Kazuki Taoka

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.

The effect of iron overload and chelation on erythroid differentiation

We investigated the mechanisms of hematopoietic disorders caused by iron overload and chelation, in particular, the inhibition of erythroblast differentiation. Murine c-kit+ progenitor cells or human CD34+ peripheral blood hematopoietic progenitors were differentiated in vitro to the erythroid lineage with free iron and/or an iron chelator. Under iron overload, formation of erythroid burst-forming unit colonies and differentiation to mature erythroblasts were significantly suppressed; these effects were canceled by iron chelation with deferoxamine (DFO). Moreover, excessive iron burden promoted apoptosis in immature erythroblasts by elevating intracellular reactive oxygen species (ROS). Interestingly, both DFO and a potent anti-oxidant agent reduced intracellular ROS levels and suppressed apoptosis, thus restoring differentiation to mature erythroblasts. Accordingly, intracellular ROS may represent a new therapeutic target in the treatment of iron overload.

Targeted gene correction of RUNX1 in induced pluripotent stem cells derived from familial platelet disorder with propensity to myeloid malignancy restores normal megakaryopoiesis

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is an autosomal dominant disease associated with a germline mutation in the RUNX1 gene and is characterized by thrombocytopenia and an increased risk of developing myeloid malignancies. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a patient with FPD/AML possessing a nonsense mutation R174X in the RUNX1 gene. Consistent with the clinical characteristics of the disease, FPD iPSC-derived hematopoietic progenitor cells were significantly impaired in undergoing megakaryocytic differentiation and subsequent maturation, as determined by colony-forming cell assay and surface marker analysis. Notably, when we corrected the RUNX1 mutation using transcription activator-like effector nucleases in conjunction with a donor plasmid containing normal RUNX1 cDNA sequences, megakaryopoiesis and subsequent maturation were restored in FPD iPSC-derived hematopoietic cells. These findings clearly indicate that the RUNX1 mutation is robustly associated with thrombocytopenia in patients with FPD/AML, and transcription activator-like effector nuclease-mediated gene correction in iPSCs generated from patient-derived cells could provide a promising clinical application for treatment of the disease.

The shortest isoform of C/EBPβ, 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 β (C/EBPβ) gene and overexpression of its protein. These findings imply that C/EBPβ is a candidate gene that collaborates with Evi1 in leukemogenesis. Cotransduction of Evi1 and the shortest isoform of C/EBPβ, 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/EBPβ (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/EBPβ as a therapeutic target.

Treatment of primary intraocular lymphoma with rituximab, high dose methotrexate, procarbazine, and vincristine chemotherapy, reduced whole-brain radiotherapy, and local ocular therapy.

Hydroxyurea. Blood, 89, 1078–1088. Ulug, P., Vasavda, N., Kumar, R., Keir, L., Awogbade, M., Cunningham, J., Rees, D.C., Menzel, S. & Thein, S.L. (2008) Hydroxyurea therapy lowers circulating DNA levels in sickle cell anemia. American Journal of Hematology, 83, 714–716. Vasavda, N., Ulug, P., Kondaveeti, S., Ramasamy, K., Sugai, T., Cheung, G., Rees, D.C., Awogbade, M., Bannister, S., Cunningham, J., Menzel, S. & Thein, S.L. (2007) Circulating DNA: a potential marker of sickle cell crisis. British Journal of Haematology, 139, 331–336. Vasavda, N., Badiger, S., Rees, D., Height, S., Howard, J. & Thein, S.L. (2008) The presence of alphathalassaemia trait blunts the response to hydroxycarbamide in patients with sickle cell disease. British Journal of Haematology, 143, 589–592. Voskaridou, E., Christoulas, D., Bilalis, A., Plata, E., Varvagiannis, K., Stamatopoulos, G., Sinopoulou, K., Balassopoulou, A., Loukopoulos, D. & Terpos, E. (2010) The effect of prolonged administration of hydroxyurea on morbidity and mortality in adult patients with sickle cell syndromes: results of a 17-year, single-center trial (LaSHS). Blood, 115, 2354–2363. Ware, R.E. & Aygun, B. (2009) Advances in the use of hydroxyurea. Hematology American Society of Hematology Education Program, 2009, 62–69.

Generation of induced pluripotent stem cells derived from primary and secondary myelofibrosis patient samples.

Induced pluripotent stem cells (iPS) derived from disease cells are expected to provide a new experimental material, especially for diseases from which samples are difficult to obtain. In this study, we generated iPS from samples from patients with primary and secondary myelofibrosis. The primary myelofibrosis cells had chromosome 13q deletions, and the secondary myelofibrosis (SMF) cells had JAK2V617F mutations. The myelofibrosis patient cell-derived iPS (MF-iPS) were confirmed as possessing these parental disease-specific genomic markers. The capacity to form three germ layers was confirmed by teratoma assay. By co-culture with specific feeder cells and cytokines, MF-iPS can re-differentiate into blood progenitor cells and finally into megakaryocytes. We found that mRNA levels of interleukin-8, one of the candidate cytokines related to the pathogenesis of myelofibrosis, was elevated predominantly in megakaryocytes derived from MF-iPS. Because megakaryocytes from myelofibrosis clones are considered to produce critical mediators to proliferate fibroblasts in the bone marrow and iPS can provide differentiated cells abundantly, the disease-specific iPS we established should be a good research tool for this intractable disease.

