ALBERT

Description: 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.

Description: RNA interference (RNAi) has rapidly evolved into an efficient tool for functional genomics in a variety of organisms. Stable expression of shRNA (short hairpin RNA) driven by pol III promoters upon retro- or lentiviral gene transfer can induce long-term gene silencing in mammalian cells, including human hematopoietic cells. We recently demonstrated that lentivirus mediated anti bcr-abl RNAi can specifically silence bcr-abl gene expression, inhibit oncogene driven cell proliferation, and eradicate leukemic cells depending on the dose of lentivirus-mediated shRNA expression (Scherr et al. Gene Therapy 2004). Since effective depletion requires a threshold of lentiviral integrations into target cell genomes, the risk of insertional mutagenesis may limit the therapeutic value of this approach. We therefore applied lentivirus-mediated RNAi for functional genomics in purified primary normal and CD34+ cells from chronic phase CML patients harvested at initial diagnosis. Several SHP-2 shRNAs were generated according to established rules and were functionally evaluated using a bicistronic reporter system as described earlier. Effective shRNA expression cassettes were subsequently cloned into lentiviral plasmids encoding RFP to track lentiviral transduction. Transduction of K562, U937, NB-4 and TF-1 cells with lentiviral supernatants results in a reduction of SHP-2 mRNA and protein by more than 90 %. Interestingly, anti-SHP-2 shRNA induced almost complete depletion of RFP+ cells in all four cell lines, demonstrating that SHP-2 expression is essential for proliferation and survival in these cells. We next transduced normal and CML-derived CD34+ cells with a puritiy of 〉 95% with control and anti-SHP-2 lentiviruses, and stimulated methylcellulose cultures of the cells with high (GM-CSF: 20 ng/ml; IL-3: 10 ng/ml) or low (GM-CSF: 0.2 ng/ml; IL-3: 0.1 ng/ml) cytokine concentrations. This assay relies on the fact that colony formation of CML-CFU is mediated by both cytokine receptor and bcr-abl signaling. Therefore differential numbers of transduced, i.e. RFP+ colonies under different cytokine stimulations reflect the role of the RNAi-target in normal or malignant CFU. Whereas anti-SHP-2 RNAi did not reduce the proliferation of normal transduced CFU (n=5), proliferation of CFU from CML patients was specifically reduced between 50 to 85 % under low cytokine concentration (n= 9). These data suggest that primary normal cells are more resistant to inhibition of SHP-2 gene expression than leukemic cell lines and CD34+ cells from CML patients and identify SHP-2 as a potential target for anti bcr-abl therapy.

Description: 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.

Description: Background: Considering the heterogeneity of leukemic cells in patients, current treatment regimens of chemotherapy and bone marrow transplantation lack specificity and are associated with frequent relapses and severe adverse effects. Hence, there is a need to develop novel therapeutics that can target the disease by its molecular fingerprint with minimal side effects.Despite the wide potential of RNA interference (RNAi) for translational therapeutics, systemic application of siRNA is hampered by rapid renal clearance, degradation by serum nucleases or associated immune responses. Lipid nanoparticles (LNPs) containing ionizable cationic lipids, when mixed with siRNA, embody the most advanced delivery platform for systemic administration of siRNA based therapeutics. Here, we established and employed the BCR-ABL dependent K562-CML xenotransplantation model as a proof of principle to validate LNP mediated siRNA functional delivery in vivo. Methods and Results: A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with anti-BCR-ABL or CTRL siRNA. To determine the delivery efficiency of LNP-siRNA formulations, human leukemic K562 cells were incubated with siRNA-containing LNPs at various concentrations. Almost 100% of cells had taken up siRNA containing LNPs even at the lowest concentration of 0.0625µg/ml with stable uptake kinetics. We also observed near 100% uptake of LNP-siRNA in hard to transfect primary patient cells (CML, AML, ALL and MDS). Looking at the on-target functional efficacy of LNP-siRNA formulations, we observed a time and dose dependent increase in apoptosis (annexin V assay) and decrease in cell viability (alamar blue assay) of K562 cells treated with anti-BCR-ABL siRNA but not CTRL siRNA. A robust knockdown in BCR-ABL mRNA levels (65-90%) at 72 hours and protein at 96 hours was observed which confirmed that cell death was an on-target effect. Colony-forming potential of primary human CD34+ CML cells was significantly reduced when treated with anti-BCR-ABL siRNA compared to CTRL siRNA and to CD34+ cells from healthy donors. To translate our findings in vivo, we evaluated the safety profile, delivery potential and functional efficacy of LNP-siRNA in mice. A total dose of 15mg/kg (3 injections of 5mg/kg at day 0, 1 and 2) in healthy NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice resulted in 100% LNP positive cells in peripheral blood up to day 10. The formulations were highly tolerable in vivo with no significant differences in body weight and complete blood counts between treated and control mice. Moreover, serum analysis showed no significant toxicity in mice following LNP-siRNA treatment. With a focus on hematopoietic tissues following systemic administration, NSG mice received transplants of human K562 cells (stably expressing GFP and luciferase) intrafemorally and were injected intravenously for 3 consecutive injections of LNP-siRNA (1 or 5mg/kg body weight) at 8 hours interval. Interestingly, almost 100% LNP uptake was observed in xenograft leukemic cells in bone marrow at 48 hours at both doses. The leukemic burden of luciferase expressing K562 cells in mice was quantified using in vivo imaging before and during treatment. Treatment with anti-BCR-ABL siRNA for 10 days (n=7) resulted in a 0.5 fold decrease, whereas CTRL siRNA (n=7) resulted in a net 5-fold increase of luciferase signal, thus proving the efficacy of our approach in vivo. Conclusion: We show a highly efficient and non-toxic delivery in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of leukemic mice. By inhibiting BCR-ABL we show a reduction of leukemic burden in our xenotransplant model, while leukemic cells expanded in CTRL siRNA treated mice. Our study provides a proof-of-principle that the combined use of lipid nanoparticles and RNAi technology can be used to target leukemia cells in vivo with promising therapeutic implications. Disclosures Walsh: Precision Nanosystems Inc.: Employment. Thomas:Precision Nanosystems Inc.: Employment. Ramsay:Precision Nanosystems Inc.: Employment. Heuser:Karyopharm: Research Funding.

