T-cell Acute Lymphoblastic Leukemia Jurkat Research Articles

CCR9 overexpression promotes T-ALL progression by enhancing cholesterol biosynthesis.

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy of the lymphoid progenitor cells, contributing to ∼ 20% of the total ALL cases, with a higher prevalence in adults than children. Despite the important role of human T-ALL cell lines in understanding the pathobiology of the disease, a detailed comparison of the tumorigenic potentials of two commonly used T-ALL cell lines, MOLT4 and JURKAT cells, is still lacking. Methodology: In the present study, NOD-Prkdc scid IL2rgd ull (NTG) mice were intravenously injected with MOLT4, JURKAT cells, and PBS as a control. The leukemiac cell homing/infiltration into the bone marrow, blood, liver and spleen was investigated for bioluminescence imaging, flow cytometry, and immunohistochemistry staining. Gene expression profiling of the two cell lines was performed via RNA-seq to identify the differentially expressed genes (DEGs). CCR9 identified as a DEG, was further screened for its role in invasion and metastasis in both cell lines in vitro. Moreover, a JURKAT cell line with overexpressed CCR9 (Jurkat-OeCCR9) was investigated for T-ALL formation in the NTG mice as compared to the GFP control. Jurkat-OeCCR9 cells were then subjected to transcriptome analysis to identify the genes and pathways associated with the upregulation of CCR9 leading to enhanced tumirogenesis. The DEGs of the CCR9-associated upregulation were validated both at mRNA and protein levels. Simvastatin was used to assess the effect of cholesterol biosynthesis inhibition on the aggressiveness of T-ALL cells. Results: Comparison of the leukemogenic potentials of the two T-ALL cell lines showed the relatively higher leukemogenic potential of MOLT4 cells, characterized by their enhanced tissue infiltration in NOD-PrkdcscidIL2rgdull (NTG) mice. Transcriptmoe analysis of the two cell lines revealed numerous DEGs, including CCR9, enriched in vital signaling pathways associated with growth and proliferation. Notably, the upregulation of CCR9 also promoted the tissue infiltration of JURKAT cells in vitro and in NTG mice. Transcriptome analysis revealed that CCR9 overexpression facilitated cholesterol production by upregulating the expression of the transcriptional factor SREBF2, and the downstream genes: MSMO1, MVD, HMGCS1, and HMGCR, which was then corroborated at the protein levels. Notably, simvastatin treatment reduced the migration of the CCR9-overexpressing JURKAT cells, suggesting the importance of cholesterol in T-ALL progression. Conclusions: This study highlights the distinct tumorigenic potentials of two T-ALL cell lines and reveals CCR9-regulated enhanced cholesterol biosynthesis in T-ALL.

P332: MIR-625-5P IS NOVEL CANDIDATE ONCOMIR IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA IMPLICATED IN REGULATION OF APOPTOSIS VIA REPRESSION OF HARAKIRI

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL, arising from T-cell precursors. miRNAs are non-coding RNAs, contributing to leukemogenesis by involvement in key cellular functions such as proliferation, apoptosis, and signaling. We previously found in miRNA transcriptome analysis that miR-625-5p is overexpressed in T-ALL patients, thus is a potential oncomiR in T-ALL. Here, we confirm the implication of miR-625-5p in the regulation of apoptosis in T-ALL cells in vitro. We show that it acts via repression of HARAKIRI (HRK). HRK was originally identified as a proapoptotic gene induced by reduced levels of cytokine in hematopoietic cells and repressed by the expression of death-repressor proteins. Although its proapoptotic function is well described, little is known about the post-transcriptional mechanisms that may participate in HRK inactivation. Aims: The aim of this study was to investigate the mechanism of miR-625-5p oncogenic action via unraveling its targetome and functional effects in T-ALL in vitro. Methods: T-ALL JURKAT cells (with high endogenous miR-625-5p level) were transduced for stable inhibition of this miRNA. To evaluate the influence of miR inhibition on growth of T-ALL cells, we used flow cytometry GFP competition assay and CCK8 proliferation assay. To identify the genes mediating the effect of miR-625-5p, we subjected JURKAT cells to Ago2-RIP-seq. We used magnetic beads coated with anti-AGO2 antibody to immunoprecipitate RNA bound to RISC complexes. Ago2-IP and total RNA fractions were sequenced (polyA RNA-seq, 150 bp reads, 60M PE reads/sample, Illumina NovaSeq 6000). Transcripts depleted in Ago2-IP fraction upon miR inhibition were screened for the presence of miR-625-5p binding sites. Luciferase assay was performed to confirm direct interaction between hsa-miR-625-5p and 3’UTR of HRK gene. The changes of HRK protein level upon miR-625-5p inhibition were evaluated via Western Blot. To estimate the apoptosis rate in T-ALL cells we used two flow cytometry assays based on Annexin V and Caspase 3/7 staining. Results: Inhibition of miR-625-5p decreased the growth of JURKAT cells. In RIP-seq analysis we identified 384 transcripts depleted in RISC by at least 20% upon miR-625-5p inhibition and having at least one putative 3’UTR binding site for this miRNA. Overrepresentation analysis performed for these genes indicated their involvement in the apoptotic process. Among transcripts depleted in RIP-seq with predicted binding sites for miR-625-5p, we found HRK gene, previously reported to have proapoptotic activity. We postulate that repression of this gene by overexpressed miR-625-5p contributes to decreased apoptosis and to growth advantage of T-ALL cells. To further confirm that miR-625-5p affects growth of T-ALL cells via negative regulation of apoptosis, we performed apoptotic assays in JURKAT cells and showed that inhibition of this miRNA increased apoptosis rate, in line with our hypothesis on the oncogenic potential of miR-625-5p. We confirmed the direct interaction between miR-625-5p and HRK 3’UTR by luciferase assay. Additionally, in Western blot a subtle (approaching statistical significance) increase of HRK protein upon miR-625-5p inhibition was observed, supporting our notion that this miRNA is indeed a negative regulator of HRK. Image:Summary/Conclusion: miR-625-5p is a novel candidate oncogenic miRNA in T-ALL, implicated in the negative regulation of apoptosis. Post-transcriptional repression of the proapoptotic HRK gene is a putative mechanism contributing to oncogenic properties of this miRNA when overexpressed in T-ALL cells.

