Role of Autophagy and Apoptosis in Acute Lymphoblastic Leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is a malignant disease characterized by an excessive number of immature lymphocytes, including immature precursors of both B- and T cells. ALL affects children more often than adults. Immature lymphocytes lead to arrested differentiation and proliferation of cells. Its conventional treatments involve medication with dexamethasone, vincristine, and other anticancer drugs. Although the current first-line drugs can achieve effective treatment, they still cannot prevent the recurrence of some patients with ALL. Treatments have high risk of recurrence especially after the first remission. Currently, novel therapies to treat ALL are in need. Autophagy and apoptosis play important roles in regulating cancer development. Autophagy involves degradation of proteins and organelles, and apoptosis leads to cell death. These phenomena are crucial in cancer progression. Past studies reported that many potential anticancer agents regulate intracellular signaling pathways. METHODS: The authors discuss the recent research findings on the role of autophagy and apoptosis in ALL. RESULTS: The autophagy and apoptosis are widely used in the treatment of ALL. Most studies showed that many agents regulate autophagy and apoptosis in ALL cell models, clinical trials, and ALL animal models. CONCLUSIONS: In summary, activating autophagy and apoptosis pathways are the main strategies for ALL treatments. For ALL, combining new drugs with traditional chemotherapy and glucocorticoids treatments can achieve the greatest therapeutic effect by activating autophagy and apoptosis.

Oncogenetic landscape and clinical impact of IDH1 and IDH2 mutations in T-ALL.

IDH1 and IDH2 mutations (IDH1/2Mut) are recognized as recurrent genetic alterations in acute myeloid leukemia (AML) and associated with both clinical impact and therapeutic opportunity due to the recent development of specific IDH1/2Mut inhibitors. In T-cell acute lymphoblastic leukemia (T-ALL), their incidence and prognostic implications remain poorly reported. Our targeted next-generation sequencing approach allowed comprehensive assessment of genotype across the entire IDH1 and IDH2 locus in 1085 consecutive unselected and newly diagnosed patients with T-ALL and identified 4% of, virtually exclusive (47 of 49 patients), IDH1/2Mut. Mutational patterns of IDH1/2Mut in T-ALL present some specific features compared to AML. Whereas IDH2R140Q mutation was frequent in T-ALL (25 of 51 mutations), the IDH2R172 AML hotspot was absent. IDH2 mutations were associated with older age, an immature phenotype, more frequent RAS gain-of-function mutations and epigenetic regulator loss-of-function alterations (DNMT3A and TET2). IDH2 mutations, contrary to IDH1 mutations, appeared to be an independent prognostic factor in multivariate analysis with the NOTCH1/FBXW7/RAS/PTEN classifier. IDH2Mut were significantly associated with a high cumulative incidence of relapse and very dismal outcome, suggesting that IDH2-mutated T-ALL cases should be identified at diagnosis in order to benefit from therapeutic intensification and/or specific IDH2 inhibitors.

Upregulated miR-96-5p inhibits cell proliferation by targeting HBEGF in T-cell acute lymphoblastic leukemia cell line.

INTRODUCTION: microRNAs (miRNAs) are critical for tumorigenesis and progression of T-cell acute lymphoblastic leukemia (T-ALL). MiR-96-5p has been shown to play important roles in the development of many cancers, but its roles in T-ALL have yet not been studied. MATERIALS AND METHODS: miR-96-5p expression was detected in T-leukemic cells from peripheral blood of 30 patients with T-ALL using real-time quantitative PCR (RT-qPCR). TargetScan database was utilized to identify the target genes for miR-96-5p, and their target relationship was verified by western blot, dual luciferase reporter assay and RT-qPCR. The effects of miR-96-5p on the viability and proliferation of T-leukemic cells (Jurkat cells) were respectively determined using MTT and BrdU incorporation assays. RESULTS: miR-96-5p presented low expression levels by qPCR in peripheral blood of T-ALL patients compared to healthy volunteers. Upregulated miR-96-5p by miR-96-5p mimic transfection markedly inhibited the viability and proliferation of Jurkat cells. Furthermore, miR-96-5p negatively regulated the expression of its target gene, HBEGF. The decreased viability and proliferation of Jurkat cells caused by miR-96-5p over-expression was suppressed after the introduction of HBEGF plasmid. CONCLUSIONS: The presented study showed that upregulation of miR-96-5p inhibited the viability and proliferation of Jurkat T-leukemic cells through suppressing HBEGF expression. Our study provides a novel sight for understanding the pathological mechanism of T-ALL and suggests that miR-96-5p may be a potential biomarker for the therapy and diagnosis of T-ALL.

