Human T-cell Acute Lymphoblastic Leukemia Cell Research Articles

Effects of NANOG gene down-regulation on the apoptosis of T-cell acute lymphoblastic leukemia cells

To explore gene expression of NANOG in T-cell acute lymphoblastic leukemia (T-ALL) cell lines and the effects of NANOG gene down-regulation on apoptosis of leukemia cells. Real-time PCR (RT-PCR) and Western blot were used to detect the expression level of NANOG gene and protein in MOLT-4, CCRF-HSB2 and Jurkat cells. To test the efficiency of RNA interference, MOLT-4 cells were firstly infected by lentiviral vectors, which were successfully constructed with NANOG specific shRNA. NANOG expression levels were subsequently re-evaluated by RT-PCR and Western blot. The percentages of early apoptotic cells (Annexin V⁺/7-AAD⁻) and late apoptotic cells (Annexin V⁺/7-AAD⁺) were analyzed by flow cytometry. The expression of apoptosis-related genes was also detected. Both NANOG gene and protein expression was positive in MOLT-4 and CCRF-HSB2 cells. The lentiviral vectors pLB-shNANOG-1, pLB-shNANOG-2, and pLB-sh control were successfully constructed, as evidenced by the viral titers (1.83-3.12)× 10⁸ IU/ml. The experimental data on infection of MOLT-4 cells with such lentiviral vectors revealed that both shRNA interfering sequences (shNANOG-1 and shNANOG-2) could stably down-regulate NANOG gene and protein expressions. The percentages of early apoptotic cells in groups of shNANOG-1[(8.06 ± 1.61)%]and shNANOG-2[(5.67 ± 1.59)%]were significantly increased as compared to that of MOLT-4 group[(1.13 ± 0.40)%]or sh-control [(1.15±0.49)%](P<0.05). However, no statistical difference among them was observed for late apoptotic cells (P>0.05). The gene expression of TP53, PMAIP1, and CASP9 of either shNANOG-1 or shNANOG-2 group was augmented as compared to that of MOLT-4 group or sh-control (P<0.05). Reversely, a significant down-regulation of Bcl-2 gene expression was observed (P<0.05). NANOG can be expressed in various human T-ALL cell lines. Down-regulation of NANOG can trigger leukemia cellular apoptosis through mitochondria-dependent apoptosis pathway.

Hsp90 inhibitor BIIB021 enhances triptolide-induced apoptosis of human T-cell acute lymphoblastic leukemia cells in vitro mainly by disrupting p53-MDM2 balance

To investigate the effects of BIIB021, an inhibitor of heat shock protein 90 (Hsp90) alone or in combination with triptolide (TPL) on T-cell acute lymphoblastic leukemia (T-ALL) and the mechanisms of action. Human T-ALL cells line Molt-4 was examined. The cell viability was measured using MTT assay. Apoptotic cells were studied with Hoechst 33258 staining. Cell apoptosis and cell cycle were analyzed using flow cytometry with Annexin V/PI staining and PI staining, respectively. The levels of multiple proteins, including Akt, p65, CDK4/6, p18, Bcl-2 family proteins, MDM2, and p53, were examined with Western blotting. The level of MDM2 mRNA was determined using RT-PCR. Treatment of Molt-4 cells with BIIB021 (50-800 nmol/L) inhibited the cell growth in a dose-dependent manner (the IC50 value was 384.6 and 301.8 nmol/L, respectively, at 48 and 72 h). BIIB021 dose-dependently induced G0/G1 phase arrest, followed by apoptosis of Molt-4 cells. Furthermore, BIIB021 increased the expression of p18, decreased the expression of CDK4/6, and activated the caspase pathway in Molt-4 cells. Moreover, BIIB021 (50-400 nmol/L) dose-dependently decreased the phospho-MDM2 and total MDM2 protein levels, but slightly increased the phospho-p53 and total p53 protein levels, whereas TPL (5-40 nmol/L) dose-dependently enhanced p53 activation without affecting MDM2 levels. Co-treatment with BIIB021 and TPL showed synergic inhibition on Molt-4 cell growth. The co-treatment disrupted p53-MDM2 balance, thus markedly enhanced p53 activation. In addition, the co-treatment increased the expression of Bak and Bim, followed by increased activation of caspase-9. The combination of BIIB021 and TPL may provide a novel strategy for treating T-ALL by overcoming multiple mechanisms of apoptosis resistance.