Apoptosis inhibition in T-cell acute lymphoblastic leukemia by UNC13B

T-cell acute lymphoblastic leukemia is a type of leukemia that is difficult to treat and has a complex pathogenesis, with no effective treatment currently available. This research group found that the mRNA expression of a new gene, UNC13B, was increased in T-cell acute lymphoblastic leukemia patients. Subsequently, we used T-cell acute lymphoblastic leukemia Jurkat cells to study the mechanism of UNC13B. We constructed a lentiviral vector expressing siRNA to target UNC13B and transfected it into the T-cell acute lymphoblastic leukemia Jurkat cell line. Using CCK-8, flow cytometry, and western blotting analyses, we found that knockdown of UNC13B inhibited the growth of T-cell acute lymphoblastic leukemia Jurkat cells via the downregulation of signaling proteins of the cell proliferation pathway and upregulation of apoptosis signaling proteins. Based on the bioinformatics analysis results, we found that the mechanism of UNC13B responsible for promoting the growth of T-cell acute lymphoblastic leukemia can be experimentally achieved by triggering AK2, MAP3K7, and PINK1. This study demonstrates that UNC13B is a new potential target for T-cell acute lymphoblastic leukemia.

Inhibition of InsP3R with Xestospongin B Reduces Mitochondrial Respiration and Induces Selective Cell Death in T Cell Acute Lymphoblastic Leukemia Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy whose chemoresistance and relapse persist as a problem despite significant advances in its chemotherapeutic treatments. Mitochondrial metabolism has emerged as an interesting therapeutic target given its essential role in maintaining bioenergetic and metabolic homeostasis. T-ALL cells are characterized by high levels of mitochondrial respiration, making them suitable for this type of intervention. Mitochondrial function is sustained by a constitutive transfer of calcium from the endoplasmic reticulum to mitochondria through the inositol 1,4,5-trisphosphate receptor (InsP3R), making T-ALL cells vulnerable to its inhibition. Here, we determine the bioenergetic profile of the T-ALL cell lines CCRF-CEM and Jurkat and evaluate their sensitivity to InsP3R inhibition with the specific inhibitor, Xestospongin B (XeB). Our results show that T-ALL cell lines exhibit higher mitochondrial respiration than non-malignant cells, which is blunted by the inhibition of the InsP3R. Prolonged treatment with XeB causes T-ALL cell death without affecting the normal counterpart. Moreover, the combination of XeB and glucocorticoids significantly enhanced cell death in the CCRF-CEM cells. The inhibition of InsP3R with XeB rises as a potential therapeutic alternative for the treatment of T-ALL.

Extracellular vesicles derived from T-cell acute lymphoblastic leukemia inhibit osteogenic differentiation of bone marrow mesenchymal stem cells via miR-34a-5p.

Reduced bone formation in patients with T-cell acute lymphoblastic leukemia (T-ALL) may be related to the interaction between tumour cells and bone marrow stromal cells (BMSCs). The miRNAs in extracellular vesicles derived from leukemia cells play an essential role in regulating the function of BMSCs; however, the regulatory mechanisms remain unclear. The expression of miR-34a-5p in T-ALL patients and cells was measured by quantitative real-time PCR. BMSCs were co-cultured with extracellular vesicles isolated from T-ALL cells in mineralization medium. The osteogenic differentiation of BMSCs was evaluated by Alizarin Red S staining, alkaline phosphatase (ALP) staining, and detection of osteogenic differentiation markers. A dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-34a-5p and Wnt family member 1 (WNT1). MiR-34a-5p expression was upregulated in T-ALL patients and Jurkat cells. After BMSCs were co-cultured with extracellular vesicles derived from T-ALL cells, osteogenic differentiation of BMSCs was inhibited, and bone mineralization and ALP activity were decreased compared to those of control cells. MiR-34a-5p knockdown in T-ALL cells restored osteogenic differentiation of BMSCs co-cultured with extracellular vesicles. In addition, miR-34a-5p targets and negatively regulates WNT1 expression. In conclusion, our results demonstrated that knockdown of miR-34a-5p in extracellular vesicles derived from T-ALL cells promoted osteogenic differentiation of BMSCs by regulating WNT1.

