T-cell Lymphoblastic Leukemia/lymphoma Research Articles

Oncogenetic landscape of T-cell lymphoblastic lymphomas compared to T-cell acute lymphoblastic leukemia

AbstractIn the latest 2016 World Health Organization classification of hematological malignancies, T-cell lymphoblastic lymphoma (T-LBL) and lymphoblastic leukemia (T-ALL) are grouped together into one entity called T-cell lymphoblastic leukemia/lymphoma (T-LBLL). However, the question of whether these entities represent one or two diseases remains. Multiple studies on driver alterations in T-ALL have led to a better understanding of the disease while, so far, little data on genetic profiles in T-LBL is available. We sought to define recurrent genetic alterations in T-LBL and provide a comprehensive comparison with T-ALL. Targeted whole-exome next-generation sequencing of 105 genes, multiplex ligation-dependent probe amplification, and quantitative PCR allowed comprehensive genotype assessment in 818, consecutive, unselected, newly diagnosed patients (342 T-LBL vs. 476 T-ALL). The median age at diagnosis was similar in T-LBL and T-ALL (17 vs. 15 years old, respectively; p = 0.2). Although we found commonly altered signaling pathways and co-occurring mutations, we identified recurrent dissimilarities in actionable gene alterations in T-LBL as compared to T-ALL. HOX abnormalities (TLX1 and TLX3 overexpression) were more frequent in T-ALL (5% of T-LBL vs 13% of T-ALL had TLX1 overexpression; p = 0.04 and 6% of T-LBL vs 17% of T-ALL had TLX3 overexpression; p = 0.006). The PI3K signaling pathway was significantly more frequently altered in T-LBL as compared to T-ALL (33% vs 19%; p < 0.001), especially through PIK3CA alterations (9% vs 2%; p < 0.001) with PIK3CAH1047 as the most common hotspot. Similarly, T-LBL genotypes were significantly enriched in alterations in genes coding for the EZH2 epigenetic regulator and in TP53 mutations (respectively, 13% vs 8%; p = 0.016 and 7% vs 2%; p < 0.001). This genetic landscape of T-LBLL identifies differential involvement of recurrent alterations in T-LBL as compared to T-ALL, thus contributing to better understanding and management of this rare disease.

Eleven‐marker 10‐color flow cytometric assessment of measurable residual disease for T‐cell acute lymphoblastic leukemia using an approach of exclusion

The study included 269 childhood T-ALL patients treated with a modified-MCP841 protocol. An 11-marker, 10-color MFC-based MRD was performed in bone marrow (BM) samples at the end-of-induction (EOI) and end-of-consolidation (EOC) time-points using Kaluza-version-1.3 software. We studied EOI-MRD in 269 and EOC-MRD in 105 childhood T-ALL patients. EOI-MRD was detectable in 125 (46.5%) samples (median, 0.3%; range, 0.0007-66.3%), and EOC-MRD was detectable in 34/105 (32.4%) samples (median, 0.055%; range, 0.0008-27.6%). Leukemia-associated immunophenotypes (LAIPs) found useful for MRD assessment were dual-negative CD4/CD8 (40.9%), dual-positive CD4/CD8 (23.3%) and only CD4 or CD8 expression (35.8%); dim/subset/dim-negative surface-CD3 (39%), dim/subset/dim-negative/negative CD5 (28.3%), dim/dim-negative/negative/heterogeneous CD45 (44.7%) and co-expression of CD5/CD56 (7.5%). EOI-MRD-positive status was found to be the most-relevant independent factor in the prediction of inferior relapse-free and overall survival. We described an 11-marker 10-color MFC-based highly sensitive MRD assay in T-ALL using an approach of exclusion. The addition of CD4 and CD8 to the pan-T-cell markers in a 10-color assay is highly useful in T-ALL MRD assessment and extends its applicability to almost all T-ALL patients.

Post-induction Measurable Residual Disease Using Multicolor Flow Cytometry Is Strongly Predictive of Inferior Clinical Outcome in the Real-Life Management of Childhood T-Cell Acute Lymphoblastic Leukemia: A Study of 256 Patients.

