ALBERT

Description: Dasatinib was approved for use in the treatment of patients (pts) with chronic myeloid leukaemia (CML) and resistance to Imatinib. We applied a rescue treatment based on Dasatinib therapy to achieve a pharmacological immunomodulation in a setting of CML-relapsed allogenic stem cell transplantated (A-HSCT) pts. Patients were required to have both resistance to Imatinib and unresponsiveness to cellular therapy such as Donor Lymphocyte Induction (DLI). We hypothesized that Dasatinib could potentially improove the disease by immunomodulatory action. Primary aim of this therapeutic design was to address, in single institution trial, therapeutic force of a innovative pharmacologic strategy to induce the cytogenetic response followed by DLI. Therefore, we investigated Dasatinib ability to achieve immuno-effects by targeting key mediators of Th1, Th2, and Treg response. Biological effects were examined on conventional diagnostic parameters such as haematological chimerism, cariotype and Bcr-Abl gene transcript. Herein, we present interim results of a pilot group of 3pts. Patients received dasatinib 70 mg twice daily(140 mg total daily dose). Dose modifications were allowed for the management of toxicity. Treatment was performed until complete cytogenetic, molecular response and haematological full donor chimerism. Materials and Methods: To investigate the immunological changes, we used a TaqMan® Low Density Array, based on comparative CTdd CT method on Applied Biosystems 7900HT, to perform relative quantification of cDNA derived from peripheral venous blood specimens harvested after DLI, before and after starting dasatinib therapy. Assumed that normal control values of all transcripts were = 1, we evaluated over or down regulation of gene expression profile (GEP) of a panel of 48 genes involved in immune response. Results: clinical changes after third month of dasatinib therapy. Case 1: responsive patient, maintained a mixed haematologic chimerism, but showed a complete cytogenetic and molecular remission. Following, patient restarted with DLI therapy. Case 2: responsive patient, showed nearly full-donor haematologic chimerism with complete cytogenetic and molecular remission. Case 3: patient no evalutable because brief treatment (only 1 month). Dasatinib caused early haematological toxicity. Patient maintained a low level of donor T cells with presence of Philadelphia chromosome associated to elevated p210 molecular signal. Gene expression profiles post-dasatinib therapy: According to in vitro experiments (Blood October 25, 2007), in all cases we observed a down regulation of IL-2 and IL-12B (Th1), IL-6 and IL- 18, IL-10 (Th2) cytokines and mediators of apoptosis such as EGR2, EGR1. By contrast, multiple pro-inflammatory factors were up-expressed: IFN-g, IL-17, IL-7. Only in case number 1, TNF-a and IFN-g molecular pathways were not influenced by the drug. In fact their elevated expression was preserved as compared to pre-dasatinib levels. Noterworthy among cases number 2 and 3 (with mixed chimerism), Dasatinib improved a marked inhibition of Th1 effectors in addition to down-regulation of several important molecular transcripts: SERPIN B3–B4, BCL2A1, SELP, PIAS 1, IRF8, IRF1, CCL7, CCL5, CXCL9, CCR4, ICAM. Regards to T regulatory cells, Foxp3 was strongly up-regulated in case number 1 and down-regulated in case2and 3. Conclusion: We think that Dasatinib represent a possibility of cure for for CML pts relapsed after A-HSCT and unresponsive to alternative treatments. For imatinib-resistant CML patients, such as in this study, there are few currently available effective options. The present results strongly emphasize the importance of immune response control to achieve the desired clinical effects.

Description: The aim of our perspective observational study was identify a fast molecular biomarker of tumorigenic-proliferative haematological disorders at on set using a non-invasive method. At this end, to better discriminate between myeloproliferative or lymphoproliferative hematological disease, from May to July 2014, we collected peripheral blood mononuclear cells (PBMCs) from patients at the first medical examination and without drug therapy. The patients were divided into groups on the basis of the diagnosis. Group 2 (n=8) included patients suffered from mixed disorders such as myeloproliferative neoplasms (MPD) associated to monoclonal gammopathy of undetermined significance (MGUS). Group 3 (n=8) included patients with only MGUS, group 4 included 9 patients with only primary myelofibrosis (M). Healthy donors (Group 1, n=16) were considered as normal subject or calibrator in molecular analysis. Their PBMCs were used to perform relativegene expression profile of a transcriptome involved in apoptotic control, stem-cell differentiation, immune network, inflammation and leukemogenesis, in order to detect whether these may serve to label the patient groups. A multigene expression assay (47 genes) was carried out with the TaqM,an® Low Density Array Fluidic card. In Group 3 (MGUS), 6 genes were differentially expressed (BMI-1, FLT3, FZD1, FZD5, ICAM1, IMP3). Also, we compared variance, main value for each gene in each group and T-test that showed a significant differential expression pattern (p

