen and no evidence suggesting primary idiopathic myelofibrosis), nausea, deep vein thrombosis, hemorrhage and pulmonary embolism, with each reported in 2 of 407 (0.5%) patients. All other serious treatment-related AEs were each reported in one patient. Eighteen patients died; 3 deaths (hemolysis, intestinal ischaema, aplastic anemia) were considered treatment-related. No neutralizing antibodies to romiplostim or TPO were reported. Approximately 90% of patients achieved each of the platelet response definitions, regardless of splenectomy status. Overall, median (Q1, Q3) time to response was 2 (1, 4) weeks for response definition 1, and 1 (1, 3) week for response definition 2. Median (Q1, Q3) baseline platelet count was 14 (8, 21) × 109/L. After 1 week of treatment median (Q1, Q3) platelet count had increased to 42 (18, 101) × 109/L. From week 8 onwards, and excluding counts within 8 weeks of rescue medication use, median platelet counts were consistently above 100 × 109/L (range 101.0–269.5 × 109/L). Median (Q1, Q3) average weekly romiplostim dose was 3.62 (1.99, 6.08) μg/kg. Summary/conclusions: This is the largest prospective study in adult ITP reported to date. The data reported here are similar to those reported for previous romiplostim studies, with romiplostim able to safely induce a rapid platelet response in adult ITP patients with low platelet counts or bleeding symptoms. Romiplostim is an important, well-tolerated, treatment option for adult ITP patients, which significantly increases and maintains platelet counts.Adverse Event Subject IncidenceN=407n (%)All AEs377 (93)Most common AEsHeadache119 (29)Arthralgia80 (20)Fatigue75 (18)Nausea72 (18)Epistaxis70 (17)Nasopharyngitis69 (17)Petechiae63 (16)Contusion62 (15)Serious AEs122 (30)Treatment-related AEs193 (47)Serious, treatment-related AEs29 (7)Platelet ResponseSplenectomisedNon-splenectomisedOverallN = 208N = 199N = 407n (%)n (%)n (%)Subjects achieving a doubling of baseline platelet count and a count ≥50 × 109/L183 (88)187 (94)370 (91)95% CI(83, 92)(90, 97)(88, 94)Subjects achieving a ≥20 × 109/L increase from baseline191 (92)189 (95)380 (93)95% CI(87, 95)(91, 98)(90, 96) Disclosures:Janssens:Amgen: Consultancy; Roche: Speakers Bureau; GSK: Membership on an entity’s Board of Directors or advisory committees. Tarantino:Cangene corporation: Research Funding; Baxter: Research Funding; Talecris: Honoraria, Speakers Bureau; Up-to-date: Patents & Royalties; The Bleeding and Clotting Disorders Institute: Board Member. Bird:Amgen: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity’s Board of Directors or advisory committees. Boccia:Amgen: Equity Ownership, Honoraria, Speakers Bureau. Lopez-Fernandez:Amgen: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Kozak:Amgen: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Steurer:Amgen: Honoraria. Dillingham:Amgen Limited: Employment, Equity Ownership. Lizambri:Amgen: Employment, Equity Ownership.

RNAi Screening Identifies BCL-XL As An Erythroid Lineage-Specific 5-Azacytidine Sensitizer While the BCL-2/BCL-XL/BCL-W Inhibitor ABT-737 Results in More Universal Sensitization in Leukemia Cells,

Abstract 3513To identify targets for rational combination therapies with 5-Azacytidine (5-Aza) in myeloid malignancies, we utilized high-throughput RNA-interference (RNAi) viability screening. A siRNA library targeting 572 kinases and a custom collection of 289 putative cancer targets, including cell cycle and apoptosis regulatory genes, were screened alone and in combination with 5-Aza in TF-1 and ML-2 myeloid leukemia cell lines to identify synergistic interactions in reducing cell viability. Of the 572 kinases that were individually silenced, less than 1% significantly increased sensitivity to 5-Aza. The kinase library was also screened in combination with 5-Aza in a third myeloid cell line, THP-1, confirming that few kinases sensitize to 5-Aza when inhibited. While few kinases sensitized to 5-Aza, the anti-apoptotic Bcl-2 family of genes emerged as potent sensitizers to 5-Aza from RNAi screens. Therefore, silencing by siRNA of BCL-XL, BCL-2, BCL-W, MCL-1 and BFL-1 was evaluated in combination with 5-Aza treatment in an expanded panel of myeloid cell lines including TF-1, HEL, THP-1, ML-2 and MDS-L. BCL-XL validated as a vulnerability and potent sensitizer to 5-Aza in erythroid leukemia cell lines TF-1 and HEL, whereas MCL-1 was a strong vulnerability in the monocytic leukemia cell line THP-1 and also a moderate sensitizer to 5-Aza in ML-2, THP-1, TF-1 and HEL. Published proteomics data from our group indicate that M6 and M7 leukemias exhibit higher levels of BCL-XL, while additional unpublished data suggest elevated levels of MCL-1 in M4 and M5 leukemias, supporting our functional observations. Additionally, data from the public database Oncomine suggest that BCL-XL expression is elevated in M6 and M7 leukemias while MCL-1 shows a trend towards elevation in M4 and M5 leukemias. Based on RNAi screening results, siRNA validation experiments and proteomic/mRNA expression data, we evaluated the BCL-2/BCL-XL/BCL-W inhibitor ABT-737 in combination with 5-Aza. ABT-737 resulted in dose-dependent sensitization to 5-Aza in all AML-derived cell lines examined (including M7, M6, M5, M4 and M2 FAB subtypes) and in the MDS cell line MDS-L; however, no sensitization was observed in the CML cell line K562. In extensive ex vivo experiments with 17 primary specimens, potent synergy between 5-Aza and ABT-737 was observed in AML, MDS and MPN samples, but not in most CML samples examined. Calculations with CalcuSyn software demonstrate synergy, with combination index values as low as 0.2, between 5-Aza and ABT-737 both ex vivo and in vitro. The combination of 5-Aza with ABT-737 resulted in substantial induction of apoptosis, measured by the induction of cleaved caspase 3 in TF-1 and HL-60 cells, as compared to either compound alone. Interestingly, although siRNA silencing of MCL-1 in combination with 5-Aza was potent across several cell lines, and silencing of BCL-XL preferentially in an erythroid differentiation background, ABT-737 with 5-Aza sensitized across a variety of cell lines and all myeloid primary specimens ex vivo. We suggest that inhibition of anti-apoptotic Bcl-2 family members is a most promising rational combination strategy with 5-Aza for the treatment of leukemias. Our results also highlight the potential utility of more specific anti-apoptotic Bcl-2 family inhibitors in the lineage-specific treatment of myeloid malignancies. Disclosures:Off Label Use: AraC in AML. Experimental Agent MK1775. Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding.