Apoptotic effect of resveratrol on human T-ALL cell line CCRF-CEM is unlikely exerted through alteration of BAX and BCL2 promoter methylation.

One of the fundamental barriers leading to failure of leukemia therapy is the resistance against conventional chemotherapies, common modality used to cure leukemia. Having the potential to trigger apoptosis in various human leukemia cell lines, resveratrol is regarded as a robust agent in chemotherapy regimens. The current study was aimed to assess whether the apoptotic effect of resveratrol on T-cell acute lymphoblastic leukemia cell line, CCRF-CEM, is exerted through DNA methylation of BAX and BCL2 gene promoters. For this purpose, the CCRF-CEM cells were treated by resveratrol under standard cell culture. To analyze the promoter DNA methylation changes, we used methylation-specific polymerase chain reaction technique following the resveratrol treatment at different dosages and time intervals. Based on our previous study, the resveratrol treatment can trigger apoptosis in CCRF-CEM cell line via upregulation of apoptotic BAX gene and downregulation of antiapoptotic BCL2 gene. Despite these alterations in gene expression, the current study reveals no changes in DNA methylation patterns of subjected genes following the resveratrol treatment. Unchanged status of DNA methylation of BAX and BCL2 genes may suggest that resveratrol causes the gene expression changes through a distinct mechanism which requires further studies to be understood.

UTX inhibition as selective epigenetic therapy against TAL1-driven T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous group of hematological tumors composed of distinct subtypes that vary in their genetic abnormalities, gene expression signatures, and prognoses. However, it remains unclear whether T-ALL subtypes differ at the functional level, and, as such, T-ALL treatments are uniformly applied across subtypes, leading to variable responses between patients. Here we reveal the existence of a subtype-specific epigenetic vulnerability in T-ALL by which a particular subgroup of T-ALL characterized by expression of the oncogenic transcription factor TAL1 is uniquely sensitive to variations in the dosage and activity of the histone 3 Lys27 (H3K27) demethylase UTX/KDM6A. Specifically, we identify UTX as a coactivator of TAL1 and show that it acts as a major regulator of the TAL1 leukemic gene expression program. Furthermore, we demonstrate that UTX, previously described as a tumor suppressor in T-ALL, is in fact a pro-oncogenic cofactor essential for leukemia maintenance in TAL1-positive (but not TAL1-negative) T-ALL. Exploiting this subtype-specific epigenetic vulnerability, we propose a novel therapeutic approach based on UTX inhibition through in vivo administration of an H3K27 demethylase inhibitor that efficiently kills TAL1-positive primary human leukemia. These findings provide the first opportunity to develop personalized epigenetic therapy for T-ALL patients.

Leukemia-initiating cell activity requires calcineurin in T-cell acute lymphoblastic leukemia.

Despite their initial efficient response to induction chemotherapy, relapse remains frequent in patients with T-cell acute lymphoblastic leukemia (T-ALL), an aggressive malignancy of immature T-cell progenitors. We previously reported sustained calcineurin (Cn) activation in human lymphoid malignancies, and showed that Cn inhibitors have antileukemic effects in mouse models of T-ALL. It was unclear, however, from these studies whether these effects resulted from Cn inhibition in leukemic cells themselves or were an indirect consequence of impaired Cn function in the supportive tumor microenvironment. We thus generated a Notch (intracellular Notch 1, ICN1)-induced T-ALL mouse model, in which conditional Cn genetic deletion is restricted to leukemic cells. Ex vivo, Cn deletion altered the adhesive interactions between leukemic cells and their supportive stroma, leukemic cell survival, proliferation, migration and clonogenic potential. In vivo, Cn activation was found to be critical for leukemia initiating/propagating cell activity as demonstrated by the failure of Cn-deficient leukemic cells to transplant the disease to syngeneic recipient mice. Importantly, combination of vincristine treatment with Cre-mediated Cn ablation cooperated to induce long-term remission of ICN1-induced T-ALL. These findings indicate that Cn is a promising target in T-ALL relapse prevention, and call for clinical trials incorporating Cn inhibitors during consolidation therapy.

