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Supplementary Methods, Figure Legends, Table Legend

Supplementary Methods

Acute leukemias are systemic malignancies associated with a dire outcome. Because of low immunogenicity, leukemias display a remarkable ability to evade immune control and are often resistant to checkpoint blockade. Here, we discover that leukemia cells actively establish a suppressive environment to prevent immune attacks by co-opting a signaling axis that skews macrophages toward a tumor-promoting tissue repair phenotype, namely the GAS6/AXL axis. Using aggressive leukemia models, we demonstrate that ablation of the AXL receptor specifically in macrophages, or its ligand GAS6 in the environment, stimulates antileukemic immunity and elicits effective and lasting natural killer cell– and T cell–dependent immune response against naïve and treatment-resistant leukemia. Remarkably, AXL deficiency in macrophages also enables PD-1 checkpoint blockade in PD-1–refractory leukemias. Finally, we provide proof-of-concept that a clinical-grade AXL inhibitor can be used in combination with standard-of-care therapy to cure established leukemia, regardless of AXL expression in malignant cells.Significance:Alternatively primed myeloid cells predict negative outcome in leukemia. By demonstrating that leukemia cells actively evade immune control by engaging AXL receptor tyrosine kinase in macrophages and promoting their alternative priming, we identified a target which blockade, using a clinical-grade inhibitor, is vital to unleashing the therapeutic potential of myeloid-centered immunotherapy.This article is highlighted in the In This Issue feature, p. 2659

Supplementary Figures and Figure legends

Supplementary Figure 1. Anti-CD3 stimulation of primary human T-ALLs. Supplementary Figure 2. TCR stimulation by in vivo administration of agonistic monoclonal antibody in a preventive setting inhibits human TCR+ T-ALL development. Supplementary Figure 3. TCR signaling is essential to the anti-leukemic effect of OKT3. Supplementary Table 1. Immunophenotypic and oncogenic characteristics of T-ALL samples.

Supplementary Methods, Tables, figures and references.

Ph+ (

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for ∼20% of ALL cases. Intensive chemotherapy regimens result in cure rates >85% in children and

International audience; Adult "T cell" acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that is associated with poor outcomes, requiring additional therapeutic options. The DNA methylation landscapes of adult T-ALL remain undercharacterized. Here, we systematically analyzed the DNA methylation profiles of normal thymic-sorted T cell subpopulations and 143 primary adult T-ALLs as part of the French GRAALL 2003–2005 trial. Our results indicated that T-ALL is epigenetically heterogeneous consisting of five subtypes (C1-C5), which were either associated with co-occurring DNA methyltransferase 3 alpha (DNMT3A)/isocitrate dehydrogenase [NADP(+)] 2 (IDH2) mutations (C1), TAL bHLH transcription factor 1, erythroid differentiation factor (TAL1) deregulation (C2), T cell leukemia homeobox 3 (TLX3) (C3), TLX1/in cis-homeobox A9 (HOXA9) (C4), or in trans-HOXA9 overexpression (C5). Integrative analysis of DNA methylation and gene expression identified potential cluster-specific oncogenes and tumor suppressor genes. In addition to an aggressive hypomethylated subgroup (C1), our data identified an unexpected subset of hypermethylated T-ALL (C5) associated with poor outcome and primary therapeutic response. Using mouse xenografts, we demonstrated that hypermethylated T-ALL samples exhibited therapeutic responses to the DNA hypomethylating agent 5-azacytidine, which significantly (survival probability; P = 0.001 for C3, 0.01 for C4, and 0.0253 for C5) delayed tumor progression. These findings suggest that epigenetic-based therapies may provide an alternative treatment option in hypermethylated T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy with few available targeted therapies. We previously reported that the phosphatase calcineurin (Cn) is required for LIC (leukemia Initiating Capacity) potential of T-ALL pointing to Cn as an interesting therapeutic target. Calcineurin inhibitors have however unwanted side effect. NFAT transcription factors play crucial roles downstream of calcineurin during thymocyte development, T cell differentiation, activation and anergy. Here we elucidate NFAT functional relevance in T-ALL. Using murine T-ALL models in which Nfat genes can be inactivated either singly or in combination, we show that NFATs are required for T-ALL LIC potential and essential to survival, proliferation and migration of T-ALL cells. We also demonstrate that Nfat genes are functionally redundant in T-ALL and identified a node of genes commonly deregulated upon Cn or NFAT inactivation, which may serve as future candidate targets for T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) arises from T cell precursors and is characterized by expression of many lineage-specific proteins. While T-cell antigen receptor (TCR) signaling and its strength are central for thymocyte development, mature T cell homeostasis and immune responses, their roles in T-ALL remain undetermined. Indeed, in contrast to mouse models, in which absence of TCR or major histocompatibility complex binding does not impact on leukemogenesis, other mouse models suggest that basal or weak signaling drives leukemia development. However, recent reports indicate that strong TCR signaling can be detrimental to leukemic cells. Indeed, sustained/high level TCR signaling, stimulated by antigen or CD3 antibody, is strongly anti-leukemic in both murine T-ALL expressing endogenous or transgenic TCR and diagnostic T-ALL cases. As discussed, further work should address the efficacy of T-ALL therapeutic targeting with either TCR/CD3 antibodies or TCR-directed chimeric antigen receptor T cells.

