Primary T-cell Acute Lymphoblastic Leukemia Cells Research Articles

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human Tcell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/β and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.

Gene expression profiles in BCL11B-siRNA treated malignant T cells.

BackgroundDownregulation of the B-cell chronic lymphocytic leukemia (CLL)/lymphoma11B (BCL11B) gene by small interfering RNA (siRNA) leads to growth inhibition and apoptosis of the human T-cell acute lymphoblastic leukemia (T-ALL) cell line Molt-4. To further characterize the molecular mechanism, a global gene expression profile of BCL11B-siRNA -treated Molt-4 cells was established. The expression profiles of several genes were further validated in the BCL11B-siRNA -treated Molt-4 cells and primary T-ALL cells.Results142 genes were found to be upregulated and 109 genes downregulated in the BCL11B-siRNA -treated Molt-4 cells by microarray analysis. Among apoptosis-related genes, three pro-apoptotic genes, TNFSF10, BIK, BNIP3, were upregulated and one anti-apoptotic gene, BCL2L1 was downregulated. Moreover, the expression of SPP1 and CREBBP genes involved in the transforming growth factor (TGF-β) pathway was down 16-fold. Expression levels of TNFSF10, BCL2L1, SPP1, and CREBBP were also examined by real-time PCR. A similar expression pattern of TNFSF10, BCL2L1, and SPP1 was identified. However, CREBBP was not downregulated in the BLC11B-siRNA -treated Molt-4 cells.ConclusionBCL11B-siRNA treatment altered expression profiles of TNFSF10, BCL2L1, and SPP1 in both Molt-4 T cell line and primary T-ALL cells.

Abstract 2809: Pathway dependence on the tyrosine kinase TYK2 in T-cell acute lymphoblastic leukemia

Abstract To identify novel oncogenic pathways that are dysregulated in T-cell acute lymphoblastic leukemia (T-ALL), we carried out an RNA interference (RNAi) screen using a retroviral library of inducible short-hairpin RNAs (shRNAs) in T-ALL cell lines. We found that loss of TYK2, a JAK family tyrosine kinase, was lethal in each of three T-ALL cell lines that we tested in this screen. An independent RNAi screen using a small-interference RNA (siRNA) library that individually silences each member of the tyrosine kinase gene family in primary T-ALL cells from a pediatric T-ALL specimens also demonstrated a dependence on TYK2 for cell viability. We confirmed by targeted knock-down analysis with multiple independent shRNAs that the loss of TYK2 induces apoptosis in T-ALL cells, whereas knock-down of other JAK proteins (JAK1, JAK2 or JAK3) had no effect. The TYK2 protein is constitutively phosphorylated on tyrosine residues in many T-ALL cell lines, and sequence analysis of the TYK2 gene in T-ALL cell lines and patient samples revealed a diversity of TYK2 point mutations in the FERM domain (V15A), pseudo-kinase domain (V731I) and kinase domain (E957D and R1027H). TYK2 cDNAs harboring each of these mutations, but not wild-type TYK2 cDNA, could transform Ba/F3 cells to factor-independent growth, and TYK2 was heavily phosphorylated in the Ba/F3 cell lines transformed with mutant cDNAs, indicating that these mutations activated the TYK2 kinase. TYK2-dependent T-ALL cell lines and the transformed Ba/F3 cells were sensitive to small-molecule JAK/TYK2 inhibitors. Moreover, we identified that inhibition of TYK2 induces downregulation of STAT1 phosphorylation, and that loss of STAT1 inhibits the growth of TYK2-dependent T-ALL cells, indicating that this transcription factor is involved in the TYK2 pathway and required for cell survival. Gene expression analysis after TYK2 or STAT1 knock-down in TYK2-dependent T-ALL cell lines revealed that BCL2 is upregulated by this pathway. In conclusion, our results establish a requirement for the TYK2-STAT1 pathway to promote T-ALL cell the survival, and establish TYK2 as a novel therapeutic target for this disease. 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 2809. doi:10.1158/1538-7445.AM2011-2809

Targeting of active mTOR inhibits primary leukemia T cells and synergizes with cytotoxic drugs and signaling inhibitors