Bortezomib enhances cytotoxicity of ex vivo-expanded gamma delta T cells against acute myeloid leukemia and T-cell acute lymphoblastic leukemia

Engagement between the natural killer group 2, member D (NKG2D) receptor and its ligands is one of the main mechanisms used by immune cells to target stressed cells for cell death. NKG2D ligands are known markers of cellular stress and are often upregulated on tumor cells. Certain drugs can further increase NKG2D ligand levels, thereby making tumor cells more susceptible to immune cell detection and destruction. However, the effectiveness of this approach appears to be limited with drug treatment alone, possibly due to immune dysregulation in the setting of malignancies. We hypothesized that a more effective approach would be a combination of NKG2D ligand-inducing drugs, such as the proteasome inhibitor bortezomib, and ex vivo-expanded peripheral blood γδ T cells (i.e., Vγ9Vδ2 T cells). Acute myeloid leukemia (AML) is a high-risk hematologic malignancy, and treatment has shown limited benefit with the addition of bortezomib to standard chemotherapy regimens. Two AML cells lines, Nomo-1 and Kasumi-1, were treated with increasing concentrations of bortezomib, and changes in NKG2D ligand expression were measured. Bortezomib treatment significantly increased expression of the NKG2D ligand UL16 binding protein (ULBP) 2/5/6 in both cell lines. Vγ9Vδ2 T cells were expanded and isolated from peripheral blood of healthy donors to generate a final cellular product with a mean of 96% CD3+/γδ T-cell receptor-positive cells. Combination treatment of the AML cell lines with γδ T cells and bortezomib resulted in significantly greater cytotoxicity than γδ T cells alone, even at lower effector-to-target ratios. Based on the positive results against AML and the generalizable mechanism of this combination approach, it was also tested against T-cell acute lymphoblastic leukemia (T-ALL), another high-risk leukemia. Similarly, bortezomib increased ULBP 2/5/6 expression in T-ALL cell lines, Jurkat and MOLT-4 and improved the cytotoxicity of γδ T cells against each line. Collectively, these results show that bortezomib enhances γδ T-cell-mediated killing of both AML and T-ALL cells in part through increased NKG2D ligand-receptor interaction. Furthermore, proof-of-concept for the combination of ex vivo-expanded γδ T cells with stress ligand-inducing drugs as a therapeutic platform for high-risk leukemias is demonstrated.

Comparative Analysis for Differential Drug Response between Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) and T-Cell Acute Lymphoblastic Leukemia (T-ALL) Patients Using the Cellworks Omics Biology Model (CBM): Mycare-021-03

Background:Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), an orphan disease, is a sub-type of T-Cell Acute Lymphoblastic Leukemia (T-ALL) with very poor prognosis and limited therapy options. ETP-ALL is a heterogeneous disease with many distinct genomic profiles, often with more myeloid than lymphoid characteristics. However, standard of care (SOC) drugs for acute myeloid leukemia (AML) have shown limited efficacy for ETP-ALL (PMID: 32733662, 25435716). The genomic profiles of ETP-ALL patients have more complex cytogenetics and larger numbers of genomic aberrations when compared to non-ETP-ALL (T-ALL) profiles (PMID: 22237106, 30641417). We present an alternative multi-gene analysis approach using the Cellworks Omics Biology Model (CBM) workflow to identify unique, intersecting protein pathways in patient-specific disease profiles. The CBM predictive workflow was used to design novel personalized therapy options for an ETP-ALL representative PEER human lymphoid cell line in comparison to a T-ALL JURKAT cell line. The predicted combination therapies were then validated in a lab model. Methods:A PEER cell line was selected to represent ETP-ALL and a JURKAT cell line was selected as a representative for non-ETP T-ALL. Next Generation Sequencing (NGS) was performed for the PEER cell line. For the JURKAT cell line, publicly available NGS whole exome sequencing from cBioPortal and Sanger, along with array CGH from Agilent, were used. The genomic data for the PEER and JURKAT cell lines were used as inputs to the CBM to generate dynamic patient-specific disease protein network maps. Biomarkers and pathway characteristics unique to the PEER and JURKAT cell lines were identified. A digital drug library of targeted FDA-approved agents was simulated on the disease models using both single drug agents and drug combinations at varying doses. The treatment impact was assessed by quantitatively measuring drug effect on a cell growth score, which is a composite of the quantified values of cell proliferation, survival and apoptosis along with impact on the patient-specific disease biomarker score. Comparative dose response studies were run to assess IC50 differences for both cell lines. Cellworks VenturaTM predicted novel therapy combinations for the ETP-ALL representative PEER cell line, which were then prospectively validated by in vitro experiments. The same therapy options were predicted to be less effective in the T-ALL representative JURKAT cell line, which was also confirmed by in vitro studies. Results:The CBM predicted three novel combination therapies for the ETP-ALL representative PEER cell line: nilotinib + cytarabine, bortezomib + cytarabine and bortezomib + idarubicin. All three therapies were predicted to be less effective in JURKAT cells. In vitro, PEER cells were sensitive to all 3 combinations, as predicted by the CBM; whereas, JURKAT cell lines were not sensitive to the first 2 combinations (as predicted), but were sensitive to bortezomib + idarubicin. The CBM analysis is supported by scientific rationales for these combinations based on the genomics-driven disease characteristics of the cell-line. The reasons for drug sensitivity and resistance were determined. These combinations were then prospectively validated in vitro on both cell lines and the experimental responses matched the predicted outcomes. Conclusion:The Cellworks Omics Biology Model integrates the multiple genomic abnormalities in a patient to identify disease network characteristics unlike other NGS analytic tools that attempt to interpret the impact of each genomic alteration in isolation. CBM identified 3 novel therapy options for ETP-ALL that were validated in vitro, similar to anecdotal experience in vivo. This predictive technology can improve clinical decision-making and identify novel treatment options. Disclosures Howard: Cellworks:Consultancy;Servier:Consultancy, Other: Speaker;EUSA Pharma:Consultancy;Sanofi:Consultancy, Other: Speaker;Boston Scientific:Consultancy.Kumar:Cellworks Research India Private Limited:Current Employment.Pampana:Cellworks Research India Private Limited:Current Employment.Ullal:Cellworks Research India Private Limited:Current Employment.Tyagi:Cellworks Research India Private Limited:Current Employment.Lala:Cellworks Research India Private Limited:Current Employment.Kumari:Cellworks Research India Private Limited:Current Employment.Joseph:Cellworks Research India Private Limited:Current Employment.Raju:Cellworks Research India Private Limited:Current Employment.Balakrishnan:Cellworks Research India Private Limited:Current Employment.Mundkur:Cellworks Group Inc.:Current Employment.Macpherson:Cellworks Group Inc.:Current Employment.Nair:Cellworks Research India Private Limited:Current Employment.Kapoor:Cellworks Research India Private Limited:Current Employment.

