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Supplementary Figures 1-4 from IL-7 Contributes to the Progression of Human T-cell Acute Lymphoblastic Leukemias

Background Intratumoral heterogeneity is crucially involved in metastasis, resistance to therapy, and cancer relapse. Amplifications of the proto-oncogene MYC display notable heterogeneity at the single-cell level and are associated with a particularly dismal prognosis in high-risk medulloblastomas (MBs). The aim of this study was to establish the relevance of interclonal cross-talk between MYC-driven and non-MYC-driven MB cells. Methods We used fluorescence in situ hybridization, single-cell transcriptomics, and immunohistochemistry, in vitro isogenic cell models, non-targeted proteomics, mass spectrometry-based metabolite quantification, HUVECs tube formation assay, and orthotopic in vivo experiments to investigate interclonal cross-talk in MB. Results We found that the release of lactate dehydrogenase A (LDHA) from MYC-driven cells facilitates metastatic seeding and outgrowth, while secretion of dickkopf WNT signaling pathway inhibitor 3 from non-MYC-driven cells promotes tumor angiogenesis. This tumor-supporting interaction between both subclones was abrogated by targeting the secretome through pharmacological and genetic inhibition of LDHA, which significantly suppressed tumor cell migration. Conclusion Our study reveals the functional relevance of clonal diversity and highlights the therapeutic potential of targeting the secretome to interrupt interclonal communication and progression in high-risk MB.

© 2023 by The American Society of Hematology., The downstream signaling of the interleukin-7 (IL-7) receptor (IL-7R) plays important physiological and pathological roles, including the differentiation of lymphoid cells and proliferation of acute lymphoblastic leukemia cells. Gain-of-function mutations in the IL-7Rα chain, the specific component of the receptor for IL-7, result in constitutive, IL-7-independent signaling and trigger acute lymphoblastic leukemia. Here, we show that the loss of the phosphoinositide 5-phosphatase INPP5K is associated with increased levels of the INPP5K substrate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) and causes an altered dynamic structure of the IL-7 receptor. We discovered that the IL-7Rα chain contains a very conserved positively charged polybasic amino acid sequence in its cytoplasmic juxtamembrane region; this region establish stronger ionic interactions with negatively charged PtdIns(4,5)P2 in the absence of INPP5K, freezing the IL-7Rα chain structure. This dynamic structural alteration causes defects in IL-7R signaling, culminating in decreased expressions of EBF1 and PAX5 transcription factors, in microdomain formation, cytoskeletal reorganization, and bone marrow B-cell differentiation. Similar alterations after the reduced INPP5K expression also affected mutated, constitutively activated IL-7Rα chains that trigger leukemia development, leading to reduced cell proliferation. Altogether, our results indicate that the lipid 5-phosphatase INPP5K hydrolyzes PtdIns(4,5)P2, allowing the requisite conformational changes of the IL-7Rα chain for optimal signaling.

The role of IL-7 and IL-7R for normal lymphoid development and an adequately functioning immune system has been recognized for long, with severe immune deficiency and lymphoid leukemia as extreme examples of the consequences of deregulation of the IL-7-IL-7R axis. In this review, we provide an update (focusing on the past couple of years) on IL-7 and IL-7R in health and disease. We highlight the findings on IL-7/IL-7R signaling mechanisms and the, sometimes controversial, impact of IL-7 and its receptor on leukocyte biology, COVID-19, acute lymphoblastic leukemia, and different solid tumors, as well as their relevance as therapeutic tools or targets.

Brain metastases (BMs) are a devastating complication of advanced cancers associated with poor prognosis. Contrarily to the current improvement in systemic therapies, BMs are still incurable and one of the main causes of death in cancer patients. We analyzed BMs from thirty patients with various primary tumor origins by RNA sequencing and identified the upregulation of UBE2C, a gene involved in the correct transition from metaphase to anaphase. Using an independent cohort of patients with BMs, we demonstrated that high protein expression of UBE2C was associated with worse survival. UBE2C-driven cancer cells promoted migration and invasion in vitro and induced an aggressive phenotype and decreased survival in mouse orthotopic xenografts. PI3K/mTOR inhibition effectively blocked cancer cell signaling and prevented the development of leptomeningeal metastases. Therefore, we have identified UBE2C as a molecular marker of worse outcome in BMs patients and pre-clinically validated an effective therapy against UBE2C-driven brain metastatic disease.

© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/., T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric cancer. Amongst the wide array of driver mutations, 10% of T-ALL patients display gain-of-function mutations in the IL-7 receptor α chain (IL-7Rα, encoded by IL7R), which occur in different molecular subtypes of this disease. However, it is still unclear whether IL-7R mutational activation is sufficient to transform T-cell precursors. Also, which genes cooperate with IL7R to drive leukemogenesis remain poorly defined. Here, we demonstrate that mutant IL7R alone is capable of inducing T-ALL with long-latency in stable transgenic zebrafish and transformation is associated with MYC transcriptional activation. Additionally, we find that mutant IL7R collaborates with Myc to induce early onset T-ALL in transgenic zebrafish, supporting a model where these pathways collaborate to drive leukemogenesis. T-ALLs co-expressing mutant IL7R and Myc activate STAT5 and AKT pathways, harbor reduced numbers of apoptotic cells and remake tumors in transplanted zebrafish faster than T-ALLs expressing Myc alone. Moreover, limiting-dilution cell transplantation experiments reveal that activated IL-7R signaling increases the overall frequency of leukemia propagating cells. Our work highlights a synergy between mutant IL7R and Myc in inducing T-ALL and demonstrates that mutant IL7R enriches for leukemia propagating potential., This work was supported by NIH grant R01CA211734 (DML), the MGH Research Scholar Award (DML), the ERC consolidator CoG-648455 and proof-of-concept PoC-862545 grants from the European Research Council, under the European Union’s Horizon 2020 research and innovation programme (JTB), and the FCT grants FAPESP/ 20015/2014, PTDC/MEC-HEM/31588/2017 and PTDC/MEC-ONC/4606/2021 (JTB)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer for which treatment options often result in incomplete therapeutic efficacy and long-term side-effects. Interleukin 7 (IL-7) and its receptor IL-7Rα promote T-ALL development and mutational activation of IL-7Rα associates with very high risk in relapsed disease. Using combinatorial phage-display libraries and antibody reformatting, we generated a fully human IgG1 monoclonal antibody (named B12) against both wild-type and mutant human IL-7Rα, predicted to form a stable complex with IL-7Rα at a different site from IL-7. B12 impairs IL-7/IL-7R-mediated signaling, sensitizes T-ALL cells to treatment with dexamethasone and can induce cell death per se. The antibody also promotes antibody-dependent natural killer-mediated leukemia cytotoxicity in vitro and delays T-cell leukemia development in vivo, reducing tumor burden and promoting mouse survival. B12 is rapidly internalized and traffics to the lysosome, rendering it an attractive vehicle for targeted intracellular delivery of cytotoxic cargo. Consequently, we engineered a B12–MMAE antibody–drug conjugate and provide proof-of-concept evidence that it has increased leukemia cell killing abilities as compared with the naked antibody. Our studies serve as a stepping stone for the development of novel targeted therapies in T-ALL and other diseases where IL-7Rα has a pathological role. ISSN:1476-5551 ISSN:0887-6924

© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/), Interleukin 7 (IL-7) and its receptor (IL-7R, a heterodimer of IL-7Rα and γc) are essential for normal lymphoid development. In their absence, severe combined immunodeficiency occurs. By contrast, excessive IL-7/IL-7R-mediated signaling can drive lymphoid leukemia development, disease acceleration and resistance to chemotherapy. IL-7 and IL-7R activate three main pathways: STAT5, PI3K/Akt/mTOR and MEK/Erk, ultimately leading to the promotion of leukemia cell viability, cell cycle progression and growth. However, the contribution of each of these pathways towards particular functional outcomes is still not completely known and appears to differ between normal and malignant states. For example, IL-7 upregulates Bcl-2 in a PI3K/Akt/mTOR-dependent and STAT5-independent manner in T-ALL cells. This is a 'symmetric image' of what apparently happens in normal lymphoid cells, where PI3K/Akt/mTOR does not impact on Bcl-2 and regulates proliferation rather than survival. In this review, we provide an updated summary of the knowledge on IL-7/IL-7R-mediated signaling in the context of cancer, focusing mainly on T-cell acute lymphoblastic leukemia, where this axis has been more extensively studied., Publication costs were supported by LISBOA-01-0145-FEDER-007391, project cofunded by FEDER, through POR Lisboa2020 Programa Operacional Regional de Lisboa, PORTUGAL 2020, and Fundação para a Ciência e a Tecnologia (FCT, Portugal). The research work in JTB's lab related to the present review was supported by the grants FAPESP/20015/2014 and PTDC/MEC-HEM/31588/2017, from FCT; and by the consolidator grant ERC CoG-648455 from the European Research Council, under the European Union's Horizon 2020 research and innovation programme. JTB is an FCT investigator (consolidator). MLO is a LisbonBioMed PhD student and received a fellowship from FCT. PA received a PhD fellowship from the EU Marie Sklodowska-Curie ITN Protein Conjugates.

Dissemination of cancer cells from primary tumors to the brain occurs in many cancer patients, increasing morbidity and death. There is an unmet medical need to develop translational platforms to evaluate therapeutic responses. Toward this goal, we established a library of 23 patient-derived xenografts (PDXs) of brain metastases (BMs) from eight distinct primary tumors. In vivo tumor formation correlates with patients' poor survival. Mouse subcutaneous xenografts develop spontaneous metastases and intracardiac PDXs increase dissemination to the CNS, both models mimicking the dissemination pattern of the donor patient. We test the FDA-approved drugs buparlisib (pan-PI3K inhibitor) and everolimus (mTOR inhibitor) and show their efficacy in treating our models. Finally, we show by RNA sequencing that human BMs and their matched PDXs have similar transcriptional profiles. Overall, these models of BMs recapitulate the biology of human metastatic disease and can be valuable translational platforms for precision medicine.

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., 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 IL2Rα 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., This work was supported by grants: (1) “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020— Operacional Program for Competitiveness and Internationalisation (POCI), PORTUGAL 2020, Portuguese funds through Portuguese Science & Technology Foundation (FCT)/Ministério da Ciência, Tecnologia e Inovação; (2) Project CANCER Ref. NORTE-01-0145-FEDER-000029 and DOCnet Ref. NORTE-01-0145-FEDER-000003), by Norte Portugal Regional Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); (3) Project Ref. PTDC/BBB-ECT/2518/2014, by FCT;(4) FCT PhD Programs and by Programa Operacional Potencial Humano (POCH), specifically by the Biotech Health Programe (Doctoral Program on Cellular and Molecular Biotechnology Applied to Health Sciences); (5) “The role of gastric cancer cell-derived extracellular vesicles”, by IPATIMUP Board of Directors; (6) GenomePT project (POCI-01-0145-FEDER-022184), by COMPETE 2020—POCI, Lisboa Portugal Regional Operational Program (Lisboa2020), Algarve Portugal Regional Operational Program (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through ERDF, and by FCT; (7) Marie Skłodowska-Curie grant agreement No. 722148 to Train-EV project by Euro-pean Union’s Horizon 2020 research and innovation program; 8) Project CANCERSTEM funded by ERDF, POCI and FCT; 9) FCT fellowship PD/BD/128406/2017 to SR, and Junior Research contractto JC (regulated by the decree-law 57/2016 emended by law 57/2017). IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT. NFCCdM has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 852832). The authors acknowledgethe support of the Advanced Light Microscopy, Bioimaging, BioSciences Screening and Histologyand Electron Microscopy i3S Scientific Platforms members of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122), and the support providedby the Translational Cytometry i3S Scientific Platforms.

