Human Acute Lymphoblastic Leukemia Research Articles

Restoration of Mismatch Repair Functions in Human Cell Line Nalm-6, Which Has High Efficiency for Gene Targeting

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20–30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.

Abstract 3598: Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele.

Abstract Mouse thymic lymphoma (TL) has been used for study of radiation carcinogenesis because of its high incidence. The lymphoma or leukemia is a mouse model of human T-cell acute lymphoblastic leukemias (T-ALL), which is a malignant clonal expansion of thymocytes that accounts for about 15% of ALL cases. Ionizing radiation can damage DNA, and the cells with damaged DNA are assumed to be the cells of origin in tumors. However, which cell in the thymus or the bone marrow leads to TL remains obscure. Bcl11b gene encodes zinc-finger transcription proteins, mutations or deletion of which has been found in 10-16% of T-ALL. Bcl11bKO/+ heterozygous mice are susceptible to thymic lymphomas when γ-irradiated, and irradiated Bcl11bKO/+ mice generate clonally expanding or premalignant thymocytes within atrophic thymus. Resembling clonal expansion of thymocytes was reported in the T-ALL model overexpressing LMO2 oncogene. Interestingly, LMO2-induced clonally expanding thymocytes persist long-term and comprise cells with the stem cell-like self-renewal property that function for the bone marrow stem cells before TL development. In this study, we developed Bcl11bflox/+;Lck-Cre and Bcl11bflox/+;CD4-Cre mice, in which loss of one Bcl11b allele occurs in thymocytes at the immature CD4−CD8− stage without expression of CD4 and CD8 cell surface markers and at the CD4+CD8+ double-positive (DP) stage, respectively. Those mice were subjected to γ-irradiation of 3Gy at 8 weeks of age and their thymocytes were characterized of clonal expansion, differentiation and cell number. The clonal expansion was observed in only the former mice, suggesting the origin of the premaligant thymocytes not in the DP cells, the cell type seen in a majority of TL. The clonally expanded thymocytes possessed common rearrangement sites at the TCRβ locus but they underwent rearrangement at various different sites at the TCRα locus, and their majority showed mature phenotypes, a higher expression of TCRβ and the CD8 expression. This suggests the presence of a subpopulation of immature thymocytes that is capable to rearrange DNA at the TCRα locus in DP cells to continuously produce further differentiated thymocytes. We also found the decrease in thymocyte number in irradiated Bcl11bflox/+;Lck-Cre mice. This probably reflects the reduced proliferation of bone marrow-derived progenitors, and this reduction may weaken the competition in niche with premaligant thymocytes and help for them to survive and proliferate. Citation Format: Ryo Kominami, Rieka Go, Satoshi Hirose, Yukio Mishima. Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3598. doi:10.1158/1538-7445.AM2013-3598

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

O-acetylated N-acetylneuraminic acid as a novel target for therapy in human pre-B acute lymphoblastic leukemia

The development of resistance to chemotherapy is a major cause of relapse in acute lymphoblastic leukemia (ALL). Though several mechanisms associated with drug resistance have been studied in detail, the role of carbohydrate modification remains unexplored. Here, we investigated the contribution of 9-O-acetylated N-acetylneuraminic acid (Neu5Ac) to survival and drug resistance development in ALL cells. A strong induction of 9-O-acetylated Neu5Ac including 9-O-acetyl GD3 was detected in ALL cells that developed resistance against vincristine or nilotinib, drugs with distinct cytotoxic mechanisms. Removal of 9-O-acetyl residues from Neu5Ac on the cell surface by an O-acetylesterase made ALL cells more vulnerable to such drugs. Moreover, removal of intracellular and cell surface-resident 9-O-acetyl Neu5Ac by lentiviral transduction of the esterase was lethal to ALL cells in vitro even in the presence of stromal protection. Interestingly, expression of the esterase in normal fibroblasts or endothelial cells had no effect on their survival. Transplanted mice induced for expression of the O-acetylesterase in the ALL cells exhibited a reduction of leukemia to minimal cell numbers and significantly increased survival. This demonstrates that Neu5Ac 9-O-acetylation is essential for survival of these cells and suggests that Neu5Ac de-O-acetylation could be used as therapy to eradicate drug-resistant ALL cells.

