Human Acute Lymphoblastic Leukemia Research Articles

Abstract 4293: Notch expression expands the pre-malignant pool of T-cell acute lymphoblastic leukemia clones without affecting self-renewal

Abstract Over 60% of human T-cell acute lymphoblastic leukemias (T-ALL) harbor NOTCH1 activating mutations, and the cMYC pathway is also commonly activated in this disease. Notch and cMyc have been shown to collaborate in mouse transgenic models of T-ALL, suggesting that these oncogenes function in parallel pathways to enhance tumor growth; however, the exact role of each in T-ALL is unknown. Here, we show that co-expression of Notch and cMyc in zebrafish T cells significantly enhances T-ALL progression compared to cMyc or Notch alone (p<0.001). However, Notch co-expression with cMyc does not enhance proliferation, alter cell cycle kinetics, or modify apoptosis in leukemic cells when compared to T-ALLs expressing cMyc alone. Clonality assays using RT-PCR analyses for T-cell receptor-beta rearrangements indicate that Notch increases the number of T-ALL clones contained within the primary tumor when compared to cMyc expressing leukemais. A large portion of T-ALL clones present in primary Notch and Notch/cMyc leukemias cannot transfer disease into transplanted animals, while all cMyc-alone expressing T-ALLs clones are capable of engraftment and reinitiation of leukemia. Large scale limiting dilution cell transplantation analyses using syngeneic zebrafish demonstrated that primary T-ALLs expressing either Notch or Notch/cMyc have 10-fold less leukemia-initiating frequency when compared to T-ALLs that express only cMyc; however, following serial passaging, both the Notch and Notch/cMyc leukemias exhibit similar leukemia-initiating frequency as cMyc-induced T-ALLs, indicating that Notch signaling does not enhance self-renewal of T-ALL initiating cells. Taken together, our data supports a model where Notch expands a pool of pre-malignant T-ALL clones within the primary tumor, a subset of which acquire additional mutations to confer a fully transformed phenotype. By contrast, cMyc alone is insufficient to increase the overall pool of pre-malignant clones but confers a fully-transformed phenotype to leukemic cells, accounting for the longer latency that likely reflects the acquisition of additional genetic changes in clones. Our data may explain why a subset of relapsed human T-ALLs develop from an underrepresented clone found in the primary leukemia, in that primary human T-ALLs likely have a large pool of pre-malignant clones resulting from NOTCH-pathway activation that are unable to self-renew and thus, cannot give rise to relapsed T-ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4293. doi:10.1158/1538-7445.AM2011-4293

Clonal selection in xenografted human T cell acute lymphoblastic leukemia recapitulates gain of malignancy at relapse

Genomic studies in human acute lymphoblastic leukemia (ALL) have revealed clonal heterogeneity at diagnosis and clonal evolution at relapse. In this study, we used genome-wide profiling to compare human T cell ALL samples at the time of diagnosis and after engraftment (xenograft) into immunodeficient recipient mice. Compared with paired diagnosis samples, the xenograft leukemia often contained additional genomic lesions in established human oncogenes and/or tumor suppressor genes. Mimicking such genomic lesions by short hairpin RNA-mediated knockdown in diagnosis samples conferred a selective advantage in competitive engraftment experiments, demonstrating that additional lesions can be drivers of increased leukemia-initiating activity. In addition, the xenograft leukemias appeared to arise from minor subclones existing in the patient at diagnosis. Comparison of paired diagnosis and relapse samples showed that, with regard to genetic lesions, xenograft leukemias more frequently more closely resembled relapse samples than bulk diagnosis samples. Moreover, a cell cycle- and mitosis-associated gene expression signature was present in xenograft and relapse samples, and xenograft leukemia exhibited diminished sensitivity to drugs. Thus, the establishment of human leukemia in immunodeficient mice selects and expands a more aggressive malignancy, recapitulating the process of relapse in patients. These findings may contribute to the design of novel strategies to prevent or treat relapse.

VentX trans-Activates p53 and p16ink4a to Regulate Cellular Senescence

Cell senescence is a process of irreversible arrest of cell proliferation and plays an important role in tumor suppression. Recent studies showed that Wnt inhibition is a trigger of cellular senescence. Using methods of reverse genetics, we recently identified VentX, a human homolog of the vertebrate Xenopus Vent family of homeobox genes, as a novel Wnt repressor and a putative tumor suppressor in lymphocytic leukemia. Here, we show that VentX is a direct transcriptional activator of p53-p21 and p16ink4a-Rb tumor suppression pathways. Ectopic expression of VentX in cancer cells caused an irreversible cell cycle arrest with a typical senescence-like phenotype. Conversely, inhibition of VentX expression by RNA interference ameliorated chemotherapeutic agent-induced senescence in lymphocytic leukemia cells. The results of our study further reveal the mechanisms underlying tumor suppression function of VentX and suggest a role of VentX as a potential target in cancer prevention and treatment.

