Flow Cytometry-Based Detection of Minimal/Measurable Residual Disease Predicts Survival Outcomes in Pediatrics, Adolescents, and Young Adults With T-acute Lymphoblastic Leukemia.

BACKGROUND: Measurable/minimal residual disease (MRD) is considered the single most powerful high-risk factor in acute leukemia, including T-cell acute lymphoblastic leukemia (T-ALL). In this study, we evaluated the impact of flow cytometry (FC)-based detection of MRD on survival outcomes in pediatrics, adolescents, and young adults (AYA) with T-ALL. METHODS: We included 139 patients, 88 pediatric patients between the ages of one and 14 years, and 51 AYA patients between 15 and 39 years of age, over a period of three years and were treated with the Indian Collaborative Childhood Leukemia Group (ICiCLe) protocol. MRD assessment was performed on post-induction (PI) bone marrow aspirate samples using a 10-color 11-antibody MRD panel on a Gallios instrument (Beckman Coulter, Miami, FL, USA). MRD value > 0.01% was considered positive. PI-MRD status was available in 131 patients. RESULTS: The five-year event-free survival (5-year EFS) in PI-MRD positive patients was inferior to those of negative patients (13.56% vs 79.06%), which was statistically significant (P < 0.001). However, the five-year overall survival (5-year OS) did not show any statistically significant difference between PI-MRD positive and negative T-ALL patients (92.93% vs 94.28%). The hazard ratio (HR) for 5-year EFS and MRD positivity was 8.03 (p-value < 0.0001). HR for 5-year EFS and early T-cell precursor ALL (ETP-ALL) was 2.63 (p = -0.02). CONCLUSIONS: PI-MRD detected using FC is a strong predictive factor of inferior survival outcomes in pediatrics, AYA patients with T-ALL. PI-MRD positivity can be used to modify the treatment of T-ALL patients, especially in resource-constrained developing countries where molecular tests are not widely available.

Chidamide combined with a modified Bu-Cy conditioning regimen improves survival in patients with T-cell acute lymphoblastic leukemia/lymphoma undergoing allogeneic hematopoietic stem cell transplantation.

This study aimed to evaluate the efficacy and safety of chidamide (Chi) combined with a modified Busulfan-Cyclophosphamide (mBuCy) conditioning regimen for T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Twenty-two patients received chidamide combined with mBuCy conditioning regimen (Chi group). A matched-pair control (CON) group of 44 patients (matched 1:2) received mBuCy only in the same period. The leukemia-free survival (LFS), overall survival (OS), cumulative incidence of relapse (CIR), and non-relapse-related mortality (NRM) were evaluated. Patients in the Chi group were associated with lower 2-year CIR (19.0 vs. 41.4%, P = 0.030), better 2-year LFS (76.1 vs. 48.1%, P = 0.014), and had no significant difference in 2-year OS (80.5 vs. 66.4%, P = 0.088). Patients with minimal residual disease (MRD) positive before HSCT in the Chi group exhibited an advantage in 2-year LFS and a trend towards better 2-year OS (75.0 vs. 10.2%, P = 0.048; 75.0 vs. 11.4%, P = 0.060, respectively). Multivariable analysis showed that the chidamide intensified regimen was independently associated with better LFS (HR 0.23; 95%CI, 0.08-0.63; P = 0.004), and showed no significant impact with OS for all patients (HR 0.34, 95%CI, 0.11-1.07; P = 0.064). The cumulative incidence rates of grade II-IV aGVHD were similar (36.4 vs. 38.6%, P = 0.858). 20 patients in Chi group evinced an elevation in γ-glutamyltransferase, as compared to the mBuCy group (90.9 vs. 65.9%, P = 0.029). No transplantation-related mortality was documented within the first 100 days after transplantation. The results demonstrate that the chidamide intensified regimen may be an effective and acceptable safety option for T-ALL/LBL undergoing allo-HSCT, and further validation is needed.

Honokiol induces apoptosis and autophagy in dexamethasone-resistant T-acute lymphoblastic leukemia CEM-C1 cells.

