ALBERT

Description: Abstract 4541 Introduction: Limited data demonstrate to what extent preventing fungal exposures is effective in preventing infection and disease. Further studies are needed to determine the optimal duration of fluconazole prophylaxis in allogeneic recipients to prevent invasive disease with fluconazole-susceptible Candida species during neutropenia. Oral, nonabsorbable antifungal drugs might reduce superficial colonization and control local mucosal candidiasis, but have not been demonstrated to reduce invasive candidiasis. Anti-fungal prophylaxis is recommended in a subpopulation of autologous recipients with underlying hematologic malignancies with prolonged neutropenia and mucosal damage. Methods: This is a retrospective study of 1007 SCT performed in our center between 1992 and 2009 in 809 consecutive patients, irrespective of diagnosis. HEPA filter and environmental monitoring (air, water, surfaces) are attributes of our transplant center. Results: The main characteristics of the patients are reported in Table 1. Systemic prophylaxis was used according to the guidelines (Table 2): fluconazole in the nineties, then itraconazole and from 2004 was either abolished or substituted with non-adsorbable prophylaxis in transplants with standard risk. Secondary prophylaxis was prescribed for high risk patients (with infectious fungal history, suggestive iconography, positive fungal biomarker). In 17 years our Center has never been colonized by mould. Only 3 probable aspergillosis infections and 4 proven fungal infections (fusarium, mucor and 2 aspergillosis) were diagnosed, all in allogeneic patients (2 haplotipical, 1 singenic, 1 sibiling, 1 MUD and 2 mismatched), resulting in death in all cases. No infection was documented in autologous setting, while the infection rate in allogenic setting was 3.6% with an incidence rate of 1.1 infection per 10000 transplants/year. These results are significantly lower than published reports. Conclusion: Systemic antifungal prophylaxis should not be performed in autologous SCT patients. The abuse of systemic prophylaxis targeting yeasts has influenced the change of epidemiology in the transplant setting with prevalence of mould infections. The identification of high risk patients is useful to select patients for systemic antifungal or secondary prophylaxis to reducing overtreatment, incidence of resistant strains and costs. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3631 Introduction: The erythroid differentiation defect observed in 5q– syndrome has been attributed to the RPS14 gene located within the CDR of the long arm of chromosome 5. We have recently demonstrated that RPS14 expression increases during lenalidomide treatment. However, haploinsufficiency of RPS14, which encodes ribosomal protein S14, does not explain clonal dominance. The expression of miRNAs, miR-145 (5q33.1) and miR-146a (5q33.3), in CD34+ bone marrow (BM) cells of individuals with MDS with deletion of the long arm of chromosome 5 (del(5q)) is lower compared to normal controls (Starczynowski et al, Nature Medicine, 2010). miRNAs are small noncoding RNAs that post-transcriptionally repress specific messenger RNA targets through interaction with the 3′ untranslated region (UTR). Loss of noncoding transcripts encoding miRNAs within the CDR may result in haploinsufficiency by loss of inhibition of their targets. Concurrent loss of both miR-145 and miR-146a resulted in activation of innate immune signalling through elevated expression of their respective targets, TIRAP and TRAF6. Furthermore, knockdown of miR-145 and miR-146a or overexpression of TRAF6 in mouse HSPC (Hematopoietic stem and progenitor cells) recapitulated features of 5q– syndrome, such as bone marrow dysplasia, anemia and thrombocytosis. We present preliminary results of changes in miRNA expression in IPSS lower-risk MDS with del(5q) during treatment with lenalidomide. Methods: A prospective single-arm trial investigating the efficacy and safety of lenalidomide in 46 patients with MDS with del(5q) with/without additional cytogenetic abnormalities and Hb 〈 10 g/dL. Lenalidomide was administered orally at a starting dose of 10 mg/day for a maximum of 12 months. When necessary, dosing was reduced to 5 mg/day or 5 mg on alternate days. Bone marrow assessments were performed at baseline and every 3 months, thereafter. For the evaluation of miRNA-145 and miRNA-146a in patient samples, 300 ng/μl of miRNAs were isolated in each purified BM sample by using mirVana™ miRNA Isolation Kit-Ambion and TaqMan miRNA Array Analysis was performed to determine the expression of miRNAs (7900HT Sequence Detection System Applied Biosystems). Patient BM-miRNAs were calibrated with miRNAs from BM of healthy volunteer donors. It was assumed that BM expression value of each calibrator miRNA was 1 unit. RPS14 gene assays were performed using TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative ddCT method, according to the manufacturer's instructions. Target gene expression levels were measured in triplicate and normalized against the expression of the 18S housekeeping gene from a BM pool of normal, healthy subjects at all timepoints. Median relative gene expression values in MDS patients were compared to healthy subjects, set as a value of 1. Results: Four patients have been evaluated (1 M, 3 F; ages 65, 66, 73 and 76 years, respectively) at baseline and after 12 weeks. At baseline, 2 patients were RBC-transfusion dependent. One patient had one additional cytogenetic abnormality (+8 in 15% metaphases). All patients obtained an erythroid response by week 12: mean Hb values significantly increased from 8.4 ± 0.9 at baseline to 11.6 ± 0.9 g/dL (p=0.01). All patients obtained a cytogenetic response, 2 of which were complete. miRNA-145 and miRNA-146a expression were both low at baseline and significantly increased by week 12 (Table). Conclusions: Preliminary results confirm that, in IPSS lower-risk MDS with del(5q), miRNA-145 and miRNA-146a expression is low. Lenalidomide treatment is associated with erythroid responses and cytogenetic remissions concurrent with significant increases in miRNA-145 and miRNA-146a expression. Disclosures: Oliva: Celgene: Consultancy.