PurposeDissemination of cancer cells from primary tumors to the brain is observed in the great majority of cancer patients, contributing to increased morbidity and being the main cause of death. Most mechanistic and preclinical studies have relied on aggressive cancer cell lines, which fail to represent tumor heterogeneity and are unsuitable to validate therapies due to fast cancer progression in vivo.Experimental designWe established a unique library of subcutaneous and intracardiac patient-derived xenografts (PDXs) of brain metastases (BMs) from eight distinct primary tumor origins. Cancer progression in mice was compared to the matched patient clinical outcome, metastatic dissemination pattern and histopathological features. Preclinical studies with FDA approved drugs were performed.ResultsIn vivo tumor formation of flank-implanted BMs correlated with patients’ poor survival and serial passaging increased tumor aggressiveness. Subcutaneous xenografts originated spontaneous metastases in 61% of the cases, including in the leptomeningeal space (21%). The intracardiac model increased the tropism to the brain and leptomeninges (46%). Strikingly, 62% of intracardiac PDXs shared metastatic sites with the donor patients, including the primary cancer organ and the central nervous system (CNS). Of therapeutic relevance, PDX-derived cultures and corresponding mouse xenografts can be effectively treated with targeted anticancer drugs.ConclusionsPatient-derived models of BMs recapitulate the biology of human metastatic disease and can be a valuable translational platform for precision medicine.TRANSLATIONAL RELEVANCESubcutaneous and intracardiac mouse xenografts of human brain metastases exhibit a spontaneous dissemination pattern that resembles patients’ metastatic disease. The preclinical testing of targeted anticancer drugs using patient-derived cultures and patient-derived xenografts of brain metastasis showed an effective therapeutic response. These translational models represent an outstanding tool to advance the understanding of the biology of brain metastases and to foster the rapid discovery of novel therapeutics.

T-cell acute lymphoblastic leukemia (T-ALL) constitutes an aggressive subset of ALL, the most frequent childhood malignancy. Whereas interleukin-7 (IL-7) is essential for normal T-cell development, it can also accelerate T-ALL development in vivo and leukemia cell survival and proliferation by activating phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin signaling. Here, we investigated whether STAT5 could also mediate IL-7 T-ALL-promoting effects. We show that IL-7 induces STAT pathway activation in T-ALL cells and that STAT5 inactivation prevents IL-7-mediated T-ALL cell viability, growth, and proliferation. At the molecular level, STAT5 is required for IL-7-induced downregulation of p27kip1 and upregulation of the transferrin receptor, CD71. Surprisingly, STAT5 inhibition does not significantly affect IL-7-mediated Bcl-2 upregulation, suggesting that, contrary to normal T-cells, STAT5 promotes leukemia cell survival through a Bcl-2-independent mechanism. STAT5 chromatin immunoprecipitation sequencing and RNA sequencing reveal a diverse IL-7-driven STAT5-dependent transcriptional program in T-ALL cells, which includes BCL6 inactivation by alternative transcription and upregulation of the oncogenic serine/threonine kinase PIM1 Pharmacological inhibition of PIM1 abrogates IL-7-mediated proliferation on T-ALL cells, indicating that strategies involving the use of PIM kinase small-molecule inhibitors may have therapeutic potential against a majority of leukemias that rely on IL-7 receptor (IL-7R) signaling. Overall, our results demonstrate that STAT5, in part by upregulating PIM1 activity, plays a major role in mediating the leukemia-promoting effects of IL-7/IL-7R.

The dissemination of cancer cells to the brain parenchyma (brain metastases - BMs) and to the leptomeninges (leptomeningeal dissemination – LD) are a late-stage complication of systemic cancers, with a poor survival. Despite advances in radiation and chemotherapy, including intrathecal administration of anticancer agents, these forms of advanced cancer are incurable. Therefore, there is an unmet clinical need for novel effective therapies that target both parenchymal and leptomeningeal disease. We analysed the transcriptomic profile of BMs from patients with diverse primary tumors, treated at CHULN, to identify genetic drivers of cancer cell dissemination to the brain and potential novel targets for therapy. The most differentially expressed gene codifies a ubiquitin conjugating enzyme (UCE). UCE levels were evaluated in tissue microarrays of BMs from an independent cohort of patients and correlated with clinical data. UCE functional role was assessed in vitro and in vivo using modulated lung and breast cancer cell lines. A high-throughput drug screening was performed to find UCE-targeting compounds. High protein levels of the UCE were associated with decreased survival in patients with BMs, independently of the primary tumor origin. High levels of UCE led to increased migration and invasion abilities in cancer cell lines in vitro, with no effect in proliferation. In vivo, high levels of UCE increased leptomeningeal dissemination and decreased survival in orthotopic models of breast cancer BMs. Leptomeningeal disease promoted by UCE was prevented by oral administration of inhibitor A, identified in our high-throughput drug screening. In conclusion, we have identified UCE as a prognostic marker in patients with BMs from different primary tumors. In orthotopic mouse models of the disease, UCE led to a worse survival and promoted leptomeningeal dissemination. Strikingly, this aggressive disease phenotype was prevented by oral therapy with inhibitor A.