P03.08 - Inhibition of Casein Kinase II (CK2) Enhances Ikaros Tumor Suppressor Activity and Shows Therapeutic Efficacy in a Preclinical Leukemia Model

Casein kinase II (CK2) promotes cell cycle progression and cellular proliferation in leukemia. Overexpression of CK2 results in the development of high-risk leukemia, that is resistant to chemotherapy. The mechanism by which CK2 promotes cellular proliferation and malignant transformation is unknown. Our published data showed that CK2 phosphorylates the Ikaros tumor suppressor protein, which results in loss of its function as a transcriptional repressor. To determine the therapeutic efficacy of CK2 inhibitors, we tested their effect on Ikaros tumor suppressor function in vitro in human pre-B acute lymphoblastic leukemia (ALL) and in vivo using a preclinical human-mouse pre-B ALL xenograft model.

KPT‐330 inhibitor of CRM1 (XPO1)‐mediated nuclear export has selective anti‐leukaemic activity in preclinical models of T‐cell acute lymphoblastic leukaemia and acute myeloid leukaemia

This study explored the anti-leukaemic efficacy of novel irreversible inhibitors of the major nuclear export receptor, chromosome region maintenance 1 (CRM1, also termed XPO1). We found that these novel CRM1 antagonists, termed SINE (Selective Inhibitors of Nuclear Export), induced rapid apoptosis at low nanomolar concentrations in a panel of 14 human T-cell acute lymphoblastic leukaemia (T-ALL) cell lines representing different molecular subtypes of the disease. To assess in vivo anti-leukaemia cell activity, we engrafted immunodeficient mice intravenously with the human T-ALL MOLT-4 cells, which harbour activating mutations of NOTCH1 and NRAS as well as loss of function of the CDKN2A, PTEN and TP53 tumour suppressors and express a high level of oncogenic transcription factor TAL1. Importantly, we examined the in vivo anti-leukaemic efficacy of the clinical SINE compound KPT-330 against T-ALL and acute myeloid leukaemia (AML) cells. These studies demonstrated striking in vivo activity of KPT-330 against T-ALL and AML cells, with little toxicity to normal murine haematopoietic cells. Taken together, our results show that SINE CRM1 antagonists represent promising 'first-in-class' drugs with a novel mechanism of action and wide therapeutic index, and imply that drugs of this class show promise for the targeted therapy of T-ALL and AML.

Dissecting the Entry Route of Saporin-based a-CD7 Immunotoxins in Human T-Cell Acute Lymphoblastic Leukaemia Cells

Elucidating the intracellular fate(s) of targeted toxins is of fundamental importance for their optimal use as anticancer drugs, since the biochemical targets of their enzymatic activity reside in the cell cytoplasm, as in the case of the plant ribosome inactivating proteins (RIP) saporin, ricin and of bacterial toxins. In this paper, we compared the cell surface binding and cytotoxic properties of the model RIP ricin to an immunotoxin constructed with a monoclonal antibody directed against the human T-cell marker CD7 covalently linked to saporin (CD7-SAP). Our results indicate that, despite the fact that internalization takes place via an apparently common entry route leading to the Golgi complex, surprisingly, the addition of an endoplasmic reticulum retrieval C-terminal signal (KDEL) to CD7-SAP does not potentiate its cytotoxicity. In addition, while ricin toxicity is clearly reduced by Brefeldin A under conditions where this fungal metabolite causes Golgi stack disruption, we paradoxically observed a potentiating effect by Brefeldin A on CD7-SAP cytotoxicity suggesting that this inhibitor interferes with retrograde route(s) other than the well established Trans-Golgi Network-ER retrograde route.

Convergence of the ZMIZ1 and NOTCH1 Pathways at C-MYC in Acute T Lymphoblastic Leukemias

Activating NOTCH1 mutations are found in 50% to 60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, these mutations generally fail to induce leukemia. This observation suggests that NOTCH1 activation must collaborate with other genetic events. Mutagenesis screens previously implicated ZMIZ1 as a possible NOTCH1 collaborator in leukemia. ZMIZ1 is a transcriptional coactivator of the protein inhibitor of activated STAT (PIAS)-like family. Its role in oncogenesis is unknown. Here, we show that activated NOTCH1 and ZMIZ1 collaborate to induce T-ALL in mice. ZMIZ1 and activated NOTCH1 are coexpressed in a subset of human T-ALL patients and cell lines. ZMIZ1 inhibition slowed growth and sensitized leukemic cells to corticosteroids and NOTCH inhibitors. Gene expression profiling identified C-MYC, but not other NOTCH-regulated genes, as an essential downstream target of ZMIZ1. ZMIZ1 functionally interacts with NOTCH1 to promote C-MYC transcription and activity. The mechanism does not involve the NOTCH pathway and appears to be indirect and mediated independently of canonical PIAS functions through a novel N-terminal domain. Our study shows the importance of identifying genetic collaborations between parallel leukemic pathways that may be therapeutically targeted. They also raise new inquiries into potential NOTCH-ZMIZ1 collaboration in a variety of C-MYC-driven cancers.