Background: Measurable/minimal residual disease (MRD) status is suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Contrary to B-cell ALL, reports on T-ALL MRD are limited and mostly based on molecular methods, mainly from developed countries. Multicolor flow cytometry (MFC)-based T-ALL studies are very few. Clinically relevant cut-off levels and ideal time-point for MRD assessment are still inconclusive. In view of lack of T-ALL MRD data from the developing world, we evaluated the prognostic value of MFC-based post-induction (PI)-MRD assessment in T-ALL in the context of standard practice.Methods: We included 256 childhood-T-ALL patients (age < 15 years) treated with a modified-MCP841 protocol, which uses high-dose cytarabine during consolidation, as a part of standard hospital practice. MRD was studied using 10-color 11-antibody MFC with any level of detectable disease being considered positive. Post-induction (PI)-MRD was available in all patients, and post-consolidation (PC) MRD was available mostly in PI-MRD-positive patients (n = 88).Results: Three years cumulative-incidence-of-relapse (3years-CIR) in PI-MRD-positive patients was inferior to negative patients (46.3% vs. 18.4%). The median relapse-free-survival (RFS), event-free-survival (EFS) and overall-survival (OS) with hazard ratio (HR) of PI-MRD-positive patients were 21.4 months vs not reached (p < 0.0001, HR-4.7), 21.6 months vs. not-reached (p = 0.0003, HR-2.01) and 37.3 months vs. not reached (p = 0.026, HR-1.64) respectively. RFS, EFS and OS of patients with PI-MRD<0.01% (n = 17) were as inferior as PI-MRD ≥ 0.01% in comparison with MRD-negative patients with HR of 4.7 (p < 0.0001), 2.45 (p = 0.0003), and 2.5 (p = 0.029), respectively. Three-years-CIR of patients with hyperleukocytosis (≥100 × 109/L) was also higher (50.5 vs. 27.6%) with inferior RFS, EFS, and OS. Among PI-MRD-positive patients, 3years-CIR, RFS, EFS, and OS of PC-MRD-positive were also inferior to that of negative patients. On multivariate analysis any-level detectable PI-MRD and hyperleukocytosis remained independently associated with inferior RFS, EFS, and OS. A combination of PI-MRD-positive status and hyperleukocytosis identified the patients with the worst clinical outcomes.Conclusion: Detectable PI-MRD using MFC was found to be the strong predictive factor of inferior clinical outcome in T-ALL patients. The combination of PI-MRD status and hyperleukocytosis provides the most influential tool for the management of T-ALL in resource constrained settings from developing world.

Current research status of diagnosis, treatment and prognostic factors of T-lymphoblastic lymphoma

T-lymphoblastic lymphoma(T-LBL) is a rare and highly aggressive lymphoma, with poor prognosis. It occurs mostly in children and male adolescents, and its main symptoms are mediastinal mass, lymphadenectasis, bone marrow invasion and central nervous system(CNS) invasion, etc.. The diagnosis of T-LBL is mainly based on lymph node biopsy, bone marrow cell morphology, immunology, cytogenetics and gene detection. After definitive diagnosis of T-LBL, the patients should be treated with extensive combined chemotherapy and sequential hematopoietic stem cell transplantation (HSCT) or maintenance treatment. There are many factors influencing the prognosis of T-LBL patients, such as age, bone marrow invasion, CNS invasion, disease stage and gene mutations. This article intends to introduce the development of diagnosis, therapy and prognostic factors of T-LBL to provide reference for the diagnosis and therapy of T-LBL. Key words: Precursor T-cell lymphoblastic leukemia-lymphoma; Genetics; Immunology; Diagnosis; Therapy

Loss of wild-type Kras promotes activation of all Ras isoforms in oncogenic Kras-induced leukemogenesis.

Despite the well-established role of oncogenic RAS in promoting tumor formation, whether and how wild-type (WT) Ras inhibits tumorigenesis under physiological conditions remains controversial. Here, we show that in a fraction of endogenous oncogenic Kras-induced hematopoietic malignancies, including acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) and myeloproliferative neoplasm (MPN), WT Kras expression is lost through epigenetic or genetic mechanisms. Using conditional Kras(G12D/-) mice, we find that WT Kras deficiency promotes oncogenic Kras-induced MPN, but not T-ALL, in a cell-autonomous manner. Loss of WT Kras rescues oncogenic Kras-mediated hematopoietic stem cell depletion and further enhances granulocyte-macrophage colony-stimulating factor signaling in myeloid cells expressing oncogenic Kras. Quantitative signaling studies reveal that oncogenic Kras but not oncogenic Nras leads to cross-activation of WT Ras, whereas loss of WT Kras further promotes the activation of all Ras isoforms. Our results demonstrate the tumor suppressor function of WT Kras in oncogenic Kras-induced leukemogenesis and elucidate its underlying cellular and signaling mechanisms.

