Development Of T-cell Leukemia Research Articles

A gain-of-function mutation in microRNA 142 is sufficient to cause the development of T-cell leukemia in mice.

MicroRNAs (miRNAs) play a crucial role in regulating gene expression. MicroRNA expression levels fluctuate, and point mutations and methylation occur in cancer cells; however, to date, there have been no reports of carcinogenic point mutations in miRNAs. MicroRNA 142 (miR-142) is frequently mutated in patients with follicular lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia (CLL), and acute myeloid leukemia/myelodysplastic syndrome (AML/MDS). To understand the role of miR-142 mutation in blood cancers, the CRISPR-Cas9 system was utilized to successfully generate miR-142-55A>G mutant knock-in (Ki) mice, simulating the most frequent mutation in patients with miR-142 mutated AML/MDS. Bone marrow cells from miR-142 mutant heterozygous Ki mice were transplanted, and we found that the miR-142 mutant/wild-type cells were sufficient for the development of CD8+ T-cell leukemia in mice post-transplantation. RNA-sequencing analysis in hematopoietic stem/progenitor cells and CD8+ T-cells revealed that miR-142-Ki/+ cells had increased expression of the mTORC1 activator, a potential target of wild-type miR-142-3p. Notably, the expression of genes involved in apoptosis, differentiation, and the inhibition of the Akt-mTOR pathway was suppressed in miR-142-55A>G heterozygous cells, indicating that these genes are repressed by the mutant miR-142-3p. Thus, in addition to the loss of function due to the halving of wild-type miR-142-3p alleles, mutated miR-142-3p gained the function to suppress the expression of distinct target genes, sufficient to cause leukemogenesis in mice.

MTG16 Variants Provide Context to Notch Signaling to Direct Leukemic Phenotype

Notch signaling guides hematopoietic stem and progenitor cells (HS/PCs) toward distinct cell fates and when dysfunctional, contributes to development of hematopoietic malignancies. Normally, Notch signaling favors T-cell fate and causes attenuated expression of myeloid-specific genes. In contrast, Notch pathway inactivation causes HS/PCs to diverge disproportionately toward a myeloid fate and is accompanied by T-lymphopenia. Mutations that constitutively activate Notch signaling trigger T-cell leukemia, while Notch inactivation is associated with myeloid leukemias. This suggests that Notch-regulated cell fate decisions are context dependent and that determinants downstream of Notch can influence Notch-dependent phenotypes in development and disease.Myeloid Translocation Gene (MTG) 16 binds the intracellular domain of Notch (NICD) and this interaction is necessary to establish CD4+/CD8+ (double positive-DP) T-cell fate. Mtg16 -/- progenitors give rise to GR1+/Mac1+ progeny in response to Notch activation. In ex vivo assays, normal DP T-cell development can be restored to Mtg16 -/- progenitors by enforced expression of MTG16, but not an MTG16 derivative which lacks NICD binding and mimics a naturally occurring Mtg16 splice variant (MTG16c). These findings suggest the status of expressed Mtg16 isoforms can determine lineage allocation in response to Notch signaling and intimates that Notch signaling could trigger alternate phenotypes depending upon the context in which it is activated.To assess impact of the NICD-MTG16 interaction as a determinant of malignant phenotype in the context of constitutive Notch signaling, we created bicistronic retroviral constructs whose NICD and MTG16 expression cassettes were separated by the “self-cleaving” 2A peptide of Foot and Mouth disease virus (FMDV). In each transduced progenitor cell, this insures concurrent stoichiometric expression of NICD and the chosen MTG16 derivative (wild type or NICD binding-deficient). Transduced Mtg16 -/- HS/PCs were then transplanted into irradiated, syngeneic recipients and leukemic phenotype established by flow cytometry. Mice transplanted with Mtg16 -/- cells transduced with vector only fail to reconstitute hematopoiesis in the recipient bone marrow due to a previously described HS/PC defect. Instead, these mice succumb to marrow failure by day 18 post-transplant. However, enforced expression of NICD alone enables survival of Mtg16 -/- progenitors, reconstitution of hematopoiesis and development of T-cell leukemia from transplanted donor elements. This same phenotype was observed when NICD was co-expressed with MTG16, indicating MTG16 is required for normal T-lymphopoiesis but is dispensible for NICD-driven T-cell leukemia. Notably, when Mtg16 -/- progenitors are transduced with NICD and a NICD binding deficient derivative of MTG16, recipients develop T-myeloid bilineal leukemia. This finding suggests that integration of NICD into transcriptional regulatory complexes involving MTG16 can alter the features of leukemic blasts along a continuum from lymphoid to myeloid phenotypes. To gain additional insights, we characterized promoter occupancy and Notch transcription complex (NTC) composition upon expression of MTG16 and its NICD binding deficient derivative. We find both MTG16 forms can exist in the same transcriptional complexes via their NHR2 oligomerization domains and can co-occupy the Notch-responsive Hes1 promoter. Given that NICD-MTG16 binding disrupts interactions between MTG16, histone deacetylases (HDACs) and other co-repressors, these data suggest that leukemic phenotype arising from constitutive Notch signaling may be influenced by combinatorial diversity among MTG16 splice variants and differential integration of the NICD into the NTC. DisclosuresNo relevant conflicts of interest to declare.