MAML1-Dependent Notch-Responsive Genes Exhibit Differing Cofactor Requirements for Transcriptional Activation.

Mastermind proteins are required for transcription of Notch target genes, yet the molecular basis for mastermind function remains incompletely understood. Previous work has shown that Notch can induce transcriptional responses by binding to promoters but more often by binding to enhancers, with HES4 and DTX1 as representative mammalian examples of promoter and enhancer responsiveness, respectively. Here, we show that mastermind dependence of the Notch response at these loci is differentially encoded in Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells. Knockout of Mastermind-like 1 (MAML1) eliminates Notch-responsive activation of both these genes, and reduced target gene expression is accompanied by a decrease in H3K27 acetylation, consistent with the importance of MAML1 for p300 activity. Add-back of MAML1 variants in knockout cells identifies residues 151 to 350 of MAML1 as essential for expression of either Notch-responsive gene. Fusion of the Notch-binding region of MAML1 to the histone acetyltransferase (HAT) domain of p300 rescues expression of HES4 but not DTX1, suggesting that an additional activity of MAML1 is needed for gene induction at a distance. Together, these studies establish the functional importance of the MAML1 region from residues 151 to 350 for Notch-dependent transcriptional induction and reveal differential requirements for MAML1-dependent recruitment activities at different Notch-responsive loci, highlighting the molecular complexity of Notch-stimulated transcription.

COTI-2 induces cell apoptosis in pediatric acute lymphoblastic leukemia via upregulation of miR-203

ABSTRACT COTI-2 is a third-generation thiosemicarbazone, which is effective against a diverse group of human cancer cell lines at nanomolar concentrations. COTI-2 also showed superior activity against tumor cells, in vitro and in vivo. As a high efficacy and low toxicity agent, it currently candidates in a phase I clinical study of gynecological malignancies and head and neck squamous cell carcinoma (HNSCC). However, its effect in pediatric T-cell acute lymphoblastic leukemia (T-ALL) is not clear. This study investigates the effect of COTI-2 on T-ALL Jurkat cells in vitro and in vivo. Jurkat cells were exposure to COTI-2 at different concentration and time. Cell apoptosis was detected by flow cytometry to examine the sensitivity of Jurkat cell lines treated with either COTI-2 alone or in combination with MiR-203 mimic or inhibitor in vitro. An orthotopic mouse model was used to examine the sensitivity of Jurkat cells treated with COTI-2 in vivo. Western blotting and RT-qPCR were performed to dissect molecular mechanisms. The results showed that COTI-2 promotes apoptosis of Jurkat cells in dose-and time-dependent way. Enforced expression of miR-203 promotes COTI-2-mediated cell apoptosis, whereas miR-203 silencing attenuates COTI-2-mediated cell apoptosis in Jurkat cells in vitro. COTI-2 is also effective against growth of Jurkat cells in vivo. Mechanistically, COTI-2 induced miR-203 upregulation and inhibited caspase-3/9 activaty leading to inhibition of cell apoptosis. Taken together, COTI-2 inhibits tumor growth in vitro and in vivo in Jurkat cells likely through miR-203-dependent mechanisms. COTI-2 may be a potential approach for T-ALL treatment.