The DN2 Myeloid-T (DN2mt) Progenitor is a Target Cell for Leukemic Transformation by the TLX1 Oncogene.

Inappropriate activation of the TLX1 (T-cell leukemia homeobox 1) gene by chromosomal translocation is a recurrent event in human T-cell Acute Lymphoblastic Leukemia (T-ALL). Ectopic expression of TLX1 in murine bone marrow progenitor cells using a conventional retroviral vector efficiently yields immortalized cell lines and induces T-ALL-like tumors in mice after long latency. To eliminate a potential contribution of retroviral insertional mutagenesis to TLX1 immortalizing and transforming function, we incorporated the TLX1 gene into an insulated self-inactivating retroviral vector. Retrovirally transduced TLX1-expressing murine bone marrow progenitor cells had a growth/survival advantage and readily gave rise to immortalized cell lines. Extensive characterization of 15 newly established cell lines failed to reveal a common retroviral integration site. This comprehensive analysis greatly extends our previous study involving a limited number of cell lines, providing additional support for the view that constitutive TLX1 expression is sufficient to initiate the series of events culminating in hematopoietic progenitor cell immortalization. When TLX1-immortalized cells were co-cultured on OP9-DL1 monolayers under conditions permissive for T-cell differentiation, a latent T-lineage potential was revealed. However, the cells were unable to transit the DN2 myeloid-T (DN2mt)-DN2 T-lineage determined (DN2t) commitment step. The differentiation block coincided with failure to upregulate the zinc finger transcription factor gene Bcl11b, the human ortholog of which was shown to be a direct transcriptional target of TLX1 downregulated in the TLX1+ T-ALL cell line ALL-SIL. Other studies have described the ability of TLX1 to promote bypass of mitotic checkpoint arrest, leading to aneuploidy. We likewise found that diploid TLX1-expressing DN2mt cells treated with the mitotic inhibitor paclitaxel bypassed the mitotic checkpoint and displayed chromosomal instability. This was associated with elevated expression of TLX1 transcriptional targets involved in DNA replication and mitosis, including Ccna2 (cyclin A2), Ccnb1 (cyclin B1), Ccnb2 (cyclin B2) and Top2a (topoisomerase IIα). Notably, enforced expression of BCL11B in ALL-SIL T-ALL cells conferred resistance to the topoisomerase IIα poison etoposide. Taken together with previous findings, the data reinforce a mechanism of TLX1 oncogenic activity linked to chromosomal instability resulting from dysregulated expression of target genes involved in mitotic processes. We speculate that repression of BCL11B expression may provide part of the explanation for the observation that aneuploid DNA content in TLX1+ leukemic T cells does not necessarily portend an unfavorable prognosis. This TLX1 hematopoietic progenitor cell immortalization/T-cell differentiation assay should help further our understanding of the mechanisms of TLX1-mediated evolution to malignancy and has the potential to be a useful predictor of disease response to novel therapeutic agents in TLX1+ T-ALL.

Potentiation of a novel palladium (II) complex lethality with bee venom on the human T-cell acute lymphoblastic leukemia cell line (MOLT-4)

BackgroundAlthough honeybee venom (BV) has been reported to induce apoptosis in different types of cancerous cells, its synergistic effects with customary anti-cancer drugs remain largely unknown. In the present study, we evaluated the cytotoxic effect of BV alone (as a natural product) and the synergistic cytological effects of this component in combination with [Pd (bpy) (Pi-Pydtc)]NO3 – a novel palladium complex on human T-cell lymphoblastic leukemia cells. To investigate the cytotoxic effect of the BV alone and in combination with palladium complex on MOLT-4 cells MTT assay was performed. In order to determine the apoptotic effects of BV separately and in combination with Pd (II) complex on these cells and its ability to induce apoptosis, morphological examination, flowcytometric analysis and caspase-3 colorimetric assay were done.ResultsWe found that BV induced morphological changes, namely nuclear shrinkage, and inhibited MOLT-4 cell proliferation; both effects were dose- and time-dependent. Flow cytometry by Annexin-V antibody demonstrated that BV induced apoptosis in MOLT-4 cells. Furthermore, BV induced apoptosis independently of caspase-3 in these cells. In addition, we proved a clear synergistic effect of BV on [Pd (bpy) (Pi-Pydtc)]NO3. The apoptotic pathway activated by BV in combination with Pd complex was caspase-3-dependent.ConclusionsThese observations provide an explanation for the anti-proliferative properties of BV, and suggest that this agent may be useful for treating lymphoblastic leukemia alone or in combination with chemotherapy drugs pending further investigations on animal models as preclinical tests.