The Potent PI3K-δ,γ Inhibitor, IPI-145, Exhibits Preclinical Activity In Murine and Human T-Cell Acute Lymphoblastic Leukemia

Abstract Phosphoinositide-3 kinases (PI3Ks) are key cellular signaling proteins that act as a central node for relaying signals from cell surface receptors to downstream mediators, such as AKT. The PI3K-δ and PI3K-γ isoforms are preferentially expressed in normal and malignant leukocytes where they play critical roles in cell differentiation, migration, and proliferation. Constitutive activity of the PI3K pathway is common in T-cell acute lymphoblastic leukemia (T-ALL) and frequently involves the deletion of PTEN, the phosphatase that negatively regulates the PI3K pathway. An important role for the PI3K-δ and PI3K-γ isoforms has been demonstrated in the Pten-deleted genetically engineered murine model of T-ALL in conjunction with PI3K-δ and/or PI3K-γ gene knock outs (Subramanian et al, Cancer Cell, 2012). IPI-145 is a potent inhibitor of PI3K-δ and PI3K-γ currently being studied in a Phase 1 trial (IPI-145-02) in patients with advanced hematologic malignancies, including T-ALL (ClinicalTrials.gov NCT01476657). We performed in vitro studies to address the sensitivity of human and murine T-ALL cell lines to IPI-145 and to additional PI3K inhibitors with defined isoform selectivity. The human T-ALL cells examined were from cell lines that lack PTEN protein expression (Loucy, MOLT-4, CCRF-CEM, CEM/C2, p12 Ichikawa, and Karpas-45) and cell lines that express PTEN protein (MOLT13 and MOLT16). In addition, two murine cell lines derived from a Pten-deleted model of T-ALL (LPN049 and LPN236) were studied. The expression levels of class I PI3K isoforms were determined by western blotting and quantitative RT-PCR, which revealed varying levels of protein and RNA expression across the cell lines. In vitro treatment of human T-ALL cells with IPI-145 resulted in variable degrees of growth inhibition, with the PTEN-deficient Loucy cell line demonstrating the greatest sensitivity with an IC50 of 245 nM. In the cell lines tested, growth inhibition to IPI-145 was only seen in PTEN-deficient human cell lines, whereas all PTEN-expressing human T-ALL cell lines were resistant to IPI-145 (IC50 &gt; 10 uM). However, not all PTEN-deficient human T-ALL cells demonstrated sensitivity to IPI-145 (e.g., CEM/C2), indicating that loss of PTEN does not confer sensitivity to PI3K inhibition in all settings. Inhibition of phospho-AKT (pAKT) correlated with growth inhibition, with an IC50 of 286 nM in the Loucy cell line. Studies to evaluate the mechanism of growth inhibition revealed that IPI-145 treatment resulted in apoptosis of sensitive cells as measured by 7-AAD and Annexin V staining. Cell lines derived from the Pten-deleted murine T-ALL model were also sensitive to IPI-145 with IC50s in the 300-600 nM range as measured by MTT assay. In addition, IPI-145 led to apoptosis, as measured by cleaved Caspase 3 and 7-AAD/Annexin V. Interestingly, Pten-deleted murine T-ALL cell lines showed down-regulation of pAKT and c-MYC expression with IPI-145 in a dose responsive manner that corresponded with increasing activated Caspase-3 expression. In NOTCH1-expressing murine T-ALL cell lines, down regulation of NOTCH1 and activated NOTCH1 was also observed in parallel with c-MYC down-regulation. To explore further the individual contributions of the varying PI3K isoforms on T-ALL cell growth, the effect of IPI-145 on tumor cell growth was compared with PI3K-δ, PI3K-δ,γ, and PI3K-β selective compounds in the Loucy PTEN-deficient T-ALL cell line. These experiments support an anti-leukemic effect for both PI3K-δ and PI3K-γ inhibition and indicate that the greatest effect is seen with combined PI3K-δ and PI3K-γ inhibition. A role for PI3K-β in T-ALL cell survival was not observed. Evaluation of the in vivo activity of IPI-145 on Loucy xenografts, as well as PTEN-expressing MOLT-13 xenografts, is ongoing. Together, these data provide a strong rationale for combined targeted inhibition of PI3K-δ and PI3K-γ in T-ALL. Disclosures: Huang: Infinity Pharmaceuticals, Inc.: Research Funding. Proctor:Infinity Pharmaceuticals, Inc.: Employment. Yang:Infinity Pharmaceuticals, Inc.: Research Funding. Gao:Infinity Pharmaceuticals, Inc.: Research Funding. Zhang:Infinity Pharmaceuticals, Inc.: Research Funding. Huang:Infinity Pharmaceuticals, Inc.: Research Funding. Changelian:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. You:Infinity Pharmaceuticals, Inc.: Research Funding.

