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In the past few years, the discovery of RNA-mediated gene silencing mechanisms, like RNA interference (RNAi), has revolutionized our understanding of eukaryotic gene expression. These mechanisms are activated by double-stranded RNA (dsRNA) and mediate gene silencing either by inducing the sequence-specific degradation of complementary mRNA or by inhibiting mRNA translation. RNAi now provides a powerful experimental tool to elucidate gene function in vitro and in vivo, thereby opening new exciting perspectives in the fields of molecular analysis and eventually therapy of several diseases such as infections and cancer. In hematology, numerous studies have described the successful application of RNAi to better define the role of oncogenic fusion proteins in leukemogenesis and to explore therapeutic approaches in hematological malignancies. In this review, we highlight recent advances and caveats relating to the application of this powerful new methodology to hematopoiesis.

Despite advances in both targeted therapies with ABL-specific tyrosine kinase inhibitors and in allogeneic stem cell transplantation, BCR-ABL positive acute lymphoblastic leukemia (ALL) remains a very high-risk disease, necessitating the development of novel treatment strategies. miRNAs are small non-coding RNAs which regulate gene expression posttranscriptionally in a sequence-specific manner. miRNAs usually repress the expression of many target genes. We hypothesized that miRNAs may help to identify potential therapeutic targets if (i) they are expressed in a disease-specific manner and if (ii) modulating their expression induces a desired phenotype, such as apoptosis of tumour cells, in appropriate experimental models. Based on our observation that miR∼17-92-encoded miRNAs are significantly less abundant in primary BCR-ABL-positive as compared to negative ALL-cells, we studied the expression and function of miRNAs encoded by the miR∼17-92 derivative miR∼17-19b in a murine pro-B-cell line with inducible BCR-ABL-expression (TonB). Induction of BCR-ABL expression in TonB cells reduced endogenous miR-17, miR-18a, and miR-19 by 2 to 3.5-fold, confirming that expression of the miR∼17-92 cluster is controlled by BCR-ABL. Interestingly, over-expression of miR∼17-19b by lentiviral gene transfer led to a substantial induction of apoptosis in TonB cells in a BCR-ABL-dependent manner. To identify potential miRNA targets, we used a proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) in miR∼17-19b transgenic TonB cells. Several apoptosis-related proteins were differentially expressed including Bcl2, an established inhibitor of mitochondrial pro-apoptotic pathways. The miRNA target prediction program RNA22 predicted several miR∼17-19b miRNA-binding sites within both murine and human Bcl2 mRNA, and we demonstrated direct miRNA binding to Bcl2 mRNA by luciferase reporter and anti-AGO2 RIP chip analyses. As with miR∼17-19b over-expression, Bcl2 specific RNAi strongly induced apoptosis in murine TonB and the human BCR-ABL-positive cell lines BV-173, Tom1 and SupB15. BCR-ABL positive human ALL-cell lines were also more sensitive than negative ones to pharmacological BCL2 inhibition with the BH3 mimetic ABT 737. In addition, inhibition of BCL2 by ABT 737 and BCR-ABL kinase activity by Imatinib exert different anti-leukemic effects with differential impact on miR∼17-92 miRNA-expression. To assess the therapeutic potential of BCL2 inhibition we used a xenotransplantation assay with real time in vivo monitoring of drug therapies by bioluminescent imaging. ABT-737 treatment substantially inhibited expansion of luciferase-expressing human primary BCR-ABL-positive ALL xenografts in NOD/LtSz-scid IL-2Rγ null (NSG) mice and significantly lengthened their median survival. Taken together, our data identify BCL2 as a therapeutic target of particular relevance in BCR-ABL-positive ALL and indicate involvement of miR∼17-92-encoded miRNAs in regulation of apoptosis in these cells. The validity of this miRNA-based approach to identify potential drug targets is demonstrated by the efficacy of the BCL2 inhibitor ABT-737 in an in vivo model of human BCR-ABL positive ALL, suggesting that BCL2 inhibition should be considered for early phase clinical testing as a strategy to improve disease outcomes. Disclosures: No relevant conflicts of interest to declare.