Zerumbone-loaded nanostructured lipid carriers: preparation, characterization, and antileukemic effect

Zerumbone, a natural dietary lipophilic compound with low water solubility (1.296 mg/L at 25°C) was used in this investigation. The zerumbone was loaded into nanostructured lipid carriers using a hot, high-pressure homogenization technique. The physicochemical properties of the zerumbone-loaded nanostructured lipid carriers (ZER-NLC) were determined. The ZER-NLC particles had an average size of 52.68 ± 0.1 nm and a polydispersity index of 0.29 ± 0.004 μm. Transmission electron microscopy showed that the particles were spherical in shape. The zeta potential of the ZER-NLC was −25.03 ± 1.24 mV, entrapment efficiency was 99.03%, and drug loading was 7.92%. In vitro drug release of zerumbone from ZER-NLC was 46.7%, and for a pure zerumbone dispersion was 90.5% over 48 hours, following a zero equation. Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human T-cell acute lymphoblastic leukemia (Jurkat) cells, the half maximal inhibitory concentration (IC50) of ZER-NLC was 5.64 ± 0.38 μg/mL, and for free zerumbone was 5.39 ± 0.43 μg/mL after 72 hours of treatment. This study strongly suggests that ZER-NLC have potential as a sustained-release drug carrier system for the treatment of leukemia.

Apoptotic events induced by synthetic naphthylchalcones in human acute leukemia cell lines

Acute leukemia is a disorder of the hematopoietic system characterized by the expansion of a clonal population of cells blocked from differentiating into mature cells. Recent studies have shown that chalcones and their derivatives induce apoptosis in different cell lines. Since new compounds with biological activity are needed, the aim of this study was to evaluate the cytotoxic effect of three synthetic chalcones, derived from 1-naphthaldehyde and 2-naphthaldehyde, on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. Based on the results, the most cytotoxic compound (A1) was chosen for further analysis in six human acute leukemia cells and in a human colon adenocarcinoma cell line (HT-29). Chalcone A1 significantly reduced the cell viability of K562, Jurkat, Kasumi, U937, CEM and NB4 cells in a concentration and time-dependent manner when compared with the control group (IC50 values between ∼1.5μM and 40μM). It was also cytotoxic to HL-29 cells. To further examine its effect on normal cells, peripheral blood lymphocytes collected from healthy volunteers were incubated with the compound. It has also been incubated with human fibroblasts cultured from bone marrow (JMA). Chalcone A1 is non-cytotoxic to PBL cells and to JMA cells. A1 caused significant cell cycle arrest in all phases according to the cell line, and increased the proportion of cells in the sub G0/G1 phase. To evaluate whether this chalcone induced cell death via an apoptotic or necrotic pathway, cell morphology was examined using fluorescence microscopy. Cells treated with A1 at IC50 demonstrated the morphological characteristic of apoptosis, such as chromatin condensation and formation of apoptotic bodies. Apoptosis was confirmed by externalization of phosphatidylserine, which was detected by the Annexin V-FITC method, and by DNA fragmentation. The results suggest that chalcone A1 has potential as a new lead compound for cancer therapy.

Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice

The CXCR4 antagonist Plerixafor (AMD3100) induces the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. AMD3100 similarly induces the mobilization of human acute lymphoblastic leukemia (ALL) cells into the blood in mice. In this study, the temporal response of pre-B ALL cells to AMD3100 was compared with that of normal hematopoietic progenitor cells (HPC) using an NOD/SCID xenograft model of ALL and BALB/c mice, respectively. ALL cells remained in the circulation up to 6 hours after AMD3100 administration, by which time normal HPCs were no longer detectable. AMD3100 also increased the proportion of actively cycling ALL cells in the peripheral blood. Together, these data suggest that ALL cells are more sensitive to the effects of bone marrow disruption than normal progenitors. Using the NOD/SCID xenograft model, we demonstrated that AMD3100 increased the efficacy of the cell cycle specific drug vincristine, resulting in reduced disease levels in the blood and spleens of animals over 3 weeks and extended the survival of NOD/SCID mice with ALL. These data demonstrate that mobilizing agents can increase the therapeutic effect of cell cycle dependent chemotherapeutic agents.