Progressive transition of Epstein–Barr virus associated lymphoproliferative disease subtypes with the development of lung cancer

Epstein-Barr virus (EBV) associated lymphoproliferative disease (LPD) comprises a wide spectrum of clinical and pathological features [1]. This variety is most systematically categorized in post-transplant lymphoproliferative disorders (PTLD) [2], in which subtypes are ordered according to disease progression from reactive polyclonal proliferation to large cell lymphoma. However, whether this categorization is applicable to LPDs other than PTLD is not well explored. Here, we present a nontransplant case of EBV-LPD that initially presented as a polyclonal self-limiting proliferation and later transited to large cell lymphoma. This transition was associated with the progression of lung cancer and its therapy. Our case demonstrates that stepwise progression of LPD is a feature that can be observed in non-PTLD cases of EBV-LPD.

Combined intravitreal methotrexate and immunochemotherapy followed by reduced-dose whole-brain radiotherapy for newly diagnosed B-cell primary intraocular lymphoma

Primary intraocular lymphoma (IOL) has a propensity for central nervous system (CNS) relapse within 2 years of initial diagnosis, affecting clinical outcome. To reduce CNS relapse, we performed the combination treatment protocols of intravitreal methotrexate injections, methotrexate‐based systemic induction chemotherapy and consolidation high‐dose cytarabine and reduced‐dose whole brain radiation therapy (rdWBRT, 23·4 Gy) for B‐cell primary IOL with or without newly diagnosed CNS involvement. All patients underwent longitudinal brain magnetic resonance imaging (MRI) and cognitive assessment for evaluation of treatment‐induced leucoencephalopathy. Seventeen patients initiated and 16 completed the protocol treatment. CNS relapse occurred in 2 patients and intraocular relapse in 3. Four‐year progression‐free survival (PFS) was 74·9% and 4‐year overall survival (OS) was 86·3%, with a median follow‐up period of 48·9 months. Of 11 patients without CNS involvement, 1 had CNS relapse and 3 intraocular relapse, and 4‐year PFS and OS was 72·7% and 88·9%, respectively. Although white matter abnormalities shown by MRI were significantly increased at 4 years after rdWBRT, only one patient developed mild cognitive impairment. The combination of intravitreal chemotherapy, prophylactic systemic chemotherapy and rdWBRT for primary IOL showed a potential to reduce CNS relapse rate and improved 4‐year PFS and OS without increase of cognitive dysfunction.

Comparison of garenoxacin with levofloxacin as antimicrobial prophylaxis in acute myeloid leukemia

OBJECTIVE Levofloxacin is widely used as antimicrobial prophylaxis against high-risk chemotherapy-induced neutropenia. Garenoxacin, a fluoroquinolone developed in Japan, shows a stronger in vitro antimicrobial activity against Gram-positive bacteria than levofloxacin. METHODS We retrospectively analyzed high-risk patients with acute myeloid leukemia who were administered garenoxacin (n = 36) or levofloxacin (n = 120) during chemotherapy. We compared the profiles of infections between these fluoroquinolones. RESULTS Febrile events occurred in 31 (86%) and 93 (78%) cases in the garenoxacin and levofloxacin group, respectively (P = 0.35). Bloodstream infections by Gram-positive bacteria were recorded in one (3%) case in the garenoxacin group and 25 (21%) cases in the levofloxacin group (P < 0.01). In contrast, bloodstream infections by Gram-negative microorganisms were identified in five (4%) cases in the levofloxacin group and eight (22%) cases in the garenoxacin group (P < 0.01). CONCLUSIONS These results indicate that there may be substantial differences in the antimicrobial spectrum between different fluoroquinolones. Although there are several biases due to rather small sample size and the retrospective nature, we should take the differences into consideration when we administer a prophylactic fluoroquinolone to a patient with hematological disease.

ADAM8 Is an Antigen of Tyrosine Kinase Inhibitor-Resistant Chronic Myeloid Leukemia Cells Identified by Patient-Derived Induced Pluripotent Stem Cells

Summary Properties of cancer stem cells involved in drug resistance and relapse have significant effects on clinical outcome. Although tyrosine kinase inhibitors (TKIs) have dramatically improved survival of patients with chronic myeloid leukemia (CML), TKIs have not fully cured CML due to TKI-resistant CML stem cells. Moreover, relapse after discontinuation of TKIs has not been predicted in CML patients with the best TKI response. In our study, a model of CML stem cells derived from CML induced pluripotent stem cells identified ADAM8 as an antigen of TKI-resistant CML cells. The inhibition of expression or metalloproteinase activity of ADAM8 restored TKI sensitivity in primary samples. In addition, residual CML cells in patients with optimal TKI response were concentrated in the ADAM8+ population. Our study demonstrates that ADAM8 is a marker of residual CML cells even in patients with optimal TKI response and would be a predictor of relapse and a therapeutic target of TKI-resistant CML cells.