Masataka Takeshita

AML1/Runx1 negatively regulates quiescent hematopoietic stem cells in adult hematopoiesis.

Transcription factor AML1/Runx1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, whereas it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. These results indicate that the number of quiescent HSCs is negatively regulated by AML1.

Reverse seroconversion of Hepatitis B virus after hematopoietic stem cell transplantation

Hepatitis B virus (HBV) reactivation in patients previously positive for hepatitis B surface antibody (HBsAb), so-called reverse seroconversion, has been considered to be a rare complication after hematopoietic stem cell transplantation (HSCT). We experienced two patients who developed reverse seroconversion among nine who were HBsAb positive and Hepatitis B core antibody (HBcAb) positive before HSCT; one after autologous bone marrow transplantation (BMT) and another after allogeneic peripheral blood stem cell transplantation (PBSCT). We reviewed the literature and considered that reverse seroconversion of HBV after HSCT is not uncommon among HBsAb positive recipients. The use of corticosteroids, the lack of HBsAb in donor, and a decrease in serum HBsAb and HBcAb levels may predict reverse seroconversion after HSCT.

Evi-1 promotes para-aortic splanchnopleural hematopoiesis through up-regulation of GATA-2 and repression of TGF-b signaling

Evi‐1 is a zinc‐finger transcriptional factor whose inappropriate expression leads to leukemic transformation in mice and humans. Recently, it has been shown that Evi‐1 regulates proliferation of hematopoietic stem/progenitor cells at embryonic stage via GATA‐2 up‐regulation; however, detailed mechanisms underlying Evi‐1‐mediated early hematopoiesis are not fully understood. We therefore evaluated hematopoietic potential of Evi‐1 mutants using a cultivation system of murine para‐aortic splanchnopleural (P‐Sp) regions, and found that both the first zinc finger domain and the acidic domain were required for Evi‐1‐mediated hematopoiesis. The hematopoietic potential of Evi‐1 mutants was likely to be related to its ability to up‐regulate GATA‐2 expression. We also showed that the decreased colony forming capacity of Evi‐1‐deficient P‐Sp cells was successfully recovered by inhibition of TGF‐b signaling, using ALK5 inhibitor or retroviral transfer of dominant‐negative‐type Smad3. Our findings suggest that Evi‐1 promotes hematopoietic stem/progenitor expansion at the embryonic stage through up‐regulation of GATA‐2 and repression of TGF‐b signaling. (Cancer Sci 2008; 99: 1407–1413)

Crk‐associated substrate lymphocyte type regulates myeloid cell motility and suppresses the progression of leukemia induced by p210Bcr/Abl

The p210Bcr/Abl and p190Bcr/Abl fusion oncoproteins are known to cause chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL). Bcr/Abl phosphorylates several proteins that can lead to leukemogenesis. Crk‐associated substrate lymphocyte type (Cas‐L)/human enhancer of filamentation‐1 (HEF1)/neural precursor cell expressed, developmentally down‐regulated 9 (NEDD9) is an adapter protein at focal adhesions known to be associated with solid tumor metastasis. Crk‐associated substrate lymphocyte type has also been reported to be tyrosine phosphorylated by p190Bcr/Abl. We demonstrated that Cas‐L was expressed in murine granulocytes, as well as in lymphocytes, and that Cas‐L‐deficient (Cas‐L−/−) granulocytes had increased migratory activity and decreased adhesiveness. To examine whether Cas‐L was involved in leukemogenesis by p210Bcr/Abl, we generated Cas‐L−/− p210Bcr/Abl transgenic mice. The mice displayed early development of myeloproliferative neoplasm seen in the chronic phase of CML, which resulted in the early death of the mice. Pathologically, increased infiltration of myeloid cells into several tissues was detected in the absence of Cas‐L. In a hematopoietic reconstitution assay, Cas‐L−/− p210Bcr/Abl transgenic cells showed a low population in the spleen, although only their myeloid cell population was normal. Thus, Cas‐L seems to regulate the progression of CML in a negative way, presumably by attenuating extramedullary hyperplasia. (Cancer Sci 2011; 102: 2109–2117)

AML1-Evi-1 specifically transforms hematopoietic stem cells through fusion of the entire Evi-1 sequence to AML1

The t(3;21) chromosomal translocation seen in blastic crisis of chronic myeloid leukemia and secondary leukemias results in a formation of a chimeric protein AML1-Evi-1, which suppresses wild-type AML1 function. Loss of AML1 function causes expansion of hematopoietic progenitor cells, whereas it is not sufficient for the development of leukemia. To identify essential mechanisms through which AML1-Evi-1 exerts full leukemogenic potential, we introduced AML1-Evi-1 and its mutants in murine bone marrow cells, and evaluated their transforming activities by colony replating assays. The transforming activity of AML1-Evi-1 was lost when any of the known functional domains of Evi-1 was deleted from the chimeric protein, and forced expression of Evi-1 did not transform the AML1-deleted bone marrow cells. Unlike the MLL-ENL and AML1-ETO leukemia-related chimeric proteins, AML1-Evi-1 could transform only the hematopoietic stem cell fraction. Moreover, AML1-Evi-1-transformed cells show a cell-marker profile distinct from that of the cells transformed by AML1-ETO, which also suppresses AML1 function. Thus, leukemogenic activity of AML1-Evi-1 may be due to activation of molecular mechanisms distinct from those activated by MLL-ENL or AML1-ETO in the hematopoietic stem cell fractions.

The Prognostic Impact of Dose-attenuated R-CHOP Therapy for Elderly Patients with Diffuse Large B-cell Lymphoma

Objective Although R-CHOP (rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisone) is a standard therapy for diffuse large B-cell lymphoma (DLBCL), the optimal dose for elderly patients remains unclear. Methods and Patients We retrospectively verified our R-CHOP dose-attenuation system implemented from 2005 for DLBCL patients. Among the 115 DLBCL patients treated during 2001-2010, 33 patients treated during 2001-2005 received R-CHOP doses adjusted according to physicians’ decisions (PHY group). Eighty-two patients treated after 2005 received adjusted R-CHOP doses according to a unified dose-attenuation system (UNI group). Patients aged <60, 60-69, 70-79, and ≥80 years received the standard R-CHOP, 100% R-CHO+P (50 mg/m2), 100% R+75% CHO+P (40 mg/m2), and 100% R+50% CHO+P (30 mg/m2), respectively. We compared the responses, survival, and treatment cessation between the PHY and UNI groups. Results The patients’ characteristics between both groups were closely comparable. All PHY patients received randomly adjusted R-CHOP doses; 94% of UNI patients received scheduled doses. The complete response rates differed significantly between the UNI (77%) and PHY patients (50%) (p=0.011). The two-year event-free survival rates were 50% and 32% in the UNI and PHY groups, respectively (p=0.0083). The two-year OS rates were 77% and 72% in the UNI and PHY group (p=0.16). Among the patients aged >70 years (n=59) overall survival was shorter in the PHY group (62%) than in the UNI group (72%; p=0.02). The UNI group received higher anti-tumor agent doses than the PHY group. The therapy discontinuation rates were 5% in the UNI group and 24% in the PHY group. Conclusion Carrying out unified dose reduction may improve the efficacy and prognosis among elderly DLBCL patients.