Objective: To study whether and, if so, how honokiol overcome dexamethasone resistance in DEX-resistant CEM-C1 cells. Methods: We investigated the effect of honokiol (0-20 µM) on cell proliferation, cell cycle, cell apoptosis and autophagy in DEX-resistant CEM-C1 cells and DEX-sensitive CEM-C7 cells. We also determined the role of c-Myc protein and mRNA in the occurrence of T-ALL associated dexamethasone resistance western blot and reverse transcription-qPCR (RT-qPCR) analysis. Results: Cell Counting Kit (CCK)-8 assay shows that DEX-resistant CEM-C1 cell lines were highly resistant to dexamethasone with IC50 of 364.1 ± 29.5 µM for 48 h treatment. However, upon treatment with dexamethasone in combination with 1.5 µM of honokiol for 48 h, the IC50 of CEM-C1 cells significantly decreased to 126.2 ± 12.3 µM, and the reversal fold was 2.88. Conversely, the IC50 of CEM-C7 cells was not changed combination of dexamethasone and honokiol as compared to that of CEM-C7 cells treated with dexamethasone alone. It has been shown that honokiol induced T-ALL cell growth inhibition by apoptosis and autophagy via downregulating cell cycle-regulated proteins (Cyclin E, CDK4, and Cyclin D1) and anti-apoptotic proteins BCL-2 and upregulating pro-apoptotic proteins Bax and led to PARP cleavage. Honokiol may overcome dexamethasone resistance in DEX-resistant CEM-C1 cell lines via the suppression of c-Myc mRNA expression. Conclusion: The combination of honokiol and DEX were better than DEX alone in DEX-resistant CEM-C1 cell lines. Honokiol may regulate T-ALL-related dexamethasone resistance by affecting c-Myc.

[Resveratrol Inhibits T-acute Lymphoblastic Leukemia in Mice by Regulating Notch1 Signaling Pathway].

OBJECTIVE: To observe the effect of resveratrol (Res) on T-acute lymphoblastic leukemia (T-ALL) mice, and further explore its mechanism on Notch1 signaling pathway. METHODS: Twenty-five 6-8 weeks old female C57BL/6 mice were randomly divided into control group, T-ALL group and Res group. Res group was further divided into low-Res, middle-Res and high-Res group. The percentage of leukemia cells in peripheral blood and spleen cell suspension were detected by flow cytometry and Wright-Giemsa staining, pathological morphology of spleen and bone marrow tissues were observed by HE staining, the expression levels of Notch1, Hes-1, c-Myc, miR-19b and PTEN mRNA in spleen tissue were detected by RT-qPCR, and the protein levels of Notch1, Hes-1, c-Myc, p-PTEN and PTEN were detected by Western blot. RESULTS: Compared with control group, the leukemia cells in peripheral blood of mice in T-ALL group were markedly increased, accompanied by diffuse infiltration of leukemia cells in spleen and bone marrow tissues, the mRNA levels of Notch1, Hes-1, c-Myc, miR-19b and the protein levels of Notch1, Hes-1, c-Myc were increased (P <0.01), while the expression of PTEN mRNA and protein were significantly decreased in the spleen tissue of T-ALL mice (P <0.01). The above indicators in the H-Res group were reversed compared with T-ALL group after administration of resveratrol. CONCLUSION: Resveratrol may play a role in anti T-ALL by inhibiting Notch1 signaling pathway in mice.

Absence of terminal deoxynucleotidyl transferase expression in T-ALL/LBL accumulates chromosomal abnormalities to induce drug resistance.

T-acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) is a malignant neoplasm of immature lymphoblasts. Terminal deoxynucleotidyl transferase (TDT) is a template-independent DNA polymerase that plays an essential role in generating diversity for immunoglobulin genes. T-ALL/LBL patients with TDT- have a worse prognosis. However, how TDT- promotes the disease progression of T-ALL/LBL remains unknown. Here we analyzed the prognosis of T-ALL/LBL patients in Shanghai Children's Medical Center (SCMC) and confirmed that TDT- patients had a higher rate of recurrence and remission failure and worse outcomes. Cellular experiments demonstrated that TDT was involved in DNA damage repair. TDT knockout delayed DNA repair, arrested the cell cycle and decreased apoptosis to induce the accumulation of chromosomal abnormalities and tolerance to abnormal karyotypes. Our study demonstrated that the poor outcomes in TDT- T-ALL/LBL might be due to the drug resistance (VP16 and MTX) induced by chromosomal abnormalities. Our findings revealed novel functions and mechanisms of TDT in T-ALL/LBL and supported that hematopoietic stem cell transplantation (HSCT) might be a better choice for these patients.

