Immunophenotype of gastric tumors unveils a pleiotropic role of regulatory T cells in tumor development

Simple SummaryThe role of regulatory T cells (Tregs) in gastric cancer (GC) is still controversial and poorly understood. GC patients have increased numbers of Tregs in peripheral blood and among tumor infiltrating lymphocytes; however, their prognostic value depends on specific tumor features (e.g., tumor location and/or microsatellite instability status). We found that Tregs might induce membrane expression of IL2R alpha in intestinal-type GC cells, which associates with MAPK signaling pathway activation and spheroid growth. Moreover, Tregs accumulate at early steps of intestinal-type GCs progression, when tumors are starting to grow through the stomach wall, and do not present vascular and perineural invasion. Our findings suggest a novel non-immunosuppressive role of Treg cells in intestinal-type GC, which may unlock novel therapeutic immuno-oncology strategies for intestinal-type GC or other tumors with similar immune context.Gastric cancer (GC) patients display increased regulatory T cell (Tregs) numbers in peripheral blood and among tumor-infiltrating lymphocytes. Nevertheless, the role of Tregs in GC progression remains controversial. Here, we sought to explore the impact of Tregs in GCs with distinct histology, and whether Tregs can directly influence tumor cell behavior and GC development. We performed a comprehensive immunophenotyping of 82 human GC cases, through an integrated analysis of multispectral immunofluorescence detection of T cells markers and patient clinicopathological data. Moreover, we developed 3D in vitro co-cultures with Tregs and tumor cells that were followed by high-throughput and light-sheet imaging, and their biological features studied with conventional/imaging flow cytometry and Western blotting. We showed that Tregs located at the tumor nest were frequent in intestinal-type GCs but did not associate with increased levels of effector T cells. Our in vitro results suggested that Tregs preferentially infiltrated intestinal-type GC spheroids, induced the expression of IL2R alpha and activation of MAPK signaling pathway in tumor cells, and promoted spheroid growth. Accumulation of Tregs in intestinal-type GCs was increased at early stages of the stomach wall invasion and in the absence of vascular and perineural invasion. In this study, we proposed a non-immunosuppressive mechanism through which Tregs might directly modulate GC cells and thereby promote tumor growth. Our findings hold insightful implications for therapeutic strategies targeting intestinal-type GCs and other tumors with similar immune context.MTG4Molecular tumour pathology - and tumour genetic

Targeting chronic lymphocytic leukemia using CIGB-300, a clinical-stage CK2-specific cell-permeable peptide inhibitor

We are grateful to all the patients that contributed to this study. We thank Dr. Riccardo Dalla-Favera for providing the MO1043 cell line and Dr. John Byrd for providing the WaC3CD5 cell line. We also thank the Helena Alaiz and Teresa Faria (Cytogenetics and Flow Cytometry sections of the Hemato-oncology lab at Instituto Portugues de Oncologia) for the cytogenetic and phenotypic characterization, respectively, of patient samples. This work was supported by the grant PIC/IC/83193/2007 from Fundacao para a Ciencia e a Tecnologia. LRM received an FCT-SFRH PhD fellowship.Chronic lymphocytic leukemia (CLL) remains an incurable malignancy, urging for the identification of new molecular targets for therapeutic intervention. CLL cells rely on overexpression and hyperactivation of the ubiquitous serine/threonine protein kinase CK2 for their viability in vitro. CIGB-300 is a cell-permeable selective CK2 inhibitor peptide undergoing clinical trials for several cancers. Here, we show that CIGB-300 promotes activation of the tumor suppressor PTEN and abrogates PI3Kmediated downstream signaling in CLL cells. In accordance, CIGB-300 decreases the viability and proliferation of CLL cell lines, promotes apoptosis of primary leukemia cells and displays antitumor efficacy in a xenograft mouse model of human CLL. Our studies provide pre-clinical support for the testing and possible inclusion of CK2 inhibitors in the clinical arsenal against CLL.publishersversionpublishe

Aberrant signaling in T-cell acute lymphoblastic leukemia: biological and therapeutic implications

T-cell acute lymphoblastic leukemia (T-ALL) is a biologically heterogeneous disease with respect to phenotype, gene expression profile and activation of particular intracellular signaling pathways. Despite very significant improvements, current therapeutic regimens still fail to cure a portion of the patients and frequently implicate the use of aggressive protocols with long-term side effects. In this review, we focused on how deregulation of critical signaling pathways, in particular Notch, PI3K/Akt, MAPK, Jak/STAT and TGF-ß, may contribute to T-ALL. Identifying the alterations that affect intracellular pathways that regulate cell cycle and apoptosis is essential to understanding the biology of this malignancy, to define more effective markers for the correct stratification of patients into appropriate therapeutic regimens and to identify novel targets for the development of specific, less detrimental therapies for T-ALL

