Apoptosis Of T-cell Acute Lymphoblastic Leukemia Cells Research Articles

Specificity protein 1/3 regulate T-cell acute lymphoblastic leukemia cell proliferation and apoptosis through β-catenin by acting as targets of miR-495-3p.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic heterogeneous disease. This study explored the mechanism of specificity protein 1/3 (Sp1/3) in T-ALL cells through β-catenin by acting as targets of miR-495-3p. Expression levels of miR-495-3p, Sp1, Sp3, and β-catenin in the serum from T-ALL children patients, healthy controls, and the T-ALL cell lines were measured. The cell proliferation ability and apoptosis rate were detected. Levels of proliferation-related proteins proliferating cell nuclear antigen (PCNA)/cyclinD1 and apoptosis-related proteins B-cell lymphoma-2 associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) were determined. The binding of Sp1/3 and β-catenin promoter and the targeted relationship between miR-495-3p with Sp1/3 were analyzed. Sp1/3 were upregulated in CD4+ T-cells in T-ALL and were linked with leukocyte count and risk classification. Sp1/3 interference prevented proliferation and promoted apoptosis in T-ALL cells. Sp1/3 transcription factors activated β-catenin expression. Sp1/3 enhanced T-ALL cell proliferation by facilitating β-catenin expression. miR-495-3p targeted and repressed Sp1/3 expressions. miR-495-3p overexpression inhibited T-ALL cell proliferation and promoted apoptosis. Conjointly, Sp1/3, as targets of miR-495-3p limit apoptosis and promote proliferation in T-ALL cells by promoting β-catenin expression.

Idasanutlin and navitoclax induce synergistic apoptotic cell death in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in which activating mutations in the Notch pathway are thought to contribute to transformation, in part, by activating c-Myc. Increased c-Myc expression induces oncogenic stress that can trigger apoptosis through the MDM2-p53 tumor suppressor pathway. Since the great majority of T-ALL cases carry inactivating mutations upstream in this pathway but maintain wildtype MDM2 and TP53, we hypothesized that T-ALL would be selectively sensitive to MDM2 inhibition. Treatment with idasanutlin, an MDM2 inhibitor, induced only modest apoptosis in T-ALL cells but upregulated the pro-apoptotic BH3 domain genes BAX and BBC3, prompting us to evaluate the combination of idasanutlin with BH3 mimetics. Combination treatment with idasanutlin and navitoclax, a potent Bcl-2/Bcl-xL inhibitor, induces more consistent and potent synergistic killing of T-ALL PDX lines in vitro than venetoclax, a Bcl-2 specific inhibitor. Moreover, a marked synergic response to combination treatment with idasanutlin and navitoclax was seen in vivo in all four T-ALL xenografts tested, with a significant increase in overall survival in the combination treatment group. Collectively, these preclinical data show that the combination of idasanutlin and navitoclax is highly active in T-ALL and may merit consideration in the clinical setting.

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.

The depletion of Circ-PRKDC enhances autophagy and apoptosis in T-cell acute lymphoblastic leukemia via microRNA-653-5p/Reelin mediation of the PI3K/AKT/mTOR signaling pathway.