Sequencing a mouse acute promyelocytic leukemia genome reveals genetic events relevant for disease progression

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). It is characterized by the t(15;17)(q22;q11.2) chromosomal translocation that creates the promyelocytic leukemia-retinoic acid receptor α (PML-RARA) fusion oncogene. Although this fusion oncogene is known to initiate APL in mice, other cooperating mutations, as yet ill defined, are important for disease pathogenesis. To identify these, we used a mouse model of APL, whereby PML-RARA expressed in myeloid cells leads to a myeloproliferative disease that ultimately evolves into APL. Sequencing of a mouse APL genome revealed 3 somatic, nonsynonymous mutations relevant to APL pathogenesis, of which 1 (Jak1 V657F) was found to be recurrent in other affected mice. This mutation was identical to the JAK1 V658F mutation previously found in human APL and acute lymphoblastic leukemia samples. Further analysis showed that JAK1 V658F cooperated in vivo with PML-RARA, causing a rapidly fatal leukemia in mice. We also discovered a somatic 150-kb deletion involving the lysine (K)-specific demethylase 6A (Kdm6a, also known as Utx) gene, in the mouse APL genome. Similar deletions were observed in 3 out of 14 additional mouse APL samples and 1 out of 150 human AML samples. In conclusion, whole genome sequencing of mouse cancer genomes can provide an unbiased and comprehensive approach for discovering functionally relevant mutations that are also present in human leukemias.

Bortezomib interacts synergistically with belinostat in human acute myeloid leukaemia and acute lymphoblastic leukaemia cells in association with perturbations in NF‐κB and Bim

Interactions between the histone deacetylase inhibitor belinostat and the proteasome inhibitor bortezomib were investigated in acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) cells. Co-administration of sub-micromolar concentrations of belinostat with low nanomolar concentrations of bortezomib sharply increased apoptosis in both AML and ALL cell lines and primary blasts. Synergistic interactions were associated with interruption of both canonical and non-canonical nuclear factor (NF)-κB signalling pathways, e.g. accumulation of the phosphorylated (S32/S36) form of IκBα, diminished belinostat-mediated RelA/p65 hyperacetylation (K310), and reduced processing of p100 into p52. These events were accompanied by down-regulation of NF-κB-dependent pro-survival proteins (e.g. XIAP, Bcl-xL). Moreover, belinostat/bortezomib co-exposure induced up-regulation of the BH3-only pro-death protein Bim. Significantly, shRNA knock-down of Bim substantially reduced the lethality of belinostat/bortezomib regimens. Administration of belinostat ± bortezomib also induced hyperacetylation (K40) of α-tubulin, indicating histone deacetylase inhibitor 6 inhibition. Finally, in contrast to the pronounced lethality of belinostat/bortezomib toward primary leukaemia blasts, equivalent treatment was relatively non-toxic to normal CD34(+) cells. Together, these findings indicate that belinostat and bortezomib interact synergistically in both cultured and primary AML and ALL cells, and raise the possibilities that up-regulation of Bim and interference with NF-κB pathways contribute to this phenomenon. They also suggest that combined belinostat/bortezomib regimens warrant further attention in acute leukaemias.

Volume-Sensitive Chloride Channels are Involved in Maintenance of Basal Cell Volume in Human Acute Lymphoblastic Leukemia Cells

Chloride channels are expressed ubiquitously in different cells. However, the activation and roles of volume-activated chloride channels under normal isotonic conditions are not clarified, especially in lymphatic cells. In this study, the activation of basal and volume-activated chloride currents and their roles in maintenance of basal cell volume under isotonic conditions were investigated in human acute lymphoblastic leukemia Molt4 cells. The patch-clamp technique and time-lapse image analysis were employed to record whole-cell currents and cell volume changes. Under isotonic conditions, a basal chloride current was recorded. The current was weakly outward-rectified and volume-sensitive and was not inactivated obviously in the observation period. A 47% hypertonic bath solution and the chloride channel blockers NPPB and tamoxifen suppressed the current. Exposure of cells to 47% hypotonic bath solution activated further the basal current. The hypotonicity-activated current possessed properties similar to those of the basal current and was inhibited by NPPB, tamoxifen, ATP and hypertonic bath solution. Furthermore, extracellular hypotonic challenges swelled the cells and induced a regulatory volume decrease (RVD). Extracellular applications of NPPB, tamoxifen and ATP swelled the cells under isotonic conditions and inhibited the RVD induced by hypotonic cell swelling. The results suggest that some volume-activated chloride channels are activated under isotonic conditions, resulting in the appearance of the basal chloride current, which plays an important role in the maintenance of basal cell volume in lymphoblastic leukemia cells. Chloride channels can be activated further to induce a regulatory volume recovery when cells are swollen.

