The IL-2/CD25 axis maintains distinct subsets of chronic myeloid leukemia-initiating cells.

Just as normal stem cells require niche cells for survival, leukemia-initiating cells (LICs) may also require niche cells for their maintenance. Chronic myeloid leukemia (CML) is caused by the activity of BCR-ABL, a constitutively active tyrosine kinase. CML therapy with tyrosine kinase inhibitors is highly effective; however, due to the persistence of residual LICs, it is not curative. Several factors are known to support CML LICs, but purification of LICs and a thorough understanding of their niche signals have not yet been achieved. Using a CML-like mouse model of myeloproliferative disease, we demonstrate that CML LICs can be divided into CD25(+)FcεRIα(-) Lineage marker (Lin)(-) Sca-1(+)c-Kit(+) (F(-)LSK) cells and CD25(-)F(-)LSK cells. The CD25(+)F(-)LSK cells had multilineage differentiation capacity, with a preference toward cytokine-producing mast cell commitment. Although cells interconverted between CD25(-)F(-)LSK and CD25(+)F(-)LSK status, the CD25(+)F(-)LSK cells exhibited higher LIC capacity. Our findings suggest that interleukin-2 derived from the microenvironment and CD25 expressed on CML LICs constitute a novel signaling axis. The high levels of CD25 expression in the CD34(+)CD38(-) fraction of human CML cells indicate that CD25(+) LICs constitute an "LIC-derived niche" that could be preferentially targeted in therapy for CML.

Intracellular signals of lung cancer cells as possible therapeutic targets.

In recent years, several molecularly targeted therapies have been developed as part of lung cancer treatment; they have produced dramatically good results. However, among the many oncogenes that have been identified to be involved in the development of lung cancers, a number of oncogenes are not covered by these advanced therapies. For the treatment of lung cancers, which is a group of heterogeneous diseases, persistent effort in developing individual therapies based on the respective causal genes is important. In addition, for the development of a novel therapy, identification of the lung epithelial stem cells and the origin cells of lung cancer, and understanding about candidate cancer stem cells in lung cancer tissues, their intracellular signaling pathways, and the mechanism of dysregulation of the pathways in cancer cells are extremely important. However, the development of drug resistance by cancer cells, despite the use of molecularly targeted drugs for the causal genes, thus obstructing treatment, is a well-known phenomenon. In this article, we discuss major causal genes of lung cancers and intracellular signaling pathways involving those genes, and review studies on origin and stem cells of lung cancers, as well as the possibility of developing molecularly targeted therapies based on these studies.

Maintenance of sweat glands by stem cells located in the acral epithelium.

The skin is responsible for a variety of physiological functions and is critical for wound healing and repair. Therefore, the regenerative capacity of the skin is important. However, stem cells responsible for maintaining the acral epithelium had not previously been identified. In this study, we identified the specific stem cells in the acral epithelium that participate in the long-term maintenance of sweat glands, ducts, and interadnexal epidermis and that facilitate the regeneration of these structures following injury. Lgr6-positive cells and Bmi1-positive cells were found to function as long-term multipotent stem cells that maintained the entire eccrine unit and the interadnexal epidermis. However, while Lgr6-positive cells were rapidly cycled and constantly supplied differentiated cells, Bmi1-positive cells were slow to cycle and occasionally entered the cell cycle under physiological conditions. Upon irradiation-induced injury, Bmi1-positive cells rapidly proliferated and regenerated injured epithelial tissue. Therefore, Bmi1-positive stem cells served as reservoir stem cells. Lgr5-positive cells were rapidly cycled and maintained only sweat glands; therefore, we concluded that these cells functioned as lineage-restricted progenitors. Taken together, our data demonstrated the identification of stem cells that maintained the entire acral epithelium and supported the different roles of three cellular classes.

The shortest isoform of C/EBPß, liver inhibitory protein (LIP), collaborates with Evi1 to induce AML in a mouse BMT model.

