Itaru Kato

Frequent inactivation of A20 in B-cell lymphomas

A20 is a negative regulator of the NF-κB pathway and was initially identified as being rapidly induced after tumour-necrosis factor-α stimulation. It has a pivotal role in regulation of the immune response and prevents excessive activation of NF-κB in response to a variety of external stimuli; recent genetic studies have disclosed putative associations of polymorphic A20 (also called TNFAIP3) alleles with autoimmune disease risk. However, the involvement of A20 in the development of human cancers is unknown. Here we show, using a genome-wide analysis of genetic lesions in 238 B-cell lymphomas, that A20 is a common genetic target in B-lineage lymphomas. A20 is frequently inactivated by somatic mutations and/or deletions in mucosa-associated tissue lymphoma (18 out of 87; 21.8%) and Hodgkin’s lymphoma of nodular sclerosis histology (5 out of 15; 33.3%), and, to a lesser extent, in other B-lineage lymphomas. When re-expressed in a lymphoma-derived cell line with no functional A20 alleles, wild-type A20, but not mutant A20, resulted in suppression of cell growth and induction of apoptosis, accompanied by downregulation of NF-κB activation. The A20-deficient cells stably generated tumours in immunodeficient mice, whereas the tumorigenicity was effectively suppressed by re-expression of A20. In A20-deficient cells, suppression of both cell growth and NF-κB activity due to re-expression of A20 depended, at least partly, on cell-surface-receptor signalling, including the tumour-necrosis factor receptor. Considering the physiological function of A20 in the negative modulation of NF-κB activation induced by multiple upstream stimuli, our findings indicate that uncontrolled signalling of NF-κB caused by loss of A20 function is involved in the pathogenesis of subsets of B-lineage lymphomas.

A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.

Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.

Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoiesis

HAX1 was identified as the gene responsible for the autosomal recessive type of severe congenital neutropenia. However, the connection between mutations in the HAX1 gene and defective granulopoiesis in this disease has remained unclear, mainly due to the lack of a useful experimental model for this disease. In this study, we generated induced pluripotent stem cell lines from a patient presenting for severe congenital neutropenia with HAX1 gene deficiency, and analyzed their in vitro neutrophil differentiation potential by using a novel serum- and feeder-free directed differentiation culture system. Cytostaining and flow cytometric analyses of myeloid cells differentiated from patient-derived induced pluripotent stem cells showed arrest at the myeloid progenitor stage and apoptotic predisposition, both of which replicated abnormal granulopoiesis. Moreover, lentiviral transduction of the HAX1 cDNA into patient-derived induced pluripotent stem cells reversed disease-related abnormal granulopoiesis. This in vitro neutrophil differentiation system, which uses patient-derived induced pluripotent stem cells for disease investigation, may serve as a novel experimental model and a platform for high-throughput screening of drugs for various congenital neutrophil disorders in the future.

Identification of Hepatic Niche Harboring Human Acute Lymphoblastic Leukemic Cells via the SDF-1/CXCR4 Axis

In acute lymphoblastic leukemia (ALL) patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc null mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.

Imatinib use immediately before stem cell transplantation in children with Philadelphia chromosome-positive acute lymphoblastic leukemia: Results from Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) Study Ph+ALL04

Incorporation of imatinib into chemotherapeutic regimens has improved the prognosis of children with Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL). We investigated a role of imatinib immediately before hematopoietic stem cell transplantation (HSCT). Children with Ph+ALL were enrolled on JPLSG Ph+ALL 04 Study within 1 week of initiation of treatment for ALL. Treatment regimen consisted of Induction phase, Consolidation phase, Reinduction phase, 2 weeks of imatinib monotherapy phase, and HSCT phase (Etoposide+CY+TBI conditioning). Minimal residual disease (MRD), the amount of BCR–ABL transcripts, was measured with the real‐time PCR method. The study was registered in UMIN‐CTR: UMIN ID C000000290. Forty‐two patients were registered and 36 patients (86%) achieved complete remission (CR). Eight of 17 patients (47%) who had detectable MRD at the beginning of imatinib monotherapy phase showed disappearance or decrease in MRD after imatinib treatment. Consequently, 26 patients received HSCT in the first CR and all the patients had engraftment and no patients died because of complications of HSCT. The 4‐year event‐free survival rates and overall survival rates among all the 42 patients were 54.1 ± 7.8% and 78.1 ± 6.5%, respectively. Four of six patients who did achieve CR and three of six who relapsed before HSCT were salvaged with imatinib‐containing chemotherapy and subsequently treated with HSCT. The survival rate was excellent in this study although all patients received HSCT. A longer use of imatinib concurrently with chemotherapy should eliminate HSCT in a subset of patients with a rapid clearance of the disease.