Cytotoxic Effect of Natural trans-Resveratrol Obtained from Elicited Vitis vinifera Cell Cultures on Three Cancer Cell Lines

trans-Resveratrol (trans-R) has been reported to be a potential cancer chemopreventive agent. Although its cytotoxic activity against different cancer cell lines has been tested, its effect on human acute leukemia cell lines has scarcely been investigated, and only a few in vitro studies were performed using human breast epithelial cell lines. Due to its potential value for human health, demand for trans-R has rapidly increased, and new biotechnological strategies to obtain it from natural edible sources have been developed. Thus, grapevine cell cultures represent a reliable system of trans-R production since they biosynthesize trans-R constitutively or in response to elicitation. In addition, there are no studies deepen on the inhibitory effect of trans-R, produced by elicited grapevine cell cultures, on growth of human tumor cell lines. In this work, the effect of trans-R extracted from the culture medium, after elicitation of grapevine cell cultures, was tested on two human acute lymphocytic and monocytic leukemia cell lines, and one human breast cancer cell line. The effect of trans-R on cell proliferation was not only dose- and time-dependent but also cell type-dependent, as seen from the different degrees of susceptibility of cancer cell lines tested. As regards the effect of trans-R on cell cycle distribution, low trans-R concentrations increased cells in the S phase whereas a high trans-R concentration increased G₀/G₁ phase in all cell lines. Perturbation of the cell cycle at low trans-R concentrations did not correlate with the induction of cell death, whereas a high trans-R concentration, cell proliferation decreased as a result of increasing apoptosis in the three cell lines. In leukemia cells, trans-R up-regulated the expression of caspase-3 while trans-R-induced apoptosis in breast cells occur through a caspase-3-independent mechanism mediated by a down-regulation of Bcl-2.

Modulation of Leukemic Stem Cell Self-Renewal and Cell Fate Decisions by Inhibition of Hedgehog Signalling in Human Acute Lymphoblastic Leukemia (ALL).

AbstractAbstract 2578The Hedgehog (Hh) pathway plays a functional role in embryonic development and promotes tumorigenesis in a diversity of cancers. Constitutive activation of Smo, an essential component of the Hh pathway, augments stem cell number and accelerates disease in BCR-ABL positive CML, whereas loss of Smo depletes CML stem cells by inhibition of self-renewal. Phase I clinical trials using Hh inhibitors have started in BCR-ABL pos ALL and CML. The role of Hh signalling on stem cell behaviour in BCR-ABL neg ALL has not yet been examined. The phenotype of leukemic stem cells (LSCs) and the target cells for transformation in ALL are controversial, but only a small subpopulation of cells seem to act as LSCs. These cells may be the most relevant targets for treatment regimens for compounds that interfere with self-renewal programs and that provide promising therapeutic options for improving treatment of adult ALL.Aims of the study are characterization of different genetically and phenotipically defined ALLs, using our twelve patient derived long term cultures (LTCs), according to their biologic behaviour including leukemia initiating capacity, assessment of the impact of Hh inhibition on proliferation, apoptosis and clonogenic capacity and LSC function and dissection of the role of different components of the Hh signalling pathway on cell fate decisions by means of single cell video microscopy. These studies are anticipated to yield information on the therapeutic potential of modulation of Hh signalling in both BCR-ABL pos and neg ALL and the potential value of combination therapy.As models of BCR-ABL pos and neg leukemias we used serum-, cytokine- and stroma-free long term cultures of primary ALL blasts. Clonogenic growth of ALL cells was assessed in semi solid methylcellulose based media. Cell subpopulations were isolated on the basis of CD20, CD34 and CD38 expression via FACS based sorting (BD FACS Aria). Cell proliferation was measured using XTT assays and Annexin V and 7 AAD FACS staining were used to quantitate apoptosis. Quantitative RT PCR of Hh signalling pathway components using predeveloped Taqman assays (Applied). Single cell video tracking to determine cell fate decisions was performed as previously described (Rieger et all, Science 2009), adapted to facilitate the analysis of ALL LTCs. Two novel Smo inhibitors being currently in clinical testing, LDE225 and BMS833923 were kindly provided by Novartis and Bristol Myers Squibb.Results: The expression pattern of surface markers varied profoundly between the different LTCs studied. In preliminary experiments designed to identify functionally distinct subpopulations of long term cultured ALL blasts, cells were isolated to greater than 90% purity based on CD20, CD34 and CD38 expression. With the exception of CD34 positive cells, the surface marker distribution rapidly reverted to an identical pattern as determined prior to culture in three cell lines studied. In two ALL LTCs, CD34 expression was associated with slower proliferation. All three cell lines displayed clonogenic capacity ranging from 0,25% to 8% and are able to engraft in NSG mice.Analysis of Hh Signalling in ALL LTCs by RT PCR demonstrated expression of Shh, Ptch, Smo, and the transcription factors Gli 1 + 3, indicating active Hh signalling in ALL. Interestingly the transcription factor Gli 2 was not expressed, the functional relevance of which remains as yet unclear. The Hh inhibitors LDE225 and BMS833923 (0,01μM to 5μM) showed no dose dependent effect on inhibition of proliferation or induction of apoptosis in ALL LTCs.In conclusion we found evidence of Hh activation in both BCR-ABL pos and neg LTC ALL cells. No impact of Smo inhibition on proliferation and apoptosis was observed in response to the Smo inhibitors LDE225 and BMS833923, consistent with the hypothesis that Hh signalling in these cells may affect primarily self-renewal mechanisms. Single cell imaging of ALL LTCs has been successfully established for up to nine days of culture and will be applied to the testing of Hh modulation on cell fate decisions. Disclosures:No relevant conflicts of interest to declare.