Wild-Type Kras Inhibits NrasQ61R/+-Induced Leukemias

Ras proteins belong to the super family of small GTPases. Although RAS mutations are oncogenic, it remains highly controversial whether wild-type (WT) RAS facilitates or inhibits tumorigenesis in the presence of oncogenic RAS. Previous studies suggest that this might depend on cell type, specific RAS isoforms, and/or stage of tumorigenesis (e.g. tumor initiation versus tumor progression). Recently, we found that deletion of WT Kras in oncogenic Kras mice promotes myeloidproliferative neoplasm (MPN) in a cell-autonomous manner. This is through promoting activation of all Ras isoforms and thus enhancing cytokine signaling in vivo. However, it remains unknown whether WT Kras plays a similar role in oncogenic Nras-induced leukemogenesis. To address this issue, we conditionally down-regulated WT Kras expression in a novel leukemia model induced by endogenous NrasQ61R/+. Detailed characterization of NrasQ61R allele reveals that NrasQ61R/+ displays an intermediate leukemogenic activity between NrasG12D/+ and NrasG12D/G12D. NrasQ61R/+ mice developed MPN (100%) and acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) (~10%) at the moribund stage with a median survival of 197 days. Therefore, NrasQ61R/+ mice provide an excellent experimental system to test genetic lesions promoting or inhibiting oncogenic Nras-induced leukemogenesis. We observed that 3-weeks after activation of NrasQ61R/+ and deletion of WT Kras, the double mutant mice displayed more severe MPN phenotypes than NrasQ61R/+ mice, as demonstrated by more prominent hepatosplenomegaly and further expanded myeloid compartment in bone marrow and spleen. Consistent with these MPN phenotypes, NrasQ61R/+; Kras-/- myeloid progenitor and precursor cells showed prolonged and hyperactivated GM-CSF signaling compared to NrasQ61R/+ cells. In addition, NrasQ61R/+; Kras-/- mice displayed further expanded common lymphoid progenitor compartment and a less differentiated phenotype in thymic T-cells than NrasQ61R/+ mice. In NrasQ61R/+; Kras-/- mice, early thymic T cell progenitors demonstrated enhanced cell proliferation and stronger ERK1/2 signaling compared to NrasQ61R/+ cells. Consequently, NrasQ61R/+; Kras-/- mice survived significantly shorter than NrasQ61R/+ mice (median survival: 97 days; P &lt; 0.001) and 100% of them developed both MPN and T-ALL at the moribund stage. Preliminary results from bone marrow transplantation assay indicate that loss of WT Kras expression promoted NrasQ61R/+-induced MPN and T-ALL in a cell-autonomous manner. Taken together, our results suggest that loss of WT Kras promotes leukemogenesis in NrasQ61R/+ mice. We are currently studying on the underlying mechanisms. Disclosures No relevant conflicts of interest to declare.

Overexpression of the shortest isoform of histone demethylase LSD1 primes hematopoietic stem cells for malignant transformation

AbstractRecent investigations indicate that epigenetic regulators act at the initial step of myeloid leukemogenesis by forming preleukemic hematopoietic stem cells (HSCs), which possess the increased self-renewal potential but retain multidifferentiation ability, and synergize with genetic abnormalities in later stages to develop full-blown acute myeloid leukemias. However, it is still unknown whether this theory is applicable to other malignancies. In this study, we demonstrate that lysine-specific demethylase 1 (LSD1) overexpression is a founder abnormality for the development of T-cell lymphoblastic leukemia/lymphoma (T-LBL) using LSD1 transgenic mice. LSD1 expression is tightly regulated via alternative splicing and transcriptional repression in HSCs and is altered in most leukemias, especially T-LBL. Overexpression of the shortest isoform of LSD1, which is specifically repressed in quiescent HSCs and demethylates histone H3K9 more efficiently than other isoforms, increases self-renewal potential via upregulation of the HoxA family but retains multidifferentiation ability with a skewed differentiation to T-cell lineages at transcriptome levels in HSCs. Transgenic mice overexpressing LSD1 in HSCs did not show obvious abnormalities but developed T-LBL at very high frequency after γ-irradiation. LSD1 overexpression appears to be the first hit in T-cell leukemogenesis and provides an insight into novel strategies for early diagnosis and effective treatment of the disease.