Ecotropic viral integration site 1 (Evi1) is one of the master regulators in the development of acute myeloid leukemia (AML) and myelodysplastic syndrome. High expression of Evi1 is found in 10% of patients with AML and indicates a poor outcome. Several recent studies have indicated that Evi1 requires collaborative factors to induce AML. Therefore, the search for candidate factors that collaborate with Evi1 in leukemogenesis is one of the key issues in uncovering the mechanism of Evi1-related leukemia. Previously, we succeeded in making a mouse model of Evi1-related leukemia using a bone marrow transplantation (BMT) system. In the Evi1-induced leukemic cells, we identified frequent retroviral integrations near the CCAAT/enhancer-binding protein ß (C/EBPß) gene and overexpression of its protein. These findings imply that C/EBPß is a candidate gene that collaborates with Evi1 in leukemogenesis. Cotransduction of Evi1 and the shortest isoform of C/EBPß, liver inhibitory protein (LIP), induced AML with short latencies in a mouse BMT model. Overexpression of LIP alone also induced AML with longer latencies. However, excision of all 3 isoforms of C/EBPß (LAP*/LAP/LIP) did not inhibit the development of Evi1-induced leukemia. Therefore, isoform-specific intervention that targets LIP is required when we consider C/EBPß as a therapeutic target.

Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms.

Acquired uniparental disomy (aUPD) is a common feature of cancer genomes, leading to loss of heterozygosity. aUPD is associated not only with loss-of-function mutations of tumour suppressor genes, but also with gain-of-function mutations of proto-oncogenes. Here we show unique gain-of-function mutations of the C-CBL (also known as CBL) tumour suppressor that are tightly associated with aUPD of the 11q arm in myeloid neoplasms showing myeloproliferative features. The C-CBL proto-oncogene, a cellular homologue of v-Cbl, encodes an E3 ubiquitin ligase and negatively regulates signal transduction of tyrosine kinases. Homozygous C-CBL mutations were found in most 11q-aUPD-positive myeloid malignancies. Although the C-CBL mutations were oncogenic in NIH3T3 cells, c-Cbl was shown to functionally and genetically act as a tumour suppressor. C-CBL mutants did not have E3 ubiquitin ligase activity, but inhibited that of wild-type C-CBL and CBL-B (also known as CBLB), leading to prolonged activation of tyrosine kinases after cytokine stimulation. c-Cbl(-/-) haematopoietic stem/progenitor cells (HSPCs) showed enhanced sensitivity to a variety of cytokines compared to c-Cbl(+/+) HSPCs, and transduction of C-CBL mutants into c-Cbl(-/-) HSPCs further augmented their sensitivities to a broader spectrum of cytokines, including stem-cell factor (SCF, also known as KITLG), thrombopoietin (TPO, also known as THPO), IL3 and FLT3 ligand (FLT3LG), indicating the presence of a gain-of-function that could not be attributed to a simple loss-of-function. The gain-of-function effects of C-CBL mutants on cytokine sensitivity of HSPCs largely disappeared in a c-Cbl(+/+) background or by co-transduction of wild-type C-CBL, which suggests the pathogenic importance of loss of wild-type C-CBL alleles found in most cases of C-CBL-mutated myeloid neoplasms. Our findings provide a new insight into a role of gain-of-function mutations of a tumour suppressor associated with aUPD in the pathogenesis of some myeloid cancer subsets.

Generation of induced pluripotent stem cells from primary chronic myelogenous leukemia patient samples.

Induced pluripotent stem cells (iPSCs) can be generated by the expression of defined transcription factors not only from normal tissue, but also from malignant cells. Cancer-derived iPSCs are expected to provide a novel experimental opportunity to establish the disease model. We generated iPSCs from imatinib-sensitive chronic myelogenous leukemia (CML) patient samples. Remarkably, the CML-iPSCs were resistant to imatinib although they consistently expressed BCR-ABL oncoprotein. In CML-iPSCs, the phosphorylation of ERK1/2, AKT, and JNK, which are essential for the maintenance of both BCR-ABL (+) leukemia cells and iPSCs, were unchanged after imatinib treatment, whereas the phosphorylation of signal transducer and activator of transcription (STAT)5 and CRKL was significantly decreased. These results suggest that the signaling for iPSCs maintenance compensates for the inhibition of BCR-ABL. CML-iPSC-derived hematopoietic cells recovered the sensitivity to imatinib although CD34(+)38(-)90(+)45(+) immature cells were resistant to imatinib, which recapitulated the pathophysiologic feature of the initial CML. CML-iPSCs provide us with a novel platform to investigate CML pathogenesis on the basis of patient-derived samples.

Multiple phosphorylation sites are important for RUNX1 activity in early hematopoiesis and T-cell differentiation.