Regulation Of Pten By Ck2 And Notch1 In Primary T-cell Acute Lymphoblastic Leukemia: Rationale For Combined Use Of Ck2- And γ-secretase Inhibitors

T-cell acute lymphoblastic leukemia (T-ALL) patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. Here, we compared patients with or without NOTCH1 mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of Notch transcriptional and CK2 post-translational inactivation of PTEN, we treated T-ALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL. © 2010 Ferrata Storti Foundation.954674678Weng, A.P., Ferrando, A.A., Lee, W., Morris JPt, Silverman LB, Sanchez-Irizarry C, et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia (2004) Science, 306 (5694), pp. 269-71Palomero, T., Sulis, M.L., Cortina, M., Real, P.J., Barnes, K., Ciofani, M., Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia (2007) Nat Med, 13 (10), pp. 1203-10Maser, R.S., Choudhury, B., Campbell, P.J., Feng, B., Wong, K.K., Protopopov, A., Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers (2007) Nature, 447 (7147), pp. 966-71Silva, A., Yunes, J.A., Cardoso, B.A., Martins, L.R., Jotta, P.Y., Abecasis, M., PTEN posttranslational inactivation and hyperactivation of the PI3K/Akt pathway sustain primary T cell leukemia viability (2008) J Clin Invest, 118 (11), pp. 3762-74Barata, J.T., Boussiotis, V.A., Yunes, J.A., Ferrando, A.A., Moreau, L.A., Veiga, J.P., IL- 7-dependent human leukemia T-cell line as a valuable tool for drug discovery in T-ALL (2004) Blood, 103 (5), pp. 1891-900Breit, S., Stanulla, M., Flohr, T., Schrappe, M., Ludwig, W.D., Tolle, G., Activating NOTCH1 mutations predict favorable early treatment response and long-term outcome in childhood precursor T-cell lymphoblastic leukemia (2006) Blood, 108 (4), pp. 1151-1157Sulis, M.L., Williams, O., Palomero, T., Tosello, V., Pallikuppam, S., Real, P.J., NOTCH1 extracellular juxtamembrane expansion mutations in T-ALL (2008) Blood, 112 (3), pp. 733-40Barata, J.T., Silva, A., Brandao, J.G., Nadler, L.M., Cardoso, A.A., Boussiotis, V.A., Activation of PI3K Is Indispensable for Interleukin 7- mediated Viability, Proliferation, Glucose Use, and Growth of T Cell Acute Lymphoblastic Leukemia Cells (2004) J Exp Med, 200 (5), pp. 659-69Barata, J.T., Cardoso, A.A., Nadler, L.M., Boussiotis, V.A., Interleukin-7 promotes survival and cell cycle progression of T-cell acute lymphoblastic leukemia cells by down-regulating the cyclin-dependent kinase inhibitor p27(kip1) (2001) Blood, 98 (5), pp. 1524-31Sharma, V.M., Calvo, J.A., Draheim, K.M., Cunningham, L.A., Hermance, N., Beverly, L., Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc (2006) Mol Cell Biol, 26 (21), pp. 8022-31Weng, A.P., Millholland, J.M., Yashiro-Ohtani, Y., Arcangeli, M.L., Lau, A., Wai, C., Et al., C-Myc is an important direct target of Notch1 in Tcell acute lymphoblastic leukemia/lymphoma (2006) Genes Dev, 20 (15), pp. 2096-109Palomero, T., Lim, W.K., Odom, D.T., Sulis, M.L., Real, P.J., Margolin, A., NOTCH1 directly regulates c-MYC and activates a feedforward- loop transcriptional network promoting leukemic cell growth (2006) Proc Natl Acad Sci USA, 103 (48), pp. 18261-18266Larson, G.A., Chen, Q., Kugel, D.S., Ge, Y., Lafiura, K., Haska, C.L., The impact of NOTCH1, FBW7 and PTEN mutations on prognosis and downstream signaling in pediatric T-cell acute lymphoblastic leukemia: A report from the Children's Oncology Group (2009) Leukemia, 23 (8), pp. 1417-25Real, P.J., Tosello, V., Palomero, T., Castillo, M., Hernando, E., de Stanchina, E., Gammasecretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia (2009) Nat Med, 15 (1), pp. 50-58Solares, A.M., Santana, A., Baladron, I., Valenzuela, C., Gonzalez, C.A., Diaz, A., Safety and preliminary efficacy data of a novel casein kinase 2 (CK2) peptide inhibitor administered intralesionally at four dose levels in patients with cervical malignancies (2009) BMC Cancer, 9, p. 146Gonzalez-Garcia, S., Garcia-Peydro, M., Martin-Gayo, E., Ballestar, E., Esteller, M., Bornstein, R., CSL-MAML-dependent Notch1 signaling controls T lineage-specific IL-7R{alpha} gene expression in early human thymopoiesis and leukemia (2009) J Exp Med, 206 (4), pp. 779-91Salmena, L., Carracedo, A., Pandolfi, P.P., Tenets of PTEN tumor suppression (2008) Cell, 133 (3), pp. 403-1