We report the cloning and characterization of a novel member of the Transcriptional Intermediary Factor 1 (TIF1) gene family, human TIF1gamma. Similar to TIF1alpha and TIF1beta, the structure of TIF1beta is characterized by multiple domains: RING finger, B boxes, Coiled coil, PHD/TTC, and bromodomain. Although structurally related to TIF1alpha and TIF1beta, TIF1gamma presents several functional differences. In contrast to TIF1alpha, but like TIF1beta, TIF1 does not interact with nuclear receptors in yeast two-hybrid or GST pull-down assays and does not interfere with retinoic acid response in transfected mammalian cells. Whereas TIF1alpha and TIF1beta were previously found to interact with the KRAB silencing domain of KOX1 and with the HP1alpha, MODI (HP1beta) and MOD2 (HP1gamma) heterochromatinic proteins, suggesting that they may participate in a complex involved in heterochromatin-induced gene repression, TIF1gamma does not interact with either the KRAB domain of KOX1 or the HP1 proteins. Nevertheless, TIF1gamma, like TIF1alpha and TIF1beta, exhibits a strong silencing activity when tethered to a promoter. Since deletion of a novel motif unique to the three TIF1 proteins, called TIF1 signature sequence (TSS), abrogates transcriptional repression by TIF1gamma, this motif likely participates in TIF1 dependent repression.

The analysis of molecular events sufficient and/or necessary to transform hematopoietic stem and progenitor cells is hampered by the lack of suitable models which allow the stepwise analysis oftransforming events. In an effort to generate such models we isolated lineage negative (lin-) murine bone marrow cells and expressed either AML1-ETO or HoxB4 along with EGFP as a quantitative reporter by retroviral gene transfer. Retrovirally transduced cells were initially cultured in the presence of SCF+IL-3+IL-6 in serum free medium, and EGFP+ cells were sorted 3 days after transduction to start long- term cultures with a ratio of about 50% EGFP + cells. Interestingly, long-term cultures for AML1-ETO remain strictly SCF-dependent wheras HOXB4 cultures only require IL-3 for cell survival and proliferation. Beside cytokine stimulation, cell survival requires expression of AML1-ETO and HoxB4, respectively, as demonstrated by RNA interference targeting EGFP which is encoded along with the respective transgene on bicistronic transcripts. Although AML1- ETO and HoxB4 cultures display similar proliferation kinetics and blast like morphology (with some degree of granulocytic maturation in HoxB4 cultures), the immunophenotype of immortalized cells differ with more than 90% of AML1-ETO cells being c-Kit and Sca-1 positive whereas HoxB4 cells are c-Kit and Sca-1 negative. Interestingly, AML1-ETO cells form only blast like colonies with very low plating efficiency (

ing Scale) in order to determine fall risk in elderly people. One hundred and fifty subjects with mild fall risk met inclusion criteria (mean age 64,85 ys) were randomized into two groups: 75 for group treated with Human Body Posturizer (HBP) and 75 for placebo group. Results.– HBP group showed a significant difference in Short Physical Performance Battery, Tinetti Scale and Pain Numeric Rating Scale (P

Introduction.–Malnutrition is a commonproblemamongolderpeople and associated with reduced functional and cognitive ability. Cognitive impairment, in turn, can determine changes in dietary habits and consequent nutritional deficiencies. Patients and methods.– We administered Mini Nutritional Assessment (MNA) and Mini Mental State Examination (MMSE) to seventy-eight patients (average age 83.2 years) admitted to rehabilitation geriatric institute. All patients were evaluated at admittance;wealsoevaluated the relationshipbetweennutritional and cognitive status and discharge (home vs residential home vs hospital vs death). Results.– On admission 58% of patients had moderate-severe cognitive impairment (MMSE

