Ruta Grebliunaite

High frequency of PTEN, PI3K, and AKT abnormalities in T-cell acute lymphoblastic leukemia.

To more comprehensively assess the pathogenic contribution of the PTEN-PI3K-AKT pathway to T-cell acute lymphoblastic leukemia (T-ALL), we examined diagnostic DNA samples from children with T-ALL using array comparative genomic hybridization and sequence analysis. Alterations of PTEN, PI3K, or AKT were identified in 47.7% of 44 cases. There was a striking clustering of PTEN mutations in exon 7 in 12 cases, all of which were predicted to truncate the C2 domain without disrupting the phosphatase domain of PTEN. Induction chemotherapy failed to induce remission in 3 of the 4 patients whose lymphoblasts harbored PTEN deletions at the time of diagnosis, compared with none of the 12 patients with mutations of PTEN exon 7 (P = .007), suggesting that PTEN deletion has more adverse therapeutic consequences than mutational disruptions that preserve the phosphatase domain. These findings add significant support to the rationale for the development of therapies targeting the PTEN-PI3K-AKT pathway in T-ALL.

Phenothiazines induce PP2A-mediated apoptosis in T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drugs antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.

Inactivation of LEF1 in T-cell acute lymphoblastic leukemia

To further unravel the molecular pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), we performed high-resolution array comparative genomic hybridization on diagnostic specimens from 47 children with T-ALL and identified monoallelic or biallelic LEF1 microdeletions in 11% (5 of 47) of these primary samples. An additional 7% (3 of 44) of the cases harbored nonsynonymous sequence alterations of LEF1, 2 of which produced premature stop codons. Gene expression microarrays showed increased expression of MYC and MYC targets in cases with LEF1 inactivation, as well as differentiation arrest at an early cortical stage of thymocyte development characterized by expression of CD1B, CD1E, and CD8, with absent CD34 expression. LEF1 inactivation was associated with a younger age at the time of T-ALL diagnosis, as well as activating NOTCH1 mutations, biallelic INK4a/ARF deletions, and PTEN loss-of-function mutations or activating mutations of PI3K or AKT genes. These cases generally lacked overexpression of the TAL1, HOX11, HOX11L2, or the HOXA cluster genes, which have been used to define separate molecular pathways leading to T-ALL. Our findings suggest that LEF1 inactivation is an important step in the molecular pathogenesis of T-ALL in a subset of young children.

Pten mediates Myc oncogene dependence in a conditional zebrafish model of T cell acute lymphoblastic leukemia.

Loss-of-function mutations in pten genes, or expression of a constitutively active version of Akt2, render T-ALL cell survival and disease progression independent of Myc.

Absence of Biallelic TCRγ Deletion Predicts Early Treatment Failure in Pediatric T-Cell Acute Lymphoblastic Leukemia

PURPOSE To identify children with T-cell acute lymphoblastic leukemia (T-ALL) at high risk of induction chemotherapy failure by using DNA copy number analysis of leukemic cells collected at diagnosis. PATIENTS AND METHODS Array comparative genomic hybridization (CGH) was performed on genomic DNA extracted from diagnostic lymphoblasts from 47 children with T-ALL treated on Childrens Oncology Group Study P9404 or Dana-Farber Cancer Institute Protocol 00-01. These samples represented nine patients who did not achieve an initial complete remission, 13 who relapsed, and 25 who became long-term, event-free survivors. The findings were confirmed in an independent cohort of patients by quantitative DNA polymerase chain reaction (DNA-PCR), an assay that is well suited for clinical application. RESULTS Analysis of the CGH findings in patients in whom induction chemotherapy failed compared with those in whom induction chemotherapy was successful identified the absence of biallelic TCRgamma locus deletion (ABD), a characteristic of early thymocyte precursors before V(D)J recombination, as the most robust predictor of induction failure (P < .001). This feature was also associated with markedly inferior event-free (P = .002) and overall survival (P < .001) rates: 25% versus 58% and 25% versus 72%, respectively. Using a rapid and inexpensive quantitative DNA-PCR assay, we validated ABD as a predictor of a poor response to induction chemotherapy in an independent series of patients. CONCLUSION Lymphoblasts from children with T-ALL should be evaluated at diagnosis for deletion within the TCRgamma locus. Patients lacking biallelic deletion, which confers a high probability of induction failure with contemporary therapy, should be assigned to alternative therapy in the context of a prospective clinical trial.

