ALBERT

Description: Multiple myeloma (MM) is a heterogeneous but incurable plasma cell malignancy which still requires new therapeutic approaches. Several molecular subsets of MM have been defined based on genetic and chromosomal aberrations. Briefly, t(4;14) or t(14;16) translocations and TP53 deletion are most frequent poor-risk genetic features of MM, while t(11;14) confers a neutral prognostic value. Because cancer cells have a high glycolytic metabolism, we investigated the efficiency of 2-deoxy-d-glucose (2-DG), a competitive inhibitor of hexokinase, to kill myeloma cells in these different molecular subsets. For this purpose we used 28 human myeloma cell lines (HMCL) representative of the molecular subsets carrying a t(11;14), t(4;14) or t(14;16) translocation, which respectively deregulate CCND1, MMSET and c-MAF. Apoptosis induced by 2DG was very heterogeneous among HMCLs, ranging from 5% to 96%. Of note, HMCLs carrying t(4;14) showed a trend to be more sensitive to 2DG (p=0.06, Mann–Whitney test) while HMCLs carrying t(11;14) appeared to be more resistant (p=0.08, Mann–Whitney test). Interestingly, 2DG sensitivity was not significantly related to the constitutive glycolytic activity of HMCLs (p=0.6). In fact, 2-DG not only strongly inhibited the glycolytic activity of HMCLs but also interfered with N-glycosylation. Indeed, addition of D-mannose, an N-linked glycosylation precursor, partly reversed 2-DG-induced cell death. However, the D-mannose efficiency was also heterogeneous among HMCLs, suggesting that the inhibition of N-glycosylation was not the only mechanism of 2-DG-induced cell death. An up-regulation of GRP78, CHOP and ATF-4 expression was induced by 2-DG in both sensitive and resistant HMCLs, suggesting that 2DG-induced cell death was independent from the UPR response. Finally, 2-DG uniformly induced Mcl-1 down-regulation in HMCLs, but only those dependent on Mcl-1 for survival (as confirmed by siRNA experiments) were killed by 2-DG. While the transcription of Mcl-1 is not affected by 2-DG, preliminary results indicate that proteosomal degradation could be involved as part of a more complex mechanism. Of note, most of t(11;14) HMCLs were resistant to cell death induced by Mcl-1 down-regulation but highly sensitive to ABT-199, which targets Bcl2 and efficiently kills t(11;14) HCMLs depending on this pathway for survival. Because 2-DG uniformly down-regulated Mcl-1, we combined it with ABT-199 in ABT-199-resistant HMCLs i.e., in HMCLs expressing a Bcl-2/Mcl-1 gene expression ratio lower to the threshold required for ABT-199 response. The combination of 2-DG and suboptimal ABT-199 dosage indeed strongly synergized in both t(4;14) and t(14;16) HMCLs. The efficiency of this combination was independent from the cells’ TP53 status. Finally, these results have been extended to primary myeloma cells. Finally our study highlights the fact that dual targeting of Mcl-1 by 2-DG and Bcl-2 by ABT-199, in MM cell lines or primary samples, is highly efficient to induce apoptosis whatever the molecular subtype, including those with the poorest prognostic value. ABT-199 is presently under evaluation in a phase I clinical trial in relapsed MM patients and the present study provides a biological rationale for evaluating 2-DG in combination with ABT-199 in MM patients. Disclosures: No relevant conflicts of interest to declare.