Abstract 4644 Aberrant activation of various oncogenes, including homeobox genes encoding fundamental transcription factors, during T-cell development contributes to T-cell acute lymphoblastic leukemia (T-ALL). Neoplastic chromosome rearrangements are known to deregulate Antennapedia-class homeobox genes of the NKL-family (TLX1, TLX3, NKX2-5) and HOXA-cluster genes of the Extended-Hox-family. Following analysis of T-ALL cell lines and primary cells, we describe leukemogenic involvement of a third homeobox gene group, the Paired (PRD)-class. Ascertainment was performed in an early stage-arrested T-ALL cell line (LOUCY) which revealed chromosomal deletion at 5q31, removing the downstream regulatory region of the PRD-homeobox gene PITX1. Comparative expression analysis confirmed ectopic PITX1 expression, consistent with aberrant activation by del(5)(q31) which removes a STAT1 binding site. STAT1 mediates repressive IL2-STAT1 signaling, implicating IL2-pathway avoidance as a possible activation mechanism. Furthermore, we detected expression of the physiologically similar PITX2 in 11/24 (46%) T-ALL cell lines, 5 with genomic PITX2 gains. Among primary T-ALL samples, 2/22 (9%) – both pediatric pre-T-ALL - ectopically expressed PITX1 but not PITX2. Forced expression of PITX1 by lentiviral transduction of JURKAT cells and subsequent analysis by expression profiling, prompted upregulation of RUNX2 and JUN and inhibition of RUNX1 and NKX3-1, indicating impaired T-cell differentiation. Taken together, our data show leukemic activation of PITX1, a novice PRD-class homeobox gene in the T-ALL repertoire, which may promote leukemogenesis by inhibiting differentiation. Disclosures: No relevant conflicts of interest to declare.

Abstract 3636 Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS-box family which is physiologically expressed in hematopoietic stem cells and during development of B-cells. Recently, we identified ectopic expression of MEF2C in T-cell acute lymphoblastic leukemia (T-ALL) cell lines activated either via chromosomally mediated ectopic expression of homeodomain protein NKX2-5 or via deletion of non-coding exon and promoter regions at 5q14, suggesting loss of negative regulatory elements. Our aim was to identify additional transcriptional regulators of MEF2C in T-ALL. Therefore, we analyzed the sequence of the MEF2C 5′-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix (bHLH) proteins, STAT5 and HOXA9/HOXA10. Overexpression studies demonstrated MEF2C activation by GFI1B (strong), LYL1 and TAL1 leukemic bHLH proteins (weak), and inhibition by STAT5 (strong) and HOXA9/HOXA10 (weak). Chromatin-Immuno-Precipitation analysis demonstrated direct binding of GFI1B, LYL1 and STAT5 but not of HOXA10 to the MEF2C 5′-region in T-ALL cells. However, HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of MEF2C regulation. Chromosomal deletion of the 5′-MEF2C STAT5 binding site in LOUCY cells by del(5)(q14), reduced expression levels of STAT5 protein in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5-signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that ectopic expression of MEF2C in T-ALL cells is mainly regulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory STAT5-signaling. Disclosures: No relevant conflicts of interest to declare.