A range of circular (Circ) RNAs have been demonstrated to be of therapeutic significance for the treatment of acute lymphoblastic leukemia (ALL). Here, we investigated the mechanisms underlying the action of Circ-PRKDC and the microRNA-653-5p/Reelin (miR-653-5p/RELN) axis in T-cell ALL (T-ALL).Clinical specimens were obtained from patients with T-ALL (n=39) and healthy controls (n=30). In each specimen, we determined the expression levels of Circ-PRKDC, miR-653-5p, and RELN. Human T-ALL cells (Jurkat) were transfected with Circ-PRKDC- or miR-653-5p-related sequences to investigate cell proliferation, apoptosis, and autophagy. We also determined the levels of Circ-PRKDC, miR-653-5p, RELN, and signaling proteins related to phosphoinositide 3-kinase (PI3K), AKT, and mammalian target of rapamycin (mTOR). Finally, we decoded the interactions between Circ-PRKDC, miR-653-5p, and RELN. The expression levels of Circ-PRKDC and RELN were upregulated in T-ALL tissues and cells while the levels of miR-653-5p were downregulated. Thereafter, then silencing of Circ-PRKDC, or the enforced expression of miR-653-5p, repressed the expression of RELN and the activation of the PI3K/AKT/mTOR signaling pathway, thus enhancing cell autophagy and apoptosis, and disrupting cell proliferation. Circ-PRKDC acted a sponge for miR-653-5p while miR-653-5p targeted RELN. The knockdown of miR-653-5p abrogated the silencing of Circ-PRKDC-induced effects in T-ALL cells. The depletion of Circ-PRKDC elevated miR-653-5p to silence RELN-mediated PI3K/AKT/mTOR signaling activation, thereby enhancing autophagy and apoptosis in T-ALL cells.

MiR-29b-3p cooperates with miR-29c-3p to affect the malignant biological behaviors in T-cell acute lymphoblastic leukemia via TFAP2C/GPX1 axis

Mounting evidence has illustrated the tumor regulatory roles of microRNAs (miRNAs) in T-cell acute lymphoblastic leukemia (T-ALL), a malignant carcinoma originated from T-cell precursors. However, the possible regulation mechanisms underlying miR-29b/29c-3p in T-ALL have not been interrogated yet. The aim of our study was to probe the association and possible molecular mechanism of miR-29b/29c-3p and Glutathione Peroxidase 1 (GPX1), a predicted highly expressed gene in acute myeloid leukemia (LAML) tissues on the cancer genome atlas (TCGA) website. In our paper, it was observed that GPX1 was relatively overexpressed in T-ALL cells, compared with normal T cells. Loss-of-function assays demonstrated that GPX1 knockdown inhibited the proliferation and activated the apoptosis in T-ALL cells. Then miR-29b/29c-3p was confirmed to regulate GPX1 mRNA and protein expression via decreasing Transcription Factor AP-2 Gamma (TFAP2C) expression. In summary, miR-29b-3p and miR-29c-3p targeted TFAP2C so as to repress GPX1 transcription, thereafter inhibiting GPXA expression. In the end, rescue experiments proved the whole regulation mechanism of miR-29b/29c-3p in T-ALL. Overall, the miR-29b/29c-3p -TFAP2C-GPX1 axis helped us to have a better understanding of T-ALL pathogenesis.