279 Methylation of Wnt antagonists and effects of AZA treatment on Wnt pathway in MDS cells

279 Methylation of Wnt antagonists and effects of AZA treatment on Wnt pathway in MDS cells

231 Azacitidine-resistant SKM1 MDS cells are defective for mitochondrial apoptosis and exhibit increased basal autophagy

231 Azacitidine-resistant SKM1 MDS cells are defective for mitochondrial apoptosis and exhibit increased basal autophagy

Induction of Autophagic Cell Death Circumvents Azacitidine-Resistance In Myelodysplastic Syndrome-Derived Cell Lines

AbstractAbstract 1817Azacitidine (AZA) is the first line treatment for IPSS (index prognostic scoring system) high-risk myelodysplastic syndrome (HR-MDS). To date, only Khan et al. (Exp Hematol. 2008) and Hollenbach et al. (PLoS One. 2009) have reported apoptosis as a mechanism of AZA effect on MDS cell lines. Nevertheless, approximately 40% of patients treated with AZA are refractory to this molecule. To investigate the possible mechanisms of AZA resistance in MDS cells, we developped AZA-resistant cell clones (AZA-R) from the well-characterized MDS cell line SKM1. The bulk resistant SKM1 cell line (AZA-R) was obtained following long time exposure of cells to iterative and increasing doses of AZA ranging from 0.1 to 8mM. We first showed that AZA triggered loss of cell metabolism in SKM1 parental cells but not in their AZA-resistant counterpart at a maximally effective dose of 1mM. AZA-mediated loss of cell metabolism accounted mainly for induction of apoptosis as judged by both an increase in caspase 9 and 3 activities triggered by this compound and by a significant protection in the presence of the pan-caspase inhibitor Z-VAD-fmk in SKM1 parental cells. Conversely, no or very few activation of caspases and apoptosis were detected in AZA-R cells strongly suggesting that apoptosis is impaired in AZA-R SKM1 cells. Finally, unlike in SKM1 cells, AZA failed to induce mitochondrial membrane permeabilization in AZA-R SKM1 cells. Importantly, basal autophagy was increased in AZA-R versus AZA-S cells as shown by LC3-I cleavage into LC3-II, p62/SQSTM1 protein expression, cathepsin B activation, mTOR and S6 ribosomal protein dephosphorylation and finally electronic microscopy experiments. In addition, Acadesine, an adenosine derivative and AMPK agonist which targets autophagy was capable to circumvent AZA resistance in both AZA-R SKM1 cells and in medullary cells from five MDS patients resistant to AZA after 6 cycles of Azacitidine. In conclusion, targeting autophagy appears as an attractive therapeutical strategy to circumvent AZA resistance in both established MDS cell lines and cells from MDS patients. Therefore, drugs capable of inducing autophagy or autophagic cell death, such as Acadesine (Robert et al., PLoS One. 2009) which is currently in phase II clinical trials for the treatment of Chronic Lymphoblastic Leukemia could be also beneficial for HR-MDS patients resistant to AZA. Disclosures:Cluzeau:Celgene: Consultancy. Raynaud:Celgene: Consultancy.

IRS2 Is Dowregulated In Primary MDS Cells and During MDS Erythroid Differentiation