RUNX1 is essential for definitive hematopoiesis and T-cell differentiation. It has been shown that RUNX1 is phosphorylated at specific serine and threonine residues by several kinase families. However, it remains unclear whether RUNX1 phosphorylation is absolutely required for its biological functions. Here, we evaluated hematopoietic activities of RUNX1 mutants with serine (S)/threonine (T) to alanine (A), aspartic acid (D), or glutamic acid (E) mutations at phosphorylation sites using primary culture systems. Consistent with the results of knockin mice, RUNX1-2A, carrying two phospho-deficient mutations at S276 and S293, retained hematopoietic activity. RUNX1-4A, carrying four mutations at S276, S293, T300, and S303, showed impaired T-cell differentiation activity, but retained the ability to rescue the defective early hematopoiesis of Runx1-deficient cells. Notably, RUNX1-5A, carrying five mutations at S276, S293, T300, S303, and S462, completely lost its hematopoietic activity. In contrast, the phospho-mimic proteins RUNX1-4D/E and RUNX1-5D/E exhibited normal function. Our study identifies multiple phosphorylation sites that are indispensable for RUNX1 activity in hematopoiesis.

Loss of AML1/Runx1 accelerates the development of MLL-ENL leukemia through down-regulation of p19ARF.

Dysfunction of AML1/Runx1, a transcription factor, plays a crucial role in the development of many types of leukemia. Additional events are often required for AML1 dysfunction to induce full-blown leukemia; however, a mechanistic basis of their cooperation is still elusive. Here, we investigated the effect of AML1 deficiency on the development of MLL-ENL leukemia in mice. Aml1 excised bone marrow cells lead to MLL-ENL leukemia with shorter duration than Aml1 intact cells in vivo. Although the number of MLL-ENL leukemia-initiating cells is not affected by loss of AML1, the proliferation of leukemic cells is enhanced in Aml1-excised MLL-ENL leukemic mice. We found that the enhanced proliferation is the result of repression of p19(ARF) that is directly regulated by AML1 in MLL-ENL leukemic cells. We also found that down-regulation of p19(ARF) induces the accelerated onset of MLL-ENL leukemia, suggesting that p19(ARF) is a major target of AML1 in MLL-ENL leukemia. These results provide a new insight into a role for AML1 in the progression of leukemia.

Notch2 signaling is required for proper mast cell distribution and mucosal immunity in the intestine.

Notch receptor-mediated signaling is involved in the developmental process and functional modulation of lymphocytes, as well as in mast cell differentiation. Here, we investigated whether Notch signaling is required for antipathogen host defense regulated by mast cells. Mast cells were rarely found in the small intestine of wild-type C57BL/6 mice but accumulated abnormally in the lamina propria of the small-intestinal mucosa of the Notch2-conditional knockout mice in naive status. When transplanted into mast cell-deficient W(sh)/W(sh) mice, Notch2-null bone marrow-derived mast cells were rarely found within the epithelial layer but abnormally localized to the lamina propria, whereas control bone marrow-derived mast cells were mainly found within the epithelial layer. After the infection of Notch2 knockout and control mice with L3 larvae of Strongyloides venezuelensis, the abundant number of mast cells was rapidly mobilized to the epithelial layer in the control mice. In contrast, mast cells were massively accumulated in the lamina propria of the small intestinal mucosa in Notch2-conditional knockout mice, accompanied by impaired eradication of Strongyloides venezuelensis. These findings indicate that cell-autonomous Notch2 signaling in mast cells is required for proper localization of intestinal mast cells and further imply a critical role of Notch signaling in the host-pathogen interface in the small intestine.

AML1/RUNX1 functions as a cytoplasmic attenuator of NF-κB signaling in the repression of myeloid tumors.

Functional deregulation of transcription factors has been found in many types of tumors. Transcription factor AML1/RUNX1 is one of the most frequent targets of chromosomal abnormalities in human leukemia and altered function of AML1 is closely associated with malignant transformation of hematopoietic cells. However, the molecular basis and therapeutic targets of AML1-related leukemia are still elusive. Here, we explored immediate target pathways of AML1 by in vitro synchronous inactivation in hematopoietic cells. We found that AML1 inhibits NF-κB signaling through interaction with IκB kinase complex in the cytoplasm. Remarkably, AML1 mutants found in myeloid tumors lack the ability to inhibit NF-κB signaling, and human cases with AML1-related leukemia exhibits distinctly activated NF-κB signaling. Furthermore, inhibition of NF-κB signaling in leukemic cells with mutated AML1 efficiently blocks their growth and development of leukemia. These findings reveal a novel role for AML1 as a cytoplasmic attenuator of NF-κB signaling and indicate that NF-κB signaling is one of the promising therapeutic targets of hematologic malignancies with AML1 abnormality.