Detection of occult cerebrospinal fluid involvement during maintenance therapy identifies a group of children with acute lymphoblastic leukemia at high risk for relapse.

We aimed at assessing the clinical significance of the levels of acute lymphoblastic leukemia (ALL) cells in samples of cerebrospinal fluid (CSF) during therapy. We studied 990 CSF samples from 108 patients, at the time of diagnosis (108) and at each time of intrathecal therapy (882). The proportions of leukemic cells in CSF samples were assessed by flow cytometry (FCM). Patients with central nervous system (CNS) involvement at diagnosis (FCM+) showed predominantly a T-ALL, and higher percentages of known negative prognostic factors: high risk group, higher white blood cell counts, normal karyotype, and the BCR-ABL fusion gene. No differences in relapse free survival (RFS) and overall survival (OS) were observed between FCM+ versus FCM- at diagnosis. Patients with CNS involvement during therapy showed significantly older age, and higher frequencies of T-cell leukemia. We found a significantly higher RFS in patients with FCM+ during therapy. The detection of subclinical CNS disease by FCM during maintenance was associated with significantly lower 3-years RFS and 3-years OS. A sensitive methodology like FCM can be applied for a close follow-up of the levels of ALL in CFS samples, and may identify a group of patients at high risk for relapse.

Divergent effects of supraphysiologic Notch signals on leukemia stem cells and hematopoietic stem cells.

The leukemia stem cell (LSC) hypothesis proposes that a subset of cells in the bulk leukemia population propagates the leukemia.We tested the LSC hypothesis in a mouse model of Notch-induced T-cell acute lymphoblastic leukemia (T-ALL) in which the tumor cells were largely CD4+ CD8+ T cells. LSC activity was enriched but rare in the CD8+ CD4 HSA(hi) immature single-positive T-cell subset. Although our murine T-ALL model relies on transduction of HSCs, we were unable to isolate Notch-activated HSCs to test for LSC activity. Further analysis showed that Notch activation in HSCs caused an initial expansion of hematopoietic and T-cell progenitors and loss of stem cell quiescence, which was followed by progressive loss of long-term HSCs and T-cell production over several weeks. Similar results were obtained in a conditional transgenic model in which Notch activation is induced in HSCs by Cre recombinase. We conclude that although supraphysiologic Notch signaling in HSCs promotes LSC activity in T-cell progenitors, it extinguishes self-renewal of LT-HSCs. These results provide further evidence for therapeutically targeting T-cell progenitors in T-ALL while also underscoring the need to tightly regulate Notch signaling to expand normal HSC populations for clinical applications.

IL-7R-mediated signaling in T-cell acute lymphoblastic leukemia.

Interleukin-7 (IL-7), a cytokine produced in the bone marrow, thymus and other organs, is mandatory for normal human T-cell development and peripheral homeostasis. Different studies, including phase I clinical trials, have indicated the potential therapeutic value of recombinant IL-7 in the context of anti-cancer immunotherapy and as a booster of immune reconstitution. However, the two main pathways activated by IL-7, JAK/STAT5 and PI3K/Akt/mTOR, have both been implicated in cancer and there is considerable evidence that IL-7 and its receptor (IL-7R), formed by IL-7Rα (encoded by IL7R) and γc, may partake in T-cell acute lymphoblastic leukemia (T-ALL) development. In this context, the most compelling data comes from recent studies demonstrating that around 10% of T-ALL patients display IL7R gain-of-function mutations leading, in most cases, to disulfide bond-dependent homodimerization of two mutant receptors and consequent constitutive activation of downstream signaling, with ensuing cell transformation in vitro and tumorigenic ability in vivo. Here, we review the data on the involvement of IL-7 and IL-7R in T-ALL, further discussing the peculiarities of IL-7R-mediated signaling in human leukemia T-cells that may be of therapeutic value, namely regarding the potential use of PI3K and mTOR pharmacological inhibitors.