International audience; Leukemias are frequently characterized by the expression of oncogenic fusion chimeras that normally a rise due to chromosomal rearrangements. Cis-splicing of adjacent genes (cis-SAGe) results in transcription of intergenically-spliced chimeric RNAs (ISCs) in the absence of structural genomic changes, and aberrant ISC expression is now recognized as a potential cancer driver. We performed high-throughput RNA-sequencing of human T-acute lymphoblastic leukemia (T-ALL)samples, and used targeted analysis pipelines to detect fusion chimeras. We identified 55 candidate T-ALL-related ISCs, with a median of 4 per patient. We performed additional in-depth characterization of the NFATC3-PLA2G15 chimera, which was expressed at variable levels in primary T-ALL cases. Experimental analysis revealed that the fusion had lower activity than wild-type NFATC3 in vitro, and that T-ALL blasts with elevated NFATC3-PLA2G15 levels had reducedtranscription of canonical NFAT pathway genes in vivo. Strikingly, we found that high expression of the NFATC3-PLA2G15 chimera in leukemic blasts correlated with aggressive disease biology in murine patient-derived T-ALL xenografts,andpoor prognosis in human T-ALLpatients treated as part of the Francophone multinational GRAALL-2003 and -2005 studies. Our results suggest that ISCs are common in T-ALL, and that expression of specific ISCs may correlate with patient outcome.

Leukemias are frequently characterized by the expression of oncogenic fusion chimeras that normally arise due to chromosomal rearrangements. Intergenically spliced chimeric RNAs (ISC) are transcribed in the absence of structural genomic changes, and aberrant ISC expression is now recognized as a potential driver of cancer. To better understand these potential oncogenic drivers, high-throughput RNA sequencing was performed on T-acute lymphoblastic leukemia (T-ALL) patient specimens (

Background: TAM receptor tyrosine kinases—Tyro3, Axl and Mertk—and their ligands, Gas6 and Pros1, have been extensively studied for their cell-intrinsic pro-oncogenic function in cancer cells, including leukemia. However, much less is known about their indirect impact on tumor growth through their function as modulators of the immune system. In particular, no study has yet explored this aspect in the context of hematological malignancies. Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is the most aggressive human ALL subtype, in particular in adults, where it represents 30% of all ALL cases. The disease responds poorly to standard chemotherapy and has a very high risk of recurrence. Treatment relies on the use of a BCR-ABL1 tyrosine kinase inhibitor (TKI) with or without chemotherapy followed by an allogenic-SCT. Despite this intensive regimen a significant fraction (40%) of adult patients fails to achieve long-term disease-free survival, clearly pointing to an unmet clinical need. Methods: To explore the importance of TAM receptors and their ligands in the immune response against leukemia, we developed a very aggressive syngeneic model of Ph+ B-ALL. In this model, leukemia is driven by the expression of the fusion oncoprotein BCR-ABL1 and the loss of the Arf gene, often altered in human Ph+ ALL. Functional studies were carried out using small-molecule inhibitors and genetic mouse models. The latter allowed us to interrogate the importance of specific TAM receptors in defined immune cellular subsets. Lastly, we used pharmacologic inhibitors to explore the potential therapeutic benefit of targeting TAMR signaling alone or in combination with standard of care treatment (TKI) and checkpoint inhibitors. Results: Our study for the first time shows that Gas6, a high-affinity Axl ligand, promotes the establishment of an immune-suppressive milieu that contributes to the aggressive phenotype associated with Ph+ B-ALL. Importantly, Gas6 is not expressed by leukemic cells but rather produced by bone marrow associated stromal cells. Using genetic approaches, we demonstrate that Gas6 primarily acts through its high-affinity receptor, Axl, specifically on myeloid cells, to inhibit the anti-leukemic immune response. This immune-suppressive effect can be effectively blocked using an orally available selective Axl inhibitor or the genetic deletion of Axl in Csf1r expressing myeloid cells, leading to reduced leukemic burden and significantly prolonged survival of leukemia-challenged mice. In a subset of long-term survivors, progressive increase in PD1 expression limited the antileukemic effects associated with Axl deficiency, an effect that can be efficiently reverted by combination treatment with anti-PD1 checkpoint inhibitor.Mechanistically, the antileukemic effects promoted by Gas6/Axl blockade are mediated by an enhanced inflammatory response, followed by a potent adaptive immune response. This can be further potentiated by combination with standard of care therapy (i.e., TKI and chemotherapy), leading to an unprecedented cure rate of over 70%. This synergistic effect was completely abolished when the combination was evaluated in immune-compromised hosts, further emphasizing the strict immune-dependent nature of this phenotype. Conclusion: Our work uncovers a novel mechanism through which leukemic cells maintain an immune-suppressive environment by co-opting the ability of stromal cells to produce Gas6, a secreted TAM receptor ligand that dampens the ability of innate immune cells to fight leukemia. Therapeutic targeting of the Gas6/Axl axis effectively unleashes the innate immune system by promoting an enhanced proinflammatory response and engagement of a productive adaptive immune response. When combined with TKI or anti-PD1 in vivo, this treatment significantly improves the outcome of leukemia-challenged mice, leading to a remarkable cure rate of over 70%, unprecedented in this agressive model of Ph+ B-ALL. Citation Format: Irene Tirado-Gonzalez, Aleksandra Nevmerzhitskaya, Arnaud Descot, Devona Soetopo, Ewelina Czlonka, Maresa Weitmann, Carolin Wachtel, Julia Slotta-Huspenina, Christine Tran-Quang, Katharina Götze, Emily Alberto, Carla Vanina Rothlin, Jacques Ghysdael, Hind Medyouf. TAM receptors targeting unleashes antileukemic immunity and enables checkpoint blockade leading to eradication of leukemic cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A092.