Clonal selection in xenografted TAM recapitulates the evolutionary process of myeloid leukemia in Down syndrome

Transient abnormal myelopoiesis (TAM) is a clonal preleukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism of leukemogenesis in this disorder, a xenograft model of TAM was established using NOD/Shi-scid, interleukin (IL)-2Rγ(null) mice. Serial engraftment after transplantation of cells from a TAM patient who developed ML-DS a year later demonstrated their self-renewal capacity. A GATA1 mutation and no copy number alterations (CNAs) were detected in the primary patient sample by conventional genomic sequencing and CNA profiling. However, in serial transplantations, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, which are clinically associated with ML-DS. Detailed genomic analysis identified minor subclones with a 16q deletion or this distinct GATA1 mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with varying leukemia-initiating potential already exist in the neonatal TAM phase, and ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model of TAM may provide unique insight into the evolutionary process of leukemia.

Iodine-131-metaiodobenzylguanidine therapy with reduced-intensity allogeneic stem cell transplantation in recurrent neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood, and iodine‐131‐metaiodobenzylguanidine (MIBG) therapy is a new approach for grade IV neuroblastoma. We describe the case history of a 3‐year‐old girl with recurrent neuroblastoma who received MIBG therapy with reduced‐intensity allogeneic stem cell transplantation (RIST) because of an extensive bone marrow involvement. The post‐transplant course was uneventful and complete chimerism was obtained. Neither acute nor chronic graft‐versus‐host disease (GVHD) was observed. The patient remained in remission for 3 months after RIST until the second relapse. MIBG therapy combined with RIST warrants further trials. Pediatr Blood Cancer 2008;50:676–678.

Bullous exudative retinal detachment due to infiltration of leukemic cells in a child with acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is known to cause several ocular involvements, but exudative retinal detachment is a rare complication. We describe a case report of a 4-year-old boy with T cell ALL who developed bilateral exudative retinal detachment caused by leukemic infiltration in the retinas after achieving hematological remission. Intravenous steroid pulse therapy and local irradiation reversed the condition, but it recurred concurrently with disease progression after a second relapse in the bone marrow. It is suggested that ophthalmic examination is crucial for ALL patients, especially for those whose white blood cell count is very high at onset.

Successful reduced-intensity stem cell transplantation for GATA2 deficiency before progression of advanced MDS.

A 13‐yr‐old boy bearing lymphedema and congenital deafness had distinct hematological abnormalities consisting of reduced monocytes, B cells, and dendritic cells in the peripheral blood as well as MDS with normal karyotype in the bone marrow. The patient was diagnosed with Emberger syndrome by sequencing of GATA2 DNA, and underwent RIST from an HLA‐matched unrelated donor. Prompt engraftment and immunological reconstitution were observed without any severe RRT. As most patients with GATA2 anomaly died due to the development of AML or active infections, RIST could be a promising treatment option before progression of advanced MDS.

In vitro expansion of CD34(+)CD38(-) cells under stimulation with hematopoietic growth factors on AGM-S3 cells in juvenile myelomonocytic leukemia.

Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34+ cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34+ cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34+ cells on AGM-S3 cells. The expansion potential of CD34+ cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34+ cells were negative for CD38 and cryopreservable. Cultured JMML CD34+CD38− cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34+ cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.