Downregulating Notch Signaling in KrasG12D/+ Mice Inhibits Both T-Cell Leukemia and Myeloproliferative Neoplasm in a Cell-Autonomous Manner

Notch signaling is implicated in diverse functions in hematopoiesis, including stem cell maintenance, cell fate specification, cell proliferation, and apoptosis. Aberrant Notch signaling is associated with the pathogenesis of various hematopoietic malignancies. NOTCH1 mutations that lead to elevated intracellular Notch1 activity are identified in 50-60% of patients with acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) and in 100% of oncogenic Ras-induced T-ALL in mice. Although Notch1 mutations contribute to the malignant transformation of normal T-cells to T-cell leukemia/lymphoma initiating cells in the KrasG12D/+-induced T-ALL model, it remains unclear whether Notch1 signaling is necessary for T-ALL genesis. Moreover, it remains controversial whether loss of Notch signaling promotes myeloproliferative neoplasms (MPN) in a cell-autonomous manner.To address these questions, we used genetic approaches to downregulate Notch signaling in KrasG12D/+ mice, which develop both T-ALL and MPN. Downregulation of Notch signaling in hematopoietic cells is achieved through Mx1-Cre–mediated conditional expression of Rosa26-GFP-dnMAML1, which inhibits canonical Notch signaling, or conditional knockout of Pofut1, which catalyzes O-fucosylation of Notch receptors and modulates Notch receptor ligand interactions. We found that either overexpression of dnMAML1 or deletion of Pofut1 significantly enhanced MPN phenotypes and shortened the survival of KrasG12D/+ mice. However, several pieces of evidence suggest that downregulation of Notch signaling in non-hematopoietic cells might influence MPN development. First, 100% of compound mice developed atopic dermatitis-like disease that is shown to promote MPN in a cell non-autonomous manner. Second, ~30% of endothelial cells were GFP-positive (expressing dnMAML1) in KrasG12D/+; Rosa26GFP-dnMAML1/+ mice.To determine whether downregulating Notch signaling prevents T-ALL and/or promotes MPN in a cell-autonomous manner, we transplanted the same number of KrasG12D/+, KrasG12D/+; Rosa26GFP-dnMAML1/+, or KrasG12D/+; Pofut1-/- bone marrow cells (CD45.2+) along with congeneic competitor cells (CD45.1+) into lethally irradiated mice (CD45.1+). As expected, inhibiting Notch signaling significantly blocked T-cell development and completely prevented T-ALL development in recipients; T-ALL that developed in a fraction of recipient mice were derived from rare donor cells that expressed oncogenic Kras and preserved intact Notch signaling. Surprisingly, we found that the percentage of donor-derived myeloid cells was significantly lower in recipients transplanted with KrasG12D/+; Rosa26GFP-dnMAML1/+ or KrasG12D/+; Pofut1-/- bone marrow cells and consequently none of them developed donor-derived MPN-like disease. In contrast, ~20% of the recipient mice transplanted with KrasG12D/+ cells developed a lethal, donor-derived MPN (P=0.02). Because the hematopoietic stem cell (HSC) frequency was significantly lower in KrasG12D/+; Rosa26GFP-dnMAML1/+ bone marrow than that in KrasG12D/+ bone marrow, we investigated whether the absence of the MPN was due to the reduced HSC reconstitution in recipients. To normalize for HSC numbers, we transplanted lethally irradiated mice with same number of KrasG12D/+ or KrasG12D/+; Rosa26GFP-dnMAML1/+ splenocytes, which contained similar numbers of HSCs mobilized from the bone marrow. Consistent with our previous observation, only 1 out of 12 recipient mice with KrasG12D/+; Rosa26GFP-dnMAML1/+ cells developed a donor-derived (KrasG12D/+; Rosa26GFP-dnMAML1/+) MPN disease, while 6 out of 12 recipient mice with KrasG12D/+ cells died with donor-derived MPN (P=0.02). Together, our results indicate that blocking Notch signaling inhibits both T-ALL and MPN development in a cell-autonomous manner. We are currently working on the underlying mechnisms. DisclosuresNo relevant conflicts of interest to declare.