Description: Bortezomib (Velcade) is used widely for the treatment of various human cancers; however, its mechanisms of action are not fully understood, particularly in myeloid malignancies. Bortezomib is a selective and reversible inhibitor of the proteasome. Paradoxically, we find that bortezomib induces proteasome-independent degradation of the TRAF6 protein, but not mRNA, in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cell lines and primary cells. The reduction in TRAF6 protein coincides with bortezomib-induced autophagy, and subsequently with apoptosis in MDS/AML cells. RNAi-mediated knockdown of TRAF6 sensitized bortezomib-sensitive and -resistant cell lines, underscoring the importance of TRAF6 in bortezomib-induced cytotoxicity. Bortezomib-resistant cells expressing an shRNA targeting TRAF6 were resensitized to the cytotoxic effects of bortezomib due to down-regulation of the proteasomal subunit α-1 (PSMA1). To determine the molecular consequences of loss of TRAF6 in MDS/AML cells, in the present study, we applied gene-expression profiling and identified an apoptosis gene signature. Knockdown of TRAF6 in MDS/AML cell lines or patient samples resulted in rapid apoptosis and impaired malignant hematopoietic stem/progenitor function. In summary, we describe herein novel mechanisms by which TRAF6 is regulated through bortezomib/autophagy–mediated degradation and by which it alters MDS/AML sensitivity to bortezomib by controlling PSMA1 expression.

Description: Background and Objectives The use of allogenic hematopoietic stem cell transplantation (HSCT) is increasing in older patients because of reduced ablative regimens and improved supportive care. An old patient usually has a old sibling healthy donors (HDs). The safety of G-CSF for mobilization in elderly HDs has not been provided. In our institution a long-term active follow-up study of G-CSF-mobilized healthy volunteer donors has been implemented. Patients and Methods After a median follow up of 94 months (7.8 years) we reviewed and analyzed safety data of our HDs database according to HDs age: HDs-1 (162), patients younger than 50 years old, HDs-2 (62), patients 50-59 years old and HDs-3 (23), patients 60 or more years old. Long-term follow-up included monitoring of neoplastic, cardiac or autoimmune diseases. Results Two hundred and forty-seven successive donors were evaluated and their characteristics are well balanced among age-groups and no statistical differences have been detected: most of them were male (55.9% ), sibling (97.2%) and HLA matching (93.1%). Mobilization failure rate has been 4.3% (7/162), 1.6% (1/62) and 13% (3/23) in HDs-1, HDs-2 and HDs-3, respectively (p=0.075). Short- and long-term safety was not different among age-groups. Bone pain was reported as the most frequent short-term adverse event (76.5%). Other commonly observed short-term symptoms included headache (31.6%), fatigue (28.7%), insomnia (21.5%), nausea (13.0%) and fever (6.1%). For long-term safety surveillance no haematological malignancies were observed. The observed rate of solid tumors, cardiovascular and autoimmune events, was the expected incidence for these diseases in the western countries. Conclusion In our study, mobilization with lenograstim in elderly HDs seems to be as safe as for young HDs safe. This data contribute to the growing body of evidence of the long-term safety of G-CSF for allogenic donor stem cell mobilization also for elderly HDs. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2452 Deletion of chromosome 5q in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients results in loss of miR-146a, which is a negative regulator of the innate immune pathway by targeting TNF receptor associated factor-6 (TRAF6). Therefore, MDS and AML patients with reduced miR-146a expression concomitantly exhibit elevated TRAF6 protein. TRAF6 is an E3 ubiquitin ligase that catalyzes K63-linked polyubiquitin chains on substrates that lead to pathway activation, one of which includes NF-kB. Mice lacking miR-146a, or with overexpression of TRAF6, develop AML- and MDS-like features. Bortezomib (Velcade©), which shows promise alone or in combination with chemotherapy in certain groups of MDS and AML patients, is a selective and reversible inhibitor of the 26S proteasome. Studies on the mechanism of action of Bortezomib have shown that pro-apoptotic proteins are stabilized following proteasome inhibition and contribute to the anti-cancer effect. In this report, paradoxically, we find that Bortezomib induces rapid and complete degradation of TRAF6 protein, but not mRNA, in MDS/AML cell lines and human CD34+ cells. A similar finding was observed when AML cells were treated with MG132, another proteasome