Long non-coding RNA signatures and related signaling pathway in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy caused by clonal proliferation of T-cell pre-cursors arising from the thymus. Although the optimized chemotherapy regimen could improve the outcome of such patients, some challenges such as higher risk for induction failure, early relapse and isolated central nervous system (CNS) relapse occurring in T-ALL patients are of great significance, leading to increased mortality rates. Long non-coding RNA (lncRNA) is a key component involved in cell signaling through a variety of mechanisms in regulating gene expression. Oncogenes and tumor suppressors are no exception and their expression can be affected by lncRNAs. In addition, accumulating researches in samples from T-ALL patients as well as pre-clinical studies in mice suggest that the expression profile of lncRNAs in T-ALL could be aberrant, resulting in deregulation of target genes and downstream signaling pathways. In addition, accumulating researches in samples from T-ALL patients as well as pre-clinical studies in mice suggest that the expression profile of lncRNAs in T-ALL could be aberrant, resulting in deregulation of target genes and downstream signaling pathways. These lncRNAs may be determinants of proliferation, apoptosis, and drug resistance observed in T-ALL. Thus, lncRNAs can be a good tool to develop novel strategies against cancer cells in the treatment of relapsed and refractory T-ALL. They can also act as promoting biomarkers in assessing T-ALL and differentiating between patients with poor prognosis and good prognosis. (AU)

Long non-coding RNA signatures and related signaling pathway in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy caused by clonal proliferation of T-cell pre-cursors arising from the thymus. Although the optimized chemotherapy regimen could improve the outcome of such patients, some challenges such as higher risk for induction failure, early relapse and isolated central nervous system (CNS) relapse occurring in T-ALL patients are of great significance, leading to increased mortality rates. Long non-coding RNA (lncRNA) is a key component involved in cell signaling through a variety of mechanisms in regulating gene expression. Oncogenes and tumor suppressors are no exception and their expression can be affected by lncRNAs. In addition, accumulating researches in samples from T-ALL patients as well as pre-clinical studies in mice suggest that the expression profile of lncRNAs in T-ALL could be aberrant, resulting in deregulation of target genes and downstream signaling pathways. In addition, accumulating researches in samples from T-ALL patients as well as pre-clinical studies in mice suggest that the expression profile of lncRNAs in T-ALL could be aberrant, resulting in deregulation of target genes and downstream signaling pathways. These lncRNAs may be determinants of proliferation, apoptosis, and drug resistance observed in T-ALL. Thus, lncRNAs can be a good tool to develop novel strategies against cancer cells in the treatment of relapsed and refractory T-ALL. They can also act as promoting biomarkers in assessing T-ALL and differentiating between patients with poor prognosis and good prognosis.

Upregulation of leukemia-induced non-coding activator RNA (LUNAR1) predicts poor outcome in pediatric T-acute lymphoblastic leukemia.

T-cell Acute Lymphoblastic Leukemia (T-ALL) accounts for around 10-15% of all lymphoblastic leukemia in children. Previous studies have proven that dysregulation of Leukemia-induced non-coding activator RNA-1 (LUNAR1) expression promotes T-ALL cell growth by enhancing the NOTCH1/IGF-1R signaling pathway. We aimed to investigate the prognostic value of LUNAR1 in pediatric T-ALL, in addition, to find out its association with NOTCH1 and IGF-1R. The LUNAR1, NOTCH1, and IGF-IR gene expression were measured in peripheral blood (PB) samples of l85 children with T-ALL and forty non-leukemic samples as a control group. Cox regression analysis revealed that overexpression of LUNAR1, NOTCH1, and IGF-IR was significantly correlated with poor prognosis, short overall survival, and progression-free survival. We concluded that LUNAR1 could serve as an independent prognostic biomarker for T-ALL in children.