Expression of the miR-150 tumor suppressor is restored by and synergizes with rapamycin in a human leukemia T-cell line

miR-150 functions as a tumor suppressor in malignancies of the lymphocyte lineage and its expression is significantly reduced in these cells. However, the mechanism of miR-150 repression is unknown and so are pharmacological interventions that can reverse it. Here, we report that reduced expression of miR-150 in human Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells is mediated by constitutive mTOR signaling, a common characteristic of T-ALL cell lines and clinical isolates. Activating mTOR signaling in non-malignant T cells also resulted in a significant miR-150 down-regulation. Conversely, treatment with a pharmacological mTOR inhibitor, rapamycin, increased miR-150 expression in a dose-dependent manner in Jurkat cells, as well as in other leukemia cells. Interestingly, ectopic over-expression of miR-150 acted in a feed-forward loop and further sensitized Jurkat cells to a rapamycin-induced cell cycle arrest by targeting a large network of cell cycle genes. These findings suggest that miR-150 is normally expressed in quiescent T lymphocytes to reinforce an anti-proliferative state, and that mTOR signaling promotes cell proliferation in part by inhibiting miR-150 expression. Restoration of the miR-150-dependent anti-proliferative loop constitutes a novel mechanism underlying the efficacy of rapamycin in a T-ALL cell line. Further investigation of this mechanism in clinical isolates of T-ALL and other hematopoietic malignancies could help better guide development of targeted therapies.

MAP3K7 is recurrently deleted in pediatric T-lymphoblastic leukemia and affects cell proliferation independently of NF-\u03baB

BackgroundDeletions of 6q15–16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which has a crucial role in innate immune signaling and was observed to be functionally and prognostically relevant in different cancer entities. Therefore, we correlated the presence of MAP3K7 deletions with clinical parameters in a cohort of 327 pediatric T-ALL patients and investigated the function of MAP3K7 in the T-ALL cell lines CCRF-CEM, Jurkat and MOLT-4.MethodsMAP3K7 deletions were detected by multiplex ligation-dependent probe amplification (MLPA). T-ALL cell lines were transduced with adeno-associated virus (AAV) vectors expressing anti-MAP3K7 shRNA or a non-silencing shRNA together with a GFP reporter. Transduction efficiency was measured by flow cytometry and depletion efficiency by RT-PCR and Western blots. Induction of apoptosis was measured by flow cytometry after staining with PE-conjugated Annexin V. In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR.ResultsMAP3K7 is deleted in approximately 10% and point-mutated in approximately 1% of children with T-ALL. In 32 of 33 leukemias the deletion of MAP3K7 also included the adjacent CASP8AP2 gene. MAP3K7 deletions were associated with the occurrence of SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and outcome. Depletion of MAP3K7 expression in T-ALL cell lines by shRNAs slowed down proliferation and induced apoptosis, but neither changed protein levels of components of NF-κB signaling nor NF-κB target gene expression after stimulation with TNF-α.ConclusionsThis study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts.

Development of an Electrochemical Impedimetric Biosensor for the Detection of Telomerase Activity in Cancer Cells

In the last decade, a great amount of effort has gone into developing methods and devices that detect early signs of cancer diseases fast and with high efficiency. Telomerase based biosensors are an attractive alternative that comply with this requirements. Telomerase serves as a cancer biomarker because it is present in the vast majority of cancers. This enzyme elongates telomeres at the end of the chromosomal DNA, which causes cancer cells to become immortal. In this work, we developed a modified inter-digitated array gold microelectrode as the sensing platform with Electrochemical Impedance Spectroscopy (EIS) as the measuring technique for the telomerase activity. The resulting DNA biosensor detects telomerase activity in total protein extracts from Jurkat T-acute lymphoblastic leukemia cells. This simple system is a good proof of concept for a fast and efficient method that can detect telomerase activity in cancer cells.

HMYH and hMTH1 cooperate for survival in mismatch repair defective T-cell acute lymphoblastic leukemia

hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH DNA glycosylase and hMTH1 for cancer cell survival by using mismatch repair defective T-cell acute lymphoblastic leukemia (T-ALL) cells. To this end, MYH and MTH1 were silenced individually or simultaneously using small hairpin RNAs. Increased sub-G1 population and apoptotic cells were observed upon concurrent depletion of both enzymes. Elevated cell death was consistent with cleaved caspase 3 accumulation in double knockdown cells. Importantly, overexpression of the nuclear isoform of hMYH could remove the G1 arrest and partially rescue the toxicity observed in hMTH1-depleted cells. In addition, expression profiles of human DNA glycosylases were generated using quantitative reverse transcriptase–PCR in MTH1 and/or MYH knockdown cells. NEIL1 DNA glycosylase, involved in repair of oxidized nucleosides, was found to be significantly downregulated as a cellular response to MTH1–MYH co-suppression. Overall, the results suggest that hMYH and hMTH1 functionally cooperate for effective repair and survival in mismatch repair defective T-ALL Jurkat A3 cells.

Effects of valproic acid and pioglitazone on cell cycle progression and proliferation of T-cell acute lymphoblastic leukemia Jurkat cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor. Administration of chemical compounds influencing apoptosis and T cell development has been discussed as promising novel therapeutic strategies. Valproic acid (VPA) as a recently emerged anti-neoplastic histone deacetylase (HDAC) inhibitor and pioglitazone (PGZ) as a high-affinity peroxisome proliferator-activated receptor-gamma (PPARγ) agonist have been shown to induce apoptosis and cell cycle arrest in different studies. Here, we aimed to investigate the underlying molecular mechanisms involved in anti-proliferative effects of these compounds on human Jurkat cells. Treated cells were evaluated for cell cycle progression and apoptosis using flowcytometry and MTT viability assay. Real-time RT-PCR was carried out to measure the alterations in key genes associated with cell death and cell cycle arrest. Our findings illustrated that both VPA and PGZ can inhibit Jurkat E6.1 cells in vitro after 24 hr; however, PGZ 400 μM presents the most anti-proliferative effect. Interestingly, treated cells have been arrested in G2/M with deregulated cell division cycle 25A (Cdc25A) phosphatase and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27) expression. Expression of cyclin D1 gene was inhibited when DNA synthesis entry was declined. Cell cycle deregulation in PGZ and VPA-exposed cells generated an increase in the proportion of aneuploid cell population, which has not reported before. These findings define that anti-proliferative effects of PGZ and VPA on Jurkat cell line are mediated by cell cycle deregulation. Thus, we suggest PGZ and VPA may relieve potential therapeutic application against apoptosis-resistant malignancies.