Molecular Mechanisms of Notch Signaling in Lymphoid Cell Lineages Development: NF-κB and Beyond.

Notch is a ligand-receptor interaction-triggered signaling cascade highly conserved, that influences multiple lineage decisions within the hematopoietic and the immune system. It is a recognized model of intercellular communication that plays an essential role in embryonic as well as in adult immune cell development and homeostasis. Four members belong to the family of Notch receptors (Notch1-4), and each of them plays nonredundant functions at several developmental stages. Canonical and noncanonical pathways of Notch signaling are multifaceted drivers of immune cells biology. In fact, increasing evidence highlighted Notch as an important modulator of immune responses, also in cancer microenvironment. In these contexts, multiple transduction signals, including canonical and alternative NF-κB pathways, play a relevant role. In this chapter, we will first describe the critical role of Notch and NF-κB signals in lymphoid lineages developing in thymus: natural killer T cells, thymocytes, and thymic T regulatory cells. We will address also the role played by ligand expressing cells. Given the importance of Notch/NF-κB cross talk, its role in T-cell leukemia development and progression will be discussed.

A fully human anti-IL-7R\u03b1 antibody promotes antitumor activity against T-cell acute lymphoblastic leukemia

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.

A Phage Display-Generated Fully Human Anti-IL-7Rα Antibody Shows Anti-Tumor Activity Against T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer that is treated with intensive multi-agent chemotherapy, often leading to long-term side-effects impacting the quality of life of survivors. Despite the therapeutic success in children, relapses still occur in 10-20% of the cases, and adults face a considerably poorer prognosis. Novel, more selective treatments that contribute to reducing toxicities and improving outcome are thus in need.Interleukin 7 (IL-7) and its receptor IL-7Rα promote leukemia development in a majority of T-ALL patients and mutational activation of IL-7Rα, which occurs in around 10% of the cases, associates with very high risk in relapsed disease. Using combinatorial scFv phage display libraries and antibody reformatting we have now generated a fully human IgG1 monoclonal antibody (named B12) against human IL-7Rα. B12 does not display cross-reactivity against the mouse receptor and recognizes both wild type and mutant forms of IL-7Rα naturally expressed in T-ALL cell lines and patient samples, as well as in Ba/F3 cells stably transduced with human, but not mouse, IL-7Rα.Interestingly, molecular dynamics simulations suggest that B12 forms a stable complex with IL-7Rα at a different site from IL-7. Nonetheless, B12 inhibits IL-7/IL-7R-mediated signaling and induces cell death per se in at least some IL-7/IL-7R-reliant T-ALL cell lines (e.g. IL-7-dependent TAIL7 cells and mutant IL7R DND4.1 cells) and patient samples. Using patient-derived xenograft (PDX) samples, HPB-ALL cells and D1 cells overexpressing a mutated gain-of-function form of IL-7Rα, we show that the antibody also promotes antibody-dependent NK-mediated leukemia cytotoxicity in vitro and delays T-cell leukemia development in vivo, reducing tumor burden and promoting mouse survival. Moreover, B12 cooperates with dexamethasone in promoting the death of both dexamethasone-resistant HPB-ALL cells and a dexamethasone-sensitive PDX sample.Notably, B12 is rapidly internalized via clathrin-coated pits to the early endosome, eventually trafficking to the lysosome - an effect that is slightly accelerated in the presence of IL-7. These characteristics render B12 an attractive vehicle for targeted intracellular delivery of a highly cytotoxic warhead. As such, we engineered a B12-mono-methyl auristatin E (MMAE) antibody-drug conjugate (ADC) in which site-specific conjugation of B12 was carried out by reducing inter-chain disulfide bonds and reacting the thiol group of the free cysteines with a Michael acceptor (carbonyl acrylic derivate) linked to a cleavable linker (valine-citrulline) and the drug (MMAE). Tested against different cell lines, primary patient cells and PDX samples, B12-MMAE ADC demonstrates increased leukemia cell killing ability in vitro as compared to the naked antibody.Altogether, our studies serve as a stepping stone towards the development of novel targeted therapeutic strategies in T-ALL and other diseases where IL-7Rα was shown to play a pathological role. DisclosuresAkkapeddi:Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents. Neri:Philochem AG: Equity Ownership. Bernardes:Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents. Barata:Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents.