The calcineurin protein phosphatase is dispensable for BCR-ABL-induced B-ALL maintenance, propagation and response to dasatinib

Cancer onset and progression involves the accumulation of multiple oncogenic hits, which are thought to dominate or bypass the physiologic regulatory mechanisms in tissue development and homeostasis. We demonstrate in T-cell acute lymphoblastic leukemia (T-ALL) that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent T-cell receptor (TCR) signaling has antileukemic properties and enforces a molecular program resembling thymic negative selection, a major developmental event in normal T-cell development. Using mouse models of T-ALL, we show that induction of TCR signaling by high-affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3ϵ chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse- or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of patients with TCR-expressing T-ALL and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. Significance: T-ALLs are aggressive malignant lymphoid proliferations of T-cell precursors characterized by high relapse rates and poor prognosis, calling for the search for novel therapeutic options. Here, we report that the lineage-specific TCR/CD3 developmental checkpoint controlling cell death in normal T-cell progenitors remains switchable to induce massive tumor cell apoptosis in T-ALL and is amenable to preclinical therapeutic intervention. Cancer Discov; 6(9); 972–85. ©2016 AACR. See related commentary by Lemonnier and Mak, p. 946. This article is highlighted in the In This Issue feature, p. 932

Intensive chemotherapy regimens have led to a substantial improvement in the cure rate of patients suffering from T-cell acute lymphoblastic leukemia (T-ALL). Despite this progress, about 15% and 50% of pediatric and adult cases, respectively, show resistance to treatment or relapse with dismal prognosis, calling for further therapeutic investigations. T-ALL is an heterogeneous disease, which presents intrinsic alterations leading to aberrant expression of transcription factors normally involved in hematopoietic stem/progenitor cell development and mutations in genes implicated in the regulation of cell cycle progression, apoptosis, and T-cell development. Gene expression profiling allowed the classification of T-ALL into defined molecular subgroups that mostly reflects the stage of their differentiation arrest. So far this knowledge has not translated into novel, targeted therapy. Recent evidence points to the importance of extrinsic signaling cues in controlling the ability of T-ALL to home, survive, and proliferate, thus offering the perspective of new therapeutic options. This review summarizes the present understanding of the interactions between hematopoietic cells and bone marrow/thymic niches during normal hematopoiesis, describes the main signaling pathways implicated in this dialog, and finally highlights how malignant T cells rely on specific niches to maintain their ability to sustain and propagate leukemia.