Description: Background: BCR-ABL+ acute lymphoblastic leukemia (ALL) in adults has a poor prognosis with allogeneic stem cell transplantation (SCT) considered the best curative option for suitable patients. BH3 mimetics induce mitochondrial outer membrane permeabilization (MOMP) linked to apoptosis induction by releasing BH3-only proteins BIM and/or BID from the anti-apoptotic factors BCL2, BCL-XL, MCL1, BCLW and BFL1. The BCL2-specific BH3 mimetic venetoclax (ABT-199) may provide an opportunity to improve pharmacotherapy of BCR-ABL+ ALL in particular for elderly patients not suitable for SCT. Aim: We aimed to rationally design optimized combination therapies for BCR-ABL+ ALL based on the molecular mechanisms of apoptosis induction by BH3 mimetics. Methods: We first biochemically characterized binding of BIM, a BH3 only activator of mitochondrial apoptosis, to BCL2, BCLXL and MCL1 and its release by BH3 mimetics in two BCR-ABL+ ALL cell lines. We next visualized and quantitated MOMP-induction by BH3 mimetics in viable cells. We then characterized the effects of dexamethasone and tyrosine kinase inhibitors (TKI) imatinib and dasatinib on BIM expression and calculated dose-effect combination indices (CI) for combination therapies in cell lines and two BCR-ABL+ ALL primograft models co-cultured with mesenchymal stem cells ex vivo. We finally used in vivo bioluminescence and survival analyses in murine xenotransplantation models to evaluate therapeutic efficacy in vivo. Results: In BCR-ABL+ BV173 and SUPB15 cells BIM but not BID binds to BCL2. BIM is rapidly released from BCL2 by venetoclax in a time and dose dependent manner. Release of BIM induces both MOMP (as defined by a decrease in mitochondrial membrane potential) and apoptosis (as defined by PARP cleavage and propidium iodide staining). Furthermore, BIM is strongly required for cytotoxicity of venetoclax, dasatinib and dexamethasone. Primary BCR-ABL+ ALL cells are more resistant against MOMP induction by venetoclax than BCR-ABL-negative ones, and BIM expression is reduced in these cells. Both, TKIs and dexamethasone augment BIM expression in BV173 and SUPB15 cells and act synergistically with venetoclax in cell lines and two BCR-ABL+ primografts (CI for the triple combination therapy of venetoclax, dexamethasone and dasatinib between 0.1 and 0.15, CI〈 1.0 considered synergistic). Triple combinations with venetoclax, dexamethasone and TKIs efficiently attenuate leukemia progression in xenotransplantation models and, notably, the dasatinib- but not the imatinib-containing combination led to treatment- and leukemia-free long-term survival in a BCR-ABL+ mouse model. Conclusions: These data demonstrate efficacy of venetoclax in ALL. Although BCR-ABL inhibits venetoclax cytotoxicity, this inhibition can be overcome by triple combination therapy with venetoclax, dexamethasone and dasatinib. Since the triple combination therapy can be curative in preclinical xenotranplatation models clinical studies with oral chemotherapy-free regimens may be considered in particular for elderly patients not suitable for allogeneic SCT. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: 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.

Description: 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.