inhibitor, indicating that degradation of TRAF6 is secondary to proteasomal inhibition. Interestingly, the reduction in TRAF6 protein coincides with Bortezomib-induced autophagy, as indicated by conversion of LC3B-I to LC3B-II and degradation of SQSTM1/p62, and subsequently with apoptosis in MDS/AML cells. Addition of an autophagy inhibitor (3-methyladenine [3-MA]) to Bortezomib-treated AML cells maintained TRAF6 protein expression and enhanced cell viability. Similarly, TRAF6 degradation was blocked by 3-MA when cells were treated with Rapamycin, an mTOR inhibitor and inducer of autophagy. These findings suggest that a mechanism of Bortezomib-induced cell death in myeloid malignancies involves elimination of TRAF6 protein by autophagosomes. Forced expression of TRAF6 in two AML cell lines partially blocked the cytotoxic effect of Bortezomib, suggesting that TRAF6 is an important target of Bortezomib. To determine whether loss of TRAF6 is sufficient to impede growth of MDS and AML, we used a genetic approach to inhibit TRAF6 in MDS/AML cell lines and bone marrow cells from MDS patients with deletion of chromosome 5q. RNAi-mediated depletion of TRAF6 in MDS and AML samples resulted in impaired malignant hematopoietic stem/progenitor function and rapid apoptosis. To uncover the molecular consequences following loss of TRAF6, we applied gene expression profiling and identified genes relevant to the survival of MDS and AML cells. In summary, these findings implicate TRAF6 in Bortezomib-induced cell death and in the maintenance of myeloid malignancies, and reveal a novel mechanism of TRAF6 regulation through autophagic degradation. Disclosures: Oliva: Celgene: Consultancy.

Description: Abstract 612 Recent work has shown that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients exhibit downregulation of miR-146a, a miRNA that negatively regulates the innate immune pathway by targeting IRAK1 and TRAF6. Mice lacking miR-146a show elevated IRAK1 protein expression, and develop AML and MDS-like features resembling the human diseases. Prior to this study, the role of IRAK1 in human myeloid malignancies was unknown. We conducted a comparison of gene expression profiles of 136 cases of MDS CD34+ cells with 17 normal CD34+ cells obtained from ArrayExpress (E-GEOD-19429; Pellagatti et al., Leukemia, 2010). According to this data set, we observed IRAK1 overexpression in MDS patients (P = 0.017). IRAK1 is a serine/threonine kinase, and after phosphorylation on threonine-209 (T209), its kinase activity is induced, thus allowing for subsequent activation of TRAF6 and eventually NF-kB. Interestingly, we observed higher basal levels of phospho-IRAK1 at T209 in MDS and AML samples as compared to normal human CD34+ cells. To investigate the potential role of IRAK1 in AML and MDS, we used genetic and pharmacological approaches to suppress IRAK1 activity in MDS/AML cell lines and bone marrow cells from MDS patients. RNAi-mediated knockdown of IRAK1 in MDS and AML samples resulted in impaired growth of malignant hematopoietic stem/progenitor cells in methylcellulose assays and rapid apoptosis in vitro. In addition, we used a small-molecule inhibitor (benzimidazole analog; Amgen Inc.) to potently inhibit IRAK1 kinase activity. MDS/AML cell lines and MDS patient samples cultured with the IRAK1 inhibitor exhibited impaired growth and increased apoptosis, which coincided with decreased phospho-IRAK1 at T209, and active versions of TRAF6 and NF-kB. Importantly, the inhibition of IRAK1 kinase function is selectively detrimental to MDS and AML samples while preserving normal CD34+ cell viability and function. Given this novel requirement of IRAK1 in MDS and AML, we examined whether Lenalidomide or Bortezomib, two treatment options for MDS/AML and reported immunosuppressors, exhibit anti-leukemic activity in part by targeting IRAK1. We observed that Bortezomib, but not Lenalidomide, inhibits IRAK1 mRNA and protein expression in MDS/AML cells. The cytotoxic effect of Bortezomib can be partly rescued by forced expression of IRAK1 in these cells. To determine the molecular and cellular basis of cell death following loss of IRAK1 function or expression, we applied microarrays to MDS cells treated with IRAK1 inhibitor or transduced with a lentiviral vector encoding an shRNA targeting IRAK1. An overlap of commonly deregulated genes imposed by loss of IRAK1 expression or by the IRAK1 inhibitor revealed unique pathways relevant to the survival of MDS and AML cells. In summary, these findings are the first to implicate IRAK1 in the maintenance of myeloid malignancies and describe the effectiveness of an IRAK1 inhibitor on suppressing MDS and AML viability. Disclosures: Oliva: Celgene: Consultancy.