The time-sequential changes of risk factors for adult T-cell leukemia development in human T-cell leukemia virus-positive patients with rheumatoid arthritis: a retrospective cohort study

Objective: This study aimed to investigate the time-sequential changes of risk factors for adult T-cell leukemia (ATL) development in human T-cell leukemia virus type 1 (HTLV-1)-positive rheumatoid arthritis (RA) patients.Methods: HTLV-1 infection was screened using particle agglutination assay and confirmed via western blotting in 365 RA patients. Twenty-three HTLV-1-positive RA patients were included in the study cohort. Blood samples were obtained from these patients at each observation time point. The values of HTLV-1 proviral load (PVL) and serum soluble IL-2 receptor (sIL2-R), which are risk factors for ATL development, were measured using real-time PCR and enzyme immunoassay, respectively.Results: The study cohort comprised 79 person-years. The median HTLV-1 PVL and sIL2-R values of the HTLV-1-positive RA patients were 0.44 copies per 100 white blood cells (WBCs) and 406 U/mL, respectively. Three HTLV-1-positive RA patients showed a high PVL value. No remarkable changes were observed in the PVL and sIL2-R values during the observation period. However, one elderly HTLV-1-positive RA patient who had a high PVL value developed ATL during treatment with methotrexate and infliximab.Conclusion: A thorough clinical assessment of the risk factors for ATL development may be necessary in daily clinical practice for RA patients in HTLV-1-endemic areas in Japan.

FoxN1-dependent thymic epithelial cells promote T-cell leukemia development.

T-cell acute lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphomas (T-LBL) are aggressive malignancies of thymocytes. The role of thymic microenvironmental cells and stromal factors in thymocyte malignant transformation and T-ALL development remains little explored. Here, using the TEL-JAK2 transgenic (TJ2-Tg) mouse model of T-ALL/LBL, which is driven by constitutive JAK/STAT signaling and characterized by the acquisition of Notch1 mutations, we sought to identify stromal cell alterations associated with thymic leukemogenesis. Immunofluorescence analyses showed that thymic lymphomas presented epithelial areas characterized by keratin (Krt) 5 and Krt8 expression, adjacently to epithelial-free areas negative for Krt expression. Both areas contained abundant laminin (extracellular matrix) and ER-TR7+ (fibroblasts) CD31+ (endothelial) and CD11c+ (dendritic) cells. Besides Krt5, Krt-positive areas harbored medullary thymic epithelial cells (TECs) labeled by Ulex europaeus agglutinin-1. By performing flow cytometry and RNA sequencing analyses of thymic lymphomas, we observed an enrichment in medullary TEC markers in detriment of cortical TEC markers. To assess whether TECs are important for T-ALL/LBL development, we generated TJ2-Tg mice heterozygous for the FoxN1 transcription factor nude null mutation (Foxn1+/nu). Strikingly, in TJ2-Tg;Foxn1+/nu compound mice, both emergence of malignant cells in preleukemic thymi and overt T-ALL onset were significantly delayed. Moreover, in transplantation assays, leukemic cell expansion within the thymus of recipient Foxn1+/nu mice was reduced as compared with control littermates. Since thymopoesis is largely normal in Foxn1+/nu mice, these results indicate that FoxN1 haploinsufficiency in TECs has a more profound impact in thymic leukemogenesis.

HOXA9 Cooperates with Activated JAK/STAT Signaling to Drive Leukemia Development.

Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/STAT5 signaling and HOXA9 overexpression, two events identified as significantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a significant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specific signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells.Significance: The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-positive leukemia cases. Cancer Discov; 8(5); 616-31. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

Effects of expressing human T-cell leukemia virus type 1 (HTLV-I) oncoprotein Tax on DOK1, DOK2 and DOK3 gene expression in mice.

Transgenic mice expressing the tax gene from human T-cell leukemia virus type 1 (HTLV-I) genome developed T-cell leukemia or histiocytic sarcoma after at least 12 months. The transgenic mice showed low expression of the downstream of tyrosine kinase (DOK) family members, DOK1, DOK2 and DOK3, which were recently reported to be tumor suppressor genes. Mice showed low DOK2 expression at 5–6 months of age, before disease onset. The expression of DOK1 and DOK3 was not significantly reduced at any age tested. These results suggest that downregulation of DOK2 by the expression of the viral tax gene is the first step in the development of T-cell leukemia or histiocytic sarcoma.

