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Leukemia-initiating/repopulating cells (LICs), also named leukemic stem cells, are responsible for propagating human acute leukemia. Although they have been characterized in various leukemias, their role in T-cell acute lymphoblastic leukemia (T-ALL) is unclear. To identify and characterize LICs in T-ALL (T-LIC), we fractionated peripheral blood cell populations from patient samples by flow cytometry into three cell fractions by using two markers: CD34 (a marker of immature cells and LICs) and CD7 (a marker of early T-cell differentiation). We tested these populations in both in vitro culture assays and in vivo for growth and leukemia development in immune-deficient mice. We found LIC activity in CD7(+) cells only as CD34(+)CD7(-) cells contained normal human progenitors and hematopoietic stem cells that differentiated into T, B lymphoid and myeloid cells. In contrast, CD34(+)CD7(+) cells were enriched in LICs, when compared with CD34(-)CD7(+) cells. These CD34(+)CD7(+) cells also proliferated more upon NOTCH activation than CD34(-)CD7(+) cells and were sensitive to dexamethasone and NOTCH inhibitors. These data show that CD34 and CD7 expression in human T-ALL samples help in discriminating heterogeneous cell populations endowed with different LIC activity, proliferation capacity and responses to drugs.

Optimized gene transfer into human primary leukemic T cell with NOD-SCID/leukemia-initiating cell activity

Understanding the pathways that regulate the human T-cell acute lymphoblastic leukemia (T-ALL) initiating cells (T-LiC) activity has been hampered by the lack of biologic assays in which this human disease can be studied. Here we show that coculture of primary human T-ALL with a mouse stromal cell line expressing the NOTCH ligand delta-like-1 (DL1) reproducibly allowed maintenance of T-LiC and long-term growth of blast cells. Human T-ALL mutated or not on the NOTCH receptor required sustained activation of the NOTCH pathway via receptor/ligand interaction for growth and T-LiC activity. On the reverse, inhibition of the NOTCH pathway during primary cultures abolished in vitro cell growth and in vivo T-LiC activity. Altogether, these results demonstrate the major role of the NOTCH pathway activation in human T-ALL development and in the maintenance of leukemia-initiating cells.

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a chimeric protein expressed in a subset of cases of anaplastic large cell lymphoma (ALCL) for which constitutive expression represents a key oncogenic event. The ALK signaling pathway is complex and probably involves functional redundancy between various signaling substrates of ALK. Despite numerous studies on signaling mediators, the molecular mechanisms contributing to the distinct oncogenic features of NPM-ALK remain incompletely understood. The search for additional interacting partners of NPM-ALK led to the discovery of AUF1/hnRNPD, a protein implicated in AU-rich element (ARE)-directed mRNA decay. AUF1 was immunoprecipitated with ALK both in ALCL-derived cells and in NIH3T3 cells stably expressing NPM-ALK or other X-ALK fusion proteins. AUF1 and NPM-ALK were found concentrated in the same cytoplasmic foci, whose formation required NPM-ALK tyrosine kinase activity. AUF1 was phosphorylated by ALK in vitro and was hyperphosphorylated in NPM-ALK-expressing cells. Its hyperphosphorylation was correlated with increased stability of several AUF1 target mRNAs encoding key regulators of cell proliferation and with increased cell survival after transcriptional arrest. Thus, AUF1 could function in a novel pathway mediating the oncogenic effects of NPM-ALK. Our data establish an important link between oncogenic kinases and mRNA turnover, which could constitute a critical aspect of tumorigenesis.

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells, we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells, whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover, long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage, strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether, these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors, and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis.

Recent efforts have been made to isolate molecular targets that could explain different outcome between histological subtypes of lymphomas and to understand the molecular mechanisms underlying oncogenic events. Using the SSH technique, we compared the transcriptome of 2 cases of Al.K + and ALK - anaplastic large cell lymphoma (ALCL) and of 2 cases of classical Hodgkin's lymphoma (cHL) with opposite behavior. Regarding ALCL, we showed that ALK-positive tumors overexpressed genes involved in different signaling pathways such as activation or signaling of T-cells, regulation of apoptosis, phospholipase Cγ and phosphatidyl inositol-3 Kinase. In addition, the characterization of a specific molecular signature may be of clinical relevance since ALK + tumors generally have a better prognosis than ALK - ones. Similar problems of differential prognosis is observed in cases of cHL, which in addition, may be morphologically and immunologically indistinguishable. Therefore, we applied the same SSH technique to 2 cHL samples from patients with favorable and poor outcome, respectively. Forty-four cDNAs were significantly overexpressed in the poor outcome case. In addition to the defender against death cell I (DAD1) gene, overexpressed clones corresponded mostly to expressed sequence tags (ESTs). Interestingly, the present study identifies new genes which may be involved in the pathogenesis and/or clinical outcome of cHL and deserve further investigations.