RUNX1 Is Required For Maintenance Of Established T-ALL Blasts

T-cell acute lymphoblastic leukemia (T-ALL) is a clinically aggressive malignancy of immature T cells. Intensive multiagent chemotherapy achieves cure in 80-90% of pediatric patients, but only 40% of adult patients survive beyond 5 years. Data from recent ChIP-seq studies has shown that RUNX1 binds throughout the T-ALL genome at sites co-occupied by known oncogenic transcription factors including TAL1 and NOTCH1. For this reason, it has been suggested that RUNX1 may be part of a transcriptional activation complex that drives an oncogenic gene expression program in this cell context. In contrast, next generation sequencing studies have recently identified heterozygous point mutations throughout the RUNX1 coding region in T-ALL including some which are predicted to encode truncated polypeptides resembling dominant negative alleles, thus raising the possibility that RUNX1 may function rather as a tumor suppressor in this context. In an effort to explore the functional role of RUNX1 in T-ALL, we examined the effect of RUNX1 knockdown in a broad panel of established human T-ALL cell lines and xenograft-expanded patient biopsy samples.Cells transduced with lentiviral shRNAs targeting coding and 3' UTR regions of RUNX1 showed a clear growth disadvantage as compared to either non-transduced cells in the same culture or cells transduced with non-silencing shRNAs in parallel cultures. As well, absolute cell counts of cultures containing only shRUNX1-transduced cells demonstrated dramatically reduced growth rates as compared to either non-transduced or non-silencing shRNA-transduced controls. BrdU incorporation and CFSE dye dilution studies showed that most cell lines exhibited reduced proliferation in response to RUNX1 knock-down, while a subset of lines also showed reduced cell viability. These phenotypes were largely consistent across a panel of over 20 T-ALL cell lines and 4 xenograft-expanded patient samples, including several which harbored either nonsense or missense RUNX1 mutations. These results support the notion that established T-ALL cells are generally dependent on RUNX1 for continued cell growth and, in some cases, also for survival.We also explored candidate RUNX1 target genes which might be responsible for mediating the observed growth/survival phenotypes. Assembling a short list of the “usual suspects” including genes known to regulate growth of T-ALL cells generally (c-MYC, PTEN), to possess a substantial RUNX1 ChIP-seq peak in T-ALL cells (IGF1R, IL7R), or to be bona fide RUNX1 targets in other cell contexts (p21/WAF1, p27/KIP1), we performed western blot or flow cytometric analyses of multiple shRUNX1-transduced cell lines. We noted consistent regulation of some, but not all of these in a manner consistent with RUNX1 positively supporting cell growth.From these results, we conclude that RUNX1 plays a pro-oncogenic role in established T-ALL cells. We surmise that the complement of presumed loss-of-function RUNX1 mutations observed in patient T-ALLs may be indicative of its known roles in regulating normal T cell development such that loss-of-function mutations may lead to differentiation arrest and consequently promote tumor initiation. For the majority of T-ALLs that express wild-type RUNX1 proteins, however, our results suggest that RUNX1 acts functionally to support maintenance of the malignant clone by promoting expression of known oncogenic factors and repressing expression known tumor suppressors. Disclosures:Aster:Cell Signaling Technology: Consultancy; Merck, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Genentech, Inc.: Honoraria.

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.

Rapamycin interacts synergistically with idarubicin to induce T-leukemia cell apoptosis in vitro and in a mesenchymal stem cell simulated drug-resistant microenvironment via Akt/mammalian target of rapamycin and extracellular signal-related kinase signaling pathways

