Notch is oncogenic dominant in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic neoplasm characterized by malignant expansion of immature T cells. Activated NOTCH (Notch(IC)) and c-MYC expression are increased in a large percentage of human T-ALL tumors. Furthermore, c-MYC has been shown to be a NOTCH target gene. Although activating mutations of Notch have been found in human T-ALL tumors, there is little evidence that the c-MYC locus is altered in this neoplasm. It was previously demonstrated that Notch and c-Myc-regulated genes have a broadly overlapping profile, including genes involved in cell cycle progression and metabolism. Given that Notch and c-Myc appear to function similarly in T-ALL, we sought to determine whether these two oncogenes could substitute for each other in T-ALL tumors. Here we report that NOTCH(IC) is able to maintain T-ALL tumors formed in the presence of exogenous NOTCH(IC) and c-MYC when exogenous c-MYC expression is extinguished. In contrast, c-MYC is incapable of maintaining these tumors in the absence of NOTCH(IC). We propose that failure of c-MYC to maintain these tumors is the result of p53-mediated apoptosis. These results demonstrate that T-ALL maintenance is dependent on NOTCH(IC), but not c-MYC, demonstrating that NOTCH is oncogenic dominant in T-ALL tumors.     

Oncogenic Kras and Notch-1 cooperate in T-cell acute lymphoblastic leukemia/lymphoma

Mutations of the Ras family are one of the most common somatic events found in all human cancers, although they are relatively rare in T-cell acute lymphoblastic leukemia (T-ALL). In mice, conditional expression of oncogenic Kras(G12D) from its endogenous promoter causes a fatal myeloproliferative disorder, and only rarely a T-ALL-like disease. In the article being evaluated, the authors demonstrate that primary mice expressing oncogenic Kras have a block in T-cell differentiation at the double-negative 1 stage. Interestingly, most secondarily transplanted mice develop a fatal T-ALL-like disease. Sequencing of NOTCH-1 showed that 50% of these mice harbored truncating mutations in the PEST domain that would be predicted to activate Notch signaling. Cell lines established from some of the mice demonstrated sensitivity to γ-secretase inhibition, suggesting that even when NOTCH-1 mutations occur as secondary collaborating events, tumors retain a dependency on this pathway that might be exploitable clinically.     

Sequential mutations in Notch1, Fbxw7, and Tp53 in radiation-induced mouse thymic lymphomas

T-cell acute lymphoblastic lymphomas commonly demonstrate activating Notch1 mutations as well as mutations or deletions in Fbxw7. However, because Fbxw7 targets Notch1 for degradation, genetic alterations in these genes are expected to be mutually exclusive events in lymphomagenesis. Previously, by using a radiation-induced Tp53-deficient mouse model for T-cell acute lymphoblastic lymphoma, we reported that loss of heterozygosity at the Fbxw7 locus occurs frequently in a Tp53-dependent manner. In the current study, we show that these thymic lymphomas also commonly exhibit activating Notch1 mutations in the proline-glutamic acid-serine-threonine (PEST) domain. Moreover, concurrent activating Notch1 PEST domain mutations and single-copy deletions at the Fbxw7 locus occur with high frequency in the same individual tumors, indicating that these changes are not mutually exclusive events. We further demonstrate that although Notch1 PEST domain mutations are independent of Tp53 status, they are completely abolished in mice with germline Fbxw7 haploinsufficiency. Therefore, Notch1 PEST domain mutations only occur when Fbxw7 expression levels are intact. These data suggest a temporal sequence of mutational events involving these important cancer-related genes, with Notch1 PEST domain mutations occurring first, followed by Fbxw7 deletion, and eventually by complete loss of Tp53.     

Aggressive Langerhans cell histiocytosis following T-ALL: clonally related neoplasms with persistent expression of constitutively active NOTCH1