The cytokine IL-7 and its receptor, IL-7R, are critical for T cell and, in the mouse, B cell development, as well as differentiation and survival of naive T cells, and generation and maintenance of memory T cells. They are also required for innate lymphoid cell (ILC) development and maintenance, and consequently for generation of lymphoid structures and barrier defense. Here we discuss the central role of IL-7 and IL-7R in the lymphoid system and highlight the impact of their deregulation, placing a particular emphasis on their 'dark side' as promoters of cancer development. We also explore therapeutic implications and opportunities associated with either positive or negative modulation of the IL-7-IL-7R signaling axis.

T-cell acute lymphoblastic leukemia (T-ALL) is often not cured by frontline chemotherapy, and efforts to improve treatment by targeting oncogenes such as NOTCH1 have been hampered by toxicity. Silva and colleagues studied primary patient samples to show that high-level interleukin 7 receptor α (IL7Rα) gene expression correlates with ongoing, oncogenic IL7R-mediated signaling. Using new in vivo models, they characterized the impact of IL7Rα expression on the pathogenesis of T-ALL and its response to various targeted therapies that reduce IL7-related signaling., Key Points Mice overexpressing IL-7Rα develop leukemia with features of human T-ALL and sensitivity to ruxolitinib, dactolisib, and venetoclax.T-ALL patients with high levels of wild-type IL7R present with evidence of ongoing, oncogenic-like IL-7R–mediated activation of signaling., Visual Abstract, Tight regulation of IL-7Rα expression is essential for normal T-cell development. IL-7Rα gain-of-function mutations are known drivers of T-cell acute lymphoblastic leukemia (T-ALL). Although a subset of patients with T-ALL display high IL7R messenger RNA levels and cases with IL7R gains have been reported, the impact of IL-7Rα overexpression, rather than mutational activation, during leukemogenesis remains unclear. In this study, overexpressed IL-7Rα in tetracycline-inducible Il7r transgenic and Rosa26 IL7R knockin mice drove potential thymocyte self-renewal, and thymus hyperplasia related to increased proliferation of T-cell precursors, which subsequently infiltrated lymph nodes, spleen, and bone marrow, ultimately leading to fatal leukemia. The tumors mimicked key features of human T-ALL, including heterogeneity in immunophenotype and genetic subtype between cases, frequent hyperactivation of the PI3K/Akt pathway paralleled by downregulation of p27Kip1 and upregulation of Bcl-2, and gene expression signatures evidencing activation of JAK/STAT, PI3K/Akt/mTOR and Notch signaling. Notably, we also found that established tumors may no longer require high levels of IL-7R expression upon secondary transplantation and progressed in the absence of IL-7, but remain sensitive to inhibitors of IL-7R–mediated signaling ruxolitinib (Jak1), AZD1208 (Pim), dactolisib (PI3K/mTOR), palbociclib (Cdk4/6), and venetoclax (Bcl-2). The relevance of these findings for human disease are highlighted by the fact that samples from patients with T-ALL with high wild-type IL7R expression display a transcriptional signature resembling that of IL-7–stimulated pro-T cells and, critically, of IL7R-mutant cases of T-ALL. Overall, our study demonstrates that high expression of IL-7Rα can promote T-cell tumorigenesis, even in the absence of IL-7Rα mutational activation.

TAL1/SCL/TCL5 is a critical transcription factor for hematopoietic stem cell maintenance and regulation of early hematopoiesis. However, aberrant expression of TAL1 in committed T-cell precursors is also directly implicated in the development of T-cell leukemia. Roughly 25 years ago TAL1 was identified in early hematopoietic cells and involved in leukemia. Here, we review the wealth of knowledge gained since then on its physiological roles and mechanisms by which TAL1 ectopic expression contributes to leukemogenesis. We emphasize recent findings that shed light into the intricacies of TAL1 (epi)genetic regulation and the transcription network orchestrated by this major T-cell oncogene. Importantly, an exciting time is coming when data using the mechanistic knowledge accumulated on TAL1 may be used to develop novel anti-leukemia targeted therapies.

Replicative stress (RS) is a cell-intrinsic phenomenon enhanced by oncogenic transformation. Checkpoint kinase 1 (CHK1) is a key component of the ATR-dependent DNA damage response pathway that protects cells from RS by preventing replication fork collapse and activating homologous DNA repair. Taking this knowledge into account, one would predict CHK1 behaves strictly as a tumor suppressor. However, the reality seems far more complex. CHEK1 loss-of-function mutations have not been found in human tumors, and transgenic expression of Chek1 in mice promotes oncogene-induced transformation through RS inhibition. Moreover, CHK1 is overexpressed in various human cancers and CHK1 inhibitors have been developed as sensitizers to enhance the cytotoxicity of DNA damage-inducing chemotherapies. Here, we summarize the literature on the involvement of CHK1 in cancer progression, including our recent observation that CHK1 sustains T-cell acute lymphoblastic leukemia (T-ALL) cell viability. We also debate the importance of identifying patients that could benefit the most from treatment with CHK1 inhibitors, taking T-ALL as a model, and propose possible markers of therapeutic response.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes and is largely driven by the NOTCH/MYC pathway. Yet, additional oncogenic drivers are required for transformation. Here, we identify protein tyrosine phosphatase type 4 A3 (PRL3) as a collaborating oncogenic driver in T-ALL. PRL3 is expressed in a large fraction of primary human T-ALLs and is commonly co-amplified with MYC. PRL3 also synergized with MYC to initiate early-onset ALL in transgenic zebrafish and was required for human T-ALL growth and maintenance. Mass-spectrometry phosphoproteomic analysis and mechanistic studies uncovered that PRL3 suppresses downstream T-cell phosphorylation signaling pathways, including those modulated by VAV1, and subsequently suppresses apoptosis in leukemia cells. Taken together, our studies have identified new roles for PRL3 as a collaborating oncogenic driver in human T-ALL and suggest that therapeutic targeting of the PRL3 phosphatase will likely be a useful treatment strategy for T-ALL.

Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.

Copyright © Ribeiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and re-production in any medium, provided the original author and source are credited., Among kinases that support the survival and turnover of tumor cells, the serine/threonine protein kinase CK2 has been shown to be frequently overexpressed or hyperactivated in solid and hematological malignancies. Our previous work on T-cell acute lymphoblastic leukemia (T-ALL) showed that CK2 maintains leukemia cell viability by phosphorylating and thereby inactivating the tumor suppressor PTEN, which results in hyperactivation of PI3K/AKT signaling. We also demonstrated the potential of using the clinical-grade CK2-specific chemical inhibitor, CX-4945 (Silmitasertib), against primary T-ALL cells. We have now extended these findings to the rare (

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer that arises from clonal expansion of transformed T-cell precursors. In this review we summarize the current knowledge on the external stimuli and cell-intrinsic lesions that drive aberrant activation of pivotal, pro-tumoral intracellular signaling pathways in T-cell precursors, driving transformation, leukemia expansion, spread or resistance to therapy. In addition to their pathophysiological relevance, receptors and kinases involved in signal transduction are often attractive candidates for targeted drug development. As such, we discuss also the potential of T-ALL signaling players as targets for therapeutic intervention.

T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease. The PHF6 gene is frequently targeted by loss-of-function mutations or deletions, with the highest prevalence in TLX1 or TLX3 rearranged T-ALLs. To gain insights into the putative function of PHF6 as a tumor suppressor in the T-cell lineage, we investigated the effects of PHF6 knock down during normal and malignant thymocytes. Notably, we observed broad effects on the investigated transcriptomes suggesting an important role for PHF6 in gene regulation. Furthermore, IL7R was identified as a common transcriptional target that was significantly upregulated upon PHF6 knockdown in both normal and malignant T cells. IL7R encodes a cytokine receptor critically involved in normal thymic development and which also acts as a bona fide oncogene in subset of primary T-ALLs. Thus, loss of PHF6 might further boost oncogenic addiction of leukemic T-cell lymphoblast to IL7-induced JAK-STAT signaling. To further explore the role of PHF6 inactivation in TLX1 driven leukemogenesis in vivo, we performed zebrafish modeling. For this, we generated a stable tg(rag2:TLX1, rag2:GFP) overexpressing as well as a phf6 knock out zebrafish line. These lines were crossed and offspring was monitored for T-ALL formation. Interestingly, three fish out of a cohort of 80 animals developed leukemia between 10 to 18 months of age. These leukemias originated from the thymus, spreaded throughout the whole body and were transplantable. Thus far, no leukemia was detected in PHF6 mutated or TLX1 overexpressing only zebrafish. Leukemic cells obtained from tumors that developed in the PHF6null TLX1rag2-TLX1/GFP animals were subjected to RNA-, ATAC- and H3K27ac ChIP-sequencing to assess the epigenetic status of the IL7R locus. In addition, exome-, and CNV-sequencing was performed to identify somatic lesions that cooperated loss of Phf6 during TLX1 driven T-cell transformation in zebrafish. Furthermore, additional injections of TLX1 in combination with an activating IL7R mutant into phf6 mutant zebrafish are currently ongoing to monitor additional effects on accelerated tumor formation. In conclusion, our data suggest that loss of PHF6 drives TLX1 mediated leukemogenesis, at least in part, by increasing surface IL7R expression. Therefore, we believe that increased addiction to oncogenic JAK-STAT signaling may render PHF6 mutant leukemic cells more sensitive to JAK inhibitors, a notion that we are currently investigating in our TLX1/PHF6 and TLX1/PHF6/IL7R zebrafish models. Citation Format: Siebe Loontiens, Kaat Durinck, Suzanne Vanhauwaert, Lisa Depestel, Mariana L. Oliveira, Givani Dewyn, Charles De Bock, João T. Barata, David Langenau, Jan Cools, Tom Taghon, Pieter Van Vlierberghe, Frank Speleman. PHF6loss drives IL7R oncogene addiction in TLX1 driven T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3696.

Novel TAL1 targets beyond protein-coding genes: identification of TAL1-regulated microRNAs in T-cell acute lymphoblastic leukemia

Interleukin-7 and interleukin-7 receptor are essential for normal T-cell development and homeostasis, whereas excessive interleukin-7/interleukin-7 receptor-mediated signaling promotes leukemogenesis. The protein kinase, casein kinase 2, is overexpressed and hyperactivated in cancer, including T-cell acute lymphoblastic leukemia. Herein, we show that while interleukin-7 had a minor but significant positive effect on casein kinase 2 activity in leukemia T-cells, casein kinase 2 activity was mandatory for optimal interleukin-7/interleukin-7 receptor-mediated signaling. Casein kinase 2 pharmacological inhibition impaired signal transducer and activator of transcription 5 and phosphoinositide 3-kinase/v-Akt murine thymoma viral oncogene homolog 1 pathway activation triggered by interleukin-7 or by mutational activation of interleukin-7 receptor. By contrast, forced expression of casein kinase 2 augmented interleukin-7 signaling in human embryonic kidney 293T cells reconstituted with the interleukin-7 receptor machinery. Casein kinase 2 inactivation prevented interleukin-7-induced B-cell lymphoma 2 upregulation, maintenance of mitochondrial homeostasis and viability of T-cell acute lymphoblastic leukemia cell lines and primary leukemia cells collected from patients at diagnosis. Casein kinase 2 inhibition further abrogated interleukin-7-mediated cell growth and upregulation of the transferrin receptor, and blocked cyclin A and E upregulation and cell cycle progression. Notably, casein kinase 2 was also required for the viability of mutant interleukin-7 receptor expressing leukemia T-cells. Overall, our study identifies casein kinase 2 as a major player in the effects of interleukin-7 and interleukin-7 receptor in T-cell acute lymphoblastic leukemia. This further highlights the potential relevance of targeting casein kinase 2 in this malignancy.