Hes1 mediates the different responses of hematopoietic stem and progenitor cells to T cell leukemic environment.

Normal hematopoiesis is suppressed during the development of leukemia. In the T-ALL leukemia mouse model described in our recent study (Hu X, et al. Blood 2009), the impacts of leukemic environment on normal hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) were distinct, in that normal HSCs were preserved in part because of increased mitotic quiescence of HSCs and resulting exhaustion of HPCs proliferation. Stem cell factor (SCF) secreted by leukemic cells in Nalm6 B-ALL model was previously suggested to force normal HSCs/HPCs out of their bone marrow niches and allow leukemic cells to occupy the niches (Colmone A, et al. Science 2008). Here we found that stem cell factor (SCF) expression in PB and BM of T-ALL model was increased, but SCF mRNA and protein levels in normal hematopoietic cells were higher than those in leukemia cells, which suggested that upregulated SCF was mainly contributed by non-leukemic cells in response to the leukemia development. To further elucidate the molecular mechanisms, microarray analysis was conducted on normal HSCs in this model and verified by real-time RT-PCR. The expression of Hes1 and its downstream target p21 were elevated in normal HSCs, whereas their expression showed no significant alteration in HPCs. Interestingly, although overexpression of Hes1 by retroviral infection inhibited the in vitro colony formation of normal hematopoietic cells, in vivo results demonstrated that normal Lin(-) cells and HSPCs were better preserved when normal Lin(-) cells with Hes1 overexpression were co-transplanted with T-ALL leukemia cells. Our results suggested that the differential expression of Hes1 between HSCs and HPCs resulted in the distinct responses of these cells to the leukemic condition, and that overexpression of Hes1 could enhance normal HSPCs in the leukemic environment.

Phenotypic and genetic characterization of adult T-cell acute lymphoblastic leukemia with del(9)(q34);SET-NUP214 rearrangement.

SET-NUP214 rearrangement is a recently recognized recurrent chromosomal translocation mostly observed in T-ALL. In order to characterize this rare entity, we performed phenotypic and genetic characterization of SET-NUP214 rearrangement through an investigation of a series of 40 consecutive samples of adult T-ALL that was selected among 229 adult ALL cases during 4 years in a single institution. Four cases (10%) of SET-NUP214 translocation were identified in our study. In all cases, diagnosis of T-ALL was established according to the World Health Organization (WHO) classification, and clonal TCR rearrangements were found. The immunophenotypic markers were indicative of the precursor nature of T lymphoblasts, and they expressed one or both of the myeloid-associated antigens (CD13, CD33). Conventional cytogenetic analysis revealed complex chromosomal aberrations in all four SET-NUP214 rearranged cases and del(12)(p13)/ETV6 was frequently involved. Array-CGH demonstrated additional genomic imbalances in addition to deletion 9q34. The genomic breakpoint sequencing identified breakpoints at SET intron 7 and NUP214 intron 17, and random nucleotide addition was found in two cases at the site of rearrangement. Our independently derived data set from a single institution confirms previous findings of SET-NUP214 rearrangement, indicates the relatively high incidence of SET-NUP214 rearrangement in adult T-ALLs, and also demonstrates comprehensive clinical, phenotypic, and genetic characteristics of this entity. Also, our report on genomic breakpoints demonstrates the homogeneity in the localization of the genomic breakpoints at 9q34. Concurrent chromosomal aberrations identified in this study should provide further areas of interest in investigation of SET-NUP214-mediated leukemogenesis.

Biphenotypic hematologic malignancy: a case report of the 8p11 myeloproliferative syndrome in a child.