Langerhans cell histiocytosis (LCH) and related entities are neoplasms of unknown pathogenesis. Here, we describe studies assessing the role of NOTCH1 mutations in LCH, which were based on a case of fatal Langerhans cell tumor after T-cell acute lymphoblastic leukemia (T-ALL). Although the two types of neoplasm in this patient were temporally and pathologically distinct, molecular analyses showed that they harbored the same T-cell receptor gene rearrangements and two activating NOTCH1 mutations involving exons 27 and 34. The exon 27 mutation altered a conserved cysteine residue in the N-terminal portion of the NOTCH1 heterodimerization domain, while the mutation in exon 34 introduced a premature stop codon that results in the deletion of C-terminal negative regulatory PEST domain. Analysis of cDNA prepared from the aggressive Langerhans cell tumor showed that the NOTCH1 mutations were aligned in cis, a configuration that caused synergistic increases in NOTCH1 signal strength in reporter gene assays. Immunohistochemistry confirmed that the Langerhans cell tumor also expressed NOTCH1 protein. Although these data suggested that NOTCH1 mutations might contribute to the pathogenesis of typical sporadic LCH and related neoplasms occurring in the absence of T-ALL, an analysis of 24 cases of LCH and Rosai-Dorfman Disease occurring in patients without an antecedent history of T-ALL revealed no mutations. Thus, activating NOTCH1 mutations appear to be unique to aggressive Langerhans cell tumors occurring after T-ALL. Persistent expression of NOTCH1 in such tumors suggests that Notch pathway inhibitors could have a role in the treatment of these unusual neoplasms.     

Notch1 mutations are important for leukemic transformation in murine models of precursor-T leukemia/lymphoma

NOTCH1 is frequently mutated in human precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). In the current study, we found that 13 of 19 cell lines and 29 of 49 primary tumors from SCL/LMO1, OLIG2/LMO1, OLIG2, LMO1, NUP98/HOXD13, and p27(-/-)/SMAD3(+/-) mice had Notch1 mutations in either the heterodimerization (HD) or the glutamic acid/serine/threonine (PEST) domain but not both. Thymocytes from clinically healthy SCL/LMO1 mice aged 5 weeks did not have Notch1 mutations, whereas thymocytes from clinically healthy SCL/LMO1 mice aged 8 to 12 weeks did have Notch1 mutations and formed tumors upon transplantation into nude mice. Remarkably, all of the HD domain mutations that we identified were single-base substitutions, whereas all of the PEST domain mutations were insertions or deletions, half of which mapped to 1 of 2 mutational "hot spots." Taken together, these findings indicate that Notch1 mutations are very frequent events that are acquired relatively early in the process of leukemic transformation and are important for leukemic cell growth.     

Notch1 inhibition targets the leukemia-initiating cells in a Tal1/Lmo2 mouse model of T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy largely caused by aberrant activation of the TAL1/SCL, LMO1/2, and NOTCH1 oncogenes. Approximately 30% of T-ALL patients relapse, and evidence is emerging that relapse may result from a failure to eliminate leukemia-initiating cells (LICs). Thymic expression of the Tal1 and Lmo2 oncogenes in mice results in rapid development of T-ALL; and similar to T-ALL patients, more than half the leukemic mice develop spontaneous mutations in Notch1. Using this mouse model, we demonstrate that mouse T-ALLs are immunophenotypically and functionally heterogeneous with approximately 1 of 10,000 leukemic cells capable of initiating disease on transplantation. Our preleukemic studies reveal expansion of Notch-active double-negative thymic progenitors, and we find the leukemic DN3 population enriched in disease potential. To examine the role of Notch1 in LIC function, we measured LIC activity in leukemic mice treated with vehicle or with a γ-secretase inhibitor. In 4 of 5 leukemias examined, Notch inhibition significantly reduced or eliminated LICs and extended survival. Remarkably, in 2 mice, γ-secretase inhibitor treatment reduced LIC frequency below the limits of detection of this assay, and all transplanted mice failed to develop disease. These data support the continued development of Notch1 therapeutics as antileukemia agents.     

K-RasG12D-induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to gamma-secretase inhibitors

To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-Ras(G12D) murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to gamma-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways.     

Idh1 mutations contribute to the development of T-cell malignancies in genetically engineered mice

Gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1) are key drivers of hematopoietic malignancies. Although these mutations are most commonly associated with myeloid diseases, they also occur in malignancies of the T-cell lineage. To investigate their role in these diseases and provide tractable disease models for further investigation, we analyzed the T-cell compartment in a conditional knock-in (KI) mouse model of mutant Idh1. We observed the development of a spontaneous T-cell acute lymphoblastic leukemia (T-ALL) in these animals. The disease was transplantable and maintained expression of mutant IDH1. Whole-exome sequencing revealed the presence of a spontaneous activating mutation in Notch1, one of the most common mutations in human T-ALL, suggesting Idh1 mutations may have the capacity to cooperate with Notch1 to drive T-ALL. To further investigate the Idh1 mutation as an oncogenic driver in the T-cell lineage, we crossed Idh1-KI mice with conditional Trp53 null mice, a well-characterized model of T-cell malignancy, and found that T-cell lymphomagenesis was accelerated in mice bearing both mutations. Because both IDH1 and p53 are known to affect cellular metabolism, we compared the requirements for glucose and glutamine in cells derived from these tumors and found that cells bearing the Idh1 mutation have an increased dependence on both glucose and glutamine. These data suggest that mutant IDH1 contributes to malignancy in the T-cell lineage and may alter the metabolic profile of malignant T cells.Somatic mutations in isocitrate dehydrogenase 1 (IDH1) are frequently observed in a number of malignancies, including glioma, cholangiocarcinoma, chondrosarcoma, and several hematological malignancies (1). IDH1 is a cytoplasmic enzyme that catalyzes the NADP-dependent conversion of isocitrate to α-ketoglutarate (αKG). Mutations in IDH1 at arginine 132 (R132) cause an enzymatic gain of function that results in the NADPH-dependent conversion of αKG to d-2-hydroxyglutarate (2HG) (2). This metabolite is normally maintained at very low levels in cells and tissues and is not part of any known productive metabolic pathway. However, in cells and tissues of patients with IDH1 mutant tumors, 2HG builds up to high levels and is thought to contribute to tumorigenesis by inhibiting a class of αKG-dependent enzymes (1). The precise effects important for driving tumorigenesis downstream of IDH1 mutations are not fully understood and may differ between disease states.In the hematopoietic system, IDH1 mutations are most often associated with myeloid diseases, where they are commonly found in myelodysplastic syndrome and acute myeloid leukemia (3). However, IDH1 mutations are also found in a small proportion of adult T-cell acute lymphoblastic leukemia (T-ALL) (4, 5). T-ALL is an aggressive malignancy of developing T cells and is responsible for ∼25% of adult ALL (6, 7). T-ALL is thought to arise via a multistep process of oncogenic mutation that leads to the transformation of immature T cells. The genetic landscape of the disease has been characterized, and a large number of driver mutations have been identified (6). The most common genetic feature of T-ALL is the presence of activating mutations in Notch1, which are present in more than 50% of patients (8). Interestingly, IDH1 mutations seem to be confined to a subset of adult patients with T-ALL bearing an immature T-cell gene expression signature and harboring other oncogenic mutations in genes more commonly associated with myeloid malignancy, including Flt3 and DNMT3A (4, 9). This subset of T-ALL has recently been recognized as a distinct disease entity called early T-cell precursor T-ALL and is associated with therapy resistance and a particularly poor outcome (10). The role of IDH1 mutations in this subset of T-ALL is not understood.Using a myeloid lineage-specific conditional Idh1-R132-KI mouse model, we previously showed that mutant IDH1 partially blocks differentiation and produces a hematopoietic phenotype similar to human myelodysplastic syndrome (11). In this study, we crossed the Idh1-R132-KI mouse with Vav-cre animals to introduce the IDH1 R132 mutation into the entire hematopoietic system to investigate the role of Idh1 mutations in T-cell malignancy.     

Functional interactions between Lmo2, the Arf tumor suppressor, and Notch1 in murine T-cell malignancies

LMO2 is a target of chromosomal translocations in T-cell tumors and was activated by retroviral vector insertions in T-cell tumors from X-SCID patients in gene therapy trials. To better understand the cooperating genetic events in LMO2-associated T-cell acute lymphoblastic leukemia (T-ALL), we investigated the roles of Arf tumor suppressor loss and Notch activation in murine models of transplantation. Lmo2 overexpression enhanced the expansion of primitive DN2 thymocytes, eventually facilitating the stochastic induction of clonal CD4(+)/CD8(+) malignancies. Inactivation of the Arf tumor suppressor further increased the self-renewal capacity of the primitive, preleukemic thymocyte pool and accelerated the development of aggressive, Lmo2-induced T-cell lympholeukemias. Notch mutations were frequently detected in these Lmo2-induced tumors. The Arf promoter was not directly engaged by Lmo2 or mutant Notch, and use of a mouse model in which activation of a mutant Notch allele depends on previous engagement of the Arf promoter revealed that Notch activation could occur as a subsequent event in T-cell tumorigenesis. Therefore, Lmo2 cooperates with Arf loss to enhance self-renewal in primitive thymocytes. Notch mutation and Arf inactivation appear to independently cooperate in no requisite order with Lmo2 overexpression in inducing T-ALL, and all 3 events remained insufficient to guarantee immediate tumor development.     