Pharmacological Inhibition of MAP2K7 Induces Apoptosis and Cell Cycle Arrest in T-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and relapsed ALL is a leading cause of cancer-related death in children. While advances in frontline therapy have led to an 85% cure rate, relapsed ALL patients face a dismal prognosis necessitating identification of novel targets and development of alternative therapies. Amongst the two sub types of ALL, T-cell acute lymphoblastic leukemia (T-ALL) occurs less frequently but T-ALL patients have worse prognosis and higher rate of relapse. Our group discovered that the transcription factor KLF4 is heavily repressed via DNA methylation coinciding with aberrant activation of MAP2K7 and the MAP2K7 pathway (Shen et al. Leukemia, 2017). Additionally, we demonstrated, as a proof of principle, that pharmacological inhibition of JNK, the only downstream target of MAP2K7, possesses anti-leukemic properties against T-ALL but presented obstacles in terms of low potency and off-target effects. In order to overcome these limitations, we hypothesize that direct pharmacological inhibition of MAP2K7 improve specificity for targeted approach to T-ALL therapeutics. To examine this hypothesis, we explore the anti-leukemic effects of the MAP2K7 pathway inhibitor, 5Z-7-oxozeaenol, against T-ALL cells because it has been shown to covalently react with a unique cysteine-218 in the ATP binding pocket of MAP2K7. We found that T-ALL cell lines exhibit increased cytotoxic sensitivity to 5Z-7-oxozeaenol (IC50 ranging 0.2-1.1 µM) compared to a non-leukemic control (1.5 µM), and more potent than JNK inhibitor IC50 of 5µM. Additionally, 5Z-7-oxozeaenol reduces the amount of phospho-JNK, in a dose dependent manner, indicative of MAP2K7 pathway inhibition. We purified MAP2K7 protein to directly address specificity of kinase inhibition and found that 5Z-7-oxozeaenol inhibits enzymatic activity in vitro of MAP2K7. In addition, 5Z-7-oxozeaenol also inhibited TAK1, which is the MAP3K upstream of MAP2K7. In a panel of T-ALL cell lines, 5Z-7-oxozeaenol treatment induced apoptosis in MOLT3, Jurkat, and KOPTK1 T-ALL cell lines. Although P12-Ichikawa, RPMI-8402, DND-41, and ALL-SIL T-ALL cell lines underwent cell cycle arrest evidenced by a reduction in percentage of S/G2/M phase cells and increase in percentage of G0 phase cells, further increasing 5Z-7-oxozeaenol dosage proved sufficient for induction of apoptosis based on increase of caspase-3 and caspase-7 cleavage. Through reverse-phase protein array analysis we identified reduced expression of several cell cycle regulator proteins, including CDC25C, Cyclin B1, Cyclin D3, and CDC2 resulting from 5Z-7-oxozeaenol treatment. Conversely, we detected increased expression of cleaved caspase-3 and caspase-7, particularly in MOLT3 and Jurkat cell lines treated with MAP2K7 inhibitor. Immunoblot analysis revealed that 5Z-7-oxozeaenol inhibits the MAP2K7 signaling pathway and induces cell cycle arrest and apoptosis in T-ALL cells. Based on these findings, we demonstrated that 5Z-7-oxozeaenol induces cell cycle arrest and apoptosis in T-ALL cells through inhibition of the MAP2K7 pathway, suggesting that MAP2K7 represents a novel pharmacological target for the development of targeted therapy for high-risk patients with T-ALL. Disclosures No relevant conflicts of interest to declare.

The NEDD8-activating enzyme inhibitor MLN4924 induces G2 arrest and apoptosis in T-cell acute lymphoblastic leukemia.

The first-in-class compound MLN4924 is a small molecule inhibitor that selectively inactivates NEDD8-activating enzyme (NAE). The anticancer effects of MLN4924 have been attributed to impaired neddylation of Cullin proteins. Here, we show that treatment of T-cell acute lymphoblastic leukemia (T-ALL) cells with MLN4924 potently suppressed the neddylation of Cullins and the oncogenic growth of T-ALL cells in-vitro. Moreover, MLN4924 induced disease regression in an in vivo xenograft model. MLN4924 also induced cell cycle arrest at G2 phase and apoptosis in T-ALL cells. However, inhibition of the neddylation of Cullins alone could not explain the effects of MLN4924 in T-ALL cells. Gene expression profiling indicated ribosome function, steroid biosynthesis, and hematopoietic cell lineage pathways were affected by MLN4924 treatment. MLN4924 also induced nucleolar disruption, suggesting nucleolar stress signaling might contribute to the anticancer effects of MLN4924 in T-ALL cells. In addition, MLN4924 treatment reduced 14-3-3ξ\\δ protein levels in T-ALL cells. Thus, MLN4924 may inhibit T-ALL cell proliferation via several pathways.

MiRNA-149* promotes cell proliferation and suppresses apoptosis by mediating JunB in T-cell acute lymphoblastic leukemia