Abstract 8 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors in which activating mutations of NOTCH1 occur in over 50% of cases and loss-of-function mutations in FBW7, which degrades activated NOTCH1, occur in 8-16% of cases. We and others have characterized c-MYC as a critical downstream transcriptional target of Notch signaling in this context, and loss of FBW7 also contributes here as intact FBW7 marks c-MYC protein for proteosomal degradation. Inhibition of Notch signaling in T-ALL cells by various means including small molecule gamma-secretase inhibitors (GSI) causes cell cycle/growth arrest and in some cases apoptosis, providing rationale for NOTCH1 as a therapeutic target. The tumor suppressor PTEN is also mutated or lost in up to 20% of T-ALL cases. PTEN activity converts PIP3 to PIP2, and thus loss of PTEN potentiates PI3K signaling. It was observed among human T-ALL cell lines that PTEN loss correlated with resistance to Notch inhibition with GSI, leading to the idea that constitutive PI3K/AKT signaling relieves T-ALL cells of their “addiction” to oncogenic Notch signaling. Since GSI and other NOTCH1 signaling inhibitors are presently under investigation as targeted therapeutic agents for T-ALL and other cancers including breast, brain, colorectal, pancreatic, and melanoma, this idea that PTEN loss could confer resistance to NOTCH1 inhibition raises concern that patients with PTEN-negative (or presumably PIK3CA-mutated) diseases may fail Notch inhibitor therapy. As the studies linking GSI-resistance/Notch-independence to PTEN loss were limited to established cell lines, we sought to address this issue using a well established and genetically defined mouse retroviral transduction/bone marrow transplantation model. Briefly, we generated primary murine T-cell acute leukemias using mutated NOTCH1 retroviruses on both wild-type and PTEN-null backgrounds and tested these tumors for response to GSI. Unexpectedly, we observed these primary murine leukemias to undergo growth arrest and cell size reduction with GSI treatment on both wild-type and PTEN-null backgrounds. We even observed wild-type background tumors which had lost PTEN spontaneously after either serial transplantation or extended culture in vitro to remain sensitive to GSI. Given that most cases of human T-ALL have also lost expression of p16INK4A/p14ARF tumor suppressors either by deletion or silencing, and since p16INK4A/p14ARF are critical regulators at the G1/S cell cycle checkpoint, we also generated primary murine tumors on a Pten, Ink4a/Arf double-null background. Even when both PTEN and Ink4a/Arf were deleted, we still observed the murine tumors to remain sensitive to GSI. To confirm these findings were not unique to murine tumors, we examined a panel of 13 primary human T-ALL cases including 7 PTEN-positive cases (6 of which were NOTCH1 mutated) and 6 PTEN-negative cases (4 of which were NOTCH1 mutated). We found 11 of these 13 cases to be GSI sensitive (6 PTEN-positive, 5 PTEN-negative) as illustrated by decreased proliferation and reduction in cell size. We found only 2 cases to be GSI resistant, including 1 PTEN-positive and 1 PTEN-negative. Our panel contained only one case with FBW7 mutation; this case was PTEN-positive and GSI-sensitive. Thus, in contrast to previous studies with established cell lines, we find in this study using primary human T-ALL samples and genetically defined primary murine leukemias that PTEN loss indeed does not confer resistance to Notch inhibition. Of note, other groups have postulated a role for FBW7 mutation in conferring resistance to Notch inhibition; however, we encountered only 2 GSI-resistant cases in our panel of primary human samples, neither of which was FBW7 mutated. In generating the murine Notch leukemias, we also had the opportunity to compare disease penetrance/latency and clonality among the various genetic backgrounds. We noted that PTEN loss was associated with accelerated disease onset and multiclonal tumors as compared to wild-type, whereas INK4A/ARF loss alone had no such effect. These findings suggest NOTCH1 activation and PTEN loss may collaborate in leukemia induction; however, technical caveats related to potentially enhanced retroviral transduction efficiency of PTEN-null bone marrow progenitors limit this conclusion, and thus more definitive experiments to address this issue are needed. Disclosures: No relevant conflicts of interest to declare.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors in which we described activating mutations of Notch1 to occur in over 50% of cases. As well, others have identified loss-of-function mutations in Sel10/Fbw7 to occur in 8–16% of cases, which also enhance Notch signaling. Notably, inhibition of Notch signaling in these cells induces growth arrest and in some cases apoptosis as well. Subsequent studies have characterized c-myc as a critical downstream target of Notch signaling in this context. More recently, mutations in PTEN (occurring in 17% of cases) were shown to potentiate PI3K/Akt signaling and proposed to confer resistance to Notch signaling inhibition, but then impose “addiction” to PI3K/Akt. Most leukemias likely derive cooperative growth/survival signals from Notch and PI3K/Akt pathways, as evidenced by synergistic effects of gamma-secretase inhibitors (GSI) which block Notch signaling and rapamycin which blocks mTOR downstream of PI3K/Akt. In mouse models, combined activation of c-myc and b-catenin with inactivation of PTEN elicited T-ALL which was devoid of Notch mutations, suggesting Notch signaling coordinately activates c-myc, Wnt, and PI3K/Akt signaling pathways. In studying the mechanism for PI3K/Akt activation in T-ALL with activated Notch signaling, we examined insulin-like growth factor receptor-1 (IGF1R) as a candidate upstream initiator of PI3K/Akt activation. We observed IGF1R expression consistently in T-ALL cells from primary human leukemias and cell lines, as well as in primary mouse leukemias derived experimentally by retroviral transduction of marrow with activated forms of Notch1. In all cases, inhibition of IGF1R signaling either with blocking antibody or small molecule kinase inhibitors resulted in growth suppression and apoptosis of leukemia cells. We further observed that inhibition of Notch signaling either by small molecule GSI or transduction with a dominant negative coactivator, Mastermind, resulted in a 2–3 fold decrease in IGF1R expression. Given that the PI3K/Akt/mTOR pathway is known to be important for growth/survival of T-ALL leukemia cells, we hypothesized that Notch might be potentiating activation of this pathway by upregulating IGF1R expression. In fact, we found leukemia cells with active Notch and higher IGF1R levels showed 20-fold greater sensitivity to IGF-1 ligand induced Akt activation as compared to leukemia cells with inactive Notch and lower IGF1R levels. This enhanced signaling output cannot be explained by Notch suppression of PTEN as this effect was noted in both PTEN wild-type and PTEN null leukemia cells. Additionally, cells with wild type PTEN demonstrated only minimal if any changes in PTEN protein levels after Notch inhibition as measured by a highly quantitative flow cytometry assay. In sum, we have identified IGF-1 signaling as being critical to growth and survival of T-ALL leukemia cells, and provide evidence that Notch may potentiate PI3K/Akt signaling by upregulating expression of IGF1R. These data are of immediate clinical relevance as several IGF1R inhibitors are currently in Phase 3 clinical trial and hopefully will provide additional therapeutic options to refractory/relapsed patients and/or in combination with front-line therapy in newly diagnosed patients.