Activating NOTCH1 mutations predict favorable early treatment response and long-term outcome in childhood precursor T-cell lymphoblastic leukemia

Activating mutations of the transmembrane receptor NOTCH1 are common in precursor T-cell lymphoblastic leukemia (T-ALL). We systematically analyzed the impact of activating NOTCH1 mutations on early treatment response and long-term outcome in 157 patients with T-ALL of the pediatric ALL-Berlin-Frankfurt-Munster (BFM) 2000 study. We confirm previous results that NOTCH1 mutations occur in more than 50% of T-ALL in children. In 82 patients (82/157; 52.2%), activating NOTCH1 mutations were identified either in the heterodimerization (55/82; 67.1%), in the PEST (13/82; 15.9%), or in both domains (14/82; 17.0%). The presence of NOTCH1 mutations was significantly correlated with a good prednisone response and favorable minimal residual disease (MRD) kinetics, which was independent from sex, age, white blood cell count, and T-cell immunophenotype at the time of diagnosis. Furthermore, activating NOTCH1 mutations specified a large subgroup of patients with an excellent prognosis. These findings indicate that in the context of the ALL-BFM 2000 treatment strategy, NOTCH1 mutations predict a more rapid early treatment response and a favorable long-term outcome in children with T-ALL.     

Notch1-dependent lymphomagenesis is assisted by but does not essentially require pre-TCR signaling

Overexpression of intracellular Notch plays an important role in the generation of human acute lymphoblastic T cell leukemia (T-ALL). In mouse models, it was shown that Notch-dependent T-ALL required pre-TCR signaling. Here we show that pre-TCR signaling is required to condition mice for Notch-dependent transformation but that it is not required to sustain malignant growth of T-ALL. In contrast to previous studies, we found that disease development does not require pre-TCR but that it can be accelerated in Rag2(-/-) mice by transient mimicking of pre-TCR signals.     

Development of Notch-dependent T-cell leukemia by deregulated Rap1 signaling

SPA-1 (signal-induced proliferation associated gene-1) functions as a suppressor of myeloid leukemia by negatively regulating Rap1 signaling in hematopoietic progenitor cells (HPCs). Herein, we showed that transplantation of HPCs expressing farnesylated C3G (C3G-F), a Rap1 guanine nucleotide exchange factor, resulted in a marked expansion of thymocytes bearing unique phenotypes (CD4/CD8 double positive [DP] CD3(-) TCRbeta(-)) in irradiated recipients. SPA-1(-/-) HPCs expressing C3G-F caused a more extensive expansion of DP thymocytes, resulting in lethal T-cell acute lymphoblastic leukemia (T-ALL) with massive invasion of clonal T-cell blasts into vital organs. The C3G-F(+) blastic thymocytes exhibited constitutive Rap1 activation and markedly enhanced expression of Notch1, 3 as well as the target genes, Hes1, pTalpha, and c-Myc. All the T-ALL cell lines from C3G-F(+) SPA-1(-/-) HPC recipients expressed high levels of Notch1 with characteristic mutations resulting in the C-terminal truncation. This proliferation was inhibited completely in the presence of a gamma-secretase inhibitor. Transplantation of Rag2(-/-) SPA-1(-/-) HPCs expressing C3G-F also resulted in a marked expansion and transformation of DP thymocytes. The results suggested that deregulated constitutive Rap1 activation caused abnormal expansion of DP thymocytes, bypassing the pre-T-cell receptor and eventually leading to Notch1 mutations and Notch-dependent T-ALL.     

PTPN2 negatively regulates oncogenic JAK1 in T-cell acute lymphoblastic leukemia

We have recently reported inactivation of the tyrosine phosphatase PTPN2 (also known as TC-PTP) through deletion of the entire gene locus in ～ 6% of T-cell acute lymphoblastic leukemia (T-ALL) cases. T-ALL is an aggressive disease of the thymocytes characterized by the stepwise accumulation of chromosomal abnormalities and gene mutations. In the present study, we confirmed the strong association of the PTPN2 deletion with TLX1 and NUP214-ABL1 expression. In addition, we found cooperation between PTPN2 deletion and activating JAK1 gene mutations. Activating mutations in JAK1 kinase occur in ～ 10% of human T-ALL cases, and aberrant kinase activity has been shown to confer proliferation and survival advantages. Our results reveal that some JAK1 mutation-positive T-ALLs harbor deletions of the tyrosine phosphatase PTPN2, a known negative regulator of the JAK/STAT pathway. We provide evidence that down-regulation of Ptpn2 sensitizes lymphoid cells to JAK1-mediated transformation and reduces their sensitivity to JAK inhibition.     