Hes1 immortalizes committed progenitors and plays a role in blast crisis transition in chronic myelogenous leukemia.

Hairy enhancer of split 1 (Hes1) is a basic helix-loop-helix transcriptional repressor that affects differentiation and often helps maintain cells in an immature state in various tissues. Here we show that retroviral expression of Hes1 immortalizes common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in the presence of interleukin-3, conferring permanent replating capability on these cells. Whereas these cells did not develop myeloproliferative neoplasms when intravenously administered to irradiated mice, the combination of Hes1 and BCR-ABL in CMPs and GMPs caused acute leukemia resembling blast crisis of chronic myelogenous leukemia (CML), resulting in rapid death of the recipient mice. On the other hand, BCR-ABL alone caused CML-like disease when expressed in c-Kit-positive, Sca-1-positive, and lineage-negative hematopoietic stem cells (KSLs), but not committed progenitors CMPs or GMPs, as previously reported. Leukemic cells derived from Hes1 and BCR-ABL-expressing CMPs and GMPs were more immature than those derived from BCR-ABL-expressing KSLs. Intriguingly, Hes1 was highly expressed in 8 of 20 patients with CML in blast crisis, but not in the chronic phase, and dominant negative Hes1 retarded the growth of some CML cell lines expressing Hes1. These results suggest that Hes1 is a key molecule in blast crisis transition in CML.

Derivation of functional mature neutrophils from human embryonic stem cells.

Human embryonic stem cells (hESCs) proliferate infinitely and are pluripotent. Only a few reports, however, describe specific and efficient methods to induce hESCs to differentiate into mature blood cells. It is important to determine whether and how these cells, once generated, behave similarly with their in vivo-produced counterparts. We developed a method to induce hESCs to differentiate into mature neutrophils. Embryoid bodies were formed with bone morphogenic protein-4, stem cell factor (SCF), Flt-3 ligand (FL), interleukin-6 (IL-6)/IL-6 receptor fusion protein (FP6), and thrombopoietin (TPO). Cells derived from the embryoid bodies were cultured on a layer of irradiated OP9 cells with a combination of SCF, FL, FP6, IL-3, and TPO, which was later changed to granulocyte-colony-stimulating factor. Morphologically mature neutrophils were obtained in approximately 2 weeks with a purity and efficiency sufficient for functional analyses. The population of predominantly mature neutrophils (hESC-Neu's) showed superoxide production, phagocytosis, bactericidal activity, and chemotaxis similar to peripheral blood neutrophils from healthy subjects, although there were differences in the surface antigen expression patterns, such as decreased CD16 expression and aberrant CD64 and CD14 expression in hESC-Neu's. Thus, this is the first description of a detailed functional analysis of mature hESC-derived neutrophils.

Notch activation induces the generation of functional NK cells from human cord blood CD34-positive cells devoid of IL-15.

The development of NK cells from hematopoietic stem cells is thought to be dependent on IL-15. In this study, we demonstrate that stimulation of human cord blood CD34(+) cells by a Notch ligand, Delta4, along with IL-7, stem cell factor, and Fms-like tyrosine kinase 3 ligand, but no IL-15, in a stroma-free culture induced the generation of cells with characteristics of functional NK cells, including CD56 and CD161 Ag expression, IFN-gamma secretion, and cytotoxic activity against K562 and Jurkat cells. Addition of gamma-secretase inhibitor and anti-human Notch1 Ab to the culture medium almost completely blocked NK cell emergence. Addition of anti-human IL-15-neutralizing Ab did not affect NK cell development in these culture conditions. The presence of IL-15, however, augmented cytotoxicity and was required for a more mature NK cell phenotype. CD56(+) cells generated by culture with IL-15, but without Notch stimulation, were negative for CD7 and cytoplasmic CD3, whereas CD56(+) cells generated by culture with both Delta4 and IL-15 were CD7(+) and cytoplasmic CD3(+) from the beginning and therefore more similar to in vivo human NK cell progenitors. Together, these results suggest that Notch signaling is important for the physiologic development of NK cells at differentiation stages beyond those previously postulated.