MiR-125b regulates differentiation and metabolic reprogramming of T cell acute lymphoblastic leukemia by directly targeting A20.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy. Although it has been reported that overexpression of miR-125b leads to T-ALL development, the underlying mechanisms of miR-125b action are still unclear. The goal of this study is to delineate the role of miR-125b in T-ALL development. We found that miR-125b is highly expressed in undifferentiated leukemic T cells (CD4-negative) while its expression is low in differentiated T cells (CD4-positive). Overexpression of miR-125b increased the CD4-negative population in T cells, whereas depletion of miR-125b by miR-125b-sponge decreased the CD4-negative cell population. We identified that A20 (TNFAIP3) is a direct target of miR-125b in T cells. Overexpression of miR-125b also increased glucose uptake and oxygen consumption in T cells through targeting A20. Furthermore, restoration of A20 in miR-125b-overexpressing cells decreased the CD4-negative population in T cell leukemia, and decreased glucose uptake and oxygen consumption to the basal level of T cells transfected with vector. In conclusion, our data demonstrate that miR-125b regulates differentiation and reprogramming of T cell glucose metabolism via targeting A20. Since both de-differentiation and dysregulated glucose metabolism contribute to the development of T-cell leukemia, these findings provide novel insights into the understanding and treatment of T-ALL.

Stem Cell Leukemia: how a TALented actor can go awry on the hematopoietic stage.

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.

Aberrant DNA methylation-induced gene inactivation is associated with the diagnosis and/or therapy of T-cell leukemias

Aberrant hypermethylation of tumor suppressor genes is known to play an important role in the development of many tumors, and aberrant DNA hypermethylation was recently identified in hematologic malignancies, where it is thought to hold relevance in leukemogenesis. Here, we report that there are differences in the DNA methylation patterns seen in normal peripheral blood and two T-cell leukemia cell lines. We identify nine genes (CLEC4E, CR1, DBC1, EPO, HAL-DOA, IGF2, IL12B, ITGA1, and LMX1B) that are significantly hypermethylated in T-cell leukemias cell lines, and suggest that aberrant hypermethylation of these normally unmethylated genes may induce their transcriptional and expressional silencing. Furthermore, we observed that the expression levels of DNMT1 and DNMT3a were significantly decreased by 5-aza-2′-deoxycytidine (5-Aza-dC), which is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells and restore the expression levels of their target genes in Jurkat cells. This result suggests that the overexpression of DNMTs could contribute to the development of T-cell leukemias by inducing hypermethylation of the target genes. Together, our results show that aberrant hypermethylation is an important molecular mechanism in the progression of T-cell leukemias, and thus could prove useful as a prognostic and/or diagnostic marker. Moreover, 5-Aza-dC might be a promising candidate for the treatment of T-cell leukemia.

SKP2 Is Dispensable for Normal T-Cell Development but Required for T-Cell Leukemogenesis

Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer. Despite the significant clinical successes in the treatment of pediatric T-ALL, leukemia relapse, refractory disease and induction failure (around 30% of patients) remain significant clinical problems, which are often life-threatening. ALL remains the second leading cause of childhood death. Thus, more effective, curative therapeutic strategies are much needed, particularly for refractory and relapse T- ALL. Recent advances in understanding the biology and the molecular alterations of acute lymphoblastic leukemias have led to identification of new molecular targets, such as the Notch signaling pathway. Constitutive activation of Notch signaling is involved in more than 50% of human T-ALL, and overexpression of activated Notch induces T-cell leukemia and lymphoma in murine tumor models. However, disruption of Notch signaling by gamma-secretase inhibitors (GSI) failed to fulfill its clinical promise and, overall, the significant advances attained in dissecting the molecular effectors in T-ALL has yet to translate into effective, curative molecular therapies for relapse patients. Furthermore, despite multiple studies on Notch signaling, little is known on the role of its downstream mediators in T-cell ALL.Previous studies in our laboratory demonstrated that Notch1 activation induces transcriptional activation of SKP2, the F-box protein of the SCF E3-ubiquitin ligase complex. SKP2 is the main F-box protein regulating cell cycle, promoting downregulation of the CKIs (p21Cip1, p27Kip1, p57Kip2 and p130) and its overexpression accelerate cell cycle progression in hematopoietic cells. SKP2 overexpression is frequently associated with cancers, in particular lymphomas and leukemias, and correlates with poor prognosis.We found that Skp2 expression is dynamically regulated during T-cell development coinciding with the Notch expression pattern. Moreover, primary thymocytes cultured in vitro, responded to Notch stimulation by the Delta1 ligand increasing their Skp2 expression and their cell cycle status, whereas the loss of SKP2 impaired their ability to mount a proliferative response to IL-7 stimulation. Importantly, we observed that SKP2 expression is increased in T-ALL patient samples and that mice with Notch-induced T-cell leukemia showed 5 fold upregulation of Skp2 expression. Our hypotheses are that Notch activation promotes T-cell leukemogenesis by altering the cell cycle control through upregulation of Skp2, and that selective targeting of SKP2 is a novel, effective therapeutic strategy for childhood T-ALL. To test whether SKP2 is a key downstream mediator of Notch in T-ALL, we transduced oncogenic Notch (ICN; the constitutive intracellular form) in hematopoietic cells lacking SKP2 from Skp2-/- null mice, and in controls and we determine their ability to induce leukemia in irradiated recipients. Loss of SKP2 significantly delayed the development of T-cell leukemia and increased animal survival by 40%. Taken together, these results demonstrate a previously unrecognized role for SKP2 in the initiation and progression of T-ALL and its potential role as a therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Absence Of The E3 Ubiquitin Ligase SKP2 Attenuates Notch-Induced T-Cell Leukemogenesis