Therapeutic Antibody Targeting of CD47 Eliminates Human Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and constitutes 15% of adult leukemias. Although overall prognosis for pediatric ALL is favorable, high-risk pediatric patients and most adult patients have significantly worse outcomes. Multiagent chemotherapy is standard of care for both pediatric and adult ALL, but is associated with systemic toxicity and long-term side effects and is relatively ineffective against certain ALL subtypes. Recent efforts have focused on the development of targeted therapies for ALL including monoclonal antibodies. Here, we report the identification of CD47, a protein that inhibits phagocytosis, as an antibody target in standard and high-risk ALL. CD47 was found to be more highly expressed on a subset of human ALL patient samples compared with normal cell counterparts and to be an independent predictor of survival and disease refractoriness in several ALL patient cohorts. In addition, a blocking monoclonal antibody against CD47 enabled phagocytosis of ALL cells by macrophages in vitro and inhibited tumor engraftment in vivo. Significantly, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. These data provide preclinical support for the development of an anti-CD47 antibody therapy for treatment of human ALL.

Inhibition of proliferation by agricultural plant extracts in seven human adult T-cell leukaemia (ATL)-related cell lines

Adult T-cell leukaemia (ATL) is caused by human T-cell leukaemia virus type I (HTLV-I) infection and is resistant to conventional chemotherapy. We evaluated the inhibitory effects of agricultural plants on the proliferation of seven ATL-related human leukaemia cells, using three ATL cell lines (ED, Su9T01 and S1T), two human T-cell lines transformed by HTLV-I infection (HUT-102 and MT-2) and two HTLV-I-negative human T-cell acute lymphoblastic leukaemia cell lines (Jurkat and MOLT-4). A total of 52 samples of 80% ethanol extracts obtained from 30 types of agricultural plants were examined. On the basis of IC(50) values, we selected samples with greater activity than genistein, which was used as a positive control. The highest inhibitory effect was observed with extracts from leaves of Vaccinium virgatum Aiton (blueberry) on four cell lines (ED, Su9T01, HUT-102 and Jurkat); seeds of Momordica charantia L. (bitter gourd) exhibited the second highest activity. The bitter gourd seeds suppressed the proliferation of three cell lines (Su9T01, HUT-102 and Jurkat). The extracts from edible parts of Ipomea batatas LAM. (sweet potato), edible parts of Colocasia esculenta (L.) Schott (taro), skin of taro and seeds of Prunus mume Sieb. et Zucc. (mume) showed markedly greater inhibitory effects on Su9T01 than genistein. These findings suggest that ATL-preventative bioactive compounds may exist in these agricultural plants, which are considered to be functional foods.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Cellular context-dependent effects of H2ax and p53 deletion on the development of thymic lymphoma

AbstractH2AX and Artemis each cooperate with p53 to suppress lymphoma. Germline H2ax−/−p53−/− mice die of T-cell receptor-β− (TCR-β−) thymic lymphomas with translocations and other lesions characteristic of human T-cell acute lymphoblastic leukemia. Here, we demonstrate that mice with inactivation of H2ax and p53 in thymocytes die at later ages to TCR-β− or TCR-β+ thymic lymphomas containing a similar pattern of translocations as H2ax−/−p53−/− tumors. Germline Artemis−/−p53−/− mice die of lymphomas with antigen receptor locus translocations, whereas Artemis−/−H2ax−/−p53−/− mice die at earlier ages from multiple malignancies. We show here that Artemis−/− mice with p53 deletion in thymocytes die of TCR-β− tumors containing Tcrα/δ translocations, other clonal translocations, or aneuploidy, as well as Notch1 mutations. Strikingly, Artemis−/− mice with H2ax and p53 deletion in thymocytes exhibited a lower rate of mortality from TCR-β− tumors, which harbored significantly elevated levels of genomic instability. Our data reveal that the cellular origin of H2ax and p53 loss impacts the rate of mortality from and developmental stage of thymic lymphomas, and suggest that conditional deletion of tumor suppressor genes may provide more physiologic models for human lymphoid malignancies than germline inactivation.

Role of Ikaros in T-cell acute lymphoblastic leukemia

Ikaros is a zinc finger transcriptional regulator encoded by the Ikzf1 gene. Ikaros displays crucial functions in the hematopoietic system and its loss of function has been linked to the development of lymphoid leukemia. In particular, Ikaros has been found in recent years to be a major tumor suppressor involved in human B-cell acute lymphoblastic leukemia. Its role in T-cell leukemia, however, has been more controversial. While Ikaros deficiency appears to be very frequent in murine T-cell leukemias, loss of Ikaros appears to be rare in human T-cell acute lymphoblastic leukemia (T-ALL). We review here the evidence linking Ikaros to T-ALL in mouse and human systems.