MicroRNA-149* (miRNA-149*) functions as an oncogenic regulator in human melanoma. However, the effect of miRNA-149* on T-cell acute lymphoblastic leukemia (T-ALL) is unclear. Here we aimed to analyze the effects of miRNA-149* on in vitro T-ALL cells and to uncover the target for miRNA-149* in these cells. The miRNA-149* level was determined in multiple cell lines and bone marrow cells derived from patients with T-ALL, B acute lymphoblastic leukemia (B-ALL), acute myelocytic leukemia (AML), and healthy donors. We found that miRNA-149* was highly expressed in T-ALL cell lines and T-ALL patients’ bone marrow samples. JunB was identified as a direct target of miR-149*. miRNA-149* mimics downregulated JunB levels in Molt-4 and Jurkat cells, while miRNA-149* inhibitors dramatically upregulated JunB expression in these cells. miRNA-149* mimics promoted proliferation, decreased the proportion of cells in G1 phase, and reduced cell apoptosis in T-ALL cells, while miRNA-149* inhibitors prevented these effects. miRNA-149* mimics downregulated p21 and upregulated cyclinD1, 4EBP1, and p70s6k in Molt-4 and Jurkat cells. Again, inhibitors prevented these effects. Our findings demonstrate that miRNA-149* may serve as an oncogenic regulator in T-ALL by negatively regulating JunB.

HSP90 inhibition leads to degradation of the TYK2 kinase and apoptotic cell death in T-cell acute lymphoblastic leukemia.

We previously found that TYK2 tyrosine kinase signaling through its downstream effector phospho-STAT1 (p-STAT1) acts to upregulate BCL2, which in turn mediates aberrant survival of T-cell acute lymphoblastic leukemia (T-ALL) cells. Here we show that pharmacologic inhibition of heat shock protein 90 (HSP90) with a small-molecule inhibitor, NVP-AUY922 (AUY922), leads to rapid degradation of TYK2 and apoptosis in T-ALL cells. STAT1 protein levels were not affected by AUY922 treatment, but p-STAT1 (Tyr 701) levels rapidly became undetectable, consistent with a block in signaling downstream of TYK2. BCL2 expression was downregulated after AUY922 treatment, and although this effect was necessary for AUY922-induced apoptosis, it was not sufficient because many T-ALL cell lines were resistant to ABT-199, a specific inhibitor of BCL2. Unlike ABT-199, AUY922 also upregulated the proapoptotic proteins BIM and BAD, whose increased expression was required for AUY922-induced apoptosis. Thus, the potent cytotoxicity of AUY922 involves the synergistic combination of BCL2 downregulation coupled with upregulation of the proapoptotic proteins BIM and BAD. This two-pronged assault on the mitochondrial apoptotic machinery identifies HSP90 inhibitors as promising drugs for targeting the TYK2-mediated prosurvival signaling axis in T-ALL cells.

Checkmate to CHK1 in T-cell ALL?