Introduction: LIM kinases 1 and 2 (LIMK1/2) are downstream effectors at the crossroads of different signaling pathways implicated in the dynamics of the cytoskeleton via phosphorylation of cofilin family proteins, degradation of the matrix by phosphorylating MT1-MMP and control of the activity of Aurora kinase A. Recently, the oncogenic role of Rho kinases (ROCK) was identified to be constitutively activated by BCR-ABL1, FLT3-ITD and KIT in hematologic malignancies via PI3 kinase and Rho GTPase mediated phosphorylation. Upon activation, ROCK phosphorylates LIMK1/2 leading to inactivation of cofilin by its phosphorylation and polymerization of actin and microtubules and possibly to other biological effects mediated by LIMK1/2, not yet fully understood. Here, we demonstrate synergy of a LIMK1/2 inhibitor with BCR-ABL1 tyrosine kinase inhibitors (TKI) in vitro and in vivo in different models for BCR-ABL1 driven ALL. Materials and Methods: Expression of LIMK1/2 was determined by RT-qPCR and WB in cell lines. Phosphorylation of cofilin was detected by WB. A small molecule inhibitor of LIMK1/2 was tested alone and in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1 positive ALL cell lines TOM-1 and BV-173. Cell viability and IC50 was assessed by MTS assays after exposure to LIMK1/2 inhibitor for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Chou-Talalay model. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) DNA intercalation. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of LIMK1/2 inhibitor. Peripheral blood (PB) nucleated cells from apharesis products of healthy donors obtained after informed consent according to Helsinki declaration were incubated with or without LIMK1/2 inhibitor for 72h, and then enriched for CD34+ cells by immuno-magnetic selection and seeded in triplicate in methylcellulose FCS and cytokines. In vivo experiments were performed in C57Bl/6 mice injected with BCR-ABL-induced B-ALL cells. These were obtained by transduction of CDKN2A-deficient B-cell progenitors with a retrovirus coding for BCR-ABL1 (P185) and GFP, followed by transplantation in sub-lethally-irradiated recipient C57Bl/6 mice. Mice were treated either with LIMK1/2 inhibitor, nilotinib or the combination of both and compared to untreated control mice. Results: Expression of the two isoforms LIMK1 and LIMK2 in TOM-1 and BV-173 cells could be detected by RT-qPCR and at the protein level by WB. IC50 after LIMK1/2 inhibitor exposure alone was 580nM in TOM-1 cells and 1000nM in BV-173 cells. All combination experiments with the LIMK1/2 inhibitor and imatinib, dasatinib, nilotinib and ponatinib yielded synergistic CI for treatment of both TOM-1 and BV-173 cell lines. Cell cycle arrest in the G1/S transition was detected and LIMK1/2 inhibition induced dose dependent apoptosis in TOM-1 and BV-173 cells up to 40% at doses 1% GFP+ cells were detected in PB. Furthermore, nilotinib+LIMK1/2 inhibitor prolonged significantly the survival of mice compared to either nilotinib (p=0.0006) or LIMK1/2 inhibitor alone and untreated mice (p Conclusion: Combination of LIMK1/2 inhibitor with BCR-ABL targeting TKI is synergistic and has significant anti-leukemic activity in BCR-ABL1+ ALL in vitro and in vivo models. Disclosures Braun: CELLIPSE: Research Funding. Prudent:CELLIPSE: Employment. Paublant:CELLIPSE: Employment. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Shire: Research Funding; Servier: Consultancy; Amgen: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Roche: Consultancy. Dombret:CELLIPSE: Research Funding.

Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here we show that homologous immunoglobulin alleles pair in a stage-specific way that mirrors the recombination patterns of these loci. The frequency of homologous immunoglobulin pairing was much lower in the absence of the RAG-1-RAG-2 recombinase and was restored in Rag1-/- developing B cells with a transgene expressing a RAG-1 active-site mutant that supported DNA binding but not cleavage. The introduction of DNA breaks on one immunoglobulin allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent biallelic recombination and chromosome breaks or translocations.

The calcineurin/NFAT signaling pathway is unique to vertebrates and clear genetic evidences show that it plays critical roles in orchestrating the intricate cellular interactions that characterize vertebrate development and morphogenesis. In this setting, the transcriptional regulators of the NFAT family function as molecular integrators of specific calcium signals with other signaling pathways, including MAPkinase, WNT or NOTCH. Deregulation of calcineurin/NFAT signaling and/or abnormal expression of its components have recently been reported in solid tumors of epithelial origin, lymphoma and lymphoid leukemia. Our studies in mouse models of human T-ALL/lymphoma shows that persistent activation of calcineurin/NFAT signaling is pro-oncogenic in vivo and can be efficiently targeted by well-characterized calcineurin inhibitors. We further discuss facts and hypotheses concerning the molecular events that may act upstream and downstream of calcineurin and/or NFAT activation in different type of cancer cells.

International audience; CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .

© 2015 John Wiley & Sons Ltd., Lymphotoxin-mediated activation of the lymphotoxin-β receptor (LTβR; LTBR) has been implicated in cancer, but its role in T-cell acute lymphoblastic leukaemia (T-ALL) has remained elusive. Here we show that the genes encoding lymphotoxin (LT)-α and LTβ (LTA, LTB) are expressed in T-ALL patient samples, mostly of the TAL/LMO molecular subtype, and in the TEL-JAK2 transgenic mouse model of cortical/mature T-ALL (Lta, Ltb). In these mice, expression of Lta and Ltb is elevated in early stage T-ALL. Surface LTα1 β2 protein is expressed in primary mouse T-ALL cells, but only in the absence of microenvironmental LTβR interaction. Indeed, surface LT expression is suppressed in leukaemic cells contacting Ltbr-expressing but not Ltbr-deficient stromal cells, both in vitro and in vivo, thus indicating that dynamic surface LT expression in leukaemic cells depends on interaction with its receptor. Supporting the notion that LT signalling plays a role in T-ALL, inactivation of Ltbr results in a significant delay in TEL-JAK2-induced leukaemia onset. Moreover, young asymptomatic TEL-JAK2;Ltbr(-/-) mice present markedly less leukaemic thymocytes than age-matched TEL-JAK2;Ltbr(+/+) mice and interference with LTβR function at this early stage delayed T-ALL development. We conclude that LT expression by T-ALL cells activates LTβR signalling in thymic stromal cells, thus promoting leukaemogenesis., Grants from Fundação para a Ciência e a Tecnologia (PTDC/SAU-OBD/103336/2008 and PEst-OE/EQB/LA0023/2013), Núcleo Regional Sul da Liga Portuguesa Contra o Cancro (NRS/LPCC-Terry Fox) and Fundação MSD to NRdS; grants from the São Paulo Research Foundation (FAPESP 08/10034-1 and 12/12802-1) to JAY; and Plan Cancer Action 29 to ED. MTF (SFRH/BD/75137/2010), MNG (SFRH/BD/80503/2011), and RKK (SFRH/BPD/70718/2010) were recipients of FCT PhD or postdoctoral fellowships. ABS and JAY are supported by PhD and Productivity Fellowships, respectively, from the Brazilian National Council for Scientific and Technological Development (CNPq). NRdS has been supported by FCT Ciência 2007 and FCT Investigator contracts (IF/00056/2012).

The TEL-JAK2 gene fusion, which has been identified in human leukemia, encodes a chimeric protein endowed with constitutive tyrosine kinase activity. TEL-JAK2 transgenic expression in the mouse lymphoid lineage results in fatal and rapid T-cell leukemia/lymphoma. In the present report we show that T-cell leukemic cells from EμSRα-TEL-JAK2 transgenic mice present an aberrant CD8+ differentiation phenotype, as determined by the expression of stage-specific cell surface markers and lineage-specific genes. TEL-JAK2 transforms immature CD4−CD8− double-negative thymocytes, as demonstrated by the development of T-cell leukemia with full penetrance in a Rag2-deficient genetic background. This disease is similar to the bona fide TEL-JAK2 disease as assessed by phenotypic and gene profiling analyses. Pre-TCR signaling synergizes with TEL-JAK2 to transform immature thymocytes and initiate leukemogenesis as shown by (1) the delayed leukemia onset in Rag2-, CD3ϵ- and pTα-deficient mice, (2) the occurrence of recurrent chromosomal alterations in pre-TCR–deficient leukemia, and (3) the correction of delayed leukemia onset in Rag2-deficient TEL-JAK2 mice by an H-Y TCRαβ transgene that mimics pre-TCR signaling. Although not affecting leukemia incidence and mouse survival, TCRαβ expression was shown to facilitate leukemic cell expansion in secondary lymphoid organs.

The transcription factor hypoxia inducible factor 1 (HIF1), an HIF1alpha-aryl hydrocarbon receptor nuclear translocator (ARNT) dimeric factor, is essential to the cellular response to hypoxia. We described a t(1;12)(q21;p13) chromosomal translocation in human acute myeloblastic leukemia that involves the translocated Ets leukemia (TEL/ETV6) and the ARNT genes and results in the expression of a TEL-ARNT fusion protein. Functional studies show that TEL-ARNT interacts with HIF1alpha and the complex binds to consensus hypoxia response element. In low oxygen tension conditions, the HIF1alpha/TEL-ARNT complex does not activate transcription but exerts a dominant-negative effect on normal HIF1 activity. Differentiation of normal human CD34+ progenitors cells along all the erythrocytic, megakaryocytic and granulocytic pathways was accelerated in low versus high oxygen tension conditions. Murine 32Dcl3 myeloid cells also show accelerated granulocytic differentiation in low oxygen tension in response to granulocyte colony-stimulating factor. Interestingly, stable expression of the TEL-ARNT in 32Dcl3 subclones resulted in impaired HIF1-mediated transcriptional response and inhibition of differentiation enhancement in hypoxic conditions. Taken together, our results underscore the role of oxygen tension in the modulation of normal hematopoietic differentiation, whose targeting can participate in human malignancies.