Rationally designed therapies aim at the specific disruption of critical signaling pathways activated by malignant transformation or signals from the tumor microenvironment. Because mammalian target of rapamycin (mTOR) is an important signal integrator and a key translational regulator, we evaluated its potential involvement in T-cell acute lymphoblastic leukemia (T-ALL) and whether mTOR blockade synergizes with chemotherapeutic agents or other signaling antagonists to inhibit primary leukemia T cells. mTOR signaling status was assessed using biochemical, immunostaining, and molecular regulation studies and functional assays performed to assess the impact of mTOR blockade on T-ALL proliferation, survival, and cell cycle. We observed that mTOR signaling is highly activated in all T-ALL patients tested, with phosphorylation of its downstream substrates eIF4G and S6 ribosomal protein. mTOR activation was detected in vivo and was further increased in vitro by stimulation with interleukin-7, a potentially leukemogenic cytokine normally produced by the bone marrow microenvironment. In T-ALL cells, mTOR blockade was associated with accumulation of the cyclin-dependent kinase inhibitor p27(kip1), which preferentially adopted a nuclear localization. Functional studies using rapamycin or CCI-779 showed a dominant inhibitory effect of mTOR blockade on interleukin-7-induced proliferation, survival, and cell-cycle progression of T-ALL cells. Furthermore, mTOR blockade markedly potentiated the antileukemia effects of dexamethasone and doxorubicin, and showed highly synergistic interactions in combination with specific inhibitors of phosphatidylinositol 3-kinase/Akt and Janus kinase 3 signaling. This study shows activation of mTOR signaling in primary T-ALL cells evolving in the leukemic bone marrow, and supports the inclusion of mTOR antagonists in current therapeutic regimens for this cancer.

Effects of down-Regulating BCL11B Expression on the Proliferation and Apoptosis of Molt-4 Cells by RNA Interference

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B /CTIP2/ RIT1/hRitα) gene, which encodes C2H2 zinc-finger transcription factors, plays critical roles during development of T lymphocytes and the central nervous system. Recently, BCL11B gene has been identified to be associated with hematopoietic malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL). However, little is known regarding the molecular mechanism by which BCL11B may contribute as a tumor-suppressor gene. In order to clarify the role of BCL11B gene in T-cell malignancies, we analyzed the effects of BCL11B target-specific small interfering RNA (siRNA) on the proliferation and apoptosis of Molt-4 cells, which is the naturally BCL11B overexpressing cell line. BCL11B-specific siRNA and scrambled non-silencing siRNA control were designed and synthesized by Invitrogen. The siRNAs were transfected into MOLT-4 cells by nucleofector techique. The level of BCL11B mRNA expression was analyzed at 48 and 72 hours after nucleofection by Real-time quantitative PCR with Taqman technique. The cells proliferation rate was assayed by MTT method at 24 and 48 hours after transfection, respectively. Cell morphology was showed by Liu's Stain. The changes of cell cycle and apoptosis rate were detected by flow cytometry. The results showed that the level of BCL11B mRNA expression was inhibited in siRNA transfected cells at 72 hours after transfection, as compared with scrambled controls. The survival rates of siRNA-transfected cells were significantly decreased at both 24 and 48 hours (P < 0.01). Apoptotic cells could be observed morphologically. The apoptosis rates of Molt-4 cells treated with BCL11BsiRNA were highly increased compared with scrambled control ( P < 0.05). However, the percentage of different phases did not show significantly difference between siRNAtransfected cells and control cells, which was suggested that the apoptosis could be induced all phases of cell cycle. In conclusion, our data indicated that, the survival of Molt-4 cells might be critically dependent on BCL11B gene. Downregulation of BCL11B expression by RNA interference selectively can inhibit the proliferation and induced the apoptosis effectively in transformed Molt-4 cells, farther investigation will be performed in primary T-ALL cells to confirm the effect of presented BCL11B-siRNA on T-ALL.

PTEN posttranslational inactivation and hyperactivation of the PI3K/Akt pathway sustain primary T cell leukemia viability

Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. Here we show that PTEN inactivation in human T cell acute lymphoblastic leukemia (T-ALL) cells is not always synonymous with PTEN gene lesions and diminished protein expression. Samples taken from patients with T-ALL at the time of diagnosis very frequently showed constitutive hyperactivation of the PI3K/Akt pathway. In contrast to immortalized cell lines, most primary T-ALL cells did not harbor PTEN gene alterations, displayed normal PTEN mRNA levels, and expressed higher PTEN protein levels than normal T cell precursors. However, PTEN overexpression was associated with decreased PTEN lipid phosphatase activity, resulting from casein kinase 2 (CK2) overexpression and hyperactivation. In addition, T-ALL cells had constitutively high levels of ROS, which can also downmodulate PTEN activity. Accordingly, both CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signaling in T-ALL cells. Strikingly, inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T cell precursors. Overall, our data indicate that T-ALL cells inactivate PTEN mostly in a nondeletional, posttranslational manner. Pharmacological manipulation of these mechanisms may open new avenues for T-ALL treatment.