© 2015 Macmillan Publishers Limited. All rights reserved., T-cell acute lymphoblastic leukemia (T-ALL) frequently involves aberrant expression of TAL1 (T-cell acute lymphocytic leukemia 1) and LMO2, oncogenic members of the TAL1 transcriptional complex. Transcriptional activity of the TAL1-complex is thought to have a pivotal role in the transformation of thymocytes and is associated with a differentiation block and self-renewal. The transcription factor Forkhead Box P3 (FOXP3) was recently described to be expressed in a variety of malignancies including T-ALL. Here we show that increased FOXP3 levels negatively correlate with expression of genes regulated by the oncogenic TAL1-complex in human T-ALL patient samples as well as a T-ALL cell line ectopically expressing FOXP3. In these cells, FOXP3 expression results in altered regulation of cell cycle progression and reduced cell viability. Finally, we demonstrate that FOXP3 binds LMO2 in vitro, resulting in decreased interaction between LMO2 and TAL1, providing a molecular mechanism for FOXP3-mediated transcriptional modulation in T-ALL. Collectively, our findings provide initial evidence for a novel role of FOXP3 as a tumor suppressor in T-ALL through modulation of TAL1 transcriptional activity., VF and JvL were supported by a grant from the Dutch Arthritis Foundation (Rheumafonds), and BAC was supported by a fellowship from Fundação para a Ciência e a Tecnologia. This work was supported by grant from the Dutch Arthritis Foundation (Rheumafonds), and a fellowship from Fundação para a Ciência e a Tecnologia.

© The Author(s) 2016. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/, Previous results indicated that miR-146b-5p is downregulated by TAL1, a transcription factor critical for early hematopoiesis that is frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL) where it has an oncogenic role. Here, we confirmed that miR-146b-5p expression is lower in TAL1-positive patient samples than in other T-ALL cases. Furthermore, leukemia T-cells display decreased levels of miR-146b-5p as compared to normal T-cells, thymocytes and other hematopoietic progenitors. MiR-146b-5p silencing enhances the in vitro migration and invasion of T-ALL cells, associated with increased levels of filamentous actin and chemokinesis. In vivo, miR-146b overexpression in a TAL1-positive cell line extends mouse survival in a xenotransplant model of human T-ALL. In contrast, knockdown of miR-146b-5p results in leukemia acceleration and decreased mouse overall survival, paralleled by faster tumor infiltration of the central nervous system. Our results suggest that miR-146b-5p is a functionally relevant microRNA gene in the context of T-ALL, whose negative regulation by TAL1 and possibly other oncogenes contributes to disease progression by modulating leukemia cell motility and disease aggressiveness., These studies were supported by Liga Portuguesa Contra o Cancro (Terry Fox Award) and by Fundação para a Ciência e a Tecnologia (project PTDC/BIM-ONC/1548/2012). N.C.C. received an FCT-SFRH PhD fellowship. R.F. and J.T.B. are supported by FCT investigator Starting and Consolidation grants, respectively.

Naïve FoxP3-expressing regulatory T-cells (Tregs) are essential to control immune responses via continuous replenishment of the activated-Treg pool with thymus-committed suppressor cells. The mechanisms underlying naïve-Treg maintenance throughout life in face of the age-associated thymic involution remain unclear. We found that in adults thymectomized early in infancy the naïve-Treg pool is remarkably well preserved, in contrast to conventional naïve CD4 T-cells. Naïve-Tregs featured high levels of cycling and pro-survival markers, even in healthy individuals, and contrasted with other circulating naïve/memory CD4 T-cell subsets in terms of their strong γc-cytokine-dependent signaling, particularly in response to IL-7. Accordingly, ex-vivo stimulation of naïve-Tregs with IL-7 induced robust cytokine-dependent signaling, Bcl-2 expression, and phosphatidylinositol 3-kinase (PI3K)-dependent proliferation, whilst preserving naïve phenotype and suppressive capacity. Altogether, our data strongly implicate IL-7 in the thymus-independent long-term survival of functional naïve-Tregs, and highlight the potential of targeting the IL-7 pathway to modulate Tregs in different clinical settings., This work was supported by Fundação para a Ciência e Tecnologia (FCT; POCI2010/IC/83068/2007 to RMMV; PTDC/SAU-MIC/109786/2009 to AES), and Gulbenkian Foundation (96526/2009 to JF; P132532/2013 to AES). SLS, ASA, RBF, ARP, PM and SMF received FCT scholarships.

The transcription factor T-cell acute lymphocytic leukemia (TAL)-1 is a major T-cell oncogene associated with poor prognosis in T-cell acute lymphoblastic leukemia (T-ALL). TAL1 binds histone deacetylase 1 and incubation with histone deacetylase inhibitors (HDACis) promotes apoptosis of leukemia cells obtained from TAL1 transgenic mice. Here, we show for the first time that TAL1 protein expression is strikingly downregulated upon histone deacetylase inhibition in T-ALL cells. This is due to decreased TAL1 gene transcription in cells with native TAL1 promoter, and due to impaired TAL1 mRNA translation in cells that harbor the TAL1(d) microdeletion and consequently express TAL1 under the control of the SCL/TAL1 interrupting locus (SIL) promoter. Notably, HDACi-triggered apoptosis of T-ALL cells is significantly reversed by TAL1 forced overexpression. Our results indicate that the HDACi-mediated apoptotic program in T-ALL cells is partially dependent on their capacity to downregulate TAL1 and provide support for the therapeutic use of HDACi in T-ALL. Leukemia (2011) 25, 1578-1586; doi:10.1038/leu.2011.140; published online 7 June 2011