Signal transducers and activator of transcription 5 (STAT5) A and B are transcriptional regulators that play a central role in cytokine signaling in the hematopoietic lineage and which are frequently activated in a persistent manner in human leukemia/lymphoma, as assessed by their constitutive tyrosine phosphorylation and DNA-binding activity. To study the intrinsic oncogenic properties of persistent STAT5 activation, we generated transgenic mice in which a constitutively activated point mutant of STAT5A, STAT5A(S711F), was expressed at physiological level in their lymphoid compartment. In this model, persistent STAT5 activation is weakly oncogenic, leading to the late emergence of clonal B-cell lymphoma/leukemia at a low incidence. In contrast, STAT5(S711F) was found to cooperate with the loss of function of the p53 tumor suppressor gene to both accelerate disease onset and to skew the large tumor spectrum that normally characterize p53-deficient mice to strongly favor B-cell lymphoma/leukemia. The emergence of STAT5A(S711F)-induced B-cell tumors is associated with the activation of STAT5 tyrosine phosphorylation and DNA-binding activity, indicating that activation of STAT5 oncogenic properties in transgenic STAT5A (TgSTAT5A) (S711F) mice involves the deregulation of STAT5 phosphorylation dynamics.

International audience; FLI-1 is a transcription factor of the ETS family that is involved in several developmental processes and that becomes oncogenic when overexpressed or mutated. As the functional regulators of FLI-1 are largely unknown, we performed a yeast two-hybrid screen with FLI-1 and identified the SUMO E3 ligase PIASxalpha/ARIP3 as a novel in vitro and in vivo binding partner of FLI-1. This interaction involved the ETS domain of FLI-1 and required the integrity of the SAP domain of PIASxalpha/ARIP3. SUMO-1 and Ubc9, the ubiquitin carrier protein component in the sumoylation pathway, were also identified as interactors of FLI-1. Both PIASxalpha/ARIP3 and the closely related PIASxbeta isoform specifically enhanced sumoylation of FLI-1 at Lys(67), located in its N-terminal activation domain. PIASxalpha/ARIP3 relocalized the normally nuclear but diffusely distributed FLI-1 protein to PIASxalpha nuclear bodies and repressed FLI-1 transcriptional activation as assessed using different ETS-binding site-dependent promoters and different cell systems. PIASxalpha repressive activity was independent of sumoylation and did not result from inhibition of FLI-1 DNA-binding activity. Analysis of the properties of a series of ARIP3 mutants showed that the repressive properties of PIASxalpha/ARIP3 require its physical interaction with FLI-1, identifying PIASxalpha as a novel corepressor of FLI-1.

Rearrangement of the FLI-1 locus and ensuing overexpression of FLI-1 protein is an early event in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. When overexpressed in primary erythroblasts, FLI-1 converts erythropoietin (Epo)-induced terminal differentiation into a proliferative response. We found that SLAP, a gene encoding a recently described negative regulator of T-cell antigen receptor function during thymocyte development, is up-regulated both at the RNA and protein levels in FLI-1-transformed erythroblasts. Src-like adaptor protein (SLAP) was found in a specific complex with erythropoietin receptor (EpoR), a cytokine receptor essential to erythroid differentiation. Constitutive expression of SLAP severely impairs hemoglobinization and late survival during Epo-induced terminal differentiation of erythroblasts. This impairment is associated with the specific inhibition of several critical Epo-dependent signaling events, including signal transducer and activator of transcription 5 (STAT5) activation and up-regulation of the expression of the antiapoptotic BCL-X gene. Our data support a model by which FLI-1 inhibits normal erythroid differentiation through the deregulation of genes encoding adaptors/effectors that modify the signaling output of cytokine receptors normally required for terminal differentiation. (Blood. 2003; 102:4555-4562)