T Cell Acute Lymphoblastic Leukemia Originates from a Differentiation Block in T Cell Development Due to Aberrant Expression of LMO2 and SCL(TAL1).

T cell acute lymphoblastic leukemia (T-ALL) is frequently associated with overexpression of the oncogenes LMO2 and SCL(TAL1) which are normally down regulated following the double negative stage of T cell development. Our goal is to decipher the molecular and cellular mechanisms leading to the onset of LMO2 associated T-ALL. We were able to isolate a complex containing the transcription factors LMO2, SCL(TAL1) and E47 from primary human T-ALL cells with proven aberrant expression of LMO2 and SCL(TAL1) by applying immunoprecipitation and Western blotting techniques. This protein complex regulates the transcription of a truncated form of RALDH2 (retinaldehyde dehydrogenase) in T-ALL cells as shown by gene transcription profiling in conjunction with RT-PCR and siRNA approaches. To monitor the effect of LMO2 expression on T cell development and leukemogenesis, lethally irradiated mice (C57BL/6) were transplanted with bone marrow cells that had been transduced with a retrovirus carrying LMO2 as the transgene. One year later, 88% of the cells in the thymus expressed LMO2 and a shift towards CD3−/CD44+/CD25+ cells was observed (an 88% increase compared to normal thymocytes), suggesting a differentiation block caused by LMO2 leading to an accumulation of immature T cells. To test and identify cooperating genes in T-ALL development, bone marrow cells of LMO2 double transgenic mice in which tet-inducible LMO2 is controlled by a thymic specific promoter, were retrovirally transduced with SCL(TAL1). So far, none of the control animals, transplanted with bone marrow cells transduced with a vector only containing EGFP, developed T-ALL. However, six out of the seven test animals developed T-ALL exhibiting enlargement of the spleen, liver and thymus between seven and nine months after transplantation. Organs and blood of the diseased animals were infiltrated with T-ALL cells of the immature phenotype CD8+/CD4+ in five cases and of the CD3−/CD44+/CD25+ phenotype in one case. This indicates that the differentiation block caused by a lack of down-regulation of LMO2 and SCL(TAL1) in maturing T cells leads to a block in T cell differentiation and precedes T-ALL. These models will be used to examine the involvement of other cooperating genes in T-ALL development as well as downstream target genes of LMO2/SCL(TAL1), such as RALDH2, in the onset of T-ALL. We conclude that aberrant expression of LMO2 in T cells leads to a block in T cell maturation and, in conjunction with up-regulation of secondary genes like SCL(TAL1), triggers deregulation of genes in immature T cells leading to impaired T cell development and the onset of T-ALL. The described model will help to identify cooperating genes in LMO2 associated T-ALL as well as the chain of events leading to malignancy.

IL-7–dependent human leukemia T-cell line as a valuable tool for drug discovery in T-ALL

AbstractThe specific targeting of critical signaling molecules may provide efficient therapies for T-cell acute lymphoblastic leukemia (T-ALL). However, target identification and drug development are limited by insufficient numbers of primary T-ALL cells and by their high rate of spontaneous apoptosis. We established a human interleukin-7 (IL-7)–dependent T-ALL cell line, TAIL7, that maintains several biologic and signaling properties of its parental leukemia cells. TAIL7 cells are pre–T-ALL cells that proliferate in response to IL-7 and IL-4. IL-7 stimulation of TAIL7 cells prevents spontaneous in vitro apoptosis and induces cell activation and cell cycle progression. The signaling events triggered by IL-7 include down-regulation of p27kip1 and hyperphosphorylation of retinoblastoma protein (Rb). Stimulation of TAIL7 cells by IL-7 leads to phosphorylation of Janus kinase 3 (JAK3), signal transducer and activator of transcription 5 (STAT5), Akt/PKB (protein kinase B), and extracellular-regulated kinase 1 and 2 (Erk1/2). Importantly, specific blockade of JAK3 by its inhibitor WHI-P131 abrogates the IL-7–mediated proliferation and survival of TAIL7 cells, suggesting that activation of JAK3 is critical for IL-7 responsiveness by these cells. Because TAIL7 cells seem to be a biologic surrogate for primary leukemia T cells, this cell line constitutes a valuable tool for the study of the signaling pathways implicated in T-ALL. Exploitation of this cell line should allow the identification of molecular targets and promote the rational design and validation of antileukemia signaling inhibitors.

Engraftment of human T-cell acute lymphoblastic leukemia in immunodeficient NOD/SCID mice which have been preconditioned by injection of human cord blood.