DNA replication ensures accurate duplication of the original genetic information present in a cell in order for it to be properly transmitted to daughter cells. However, replication can be perturbed, for instance in rapidly dividing cancer cells, in a process referred to as replication stress (RS). Checkpoint kinase 1 (CHK1) is an essential component of the ATR-dependent DNA damage-response pathway that protect cells from RS by preventing replication fork collapse and activating homologous DNA repair. The ATR-CHK1 pathway is triggered upon exposure of single-stranded DNA that arises with the stalling of replication forks [1], and it is required to reset proper origin firing, and to promote fork stability and checkpoint activation, delaying mitosis until replication is completed and thereby avoiding mitotic catastrophe [2]. Whereas these functions point towards a tumor suppressor role for CHK1, mouse models modulating ATR-CHK1 expression and genetic evidence from human tumors suggest otherwise: Atr and Chk1 knock-out models do not display higher tumor frequency; Chk1 favors oncogene-induced transformation in mice; CHK1 is frequently overexpressed in human cancers, while loss-of-function mutations are rare [2, 3]. Moreover, CHK1 affords protection against DNA damaging agents, a fact that prompted the use of CHK1 inhibitors as chemosensitizers [4]. Similarly, tumors whose oncogenic profile fuels RS were proposed to become addicted to ATR-CHK1 response [1, 2]. In our recent study published in Oncogene [5], we hypothesized that T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological cancer arising from T-cell precursor clonal expansion, could be one of such tumors and showed that CHK1 plays a key role in T-ALL cell maintenance. T-ALL cells tend to be highly proliferative due to a myriad of genetic lesions that culminate in cyclin-dependent kinase hyperactivation, and deregulated progression of S-phase that may impact on DNA replication [5, 6]. We found that T-ALL cells overexpressed CHK1 mRNA and protein as compared to normal hematopoietic progenitors. This was accompanied by aberrantly high CHK1 kinase activity, likely triggered by high basal levels of RS [5]. Experimental inactivation of CHK1, by a CHK1 selective inhibitor (PF-00477736) or by gene silencing, demonstrated that CHK1 is essential to control the accumulation of RS and to prevent apoptosis of T-ALL cells that appear to enter mitosis without having concluded DNA replication. Furthermore, accumulation of DNA damage in the context of CHK1 loss induced the activation of the ATM-CHK2 DNA double-strand break (DSB) response pathway, likely due to DSB formation upon the collapse of stalled replication forks. T-ALL apoptosis upon CHK1 inactivation was in the first instance dependent on ATM and caspase-3, since ATM inhibition prevented caspase-3 cleavage and rescued T-ALL cell viability despite sustained elevated amounts of RS markers [5]. Following the demonstration that T-ALL cells were eliminated using a CHK1 small molecule inhibitor as single agent, we showed that this effect was leukemia-specific, since normal T-cell progenitors were not sensitive to the low doses of PF-00477736 that killed primary T-ALL patient cells. Moreover, the in vitro anti-leukemia effect of PF-00477736 was not prevented by microenvironment pro-survival factors, and the potential clinical value of CHK1 inhibition was further demonstrated by the fact that PF-00477736 limited the growth of xenografted T-ALL tumors [5]. Interestingly, our preliminary analyses indicated that T-ALL cells expressing higher CHK1 levels appeared more sensitive to CHK1 pharmacological inhibition, suggesting that CHK1 expression could be a suitable drug response marker in T-ALL patients. As clinical trials against ATR-CHK1 pathway may be envisaged, this issue warrants extended T-ALL patient analysis. T-ALL constitutes only a fraction of all ALL cases, but it associates with high-risk. Therapeutic options with less detrimental side-effects and/or effective upon relapse are most desired. Our findings defining CHK1 as a ‘subverted’ tumor suppressor that stands in T-ALL as a major guardian of leukemia cell survival, thereby formally acting as an oncogene, reinforce a new way of viewing the mechanisms of cancer progression [2] and may set the ground for anti-leukemia breakthrough approaches. In this context, it is important to understand the mechanisms of CHK1 upregulation in T-ALL. We thoroughly documented CHK1 transcript overexpression in primary T-ALL [5]. However, how this occurs remains undetermined. Maybe transcription factors known to activate CHK1, such as E2F (downstream of G1/S-phase CDK activity) or MYC (downstream of NOTCH1), are involved in CHK1 overexpression in T-ALL. Or maybe as yet unidentified CHK1 regulatory elements are mutated or epigenetically altered. Curiously, in contrast to our findings, Chk1 mRNA downregulation was documented in a murine T-ALL model [7]. A more integrative view of the role of CHK1 in T-ALL is therefore required. We believe CHK1 downregulation may occur at T-ALL initiation, driving genomic instability secondary to an increase in RS. As the pro-proliferative oncogenic program establishes and RS rises, leukemic cells are naturally selected for their ability to upregulate CHK1 as a means to maintain RS levels compatible with cell viability.

Genome-wide analyses identify KLF4 as an important negative regulator in T-cell acute lymphoblastic leukemia through directly inhibiting T-cell associated genes.