Cannabinoid CP55940 selectively induces apoptosis in Jurkat cells and in ex vivo T-cell acute lymphoblastic leukemia through H2O2 signaling mechanism.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous malignant hematological disorder arising from T-cell progenitors. This study was aimed to evaluate the cytotoxic effect of CP55940 on human peripheral blood lymphocytes (PBL) and on T-ALL cells (Jurkat). PBL and Jurkat cells were treated with CP55940 (0-20 µM), and morphological changes in the cell nucleus/ DNA, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species levels were determined by fluorescence microscopy and flow cytometry. Cellular apoptosis markers were also evaluated by western blotting, pharmacological inhibition and immunofluorescence. CP55940 induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner with increasing fragmentation of DNA, arrest of cell cycle and damage of ΔΨm. CP55940 increased dichlorofluorescein fluorescence (DCF) intensity, increased DJ-1 Cys106- sulfonate, a marker of intracellular stress, induced the up-regulation of p53 and phosphorylation of transcription factor c-JUN. It increased the expression of BAX and PUMA, up-regulated mitochondrial proteins PINK1 and Parkin, and activated CASPASE-3. Antioxidant NAC, pifithrin-α, and SP600125 blocked CP55940 deleterious effect on Jurkat cells. However, the potent and highly specific cannabinoid CB1 and CB2 receptor inverse agonist SR141716 and SR144528 were unable to blunt CP55940-induced apoptosis in Jurkat cells. Conclusively CP55940 provokes cell death in Jurkat through CBR-independent mechanism. Interestingly, CP55940 was also cytotoxic to ex vivo T-ALL cells from chemotherapy-resistant pediatric patients. In conclusion, CP55940 selectively induces apoptosis in Jurkat cells through a H2O2-mediated signaling pathway. Our findings support the use of cannabinoids as a potential treatment for T-ALL cells.

The frequency of NOTCH1 variants in T-acute lymphoblastic leukemia/lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma among Jordanian patients.

The transmembrane receptor NOTCH1 is thought to be associated with the development and progression of T-acute lymphoblastic leukemia (T-ALL)/T-lymphoblastic lymphoma (T-LBL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). The current study aimed to characterize NOTCH1 expression and elucidate the variants in the functional PEST domain of the receptor in T-ALL/LBL and CLL/SLL. The nuclear expression of NOTCH1 protein was detected in 25% and 5% of cases of T-ALL/LBL and CLL/SLL, respectively, whereas cytoplasmic expression was detected in 33.3% and 15% cases, respectively. The frequency of variants in T-ALL/LBL was 33%, whereas 40% of CLL/SLL cases possessed variants. Four novel variants were identified; three of which were non-synonymous and one common variant c.7280_7280delG between T-ALL/LBL and CLL/SLL cases. The previously described variant, c.7541_7542delCT, was detected in 3 cases of CLL/SLL. These results provide support for the contribution of NOTCH1 in the etiology of these types of cancers.

Overexpression of CD59 inhibits apoptosis of T-acute lymphoblastic leukemia via AKT/Notch1 signaling pathway.

BACKGROUND: T-acute lymphoblastic leukemia (T-ALL) was a hematological malignancy characterized by the accumulation of immature T cells in bone marrow and peripheral blood. In this study, we tried to explore the physiological role of CD59 in T-ALL. METHODS: In this study, we collected the bone marrow samples from 17 T-ALL patients and 38 healthy participants to find differences in CD59 expression patterns. Then, CD59 was over-expressed in T-ALL cell line Jurkat, and its biological functions were detected. In addition, in order to understand the active site of CD59, the Trp40 was mutated. Further, we constructed a mouse model by transplanting Jurkat cells into the nude mice to verify the function of CD59 in vitro. At last, mechanism studies were performed by western blot. RESULTS: We found that the proportion of T lymphocytes expressing CD59 in bone marrow of T-ALL patients was significantly higher than that of healthy individuals. Then, we found that the overexpression of CD59 in Jurkat cells was beneficial to the cell survival by inhibiting apoptosis and promoting IL-2 secretion. In this process, Trp40 of CD59 was a key functional site. Further, the high expression of CD59 inhibited apoptosis of bone marrow and peripheral blood cells, and promoted IL-2 secretion in mouse model. At last, mechanism studies showed that the activation of AKT, STAT5 and Notch1 signaling pathways in Jurkat cells, may be involved in the regulation of apoptosis by CD59; and mutation in the Trp40 affect the interaction of CD59 with these signaling pathways. CONCLUSIONS: In conclusion, CD59 inhibited apoptosis of T-ALL by regulating AKT/Notch1 signaling pathway, providing a new perspective for the treatment of T-ALL.

Prognostic relevance of genetic variations in T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma.

T-cell acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) are highly aggressive malignant tumors. With the current intensive treatment regimens, event-free survival (EFS) rates of up to 60-90% can be achieved, but the survival rate of relapsed patients remains poor-only approximately 3-12%. Therefore, precise and effective prognostic parameters are highly needed to further improve survival rates along with reduced acute and long-term toxicities, including the rate of secondary malignancies. In addition, gene mutations can be used as therapeutic targets. This review highlights several gene mutations with a high frequency or a strong influence associated with favorable or unfavorable aspects of prognosis-NOTCH1, FBXW7, PTEN, LOH6q, CASP8AP2, c-MYC, IL-7, CALM-AF10, and CDKN2A/B-and indicates that the actual incidence of mutations in T-ALL/LBL is much higher than currently recognized. The development of multicenter clinical trials and molecular genetics research aimed at understanding the biology of these diseases offers promise for targeted and more effective therapy.