TEL is a frequent target of chromosomal translocations in human cancer and an alleged tumor suppressor gene. TEL encodes two isoforms: a major TEL-M1 isoform as well as TEL-M43, which lacks the first 42 amino acid residues of TEL-M1. Both isoforms are potent transcriptional repressors that can inhibit RAS-induced transformation. Here we show that the v-SRC proteintyrosine kinase relieves the repressive activity of TEL-M1, an activity that is associated with the v-SRC-induced delocalization of TEL-M1 from the nucleus to the cytoplasm. TEL-M1 delocalization requires the kinase activity of v-SRC and is not induced by oncogenic RAS or AKT. Cytoplasmic delocalization of TEL-M1 in response to v-SRC critically depends upon its unique amino-terminal domain (SRCD domain) because (i) v-SRC did not inhibit the repressive properties of TEL-M43, nor affected TEL-M43 nuclear localization; (ii) fusion of the first 52 amino acid residues of TEL-M1 to FLI-1, an ETS protein insensitive to v-SRC-induced delocalization, is sufficient to confer v-SRC-induced delocalization to this TEL/FLI-1 chimeric protein. The v-SRC-induced nucleo-cytoplasmic delocalization of TEL-M1 does not involve phosphorylation of the SRCD and does not require TEL self-association and repressive domains. Finally, enforced expression of the v-SRC-insensitive TEL-M43, but not of TEL-M1, inhibits v-SRC-induced transformation of NIH3T3 fibroblasts. These results identify a regulatory domain in TEL that specifically impinges on the subcellular localization of its major TEL-M1 isoform. They, furthermore, indicate that inhibition of TEL-M1 nuclear function is required for v-SRC to induce cellular transformation.

The p16INK4a–RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a–RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a–RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a–RB pathway.

Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic retrovirus whose transcriptional promoter is located in the viral 5' long terminal repeat (LTR). To date, no B-lymphocyte-specific cis-regulatory element has been identified in this region. Since ETS proteins are known to regulate transcription of numerous retroviruses, we searched for the presence in the BLV promoter region of binding sites for PU.1/Spi-1, a B-cell- and macrophage-specific ETS family member. In this report, nucleotide sequence analysis of the viral LTR identified a PUbox located at -95/-84 bp. We demonstrated by gel shift and supershift assays that PU.1 and the related Ets transcription factor Spi-B interacted specifically with this PUbox. A 2-bp mutation (GGAA-->CCAA) within this motif abrogated PU.1/Spi-B binding. This mutation caused a marked decrease in LTR-driven basal gene expression in transient transfection assays of B-lymphoid cell lines, but did not impair the responsiveness of the BLV promoter to the virus-encoded transactivator Tax(BLV). Moreover, ectopically expressed PU.1 and Spi-B proteins transactivated the BLV promoter in a PUbox-dependent manner. Taken together, our results provide the first demonstration of regulation of the BLV promoter by two B-cell-specific Ets transcription factors, PU.1 and Spi-B. The PU.1/Spi-B binding site identified here could play an important role in BLV replication and B-lymphoid tropism.

SummaryImpaired cell migration has been demonstrated in T cell acute lymphoblastic leukemia (T-ALL) cells upon calcineurin inactivation, among other phenotypic traits including increased apoptosis, inhibition of cell proliferation, and ultimately inhibition of leukemia-initiating cell (LIC) activity. Herein we demonstrate that the chemokine receptor CXCR4 is essential to the LIC activity of T-ALL leukemic cells both in NOTCH-induced mouse T-ALL and human T-ALL xenograft models. We further demonstrate that calcineurin regulates CXCR4 cell-surface expression in a cortactin-dependent manner, a mechanism essential to the migratory properties of T-ALL cells. Because 20%–25% of pediatric and over 50% of adult patients with T-ALL do not achieve complete remission and relapse, our results call for clinical trials incorporating CXCR4 antagonists in T-ALL treatment.

International audience; Development of novel therapies is critical for T-cell acute leukae-mia (T-ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T-ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T-ALL cell samples cultured on stromal cells independently of NOTCH activa-tion and maintained their ability to propagate in vivo. Similar results were obtained when T-ALL cells were cultured with ERK1/ 2-knockdown stromal cells or with conditioned medium from MEKi-treated stromal cells. Microarray analysis identified interleu-kin 18 (IL-18) as transcriptionally up-regulated in MEKi-treated MS5 cells. Recombinant IL-18 promoted T-ALL growth in vitro, whereas the loss of function of IL-18 receptor in T-ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL-18R was activated by IL-18 in blast cells. IL-18 circulating levels were increased in T-ALL-xeno-grafted mice and also in T-ALL patients in comparison with controls. This study uncovers a novel role of the pro-inflammatory cytokine IL-18 and outlines the microenvironment involvement in human T-ALL development.

In epithelial and stem cells, lethal giant larvae (Lgl) is a potent tumour suppressor, a regulator of Notch signalling, and a mediator of cell fate via asymmetric cell division. Recent evidence suggests that the function of Lgl is conserved in mammalian haematopoietic stem cells and implies a contribution to haematological malignancies. To date, direct measurement of the effect of Lgl expression on malignancies of the haematopoietic lineage has not been tested. In Lgl1⁻/⁻ mice, we analysed the development of haematopoietic malignancies either alone, or in the presence of common oncogenic lesions. We show that in the absence of Lgl1, production of mature white blood cell lineages and long-term survival of mice are not affected. Additionally, loss of Lgl1 does not alter leukaemia driven by constitutive Notch, c-Myc or Jak2 signalling. These results suggest that the role of Lgl1 in the haematopoietic lineage might be restricted to specific co-operating mutations and a limited number of cellular contexts.