MOHITO, a novel mouse cytokine-dependent T-cell line, enables studies of oncogenic signaling in the T-cell context

The mouse pro-B cell line Ba/F3 has gained major interest as a model system to investigate oncogenic tyrosine kinases and to determine the efficacy of kinase inhibitors. While Ba/F3 cells are suitable to study oncogenic kinases derived from various cell types, the signaling networks in Ba/F3 cells are B-cell specific. We have established a mouse CD4+CD8+ double positive T-cell line (named MOHITO, for MOuse Hematopoietic Interleukin-dependent cell line of T-cell Origin) that has many features of human T-cell acute lymphoblastic leukemia (Notch1 and Jak1 mutation, TCR rearrangement) and is dependent on interleukin-7. The MOHITO cell line can be transformed to cytokine independent proliferation by BCR-ABL1 or mutant JAK1. This mouse T-cell line is a novel model system to investigate protein signaling and inhibition in a T-cell specific context and is a valuable tool to study and verify oncogenic capacity of mutations in the kinome and phosphatome in T-cell malignancies.

Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1

AbstractPoint mutations that trigger ligand-independent proteolysis of the Notch1 ectodomain occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL) but are rare in murine T-ALL, suggesting that other mechanisms account for Notch1 activation in murine tumors. Here we show that most murine T-ALLs harbor Notch1 deletions that fall into 2 types, both leading to ligand-independent Notch1 activation. Type 1 deletions remove exon 1 and the proximal promoter, appear to be RAG-mediated, and are associated with mRNA transcripts that initiate from 3′ regions of Notch1. In line with the RAG dependency of these rearrangements, RAG2 binds to the 5′ end of Notch1 in normal thymocytes near the deletion breakpoints. Type 2 deletions remove sequences between exon 1 and exons 26 to 28 of Notch1, appear to be RAG-independent, and are associated with transcripts in which exon 1 is spliced out of frame to 3′ Notch1 exons. Translation of both types of transcripts initiates at a conserved methionine residue, M1727, which lies within the Notch1 transmembrane domain. Polypeptides initiating at M1727 insert into membranes and are subject to constitutive cleavage by γ-secretase. Thus, like human T-ALL, murine T-ALL is often associated with acquired mutations that cause ligand-independent Notch1 activation.

Therapeutic Efficacy of Natural Compounds From Viscum Albuml. In Acute Lymphoblastic Leukemia

AbstractAbstract 3261Viscum album L. (mistletoe) is one of the most widely used complementary cancer therapies. Due to their low solubility, aqueous extracts contain hardly any triterpenes which are known to possess anti-tumoral properties. Using cyclodextrins it was possible to solubilisate mistletoe triterpenes (mainly oleanolic acid (OA)) and achieve a plant extract with high levels of OA and mistletoe lectins (ML).In the present study, we determined for the first time the effect of clearly defined mistletoe extracts against human acute lymphoblastic leukemia (ALL) in vitro and in vivo. These mistletoe extracts contain either lectins (aqueous extract, viscum) or cyclodextrin solubilised triterpenes (STE) such as oleanolic - and betulinic acid and combinations thereof (viscumTT). We used the C.B-17/SCID mouse model and tested efficacy and mechanisms of the treatment with these preparations in vitro and in vivo. The human leukemia cell line NALM-6 was incubated with increasing concentrations of mistletoe preparations (10-60 μg/ml OA; 0.8–8 ng/ml ML) and tested for their cytotoxicity in vitro. Apoptosis was determined using mitochondrial potential, DNA fragmentation and Annexin/PI assays. In vivo efficacy was determined in the C.B-17/SCID mouse model. For this purpose, 1×106 NALM-6 cells were injected IV into groups of C.B-17/SCID mice (n=8) and STE extracts were administered three times per week for 14 days by intraperitoneal (IP) injection.Viscum album L. extracts inhibited cell proliferation and show cytotoxic properties in vitro. The highest level of apoptosis with a decrease of the mitochondrial potential was observed with STE preparation at a concentration of 50 μg/ml OA and for lectin-treated cells for 4.7 ng/ml (IC50). To exclude an unwanted cell death via necrosis, LDH release was measured after 4h of incubation with different doses and extracts of Viscum album L without significant LDH release.Based on these data, we investigated the effect of Viscum album L. extracts in vivo. For this purpose 40 mg/kg/day oleanolic acid (STE), 3 μg/kg/day lectin (viscum) or a combination thereof (viscumTT) were administered IP. In line with the in vitro results, mice treated with viscumTT showed a significant longer survival. Mice receiving PBS had a mean survival time of 38 days whereas mice treated with viscumTT had a mean survival of 50,5 days (p=0,005). In summary, we demonstrate for the first time that either solubilised triterpenes or lectins and combinations thereof induce dose- and time-dependent apoptosis in the ALL cell line NALM-6. Based on the in vivo data we believe that triterpene containing Viscum album L. extracts may possess an impressive therapeutic potential. Thus, our investigations provide an important base line for the design of further experimental studies and clinical trials to investigate the effects of individual components and potential synergisms in ALL. Disclosures:No relevant conflicts of interest to declare.

Notch Expression Expands the Pre-Malignant Pool of T-Cell Acute Lymphoblastic Leukemia Clones without Affecting Self-Renewal

AbstractAbstract 316160% of human T-cell acute lymphoblastic leukemias (T-ALL) harbor NOTCH1 activating mutations, making it the most commonly mutated oncogene in T-ALL. Notch signaling is critical for T cell development, and activating Notch mutations are found in all subtypes of T-ALL, suggesting that Notch deregulation may be a dominant initiating event in human disease. In human and rodent models of T-ALL, Notch directly induces cMyc expression. However, cMyc over expression cannot completely rescue Notch inhibition, suggesting that Notch may have other important roles in T-ALL progression. Classic viral insertion screens in mice have indentified that insertional activation of Notch1 is common in Myc induced T-cell malignancies, suggesting that Notch imparts a distinct advantage to leukemic clones independent of cMyc.Notch-induced transgenic zebrafish models of T-ALLs are unique in that Notch signaling does not induce cMyc expression, allowing new opportunities to determine the function of Notch which are independent of cMyc. As with rodent models, the co-expression of Notch and cMyc in zebrafish T cells significantly enhanced T-ALL progression compared to cMyc or Notch alone (p<0.001). However, Notch co-expression with Myc did not enhance proliferation, alter cell cycle kinetics, or modify apoptosis in leukemic cells when compared to Myc alone expressing T-ALLs. Moreover, clonality assays using RT-PCR analyses for T-cell receptor-beta rearrangements indicate that Notch collaborates with Myc to increase the number of T-ALL clones contained within the primary tumor by 2–4 fold when compared to single transgenic animals that express only cMyc. Following serial transplantation, a large portion of T-ALL clones present in primary Notch/Myc leukemias are not found in transplanted animals. This starkly contrasts to results seen in Myc-alone expressing T-ALLs where all primary clones are capable of engraftment and reinitiation of leukemia. Paradoxically, transplant animals developing T-ALL from leukemias that coexpress Notch and Myc have similar numbers of clones as found in primary Myc-induced leukemias. Primary Myc-induced T-ALLs express high levels of endogenous scl and lmo2, recapitulating the most common and treatment resistant subtype in human T-ALL. By contrast, T-ALLs that co-express Notch and Myc fail to upregulate any of the T-ALL oncogenes; however, following transplantation into recipient animals, double transgenic Notch/Myc leukemias now express high levels of scl and lmo2. Finally, large scale limiting dilution cell transplantation analyses using syngeneic zebrafish demonstrated that Notch does not collaborate with Myc to increase self-renewal of leukemia initiating cells (LICs). Primary T-ALLs expressing both Notch and Myc have 10-fold less leukemia-initiating frequency when compared to T-ALLs that express only cMyc; however, following serial passaging, these Notch/Myc leukemias exhibit similar leukemia-initiating frequency as Myc-induced T-ALLs. Taken together, our data supports a model where Notch expands a pool of pre-malignant T-ALL clones within the primary tumor, a subset of which acquire additional mutations to confer a fully transformed phenotype. By contrast, Myc alone is insufficient to increase the overall pool of pre-malignant clones but confers a fully-transformed phenotype to leukemic cells accounting for the longer latency likely reflecting acquisition of additional genetic changes in clones. Our data may explain why a subset of relapse human T-ALLs develop disease from an underrepresented clone found in the primary leukemia. Primary human T-ALLs likely have a large pool of premalignant clones resulting from NOTCH-pathway activation that are unable to self-renew and thus, cannot give rise to relapsed T-ALL. Disclosures:No relevant conflicts of interest to declare.

Connective Tissue Growth Factor (CTGF) Is Essential for Self Renewal and Proliferation of Mesenchymal Stromal Cells (MSCs) and Affects Leukemia-Stromal Interactions.