Description: Myelodysplastic syndromes (MDS) comprise a heterogeneous group of myeloid neoplasms characterized by peripheral cytopenia, bone marrow (BM) dysplasia in one or more hematopoietic lineages, and clonal instability with an increased risk to transform to secondary acute myeloid leukemia (AML). In most patients, the erythroid lineage is affected. During the past few years our knowledge about the pathogenesis of MDS, has increased substantially. However, most of these studies focused on CD34+ stem and progenitor cells or granulomonocytic cells, whereas only little is known about genes aberrantly expressed in dysplastic erythroid cells in MDS. In an attempt to define abnormal gene expression patterns and molecular pathways contributing to erythroid dysplasia in MDS and to identify disease-specific phenotypic and molecular abnormalities in dysplastic erythoid cells, we compared gene expression profiles of erythroid progenitor cells (EryPC) in 10 patients with low-risk MDS (IPSS-R score≤7) and 15 control BM samples. EryPC were defined as CD45low/CD105+ cells and purified from BM samples by multicolor flow cytometry and cell sorting (purity〉95%). Gene expression levels were analyzed by Affymetrix array technology (GeneChip U133 Plus 2.0 arrays) and confirmed for a panel of select genes by qPCR. A stringent cut-off (p3.0) was used to define differences in mRNA expression levels. A total number of 1,180 mRNA species were found to be differently expressed in EryPC in MDS (72 up-regulated, 1,108 down regulated) compared to normal BM EryPC. Among these, a number of genes regulating proliferation and differentiation in lymphohematopoietic cells, including TCEB1 transcription elongation factor B (SIII), the transcriptional repressor YY1, E2F3, and the ATP-dependent RNA helicase DDX5, were found to be downregulated in EryPC in MDS compared to normal BM. In the validation phase of our project, we identified the major Coxsackie-Adenovirus Receptor (CAR) as a rather specifically regulated protein-product in dysplastic EryPC. In fact, as assessed by flow cytometry, a significantly lower or even absent expression of CAR was found on EryPC in 20 of 30 patients with MDS (67%), including 3/4 patients with RCUD, 1/4 with RARS, 6/10 with RCMD, 8/9 with RAEB, and 2/3 with 5q- syndrome, compared to normal BM or non-neoplastic controls (p

Description: Introduction The treatment of Adult Acute Lymphoblastic Leukemia (ALL) has improved by the use of pediatric-like approaches, erasing the poor prognostic impact of numerous variables. New markers are however still needed to identify poor prognosis patients in prospective trials. The p16INK4A/CDK4-6/pRb pathway and telomerase activity (TA), markers of cell activation and aging, were analyzed in 175 adult ALLs, treated between November 2003 and January 2007 according to the GRAALL/GRAAPH trials, to investigate their prognostic value in this context. Methods The cohort comprised 105 males and 70 females aged between 16 and 59 years (median: 36). Immunophenotype by flow-cytometry allowed for the characterization of 123 B- and 52 T-lineage ALLs, 163 of them being subdivided according to EGIL criteria. Cytogenetic and/or molecular analyses allowed for the detection of BCR-ABL, MLL-AF4 or E2A-PBX1 fusion transcripts, NOTCH1 mutations in T-ALLs, and IKAROS deletions in BCR-ABL-negative B-ALLs. In all cases, cell samples were obtained at diagnosis, before any treatment, from bone marrow aspiration after the patients provided informed consent. Flow cytometric analysis of the DNA content was performed for evaluation of the percentages of cells in S/G2/M phases of the cell cycle. Cell cycle regulatory proteins were examined in 135 samples, by western blot. The TA assay was performed on 156 samples according to a telomeric repeat amplification protocol. Furthermore, in vitro analyses of the p16INK4A/CDK4-6/pRb pathway and TA were carried out in normal peripheral blood lymphocytes before and after stimulation and during lymphocyte long-term culture. Results The p16INK4A/CDK4-6/pRb pathway and TA were analyzed according to the immunological phenotype and molecular characteristics of ALLs. Leukocytosis (p 0) (p = 0.042), paradoxically, with high expression levels of p16INK4A (p = 0.029), and also with above median TA (p=0.040). Patients with the highest TA (over quartile 75) showed a significantly shorter OS (p = 0.018) especially when considering only disease-related death (p = 0.007). The poor prognostic value of above median TA for OS (p = 0.026) and disease–related OS (p = 0.009) was also confirmed in the non-Allo SCT group. Considering that IKAROS is deleted in most of BCR-ABL1+ ALLs and since survival was similar in BCR-ABL1+ ALLs and BCR-ABL1-/IKAROSdel B-ALLs, these two groups of patients were pooled for analysis (42 cases). A shorter DFS was linked to pRb phosphorylation (p = 0.036), while both a shorter DFS (p = 0.026) and OS (p = 0.055) were noted for patients with high TA. In vitro analyses in normal lymphocytes demonstrated that increased expression of p16INK4A, CDK4, p-pRb and TA is related to cell activation, suggesting that ALL blasts with these criteria could be in an activated stage. Conclusions These data bring new perspectives to the biological characterization of ALLs and associate a poor prognosis in BCR-ABL1+ ALLs with enhanced cell activation. Additional investigations could focus, in a prospective series, on the analysis of the cell activation markers described here and on the development of new therapeutic strategies by proposing the association of lymphocyte activation inhibitors. Disclosures: No relevant conflicts of interest to declare.