Andrographolide inhibits growth of human T-cell acute lymphoblastic leukemia Jurkat cells by downregulation of PI3K/AKT and upregulation of p38 MAPK pathways.

T-cell acute lymphoblastic leukemia (T-ALL) as a prevalent hematologic malignancy is one of the most common malignant tumors worldwide in children. Andrographolide (Andro), the major active component from Andrographis paniculata, has been shown to possess antitumor activities in several types of cancer cells. However, whether Andro would inhibit T-ALL cell growth remains unclear. In this study, we investigated the cytotoxic effect of Andro on human T-ALL Jurkat cells and explored the mechanisms of cell death. Cell apoptosis was assayed by flow cytometry, and the signaling transduction for Andro was analyzed by Western blotting. The results indicated 10 μg/mL Andro could significantly induce Jurkat cells’ apoptosis, depending on the inhibition of PI3K/AKT pathway. Moreover, Andro-induced apoptosis is enhanced by AKT-selective inhibitor LY294002. ERK- or JNK-selective inhibitors PD98059 and SP600125 had no effect on Andro-induced apoptosis. In addition, p38 inhibitor SB203580 could reverse Andro-induced apoptosis in Jurkat cells. We also found that the protein expression of p-p53 and p-p38 were increased after Andro treatments. The result of an in vivo study also demonstrated Andro’s dose-dependent inhibition in subcutaneous Jurkat xenografts. In conclusion, our findings explained a novel mechanism of drug action by Andro in Jurkat cells and suggested that Andro might be developed into a new candidate therapy for T-ALL patients in the coming days.

Abstract B25: BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia

Abstract Introduction: Despite the improved survival of patients with T-cell acute lymphoblastic leukemia (T-ALL), therapy-resistant or refractory T-ALL remains a major clinical challenge. Recently, we and others reported promising therapeutic activity for ABT-199, a highly specific inhibitor of the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), especially in immature T-ALL. Nevertheless, the occurrence of dose-limiting toxicities and the emergence of therapy resistance provides a rationale for the evaluation of ABT-199 as part of combination therapies. Indeed, previous studies have shown that ABT-199 can synergize with conventional chemotherapeutic agents in human T-ALL. BRD4 is an important bromodomain and extra-terminal (BET) protein that facilitates the recruitment of the transcriptional apparatus to acetylated chromatin. Small molecule inhibitors that target BET-bromodomain proteins, such as JQ1, can broadly affect oncogenic gene transcription and have recently emerged as powerful anti-cancer agents in a variety of hematological malignancies, including T-ALL. Here, we evaluated JQ1 and ABT-199 combination therapy and provide in vitro and in vivo evidence that combined BCL-2 and BRD4 inhibition synergistically affects leukemic tumor growth in T-ALL. Experimental Procedures: In vitro synergism between ABT-199 and JQ1 in a panel of T-ALL cell lines was determined by the combination index (CI) based on cell viability 48h after compound administration. For in vivo evaluation of the ABT-199/JQ1 combination therapy, luciferase-labeled T-ALL cells were administered by tail vein injection in immune compromised NOD scid gamma mice. Upon engraftment of leukemic cells, mice were divided into four groups and treated daily with 50mg ABT-199/kg body weight and/or 50mg JQ1/kg body weight for 13 days. The percentage of leukemic cells in the blood and bone marrow after treatment was analyzed using FACS analysis upon staining with an anti-human CD45 antibody. Results: In vitro evaluation of ABT-199 and JQ1 combination therapy revealed strong synergism (CI<0.5) in the T-ALL cell lines LOUCY, DND-41, ALL-SIL and TALL-1. In contrast, no or only slight synergistic effects (CI>0.5) were observed in other T-ALL lines, including KOPTK1, JURKAT, PEER and HPB-ALL. Notably, the degree of synergism was not defined by the IC50 values for ABT-199 or JQ1, but was significantly correlated with BCL-2 expression levels. Next, in vivo drug treatment experiments using luciferase-positive LOUCY T-ALL cells confirmed the synergistic activity of the JQ1/ABT-199 combination therapy as evaluated by bioluminescence, percentage of leukemic blasts in blood or bone marrow and spleen size. Interestingly, although ABT-199 strongly reduced the leukemic burden during the first days of treatment, the therapeutic response of single ABT-199 therapy was not durable. In contrast, JQ1/ABT-199 combination therapy resulted in a sustained therapeutic response associated with tumor regression. As expected, combination therapy resulted in enhanced levels of cleaved PARP and increased caspase activity in synergistic T-ALL cell lines, in line with induction of cellular apoptosis upon ABT-199/JQ1 treatment. To better understand the molecular mechanism by which JQ1 enhances the anti-leukemic properties of ABT-199, we defined its effect on gene expression in T-ALL cell lines. Notably, this analysis showed that the balance of pro- and anti-apoptotic BCL2 family factors was affected by JQ1 treatment. For example, JQ1 treatment caused a significant reduction in BCL2 levels, but also induced a concomitant increase in anti-apoptotic MCL1. In addition, pro-apoptotic factors, such as BIM, were also upregulated upon JQ1 treatment. Conclusions: Here, we show that BET-bromodomain inhibition can sensitize BCL2-positive human T-ALL to ABT-199 treatment by interfering with the balance of pro- and anti-apoptotic BCL2 family members. Citation Format: Sofie Peirs, Filip Matthijssens, Tim Pieters, Niels Vandamme, Geert Berx, Bruce Poppe, Pieter Van Vlierberghe. BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B25.