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in childhood, accounting for almost 30% of pediatric cancers. In pediatric T-cell acute lymphoblastic leukemia (T-ALL), around 30% of the children still undergo disease relapse, which is associated with poor prognosis. Hence, novel therapeutic strategies for the treatment of T-ALL are necessary, since conventional therapies still fail to cure a significant number of patients. Notch signaling contributes to the regulation of normal T-cells homeostasis, whereas oncogenic, activated form of Notch is present in the majority of the T-ALL cases.Previous studies in our laboratory demonstrated that Notch1 signaling induces transcriptional activation of SKP2, the F-box protein of the SCF E3-ubiquitin ligase complex. SKP2 targets the major cell cycle inhibitors, the CKIs p21Cip1, p27Kip1, p57Kip2 and p130, for proteasome-mediated degradation. Increased Skp2 expression accelerates cell cycle progression in hematopoietic cells, and its overexpression is frequently found in cancers, in particular lymphomas and leukemias, in which is associated with poor prognosis.We found that Skp2 expression is dynamically regulated during T-cell differentiation coinciding with the Notch expression pattern. Moreover, primary thymocytes cultured in vitro responded to Notch stimulation by the Delta1 ligand increasing their Skp2 expression and their cell cycle activity. As anticipated, we found that Skp2 expression is increased in T-ALL patient samples. Our hypothesis is that Notch activation promotes T-cell leukemogenesis by altering the cell cycle control through upregulation of SKP2. To better define the role of SKP2 in T-ALL, we analyzed the Skp2 expression in a murine model of Notch-induced T-cell leukemia. Mice transplanted with stem/progenitor cells transduced with the constitutive active form of Notch (ICN), developed T-ALL and died by week 12 after transplant. Analysis of T-ALL cells revealed a 5 fold upregulation of Skp2 expression compared to controls. Next, we tested whether SKP2 was required for T-ALL development. To this end, we evaluated leukemia development in mice transplanted with SKP2 deficient cells overexpressing ICN. Stem/progenitor cells derived from Skp2+/+, Skp2+/- or Skp2-/- mice were transduced with ICN and then transplanted into WT recipients. Loss of SKP2 significantly delayed the development of T-cell leukemia in transplanted mice and increased their survival by 60% at 12 weeks.Taken together, these results demonstrate a previously unrecognized role for SKP2 in the initiation and progression of T-ALL and its potential role as a therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Identification Of Genes/Signaling Pathways That Predispose To Therapy Related MDS (t-MDS)

Myelodysplastic syndrome (MDS) constitutes a complex set of hematopoietic stem cell diseases that involves defective differentiation into either one, some or all of the hematopoietic lineages. MDS is caused due to the accumulation of mutations in hematopoietic stem/progenitor cells (HSPCs). Therapy related MDS (t-MDS) is a secondary complication associated with prior chemo-radiation therapy for either cancer or non-cancer related diseases and accounts about 20% of the total MDS cases. Genes predisposing t-MDS in response to chemo-radiation therapy remain enigmatic. We combined a retroviral insertional mutagenesis approach with a radiation treatment regimen in the mouse in an attempt to identify novel t-MDS predisposing genes and signaling pathways. To this end, C57BL/6 low-density bone marrow (LDBM) cells were transduced with the SFβ91 EGFP+ retrovirus, which results in a very high level of insertional mutagenesis (dysregulation of genes in the vicinity of the integration site). Transduced LDBM cells were transplanted into BoyJ recipient mice. After confirming successful transplantation of donor cells into recipients, animals were treated 3-4 weeks post transplant with either 0, 0.5 or 2Gy radiation once a day for 3 days. Mice were monitored for aberrations in any of the hematopoietic lineages in both peripheral blood (PB) and bone marrow (BM, intra-femural aspirates) at frequent intervals thereafter. Mice irradiated with 2 Gy exhibited cytotoxic effects on different hematopoietic lineages 1-week post radiation. Currently at 35 weeks post-radiation, 4 mice treated with 0.5 Gy and 1 mouse treated with 2 Gy developed a tMDS/AML-like disease. In these animals, BM was saturated with transduced donor cells (measured by GFPpos cells, ranging from 82-97% in BM of different mice) and mice exhibited anemia, thrombocytopenia, splenomegaly and myeloid blasts in BM at different time points. Additionally, 1 mouse treated with 2 Gy developed T-cell leukemia with 95.5% GFPpos T cells in PB and lymphoid blasts in BM and 1 mouse with 98.7% GFPneg cells in BM, with 75% of contribution from Mac1/Gr1. Mice developing t-MDS/AML like disease were sacrificed and bone marrow (BM) cells were collected for analysis of retroviral integration sites. We performed ligation-mediated PCR (LM-PCR) using retroviral long terminal repeats (LTR) sequence based primers and amplifying into the mouse genomic region to identify the unique insertion sites. We observed unique band patterns for each of the four t-MDS/AML mice indicating that the clones driving the disease are probably unique, which were confirmed by sequencing of LM-PCR products to identify the genome integration sites. Further gene expression analysis will be performed on the genes located around the integration site to identify individual genes and/or signaling pathways that are altered in these mice. Validation of such genes or pathways in mice might facilitate the identification of novel genes that predispose to t-MDS/AML. Disclosures:No relevant conflicts of interest to declare.