RANTES (regulated upon activation normal T cell expressed and secreted) is a chemoattractant cytokine important in the generation of inflammatory responses and human immunodeficiency virus resistance. In hematopoietic cells, RANTES is over-expressed by type I interferons (IFN-alpha and IFN-beta). The upstream region of the RANTES gene promoter contains a distal low affinity IFN-stimulated response element (ISRE). Specific mutagenesis in this ISRE-like motif abolished the activation of RANTES transcription by type I IFNs. Examination of the ISRE binding factors strongly suggested that signal transducer and activator of transcription (Stat)-2 and p48/IFN-stimulated gene factor 3gamma (ISGF3gamma) are not required for the induction of RANTES by type I IFNs. The specific requirement of Stat-1 was demonstrated using Stat-1-deficient U3A cells. These results revealed a non-classical ISRE/ISGF3 signal transduction pathway for the induction of RANTES by type I IFNs.

ETS proteins form one of the largest families of signal-dependent transcriptional regulators, mediating cellular proliferation, differentiation and tumorigenesis. Most of the known ETS proteins have been shown to activate transcription. However, four ETS proteins (YAN, ERF, NET and TEL) can act as transcriptional repressors. In three cases (ERF, NET and TEL) distinct repression domains have been identified and there are indications that NET and TEL may mediate transcription via Histone Deacetylase recruitment. All four proteins appear to be regulated by MAPKs, though for YAN and ERF this regulation seems to be restricted to ERKs. YAN, ERF and TEL have been implicated in cellular proliferation although there are indications suggesting a possible involvement of YAN and TEL in differentiation as well. Other ETS-domain proteins have been shown to repress transcription in a context specific manner, and there are suggestions that the ETS DNA-binding domain may act as a transcriptional repressor. Transcriptional repression by ETS domain proteins adds an other level in the orchestrated regulation by this diverse family of transcription factors that often recognize similar if not identical binding sites on DNA and are believed to regulate critical genes in a variety of biological processes. Definitive assessment of the importance of this novel regulatory level will require the identification of ETS proteins target genes and the further analysis of transcriptional control and biological function of these proteins in defined pathways.

We previously reported a fusion between TEL and JAK2 in a t(9;12)(p24;p13) chromosomal translocation in childhood acute T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific self-association domain, are fused to the kinase domain of JAK2. TEL-JAK2 exhibits constitutive activation of its tyrosine kinase activity which, in turn, confers growth factor-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line. To elucidate the properties of TEL-JAK2 in primary cells and to create an animal model for TEL-JAK2-induced leukemia, we generated transgenic mice in which the TEL-JAK2 complementary DNA was placed under the transcriptional control of the EmuSRalpha enhancer/promoter. TEL-JAK2 founder mice and their transgenic progeny developed fatal leukemia at 4 to 22 weeks of age. Selective amplification of CD8-positive T cells was observed in blood, lymph nodes, thymus, spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells. Leukemic organs of founder and transgenic progeny contained a monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCRbeta locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that the TEL-JAK2 fusion is an oncogene in vivo and that its expression in lymphoid cells results in the preferential expansion of CD8-positive T cells. (Blood. 2000;95:3891-3899)

The TEL / ETV6 gene is located at 12p13 and encodes a member of the ETS family of transcription factors. Translocated ETS leukemia (TEL) is frequently involved in chromosomal translocations in human malignancies, usually resulting in the expression of fusion proteins between the amino-terminal part of TEL and either unrelated transcription factors or protein tyrosine kinases. We have characterized a t(1;12)(q21;p13) translocation in an acute myeloblastic leukemia (AML-M2). At the protein level, the untranslocated TEL copy and, as a result of the t(1;12) translocation, a fusion protein between TEL and essentially all of aryl hydrocarbon receptor nuclear translocator (ARNT) are expressed. The involvement of ARNT in human leukemogenesis has not been previously described. The ARNT protein belongs to a subfamily of the “basic region helix–loop–helix” (bHLH) protein that shares an additional region of similarity called the PAS (Per, ARNT, SIM) domain. ARNT is the central partner of several heterodimeric transcription factors, including those containing the aryl hydrocarbon (dioxin) receptor (AhR) and the hypoxia-inducible factor 1α (HIF1α). Our results show that the TEL-ARNT fusion protein is the crucial product of the translocation and suggest that interference with the activity of AhR or HIF1α can contribute to leukemogenesis.