AbstractAbstract 3845Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors that are critical regulators of vertebrate development. CTGF is a secreted protein that promotes extracellular matrix production, chemotaxis, cell proliferation and integrin expression. Since CTGF is highly expressed in acute lymphoblastic leukemia (ALL) and CTGF expression levels are related to ALL patient survival (Olga ST et al., Blood, 2007), we hypothesized that CTGF plays a role in regulating stromal cell proliferation and leukemia-stroma interaction. Our first goal was to characterize multipotent mesenchymal stromal cells (MSCs) from CTGF-/- mice, which die soon after birth from respiratory failure due to abnormal skeletal growth. We first attempted to isolate MSCs from BALB/C wild type and CTGF-/- newborn mice (Lvkovic S et al., Development, 2003). While we had no difficulty isolating MSCs from wild type mice, we failed to generate MSCs from CTGF-/- newborns (bone marrow, liver, spleen and thymus). As an alternative approach, we suppressed CTGF in bone marrow derived human MSCs with lenti-virus delivered CTGF shRNA (CTGF-KD-MSCs) and achieved knockdown of ≂f65% compared to vector control cells as determined by RT-PCR. Shortly after transduction (4 days) we started to observe major changes in the phenotype of CTGF-KD-MSCs, which grew 6–7 fold slower compared to vector control MSCs. CTGF-KD-MSCs displayed a significant decrease in the number of cells in S phase (from 14.7% ± 0.8% to 3.5%± 0.4%) and a concomitant increase in the number of G0/1 cells (from 68.8%±1.8% to 82.4%± 1.3%) suggesting a G0 or G1 block. To investigate if CTGF affects expression of MSC surface proteins, we analyzed standard MSC markers including CD105, CD90, CD73, CD44, CD140b, CD166 and CD45 (as a negative marker). Surprisingly, no differences in the expression of these markers in CTGF-KD-MSCs compared to vector control MSCs were observed. To determine if CTGF might regulate gene expression in MSCs, we performed a microarray analysis using Illumina arrays. By gene set analysis methods, we observed significant down regulation (p < 10-9 by the hypergeometric distribution test) in CTGF-KD-MSCs of genes involved in cell cycle progression, most notably in the Gene Ontology lists for ribosomal biogenesis and mitosis. As MSCs are known to differentiate into mesodermal cell lineages, we next tested the differentiation potential of CTGF-KD-MSCs as compared to the vector control MSCs. Following standard differentiation protocols, both CTGF-KD- and vector control-MSCs differentiated into osteoblasts and chondrocytes with no differences in their ability to differentiate into these lineages; whereas, CTGF-KD-MSCs showed 6–7 fold increase in their adipocyte differentiation potential compared to vector control MSCs. Another property of MSCs is to self-renew and generate colony forming units-fibroblast like (CFU-F). Fifty or 100 cells of either CTGF-KD-MSCs or vector control MSCs were seeded in alpha-MEM with 10% serum. After 2 weeks of culture in alpha-MEM with 10% serum, the resulting fibroblast-like colonies were counted. Vector control MSCs generated ≂f30-fold higher CFU-F compared to CTGF-KD-MSCs indicating that CTGF-KD-MSCs lack the ability to self renew. Next we determined if suppression of CTGF might impede leukemia cell migration to MSCs. Using standard trans-well assays, we examined human pre-B acute lymphoblastic leukemia (ALL) derived REH cells and found that they migrated 45% ± 4% less to CTGF-KD-MSCs compared to control MSCs. These findings suggest that CTGF plays a major role in stromal cell proliferation, self renewal and adipocyte differentiation of MSCs. In addition, CTGF is involved in leukemia cell migration towards MSCs and inhibition of CTGF impairs the migration of leukemic cells towards stromal cells and thereby provides opportunities to prevent homing of leukemic cells and sensitize them to chemotherapy. Disclosures:Andreeff:MyeloRx LLC: Consultancy, participant in research under an NCI SBIR Contract to MyeloRx LLC.

Inhibition of BCL11B Expression Leads to Apoptosis of Malignant T Cells but Not CD34+ Cells.

AbstractAbstract 3755Altered expression, mutation, disruption, or rearrangement of the B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) has been associated with T-cell malignancies. BCL11B overexpression occurs primarily in T-cell malignancies, suggesting that it may be a target gene for cancer therapy. Our previous study showed that the inhibition of BCL11B expression by siRNA led to apoptosis in human T-cell acute lymphoblastic leukemia cell lines (Jurkat, huT78, and Molt-4), but not in normal, mature T cells. To confirm the potential of BCL11B siRNA as a therapeutic agent and to determine its safety, the present study further analyzed the effects of BCL11B suppression on hemopoietic stem/progenitor cells, CD34+ cells were collected from three samples of umbilical cord blood using magnetic-activated cell sorting. BCL11B expressions in mRNA level of umbilical cord blood CD34+ cells were analyzed by the real-time quantitative PCR with TaqMan technique. For nucleofection of CD34+ cells, BCL11B-siRNA935 and the U-001 program were used of a Nucleofection Device II. Mock-transfected cells nucleofected without siRNA were used as a negative control. To evaluate the role of differentiation and proliferation in CD34+ cells after BCL11B-siRNA935 transfection, erythroid burst-forming units (BFU-E), granulocyte/macrophage colony-forming units (CFU-GM), and megakaryocyte colony-forming units (CFU-Meg) from BCL11B siRNA935-, mock-CD34+ cells were performed using methylcellulose assays. Colonies were assessed 14 days after plating. The BCL11B mRNA expression level in the CD34+ cells was significantly lower than that in Molt-4 cells and in peripheral blood mononuclear cells from eight healthy individuals. There was no significant difference between BCL11B siRNA935-transfected and mock-transfected CD34+ cells with respect to the formation of BFU-E, CFU-GM, or CFU-Meg (P > 0.05). We speculate that the BCL11B gene, because of its very low expression level, may not play a major role in proliferation and differentiation of hematopoietic stem/progenitor cells, but we cannot rule out another anti-apoptotic mechanism. Nevertheless, BCL11B gene silencing alone does not affect the proliferation and differentiation of hematopoietic stem/progenitor cells in vitro. In conclusion, our findings provide evidence for an anti-apoptotic function of BCL11B in T-cell malignancies, but not in hemopoietic stem/progenitor cells, suggesting that BCL11B siRNA is safe and may be considered a new targeted therapeutic strategy for T-cell malignancies.* National Natural Science Foundation(30771980)& Guangdong Science and Technology Project (2007B030703008) Grants Disclosures:Li:National Natural Science Foundation (30771980) and Guangdong Science and Technology Project (2007B030703008, 2009B050700029) Grants: Research Funding.