Abstract B39: BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia

Abstract Introduction: Despite the improved survival of patients with T-cell acute lymphoblastic leukemia (T-ALL), therapy-resistant or refractory T-ALL remains a major clinical challenge. Recently, we and others reported promising therapeutic activity for ABT-199, a highly specific inhibitor of the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), especially in immature T-ALL. Nevertheless, the occurrence of dose-limiting toxicities and the emergence of therapy resistance provides a rationale for the evaluation of ABT-199 as part of combination therapies. Indeed, previous studies have shown that ABT-199 can synergize with conventional chemotherapeutic agents in human T-ALL. BRD4 is an important bromodomain and extra-terminal (BET) protein that facilitates the recruitment of the transcriptional apparatus to acetylated chromatin. Small molecule inhibitors that target BET-bromodomain proteins, such as JQ1, can broadly affect oncogenic gene transcription and have recently emerged as powerful anti-cancer agents in a variety of hematological malignancies, including T-ALL. Here, we evaluated JQ1 and ABT-199 combination therapy and provide in vitro and in vivo evidence that combined BCL-2 and BRD4 inhibition synergistically affects leukemic tumor growth in T-ALL. Experimental Procedures: In vitro synergism between ABT-199 and JQ1 in a panel of T-ALL cell lines was determined by the combination index (CI) based on cell viability 48h after compound administration. For in vivo evaluation of the ABT-199/JQ1 combination therapy, luciferase-labeled T-ALL cells were administered by tail vein injection in immune compromised NOD scid gamma mice. Upon engraftment of leukemic cells, mice were divided into four groups and treated daily with 50mg ABT-199/kg body weight and/or 50mg JQ1/kg body weight for 13 days. The percentage of leukemic cells in the blood and bone marrow after treatment was analyzed using FACS analysis upon staining with an anti-human CD45 antibody. Results: In vitro evaluation of ABT-199 and JQ1 combination therapy revealed strong synergism (CI<0.5) in the T-ALL cell lines LOUCY, DND-41, ALL-SIL and TALL-1. In contrast, no or only slight synergistic effects (CI>0.5) were observed in other T-ALL lines, including KOPTK1, JURKAT, PEER and HPB-ALL. Notably, the degree of synergism was not defined by the IC50 values for ABT-199 or JQ1, but was significantly correlated with BCL-2 expression levels. Next, in vivo drug treatment experiments using luciferase-positive LOUCY T-ALL cells confirmed the synergistic activity of the JQ1/ABT-199 combination therapy as evaluated by bioluminescence, percentage of leukemic blasts in blood or bone marrow and spleen size. Interestingly, although ABT-199 strongly reduced the leukemic burden during the first days of treatment, the therapeutic response of single ABT-199 therapy was not durable. In contrast, JQ1/ABT-199 combination therapy resulted in a sustained therapeutic response associated with tumor regression. As expected, combination therapy resulted in enhanced levels of cleaved PARP and increased caspase activity in synergistic T-ALL cell lines, in line with induction of cellular apoptosis upon ABT-199/JQ1 treatment. To better understand the molecular mechanism by which JQ1 enhances the anti-leukemic properties of ABT-199, we defined its effect on gene expression in T-ALL cell lines. Notably, this analysis showed that the balance of pro- and anti-apoptotic BCL2 family factors was affected by JQ1 treatment. For example, JQ1 treatment caused a significant reduction in BCL2 levels, but also induced a concomitant increase in anti-apoptotic MCL1. In addition, pro-apoptotic factors, such as BIM, were also upregulated upon JQ1 treatment. Conclusions: Here, we show that BET-bromodomain inhibition can sensitize BCL2-positive human T-ALL to ABT-199 treatment by interfering with the balance of pro- and anti-apoptotic BCL2 family members. Citation Format: Sofie Peirs, Filip Matthijssens, Tim Pieters, Niels Vandamme, Geert Berx, Bruce Poppe, Pieter Van Vlierberghe. BRD4 inhibition improves the efficacy of ABT-199 in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B39.

Ribonuclease binase induces death in T-cell acute lymphoblastic leukemia cells by apoptosis

Bacterial ribonuclease binase is a potential anticancer agent. In the present study, we have determined the toxic effect of binase towards cell lines of T-cell acute lymphoblastic leukemia Jurkat and CEMss. We have shown that binase induces apoptosis in these cells. At the same time, binase does not cause toxic effects in leukocytes of healthy donors, which suggests that binase activity towards leukemic cells is selective. We have found that the treatment of cancer cells with binase leads to a reduction in reactive oxygen species and transcription factor NFκB levels, and demonstrated that these effects are a common feature of the action of RNases on cancer cells.