Abstract 3761 Poster Board III-697 Farnesylation is a post-translational modification critical for the proper function of multiple physiologically important proteins, including small G-proteins, such as RAS. Methods allowing rapid and selective detection of protein farnesylation are fundamental for the understanding of farnesylated protein function and for monitoring efficacy of farnesyltransferase inhibitors (FTI). While the natural substrates for prenyltransferases are farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), FTase has been shown to incorporate isoprenoid analogues into protein substrates. Materials and Methods FTase targets in three different myeloid leukemia cell lines (HL-60, K562 and NB-4) were labeled using the unnatural FPP analogue 8-anilinogeranyl diphosphate (AGPP) in a tagging-via-substrate approach. Antibodies specific for the anilinogeranyl moiety were used to detect AG-modified proteins. This highly effective labeling/detection method was coupled with two-dimensional electrophoresis (2-DE) and subsequent Western blotting. Identities of individual protein spots were determined by overlaying anti-AG immunoblots and immunoblots probed with antibodies specific for different known prenylation target proteins, such as RAS-family proteins. The clinically tested FTIs BMS-214,662 and L-778,123 as well as the nitrogen-containing bisphosphonate zoledronate were used to further validate the specificity of this labeling technique. As a proof of principle of the ability of this approach to identify target proteins important for FTI resistance, RNA interference (RNAi) was applied to investigate the role of one of the specific protein targets (K-RAS) which remained AG-labelled in the presence of FTI BMS-214,662. Following confirmation of K-RAS silencing by Western blotting, flow cytometric analysis of Annexin-V stained cells was used to quantify the effect of K-RAS silencing on BMS-214,662-induced apoptosis. Results Metabolic labeling of prenylated proteins with anilinogeranyl (AG) occurred in a time-dependent manner. This method provided increased resolution of the prenylated proteome as we were able to detect as many as ten distinct protein spots corresponding to a single band at approximately 20 kDa observed by 1D SDS-PAGE. Some of the proteins we have identified using this overlay method include H-RAS, K-RAS, N-RAS, Rap1, RhoB, RhoC, pre-Lamin A, Lamin B, and LKB1. AG-specific signals decreased upon FTI treatment, further substantiating the specificity of this method. Additional evidence of the specificity of this approach was the observation that the bisphosphonate inhibitor zoledronate did not inhibit protein AG-labeling. Interestingly, this method allowed direct evaluation of specific FTI targets as demonstrated by the different efficacies we observed between the two FTIs studied here – BMS-214,662 and L-778,123. This method even allowed identification of subtle differences among the leukemia cell lines tested. Closer evaluation of the farnesylated proteome demonstrated clear differences between BMS-214,662 and L-778,123, consistent with earlier reports that FTIs elicit numerous cellular effects, including induction of apoptosis and cytostatic effects. While BMS-214,662 effectively inhibited farnesylation of the majority of larger molecular weight proteins, L-778,123 also blocked prenylation of smaller molecular weight proteins such as N-RAS and K-RAS. However, RhoB and RhoC remained farnesylated regardless of FTI treatment. BMS-214,662-induced apoptosis was substantially potentiated by RNAi knock-down of K-RAS expression. Conclusions This snapshot approach allowed simple, rapid and dynamic analysis of the complex farnesylated proteome in leukemia cells. Our results demonstrate that this method can be used to identify and validate specific inhibitor targets. Importantly, this approach also successfully identified proteins (e.g. K-RAS) which may be important for resistance to some FTIs, and thus may be useful in directing development of more efficient therapeutic regimens. Disclosures: No relevant conflicts of interest to declare.

Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by specific hybridization to complementary sequences in the 3′-untranslated region of corresponding mRNAs resulting in inhibition of mRNA translation or mRNA degradation. Aberrant expression of specific miRNAs has been described in a variety of human malignancies but its functional consequences are currently largely unknown. The miR-17- 92 cluster (encoding miR-17-5p, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92) has been shown to be over-expressed in chronic phase but not in blast crisis CML CD34+ cells in a BCR-ABL- and c-Myc-dependent manner (Venturini et al. 2007). Interestingly, the knock-out of the polcystronic miR-17-92 cluster in mice recently revealed an essential role in lung development and B-lymphopoiesis at the stage of pre-B-cells also affected in pre- B-ALL which often expresses the BCR-ABL oncogene. To study miR-17-92 expression in BCR-ABL + myeloid and lymphoid cells we performed miRNA specific quantitative RT-PCR (miR-qRT-PCR) in myeloid human K562 and murine 32D/BCR-ABL cells and in lymphoid human BV173 and IL-3-dependent murine TonB cells which can be induced to express BCR-ABL. We found reduced expression of both the miR-17-92 cluster and c-MYC in lymphoid as compared to myeloid cells. Inhibition of BCR-ABL tyrosine kinase activity by imatinib reveals that miR-17-92 expression depends on BCR-ABL in myeloid K562 and 32D/BCR-ABL but not in lymphoid BV173 and TonB cells. Similarly, RNAi against c-MYC reduces miR-17-92 expression in the myeloid but not in lymphoid cell lines demonstrating lineage-specific effects on BCR-ABL-mediated regulation of miR17-92 expression. Furthermore, preliminary chromatin immunoprecipitation analyses in the presence and absence of imatinib demonstrate BCR-ABL tyrosine kinase dependent recruitment of c-MYC to the miR-17-92 5′-regulatory region in myeloid, but not in lymphoid cells. Correspondingly, transcription of the miR-17-92 pri-miRNA is reduced by imatinib treatment in myeloid cell lines. To study the function of individual miRNAs encoded within the polycistronic miR-17-92 cluster in the presence and absence of BCR-ABL, TonB cells were lentivirally transduced with miR-17-19b, a variant of miR- 17-92 selected for efficient transgenic miRNA expression, and with individual miRNAs embedded within miR-30-derived sequences to induce stable miRNA-specific gain-offunction phenotypes (Scherr et al. 2007). Transduction rates were about 98%, and miRqRT- PCR demonstrated increased miRNA-expression between 2- and 14-fold for miR-17- 5p, miR-19b, and miR-20a, respectively. Over-expression of these three miRNAs and the miR-17-19b polycistron had no effect on cell proliferation of TonB cells under stimulation with IL-3 as compared to controls. In contrast, BCR-ABL-dependent proliferation in the absence of IL-3 was reduced by miR-17-5p by about 80% and completely by miR-17-19b in two experiments, whereas miR-19b and miR-20a had only minor inhibitory effects. Interestingly, addition of IL-3 to BCR-ABL+ TonB cells can rescue cell proliferation for all these miRNAs. These data demonstrate lineage-specific effects of BCR-ABL on expression of the miR-17-92 cluster and may suggest that miRNAs encoded within this polycistron encompass target genes whose expression is required for BCR-ABL-mediated cell transformation.

Three NK-like (NKL) homeobox genes, TLX1/HOX11, TLX3/HOX11L2 and NKX2- 5/CSX, have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Here we screened further NKL genes in 24 T-ALL cell lines by RT-PCR and identified common expression of MSX2, highlighting this homeobox gene as a potential physiological family member in T-cells. Subsequent quantification of MSX2 confirmed expression in primary hematopoietic cells demonstrating higher levels in CD34+ stem cells when compared to peripheral blood cells or mature CD3+ T-cells. Analysis of core thymic factors in T-ALL cell lines, including IL7, BMP4, TGFbeta, NOTCH and T-cell receptor signaling, suggests their involvement in MSX2 regulation during T-cell differentiation. Chromosomal and genomic analysis of the MSX2 locus (at 5q35) uncovered deletion in t(5;14)(q35;q32) positive T-ALL cell lines associated with low expression levels of MSX2 and ectopic activation of TLX3 or NKX2-5, respectively. For functional analysis we lentivirally transduced T-ALL cells for overexpression of either MSX2 or oncogenic TLX1 and NKX2-5. These cells displayed transcriptional activation of NOTCH3-signaling, as indicated by expression array profiling and real-time PCR analysis of NOTCH3, HES1 and HEY1. The sensitivities to gamma-secretase inhibitor analyzed by MTT-assay of cells overexpressing MSX2, TLX1 or NKX2-5, respectively, were consistently decreased. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with repressor proteins of the NOTCH-pathway, SPEN/MINT and TLE1/GRG1, as shown by co-immunoprecipitation, probably representing one mechanism of (de)regulation. Elevated expression of NOTCH3 and HEY1 mRNA was detected in TLX1/3 positive T-ALL patients, confirming data obtained from cell lines. In conclusion, we have defined expression patterns, regulation and targets of MSX2 in hematopoietic cells, to reveal a novel modulatory activity in T-cell differentiation operating via NOTCH-signaling, and in leukemogenesis when replaced or supplemented by oncogenic NKL homeodomain proteins.

Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by specific hybridization to complementary sequences in the 3′ untranslated region of corresponding mRNAs. Concomitant recruitment of specific multi-protein complexes results either in inhibition of mRNA translation or mRNA degradation. miRNAs are processed in a regulated multi-step process from primary transcripts into mature miRNAs by cellular components which are also at least partially involved in the process of RNA interference (RNAi). Aberrant expression of specific miRNAs has recently been described in human lymphoma and leukemia. In particular, BCR-ABL and c-MYC dependent over-expression of the polycistronic and oncogenic miR-17-92 cluster (encoding miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92) has been described in chronic myeloid leukemia (CML) cell lines, primary CD34+ cells from CML patients (Venturini et al. 2007), and in lung cancer. In BCR-ABL positive K562 cells, miR-17-92 encoded miRNAs repress luciferase activity in miRNA-specific reporter assays. In addition, lentivirus-mediated over-expression of miR-17-92 increases both cell proliferation and sensitivity to imatinib induced cell death. To analyse the function of individual miRNAs of the miR-17-92 polycistron, we generated lentivirus-based strategies to induce stable miRNA-specific loss- and gain-of function phenotypes for miR-18a, miR-19b, and miR-20a, respectively. Over-expression of miRNAs embedded within miR-30-derived sequences from an internal SFFV-LTR promoter allows isolation of K562 cells with increased miRNA expression. In contrast, expression of complementary oligonucleotides (antagomirs) from a H1 promoter located in the lentiviral 3′LTR can induce stable hypomorphic miRNA-phenotypes. In lentivirally transduced K562 cells, individual silencing of miR-18a, miR-19b, and miR-20a by the corresponding antagomirs (ant-miR-18a, ant-miR-19b, ant-miR-20a) specifically relieves miRNA-mediated reporter gene repression. Correspondingly, inhibition of miRNA-function correlates to reduced ‘miRNA’-amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals specific induction of cell proliferation by miR-18a but strong inhibition by miR-20a in K562 cells, respectively. In contrast, anti-miR-18a, but not anti-miR-19b, anti-miR-20a, or control antagomirs inhibits proliferation of K562 cells. These data demonstrate individual and complementary functions of miR-17-92 encoded miRNAs in CML and identify potential targets for specific therapeutic intervention on the miRNA level.

Homeobox genes of the NK-like familiy, including TLX1, TLX3 and NKX2-5, are ectopically activated in T-cell acute lymphoblastic leukemia (T-ALL) cells mostly via chromosomal aberrations. The pathologic function of these closely related genes is still unclear. Here we analyzed their effect on the C13ORF25 gene, containing the miR-17-92 cluster. Micro RNAs (miRNAs) are a class of small non-coding RNAs which are part of an evolutionarily highly conserved intracellular mechanism, regulating gene expression by hybridization to complementary sequences usually located in the 3′untranslated region of coding transcripts. The primary transcripts (pri-mRNA) are processed to short mature miRNAs, mediating either inhibition of mRNA translation or mRNA cleavage. Aberrant expression of specific miRNAs is involved in oncogenesis as recently described for several human malignancies. The miR-17-92 polycistron encodes miRNAs which decrease E2F1 protein expression. Transcription of both E2F1 and miR-17-92 is induced by MYC, itself a target of E2F1, generating a highly regulated interactive network. Depending on the cellular context, E2F1 performs conflicting tasks by triggering proliferation or inducing apoptosis. We investigated the expression of the miR-17-92 cluster in T-ALL cell lines. Real-time RT-PCR analysis of both miR-17-92 pri-mRNA and mature miRNAs revealed different expression levels in these cells, suggesting a possible implication of the NK-like homeodomain proteins in the regulation of the miR-17-92 cluster in T-ALL. HELA cells transfected with TLX1 or NKX2-5 expression constructs showed elevated miR-17-92 pri-mRNA expression, demonstrating an activating effect. Lentiviral-mediated overexpression of NKX2-5 in the T-ALL cell line MOLT-4 consistently showed increased miR-17-92 pri-mRNA levels and decreased E2F1 protein amounts. For functional analysis of these downstream targets, another T-ALL cell line (PEER) was lentivirally transduced with expression constructs for either miR-17-92 or E2F1, resulting in reduced or elevated E2F1 protein levels, respectively. Overexpression of miR-17-92 or E2F1 did not significantly influenced the cell proliferation. However, induction of apoptosis by treating these cells with etoposide, an inhibitor of topoisomerase II, indicated that overexpression of miR-17-92 and E2F1 resulted in enhanced and reduced cell viability, respectively, as analyzed by MTT assay. In summary, these data indicate an activatory effect of oncogenic NK-like homeodomain proteins on miR-17-92 expression, reducing E2F1 protein levels and thereby enhancing survival of leukemic T-cells.