AbstractAbstract 1886Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia (AML). The abnormal differentiation of myeloid and erythroid cells is probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling, from upstream activators, to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R) and Erythropoietin receptor (EPOR). It is upregulated and phosphorylated by EPO in normal bone marrow erythroblasts and in UT-7 leukemic cells, as well as in HL60 leukemic cells during granulocytic-differentiation upon DMSO induction. IGF1 signaling is capable of inducing differentiation in several cell types and plays an important role in the regulation of human erythropoiesis. EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. In view of the role of IRS2 in the erythroid and granulocytic differentiation process, we hypothesized that IRS2 might be related to the deficient differentiation of MDS cells and disease progression. The aim of the present study was to characterize the mRNA and protein expression levels of IRS2 in cells of MDS patients and normal donors and to analyze the IRS2 expression levels between low-risk and high-risk of MDS. We also elucidated the expression levels of IRS2 transcripts during erythroid differentiation of CD34+ cells from normal donors and MDS patients. We studied 12 healthy donors and 29 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification; 15 low-risk [RCUD/RCMD/RARS] and 11 high-risk [RAEB-1/RAEB-2] according to WHO classification; 22 low/INT-1 risk and 7 INT-2/high risk according to IPSS; 25 low risk and 4 intermediate/high risk cytogenetic). RT-PCR was performed in total cell from bone marrow samples for gene expression studies. Protein expression was evaluated by Western blot in bone marrow mononuclear cells from MDS patients or in peripheral blood CD34+ from normal donors. Erythroid-differentiation was performed in CD34+ bone marrow cells from 4 normal donors and 4 MDS patients. IRS2 gene expression was significantly decreased in primary MDS cells compared with normal cells (0.74 [4.06-0.15] vs. 4.71 [11.78-0.66], P<0.0001). Western blot analysis corroborated these findings. According to FAB and WHO classifications, real time RT-PCR demonstrated a significantly lower expression of IRS2 in high-risk MDS samples when compared with low-risk: FAB, 0.34 [0.15–1.56] vs. 1.05 [0.19–4.06], P=0.0172; WHO, 0.30 [0.15–1.44] vs. 1.00 [0.19–4.06], P=0.0204. Based on the IPSS classification and cytogenetic risk group, the expression levels of IRS2 were similar between the low and high-risk groups. During erythroid differentiation, we evaluated IRS2 gene expression of CD34+ cells on days 6, 8 and 12 of culture. On day 12 of normal CD34+ erythroid differentiation, there was an 8.25-fold increased in IRS2 expression, compared to day 6. Interesting, MDS CD34+ cells showed a lower increment in IRS2 transcripts at the same time point (3.89-fold increase only). In conclusion, the down regulation of IRS2 in primary MDS cells and the lower increase in its expression during MDS erythroid differentiation, suggest a role of IRS2 to maintain the effective hematopoiesis. Moreover, IRS2 lower expression in high-risk group, suggests that IRS2 plays a role in the MDS pathophysiology and disease progression. Supported by FAPESP and CNPq. Disclosures:No relevant conflicts of interest to declare.

Erlotinib Increases Efficacy of 5-Azacitidine (AZA) by Inhibiting Drug Efflux Via ABC-Transporters

AbstractAbstract 974 Background:We and others showed that the TKI erlotinib (Erlo) has in vivo and in vitro efficacy in MDS and AML (Boehrer et al., Blood, 2008) and clinical studies defining more closely its therapeutic benefit are underway in MDS, including by our group (NCT00977548, NCT01085838). We tried to define in this preclinical study the potential interest of combining Erlo to hypomethylating agents, which have become reference treatments in MDS. Methods:Erlo (10mM) was combined to AZA, (2mM) or decitabine (DAC, 2mM) and apoptosis over-time (24, 48, 72h) quantified by DioC3(6)/PI staining in MDS- and AML-derived cell lines (MOLM-13, KG-1, HL-60) and ex vivo patient (pt) MDS and AML cells. To fully evaluate the extent of Erlo-induced sensitization, efficacy was compared to apoptosis induced by AraC (100nM), doxorubicine (Dox, 100nM), VP-16 (1mM) +/− Erlo. Single drug dosages had been chosen since they caused little to no apoptosis. Efflux via P-gp (by staining with DioC23), MRP (by calcein) and was quantified by FACS, specific biochemical efflux inhibitors (CSA, MK-571, KO143) served as controls, intracellular retention of mitoxantrone (MTZ) assessed overall functionality of ABC-transporters. Surface expression of P-gp was quantified by FACS. Results:Whereas co-incubation of Erlo with DAC did not increase apoptosis in any of the myeloid cell lines, combination of Erlo with AZA exhibited synergistic effects already observed at 24h (HL-60: Erlo: 3%, AZA: 10%, Erlo+ AZA: 30%), and increasing over-time (72h: Erlo: 6%, AZA: 15%, Erlo+ AZA: 40%). Noteworthy, the degree of apoptosis observed by combining Erlo+ AZA was comparable to cell death induced by 1mM VP-16, and largely exceeded apoptosis observed with 100nM Dox and 100nM AraC in the respective cell lines. To determine if Erlo also impacts on apoptosis in MDS- and AML-derived cells, we first screened 5 pt samples (with MDS or AML) for apoptosis observed under AZA and DAC, and demonstrated that, in all samples, AZA-induced apoptosis exceeded DAC-induced apoptosis (by up to 40% at 24h). Noteworthy, the “peak” of apoptosis in sensitive pt cells occurred at 24h and – under single agents - did not increase over-time. Co-incubation with Erlo and AZA was able to induce synergistic effects already at 24h (AML post MDS: Erlo: 10%, AZA: 33%, Erlo+ AZA: 55%) and apoptosis increased over-time. In addition, whereas malignant CD34- cells already showed a high rate of “spontaneous” apoptosis (that is in the absence of any drug, about 50% of apoptosis at 48h), CD34+ blasts not only exhibited a lower rate of spontaneous apoptosis (remaining below 15% at 72h), but also a considerable degree of chemoresistance (48h: no increase of apoptosis under AraC, Dox or VP-16). Testing if the combination of Erlo+ AZA also targets this resistant population, we showed that it is particularly efficient in CD34+ progenitors (inducing 80% cell death at 72h), exceeding efficiency of all other tested agents/combinations (AraC, Dox, VP-16, Dec+/−Erlo). Hypothesizing that this pattern of sensitization might be due to a decreased drug efflux via ABC-transporters (overexpressed on malignant myeloid progenitors), we tested this concept on cell lines and ex vivo pt cells. We found that erlotinib can inhibit drug efflux via P-gp (as evidenced by increased intracellular retention of DioC23) and MRP (increased retention of calcein). Notably, Erlo increased intracellular accumulation of MTZ in MDS- and AML-derived CD34- and CD34+ blasts, and exhibited - as single agent - a comparable degree of efflux inhibition as the combination of all three biochemical inhibitors. To more closely define the advantageous effect of Erlo+AZA in pt cells, we quantified drug-induced changes in P-gp expression separately in CD34+CD38dim and CD34dimCD38+ blasts. We found that Erlo down-regulates P-gp surface expression (decrease of 50% at 48h), and that this down-regulation is more pronounced in the immature CD34+CD38dim population than in the CD34dimCD38+ blasts. Conclusions:Our study shows erlotinib's ability to reverse the drug-resistance phenotype in malignant blasts and its interesting combination with AZA. We also provide novel evidence that Erlo (combined to AZA) can target chemoresistant myeloid progenitors by diminishing functionality as well as surface expression of ABC-transporters. Those results suggest a potential clinical interest of combining erlotinib to AZA in MDS Disclosures:Fenaux:CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.