Salinomycin Enhances the Apoptosis of T-cell Acute Lymphoblastic Leukemia Cell Line Jurkat Cells Induced by Vincristine

This study was aimed to investigate the effect of salinomycin combined with vincristine on the proliferation and apoptosis of Jurkat cells and its possible mechanisms. The proliferation of Jurkat cells was examined by CKK-8 assay. Flow cytometry was used to assess cellular apoptosis. Levels of BCL-2, caspase-3, and caspase- 8 were measured by Western blot. The salinomycin or vincristine, either alone or in combination, inhibited the proliferation of Jurkat cells in a dose-dependent manner. Salinomycin combined with vincristine produced more obveous inhibition of cell proliferation than either compound used alone (P<0.05). Western blot analysis showed that the combined use of Sal and VCR reduced the expression of BCL-2 protein, and increased expression of caspase 3 and caspase 8 protein, more significantly. Furthermore, combination of Sal and VCR synergistally promoted apoptosis of the Jurkat cells (P<0.05). The combination of salinomycin and vincristine synergistically inhibits proliferation and promotes apoptosis of T-cell acute lymphoblastic leukemia Jurkat cells.

­­­­­­N-Me, a Long Range T-Cell Specific Oncogenic Enhancer in T-ALL

Over the last years, numerous studies have dissected the mutational landscape of T-cell acute lymphoblastic leukemia (T-ALL) resulting in the identification of numerous oncogenes and tumor suppressors implicated in T-cell transformation. However, most genetic abnormalities found in cancer are located in intergenic regions, whose role in cancer development, if any, remains poorly understood. Here we hypothesized that recurrent cancer-associated intergenic mutations, amplifications and deletions may implicate strong transcriptional regulatory sequences responsible for the activation of key oncogenic factors in the pathogenesis of T-ALL. To address this question, we first used ChIPseq analysis to map the genomic landscape of enhancer sequences controlled by NOTCH1, a critical T-ALL oncogene activated by mutations in over 60% of human T-ALLs. In addition, we performed high resolution aCGH analysis of somatic chromosomal amplifications and deletions in a comprehensive series of 160 T-ALL samples. These analyses revealed recurrent focal duplications at chromosome 8q24 in 8/160 (5%) T-ALL cases in an area devoid of protein-coding genes containing a prominent 1 kb NOTCH1-binding peak. Strikingly, this putative oncogenic element is located +1,427 kb 3’ from the MYC locus and chromatin configuration 3C analysis demonstrated its direct association with the MYC proximal promoter. Multispecies DNA sequence alignment revealed remarkable conservation of this region in mammals, birds and reptiles and local ChIP analysis revealed bona fide active enhancer features including P300 occupancy and high levels of H3K4me1 with low levels of H3K4me3. In addition, detailed analysis of epigenetic marks across 64 hematopoietic and non-hematopoietic cell lines and tissues revealed that his regulatory element is located within a major superenhancer specifically active in T-cells. Based on these results, we proposed that this regulatory sequence, hereby named N-Me for NOTCH-bound MYCenhancer, could function as an important regulatory element driving the activation of MYC downstream of NOTCH1 in T-ALL. Consistently, luciferase reporter assays showed strong, orientation-independent and NOTCH-dependent activation of reporter constructs containing N-Me in association with a -2.5 kb MYC proximal promoter in JURKAT T-ALL cells.Next and to formally test the functional relevance of this enhancer in T-cell development and transformation we generated and characterized N-Me knockout and conditional knockout mice. N-Me null animals were viable and showed a marked and selective reduction in thymus size and cellularity as their only developmental alteration. Detailed immunophenotypic analysis of N-Me knockout thymocytes demonstrated accumulation of double negative 3 (DN3) T cells and marked reductions in double positive and CD4+ and CD8+ single positive cells. Mechanistically, this phenotype was associated with a marked reduction in Myc expression in DN3, DN4 and ISP cells. Moreover, transplantation of N-Me knockout bone marrow hematopoietic progenitors demonstrated that this phenotype is cell autonomous, and can be rescued upon retroviral expression of MYC. Next, we analyzed the role of N-Me in the induction of NOTCH-driven leukemias by transplanting mice with N-Me wild-type and knockout hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of NOTCH1 (ΔE-NOTCH1). In this context, mice transplanted with ΔE-NOTCH1-infected N-Me wild type cells developed T-ALL with 100% penetrance 6 weeks after transplant. In contrast, mice transplanted with ΔE-NOTCH1-N-Me knockout cells showed complete resistance to NOTCH1-induced transformation remaining 100% leukemia-free at 15 weeks post-transplant. In addition, secondary deletion of N-Me in established NOTCH1 induced leukemias from tamoxifen-inducible N-Me conditional knockout mice (Rosa26TMCre N-Meflox/flox) induced profound antileukemic effects with extended survival and almost complete suppression of leukemia initiating cell activity. Altogether, these results identify N-Me as the first long range oncogenic enhancer directly implicated in the pathogenesis of human leukemia. DisclosuresNo relevant conflicts of interest to declare.