Negative Prognostic Impact Of Pten Mutation In Pediatric T-cell Acute Lymphoblastic Leukemia

[No abstract available]241239242Silva, A., Yunes, J.A., Cardoso, B.A., Martins, L.R., Jotta, P.Y., Abecasis, M., PTEN posttranslational inactivation and hyperactivation of the PI3K/Akt pathway sustain primary T cell leukemia viability (2008) J Clin Invest, 118, pp. 3762-3774Maser, R.S., Choudhury, B., Campbell, P.J., Feng, B., Wong, K.K., Protopopov, A., Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers (2007) Nature, 447, pp. 966-971Palomero, T., Sulis, M.L., Cortina, M., Real, P.J., Barnes, K., Ciofani, M., Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia (2007) Nat Med, 13, pp. 1203-1210Gutierrez, A., Sanda, T., Grebliunaite, R., Carracedo, A., Salmena, L., Ahn, Y., High frequency of PTEN, PI3K and AKT abnormalities in T-cell acute lymphoblastic leukemia (2009) Blood, 114, pp. 647-650Remke, M., Pfister, S., Kox, C., Toedt, G., Becker, N., Benner, A., High-resolution genomic profiling of childhood T-ALL reveals frequent copy-number alterations affecting the TGF-{beta} and PI3K-AKT pathways and deletions at 6q15-16.1 as a genomic marker for unfavorable early treatment response (2009) Blood, 114, pp. 1053-1062Larson, G.A., Chen, Q., Kugel, D.S., Ge, Y., Lafiura, K., Haska, C.L., The impact of NOTCH1, FBW7 and PTEN mutations on prognosis and downstream signaling in pediatric T-cell acute lymphoblastic leukemia: A report from the Children's Oncology Group (2009) Leukemia, 23, pp. 1417-1425Cortez, M.A., Scrideli, C.A., Yunes, J.A., Valera, E.T., Toledo, S., Pavoni-Ferreira, P.C.B., MRNA expression profile of multidrug resistance genes in childhood acute lymphoblastic leukemia. Low expression levels associated with a higher risk of toxic death (2009) Pediatric Blood Câncer, 53, pp. 996-1004Georgescu, M.M., Kirsch, K.H., Akagi, T., Shishido, T., Hanafusa, H., The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region (1999) Proc Natl Acad Sci USA, 96, pp. 10182-1018

Activity of the pan-class I phosphoinositide 3-kinase inhibitor NVP-BKM120 in T-cell acute lymphoblastic leukemia

Constitutively active phosphoinositide 3-kinase (PI3K) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival and drug resistance. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the pan-PI3K inhibitor NVP-BKM120 (BKM120), an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in G(2)/M phase cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T lymphoblasts, and promoting a dose-and time-dependent dephosphorylation of Akt and S6RP. BKM120 maintained its pro-apoptotic activity against Jurkat cells even when cocultured with MS-5 stromal cells, which mimic the bone marrow microenvironment. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. Moreover, in vivo administration of BKM120 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth, thus prolonging survival time. Taken together, our findings indicate that BKM120, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment for T-ALLs that have aberrant upregulation of the PI3K signaling pathway

Cytotoxic activity of the casein kinase 2 inhibitor CX-4945 against T-cell acute lymphoblastic leukemia: targeting the unfolded protein response signaling

Constitutively active casein kinase 2 (CK2) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). CK2 phosphorylates PTEN (phosphatase and tensin homolog) tumor suppressor, resulting in PTEN stabilization and functional inactivation. Downregulation of PTEN activity has an impact on PI3K/Akt/mTOR signaling, which is of fundamental importance for T-ALL cell survival. These observations lend compelling weight to the application of CK2 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of CX-4945-a novel, highly specific, orally available, ATP-competitive inhibitor of CK2 alpha. We show that CX-4945 treatment induced apoptosis in T-ALL cell lines and patient T lymphoblasts. CX-4945 downregulated PI3K/Akt/mTOR signaling in leukemic cells. Notably, CX-4945 affected the unfolded protein response (UPR), as demonstrated by a significant decrease in the levels of the main UPR regulator GRP78/BIP, and led to apoptosis via upregulation of the ER stress/UPR cell death mediators IRE1 alpha and CHOP. In vivo administration of CX-4945 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth. Our findings indicate that modulation of the ER stress/UPR signaling through CK2 inhibition could be exploited for inducing apoptosis in T-ALL cells and that CX-4945 may be an efficient treatment for those T-ALLs displaying upregulation of CK2 alpha/PI3K/Akt/mTOR signaling