SUMMARY: The 8p11 myeloproliferative syndrome, also known as stem cell leukemia/lymphoma, is a rare, atypical, myeloproliferative disorder and lymphoid malignancy associated with chromosomal abnormalities involving the 8p11 chromosomal band. Translocations associated with this syndrome result in the fusion of the fibroblast growth factor receptor 1 (FGFR 1) gene with various partners, resulting in ligand-independent FGFR activity. To date, 8 partner genes have been identified in association with FGFR1 rearrangements. The most frequent FGFR1 translocation partner is the zinc finger gene ZNF198 located at 13q11. Disease phenotypes associated with this translocation include poor prognosis and transformation to acute leukemia and non-Hodgkin lymphoma. In common with a T-cell phenotype, obtaining and maintaining remission is difficult by conventional chemotherapy. This study describes an illustrative case of 8p11 myeloproliferative syndrome/stem cell leukemia/lymphoma outlining its chief features and historical developments.

Pre T-cell receptor alpha (pTalpha) expression patterns and functional analysis in human T-cell lymphoblastic leukemia.

BACKGROUND: The pTalpha/preTCR regulates the beta-selection, a crucial T-cell developmental checkpoint, providing a most potent survival advantage to thymocytes mediated by the src-kinase p56(Lck). METHODS: To define the relevance of pTalpha in human T-cell lymphoblastic leukemia (T-ALL), we analyzed in T-ALL cell lines (n=14) pTalpha and p56(Lck) mRNA and protein expression as also the tyrosine-phosphorylation. The p56(Lck) specific src-protein-tyrosine kinase inhibitor (PTK-I) PP1 was used in growth inhibition assays. IC(50) value determination, cell cycle- and apoptosis analyses were performed in T-ALL-, non-T-ALL- and murine transgenic cell lines. RESULTS: pTalpha expression patterns were markedly different in T-ALL cell lines as compared to those reported for normal lymphoid counterparts. PP1 induced in 6/11 T-ALL cell lines a survival disadvantage resulting from a cell cycle arrest in the G(1/0) phase in thymic lymphoblastic cells and apoptosis induction in the immature cell line HSB-2, respectively. PP1 sensitive cell lines expressed the target protein p56(Lck) and showed a corresponding P-Tyr signal. CONCLUSION: Sensitivity of thymic T-ALLs to PP1 clearly underlines the impact of pTalpha mediated proliferation in this leukemic sub-type. In addition, p56(Lck) represents also independently of pTalpha a promising therapeutical target for the src-kinase inhibitors in neoplastic lymphoid diseases.

Overexpression of human CAP10-like protein 46 KD in T-acute lymphoblastic leukemia and acute myelogenous leukemia.

AIMS: We earlier identified a novel gene human CAP10-like protein 46 KD (hCLP46) from human acute myelogenous leukemia (AML) transformed from myelodysplastic syndrome CD34(+) cells, but the function of this gene remains unclear. In this study, a real-time polymerase chain reaction-based assay was developed to quantify expression of hCLP46 in the peripheral blood of AML and T-acute lymphoblastic leukemia (T-ALL) primary samples and in six leukemic cell lines. Also, we investigated expression of CDKN2A/B and the apoptosis in U937 cells when hCLP46 is downregulated in vitro. RESULTS: Our findings showed that hCLP46 was overexpressed in AML, T-ALL, and the leukemic cell lines. Suppressing hCLP46 overexpression had no effect on expression of CDKN2A/B and apoptosis of U937 cells. CONCLUSION: Considering that hCLP46 has the capability of modifying the Notch pathway, our finding adds weight to the importance of Notch signaling in hematopoiesis and suggests that overexpression of hCLP46 might be an early event in the pathogenesis of AML and T-ALL.

Targeted therapy in T-cell malignancies: dysregulation of the cellular signaling pathways.

T-cell malignancies, mainly known as T-cell acute lymphoblastic leukemia (T-ALL) and T-cell non-Hodgkin's lymphoma (T-NHL), are aggressive tumors. Although the clinical outcome of the patients has improved dramatically with combination chemotherapy, significant challenges remain, including understanding of the factors that contribute to the malignant behavior of these tumor cells and developing subsequently optimal targeted therapy. Aberrant cell signal transduction is generally involved in tumor progression and drug resistance. This review describes the pathogenetic role of multiple cellular signaling pathways in T-cell malignancies and the potential therapeutic strategies based on the modulation of these key signaling networks.