BackgroundKruppel-like factor 4 (KLF4) induces tumorigenesis or suppresses tumor growth in a tissue-dependent manner. However, the roles of KLF4 in hematological malignancies and the mechanisms of action are not fully understood.MethodsInducible KLF4-overexpression Jurkat cell line combined with mouse models bearing cell-derived xenografts and primary T-cell acute lymphoblastic leukemia (T-ALL) cells from four patients were used to assess the functional role of KLF4 in T-ALL cells in vitro and in vivo. A genome-wide RNA-seq analysis was conducted to identify genes regulated by KLF4 in T-ALL cells. Chromatin immunoprecipitation (ChIP) PCR was used to determine direct binding sites of KLF4 in T-ALL cells.ResultsHere we reveal that KLF4 induced apoptosis through the BCL2/BCLXL pathway in human T-ALL cell lines and primary T-ALL specimens. In consistence, mice engrafted with KLF4-overexpressing T-ALL cells exhibited prolonged survival. Interestingly, the KLF4-induced apoptosis in T-ALL cells was compromised in xenografts but the invasion capacity of KLF4-expressing T-ALL cells to hosts was dramatically dampened. We found that KLF4 overexpression inhibited T cell-associated genes including NOTCH1, BCL11B, GATA3, and TCF7. Further mechanistic studies revealed that KLF4 directly bound to the promoters of NOTCH1, BCL2, and CXCR4 and suppressed their expression. Additionally, KLF4 induced SUMOylation and degradation of BCL11B.ConclusionsThese results suggest that KLF4 as a major transcription factor that suppresses the expression of T-cell associated genes, thus inhibiting T-ALL progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-014-0285-x) contains supplementary material, which is available to authorized users.

Resveratrol Induces Apoptosis and Autophagy in T-cell Acute Lymphoblastic Leukemia Cells by Inhibiting Akt/mTOR and Activating p38-MAPK

ObjectiveTo explore the effects of resveratrol-induced apoptosis and autophagy in T-cell acute lymphoblastic leukemia (T-ALL) cells and potential molecular mechanisms. MethodsThe anti-proliferation effect of resveratrol-induced, apoptosis and autophagy on T-ALL cells were detected by using MTT test, immunofluorescence, electronic microscope, and flow cytometry, respectively. Western blotting was performed for detecting changes of apoptosis-associated proteins, cell cycle regulatory proteins and state of activation of Akt, mTOR, p70S6K, 4E-BP1, and p38-MAPK. ResultsResveratrol inhibited the proliferation and induced apoptosis and autophagy in T-ALL cells in a dose and time-dependent manner. It also induced cell cycle arrest at G0/G1 phase via up regulating cyclin-dependent kinase (CDK) inhibitors p21 and p27 and down regulating cyclin A and cyclin D1. Western blotting revealed that resveratrol significantly decreased the expression of antiapoptotic proteins (Mcl-1 and Bcl-2) and increased the expression of proapoptotic proteins (Bax, Bim, and Bad), and induced cleaved-caspase-3 in a time-dependent manner. Significant increase in ratio of LC3-II/LC3-I and Beclin 1 was also detected. Furthermore, resveratrol induced significant dephosphorylation of Akt, mTOR, p70S6K, and 4E-BP1, but enhanced specific phosphorylation of p38-MAPK which could be blocked by SB203580. When autophagy was suppressed by 3-MA, apoptosis in T-ALL cells induced by resveratrol was enhanced. ConclusionOur findings have suggested that resveratrol induces cell cycle arrest, apoptosis, and autophagy in T-ALL cells through inhibiting Akt/mTOR/p70S6K/4E-BP1 and activating p38-MAPK signaling pathways. Autophagy might play a role as a self-defense mechanism in T-ALL cells treated by resveratrol. Therefore, the reasonable inhibition of autophagy in T-ALL cells may serve as a promising strategy for resveratrol induced apoptosis and can be used as adjuvant chemotherapy for T-ALL.

Cytotoxic activity of the casein kinase 2 inhibitor CX-4945 against T-cell acute lymphoblastic leukemia: targeting the unfolded protein response signaling