The Role of IL7R in the Induction and Maintenance of T-Cell Leukemia/Lymphoma in Vivo

AbstractAbstract 116Interleukin 7 (IL-7) and its receptor, a heterodimer constituted by IL-7Rα (hereafter referred to as IL7R) and γc subunits, are essential for normal T-cell development and homeostasis. While it is known for long that IL7R genetic inactivation leads to severe combined immunodeficiency, only recently we and others have found IL7R gain-of-function mutations in T-cell acute lymphoblastic leukemia (T-ALL). In agreement with an oncogenic role for deregulated IL-7/IL7R signaling, IL-7 transgenic mice develop lymphomas and we have shown that xenotransplant models of human T-ALL rely on microenvironmental IL-7 for tumor acceleration. These studies have highlighted the importance of keeping IL7R-mediated signaling within physiological levels. Indeed, throughout normal T-cell development IL7R expression at the cell surface is tightly controlled and there is correlative evidence, from studies in AKR/J mice, that high IL7R levels in developing thymocytes can promote the development of T-cell leukemia. However, no studies have yet directly evaluated the impact of IL7R on T-cell oncogenesis.In the present study, we used a mouse model in which expression of an IL7R transgene is controlled by a tetracycline responsive promotor (TreIL7R rtTAhuCD2Il7r−/− mice) to analyze the role of IL7R-mediated signaling in T-cell leukemogenesis. Continuous expression of TreIL7R upon doxycycline feeding induced thymus hypertrophy and hyperplasia due to increased size and hyperproliferation of T-cells, which subsequently infiltrated lymph nodes, spleen and bone marrow, ultimately leading to leukemia/lymphoma-associated death. Adoptive transfer of thymic TreIL7R cells to immunodeficient mice confirmed their malignant origin. Surprisingly, tumors developed in recipient animals even in the absence of doxycycline administration, indicating that they eventually become independent of continuous IL7R expression. Remarkably, the tumors mimicked several features of human T-ALL. First, their immunophenotype varied considerably between animals (from CD4 CD8 double-negative to double positive to CD4 or CD8 single positive cells), reflecting the heterogeneity of human disease. Second, similar to the majority of primary T-ALL cases, most tumors displayed hyperactivation of PI3K/Akt pathway, which sometimes associated with absence or decreased PTEN protein expression. Third, the cell cycle inhibitor p27Kip1 was frequently downregulated, a molecular characteristic associated with some T-ALLs.We next sought to determine the contribution of TCR diversity to IL7R-mediated tumorigenesis by crossing mice to the F5 TCR transgenic background. Tumor development in these mice occurred with similar timing and incidence, suggesting that the tumors arise irrespectively of whether a clonal or polyclonal TCR repertoire exists. Finally, the role of Rag recombinase dependent genomic instability in tumor development was assessed by further crossing mice F5 TreIL-7R rtTAhuCD2Il7r−/− mice to Rag1−/− background. Tumor development occurred independently of Rag1 expression, indicating a dominant role for IL7R elicited signals in tumorigenesis.Altogether, our results reveal that continuous IL7R-mediated signaling promotes T-cell tumorigenesis in vivo, providing further indication that IL7R can act as a critical T-cell oncogene. Disclosures:No relevant conflicts of interest to declare.