T-cell acute lymphoblastic leukemias (T-ALLs) are clonal lymphoid malignancies with a poor prognosis, and still a lack of effective treatment. Here we examined the interactions between the mammalian target of rapamycin (mTOR) inhibitor rapamycin and idarubicin (IDA) in a series of human T-ALL cell lines Molt-4, Jurkat, CCRF-CEM and CEM/C1. Co-exposure of cells to rapamycin and IDA synergistically induced T-ALL cell growth inhibition and apoptosis mediated by caspase activation via the intrinsic mitochondrial pathway and extrinsic pathway. Combined treatment with rapamycin and IDA down-regulated Bcl-2 and Mcl-1, and inhibited the activation of phosphoinositide 3-kinase (PI3K)/mTOR and extracellular signal-related kinase (ERK). They also played synergistic pro-apoptotic roles in the drug-resistant microenvironment simulated by mesenchymal stem cells (MSCs) as a feeder layer. In addition, MSCs protected T-ALL cells from IDA cytotoxicity by up-regulating ERK phosphorylation, while rapamycin efficiently reversed this protective effect. Taken together, we confirm the synergistic antitumor effects of rapamycin and IDA, and provide an insight into the potential future clinical applications of combined rapamycin–IDA regimens for treating T-cell malignancies.

RNA interference-mediated silencing of NANOG leads to reduced proliferation and self-renewal, cell cycle arrest and apoptosis in T-cell acute lymphoblastic leukemia cells via the p53 signaling pathway

NANOG is critical for maintaining the self-renewal and proliferative properties of embryonic stem cells. Here we found that cultured T-cell acute lymphoblastic leukemia (T-ALL) cells, as well as human primary T-ALL cells, express a functional variant of NANOG. NANOG mRNA is derived predominantly from a retrogene locus termed NANOGP8. Furthermore, we showed that RNA interference-mediated NANOG knockdown inhibited cell proliferation, reduced self-renewal, promoted apoptosis and arrested the cell cycle through a p53-mediated pathway in leukemic cells. These findings demonstrate the oncogenic potential of this pluripotent gene in human T-ALL cells.

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 4448: Notch1 inactivation contributes to PI3Ki resistance in T-ALL.

Abstract Tyrosine kinases inhibitors induce genotype-specific regression in advanced human cancers. However, these responses are almost invariably transient and patients ultimately relapse due to outgrowth of drug-resistant clones. Thus, increased knowledge of the mechanisms underlying de novo and acquired resistance is essential for enhancing clinical efficacy. T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer for which current therapies are toxic and frequently ineffective. Mutations in genes encoding components of the Notch1 and Ras/PI3K/PTEN/Akt signaling networks are prevalent in T-ALL and frequently coexist in individual patients. Thus, inhibiting these pathways alone and in combination is a rational therapeutic strategy. Notch1 inhibition with a γ-secretase inhibitor (GSI) was recently evaluated in refractory T-ALL, with disappointing results that may have been due to dose-limiting gastrointestinal toxicity. We are not aware of clinical trials of Ras/PI3K/Akt/mTOR pathway inhibitors in T-ALL. Retroviral insertional mutagenesis (RIM) in mice is a robust strategy for modeling both the clonal heterogeneity that exists within individual tumors and the genetic diversity found across a population of patients with the same cancer. We generated transplantable T-ALLs by performing RIM in mice expressing oncogenic KrasG12D from its endogenous genetic locus (Mx1-Cre, KrasG12D) and in wild-type (WT) littermates. These aggressive multi-step cancers are characterized by recurring viral integrations in known oncogenes such as Ikzf1 and late occurring acquired Notch1 PEST domain mutations. We have extensively investigated patterns of response and resistance to targeted inhibitors of PI3K (PI3Ki) and MEK (MEKi) in this system. Whereas KrasWT T-ALLs respond to either PI3Ki or to a combination regimen that also include a MEKi, KrasG12D T-ALLs are largely refractory to PI3Ki, but are sensitive to the MEKi + PI3Ki combination. T-ALLs invariably relapsed despite ongoing treatment, and we verified phenotypic drug resistance in relapse leukemias by transplanting and retreating them in vivo. We unexpectedly identified multiple independent PI3Ki resistant T-ALLs with markedly reduced levels of activated Notch1, including some in which Notch1 mutations that were present at enrollment were absent at relapse. Expression of activated Notch1 increases the sensitivity of mouse and human T-ALL cells to a PI3Ki, and loss of activated Notch promotes resistance and enhances PI3K signaling. Thus, oncogenic Notch1 mutations that promote outgrowth during transformation unexpectedly “switch” to become deleterious during in vivo treatment with a PI3Ki. These data suggest that aberrant Ras/PI3K signaling plays a dominant role in T lineage leukemogenesis and also raise the unexpected possibility that administering Notch1 and PI3K inhibitors sequentially or in combination might inadvertently promote resistance. Citation Format: Monique Dail, Jason Wong, Daniel O'Connor, Joy Nakitandwe, Shann-Ching Chen, Jessica Lawrence, James R. Downing, Deepak Sampath, Kevin Shannon. Notch1 inactivation contributes to PI3Ki resistance in T-ALL. [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 4448. doi:10.1158/1538-7445.AM2013-4448