Constitutively active casein kinase 2 (CK2) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). CK2 phosphorylates PTEN (phosphatase and tensin homolog) tumor suppressor, resulting in PTEN stabilization and functional inactivation. Downregulation of PTEN activity has an impact on PI3K/Akt/mTOR signaling, which is of fundamental importance for T-ALL cell survival. These observations lend compelling weight to the application of CK2 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of CX-4945-a novel, highly specific, orally available, ATP-competitive inhibitor of CK2α. We show that CX-4945 treatment induced apoptosis in T-ALL cell lines and patient T lymphoblasts. CX-4945 downregulated PI3K/Akt/mTOR signaling in leukemic cells. Notably, CX-4945 affected the unfolded protein response (UPR), as demonstrated by a significant decrease in the levels of the main UPR regulator GRP78/BIP, and led to apoptosis via upregulation of the ER stress/UPR cell death mediators IRE1α and CHOP. In vivo administration of CX-4945 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth. Our findings indicate that modulation of the ER stress/UPR signaling through CK2 inhibition could be exploited for inducing apoptosis in T-ALL cells and that CX-4945 may be an efficient treatment for those T-ALLs displaying upregulation of CK2α/PI3K/Akt/mTOR signaling.

HSP90 Inhibition Has Potent Activity Against T-Cell Acute Lymphoblastic Leukemia (T-ALL) Through Degradation Of TYK2 Kinase

The intensification of therapy for patients with T-cell acute lymphoblastic leukemia (T-ALL) has improved clinical outcomes substantially. However, first-line therapy still continues to fail in approximately 25% of children and in more than 50% of adults, clearly indicating that further therapeutic improvement is urgently needed. Recently, we identified a novel oncogenic pathway that involves aberrant activation of the TYK2 tyrosine kinase and its downstream substrate, STAT1, which ultimately promotes T-ALL cell survival through the upregulation of BCL2 expression (Sanda et al. Cancer Discovery 2013). This finding indicates that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, suggesting that drugs able to potently inhibit or degrade the TYK2 kinase are likely to provide a therapeutic advantage in patients with T-ALL. However, there are no kinase inhibitors with potent activity to TYK2 under current clinical trials. Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone that is exploited by cancer cells to support activated oncoproteins including many cancer-associated kinases, and recent reports on the early clinical efficacy of HSP90 inhibitors are encouraging in some tumors. Based on the finding that TYK2 is related to JAK2 and the reports implicating JAK2 as an HSP90 client protein (Weigert et al. JEM 2012 and Marubayashi et al. J Clin Invest 2010), we investigated the therapeutic efficacy of HSP90 inhibition in T-ALL. First, we tested the effect of two HSP90 inhibitors (AUY922 and HSP990) on the cell viability of T-ALL cell lines. The growth of 10 T-ALL lines, which harbor different genetic aberrations, was dramatically reduced in response to treatment with both compounds, with IC50 values of 5-280 nM (AUY922) and 18-221 nM (HSP990) after 72 hours of exposure. Treatment with AUY922 induced significant apoptosis in KOPT-K1, HPB-ALL and Jurkat “TYK2-dependent” T-ALL cell lines, which are sensitive to shRNA knockdown of the TYK2 gene, as indicated by increased levels of Annexin V staining, which was not observed in Loucy and TALL-1 “TYK2-independent” cells. AUY922 also induced strong cell-cycle arrest at the G2/M phase in some of T-ALL cell lines. Western blotting analysis using T-ALL cell lines after exposure to AUY922 demonstrated rapid reduction of the TYK2 kinase and dephosphorylation of STAT1 in concentrations less than 30 nM of AUY922. The decrease of BCL2 expression after AUY922 treatment was seen only in “TYK2-dependent” T-ALL cell lines, not in “TYK2-independent” lines. These results indicate that pharmacological HSP90 inhibition resulted in TYK2 degradation and subsequent downregulation of its downstream pathway, which includes phosphorylation of STAT1 and activation of BCL2, and this effect is critical especially for the survival of “TYK2-dependent” T-ALL cells. To assess the significance of BCL2 on the cell viability of T-ALL cells after HSP90 inhibition, we tested AUY922 on specific Jurkat cell lines overexpressing each of the pro-survival BCL2 family proteins BCL2, BCLXL, or MCL1. The growth curve after 72 hours of treatment demonstrated that BCL2 overexpression could partially rescue the AUY922-induced decrease of cell viability, but overexpression of BCLXL or MCL1 had no effect. Immunoprecipitation assay using Jurkat cells overexpressing BCL2 indicated that treatment with AUY922 resulted in increased interaction between overexpressed BCL2 and the pro-apoptotic BH3-only protein, Bim. These results suggest that decreasd BCL2 expression is at least partially responsible for the ability of HSP90 inhibitors to induce apoptosis in T-ALL cells. In conclusion, these results provide preclinical evidence of the therapeutic potential of HSP90 inhibitors against T-ALL, providing a rationale for the evaluation of these inhibitors in clinical trials of patients with T-ALL. Disclosures:No relevant conflicts of interest to declare.