Spatiotemporal analysis of illegitimate V(D)J deletions in Notch1 and Bcl11b genes (162.14)

Abstract Aberrant V(D)J recombination events in oncogenes and tumor suppressor genes are implicated in development of T-cell leukemias and lymphomas. Previously, others identified hotspots for deletional DNA rearrangements adjacent to cryptic recombination signal sequences in Notch1 and Bcl11b genes in thymus of adult mice with radiation-induced thymic lymphomas. Interestingly, these rearrangements were also found at detectable frequencies in thymus of unirradiated WT animals suggesting that factors in addition to deletions contribute to development of lymphomas, but the frequency of deletions at different developmental stages or in peripheral lymphoid organs was not analyzed. To determine the timing, frequency, and tissue distribution of Notch1 and Bcl11b deletions during development, we are examining tissues harvested at different developmental stages from C57BL/6 mice. We detect these deletions in thymus at all ages examined from fetus to 6 months. Deletions are also detected in spleen, consistent with the presence of T cells bearing deletions in the circulating lymphocyte pool. We are analyzing sequences of deletion junctions for clonal relationships in individual animals and performing broken DNA end analysis for ongoing deletions. We are also using an inducible tetracycline-regulated RAG transgenic mouse system to determine if these rearrangements can occur at all ages with induced RAG expression or are limited to specific developmental windows.

STAT3 Is Required for Notch-Induced Leukemogenesis, and Functions As a Critical Survival Effector in T-Cell Leukemia

Abstract 920The disruption of main signaling mediators of critical leukemia cell functions represents the most promising approach for the development of more effective targeted therapies for acute lymphoblastic leukemia (ALL). Increasing evidence suggests an important combinatorial role for cell autonomous, epigenetic and extrinsic factors in tumorigenesis and tumor progression. However, little is known on how oncogenic and microenvironmental signals are integrated within leukemia T-cells and how they influence T-cell leukemogenesis. Our studies in patients’ leukemia T-cells showed that oncogenic Notch enhances their fitness by regulating the transcription of IL-7Rα, thus increasing the sensitivity of leukemia cells to IL-7, which triggers survival and mitogenic pathways. Interestingly, analysis of the Notch1/CBF-1 complex regulating IL7-Rα transcription show that it contains STAT3, but not STAT5, the main STAT molecule implicated in lymphoid cell signaling. Further analyses showed that STAT3 signaling is active in patients’ T-ALL specimens and in leukemia cells from mice with Notch-induced leukemia. We observed that IL-7 signaling triggers STAT3 phosphorylation and transcriptional activity, regulating the expression of the STAT3 target genes Bcl-xL, survivin, Cyclin D3 and miR-21.We then performed studies using a genetic model of conditional STAT3 deletion in the hematopoietic system. We observed that STAT3 loss-of-function markedly decreased the response of T-lineage cells (including thymocytes) to IL-7 stimulation, even if these cells exhibit functional STAT5 signaling. Importantly, in comparison to wild-type animals, STAT3-null mice did not exhibit defects in common lymphoid progenitors (CLP) in the bone marrow (BM) or in T-cell development, namely on DN thymocyte subsets. Also, no changes were observed in STAT3-null mice in the surface expression or transcriptional levels of IL-7Rα in CLP or thymocytes. To investigate the putative role of STAT3 in leukemia development, we performed the Notch-induced tumor model using BM primitive hematopoietic cells from STAT3-null mice, which could be effectively transduced with the constitutively-active Notch (ICN) construct. We observed that STAT3 deletion significantly inhibited the development of T-cell leukemia, with animals surviving for long periods without evidence of leukemia. Importantly, this was not due to defects in homing or in early engraftment of transplanted cells, which was further documented using intravital microscopy.Finally, pre-clinical studies were performed to evaluate the therapeutic potential of STAT3 pharmacological targeting on leukemia T-cells. STAT3 blockade by the selective inhibitors STATTIC or S3I-201 or the JAK2 inhibitor WP-1066 abrogated STAT3 phosphorylation, inhibiting the transcription of Bcl-xL and Survivin. This inhibition resulted in marked inhibition of leukemia T-cells, which was observed in primary T-ALL cells, in a IL-7-dependent T-ALL line and in leukemia cells from mice carrying ICN-induced leukemia. Mechanistically, we observed that STAT3 blockade results in and massive leukemia cell apoptosis, which is associated with activation of the pro-apoptotic p53(Ser46), and PARP cleavage. Also, STAT3 silencing by shRNA prevented leukemia cell expansion. Strong anti-leukemia synergism was observed between low doses of STATTIC and the frontline therapeutic drug Dexamethasone, or selective PI3K or mTOR inhibitors.In summary, our studies indicate that STAT3 signaling plays an important role in the integration of IL-7 and Notch signals in T-cell leukemia, and unveiled an unexpected role for STAT3 in T-cell leukemogenesis. Finally, our work suggests that the targeted disruption of STAT3 represents a valid therapeutic approach in this cancer. Disclosures:No relevant conflicts of interest to declare.