Convergence of the ZMIZ1 and NOTCH1 Pathways at C-MYC in Acute T Lymphoblastic Leukemias

Activating NOTCH1 mutations are found in 50% to 60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, these mutations generally fail to induce leukemia. This observation suggests that NOTCH1 activation must collaborate with other genetic events. Mutagenesis screens previously implicated ZMIZ1 as a possible NOTCH1 collaborator in leukemia. ZMIZ1 is a transcriptional coactivator of the protein inhibitor of activated STAT (PIAS)-like family. Its role in oncogenesis is unknown. Here, we show that activated NOTCH1 and ZMIZ1 collaborate to induce T-ALL in mice. ZMIZ1 and activated NOTCH1 are coexpressed in a subset of human T-ALL patients and cell lines. ZMIZ1 inhibition slowed growth and sensitized leukemic cells to corticosteroids and NOTCH inhibitors. Gene expression profiling identified C-MYC, but not other NOTCH-regulated genes, as an essential downstream target of ZMIZ1. ZMIZ1 functionally interacts with NOTCH1 to promote C-MYC transcription and activity. The mechanism does not involve the NOTCH pathway and appears to be indirect and mediated independently of canonical PIAS functions through a novel N-terminal domain. Our study shows the importance of identifying genetic collaborations between parallel leukemic pathways that may be therapeutically targeted. They also raise new inquiries into potential NOTCH-ZMIZ1 collaboration in a variety of C-MYC-driven cancers.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Phenothiazines Induce Apoptosis in T-Cell Acute Lymphoblastic Leukemia by Activating the Phosphatase Activity of the PP2A Tumor Suppressor

AbstractAbstract 3558Despite the use of intensive and toxic treatment regimens, T-cell acute lymphoblastic leukemia (T-ALL) remains fatal in 25% of children and 60% of adults, highlighting the need for novel therapeutic strategies. We performed two complementary small molecule screens to identify novel T-ALL therapeutic agents: i) an in vivo zebrafish screen for small molecules toxic to MYC-overexpressing thymocytes, and ii) an in vitro screen for drugs that synergize with NOTCH inhibitors in human T-ALL cell lines. Hits common to both screens included perphenazine, an FDA-approved phenothiazine antipsychotic. Perphenazine potently induced mitochondrial apoptosis and had antileukemic activity in zebrafish T-ALL, in human T-ALL cell lines, and in primary human T-ALL lymphoblasts. However, the molecular target mediating the antileukemic activity of perphenazine appeared to be unrelated to any of its known cellular targets. To identify the relevant biologic target of perphenazine, we developed a novel method for drug target discovery, termed activity correlation proteomics, which combines native protein extraction of isotope-labeled proteomes, fluorous affinity chromatography, and quantitative mass spectrometry proteomics to relate phenotypic activities of drugs with their binding targets. Using activity correlation proteomics, we identified protein phosphatase 2A (PP2A) as the protein whose binding affinity for different phenothiazines most closely correlated with their antileukemic activity. Perphenazine was shown to activate PP2A in T-ALL cells, as assessed by rapid drug-induced dephosphorylation of multiple PP2A substrates including MYC, AKT, p70S6K, ERK, and BAD, and its effects were phenocopied by FTY720, a PP2A activator. Moreover, shRNA knockdown of PP2A scaffolding or catalytic subunits attenuated the activity of perphenazine, indicating that PP2A is required for its antileukemic activity. Our findings thus establish a novel strategy for pharmacologic activation of the PP2A tumor suppressor, and highlight its therapeutic potential in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates. Disclosures:Off Label Use: Discovery of antileukemic activity of perphenazine mediated via activation of PP2A.