Par-4/THAP1 complex and Notch3 competitively regulated pre-mRNA splicing of CCAR1 and affected inversely the survival of T-cell acute lymphoblastic leukemia cells

Although the intensification of therapy for children with T-cell acute lymphoblastic leukemia (T-ALL) has substantially improved clinical outcomes, T-ALL remains an important challenge in pediatric oncology. Here, we report that the cooperative synergy between prostate apoptosis response factor-4 (Par-4) and THAP1 induces cell cycle and apoptosis regulator 1 (CCAR1) gene expression and cellular apoptosis in human T-ALL cell line Jurkat cells, CEM cells and primary cultured neoplastic T lymphocytes from children with T-ALL. Par-4 and THAP1 collaborated to activate the promoter of CCAR1 gene. Mechanistic investigations revealed that Par-4 and THAP1 formed a protein complex by the interaction of their carboxyl termini, and THAP1 bound to CCAR1 promoter though its zinc-dependent DNA-binding domain at amino terminus. Par-4/THAP1 complex and Notch3 competitively bound to CCAR1 promoter and competitively modulated alternative pre-mRNA splicing of CCAR1, which resulted in two different transcripts and played an opposite role in T-ALL cell survival. Despite Notch3 induced a shift splicing from the full-length isoform toward a shorter form of CCAR1 mRNA by splicing factor SRp40 and SRp55, Par-4/THAP1 complex strongly antagonized this inductive effect. Our finding revealed a mechanistic rationale for Par-4/THAP1-induced apoptosis in T-ALL cells that would be of benefit to develop a new therapy strategy for T-ALL.

Abstract 4487: Synergistic effects of resveratrol and doxorubicin on inducing the apotosis of mouse and human PTEN deficient T-cell acute lymphoblastic cells.

Abstract Resveratrol (3,5,4’-trihydroxy-trans-stilbene), a plant antibiotic produced in a wide variety of plants in response to stress, injury, UV irradiation, and fungal infection, has been shown to inhibit cell growth of several types of cancer. The aim of the present study was to determine the effects of Resveratrol alone and its combination with Doxorubicin, a commonly used chemotherapeutics for T-cell acute lymphoblastic leukemia (T-ALL), on the proliferation and apoptosis of T-ALL cells. Mouse T-ALL cell lines were derived from T-cell-specific deletion of Pten tumor suppressor gene, and human T-ALL cells were obtained from ATCC. We treated T-ALL cells with different concentrations of Resveratrol (0 μM to 200 μM) alone and in combination with various concentrations of Doxorubicin (0 μM to 1 μM) for different durations (0, 12, 24, 48 and 72 hours, respectively). We showed mouse and human Pten deficient T-ALL cell lines were sensitive to the treatment of Resveratrol in dose- and time-dependent manners. In addition, low dose Resveratrol (3.125 μM) in combination with very low dose of o (0.01 μM) showed a remarkable synergistic effect on the cell proliferation and apoptosis. Flow cytometric analysis with Annexin V and Western blot studies on Caspase 3 confirmed that Resveratrol induced apoptosis, especially in combination with Doxorubicin. This study establishes a potential role of Resveratrol in the treatment of Pten deficient T-ALL. Citation Format: Ling Chen, Yaling Yang, M. James You. Synergistic effects of resveratrol and doxorubicin on inducing the apotosis of mouse and human PTEN deficient T-cell acute lymphoblastic cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4487. doi:10.1158/1538-7445.AM2013-4487

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