The BCL11B Tumor Suppressor Is Mutated In Human T-Cell Acute Lymphoblastic Leukemia

AbstractAbstract 4177The BCL11B transcription factor, previously identified as a central player in normal α/β T-cell development, has recently been found to play critical roles in the maintenance of T-cell identity, with biallelic Bcl11b inactivation in T-cell precursors severely impairing their continued development into the T-cell lineage. Previous work has demonstrated that Bcl11b haploinsufficiency accelerates the onset of thymic lymphomas in p53-mutant mice, as well as T-lymphoid blast crisis in a mouse model of chronic myeloid leukemia induced by BCR-ABL. Furthermore, we have recently identified recurrent monoallelic Bcl11b deletions in 91% of T-cell acute lymphoblastic leukemias (T-ALL) arising in Atm-deficient mice, further supporting a role for Bcl11b haploinsufficiency in T-cell leukemogenesis. In order to determine whether BCL11B inactivation plays a role in the molecular pathogenesis of human T-ALL, we analyzed BCL11B status in primary T-ALL patient samples by array CGH and sequencing analysis. Monoallelic BCL11B deletions were identified in 6% of cases (n = 3 of 47) analyzed by array CGH, including one microdeletion within the BCL11B locus, one small deletion involving BCL11B and 6 additional genes, and one large 26 Mbp deletion of the distal arm of chromosome 14. BCL11B sequencing revealed heterozygous missense mutations in an additional 9% (n = 4 of 43) of primary T-ALL patient samples and in 19% (n = 3 of 16) of T-ALL cell lines. Structural homology modeling revealed that many of the mutations identified, including 3 of the 4 in the primary patient samples, disrupted key amino acids within BCL11B zinc finger domains that are involved in DNA recognition or structural stabilization required for zinc finger domain-mediated transcriptional activity. Analysis of TCRγ rearrangement status and gene expression data revealed that most cases with BCL11B inactivation were characterized by biallelic TCRγ rearrangements together with an early thymocyte precursor (ETP) gene expression signature, indicating developmental arrest at a prethymocyte stage of T-cell development, which we and others have shown confers an increased risk of treatment failure (Coustan-Smith et al. Lancet Oncol 2009; Gutierrez et al. J Clin Oncol 2010). Given that BCL11B expression during normal T-cell development increases markedly at the prethymocyte stage, our findings suggest that BCL11B inactivation may be directly responsible for developmental arrest at a prethymocyte stage during thymocyte transformation. Our findings provide compelling evidence that BCL11B is a tumor suppressor in a subset of human T-ALLs with a high risk of treatment failure. Disclosures:No relevant conflicts of interest to declare.

Evolution of Human BCR-ABL1lymphoblastic Leukaemia-Initiating Cells.

AbstractAbstract 1023Many tumour types are composed of genetically diverse cells, however little is known of how diversity evolves or the impact that diversity has on functional properties. Here, using xenografting and DNA copy number alteration (CNA) profiling of human BCR-ABL1 acute lymphoblastic leukaemia, we demonstrate that genetic diversity occurs in functionally defined leukaemia-initiating cells and that many diagnostic patient samples contain multiple genetically distinct subclones. Reconstruction of the subclonal genetic ancestry of several samples by CNA profiling demonstrated a branching multiclonal evolution model of leukaemogenesis, rather than linear succession. For some patient samples, the predominant diagnostic clone repopulated xenografts, while in others it was outcompeted by minor subclones. Reconstitution with the predominant diagnosis clone was associated with more aggressive growth properties in xenografts, deletion of CDKN2A/CDKN2B, and poor patient outcome. Our findings link clonal diversity with function and underscore the importance of developing therapies that eradicate all subclones. Disclosures:No relevant conflicts of interest to declare.