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

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human Tcell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Highlights of This Issue

Gamma-secretase inhibitors (GSI) have been proposed as anti-NOTCH-targeted therapy in T-cell acute lymphoblastic leukemia (T-ALL). Samon and colleagues characterized the interaction between the clinically relevant GSI PF-03084014 and glucocorticoids in preclinical models of glucocorticoid-resistant T-ALL. The combination of PF-03084014 and dexamethasone induced a synergistic antileukemic response in human T-ALL cell lines, primary patient samples, and in a xenograft model of T-ALL. Moreover, dexamethasone treatment also reversed the gastrointestinal toxicity associated with systemic inhibition of NOTCH signaling by GSIs. These results substantiate the clinical evaluation of PF-03084014 and dexamethasone in combination for the treatment of glucocorticoid-resistant T-ALL.Targeted cancer therapies that inhibit the IGF-I signaling pathway are under investigation. Dietary fat reduction may have anticancer effects, in part, through inhibition of the IGF-I axis. Konijeti and colleagues reported that combination therapy (low-fat diet and IGF-1 receptor blockade) did not have additive effects on xenograft growth, whereas there was a reduction in tumor proliferation and serum insulin and TNF-α levels. The approach of combining pharmacological and nutritional interventions offers the potential for improving anticancer therapy and limiting the untoward metabolic effects of targeted therapies.Stem cell pathways have been implicated in tumorigenesis and survival multiple malignancies. Steg and colleagues examined the effects of hedgehog pathway targeting on chemotherapy response. The smoothened inhibitors cyclopamine and LDE225 both sensitized ovarian cancer cells to paclitaxel, but not carboplatin, as did selective knockdown of individual hedgehog signaling components. Combined LDE225 and paclitaxel also significantly reduced tumor burden compared to vehicle or single agents. The increased taxane sensitivity was due at least in part to decreased P-glycoprotein (MDR1) expression. These findings suggest that anti-hedgehog therapies are attractive in ovarian cancer, especially in combination with taxanes.This is the first study to quantify signaling output of the PI3K pathway in ovarian cells isolated from ascites and to correlate this with response to subsequent chemotherapy. This study also looked for associations between signaling output and known drivers of the PI3K pathway. A raised p-S6K was negatively associated with response to subsequent chemotherapy and interestingly, there was no clear association between PI3K pathway signaling output and PIK3CA/AKT mutation or amplification, or PTEN loss. This reinforces the need to look wider for drivers of the PI3K pathway in ovarian cancer while designing clinical trials of PI3K pathway inhibitors.

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/β and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.

Reversal of glucocorticoid resistance by the g-secretase inhibitor PF-03084014 in T-cell acute lymphoblastic leukemia.

9553 Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer frequently characterized by activating mutations in NOTCH1. Early studies identified NOTCH1 as an attractive therapeutic target for the treatment of T-ALL through the use of g-secretase inhibitors (GSIs), but subsequent work found that inhibition of NOTCH signaling is characterized by a lack of cytotoxicity of human T-ALL cells and severe gastrointestinal toxicity. Our preliminary results show that combination therapy of generic GSIs plus glucocorticoids has improved antileukemic effects in vitro and in vivo and reduced gut toxicity. Here we characterized the interaction between PF-03084014, a clinically relevant GSI, and dexamethasone in preclinical models of glucocorticoid resistant T-ALL. Methods: Studies were performed using a panel of human T-ALL cell lines and primary patient samples. The effect of GSI treatment plus glucocorticoids was assessed using cell viability, cell cycle, and proliferation assays....

Abstract 2603: Inhibition of Notch and mTOR by nelfinavir as a novel approach for T cell acute lymphoblastic leukemia (T-ALL)