Within the human genome nearly 200 homeobox genes have been identified. These are functionally involved in distinct developmental processes during embryogenesis and in the adult. Many have been implicated in cancer mostly following ectopic expression. In this study, we analyzed homeobox gene expression in Hodgkin lymphoma (HL) cell lines using degenerate primers for RT-PCR and Affymetrix U133A expression arrays followed by extended RT-PCR analysis of candidate homeobox genes in additional hematopoietic cell lines. We identified upregulated HOXB9 expression in HL, as well as in anaplastic large cell lymphoma and mediastinal B-cell lymphoma cell lines. Analysis of HOXB9 regulation in HL cells revealed activation by E2F3A, and repression by BMI1, respectively. Constitutive ERK5 pathway activation was identified in all HL cell lines analyzed together with primary HL lymph nodes biopsies. Our data show that ERK5 regulates HOXB9 expression, probably mediated via BMI1 repression. Expression analysis of microRNA gene mir-196a1 which is located upstream of HOXB9 indicated coregulation together with HOXB9. Functional analysis of HOXB9 by knockdown or overexpression assays revealed its influence in both proliferation and apoptosis in HL cells. In summary we identified upregulated expression of HOXB9 in HL, and its regulation mediated by constitutively active ERK5 signalling, thus representing novel therapeutic targets in both HL and NHL subtypes.

Micro RNAs (miRNAs) are small non-coding, single-stranded RNAs regulating gene expression by hybridization to complementary sequences in target mRNAs. Aberrant miRNA expression has been found in a variety of human malignancies including B-cell leukemias and lymphomas. The polycistronic miR-17–92 cluster, which includes seven miRNAs (miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-92-1), resides in intron 3 of the C13orf25 gene located on human chromosome 13 and has recently been described to harbor oncogenic potential in a murine transplantation model. In addition, expression of the polycistron can be regulated by c-myc, which is also activated by the Bcr-Abl oncogene product. To study miRNA expression in CML, K562 cells were analyzed by microarray analysis (miCHIP) for normalized expression of 244 miRNAs following treatment either with imatinib to inhibit Bcr-Abl kinase activity or with specific anti bcr-abl shRNA to knock-down gene expression. Both of these treatment strategies led to a 2–4-fold reduction of six mature miRNAs (miR-17-5p, miR-17-3p, miR-18a, miR-20a miR-19b-1, miR-92–1) as compared to untreated control cells. The reduction of the miR-17–92 miRNA cluster expression was confirmed for each individual mature miRNA species encoded in the cluster by real-time RT-PCR. Treatment with either imatinib or anti-bcr-abl shRNA also down-regulates expression of mature miRNA miR-19a (>3-fold) in K562 cells as quantified by real-time RT-PCR. We next used PCR based quantification to analyze miR-17–92 expression in purified normal (n=4) and CML CD34+ cells harvested at diagnosis of chronic phase disease (n=16). Individual mature miRNAs of the cluster were up-regulated (2–6-fold) in CML as compared to normal CD34+ cells. Additionally, the pri-miRNA for the miR-17–92 cluster was up-regulated in 70% (7/10) of CML samples. Interestingly, retrovirus-mediated over-expression of the miR-17-19b-1 cluster in K562 cells mediates enhanced colony-formation of transduced cells. Furthermore, K562 cells expressing high levels of the miR-17-19b-1 cluster are more resistant to proliferation inhibition induced by specific anti-myc RNAi, indicating some functional role of the miR-17-92 cluster on proliferation and survival in this model. In summary, the polycistronic miR-17–92 cluster is up-regulated in early chronic phase CML CD34+ cells and may contribute to cellular transformation by the Bcr-Abl oncogene product.