Coordinated regulation of transcription factors through Notch2 is an important mediator of mast cell fate.

Mast cells are thought to participate in a wide variety of pathophysiological conditions. Mechanisms of regulation, however, of mast cell production and maturation are still to be elucidated. Mast cell developmental process is likely to be profoundly affected by cell-autonomous transcriptional regulators such as the GATA family and CCAAT/enhancer binding protein (C/EBP) family members. Extracellular regulators such as stem cell factor and IL-3 have essential roles in basal and inducible mast cell generation, respectively. The relationship, however, between the extracellular signaling and cellular transcriptional control is unclear, and the trigger of the mast cell development remains elusive. Notch signaling plays a fundamental role in the lymphopoietic compartment, but its role in myeloid differentiation is less clear. Here, we demonstrate that Notch signaling connects environmental cues and transcriptional control for mast cell fate decision. Delta1, an established Notch ligand, instructs bone marrow common myeloid progenitors and granulocyte-macrophage progenitors toward mast cell lineage at the expense of other granulocyte-macrophage lineages, depending on the function of the Notch2 gene. Notch2 signaling results in the up-regulation of Hes-1 and GATA3, whereas simultaneous overexpression of these transcription factors remarkably biases the progenitor fate toward the mast cell-containing colony-forming cells. C/EBPalpha mRNA was down-regulated in myeloid progenitors as a consequence of Hes-1 overexpression, in agreement with the recent proposal that the down-regulation of C/EBPalpha is necessary for mast cell fate determination. Taken together, signaling through Notch2 determines the fate of myeloid progenitors toward mast cell-producing progenitors, via coordinately up-regulating Hes-1 and GATA3.

Cellular basis of omentum activation and expansion revealed by single-cell RNA sequencing using a parabiosis model.

Although the physiological function of the omentum remains elusive, it has been proposed that it plays an important role in fat storage, immune regulation, and regeneration of injured tissues and organs. Although the omentum undergoes expansion upon activation, reports on the accurate assessment of increased cell types and the origin of the increased cells remain limited. To investigate this aspect, the omenta of parabiotic mice were subjected to activation using distinct fluorescent markers and single-cell RNA sequencing (scRNA-seq) was performed to identify circulation-derived omental cells. We found that a considerable number of circulating cells contributed to the activation of the omentum. The omental cells derived from circulating cells exhibited morphological features similar to those of fibroblasts. scRNA-seq revealed the existence of a novel cell population that co-expressed macrophage and fibroblast markers in the activated omentum, suggesting that it corresponded to circulating macrophage-derived fibroblast-like cells. Lineage tracing experiments revealed that most fibroblasts in the activated omentum were not derived from WT1-positive mesenchymal progenitors. The cell cluster also expressed various chemokine genes, indicating its role in the activation and recruitment of immune cells. These results provide important information regarding the activation of the omentum.

The role of Runx1/AML1 and Evi-1 in the regulation of hematopoietic stem cells.

Lineage-specific transcription factors must be precisely regulated during stem cell self-renewal and lineage commitment decisions. The role of specific transcription factors in hematopoietic stem cell (HSC) fate decisions has derived largely from genetic strategies, primarily gene-targeting and transgenic or retroviral overexpression experiments. From the previous experimental results, several transcription factors have been found to play critical roles in HSC physiology. Among them, we focus two transcription factors, Runx1/AML1 and Evi-1, in this review. During embryogenesis, both Runx1 and Evi-1 are essential for HSCs whereas in the adult, Runx1 and Evi-1 regulate HSCs negatively and positively, respectively.

Clinical efficacy of haematopoietic stem cell transplantation for adult adrenoleukodystrophy.