Alternative Super-Enhancer States Determine MYC Sensitivity to Notch and Brd4 Inhibitors in T Lymphoblastic Leukemia/Lymphoma

Gain-of-function NOTCH1 mutations are oncogenic drivers in a high fraction of T-cell lymphoblastic leukemia/lymphoma (T-LL). These mutations variously cause increased production or stabilization of the free intracellular domain of NOTCH1, which regulates gene expression by forming a transcription complex with the DNA-binding factor RBPJ and coactivators of the MAML family. Using expression profiling and ChIP-seq, we have shown that NOTCH1/RBPJ complexes activate most target genes by binding to super-enhancers, large regulatory elements that switch on transcription through long-range interactions with gene promoters. MYC is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates MYC is unknown. To understand which regulatory element(s) regulate MYC expression, we used chromatin conformation capture (3C) assays to test the interaction between putative enhancer(s) and the MYC promoter in T-LL cell lines, and reporter gene assays to confirm enhancer function of candidate sites. We identified a distal site located >1 Mb 3’ of human and murine MYC termed the Notch-dependent MYC enhancer (NDME) that binds Notch transcription complexes and physically interacts with the MYC proximal promoter. An ~1 kb DNA fragment containing this site activates a luciferase reporter gene in a Notch-dependent fashion in T-LL cells but not in heterologous cell types. The Notch binding site lies within a large enhancer region (>600 kb in breadth) containing multiple discrete H3K27ac peaks. Remarkably, acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer region and the MYC promoter that correlate with NOTCH1/RBPJ complex binding and MYC expression. T-LL cells selected for resistance to gamma-secretase inhibitors (GSIs) exhibit epigenetic silencing of the NDME and loss of NDME looping interactions with the MYC promoter, yet maintain MYC expression. 3C analysis of GSI resistant cells shows preferential interaction between the MYC promoter and a more 3’ enhancer element recently described as a BRD4-dependent regulator of MYC expression in acute myeloid leukemia cells. In line with this observation, BRD4 antagonists are potent inhibitors of MYC expression in GSI resistant T-LL cells but not GSI-sensitive cells. We also studied a case of Notch-mutated early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL). ChIP-Seq analysis of the leukemic blasts revealed an “AML-like” MYC enhancer chromatin state, and as predicted from our analysis of cell lines, the blasts rapidly down-regulated MYC in response to BRD4 inhibitor but not in response to GSI. These findings suggest that specific MYC chromatin states predict responsiveness to Notch and BRD4 inhibitors, and provide a rationale for use of Notch and BRD4 inhibitor combinations in Notch-mutated leukemias. DisclosuresNo relevant conflicts of interest to declare.

Abstract A56: Loss of wild-type Kras promotes oncogenic Kras-induced leukemogenesis

Abstract Although the Ras genes have long been established as proto-oncogenes, substantial evidence indicates that wild-type (WT) Ras genes act as tumor suppressors in a few solid tumor models. However, recent study by Shannon group discovers that loss of WT Nras does not further promote oncogenic Nras-induced leukemias (Xu et al., 2013), raising the possibility that the earlier finding of WT Ras genes as tumor suppressors restricts to specific Ras isoform(s) and/or specific cell types. Here, we show that in mouse cell lines established from oncogenic Kras-induced acute T-cell lymphoblastic leukemia/lymphoma (T-ALL), WT Kras expression is absent or significantly downregulated due to deletion or epigenetic silencing of the locus. Moreover, WT Kras allele is deleted in ∼30% of primary KrasG12D T-ALL samples but not in pre-leukemia specimens. Our results are consistent with a recent study that WT KRAS allele is deleted in early-stage progenitor ALL patients with an oncogenic KRAS mutation (Zhang, et al., 2012), suggesting that loss of WT Kras expression is a pathological mechanism contributing to T-ALL progression. To test our hypothesis, we generated compound mice carrying a conditional KrasG12D allele, a floxed WT Kras allele and the Mx1-Cre allele. In the absence of pIpC induction, the leaky expression of Cre is sufficient to induce both oncogenic KrasG12D activation and WT Kras deletion in ∼50% of HSCs. We find that KrasG12D/- mice have a much shorter life span than KrasG12D/+ mice and display more severe leukemia phenotypes, including splenomegaly, leukocytosis, thrombocytopenia, and increase of monocytic cells in peripheral blood and spleen. Bone marrow transplantation experiment confirms that loss of WT Kras promotes oncogenic Kras-induced leukemias in a cell-autonomous manner. Further studies demonstrate that loss-of-WT Kras attenuates KrasG12D HSC depletion and enhances GM-CSF signaling in KrasG12D hematopoietic stem/progenitor cells (HSPCs). We are continuously working on the underlying mechanisms. Taken together, our study elucidates the cross-talk between WT and oncogenic Kras in leukemogenesis and provides mechanistic insights of the WT Kras function as a tumor suppressor. Citation Format: Yuan-I Chang, Alisa Damnernsawad, Guangyao Kong, Yangang Liu, Qiang Chang, Jing Zhang. Loss of wild-type Kras promotes oncogenic Kras-induced leukemogenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A56. doi: 10.1158/1557-3125.RASONC14-A56