Erlotinib for Treatment of Myelodysplastic Syndromes: A Phase II Clinical Study

AbstractAbstract 1854 Backgound:Erlotinib is an oral small molecule tyrosine kinase inhibitor that inhibits intracellular EGFR tyrosine kinase. Preclinical data suggest that erlotinib has in vivo and in vitro efficacy in MDS and AML (Boehrer et al., Blood, 2008), inducing apoptosis in MDS and AML cell lines and primary myeoblasts, and promoting myeloid differentiation. The antitumor effect of erlotinib was demonstrated in AML xenograft mouse model. Isolated case reports documented the haematological activity of erlotinib in patients who had lung cancer and concomitant MDS or AML. The precise non-EGFR kinase targets responsible for erlotinib's activity in myeloid malignancies are not clear, but may involve aberrant Lyn, Syk and mTOR-mediated signaling. We report interim analysis results of a phase II trial examining the activity of erlotinib in the treatment of myelodysplastic syndromes. Methods:This is a single-institution two-stage phase 2 study. Eligibility includes confirmed int-2 or high risk MDS, low or int-1 risk patients with symptomatic anemia/transfusion dependent anemia or patients with platelet counts < 50 × 109/L or a significant clinical hemorrhage requiring platelet transfusions, ANC < 1 × 109/L, and RAEB-t by FAB classification are also eligible. All patients signed written infomred consent. Prior intensive induction chemotherapy, malignancy in the past 2 years, known history of HIV infection, ECOG PS 3–4 were exclusions. Erlotinib was given as an oral 150 mg daily dose for 16 weeks. The dose was adjusted for diarrhea, rash and pulmonary toxicty. Study assessments included baseline and weekly complete blood counts and repeat bone marrow aspirate and biopsy at weeks 8 and 16; non-responders were removed from study after 16 weeks. Responding patients (at least hematological response) continued study treatment until evidence of disease progression or relapse. The primary endpoint was the overall response rate ORR (CR, PR, marrow CR, or HI) as defined by the IWG 2006 criteria. ORR in at least two of the first 20 patients (stage 1) was necessary to proceed with expanded accrual of 15 additional patients. The study was approved by scientific review committee and IRB, and monitored by independent internal montioring committee. Results:Between September 2009 and March 2010, 25 patients were enrolled at Moffitt Cancer Center (two patients were found to be ineligible after signing informed consent, the first had myeloblasts >30% and the second patient had low grade MDS progressed to CML, but treated). For the 24 patients treated on protocol, the median age was 71 (range: 64–80) years, 18 patients (75%) were male. Majority were Caucasians (n=23) (96%). According to the WHO classification cases included RCMD 1 (4.2%), RAEB-I 8 (33.3%), RAEB-II 8 (33.3%), CMML 2 (8.3%), AML 4 (16.7%), and MDSU 1 (4.2%). The IPSS risk group was low in 2 (8.3%) patients, int-1 in 7 (29.2%), int-2 in 8 (33.3%) and high risk in 7 (29.2%). The median number of prior treatments was 2 (range: 1–4); all patients (100%) had received a hypomethylating agent (azacitidine or decitabine).The best responses for all 23 eligible subjects treated on study by IWG 2006 criteria were 3 marrow CR (13.0%), one HI (4.4%), and 6 (26.1%) stable disease, for a combined ORR of 4/23 (17.4%, 95% CI: 5%-39%). Four deaths occurred on study (sepsis=1, intracranial hemorrhage=1, sudden death=1, AML=1). The most common observed grade 3/4 toxicity according to CTCAE v3 in 24 treated patients were diarrhea (5, 20.8%), platelets (4, 16.7%), and rash: acne/acneiform (4, 16.7%). Conclusion:Erlotinib has acceptable toxicity profile in MDS patients and anti-leukemia activity in higher risk MDS patients after hypomethylating agents' failure. The study has proceeded to the stage 2 expansion. Disclosures:Komrokji:Genentech: Research Funding. Off Label Use: Erlotinib use for MDS is investigational.