Aberrant AKT activation drives well-differentiated liposarcoma

Well-differentiated liposarcoma (WDLPS), one of the most common human sarcomas, is poorly responsive to radiation and chemotherapy, and the lack of animal models suitable for experimental analysis has seriously impeded functional investigation of its pathobiology and development of effective targeted therapies. Here, we show that zebrafish expressing constitutively active Akt2 in mesenchymal progenitors develop WDLPS that closely resembles the human disease. Tumor incidence rates were 8% in p53 wild-type zebrafish, 6% in p53 heterozygotes, and 29% in p53-homozygous mutant zebrafish (P = 0.013), indicating that aberrant Akt activation collaborates with p53 mutation in WDLPS pathogenesis. Analysis of primary clinical specimens of WDLPS, and of the closely related dedifferentiated liposarcoma (DDLPS) subtype, revealed immunohistochemical evidence of AKT activation in 27% of cases. Western blot analysis of a panel of cell lines derived from patients with WDLPS or DDLPS revealed robust AKT phosphorylation in all cell lines examined, even when these cells were cultured in serum-free media. Moreover, BEZ235, a small molecule inhibitor of PI3K and mammalian target of rapamycin that effectively inhibits AKT activation in these cells, impaired viability at nanomolar concentrations. Our findings are unique in providing an animal model to decipher the molecular pathogenesis of WDLPS, and implicate AKT as a previously unexplored therapeutic target in this chemoresistant sarcoma.

Abstract IA8: A new class of drugs active in T-ALL is revealed in a zebrafish screen

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer frequently associated with activating NOTCH1 mutations and dysregulation of MYC. We performed two complementary screens to identify novel agents with activity against T-ALL: i) a zebrafish screen for small molecules toxic to MYC-overexpressing thymocytes, and ii) a human T-ALL cell line screen for small molecules that synergize with Notch inhibitors. “Hits” common to both screens included perphenazine, a phenothiazine antipsychotic that induced apoptosis of fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled to mass spectrometry, we identified protein phosphatase 2A (PP2A) as the critical perphenazine target. In line with this finding, T-ALL cell lines treated with perphenazine underwent apoptosis associated with rapid dephosphorylation of multiple PP2A substrates, indicating that perphenazine binds and activates the PP2A tumor supressor. Moreover, shRNA knockdown of the scaffolding or catalytic subunits of PP2A attenuated the activity of perphenazine, indicating that PP2A is required for its antileukemic activity. Finally, treatment of primary human T-ALLs with pherphenazine suppressed cell growth and caused dephosphorylated of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurrent “rediscovery” of phenothiazines as a class of drugs with anti-cancer effects and highlight the therapeutic potential of pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates. Citation Format: Alejandro Gutierrez, Li Pan, Richard Groen, Frederic Baleydier, Alex Kentsis, Jason Marineau, Ruta Grebliunaite, Elena Kozakewich, Casie Reed, Francoise Pflumio, Sandrine Poglio, Benjamin Uzan, Paul Clemons, Lynn Verplank, Frank An, Jason Burbank, Stephanie Norton, Nicola Tolliday, Hanno Steen, Andrew P. Weng, Huipin Yuan, James E. Bradner, Constantine Mitsiades, A. Thomas Look, Jon C. Aster. A new class of drugs active in T-ALL is revealed in a zebrafish screen. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr IA8.