Notch1 Gene Mutations Target KRAS G12D-expressing CD8+ Cells and Contribute to Their Leukemogenic Transformation

Acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive hematopoietic malignancy affecting both children and adults. Previous studies of T-ALL mouse models induced by different genetic mutations have provided highly diverse results on the issues of T-cell leukemia/lymphoma-initiating cells (T-LICs) and potential mechanisms contributing to T-LIC transformation. Here, we show that oncogenic Kras (Kras G12D) expressed from its endogenous locus is a potent inducer of T-ALL even in a less sensitized BALB/c background. Notch1 mutations, including exon 34 mutations and recently characterized type 1 and 2 deletions, are detected in 100% of Kras G12D-induced T-ALL tumors. Although these mutations are not detected at the pre-leukemia stage, incremental up-regulation of NOTCH1 surface expression is observed at the pre-leukemia and leukemia stages. As secondary genetic hits in the Kras G12D model, Notch1 mutations target CD8(+) T-cells but not hematopoietic stem cells to further promote T-ALL progression. Pre-leukemia T-cells without detectable Notch1 mutations do not induce T-ALL in secondary recipient mice compared with T-ALL tumor cells with Notch1 mutations. We found huge variations in T-LIC frequency and immunophenotypes of cells enriched for T-LICs. Unlike Pten deficiency-induced T-ALL, oncogenic Kras-initiated T-ALL is not associated with up-regulation of the Wnt/β-catenin pathway. Our results suggest that up-regulation of NOTCH1 signaling, through either overexpression of surface NOTCH1 or acquired gain-of-function mutations, is involved in both T-ALL initiation and progression. Notch1 mutations and Kras G12D contribute cooperatively to leukemogenic transformation of normal T-cells.

Kruppel-Like Factor, Klf9, Promotes Proliferation of Notch-Independent Precursor T-Cell Lymphoblastic Lymphomas

AbstractAbstract 3496Notch1 signaling is required at multiple stages of normal T-lymphocyte development. Notch1 is a transmembrane receptor that is physiologically activated when Notch ligands induce conformational changes that allow Notch1 cleavage by the intramembranous γ-secretase complex, releasing active intracellular Notch1 (ICN1) fragment from the plasma membrane. Activating NOTCH1 mutations are very frequent in human and mouse T-cell lymphoblastic leukemia/lymphoma (T-LL). Typically, these mutations promote ligand-independent Notch1 cleavage by γ-secretase or increase ICN1 stability by truncating the C-terminal PEST domain. Understandably, much effort has focused on elucidating mechanisms of normal and oncogenic Notch1 signaling. However, some studies suggest that the absence of NOTCH1 mutations portends a worse prognosis for human T-LL. Therefore, we set out to define signals that promote proliferation and survival of T-LL cells lacking activated Notch1. We used Western blotting to detect γ-secretase cleaved ICN1 protein in a cohort of 35 primary T-LLs that developed spontaneously in mice lacking the Ataxia telangiectasia mutated (Atm) tumor suppressor. We identified 3 ICN1 subgroups: 63% expressed PEST-truncated ICN1 (T-ICN1); 17% expressed non-truncated ICN1 (NT-ICN1); and 20% had undetectable ICN1 (UD-ICN1), most lacked Notch1 mRNA. We confirmed the difference in Notch transcriptional activity and functional dependence between the UD-ICN1 and T-ICN1 subgroups and then compared their gene expression profiles to define pathways unique to the UD-ICN1 group. Gene set enrichment analyses revealed that UD-ICN1 T-LLs expressed higher levels of Klf9 and other transcription factors associated with a highly proliferative stage of normal T-cell development. siRNA knock-down studies demonstrated that Klf9 promoted proliferation of UD-ICN1 but not T-ICN1 T-LL cells. Collectively, these data demonstrate that Klf9 can regulate proliferation of Notch-independent T-LLs and suggest that Klf9 may provide a novel therapeutic target for human T-LLs lacking activating NOTCH1 mutations. Disclosures:No relevant conflicts of interest to declare.