The TAL1/SCL gene encodes a bHLH (basic Helix-Loop-helix) protein that acts as a master gene in hematopoiesis. The TAL1/SCL gene is also the most frequently activated gene in human T-ALL but the oncogenic transcriptional programs, downstream of TAL1 in human T-ALL, are not well characterized. Using RNA interference to knockdown TAL1 expression, we show that TAL1 regulates both cell proliferation and death of human T-ALL cells. To determine the TAL1 target genes in human T-ALL, we combine TAL1 knockdown and gene expression profiling and show that TAL1 activates and repress a common subset of genes in cell lines. This subset includes known TAL1 target genes but also the NKX3.1 gene that is a homeobox gene, specifically expressed in the prostate epithelium during prostate development and in adulthood. NKX3.1 gene inactivation is one of the earliest events that occur in prostate cancer initiation, defining NKX3.1 as a major tumor suppressor gene of this cancer. TAL1 expression is associated with NKX3.1 activation in human T-ALL cell lines and NKX3.1 is expressed in TAL1 expressing human T-ALL blasts. TAL1 and GATA-3 are specifically bound in vivo to the [−870/−570] region of the human NKX3.1 gene promoter, and ex vivo, TAL1 can either directly binds an E-box [position −738] or be recruited by GATA-3 on a GATA binding site [position −697]. Finally, functional analyses of the NKX3.1 promoter indicate that these binding sites mediate the transcriptional activity of this promoter in T-cell lines. Sequences analysis of the human and mouse NKX3.1 promoters show that the regulatory sequences involved in the TAL1 activation of the human NKX3.1 gene are not conserved in the mouse gene, indicating why the NKX3.1 gene is not expressed in mouse models of TAL1 mediated leukemogenesis. NKX3.1 knockdown shows that NKX3.1 is necessary for the proliferation of TAL1 expressing T-ALL cell lines and NKX3.1 overexpression can complement the proliferation defects associated with TAL1 knockdown in T-ALL cell lines. Microarray analyses show that TAL1 and NKX3.1 regulate a common subset of genes in T-ALL that includes numerous genes encoding proteins known to be involved in T-cell proliferation and/or signaling. Finally, using a new culture system that enables proliferation of primary human leukemic cells, we show that the NKX3.1 gene is specifically activated in human TAL1 expressing T-ALL together with the defined potential TAL1 and/or NKX3.1 target genes. These results characterize NKX3.1 as the first gene directly activated by TAL1 and involved in the TAL1 dependent proliferation of human T-cell Acute Lymphoblastic Leukemia.