T-Cell Receptor Rearrangements Determined Using Fragment Analysis in Patients With T-Acute Lymphoblastic Leukemia.

BACKGROUND: Chromosomal abnormalities and common genetic rearrangements related to T-acute lymphoblastic leukemia (T-ALL) are not clear. We investigated T-cell receptor (TCR) rearrangement in Korean T-ALL patients by fragment analysis, examining frequency, association between clinicopathologic characteristics and TCR clonality, and feasibility for detecting minimal residual disease (MRD). METHODS: In 51 Korean patients diagnosed as having T-ALL, TCR rearrangement was analyzed using the IdentiClone TCR gene clonality assay (InVivoScribe Technologies, San Diego, CA, USA) from archived bone marrow specimens. Limit of detection (LOD) and clonal stability at relapse were evaluated. The association between clinical prognosis and TCR clonality was examind by age and immunophenotypic classification. RESULTS: Thirty-eight patients (74.5%) had 62 clonal products of TCRß, TCRγ, and/or TCRδ rearrangements at diagnosis. Children with T-ALL (<12 years) showed a higher frequency of clonality (93.8%) than adolescents/adults (65.7%; ≥12 years). Patients with a mature immunophenotype (84.4%) showed a relatively higher frequency of clonality than those with the immature immunophenotype (57.9%). Survival and event-free survival were not influenced by immunophenotype or TCR clonality. The LOD was 1%. Clonal evolution at the relapse period was noted. CONCLUSIONS: The overall detection rate of TCR clonality was 74.5%. Survival did not differ by TCR clonality or immunophenotype and age group. Fragment analysis of TCR rearrangement cannot be used to assess MRD due to low sensitivity. Further research on the relationship between prognosis and frequency of TCR rearrangements is needed, using more sensitive methods to detect clonality and monitor MRD.

EB-3D a novel choline kinase inhibitor induces deregulation of the AMPK-mTOR pathway and apoptosis in leukemia T-cells.

Choline kinase alpha 1 (ChoKα1) has recently become an interesting therapeutic target since its overexpression has been associated to tumorigenesis in many cancers. Nevertheless, little is known regarding hematological malignancies. In this manuscript, we investigated the effect of a novel and selective ChoKα inhibitor EB-3D in T acute lymphoblastic leukemia (T-ALL). The effect of EB-3D was evaluated in a panel of T-leukemia cell lines and ex-vivo primary cultures derived from pediatric T-ALL patients. We also evaluated in detail, using Reverse Phase Protein Array (RPPA), protein phosphorylation level changes in T-ALL cells upon treatment. The drug exhibits a potent antiproliferative activity in a panel of T-leukemia cell lines and primary cultures of pediatric patients. Moreover, the drug strongly induces apoptosis and more importantly it enhanced T-leukemia cell sensitivity to chemotherapeutic agents, such as dexamethasone and l-asparaginase. In addition, the compound induces an early activation of AMPK, the main regulator of cellular energy homeostasis, by its phosphorylation at residue T712 of catalytic subunit α, and thus repressing mTORC1 pathway, as shown by mTOR S2448 dephosphorylation. The inhibition of mTOR in turn affects the activity of several known downstream targets, such as 4E-BP1, p70S6K, S6 Ribosomal Protein and GSK3 that ultimately may lead to a reduction of protein synthesis and cell death. Taken together, our findings suggest that targeting ChoKα may be an interesting option for treating T-ALL and that EB-3D could represent a valuable therapeutic tool.

Novel biallelic ATM mutations coexist with a mosaic form of triple X syndrome in an 11-year-old girl at remission after T cell acute leukemia.