The TAL1 Complex Represses the FBXW7 Tumor Suppressor Through Mir-223 in Human T-Cell Acute Lymphoblastic Leukemia

Abstract 1296The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL) and causes T-ALL in murine transgenic models, emphasizing its importance in the molecular pathogenesis of this disease. However, the mechanism by which TAL1 leads to transformation of thymocytes is unclear. Dysregulation of miRNAs play an important role in tumorigenesis in diverse cancer types. A recent study identified miR-223 as the most abundant miRNA in T-ALL patient samples and was oncogenic by virtue of its ability to accelerate Notch-induced T-ALL in a murine model (Mavrakis et al. Nature Genetics 2011). However, the underlying mechanisms leading to dysregulated miRNA expression in T-ALL remain poorly understood.In order to explore the hypothesis that aberrant expression of miRNAs is mediated by the TAL1 oncogene in T-ALL, we generated high-resolution maps of the genome-wide occupancy of the TAL1 complex, including E2A, HEB, GATA3, LMO2 and RUNX1 by chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq). Analysis of binding sites in two TAL1-positive T-ALL cell lines (Jurkat and CCRF-CEM cells) and two primary T-ALL samples identified 54 miRNAs where binding of the TAL1 complex was within 10 kb of either the transcriptional start sites or the start sites of genes that contain miRNAs in their intronic regions. To determine which of these miRNAs were not only directly bound, but also regulated by the TAL1 complex, we analyzed global changes in miRNAs after knockdown of TAL1 in Jurkat cells using two independent shRNAs. By miRNA expression profiling, we identified significant changes in expression of 25 miRNAs, of which nine were down-regulated on TAL1 knockdown (and thus positively regulated by TAL1) and 16 were up-regulated on TAL1 knockdown (and thus negatively regulated by TAL1). Of these 25 miRNAs, four (miR-223, miR181a*, miR-26a and miR-29c) were shown to be direct targets of the TAL1 complex based on our ChIP-seq data.We chose to focus on miR-223 because it exhibited the most dynamic down-regulation after TAL1 knockdown. ChIP-qPCR validated binding of the TAL1 complex to a region within 4 kb of the miR-223 transcriptional start site. Analysis of RNA polymerase II and CBP binding showed significant enrichment, and high levels of H3K4M3 and H3K79M2 modification were detected indicative of transcriptional initiation and elongation of this locus. Furthermore, expression of miR-223 was significantly higher in the TAL1-positive cell lines (n=13) as compared to the TAL1-low cells (n=10) (P<0.0001). miR-223 levels also closely mirrored TAL1 levels in murine thymic subsets, with marked down-regulation after the DN2 stage, suggesting miR-223 is a physiological target of TAL1 during normal thymic development, and that its overexpression in TAL1-positive T-ALL cells, arrested at the double-positive (DP) stage, is aberrant compared to their normal DP counterpart.To test the hypothesis that the growth inhibition observed after TAL1 knockdown is mediated by decreases in miR-223 expression, we retrovirally infected Jurkat and RPMI-8402 T-ALL cell lines with a miR-223 construct, such that miR-223 expression was no longer under the control of TAL1 in these cells. Forced expression of miR-223 partially rescued the growth inhibitory effects induced by TAL1 knockdown, in both a lentiviral and doxycycline-inducible shRNA system. Additionally, inhibition of mature miR-223 by lentiviral infection of a miR-223 shRNA construct led to significant growth inhibition of TAL1-positive cell lines through the induction of apoptosis. Thus, maintenance of miR-223 expression is required for optimal growth of TAL1-positive T-ALL cells.The highest ranked predicted target of miR-223 by Targetscan is the FBXW7 tumor suppressor, a ubiquitin ligase that is mutated in a significant proportion of T-ALL patients and targets oncogenes such as c-MYC, NOTCH and mTOR for degradation. Accordingly, overexpression of miR-223 in TAL1-low miR-223-low T-ALL cells markedly down-regulated FBXW7 protein expression. Furthermore, the up-regulation of FBXW7 protein expression observed on knockdown of TAL1 in TAL1-positive cell lines could be prevented by retroviral miR-223 expression. Thus, miR-223 is an important target of TAL1 and links the TAL1 oncogene to repression of the FBXW7 tumor suppressor. Disclosures:No relevant conflicts of interest to declare.