Childhood T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers. Study of leukemia biology, as well as preclinical testing of potential therapeutic regimens directed at T-ALL, has been impeded by the lack of an efficient in vivo model of primary leukemia. We have reported elsewhere some observations that human cord blood conditioned medium enhances leukemia colony formation in vitro and preconditioning of sublethally irradiated nonobese diabetic/ severe combined immunodeficient (NOD/SCID) mice with cord blood mononuclear cells (MNCs) facilitates the subsequent engraftment of primary T-ALL cells in these mice. Here we characterize in greater detail this in vivo xenograft model of human leukemia in NOD/SCID mice. Consistent with the thesis that cord blood facilitates engraftment, the engraftment of human leukemia can be shown to increase with increasing number of cord blood MNCs injected. In addition, we documented the expression of chemokine receptor CXCR4 by primary T-ALL from patients and found that the presence of these receptors did not result in the transmigration of T-ALL cells induced by stromal cell-derived factor-1alpha. Finally, we show that in this xenograft system T-ALL cells recovered from engrafted bone marrow are characterized by upregulated expression of interleukin 2 receptor gamma chain, suggesting that cord blood preconditioning may function in part to increase T-ALL responsiveness to growth factor(s).

Preconditioning with fetal cord blood facilitates engraftment of primary childhood T-cell acute lymphoblastic leukemia in immunodeficient mice

AbstractChildhood T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers. It is reported that preconditioning sublethally irradiated immunodeficient NOD/SCID (nonobese diabetic/X-linked severe combined immunodeficient) mice with human cord blood mononuclear cells facilitates the engraftment, expansion, and dissemination in these mice of primary T-ALL cells obtained from patients at the time of diagnosis. Cells recovered from mouse bone marrow or spleen resembled the original leukemia cells from patients with respect to surface lineage markers and T-cell receptor Vβ gene rearrangements. Moreover, the pattern of leukemia dissemination in mouse tissues, resulting in universally fatal leukemia, is reminiscent of the human clinical disease. In addition, the fidelity of the model to the human disease is documented with regard to the presence of morphologically identifiable human leukemia cells in mouse bone marrow and blood and the maintenance of leukemia-initiating capacity within the leukemia-engrafted mouse. Therefore, several lines of independent approaches are used to suggest that the engrafted cells are of human leukemia origin and are not derived from cord blood. The in vivo model described here should enable the study of the growth properties of primary T-ALL cells obtained from patients and should prove useful in evaluating the potential efficacy of therapeutic strategies directed toward T-ALL.

Human cord blood conditioned medium enhances leukemia colony formation in vitro

Childhood T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers. Recently, we observed that pre-conditioning sub-lethally irradiated immunodeficient mice with human cord blood mononuclear cells facilitates in these mice high level engraftment of primary T-ALL cells obtained from patients. Here we report that human cord blood cells secrete a factor(s) which markedly enhances in vitro both colony number and burst size of the T-ALL clonogenic progenitors from patients. The enhancing activity does not correspond to IL-2, IL-15, nor to several other cytokines implicated in T cell proliferation/activation. Thus, it is possible cord blood may secrete an as yet unidentified factor(s) acting on leukemia clonogenic progenitors of T-ALL. Collectively, these studies should prove invaluable in addressing the growth properties of primary T-ALL cells from patients.

Does Activation of the TAL1 Gene Occur in a Majority of Patients With T-Cell Acute Lymphoblastic Leukemia? A Pediatric Oncology Group Study

Almost 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL) have tumor-specific rearrangements of the TAL1 gene. Although TAL1 expression has not been observed in normal lymphocytes, TAL1 gene products are readily detected in leukemic cells that harbor a rearranged TAL1 allele. Hence, it has been proposed that ectopic expression of TAL1 promotes the development of T-ALL. In this report, we show that TAL1 is expressed in the leukemic cells of most patients with T-ALL, including many that do not display an apparent TAL1 gene alteration. A polymorphic dinucleotide repeat in the transcribed sequences of TAL1 was used to determine the allele specificity of TAL1 transcription in primary T-ALL cells. Monoallelic expression of TAL1 was observed in the leukemic cells of all patients (8 of 8) bearing a TAL1 gene rearrangement. In the leukemic cells of patients without detectable TAL1 rearrangements, TAL1 transcription occurred in either a monoallelic (3 of 7 patients) or a biallelic (4 of 7 patients) fashion. Thus, TAL1 activation in these patients may result from subtle alterations in cis-acting regulatory sequences (affecting expression of a single TAL1 allele) or changes in trans-acting factors that control TAL1 transcription (affecting expression of both TAL1 alleles).