Abstract Background: T-ALL accounts for 10-15% of childhood ALL and 25% of adult ALL. Although intensive chemotherapy improves disease free survival, patients who relapse or progress have a dismal prognosis. Notch1 is a promising target in T-ALL because it is commonly activated, and inhibitors of γ-secretase (GSI), an aspartic protease that processes Notch1, are in clinical trials. Nelfinavir is an HIV protease inhibitor with anti-cancer activity through its inhibition of the Akt/mTOR pathway as well as unknown mechanisms. Because the HIV protease is an aspartic protease, we hypothesized that a target for nelfinavir in cancer cells could be γ-secretase. Methods: Nelfinavir was compared to compound E, a commercially available GSI, in in vitro studies. Notch1 and mTOR signalling was evaluated in human and murine T-ALL cell lines by immunoblotting. γ-secretase activity was assessed in cell-free in vitro assays. Cell death was measured by flow cytometry after annexin V and propidium iodide staining. Nelfinavir was also tested in a transgenic model of T-ALL (established by overexpression of SCL and LMO1 in the mouse thymus) using microCT scans, flow cytometry and IHC. Results: Among 7 HIV protease inhibitors evaluated, only nelfinavir inhibited expression of the Notch1 transmembrane and intracellular domains as well as Hes1 and survivin, well-established downstream targets of Notch1. At clinically achievable concentrations, nelfinavir rapidly inhibited Notch1, which was sustained to 16 hr when death was evident. Cell-free assays showed that although compound E was a more potent inhibitor of γ-secretase, the maximal enzymatic inhibition by nelfinavir was equivalent at higher doses. Nelfinavir was far more effective than compound E in reducing expression of Notch targets such as phospho-Rb, c-Myc, and Hrb, Hes1, and survivin. Nelfinavir induced apoptosis to levels equivalent to 20 µM etoposide, but compound E was only cytostatic. To explore other mechanisms that could explain the decreased protein expression and higher levels of cell death with nelfinavir, we assessed the Akt/mTOR pathway. Nelfinavir rapidly induced AMPK activation and mTOR inhibition, coincident with Notch1 inhibition. In SCL-LMO1 double transgenic mice, nelfinavir decreased thymic size and markedly increased apoptotic cells in the thymus. It also decreased the CD3+ populations in spleen and bone marrow and decreased CD4+CD8+ double positive leukemic cells. Conclusion: These studies show that nelfinavir is superior to a GSI and highly active in preclinical studies of T-ALL. Clinical testing of nelfinavir in T-ALL patients is planned. 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 2603. doi:10.1158/1538-7445.AM2011-2603

Mcl-1 Ubiquitination and Destruction

Loss of the Fbw7 tumor suppressor is common in diverse human cancer types, including T-Cell Acute Lymphoblastic Leukemia (T-ALL), although the mechanistic basis of its anti-oncogenic activity remains largely unclear. We recently reported that SCFFbw7 regulates cellular apoptosis by controlling the ubiquitination and destruction of the pro-survival protein, Mcl-1, in a GSK3 phosphorylation-dependent manner. We found that human T-ALL cell lines displayed a close relationship between Fbw7 loss and Mcl-1 overexpression. More interestingly, T-ALL cell lines that are deficient in Fbw7 are particularly sensitive to sorafenib, a multi-kinase inhibitor that has been demonstrated to reduce Mcl-1 expression through an unknown mechanism. On the other hand, Fbw7-deficient T-ALL cell lines are much more resistant to the Bcl-2 antagonist, ABT-737. Furthermore, reconstitution of Fbw7 or depletion of Mcl-1 in Fbw7-deficient cells restores ABT-737 sensitivity, suggesting that elevated Mcl-1 expression is important for Fbw7-deficient cells to evade apoptosis. Therefore, our work provides a novel molecular mechanism for the tumor suppression function of Fbw7. Furthermore, it provides the rationale for targeted usage of Mcl-1 antagonists to treat Fbw7-deficient T-ALL patients.

SCFFBW7 regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction

The effective use of targeted therapy is highly dependent upon the identification of responder patient populations. Loss of the Fbw7 tumor suppressor is frequently found in various types of human cancers including breast cancer, colon cancer 1 and T-cell acute lymphoblastic leukemia (T-ALL)2. In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL3–5, validating Fbw7 as a T-ALL tumor suppressor. The precise molecular mechanisms by which Fbw7 exerts anti-tumor activity remain areas of intensive investigation and are thought to relate in part to Fbw7-mediated destruction of key cancer relevant proteins including c-Jun6, c-Myc 7, Cyclin E 8 and Notch-19, all of which possess oncogenic activity and are overexpressed in various human cancers including leukemia. Besides accelerating cell growth 10, overexpression of either c-Jun, c-Myc or Notch-1 can also provoke programmed cell death 11. Thus, considerable uncertainty surrounds how Fbw7-deficient cells evade cell death in the setting of upregulated c-Jun, c-Myc and/or Notch-1. Here we report that SCFFbw7 governs cellular apoptosis by targeting the pro-survival Bcl-2 family member, Mcl-1, for ubiquitination and destruction in a GSK3 phosphorylation-dependent manner. Human T-ALL cell lines showed a close relationship between Fbw7 loss and Mcl-1 overexpression. Correspondingly, T-ALL cell lines with defective Fbw7 are particularly sensitive to the multi-kinase inhibitor, sorafenib, but resistant to the Bcl-2 antagonist, ABT-737. On the genetic level, Fbw7 reconstitution or Mcl-1 depletion restores ABT-737 sensitivity, establishing Mcl-1 as a therapeutically relevant bypass survival mechanism for Fbw7-deficient cells to evade apoptosis. Therefore, our work provides novel molecular insight into Fbw7-direct tumor suppression with direct implications for the targeted treatment of Fbw7-deficient T-ALL patients.