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy that presents with poor prognosis. Treatment relies on the application of aggressive therapies that produce deleterious side-effects, justifying the quest for novel, more efficient and selective molecular targeting agents. Mutations leading to abnormal Notch-1 activity are present in more than half of the T-ALL patients, underscoring the potential therapeutic relevance of targeting Notch-1 inhibition and further reinforcing the need to better comprehend the mechanisms by which Notch-1 drives T cell leukemogenesis. Clinical application of γ-secretase inhibitors to block Notch signaling in T-ALL revealed new challenges that involve improvement of the therapeutic benefit and reduction of intestinal toxicity. Here, we review the latest advances in the development and use of Notch antagonists and summarize the current knowledge on Notch function in T-ALL to understand how it may translate into novel therapeutic strategies that increment the efficiency of Notch inhibition.

Interleukin-7 (IL-7) activates phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, thereby mediating viability, proliferation and growth of T-cell acute lymphoblastic leukemia (T-ALL) cells. Reactive oxygen species (ROS) can be upregulated by growth factors and are known to regulate proliferation and viability. Here, we show that IL-7 upregulates ROS in T-ALL cells in a manner that is dependent on PI3K/Akt/mTOR pathway activity and that relies on both NADPH oxidase and mitochondrial respiratory chain. Conversely, IL-7-induced activation of PI3K signaling pathway requires mitochondrial respiration and ROS. We have previously shown that IL-7-mediated activation of PI3K pathway drives the upregulation of the glucose transporter Glut1, promoting glucose uptake in T-ALL cells. Using phloretin to inhibit Glut function, we demonstrate that glucose uptake is mandatory for ROS upregulation in IL-7-treated T-ALL cells, suggesting that IL-7 stimulation leads to increased ROS via PI3K pathway activation and consequent upregulation of Glut1 and glucose uptake. Overall, our data reveal the existence of a critical crosstalk between PI3K/Akt signaling pathway and ROS that is essential for IL-7-mediated T-ALL cell survival, and that may constitute a novel target for therapeutic intervention.

Gene alterations affecting elements of PI3K signaling pathway do not appear to be sufficient to explain the extremely high frequency of PI3K signaling hyperactivation in leukemia. It has been known for long that PTEN phosphorylation at the C-terminal tail, in particular by CK2, contributes to the stabilization and simultaneous inhibition of this critical tumor suppressor. However, direct evidence of the involvement of this mechanism in cancer has been gathered only recently. It is now known that CK2-mediated posttranslational, non-deleting, inactivation of PTEN occurs in T-ALL, CLL and probably other leukemias and solid tumors. To explore this knowledge for therapeutic purposes remains one of the challenges ahead.

T-cell acute lymphoblastic leukemia (T-ALL), an aggressive and common childhood hematological malignancy, arises from clonal expansion of T-cell progenitors. Autophagy is a homeostatic process characterized by the sequestration of cytoplasmic compartments within double-membrane vesicles (autophagosomes) to promote their degradation. Importantly, autophagy is upregulated during starvation and cellular stress, as a compensatory mechanism to provide nutrients and stress relief. By mitigating stress and allowing cell survival, autophagy may serve as a pro-tumoral mechanism. On the other hand, persistent autophagy can lead to cell death and thereby prevent tumor growth. Interleukin-7 (IL-7), a cytokine produced by the bone marrow and thymic stroma, is essential for normal T-cell development. However, IL-7-mediated signaling can also contribute to leukemogenesis. A majority of T-ALL patients (~70%) expresses the IL-7 receptor and IL-7 accelerates T-ALL progression in vivo and promotes T-ALL cell proliferation, survival and metabolic activation in vitro via PI3K/Akt/mTOR pathway (a master negative regulator of autophagy). IL-7 can also activate MEK/Erk pathway (which has been implicated in promotion of autophagy). Because IL-7 has the ability to activate signaling pathways with potentially opposing roles in autophagy regulation, we explored whether IL-7 impacted on the autophagic process in T-ALL cells and sought to elucidate the molecular mechanisms and functional consequences of IL-7-mediated autophagy regulation under different culture conditions: a) medium with 10% serum - a scenario where cells have optimal growth conditions, b) medium with low (1%) serum - to mimic a milieu with nutrient stress (which may happen in densely populated leukemic niches in vivo), and c) regular medium plus asparaginase (ASNase) - to induce treatment-imposed stress. Using IL-7-responsive T-ALL cell lines and patient-derived xenograft (PDX) samples, we show that, in optimal culture conditions, IL-7 leads to a decrease in LC3-I/-II conversion and a reduction in both LC3 puncta and autophagosome/autolysosome formation, as determined by immunoblot, confocal microscopy, flow cytometry and electron microscopy - indicating that IL-7 inhibits autophagy in T-ALL. Using signaling-specific small molecule inhibitors (UO126 for MEK/Erk; LY294002 and rapamycin for PI3K/Akt/mTOR) we found that IL-7-mediated regulation of autophagy occurs in a complex manner that involves concomitant triggering of both pro- (via MEK/Erk) and anti- (via PI3K/Akt/mTOR) autophagic signaling, with the effects of the latter prevailing over the former. In this scenario, IL-7-mediated viability relies on PI3K/Akt/mTOR pathway and, as expected, autophagy inhibition (using MRT68921) does not prevent the ability of IL-7 to promote leukemia cell survival. In contrast, under serum starvation IL-7 promotes autophagy, and IL-7-mediated leukemia cell viability partially relies on autophagy activation, and strictly requires MEK/Erk activation. Mechanistically, we provide evidence that depending on the culture conditions, IL-7 can balance the relative activation of PI3K/Akt/mTOR and MEK/Erk pathways, inhibiting or facilitating autophagy, in order to consistently promote T-ALL cell viability. We further extended our studies to a therapy-related scenario and found that under ASNase treatment, IL-7 still promotes increased survival of leukemic cells, a possible mechanism of treatment resistance. Functionally, we demonstrate that the IL-7-mediated increase in survival under ASNase treatment is, in part, mediated via activation of autophagy, suggesting that combining ASNase administration with autophagy inhibitors may be an attractive strategy to prevent resistance. In summary, our results indicate that IL-7 makes use of a 'flexible strategy' to promote T-ALL cell viability by activating both pro- and anti-autophagic pathways, which are differentially recruited, depending on the microenvironmental conditions, to prevent tumor cell death. Moreover, our findings strengthen the notion that combination therapies against PI3K/Akt/mTOR and MEK/Erk pathways may constitute a valid therapeutic avenue and highlight the potential of using autophagy inhibitors to prevent microenvironment-induced chemotherapy resistance in T-ALL. Disclosures Barata: Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents.