Alzheimer's disease (AD) is a multifactorial disorder characterized by the progressive deterioration of neuronal networks. The primary cause and sequence of its progression are only partially understood but abnormalities in folding and accumulation of insoluble proteins such as beta-amyloid and Tau-protein are both associated with the pathogenesis of AD. Mitochondria play a crucial role in cell survival and death, and changes in mitochondrial structure and/or function are related to many human diseases. Increasing evidence suggests that compromised mitochondrial function contributes to the aging process and thus may increase the risk of AD. Dysfunctional mitochondria contribute to reactive oxygen species which can lead to extensive macromolecule oxidative damage and the progression of amyloid pathology. Oxidative stress and amyloid toxicity leave neurons chemically vulnerable. The mitochondrial toxicity induced by beta-amyloid is still not clear but may include numerous mechanisms, such as the increased permeability of mitochondrial membranes, the disruption of calcium homeostasis, the alteration of oxidative phosphorylation with a consequent overproduction of reactive oxygen species. Other mechanisms have been associated with the pathophysiology of AD. Inflammatory changes are observed in AD brain overall, particularly at the amyloid deposits, which are rich in activated microglia. Once stimulated, the microglia release a wide variety of pro-inflammatory mediators including cytokines, complement components and free radicals, all of which potentially contribute to further neuronal dysfunction and eventually death. Clinically, novel approaches to visualize early neuroinflammation in the human brain are needed to improve the monitoring and control of therapeutic strategies that target inflammatory and other pathological mechanisms. Similarly, there is growing interest in developing agents that modulate mitochondrial function.

Chronic Fatigue Syndrome (CFS), also referred to as Myalgic Encephalomyelitis (ME), is a disease of unknown origin. It is classified as Post Viral Fatigue Syndrome (PVFS) in the WHO International Classification of Diseases (ICD) and listed as sub-category at G93.3 under chapter G93, “other disorders of the brain“. ME/CFS is primarily an endemic disorder but occurs in both epidemic and sporadic forms. It affects all racial/ethnic groups and is seen in all socioeconomic strata. A diagnosis of CFS is a diagnosis of exclusion, meaning other medical conditions, including psychiatric disorders, must be first ruled out. CFS is diagnosed if there is no other explanation for the fatigue and if the other symptoms did not develop before the fatigue. The estimated worldwide prevalence of CFS is 0.4–1%. The disease predominantly affects young adults, with a peak age of onset of between 20 and 40 years, and women, with a female to male ratio of 6:1. Mean illness duration ranges from 3 to 9 years. The patho-physiological mechanism of CFS is unclear but the immunological pattern of CFS patients gleaned from various studies indicates that the immune system is chronically activated. Besides the role of environmental insults (xenobiotics, infectious agents, stress) the genetic features of patients are studied to evaluate their role in triggering the pathology. At present there are no specific pharmacological therapies to treat the disease but a variety of therapeutic approaches have been described as benefiting patients. Treatment programs are directed at relief of symptoms, with the goal of the patient regaining some level of preexisting function and well-being.