The Pias-Like Coactivator ZMIZ1 Drives C-MYC Induction in Collaboration with NOTCH1 in T-Cell Acute Lymphoblastic Leukemia.

Abstract 2380Activating NOTCH1 mutations are found in 50–60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, these mutations generally fail to induce leukemia. Cooperating oncogenes must be recruited by NOTCH1 to fully induce leukemia. Murine insertional mutagenesis screens previously implicated ZMIZ1 as a possible NOTCH1 collaborator in leukemia (Uren et al., Cell, 2008; Dupuy et al., Nature, 2005; Berquam-Vrieze et al., Blood, 2011). ZMIZ1 is a transcriptional co-activator of the Protein Inhibitor of Activated STAT (PIAS)-like family. It shares a zinc finger domain, the MIZ domain, with PIAS proteins. The MIZ domain mediates interactions with DNA-binding transcription factors and sumoylation. Previously, we showed that ZMIZ1 promotes T-ALL in collaboration with leukemia-associated NOTCH1 alleles in mouse models. ZMIZ1 and activated NOTCH1 were co-expressed in a subset of human patients. Genetic ZMIZ1 inhibition slowed leukemic cell growth and overcame resistance of some T-ALL cell lines to NOTCH inhibitors. ZMIZ1 may be a new clinically relevant oncogene. Here we sought to determine the downstream target genes of ZMIZ1 in leukemia. Validation of gene expression profiling data identified C-MYC and IL7RA as downstream targets of ZMIZ1. Targeting the C-MYC or IL-7 pathways using genetic and pharmacological inhibitors partly phenocopied the growth inhibitory effects we previously saw with ZMIZ1 inhibition. In order to determine whether these genes are direct or indirect targets of ZMIZ1, we generated an estrogen fusion protein, ZMIZ1-ER. ZMIZ1-ER induced C-MYC and IL7RA expression in the presence of tamoxifen, but failed to induce these genes with the addition of cycloheximide. These data suggest that C-MYC and IL-7RA are indirect targets. Like the PIAS proteins, ZMIZ1 appeared to have a broad effect on transcription to exert its functions. We next sought to elucidate the biochemical mechanism of ZMIZ1. Ectopic expression of ZMIZ1 or NOTCH1 had weak effects on endogenous c-Myc expression and failed to rescue a C-MYC-dependent T-ALL cell line after withdrawal of ectopic C-MYC. In contrast, ZMIZ1 in combination with NOTCH1 dramatically induced C-MYC expression by several fold and rescued the C-MYC dependent cell line. ZMIZ1 enhanced the ability of even weak NOTCH1 mutants to induce C-MYC, suggesting a mechanism by which ZMIZ1 may increase resistance to NOTCH inhibitors. ZMIZ1 did not influence C-MYC expression post-transcriptionally. It functioned primarily as a transcriptional activator. Although both C-MYC and IL7RA are both NOTCH1 target genes, ZMIZ1 did not directly interact with NOTCH1 or influence the expression of several other NOTCH1 target genes such Ptcra, Hes1, Dtx1, and Cd25. Thus, ZMIZ1 did not pan-activate NOTCH signaling. Based on bioinformatic analysis, we generated mutants that deleted individual domains of ZMIZ1. All mutants expressed at high levels by Western blot. Deletion of the transcriptional activation domain or the N-terminal domain (NTD) abolished the ability of ZMIZ1 to induce c-Myc and drive proliferation. Surprisingly, deletion of the PAT-like, Proline-rich, and MIZ domains or all three domains simultaneously had no effect on ZMIZ1 function. The 120-amino acid NTD has a predicted helical structure without significant sequence homology to any known domain. It is not found in ZMIZ2 or PIAS proteins. In summary, the mechanism of ZMIZ1 appears to be novel, indirect, transcriptional, and independent of canonical NOTCH and PIAS functions. Our study demonstrates the importance of characterizing genetic collaborations between parallel leukemic pathways that may be therapeutically targeted. They also raise new inquiries into potential NOTCH-ZMIZ1 collaboration in a variety of C-MYC-driven cancers. Disclosures:No relevant conflicts of interest to declare.

Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

AbstractAbstract 4295Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures:No relevant conflicts of interest to declare.

BCL11B suppression does not influence CD34+ cell differentiation and proliferation

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B) gene plays a critical role in T-cell differentiation and proliferation. However, little is understood about the role of BCL11B in human hematopoietic stem/progenitor cells. Small interfering RNA (siRNA)-mediated suppression of the BCL11B was shown to induce apoptosis in human T-cell acute lymphoblastic leukemia cells. To further characterize the role of BCL11B in hematopoietic stem/progenitor cells and assess the safety of siRNA-mediated targeted therapy, the in vitro differentiation and proliferation of CD34+ cells after BCL11B-siRNA935 treatment were studied. CD34+ cells were sorted from three cases of umbilical cord blood by the magnetic activated cell sorting technique, and the purity was identified by flow cytometry. BCL11B-siRNA935 was delivered into CD34+ cells by nucleofection and the BCL11B expression level was analyzed by quantitative real-time polymerase chain reaction. Erythroid burst-forming units (BFU-E), granulocyte/macrophage colony-forming units (CFU-GM), and megakaryocyte colony-forming units (CFU-Meg) were assessed using BCL11B-siRNA935-treated CD34+ cells by the methylcellulose semi-solid culture method. The BCL11B expression level in CD34+ cells was significantly lower than that in Molt-4 cells and peripheral blood mononuclear cells from healthy individuals. An approximate one-fold reduction in the BCL11B mRNA level was observed 24 hours post-transfection with BCL11B-siRNA935. However, there was no significant difference on the colony formation ability of BFU-E, CFU-GM, and CFU-Meg for CD34+ cells between the BCL11B-siRNA935-treated and mock-transfected groups (P > 0.05). BCL11B suppression by RNA interference had no significant influence on the differentiation and proliferation of CD34+ cells. In conclusion, the BCL11B-siRNA935 used in this study may be safe, and BCL11B may be considered a new candidate for targeted gene therapy in T-cell malignancies.

Antitumor activity of L-asparaginase from Erwinia carotovora against different human and animal leukemic and solid tumor cell lines

The dose- and time-dependent antitumor and cytotoxic effects of L-asparaginases from Erwinia carotovora (ECAR LANS) and Escherichia coli (MEDAC) have been investigated using human leukemic cells and human and animal solid tumor cells. These included human T-cell acute lymphoblastic leukemia cell lines (Jurkat, Jurkat/A4, Molt-4), human chronic myeloid leukemia K562 cells, human promyelocytic leukemia HL-60, and also human solid tumor cells (prostate carcinoma LnCap, breast adenocarcinoma MCF7, ovarian adenocarcinoma SCOV-3 and carcinoma CaOV, hepatocarcinoma Hep G2, fibrosarcoma HT-1080) and animal solid tumor cells (rat Gasser’s ganglion neurinoma cells GGNC-1, mouse glioblastoma EPNT-5). We investigated sensitivity of tumor cells (seeded at different density) to L-asparaginases, as well the effect of L-asparaginases on cell growth rate, protein and DNA synthesis in the presence of various cytostatics. Cell cycle analysis by flow cytofluorimetry and detection of apoptotic cells before and after treatment with L-asparaginases indicate that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division. The cytofluorometric study of solid and leukemic cells revealed that except HL-60 cells the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. Combined treatment of cells using a combination of L-asparaginase and doxorubicin significantly increased the number of apoptotic cells up to 60% (MCF-7 cells), 40% (Jurkat cells) and even 99% (HL-60). High levels of DNA and protein synthesis rates in asparaginase-treated tumor cells suggest lack of massive entry of tumor cells to apoptosis. This conclusion is based on the fact of sensitivity of multi-resistant Jurkat/A4 cells to L-asparaginases (it is nearly impossible to induce apoptosis in these cells). Since ECAR LANS did not influence growth of normal human fibroblasts it appears that the enzyme cytotoxicity is associated only asparagine deficiency.