MTOR complex 2 mediates Notch signaling in thymocyte and T-cell leukemia development (64.8)

Abstract Mammalian target of rapamycin (mTOR) is an important component of the PI3-kinase (PI3K) pathway that regulates cell growth and proliferation. PI3K signaling cascades can be initiated by activation of Notch, a critical regulator for T lineage commitment and leukemia development. Here we show that mTOR complex 2 (mTORC2) is functionally vital as a downstream target of Notch in thymocytes and T-cell acute lymphoblastic leukemia (T-ALL). Conditional deletion of rictor, an essential component of mTORC2, results in impaired proliferation and differentiation of pre-T cells driven by Notch ligation. Moreover, mTORC2 is a primary kinase for Akt S473 and enhances Akt activity in thymocytes. Furthermore, mTORC2 regulates NF-κB activity, such that constitutively active Akt can restore nuclear NF-κB levels in the rictor-depleted pre-T cells, as well as normalizing Notch-initiated proliferation and differentiation. Strikingly, interruption of mTORC2 significantly delays T-ALL induced by Notch1 activating mutation and suppresses infiltration of leukemic cells into the non-lymphoid organs. Our study provides evidence of a key role of mTORC2 in relaying Notch signaling in thymocyte development, and reveals that mTORC2 promotes Notch-driven leukemia progression.

Dosage-Sensitive Role of Growth Factor Independence 1(Gfi1) In the Development of T-Cell Leukemia

AbstractAbstract 706Gfi1 is a transcriptional repressor with important roles in the development and malignant transformation of T-cells. Gfi1 deficient mice have reduced thymic cellularity and show a partial block of differentiation of the double negative (CD4/CD8) pre-T cells into more mature stages. It is also known that Gfi1 is preferably selected as a proviral insertion site in retrovirally induced T-cell tumors in mice, which led to the concept that high-level expression of Gfi1 may be required or may drive T-cell leukemogenesis. This was consistent with findings reporting the emergence of T-cell tumors in transgenic mice over expressing Gfi1. However, lower levels of GFI1 expression was shown to be a strong indicator for worse prognosis in a group of T-ALL patients, although higher levels would have been expected. To find a solution for this discrepancy, we investigated whether Gfi1 dosage may be important in T-cell leukemogenesis or even in normal T-cell development. We have generated a new strain of knockin mice, which express a human GFI1 gene either at the same level as the endogenous wild type Gfi1 gene (Gfi1KI/KI) or at reduced levels (Gfi1KD/KD). We observed that low-level expression of Gfi1 leads to a reduction of thymocyte numbers (3 fold), and neutropenia in bone marrow and blood (p≤0.01). This correlates well with the phenotype of previously described Gfi1 knockout mice (Gfi1KO/KO). Interestingly however, unlike Gfi1KO/KO mice, Gfi1KD/KD animals showed a normal B-cell development, suggesting that low Gfi1 levels disturb T-cell and myeloid development but are sufficient to support the differentiation of B-cells. Next we induced T-ALL in wt, Gfi1KO/KO or Gfi1KD/KD mice by injecting them with a single dose of N-ethyl-N-Nitroso-urea. About 15 % of wt mice developed T-cell tumors with a median latency of 140 days as was expected. Absence of Gfi1 (Gfi1KO/KO) impeded tumor development with only 5 % of mice developing tumors and this within a longer latency period. In contrast, Gfi1KD/KD mice developed tumors with a higher incidence (57%) and shorter latency period (100 days) than wt or Gfi1KO/KO mice (p≤0.01 for incidence and latency). Also Gfi1KD/KD mice developing a tumor showed a 3 fold higher number of blasts in the peripheral blood that wt mice developing T-cell leukemia (p≤0.01). Both the shorter latency period and the higher number of blasts is indicative of a more aggressive disease course in animals with low levels of Gfi1 expression. Interestingly, not only tumors arising in Gfi1KD/KD showed lower level of Gfi1 expression but also T-ALL cells from ENU treated wt mice exhibited a 4–8 fold lower expression level of Gfi1 than normal non-leukemic wt thymocytes suggesting that Gfi1 is downregulated upon development of leukemia. Evasion of apoptosis is one hallmark of leukemia initiation and thus we studied how the different level of Gfi1 might influence apoptotic answer. We observed that Gfi1KO/KO thymocytes were more sensitive to DNA induced cell death than wt thymocytes and underwent 2–3 times more apoptosis (p≤0.01) after DNA damage induction, which might explain why absence of Gfi1 impedes tumor initiation. In contrast Gfi1KD/KD thymocytes showed a similar rate of apoptosis as wt thymocytes. We conclude that reduced levels of Gfi1 are not sufficient to sustain a fully normal pre-T cell development, but protect against apoptosis. Thus, the differentiation block, which is a prerequisite in any leukemia development, and a protection against apoptosis might accelerate T-cell leukemia development in Gfi1KD/KD mice. This would indicate that the role of Gfi1 in the malignant transformation of T-cell is dose dependent and that low level of Gfi1 expression (but not absence) favors leukemia development and this might be one explanation why lower level of Gfi1 is associated with worse prognosis in T-ALL patients. Disclosures:No relevant conflicts of interest to declare.