IRS1 and SHP2 Signaling in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

Abstract 1779Poster Board I-805 IntroductionMyelodysplastic syndrome (MDS) encompasses a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia (AML). The occurrence of acute leukemia results from a combination of mutations and changes in protein functions that confer the ability of proliferation, defects in cell differentiation and apoptosis. The deregulation of multiple cellular signaling pathways in MDS and acute leukemia hinders the development of an effective drug for treatment. SHP2 is a cytoplasmic tyrosine phosphatase encoded by the gene PTPN11. The SHP2 signaling pathway is involved in important functions as regulation of proliferation, differentiation and cell migration and has been described as overexpressed and constitutively phosphorylated in acute and chronic leukemic cells. The IGF1/IRS1 signaling pathway has an important role in the development of various cancers such as breast, colon and prostate cancer and multiple myeloma. Increased expression of IRS1 has been described in neoplastic transformation, and reduction of IRS1 expression has been related to the level of differentiation of neoplastic cells and with a poor prognosis in solid tumors. In acute lymphoblastic leukemia (ALL), IRS1 expression was noted to be overexpressed and correlated with prognosis. The expression of SHP2 and IRS1 in MDS has not yet been elucidated, as well the expression of IRS1 in AML. The aim of the present study was to investigate the gene and protein expression of IRS1 and SHP2 in MDS and AML patients, and also PTPN11 exon 3 mutations. Patients and MethodsWe studied 06 healthy donors, 26 patients with MDS (18 low-risk [RA/RARS] and 07 high-risk [RAEB/RAEBt] according to FAB classification), 24 with AML and 5 with ALL. Samples were obtained from bone marrow. Gene expression was evaluated by real time RT-PCR in total cells. Western blot of mononuclear cells was performed for protein expression. PTPN11 mutations were analyzed by PCR with specific primers and sequencing. ResultsReal time RT-PCR demonstrated a reduced expression of IRS1 in MDS cells (0.13 [0.01-1.19], P=0.01) and AML cells (0.10 [0.009-7.14], P=0.02), when compared to normal cells (0.62 [0.10-1.41]). On the other hand, IRS1 expression was significantly increased in LLA samples (2.35[0.73-11.64] P=0.03) when compared to normal cells. Western blot analysis confirmed the gene expression pattern: IRS1 expression and phosphorylation were down-regulated in MDS and AML and upregulated in ALL when compared to normal hematopoietic cells. Regarding PTPN11 expression, no difference was detected in MDS (1.03 [0.07-4.96], P >0.05) and AML cells (0.48 [0.17-2.29], P >0.05) when compared with normal hematopoietic cells (0.56 [0.2-1.0]). Interesting, PTPN11 expression was increased in low risk MDS (1.35 [0.12-4.96], P=0.048) or total MDS (1.03 [0.07-4.96], P =0.039) when compared to AML cells. Western blot analysis confirmed the SHP2 expression pattern. However, SHP2 phosphorylation was marked increased in both MDS and AML compared to normal hematopoietic cells. PTPN11 mutation was not detected in the samples. ConclusionsOur results show that IRS1 expression and phosphorylation is down-regulated in MDS and AML patients compared to normal hematopoietic cells, suggesting that IGF1/IRS1 signaling pathway is differently regulated in normal cells, MDS, AML and ALL. IRS1 overexpression in ALL is in agreement with previous authors and indicates IRS1 as a possible target in ALL, thought not MDS and AML. SHP2 activation is increased in MDS and AML, and was independent from PTPN11 mutation. The increased activation of SHP2 in MDS, not previously described, indicates that this tyrosine phosphatase may play a role in MDS pathophysiology and leukemia transformation.Keywords: IRS1, SHP2, leukemia, myelodysplasia DisclosuresNo relevant conflicts of interest to declare.

Role of GATA-1 and HSP70 in the Dyserythropoiesis of Early Myelodysplastic Syndromes.

Abstract 3823Poster Board III-759Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell disorders characterized by a hypercellular dysplastic bone marrow (BM) with peripheral blood cytopenias, mainly anemia. Early MDS with less than 10% BM blasts which belong in most cases to low and intermediate-1 (int-1) risk groups according to the International Prognostic Scoring System (IPSS), usually demonstrate dyserythropoiesis. The growth of erythroid progenitors is altered, with increased caspase activation leading to excessive cell death, and cellular dysplasia characterized, in liquid culture of CD34+-derived erythroid progenitors, by a delayed expression of the glycophorin A (GPA) marker. The erythroid/megakaryocytic transcription factor GATA-1, required for erythroid commitment, is a target protein for proteases of the caspase family. Accordingly, caspase-3 is activated and GATA-1 is cleaved when erythroid cells undergo apoptosis, e.g. when starved from cytokines or exposed to Fas receptor agonists. PARP-1, Acinus and Lamin B are also cleaved in this setting. On the other hand, the limited activation of caspase-3 that is required for the normal maturation of basophilic to polychromatophilic erythroblasts does not lead to GATA-1 cleavage. We have shown previously that, during normal erythroid differentiation, the ATP-dependent chaperone protein HSP70 re-localized from the cytoplasm to the nucleus to protect GATA-1 from caspase-mediated proteolysis. The present study was undertaken to determine whether alteration of this pathway could play a role in the dyserythropoiesis observed in 22 patients with MDS. In cultured MDS erythroblasts, we observed that concomitantly to the burst of caspase activation, GATA-1 expression spontaneously decreased despite the presence of cytokines. The addition of pan-caspase inhibitors, i.e. zVAD or qVD rescued the expression of GATA-1, suggesting that GATA-1 downregulation depended on caspase-mediated proteolysis. Immuno-fluorescence or immunoblot experiments (n=11) demonstrated a defect in the nuclear relocalization of HSP70 in MDS erythroblasts when caspases were activated. On the contrary to the observations made in normal cells, HSP70 did not co-localize with GATA-1 or activated caspase-3 in MDS cell nuclei. Ectopic expression, through retroviral transduction of MDS progenitors (n=8), of a μGATA-1 mutant protein uncleavable by caspases, induced GATA-1 target mRNAs (PBGDe, GPA, EPO-R, b- and g-globins), and rescued erythroid differentiation with an increased number of morphologically differentiated cells and a GPA up-regulation. μGATA-1 had no effect on the apoptotic phenotype. These results indicate that the re-localization of HSP70 to the nucleus in differentiating erythroblasts is altered in MDS. This contributes to dyserythropoiesis through GATA-1 proteolysis. Ongoing studies explore the pathways involved in HSP70 nuclear distribution to identify which of these pathways is deregulated in MDS cells. Disclosures:No relevant conflicts of interest to declare.