Accumulated experience supports the efficacy of allogenic haematopoietic stem cell transplantation in arresting the progression of childhood-onset cerebral form of adrenoleukodystrophy in early stages. For adulthood-onset cerebral form of adrenoleukodystrophy, however, there have been only a few reports on haematopoietic stem cell transplantation and the clinical efficacy and safety of that for adulthood-onset cerebral form of adrenoleukodystrophy remain to be established. To evaluate the clinical efficacy and safety of haematopoietic stem cell transplantation, we conducted haematopoietic stem cell transplantation on 12 patients with adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy in a single-institution-based prospective study. Through careful prospective follow-up of 45 male adrenoleukodystrophy patients, we aimed to enrol patients with adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy at early stages. Indications for haematopoietic stem cell transplantation included cerebral form of adrenoleukodystrophy or cerebello-brainstem form of adrenoleukodystrophy with Loes scores up to 13, the presence of progressively enlarging white matter lesions and/or lesions with gadolinium enhancement on brain MRI. Clinical outcomes of haematopoietic stem cell transplantation were evaluated by the survival rate as well as by serial evaluation of clinical rating scale scores and neurological and MRI findings. Clinical courses of eight patients who did not undergo haematopoietic stem cell transplantation were also evaluated for comparison of the survival rate. All the patients who underwent haematopoietic stem cell transplantation survived to date with a median follow-up period of 28.6 months (4.2-125.3 months) without fatality. Neurological findings attributable to cerebral/cerebellar/brainstem lesions became stable or partially improved in all the patients. Gadolinium-enhanced brain lesions disappeared or became obscure within 3.5 months and the white matter lesions of MRI became stable or small. The median Loes scores before haematopoietic stem cell transplantation and at the last follow-up visit were 6.0 and 5.25, respectively. Of the eight patients who did not undergo haematopoietic stem cell transplantation, six patients died 69.1 months (median period; range 16.0-104.1 months) after the onset of the cerebral/cerebellar/brainstem lesions, confirming that the survival probability was significantly higher in patients with haematopoietic stem cell transplantation compared with that in patients without haematopoietic stem cell transplantation (P = 0.0089). The present study showed that haematopoietic stem cell transplantation was conducted safely and arrested the inflammatory demyelination in all the patients with adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy when haematopoietic stem cell transplantation was conducted in the early stages. Further studies are warranted to optimize the procedures of haematopoietic stem cell transplantation for adolescent-/adult-onset cerebral form/cerebello-brainstem form of adrenoleukodystrophy.

Dual antitumor mechanisms of Notch signaling inhibitor in a T-cell acute lymphoblastic leukemia xenograft model.

Constitutive activation of Notch signaling is required for the proliferation of a subgroup of human T-cell acute lymphoblastic leukemias (T-ALL). Previous in vitro studies have demonstrated the therapeutic potential of Notch signaling inhibitors for treating T-ALL. To further examine this possibility, we applied a gamma-secretase inhibitor (GSI) to T-ALL xenograft models. Treatment of established subcutaneous tumors with GSI resulted in partial or complete regression of tumors arising from four T-ALL cell lines that were also sensitive to GSI in vitro. To elucidate the mechanism of action, we transduced DND-41 cells with the active form of Notch1 (aN1), which conferred resistance to in vitro GSI treatment. Nevertheless, in vivo treatment with GSI induced a partial but significant regression of subcutaneous tumors that developed from aN1-transduced DND-41 cells, whereas it induced complete regression of tumors that developed from mock-transduced DND-41 cells. These findings indicate that the remarkable efficacy of GSI might be attributable to dual mechanisms, directly via apoptosis of DND-41 cells through the inhibition of cell-autonomous Notch signaling, and indirectly via disturbance of tumor angiogenesis through the inhibition of non-cell-autonomous Notch signaling.

Gain-of-function mutations and copy number increases of Notch2 in diffuse large B-cell lymphoma.

Signaling through the Notch1 receptor has a pivotal role in early thymocyte development. Gain of Notch1 function results in the development of T-cell acute lymphoblastic leukemia in a number of mouse experimental models, and activating Notch1 mutations deregulate Notch1 signaling in the majority of human T-cell acute lymphoblastic leukemias. Notch2, another member of the Notch gene family, is preferentially expressed in mature B cells and is essential for marginal zone B-cell generation. Here, we report that 5 of 63 (approximately 8%) diffuse large B-cell lymphomas, a subtype of mature B-cell lymphomas, have Notch2 mutations. These mutations lead to partial or complete deletion of the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain, or a single amino acid substitution at the C-terminus of Notch2 protein. Furthermore, high-density oligonucleotide microarray analysis revealed that some diffuse large B-cell lymphoma cases also have increased copies of the mutated Notch2 allele. In the Notch activation-sensitive luciferase reporter assay in vitro, mutant Notch2 receptors show increased activity compared with wild-type Notch2. These findings implicate Notch2 gain-of-function mutations in the pathogenesis of a subset of B-cell lymphomas, and suggest broader roles for Notch gene mutations in human cancers.