Ataxia-telangiectasia (AT) is a rare neurodegenerative disease characterized by an early onset ataxia, oculocutaneous telangiectasia, immunodeficiency, recurrent infections, radio-sensitivity, and a predisposition to malignancy. We present the case of a child with coexistent AT and trisomy X (47,XXX). We used fluorescent in situ hybridization (FISH) to confirm that this person had 47,XXX karyotype in blood cells, bone marrow, fibroblasts, and buccal smear. Standard cytogenetic studies (not banded) were conducted on blood cells. G-banding analysis was performed on bone marrow cells at the time of the leukemia diagnosis. Flow cytometric investigation of lymphocytes and Sanger sequencing of the ATM gene were used for diagnosis confirmation and description. We report the case of an 11-year-old girl at remission after having T cell acute leukemia for 7 years with progressive signs of ataxia-telangiectasia and with additional X chromosome since birth. At the age of 2 years and 7 months, she was diagnosed with pre-T acute leukemia. From the age of four, she had gait abnormalities. AT was established at the age of seven based on clinical signs and laboratory findings (increased alpha fetoprotein-AFP [227]) and confirmed by detecting compound heterozygous truncating mutations in the ATM gene (p.Y705X and p.L2312I). These genetic findings have not been previously reported in AT and our "double hit" case demonstrates the value of careful clinical evaluation of children with an established genetic diagnosis. Measurement of AFP levels should be considered in patients with neurologic abnormalities after leukemia treatment.

Gene expression ratio as a predictive determinant of nelarabine chemosensitivity in T-lymphoblastic leukemia/lymphoma.

BACKGROUND: Nelarabine has been used for the treatment of T-cell malignancies including T-acute lymphoblastic leukemia (T-ALL)/T-lymphoblastic lymphoma. However, the mechanisms that underlie the susceptibility or resistance to nelarabine have not been fully elucidated. The aim of this study was to determine the significance of nelarabine transport and metabolism in the context of nelarabine cytotoxicity. PROCEDURE: The expression profiles of six genes in the nelarabine pathway were analyzed in blast cells from six patients with T-ALL as well as in three T-ALL cell lines. In vitro cytotoxicity (LC50 of 9-ß-d-arabinofuranosylguanine [ara-G]) was evaluated. RESULTS: The mRNA expression of ENT1, DCK, CDA, NT5C2, RRM1, and RRM2 in patients showed inter-individual variability and was not correlated with the LC50 of ara-G. However, the ratio of (ENT1 × DCK)/(CDA × RRM1) expression was significantly correlated with LC50 (r = -0.831, P = 0.0405). CONCLUSIONS: Chemosensitivity to nelarabine is influenced by the balance of the expression of these four genes, and the ratio of their expression predicts the response of T-cell malignancies to nelarabine.

Synergistic effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway or NUP214-ABL1 fusion protein in human Acute Lymphoblastic Leukemia.

Philadelphia chromosome-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) accounts for 25-30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes are markers of poor prognosis and amplification of the NUP214-ABL1 oncogene can be detected mainly in patients with T-ALL. T cell malignancies harboring the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been achieved. The PI3K/Akt/mTOR signaling pathway is often activated in leukemias and plays a crucial role in leukemogenesis.We analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on three NUP214-ABL1 positive T-ALL cell lines that also displayed PI3K/Akt/mTOR activation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis. Four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226, ZSTK474 and Torin-2, showed marked cytotoxic effects on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of these compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL.

Healthy CD4+ T lymphocytes are not affected by targeted therapies against the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia.

An attractive molecular target for novel anti-cancer therapies is the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway which is commonly deregulated in many types of cancer. Nevertheless, the effects of PI3K/Akt/mTOR inhibitors on T lymphocytes, a key component of immune responses, have been seldom explored. In this study we investigated the effects on human CD4+ T-cells of a panel of PI3K/Akt/mTOR inhibitors: BGT226, Torin-2, MK-2206, and ZSTK474. We also assessed their efficacy against two acute leukemia T cell lines. T lymphocytes were stimulated with phytohemagglutinin. Inhibitor effects on cell cycle and apoptosis were analyzed by flow cytometry, while cytotoxicity was assessed by MTT assays. In addition, the activation status of the pathway as well as induction of autophagy were analyzed by Western blotting. Quiescent healthy T lymphocytes were unaffected by the drugs whereas mitogen-stimulated lymphocytes as well as leukemic cell lines displayed a cell cycle block, caspase-dependent apoptosis, and dephosphorylation of key components of the signaling pathway. Autophagy was also induced in proliferating lymphocytes and in JURKAT and MOLT-4 cell lines. When autophagy was inhibited by 3-methyladenine or Bafilomycin A1, drug cytotoxicity was increased, indicating that autophagy is a protective mechanism. Therefore, our findings suggest that PI3K/Akt/mTOR inhibitors preserve lymphocyte viability. This is a valuable result to be taken into account when selecting drugs for targeted cancer therapy in order to minimize detrimental effects on immune function.