Quantitative Assessment of Myeloid Nuclear Differentiation Antigen Distinguishes Myelodysplastic Syndrome From Normal Bone Marrow.

Abstract 1754▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-780 IntroductionDefinitive diagnosis and classification of MDS are often difficult because of variable presence of diagnostic criteria and imprecision and ambiguities of interpretation of both morphologic and ancillary data. An objective criterion that reliably distinguishes MDS from normal marrow would greatly facilitate diagnosis, and might contribute to subclassification of MDS. Gene expression profiling has identified marked (7 fold) down-regulation of myeloid nuclear differentiation antigen (MNDA) message in cases of MDS (Hofmann WK, et al, Blood 2002;100:3553-60), and we have previously shown down-regulation of MNDA protein expression in random MDS cases using immunohistochemistry and flow cytometry (Briggs RC, et al. Cancer Research 2006;66:4645-51). To continue our previous analyses, we evaluated MNDA expression in myeloid progenitors using quantitative flow cytometric analysis in MDS and normal control marrow samples. Patients and MethodsThe study included 20 MDS patients receiving only supportive care undergoing bone marrow sampling for clinical purposes, and 19 frequency age-matched normal controls undergoing orthopedic surgery with no antecedent primary hematologic abnormalities. MDS diagnosis was based on 2008 WHO criteria. Quantitative flow cytometric analysis was performed with an FC500 flow cytometer (Beckman Coulter, Fullerton, CA). Cells were treated with CD45-PE and CD34-ECD (Beckman Coulter), washed in PBS with 2% FCS, and permeabilized with PermiFlow. MNDA-Alexa-488 was added at a granulocyte-monocyte specific concentration, with analysis using Winlist 5.0 software (Verity Software, Topsham, ME) with DDE links to ModFitLT 3.0 using modifications of published methods. Differentiating myeloid progenitors were identified as high side scatter/intermediate CD45/CD34-negative cells, with lymphocytes (low side scatter/high CD45/CD34-negative cells) serving as an internal dim MNDA control in each sample. ResultsMDS cases consisted of 12 patients with refractory cytopenia with multilineage dysplasia (RCMD), 3 with refractory anemia with excess blasts-1(RAEB-1), 4 with RAEB-2, and 1 with therapy-related MDS. In myeloid progenitors in MDS patients, the median percent of MNDA-dim cells was 67.4% (range 0.7-97.5%, interquartile range 44.9-82.7%). The analogous median percent of MNDA-dim cells in control patients was 1.2% (range 0.2-13.7%, interquartile range 0.6-2.7%). The area under the ROC curve was 0.96 (p = 9×10-7), indicating almost complete discrimination between cases and controls. 19 of 20 MDS patients demonstrated bimodal distribution of MNDA expression comprising a distinct MNDA-dim population and a separate MNDA-normal population, suggesting an admixture of MDS and normal cells. 18 of 19 control patients demonstrated a single population of MNDA-normal cells without evidence of a bimodal distribution. The single MDS patient with normal MNDA expression had prior clinical and laboratory features suggestive of refractory anemia with ringed sideroblasts. The single pediatric MDS patient had reduced MNDA expression similar to other high grade MDS samples. Patients showed trends for MDS subtype (p = 0.21) and IPSS score (p = 0.07) versus percent MNDA-dim myeloid progenitors. These data demonstrate remarkable sensitivity and specificity for use of MNDA expression to detect MDS. ConclusionsQuantitative flow cytometric analysis of MNDA expression in marrow myeloid progenitors is a promising objective test for diagnosis of MDS, demonstrating a striking difference of MNDA expression in myeloid progenitors in MDS versus control patients. Our results require elaboration with analysis of low grade cases. A single possible low grade MDS case in this series demonstrated MNDA expression identical to normal control samples. Our results also require elaboration in cases constituting the differential diagnosis of MDS to evaluate the clinical utility of MNDA evaluation. The biological significance of down-regulation of MNDA in MDS is uncertain, although MNDA has been implicated in regulation of programmed cell death. Our results suggest a mix of normal and abnormal myelopoiesis in MDS patients, as predicted by cytogenetics in many cases of MDS. In summary, testing of MNDA expression in myeloid progenitors shows great promise as an objective test for diagnosis of MDS in marrow samples. DisclosuresNo relevant conflicts of interest to declare.

Cyclosporine Levels and Rate of Graft Rejection Following Reduced Intensity Conditioning.