Deletion-Based Mechanisms of Notch1 Activation In T-ALL: Key Roles for RAG Recombinase and A Conserved Internal Translational Start Site In Notch1.

AbstractAbstract 3367Point mutations that trigger ligand-independent proteolysis of the Notch1 ectodomain occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL) but are rare in murine T-ALL, suggesting that other mechanisms account for Notch1 activation in murine tumors. Using a panel of 13 murine T-ALL cell lines arising in a variety of T-ALL prone genetic backgrounds, we identified two types of acquired somatic deletions involving the 5' end of Notch1 in all cell lines screened. Type 1 deletions were found in 10 cell lines. These rearrangements remove exon 1 and the proximal promoter of Notch1, yet paradoxically activate transcription from a conserved initiator element positioned adjacent to a cryptic internal promoter in exon 25, roughly 40 kb downstream from the normal transcriptional start site. Expression of type 1 transcripts is associated with markedly increased histone H3 acetylation in the 3' end of Notch1, indicating that deletion of the proximal promoter and exon 1 leads to global remodeling of chromatin within the locus. Type 1 transcripts lack the normal Notch1 translation start site and initiate translation from a conserved methionine, M1727, which lies within the transmembrane domain of Notch1. A Notch1 polypeptide with M1727 for its N-terminus scored as a strong gain-of-function molecule in reporter gene assays and required gamma-secretase cleavage for activation. Cloning and sequencing of alleles with type 1 rearrangements showed that the responsible DNA breaks occurred immediately adjacent to germ line sequences resembling RAG recombinase signal sequences (RSSs), and that the DNA sequences at the point of joining bear all of the structural hallmarks of RAG-mediated rearrangements. Moreover, ChIP-Seq analyses showed that RAG2 associates with the proximal Notch1 promoter close to the ectopic RSSs in normal thymocytes in a distribution similar to that of the histone mark H3K4-me3, placing RAG at the “scene of the crime” in normal T cell progenitors. The conserved nature of the DNA breaks that produce type 1 deletions allowed for design of a PCR assay that detected this type of mutation in the majority of primary murine tumors, which also expressed high amounts of aberrant 3' Notch1 transcripts, as judged by a ratiometric q-RT-PCR assay capable of comparing the levels of 5' and 3' Notch1 transcripts. The remaining three cell lines had type 2 deletions, which removed sequences between exon 1 and exons 26–28 of Notch1, appeared to be created by non-homologous or microhomology mediated end joining, and were associated with the expression of transcripts in which exon 1 is spliced out-of-frame to 3' Notch1 exons. Translation of type 2 transcripts also initiated at M1727. Both types of deletions in murine tumors often occur concomitantly with mutations in the 3' end of Notch1 that remove a C-terminal PEST degron domain, and the combination of 5' deletions and 3' PEST mutations produced synergistic increases in Notch1 signal strength in reporter gene assays. The absence of intact Notch1 ectodomains in tumors with type 1 and type 2 deletions render murine T-ALLs resistant to agents such as inhibitory antibodies that target the extracellular domain of Notch1. Unlike murine T-ALL, 5' deletions were not detected by comparative genomic hybridization on SNP arrays in nearly 200 human T-ALLs, suggesting that activating deletions are rare in human disease. The absence of 5' Notch1 deletions in human T-ALL may be related to evolutionary divergence of the RSS sequences, which are not highly conserved in man. One human mutation that bears some similarities to the Notch1 deletions in murine T-ALL was found in the human T-ALL cell line CUTLL1. CUTLL1 contains a t(7;9) that fuses TCR-beta with the 3' end of Notch1. The fusion gene expresses a TCR J-beta 2.4-Notch1 fusion transcript that initiates translation from M1738, which is homologous to murine M1727. We conclude that like human T-ALL, murine T-ALL is often associated with acquired mutations that cause ligand-independent Notch1 activation. These findings further reinforce the central oncogenic role of aberrant Notch1 signaling in both human and murine T-ALL, and also point out some limitations in using murine T-ALL models for development of therapeutic agents that target Notch1. Disclosures:No relevant conflicts of interest to declare.