Description: Deletions of the long arm of chromosome 5 (del(5q)) are common cytogenetic alterations in myelodysplastic syndrome (MDS), a disease characterized by refractory anemia, megakaryocyte dysplasia, and thrombocytosis. The thalidomide analogue lenalidomide (LEN) produces durable erythroid responses in ~60% of del(5q) MDS patients, including a majority of cytogenetic responses in which the del(5q) clone becomes undetectable in the bone marrow. Despite high response rates, clinical and cytogenetic relapse occur within 2-3 years. Mechanisms of clinical response, resistance and relapse with LEN therapy remain to be elucidated. The target of LEN has recently been identified as the cereblon (CRBN) component of the cullin 4 RING E3 ubiquitin ligase complex (CRL4-CRBN). Upon LEN binding, the substrate-specificity of the CRL4-CRBN complex is altered, and LEN-regulated substrates are beginning to be identified. An RNA interference screen was performed to identify genes/pathways that mediate LEN sensitivity and resistance in del(5q) MDS. The LEN-sensitive del(5q) MDS patient-derived cell line MDSL was screened with a genome-wide shRNA library (SBI GeneNet Human 50K Library) in the presence and absence of LEN treatment (0 and 10 μM) for 7 days. Three independent shRNAs targeting the proton-sensing G protein-coupled receptor 1 (GPR68 or OGR1) were among the most enriched shRNAs in LEN-treated cells, suggesting that loss of GPR68 expression conferred resistance to LEN. This finding was validated in MDSL cells, using an independent set of shRNAs. Conversely, a GPR68 agonist (N-cyclopropoyl-5-[thiophen-2-yl]-isoxazole-3-carboxamid) enhanced LEN-induced cytotoxicity to MDSL cells. GPR68 is a proton-sensing G-protein coupled receptor that stimulates inositol phosphate production and/or intracellular calcium (Ca2+) mobilization. Curiously, CRBN was originally identified as a binding protein of calcium-activated potassium channels. These data led us to hypothesize that Ca2+ signaling may be responsible for LEN-mediated cytotoxic effect in MDS cells. Reducing intracellular Ca2+ level with chelators reversed LEN’s cytotoxic effects, while increasing intracellular Ca2+ level with ionomycin enhanced LEN’s cytotoxic effect, indicating that intracellular Ca2+ levels determine cellular responsiveness to LEN. Although LEN did not induce an instant burst of Ca2+ influx, a gradual increase of basal intracellular free Ca2+ was observed following LEN treatment in LEN-sensitive cell lines and primary MDS marrow cells, but not in LEN-resistant cells, suggesting that LEN cytotoxicity was dependent on the cell’s ability to release Ca2+ from intracellular stores. GPR68 and CRBN were both necessary for the LEN-induced increase in Ca2+, as knockdown of GPR68 or CRBN in LEN-sensitive cells prevented the Ca2+increase. To identify the Ca2+-dependent signaling pathway responsible for mediating the cytotoxic effect of LEN, a panel of seven inhibitors that blocked mitochondrial/caspase-, calpain-, autophagy-, or lysosomal-dependent cell death pathways was tested in combination with LEN on MDSL cells. Only the inhibitor of calpains (PD150606) prevented LEN-induced cytotoxic effects in MDSL cells, indicating that calpain activation was necessary for mediating cell death in LEN-treated cells. Calpains are Ca2+-dependent cysteine proteases that can induce apoptotic and necrotic cell death by proteolytic cleavage of protein substrates. Calpastatin, the only endogenous calpain inhibitor, is localized to 5q15 and its expression is haploinsufficient in del(5q) MDS as compared to normal karyotype MDS. Taken together, our results show that LEN increased intracellular Ca2+ levels by a CRBN- and GPR68-dependent mechanism, leading to calpain-mediated cytotoxicity in del(5q) MDS cells. We propose a model in which haploinsufficient expression of calpastatin in del(5q) MDS sensitizes cells to cytotoxic effects of LEN. Further studies are required to identify the direct LEN-modulated substrates of CRBN that mediate this effect. Disclosures Oliva: Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria. MacBeth:Celgene: Employment, Equity Ownership. Starczynowski:Celgene: Research Funding.

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.