Abstract 3337Poster Board III-225In the past decade there has been a significant increase in the use of reduced intensity conditioning (RIC) regimens for allogeneic hematopoietic stem cell transplantation (HSCT). RIC HSCT is associated with reduced transplant related mortality, but also with an increased risk of relapse and graft rejection. Little is known about factors associated with graft rejection. Based on early animal models, intensified cyclosporine (CyA) administration is considered an essential component of RIC HSCT with minimal conditioning. We were interested in analyzing the role of CyA in graft rejection following RIC HSCT. Between October 1999 and December 2008, 85 patients received an allogeneic RIC HSCT with fludarabine 3 × 30 mg/m2 and 2 Gy TBI as conditioning at our institution. 6 patients were excluded from the analysis due to graft loss associated with relapse or progressive disease, 2 were excluded for missing data. Diseases were acute leukemia (N=19), MDS (N=11), CML/MPN (N=10), lymphoma (N=24), multiple myeloma (N=12), and pure white cell aplasia (N=1). Donors were HLA-identical siblings (N=54) or unrelated donors (N=23). Graft source was peripheral blood (N=74) or bone marrow (N=3). Graft-versus-host disease (GvHD) prophylaxis consisted of CyA and mycophenolate mofetil (MMF). CyA was started at a dose of 6.25mg/kg p.o. twice daily and adjusted according to trough levels (measured weekly) and toxicity. MMF was administered at a dose of 15mg/kg twice daily; trough levels were not analyzed systematically. For the analysis of impact on graft rejection, CyA levels between day 1 and 28 where summarized as mean trough levels. Graft rejection was defined as <5% donor cells at any time on day 28 or later or application of donor lymphocyte infusion for declining donor chimerism. Of the 77 evaluable patients, 16 (21%) experienced graft rejection at a median of 69 days after transplantation. There was no significant difference in disease stage (early vs. advanced), median age at transplant (56 and 57 years, respectively), EBMT risk score, graft cell count (nucleated and CD34+ cells) and history of previous transplants between patients with or without rejection. In contrast, there was a significant difference in underlying disease, with a higher proportion of CML/MPN and a lower proportion of multiple myeloma in rejecting patients. Mean CyA trough levels were clearly associated with graft rejection with a cumulative incidence of rejection of 8%, 29% and 56% with trough levels of <300 ng/ml (N=40), 300-600 ng/ml (N=28) and >600 ng/ml (N=9) (p=0.002) (Figure A) and a median donor chimerism at day 28 of 99%, 84%, and 72% respectively (p=0.004). In a multivariate analysis, two factors remained significantly associated with graft rejection: unrelated donor (HR vs. sibling donor 4.52, p = 0.03), and mean trough level of CyA for days 1-28 (HR 1.006 per ng/ml increase, p=0.001). Patients with CML/MPN had an increased risk of rejection (HR 1.36 versus other patients combined, p=0.65) which did not reach statistical significance. The overall survival of the entire patient group was 55% at five years; patients with stable engraftment had a five-year survival rate of 61%, those rejecting their graft 36% (p=0.04). These data indicate that elevated CyA levels during the first month posttransplant might be associated with an increased risk of graft rejection. The potentially detrimental effects of high CyA levels have been described previously in the myeloablative setting, where a correlation between CyA levels and risk of relapse has been shown. Further studies will be needed to better define the exact role of CyA following RIC regimens. [Display omitted] DisclosuresGratwohl:Amgen: Research Funding; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding.

Alpha-Catenin, ARHGAP21 and β-Catenin Are Abnormally Expressed in Bone Marrow Cells From Patients with Myelodysplatic Syndromes and Are a Possible Target for Decitabine Treatment.

Abstract 1778Poster Board I-804 IntroductionMyelodysplastic syndrome (MDS) encloses a group of clonal hematopoietic disorders clinically and morphologically characterized by ineffective hematopoiesis. The gene encoding alpha-catenin (CTNNA1) is expressed at a much lower level in leukemia-initiating stem cells from individuals with MDS del(5q). Thus, loss of alpha-catenin tumor suppressor expression in hematopoietic cells may provide a growth advantage that collaborates MDS pathogenesis. ARHGAP21, a negative regulator of RhoGTPase signaling pathways, is a partner of alpha-catenin that controls its recruitment to the adherens junctions. ARHGAP21 is upregulated during myeloid differentiation, and could be involved in the malignant process of hematopoietic cells. In addition, alpha-catenin is a target for decitabine (DAC) treatment, a demethylating agent with potent antitumorigenic properties against MDS. The aim of this work was to evaluate the expression of alpha-catenin, ARHGAP21 and beta-catenin (gene CTNNB1) in bone marrow cells from MDS patients with or without del(5q) and to analyze CTNNA1, ARHGAP21 and CTNNB1 expression after DAC treatment. PATIENTS AND METHODScells were isolated from bone marrow of 6 MDS patients, including 5 refractory anemia (RA), being two with del(5q), and 1 refractory anemia with excess blasts (RAEB), based on the French-American-British classification, and 4 control subjects (normal hematopoietic tissues were obtained from healthy donors). The study was approved by the National Ethical Committee Board and bone marrow samples were collected at the Hematology and Hemotherapy Center, University of Campinas, after all participants provided informed written consent. Alpha-catenin, ARHGAP21 and beta-catenin localization in CD34+ cells was obtained using confocal microscopic analysis. ARHGAP21 localization was also analyzed in HS-5 stromal cells that were submitted to a CTNNA1 RNA interference (RNAi) approach. Leukemia cells lines (HL-60 and P-39) and bone marrow mononuclear cells obtained from 7 MDS patients, 5 RA and 2 refractory anemia with ringed sideroblasts (RARS), were treated with DAC for 72 hours; then mRNA expression of CTNNA1, ARHGAP21 and CTNNB1 was analyzed by Real-time PCR (normalized by GAPDH and beta-actin). RESULTSalpha-catenin, ARHGAP21 and beta-catenin are preferentially localized in the nucleus of CD34+ cells from MDS patients in contrast to the preferential cytoplasm and membrane localization in healthy donors and in MDS patients with del(5q). In del(5q) patients and healthy donors, ARHGAP21 and alpha-catenin co-localizated in the cell membrane. ARHGAP21 was abnormally expressed in cells with decreased CTNNA1 expression: in HS-5 stromal cells, ARHGAP21 was localized at the cytoplasm (mainly in the perinuclear region) and at the nucleus, in contrast, ARHGAP21 was poorly detectable in the nucleus of CTNNA1-RNAi treated cells. DAC treatment of MDS cells and leukemia cell lines induced CTNNA1, ARHGAP21, and CTNNB1 expression in a dose-dependent way. In HL60 and P39 cells, ARHGAP21 relocate to the cell membrane after DAC treatment. CONCLUSIONThe abnormal localization of alpha-catenin, ARHGAP21 and beta-catenin in MDS may compromise the reorganization of actin dynamics at sites of cell–cell contact that stabilizes cadherin-mediated cell–cell adhesion; moreover, these results also suggest a deficient recruitment of alpha-catenin to the cell membrane and an aberrant signaling in the Wnt pathway. In addition, ARHGAP21, alpha-catenin and beta-catenin are a target for DAC treatment in MDS. Supported by: FAPESP.Keywords: alpha-catenin, ARHGAP21, beta-catenin, myelodysplastic syndrome, Rho-GAP, decitabine DisclosuresNo relevant conflicts of interest to declare.