Background: T-cell acute lymphoblastic leukemia (T-ALL) constitutes an aggressive subset of ALL, the most frequent childhood malignancy. Although risk-adjusted chemotherapeutic regimens are currently extremely effective, their efficacy is associated with significant long-term side effects. Moreover, a significant fraction of the patients still relapse despite intensive chemotherapy, prompting the need for a deeper understanding of T-ALL biology in order to develop novel therapies. Interleukin-7 is a cytokine essential for normal T-cell development, where it has a pivotal role in promoting thymocyte survival via Bcl-2 upregulation. In this normal setting, Bcl-2 is under the control of STAT5 mediated signaling. Previously, we have shown that IL-7 promotes T-ALL expansion in vivo and leukemia cell survival and proliferation in vitro by activating PI3K/Akt/mTOR signaling pathway, consequently leading to p27kip1 downregulation and Bcl-2 upregulation. However, it is also known that T-cell lymphomas arising spontaneously in IL-7 transgenic mice depend on STAT5 activity and that leukemias displaying IL7R gain-of-function mutations are sensitive to JAK and STAT5 inhibitors. Thus, we investigated whether STAT5 could also be involved in the IL-7 pro-leukemia effects in human T-ALL cells. Methods: We used an IL-7-dependent leukemia T-cell line (TAIL7), an IL-7-responsive T-ALL cell line (HPB-ALL, with or without shRNA-mediated STAT5 silencing), primary T-ALL samples collected at diagnosis and patient-derived xenografts (PDX) and treated them with inhibitors of STAT5 (N-((4-Oxo-4H-chromen-3-yl) methylene) nicotinohydrazide) and PIM (AZD1208). Analysis of viability, cell size, cell cycle, surface CD71 and Bcl-2 expression was performed by flow cytometry. Signaling pathway activation, STAT5, PIM1, Bcl-xL, Mcl-1 and cell cycle protein expression was performed by immunoblot analysis. Proliferation was assessed by 3H-Thymidine incorporation. STAT5 ChIP-seq and RNA-seq were performed on TAIL7 cells. ChIP-PCR of histone marks H3K4me3, H3K27me, H3K27ac was performed in TAIL7 and HPB-ALL. Results: IL-7 induces JAK/STAT5 pathway activation in T-ALL cells and STAT5 genetic or pharmacological inactivation prevents IL-7-mediated T-ALL cell viability, growth and proliferation. At the molecular level, STAT5 is required for IL-7-induced downregulation of p27kip1, and upregulation of Cyclin A and TfR/CD71. Surprisingly, STAT5 inhibition does not significantly affect IL-7-mediated Bcl-2 upregulation, suggesting that, contrary to normal T-cells, STAT5 promotes leukemia cell survival via a Bcl-2-independent mechanism. In addition, IL-7-mediated increase in transcription of BCL2, BCL2L1 (Bcl-xL) and MCL1 is not affected by STAT5 silencing. To understand how STAT5 mediates the survival effects of IL-7 in T-ALL cells without affecting BCL2 transcription, we performed STAT5 ChIP-seq together with RNA-seq. Data cross-analysis reveal a diverse IL-7-driven STAT5-dependent transcriptional program in T-ALL cells, which includes transcription of the serine/threonine kinase PIM1. PIM1 is involved in cell cycle regulation and apoptosis, thereby constituting a possible alternative to Bcl-2-dependent prevention of apoptosis. We show that STAT5 silencing prevents IL-7-mediated PIM1 expression and the upregulation of active chromatin marks, H3K4me3 and H3K27ac, at the STAT5 binding region in the PIM1 gene. Notably, pharmacological inhibition of PIM kinase abrogates IL-7-mediated T-ALL cell growth and proliferation, however, without affecting cell survival. In agreement, PIM inhibition does not affect expression of Bcl-2 or Bcl-2 family anti-apoptotic members Bcl-xL and MCL1. Conclusion: Here we present evidence that T-ALL cells may have an alternative wiring of signaling networks downstream of IL-7 to that present in normal T-cells. In contrast to healthy lymphoid cells, IL-7-mediated control of survival of T-ALL cells via STAT5 does not rely on modulation of Bcl-2. Moreover, exploration of STAT5 downstream signaling reveals that PIM1 is required for IL-7-mediated proliferation of human T-ALL cells, indicating that strategies involving the use of PIM kinase small molecule inhibitors may have therapeutic potential against leukemias that rely on IL-7R and STAT5 signaling. Disclosures Barata: Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents.