A genome-wide CRISPR/Cas9 screen reveals novel positive regulators of FTY720 sensitivity in acute lymphoblastic leukemia cells.

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

Introduction: Glucocorticoid (GC) resistance is a negative prognostic factor in pediatric acute lymphoblastic leukemia (ALL) treatment. The tumor suppressor PTEN, when inactivated, may lead to increased GC resistance through aberrant activation of the PI3K/Akt pathway and inhibition of apoptosis. We investigated the effects of inhibiting the PI3K/Akt pathway with the dual PI3K/mTOR inhibitor NVP-BEZ235 on the enhancement of dexamethasone apoptosis activity in a panel of T-cell and B-cell ALL cell lines and the role PTEN plays in resistance to GCs. Methods: Cytotoxicity of NVP-BEZ235, dexamethasone, and the combination were evaluated using the fluorescence based microscopy system, DIMSCAN, in in vitro models of T- and B-cell ALL and combination indices (CI) were calculated using CalcuSyn. Apoptotic activity for each single agent and the combination was determined using the Annexin-V/PI staining method and measured by flow cytometry. Protein and mRNA expression were measured by immunoblotting and RT-PCR, respectively. Knockdown of PTEN was accomplished using siRNA. Results: The combination of dexamethasone plus NVP-BEZ235 showed strong synergistic activity (CI<0.3) in 6/6 T-cell ALL models tested, while only 1/6 B-cell ALL models, were characterized by such activity. In COG-LL-317h, a T-cell ALL cell line, the percentage of apoptotic cells were significantly greater in cells treated with the combination (17.3±1.0%) compared to vehicle control (4.0±1.7%, p=0.01), dexamethasone (4.5±0.4%, p=0.004), and NVP-BEZ235 (2.3±0.3%, p=0.002). In COG-LL-317h and another T-cell ALL cell line CCRF-CEM, the pro-apoptotic Bcl-2 family protein Bim was substantially increased at both the mRNA and protein levels when treated with the combination compared to either single agent alone, while the B-cell ALL models COG-LL-319h and RS4;11 did not show any enhancement with the combination. Knockdown of PTEN in dexamethasone-sensitive RS4;11 showed a significant reduction in dexamethasone cytotoxicity (non-targeting siRNA: 88.3±1.5% vs. PTEN siRNA: 82.6±2.6%; p<0.001), and activity was restored when dexamethasone was in combination with NVP-BEZ235 (non-targeting siRNA: 91.9±1.8% vs. PTEN siRNA: 92.1±1.6%, p=0.77). Conclusion: These data suggest PTEN dysfunction and subsequent PI3K/Akt activation play a significant role in altering GC responses and use of the combination may offset the negative consequences of PTEN loss by increasing Bim expression and apoptosis. Our results show that NVP-BEZ235 enhances dexamethasone apoptotic activity in models of T-cell ALL which have loss of PTEN function, and may provide a new adjunct to increase response in current T-cell ALL treatment protocols. Citation Format: Connor Hall, Min Kang. The dual PI3K/mTOR inhibitor NVP-BEZ235 enhances dexamethasone induced apoptosis in models of T-cell ALL with PTEN dysfunction and hyperactivated PI3K/Akt pathway. [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 2757. doi:10.1158/1538-7445.AM2013-2757

Ubiquitin-Mediated Degradation of MORF4L1 By FBXO11 Suppresses Cholesterol Biosynthesis in T Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) occasionally develops before or after the onset of Langerhans cell histiocytosis (LCH). The mechanism of LCH developing after ALL remains unclear; thus the clonality of LCH developing during maintenance chemotherapy for T-cell ALL (T-ALL) was investigated. The T-ALL and LCH cells tested had the same T-cell receptor (TCR) gamma rearrangement. Mutation analysis of the NOTCH1 gene revealed 7213C>T (Q2405X) in exon 34 in T-ALL and LCH cells, but 5156T>C (I1719T) in exon 27 only in T-ALL. Polymerase chain reaction-restriction fragment length polymorphism analysis revealed three patterns of NOTCH1 mutations in T-ALL cells. The results suggest that the T-ALL and LCH cells were derived from a common precursor with TCR rearrangement and a single NOTCH1 mutation, rather than LCH cells developing from a minor clone of T-ALL with single NOTCH1 mutation.

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.

The CXCR4 Antagonist, BL8040, Is Highly Active Against Human T-ALL in Preclinical Models

Homoharringtonine Exhibits Anti-Tumor Effect in T-Cell Acute Lymphoblastic Leukemia By Targeting Notch1/Myc Pathway

CXCR4 Blockade By BL-8040 in T Cell Acute Lymphoblastic Leukemia Decreases Mitochondrial Mass and Induces Non-Apoptotic Cell Death

The Function and Autophagy Regulation of PLEKHA8 in T Cell Acute Lymphoblastic Leukemia

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

Tumor-associated myeloid cells provide critical support for T-ALL

Metastatic disease remains the primary cause of death for individuals with T cell acute lymphoblastic leukemia (T-ALL). microRNAs (miRNAs) play important roles in the pathogenesis of T-ALL by inhibiting gene expression at posttranscriptional levels. The goal of the current project is to identify any significant miRNAs in T-ALL metastasis. We observed miR-146b-5p to be downregulated in T-ALL patients and cell lines, and bioinformatics analysis implicated miR-146b-5p in the hematopoietic system. miR-146b-5p inhibited the migration and invasion in T-ALL cells. Interleukin-17A (IL-17A) was predicted to be a target of miR-146b-5p; this was confirmed by luciferase assays. Interestingly, T-ALLpatients and cell lines secreted IL-17A and expressed the IL-17A receptor (IL-17RA). IL-17A/IL-17RA interactions promoted strong T-ALL cell migration and invasion responses. Gene set enrichment analysis (GSEA) and quantitative polymerase chain reaction (qPCR) analysis indicated that matrix metallopeptidase-9 (MMP9), was a potential downstream effector of IL-17A activation, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was also implicated in this process. Moreover, IL-17A activation promoted T-ALL cell metastasis to the liver in IL17A -/- mouse models. These results indicate that reduced miR-146b-5p expression in T-ALL may lead to the upregulation of IL-17A, which then promotes T-ALL cell migration and invasion by upregulating MMP9 via NF-κB signaling.

Transcriptional Repression of the LMO2 Oncogene By Ikaros in T-Cell Acute Lymphoblastic Leukemia

Background:T-cell acute lymphoblastic leukemia is a hematologic malignancy characterized by T-cell proliferation, and in many cases, the ectopic expression of the oncogenic transcription factor T-cell acute lymphocytic leukemia protein 1 (TAL1). MicroRNA-7 has been shown to play a critical role in proliferation, migration, and treatment sensitivity in a diverse array of cancers. In this study, we sought to establish a novel link between microRNA-7 and T-cell acute lymphoblastic leukemia oncogenesis.Material and Method:To do so, we characterized gene expression of microRNA-7 as well as TAL1 in both T-cell acute lymphoblastic leukemia patient-derived tissue and cell lines, as well as performing functional luciferase assays to assess microRNA-7 binding to the TAL1 3′-untranslated region. We also performed growth, apoptosis, and migration experiments using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide, Annexin V, and transwell assays in the context of microRNA-7 overexpression.Results:We found that microRNA-7 expression is attenuated and inversely correlated with TAL1 expression in TAL1 + T-cell acute lymphoblastic leukemia cells. Additionally, microRNA-7 directly targets and suppresses TAL1 levels. Finally, microRNA-7 overexpression reduces growth, motility, and migration while inducing apoptosis in T-cell acute lymphoblastic leukemia cells, phenotypes that can be rescued by concomitant overexpression of TAL1.Conclusions:These results indicate that microRNA-7 functions as a potent tumor suppressor by inhibiting the oncogene TAL1 and suggest microRNA-7 could function as a prognostic biomarker and possible therapeutic in the clinical management of T-cell acute lymphoblastic leukemia.

The NF-κB signaling pathway is an important downstream pathway of oncogenic Notch1 in T cell acute lymphoblastic leukemia (T-ALL) cells. However, the molecular mechanisms underlying the cascade activation of Notch1 in T-ALL cells are poorly understood. Here, we evaluated the role of CARMA1 in Notch1-induced NF-κB activation in T-ALL cells. CARMA1 was highly and specifically expressed in T-ALL cells and correlated with the prognosis of T-ALL patients. Interestingly, CARMA1 knockdown only inhibited the growth and proliferation of SIL-TAL1 fusion gene-negative T-ALL cells. In addition, CARMA1 knockdown arrested T-ALL cells at the G1 phase. Furthermore, CARMA1 knockdown significantly inhibited the proliferation of T-ALL cells in vivo and prolonged the survival of mice. Mechanistically, CARMA1 deficiency abolished Notch1-induced NF-κB transcriptional activation and significantly reduced expression levels of the NF-κB target genes c-Myc, Bcl-2, and CCR7. Taken together, these results of our study identify CARMA1 as one of the crucial mediators of Notch1-induced transformation of T-All cells, suggesting that CARMA1 is a promising therapeutic target for T-ALL due to its specific expression in lymphocytes. KEY MESSAGES: CARMA1 contributes to cell survival only in SIL-TAL1 negative T-ALL cells. CARMA1 is a crucial mediator of Notch1-induced activation of NF-κB pathway. CARMA1 is a promising therapeutic target for T-ALL.