Takashi Sonoki

Eμ/miR-125b transgenic mice develop lethal B-cell malignancies

MicroRNA-125b-1 (miR-125b-1) is a target of a chromosomal translocation t(11;14)(q24;q32) recurrently found in human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation results in overexpression of miR-125b controlled by immunoglobulin heavy chain gene (IGH) regulatory elements. In addition, we found that six out of twenty-one BCP-ALL patients without t(11;14)(q24;q32) showed overexpression of miR-125b. Interestingly, four out of nine patients with BCR/ABL-positive BCP-ALL and one patient with B-cell lymphoid crisis that had progressed from chronic myelogenous leukemia overexpressed miR-125b. To examine the role of the deregulated expression of miR-125b in the development of B-cell tumor in vivo, we generated transgenic mice mimicking the t(11;14)(q24;q32) (Eμ/miR-125b-TG mice). Eμ/miR-125b-TG mice overexpressed miR-125b driven by IGH enhancer and promoter and developed IgM-negative or IgM-positive lethal B-cell malignancies with clonal proliferation. B cells obtained from the Eμ/miR-125b-TG mice were resistant to apoptosis induced by serum starvation. We identified Trp53inp1, a pro-apoptotic gene induced by cell stress, as a novel target gene of miR-125b in hematopoietic cells in vitro and in vivo. Our results provide direct evidence that miR-125b has important roles in the tumorigenesis of precursor B cells.

Aberrant expression of RasGRP1 cooperates with gain-of-function NOTCH1 mutations in T-cell leukemogenesis

Ras guanyl nucleotide-releasing proteins (RasGRPs) are activators of Ras. Previous studies have indicated the possible involvement of RasGRP1 and RasGRP4 in leukemogenesis. Here, the predominant role of RasGRP1 in T-cell leukemogenesis is clarified. Notably, increased expression of RasGRP1, but not RasGRP4, was frequently observed in human T-cell malignancies. In a mouse bone marrow transplantation model, RasGRP1 exclusively induced T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) after a shorter latency when compared with RasGRP4. Accordingly, Ba/F3 cells transduced with RasGRP1 survived longer under growth factor withdrawal or phorbol ester stimulation than those transduced with RasGRP4, presumably due to the efficient activation of Ras. Intriguingly, NOTCH1 mutations resulting in a gain of function were found in 77% of the RasGRP1-mediated mouse T-ALL samples. In addition, gain-of-function NOTCH1 mutation was found in human T-cell malignancy with elevated expression of RasGRP1. Importantly, RasGRP1 and NOTCH1 signaling cooperated in the progression of T-ALL in the murine model. The leukemogenic advantage of RasGRP1 over RasGRP4 was attenuated by the disruption of a protein kinase C phosphorylation site (RasGRP1(Thr184)) not present on RasGRP4. In conclusion, cooperation between aberrant expression of RasGRP1, a strong activator of Ras, and secondary gain-of-function mutations of NOTCH1 have an important role in T-cell leukemogenesis.

Establishment of a novel CD20 negative mature B-cell line, WILL2, from a CD20 positive diffuse large B-cell lymphoma patient treated with rituximab.

Rituximab is a chimeric monoclonal antibody that binds to human CD20 antigen expressed on cell surface of mature B-cells, and depletes CD20 positive cells through direct signaling of apoptosis, complement activation and cellmediated cytotoxicity [1]. Use of this antibody accompanied with conventional chemotherapies resulted in better clinical outcome than chemotherapies only in various types of mature B-cell malignancies [2]. However, proliferation of CD20 negative B-cell clone after rituximab treatment is an emerging issue [3–6]. Here, we report the establishment of a novel CD20 negative mature B-cell line, WILL2, which was derived from a CD20 positive diffuse large B-cell lymphoma patient treated with rituximab repeatedly. A 62-year-old Japanese woman was admitted to our hospital because of malaise and liver dysfunction. Pathological examination of her lymph node biopsy specimen showed diffuse proliferation of CD20 positive B-cells (Fig. 1a); thus she was diagnosed as diffuse large B-cell lymphoma with CD10 and CD20 expression. Abnormal lymphocytes appeared in her peripheral blood (PB), and they were positive for CD20 as detected by flow cytometry (Fig. 1b). After eight courses of chemotherapy including rituximab, cyclophosphamide, doxorubicine, vincristine, and prednisolone (R-CHOP regimen), she received autoperipheral hematopoietic stem cell transplantation after high dose chemotherapy including melphalan, etoposide, cyclophosphamide dexamethazone, and rituximab (R-LEED regimen). She achieved complete remission for 3 months; however, she was died of relapsed disease sixteen months after initial diagnosis. A postmortem study revealed that the tumor cells infiltrated into multiple organs, pleura, and peritoneum. Pathological examination of a pleural tumor revealed diffuse proliferation of lymphoma cells lacking CD20, though CD10 remained to be positive (Fig. 1a). The WILL2 cell line was established from ascites obtained at the autopsy. The WILL2 cells were positive for CD10 and CD19 but negative for CD20 expression (Fig. 1b). Reverse transcription PCR (RT-PCR) analysis suggested that this cell line expresses less amount of CD20 mRNA than other CD20 positive B-cell lines (Fig. 1c). Karyotypic analysis of the WILL2 cells showed multiple structural abnormalities; 46,X,add(X)(q13),dup(1)(q32 q21),ins(1;?)(q21;?),der(4)t(4;6)(p16;p21),t(5;9)(q13;p22), t(8;22)(q24;q11),t(14;18)(q32;q21) (Fig. 1d). Fluorescent in situ hybridization (FISH) demonstrated a fusion of immunoglobulin lambda and C-MYC loci; indicating that t(8;22)(q24;q11) occurred (Fig. 1d). The t(14;18)(q32;q21) and 8q24 translocation were also found in the tumor cells in ascites at the autopsy as detected by FISH. (data not shown). Thus, the patient suffered from a diffuse large B-cell lymphoma with t(14;18)(q32;q21) and t(8;22) (q24;q11). A PCR method for amplification of T. Sonoki (&) N. Hanaoka H. Matsuoka H. Nakakuma Department of Hematology/Oncology, Wakayama Medical University, 811-1 Kimi-idera, Wakayama 633-1611, Japan e-mail: sonoki@wakayama-med.ac.jp