A Dominantly Acting Murine Allele of Mcm4 Causes Chromosomal Abnormalities and Promotes Tumorigenesis

Here we report the isolation of a murine model for heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) called Spontaneous dominant leukemia (Sdl). Sdl heterozygous mice develop disease with a short latency and high penetrance, while mice homozygous for the mutation die early during embryonic development. Sdl mice exhibit an increase in the frequency of micronucleated reticulocytes, and T-ALLs from Sdl mice harbor small amplifications and deletions, including activating deletions at the Notch1 locus. Using exome sequencing it was determined that Sdl mice harbor a spontaneously acquired mutation in Mcm4 (Mcm4D573H). MCM4 is part of the heterohexameric complex of MCM2–7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at replication forks. Previous studies in murine models have discovered that genetic reductions of MCM complex levels promote tumor formation by causing genomic instability. However, Sdl mice possess normal levels of Mcms, and there is no evidence for loss-of-heterozygosity at the Mcm4 locus in Sdl leukemias. Studies in Saccharomyces cerevisiae indicate that the Sdl mutation produces a biologically inactive helicase. Together, these data support a model in which chromosomal abnormalities in Sdl mice result from the ability of MCM4D573H to incorporate into MCM complexes and render them inactive. Our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences by causing chromosomal abnormalities.

EPHA7 , a new target gene for 6q deletion in T-cell lymphoblastic lymphomas

Cryptic deletions at chromosome 6q are common cytogenetic abnormalities in T-cell lymphoblastic leukemia/lymphoma (T-LBL), but the target genes have not been formally identified. Our results build on detection of specific chromosomal losses in a mouse model of γ-radiation-induced T-LBLs and provide interesting clues for new putative susceptibility genes in a region orthologous to human 6q15-6q16.3. Among these, Epha7 emerges as a bona fide candidate tumor suppressor gene because it is inactivated in practically all the T-LBLs analyzed (100% in mouse and 95.23% in human). We provide evidence showing that Epha7 downregulation may occur, at least in part, by loss of heterozygosity (19.35% in mouse and 12.5% in human) or promoter hypermethylation (51.61% in mouse and 43.75% in human) or a combination of both mechanisms (12.90% in mouse and 6.25% in human). These results indicate that EPHA7 might be considered a new tumor suppressor gene for 6q deletions in T-LBLs. Notably, this gene is located in 6q16.1 proximal to GRIK2 and CASP8AP2, other candidate genes identified in this region. Thus, del6q seems to be a complex region where inactivation of multiple genes may cooperatively contribute to the onset of T-cell lymphomas.

Abstract A37: A dominantly acting allele of murine Mcm4 causes chromosome instability and promotes leukemiogenesis

Abstract Mouse models have been invaluable tools for studying human cancer. Given the contribution of mouse models to understanding tumorigenesis, when a spontaneous mouse mutant that developed heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) arose in our colony, we pursued studies to both characterize the disease in these mice and to identify the mutant gene responsible for the phenotype. Because the mutation was spontaneous and the phenotype was dominant, we named the mutant Spontaneous dominant leukemia (Sdl). Sdl heterozygous mice develop disease with short latency and high penetrance; while mice homozygous for the mutation die early during embryonic development. We have discovered a spontaneously acquired mutation in Mcm4 in Sdl mice by whole exome sequencing. MCM4 is part of the heterohexameric complex of MCM2–7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at the replication fork. Previous studies in murine models have discovered that reduced levels of MCM complexes resulting from hypomorphic Mcm alleles promote tumor formation by causing genomic instability. However, Sdl mice possess normal levels of MCMs and there is no evidence for loss-of-heterozygosity at the Mcm4 locus in Sdl leukemias; indicating that the Sdl Mcm4 allele is not a hypomorphic or loss-of-function allele. Sdl mice harbor increased numbers of spontaneous micronuclei and Sdl T-ALLs are characterized by intragenic deletions at the Notch1 locus. Therefore, our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences caused by chromosome instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A37.