Nonhereditary p53 mutations in T-cell acute lymphoblastic leukemia are associated with the relapse phase

We have previously reported that greater than 60% of human leukemic T- cell lines possess mutations in the p53 tumor suppressor gene. To determine whether T-cell acute lymphoblastic leukemia (T-ALL) patient samples possess p53 mutations, we screened peripheral blood-and bone marrow-derived leukemia samples, taken at diagnosis and at relapse, for p53 mutations. Exons 4 through 9 and selected intron regions of the p53 gene were analyzed using polymerase chain reaction-single-strand conformation polymorphism and direct sequencing. p53 mutations were found in 0 of 15 T-ALL diagnosis samples, as compared with 10 of 36 (28%) T-ALL relapse samples. To determine whether p53 mutations play a role in the recurrence (relapse) of T-ALL, two special groups of T-ALL patients were studied: (1) a group of 8 relapse patients whose disease was refractory to chemotherapeutic treatment, and (2) a group of 6 "paired" T-ALL cell samples from patients for whom we possess both diagnosis and relapse samples. Three of 8 relapsed patients (37.5%) whose disease was refractory to the reinduction of remission by chemotherapy possessed missense mutations of the p53 gene. All 3 cases had mutations in exon 5. Among the paired samples, 3 of 6 patients harbored p53 mutations at disease recurrence, but possessed only wild- type p53 alleles at diagnosis. One case had mutation on exon 4, 1 case in exon 5, and 1 case in exon 8 with loss of heterozygosity. These data clearly indicate that recurrence of T-ALL is associated with missense mutations in p53. Our results indicate that (1) mutations of p53 do occur in T-ALL in vivo, and such mutations are associated with the relapse phase of the disease; and (2) p53 mutation is involved in the progression of T-ALL. This conclusion is supported by our observation that the introduction of T-ALL-derived mutant p53 expression constructs into T-ALL cell lines further increases their growth rate in culture, enhances cell cloning in methylcellulose, and increases tumor formation in nude mice.     

Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia

Efforts to model pancreatic cancer in mice have focused on mimicking genetic changes found in the human disease, particularly the activating KRAS mutations that occur in pancreatic tumors and their putative precursors, pancreatic intraepithelial neoplasia (PanIN). Although activated mouse Kras mutations induce PanIN lesions similar to those of human, only a small minority of cells that express mutant Kras go on to form PanINs. The basis for this selective response is unknown, and it is similarly unknown what cell types in the mature pancreas actually contribute to PanINs. One clue comes from the fact that PanINs, unlike most cells in the adult pancreas, exhibit active Notch signaling. We hypothesize that Notch, which inhibits differentiation in the embryonic pancreas, contributes to PanIN formation by abrogating the normal differentiation program of tumor-initiating cells. Through conditional expression in the mouse pancreas, we find dramatic synergy between activated Notch and Kras in inducing PanIN formation. Furthermore, we find that Kras activation in mature acinar cells induces PanIN lesions identical to those seen upon ubiquitous Kras activation, and that Notch promotes both initiation and dysplastic progression of these acinar-derived PanINs, albeit short of invasive adenocarcinoma. At the cellular level, Notch/Kras coactivation promotes rapid reprogramming of acinar cells to a duct-like phenotype, providing an explanation for how a characteristically ductal tumor can arise from nonductal acinar cells.     

PML/RARalpha and FLT3-ITD induce an APL-like disease in a mouse model

Acute promyelocytic leukemia (APL) cells invariably express aberrant fusion proteins involving the retinoic acid receptor alpha (RARalpha). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARalpha in the balanced reciprocal chromosomal translocation, t(15;17)(q22:q11). Expression of PML/RARalpha from the cathepsin G promoter in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is FLT3, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3, including internal tandem duplication (ITD) in the juxtamembrane domain, transform hematopoietic cell lines to factor independent growth. FLT3-ITDs also induce a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML. Here, we test the hypothesis that PML/RARalpha can cooperate with FLT3-ITD to induce an APL-like disease in the mouse. Retroviral transduction of FLT3-ITD into bone marrow cells obtained from PML/RARalpha transgenic mice results in a short latency APL-like disease with complete penetrance. This disease resembles the APL-like disease that occurs with long latency in the PML/RARalpha transgenics, suggesting that activating mutations in FLT3 can functionally substitute for the additional mutations that occur during mouse APL progression. The leukemia is transplantable to secondary recipients and is ATRA responsive. These observations document cooperation between PML/RARalpha and FLT3-ITD in development of the murine APL phenotype.