Single Agent Activity of the Cyclin-Dependent Kinase (CDK) Inhibitor Dinaciclib (SCH 727965) In Acute Myeloid and Lymphoid Leukemia Cells

AbstractAbstract 3981Dinaciclib (SCH 727965) is a selective and potent inhibitor of CDK 1, 2, 5 and 9 (IC50 < 5 nM) that has demonstrated in vitro and in vivo anti-tumor activity against a variety of tumor cell lines and human tumor xenograft models. The concentration of dinaciclib required to achieve these effects (< 100 nM) is achieved in clinical trials, and dinaciclib was found to have a more favorable therapeutic index, in preclinical murine models, than other CDK inhibitors. We have studied the effect of dinaciclib on human acute myelogenous (AML) and acute lymphoblastic (ALL) leukemia cell lines, including HL-60, K562 and Molt-4, and primary leukemia cells.Dose response curves (0.0004-10 μM) were generated for different exposure times (2, 24 and 72 h), and data from cell proliferation assay (WST-1) were used to calculate the IC50 values. Short 2 h exposure to dinaciclib followed by 24 h culture without drug resulted in different responses between the cell lines (IC50 values of 0.13 μM, 2.17 μM and ND; and viability at 10 μM 62%, 76% and 95%, for HL-60, Molt-4, and K562, respectively). With longer exposure times (24 and 72 h), the IC50 was similar between the cell lines (IC50 24 h values of 0.017, 0.015, and 0.019 μM for HL-60, Molt-4, and K562, respectively). However, even in the presence of the highest drug concentration tested (10 μM), approximately 5–25% of cells remained metabolically active after 24 h culture, and in a colony forming assay were able to proliferate and form colonies after removal of the drug. Longer 72 h exposure to dinaciclib (0.2-10 μM) completely inhibited cell proliferation in all cell lines and prevented colony formation.Next, we examined the effect of dinaciclib (2-200 nM) on cell cycle in HL-60 and K562 cells (2, 6, 9, 24 h). While lower drug concentrations and shorter exposures resulted in a minor increase in the proportion of cells in the G2 phase, a considerable increase of cells in the sub-G1 phase was observed with prolonged exposures and higher drug concentrations, most prominently in HL-60 cells (4h 200 nM 38%; 6h 20 nM 53% or 200 nM 71%, and 24 h 20 nM 84%), which is consistent with cell viability assay data. These findings were also confirmed by Annexin V/PI staining. To characterize the molecular mechanisms behind the induction of cell cycle arrest and apoptosis by dinaciclib, we measured the changes in protein expression of Mcl-1, phosphorylation of retinoblastoma (Rb) protein, and cleavage of PARP by Western blotting. Dinaciclib treatment in a dose- and time-dependent manner (6 and 24 h; 10–500 nM) significantly decreased the expression of anti-apoptotic protein Mcl-1, Rb phosphorylation at Ser 811/817, and induced cleavage of the PARP protein in the three cell lines tested. For HL-60 cells, even 2 h exposure to dinaciclib was able to induce these effects when cells were examined 4 h after treatment; however, both Mcl-1 and p-Rb returned to baseline 24 h later, suggesting that the cells were able to recover. Using HL-60 cells, we were also able to demonstrate that a decrease in Mcl-1 correlates with the decrease in phosphorylation of the carboxy-terminal domain of RNA polymerase II, suggesting that dinaciclib successfully inhibits CDK9 which may lead to transcriptional down-regulation of Mcl-1. Dinaciclib treatment also down-regulated the expression of XIAP, Bcl-xl, and phosphorylation of Bad at Ser 112 (the pro-survival form of Bad), while Bak and Bax levels remained unaffected. The cleavage of PARP correlated with the activation of the caspase-3 and -9, suggesting the involvement of the intrinsic pathway of apoptosis.We confirmed our findings in primary leukemia cells. Dinaciclib was able to induce growth inhibition in all 7 primary AML samples (IC50 for 24 h exposure ranging from 0.008 to 0.017 μM) and apoptosis (Annexin V/PI staining). Treatment with dinaciclib also resulted in down-regulation of Mcl-1, cleavage of PARP, and dephosphorylation of Rb in all primary leukemia cells examined. In summary, dinaciclib potently inhibits the growth and induces apoptosis of human leukemia cells in vitro. Prolonged exposure times may be required for its maximum efficacy, and given its short half-life in humans (1.5 to 3.3 hours), this should be considered when designing the clinical studies for patients with acute leukemias. Disclosures:Sadowska:Merck & Co: Research Funding. Muvarak:Merck & Co: Research Funding. Lapidus:Merck & Co: Equity Ownership, Research Funding. Bannerji:Merck & Co: Employment, Equity Ownership. Gojo:Merck & Co.: Research Funding.