Endogenous Oncogenic Nras Mutation Initiates T-Cell Leukemia/Lymphoma In a Dose-Dependent Manner

AbstractAbstract 4183The oncogenic NRAS mutations are frequently identified in myeloid diseases but rare in lymphoid diseases. They occur in 4% of acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) patients and 22% of human T-ALL cell lines. Its differential roles in myeloid versus lymphoid disease development remain unclear. Here we examine the tumorigenic potential of oncogenic Nras in T-cells using two conditional Nras G12D murine knock-in models that either hypomorphically (NrasG12D Hypo) or normally (NrasG12D Norm) expresses oncogenic Nras G12D from its endogenous locus. Mice expressing monoallelic or biallelic NrasG12D Hypo develop normally and are tumor free. However, NrasG12D Norm leads to acute T-cell leukemia/lymphoma (TAL/L) in a bone marrow transplantation model, with a low incidence (∼8%) when expressing one allele (TAL/L-het) and a complete penetrance when expressing two alleles (TAL/L-homo). TAL/L-het tumors are associated with spontaneous up-regulation of oncogenic Nras in ∼67% of animals, and tumor cells are TdT positive, suggesting that they are transformed at an immature stage. In contrast, TAL/L-homo tumors express comparable levels of Nras to control thymocytes, and tumor cells are TdT negative, suggesting that they are transformed at a more mature stage. Both TAL/L-het and TAL/L-homo tumors are oligoclonal or polyclonal. Above 70% of these tumors contain clonal Notch1 mutations and are sensitive to gamma-secretase inhibitor. These data indicate that Notch1 mutations are acquired at an early stage and play an important role in the development of TAL/L-het and TAL/L-homo tumors. Together, our results show that engdogenous oncogenic Nras mutation leads to TAL/L in a dose-dependent manner, and thus explain the low incidence of oncogenic NRAS mutations in human T-cell diseases. Disclosures:No relevant conflicts of interest to declare.

Oncogenic Kras-induced leukemogeneis: hematopoietic stem cells as the initial target and lineage-specific progenitors as the potential targets for final leukemic transformation

AbstractKRAS is often mutated in human hematopoietic malignancies, including juvenile myelomonocytic leukemia (JMML) and T-cell lymphoblastic leukemia/lymphoma (TLL/L). However, the exact role and function of oncogenic KRAS mutations in the initiation and progression of JMML and TLL/L remain elusive. Here, we report the use of a mouse bone marrow transplantation model to study oncogenic Kras-induced leukemogenesis. We show that as the first genetic hit, oncogenic Kras mutations initiate both JMML and TLL/L, but with different efficiencies. Limiting dilution analyses indicated that an oncogenic Kras mutation alone is insufficient to produce frank malignancy. Instead, it cooperates with additional subsequent genetic event(s). Moreover, transplantation of highly purified hematopoietic stem cells (HSCs) and myeloid progenitors identified HSCs as the primary target for the oncogenic Kras mutation. Karyotypic analysis further indicated that secondary genetic hit(s) target lineage-specific progenitors rather than HSCs for terminal tumor transformation into leukemic stem cells. Thus, we propose the cellular mechanism underlying oncogenic Kras-induced leukemogenesis, with HSCs as the primary target by the oncogenic Kras mutations and lineage-committed progenitors as the final target for cancer stem cell transformation. Our model might be also applicable to other solid tumors harboring oncogenic Kras mutations.

Adult Precursor T-Lymphoblastic Leukemia/Lymphoma with Myeloid-Associated Antigen Expression Is Associated with a Lower Complete Remission Rate following Induction Chemotherapy

Prognostic studies of T-cell lymphoblastic leukemia/lymphoma (T-ALL) have been performed in small patient cohorts with conflicting results. We systematically reviewed 67 adult T-ALL patients diagnosed and treated at our institute to identify clinical and pathologic prognostic factors. The median initial WBC was 21.3 × 10⁹/l. Blasts expressed at least one myeloid-associated antigen in 33%. Karyotypes were abnormal in 32% of the cases. Fifty-six of 64 patients (88%) achieved complete remission (CR). In univariate analysis, age, gender, initial WBC, CD10, CD34 and abnormal karyotype did not predict CR. Patients expressing at least one myeloid-associated antigen had a CR of 74% compared to 94% (p = 0.04) for those not expressing myeloid antigens. None of the above factors affected relapse-free or overall survival in this cohort. Our study indicates that expression of myeloid-associated antigens is associated with a lower CR rate in adult T-ALL and may be considered in risk stratification for induction chemotherapy.