PI3K/AKT Activity and Functional Relevance in Deguelin Induced Apoptosis in MDS Cell Line.

Abstract 4977 IntroductionPI3K/AKT signalling pathway is involved in cell growth, proliferation and apoptosis. PI3K/AKT constitutive activation is observed in several solid tumors and leukemic cells. Inhibition of PI3K/AKT activity using specific inhibitor, LY294002, results in apoptosis. Deguelin is a natural product isolated from the leguminous, Mundulea sericea, with antitumorigenic effect in vitro and in vivo. The inhibition effect of Deguelin in PI3K/AKT signalling pathway in leukaemia cells was also observed in vitro. AimsWe evaluated PI3K/AKT activation using p-AKT expression by flow cytometry in P39 cell line and CD34+ hematopoietic progenitors. PI3K/AKT activity inhibition mediated by deguelin and its proapoptotic effect was tested in P39 cell line. Material and MethodsThe high-risk MDS cell line P39 and leukaemia cell lines, Jurkat and HL60, were maintained in RPMI1640 and FBS 10%. Jurkat and HL60 were used as positive and negative control for p-AKT expression, respectively. Cell lines and CD34+ cells from bone marrow healthy donors (n=5) were assessed for p-AKT expression. Flow cytometry detection of intracellular Ser 473 p-AKT was performed after permeabilization and fixation with a saponin based solution with Tween20 and FACSlysing solution (Becton Dickinson-BD). An alexa-fluor 488-conjugated rabbit antibody to Ser 473 p-AKT (Cell Signalling Technology) was employed. A double immunostaining procedure using CD45-PerCP and CD34+PE was performed to analyse p-AKT expression in CD34+ cells from bone marrow healthy donors. After incubation, cells were analysed on a FACSCalibur (BD). The p-AKT activity was determined using Kolmogorov-Smirnov test (D). Difference between groups was analyzed using the Mann Whitney test. For treatment and determination of apoptosis, cells were exposed to deguelin (at concentrations of 10-300nM) and LY294002 (15-50uM) for 24-48h. To determine apoptotic changes, cells were stained withy Annexin-V/FITC and propidium iodide and examined on a FACScalibur (BD). ResultsJurkat cells showed constitutive PI3K/AKT activation with a mean D value for p-AKT expression of D=0,84 ± 0,02. P39 cells also showed a constitutive PI3K/AKT activation with p-AKT expression as high as in Jurkat cells (mean D= 0,76±0,07). P-AKT expression was significant lower in CD34+cells from health donors (D=0,38±0,06) than in Jurkat and P39, respectively (p=0,0043 and p=0,015). Inhibition of p-AKT was observed using different concentrations of LY294002 (15-50uM) in Jurkat cells and apoptosis was observed after 24 and 48h when cells were treated with 50uM. For P39, although apoptosis was observed, no p-AKT inhibition was detected after LY294002 treatment. Treatment with deguelin induced apoptosis in Jurkat via p-AKT inhibition. No effect on apoptosis or p-AKT expression was observed in P39 cell line after deguelin treatment. ConclusionsPI3/AKT constitutive activation was observed in P39 cell line and suggests that PI3/AKT can play a role in MDS progression. Deguelin effect in PI3K/AKT pathway can be cell specific, as observed in Jurkat but not in P39 cell line. Thus, deguelin in combination with other agents should be tested as a potential therapeutic option for MDS via PI3K/AKT inhibition. DisclosuresNo relevant conflicts of interest to declare.

Inducible expression of EVI1 in human myeloid cells causes phenotypes consistent with its role in myelodysplastic syndromes

The oncogene EVI1 has been implicated in the etiology of AML and MDS. Although AML cells are characterized by accelerated proliferation and differentiation arrest, MDS cells hyperproliferate when immature but fail to differentiate later and die instead. In agreement with its roles in AML and in immature MDS cells, EVI1 was found to stimulate cell proliferation and inhibit differentiation in several experimental systems. In contrast, the variant protein MDS1/EVI1 caused the opposite effect in some of these assays. In the present study, we expressed EVI1 and MDS1/EVI1 in a tetracycline-regulable manner in the human myeloid cell line U937. Induction of either of these proteins caused cells to accumulate in the G0/G1-phase of the cell cycle and moderately increased the rate of spontaneous apoptosis. However, when EVI1- or MDS1/EVI1-expressing cells were induced to differentiate, they massively succumbed to apoptosis, as reflected by the accumulation of phosphatidylserine in the outer leaflet of the plasma membrane and increased rates of DNA fragmentation. In summary, these data show that inducible expression of EVI1 in U937 cells causes phenotypes that may be relevant for its role in MDS and provides a basis for further investigation of its contribution to this fatal disease.