Pretreatment of leukemic cells with low-dose decitabine markedly enhances the cytotoxicity of gemtuzumab ozogamicin

Prognosis in relapsed/refractory acute myeloid leukemia (AML) is poor due to drug resistance. Gemtuzumab ozogamicin (GO) is a molecularly targeted drug, which consists of anti-CD33 antibody and calicheamicin. The high expression of CD33 antigen on most AML cells and exceptionally marked cytotoxicity of calicheamicin have attracted strong interest in GO, but clinical results on GO monotherapy have been disappointing, and methods of overcoming resistance to GO have accordingly been sought.

MicroRNA - 125b - 1 accelerates a C-terminal mutant of C/EBPα (C/EBPα-C m )-induced myeloid leukemia

MicroRNA-125b-1 (miR-125b-1) is a target of the chromosomal translocations t(11;14)(q24;q32) and t(2;11)(p21;q23), which are found in human B-lymphoid and myeloid malignancies, respectively. These translocations result in overexpression of mature miR-125b, consisting of 22 nucleotides. To analyze the role of miR-125b-1 in leukemogenesis, we created a bone marrow transplantation model using a retrovirus vector containing GFP expression elements. Sole transduction of miR-125b-1 into bone marrow cells resulted in expansion of hematopoietic cells expressing GFP. Compared with cells lacking GFP expression, we observed that GFP+/CD11b+ or GFP+/Gr−1+ cells were increased in the bone marrow and spleen. Although previous studies reported sole induction of miR-125b-induced leukemia, we did not find leukemic transformation in our model. Transduction of miR-125b-1 did accelerate myeloid tumors induced by a C-terminal mutant of CAAT-enhancer binding protein (C/EBPα-Cm), a class II-like mutation. As miR-125b has been shown to hasten the development of leukemia in a BCR/ABL-transduced animal model, our present results support the conclusion that overexpression of miR-125b cooperates with other genetic alterations in the pathogenesis of myeloid malignancies.

Molecular cloning of IGλ rearrangements using long‐distance inverse PCR (LDI‐PCR)

Malignant cells of mature B‐cell origin show tumor‐specific clonal immunoglobulin gene (IG) rearrangements, including V(D)J recombinations, nucleotide mutations, or translocations. Rapid molecular cloning of the breakpoint sequence by long‐distance inverse PCR (LDI‐PCR) has so far been applied to rearrangements targeted to IGH joining, IGH switch, and IGκ regions. We tended to apply LDI‐PCR method for cloning of IGλ rearrangements.

EphA4-deleted microenvironment regulates cancer development and leukemoid reaction of the isografted 4T1 murine breast cancer via reduction of an IGF1 signal

EphA4 belongs to the largest family of receptor tyrosine kinases (RTKs). Although EphA4 is highly expressed in the central nervous system, EphA4 has also been implicated in cancer progression. Most of the studies focus on the expression and function in tumor cells. It is unknown whether EphA4‐deleted microenvironment affects tumor progression. Some of cancers in animals and humans, such as 4T1 cancer cells, are known to produce a large amount of granulocyte colony‐stimulating factors (G‐CSF/Csf3) which can stimulate myeloproliferation, such as myeloid‐derived suppressor cells (MDSCs) leading to a poor recipient prognosis. We isografted 4T1 breast cancer cells into both EphA4‐knockout and control wild‐type female littermate mice. The results showed that the EphA4‐deleted host could inhibit primary tumor growth and tumor metastasis mainly by decreasing the amount of IGF1 synthesis in the circulation and locally tissues. The EphA4‐deleted microenvironment and delayed tumor development reduced the production of G‐CSF resulting in the decrease of splenomegaly and leukemoid reaction including MDSCs, which in turn inhibit the tumor progression. This inhibition can be reversed by supplying the mice with IGF1. However, an excess of IGF1 supply over demand to the control mice could not further accelerate the tumor growth and metastasis. A better understanding and re‐evaluation of the main role of IGF1 in regulating tumor progression could further enhance our cognition of the tumor development niche. Our findings demonstrated that EphA4‐deleted microenvironment impairs tumor‐supporting conditions. Conclusion: Host EphA4 expression regulates cancer development mainly via EphA4‐mediated IGF1 synthesis signal. Thus, targeting this signaling pathway may provide a potential therapeutic option for cancer treatment.

Bone marrow large B cell lymphoma bearing cyclin D3 expression: clinical, morphologic, immunophenotypic, and genotypic analyses of seven patients

We report seven large B cell lymphoma patients showing the involvement of tumor cells with cyclin D3 (CCND3) expression in bone marrow (BM) at the initial diagnosis. All patients presented with B symptoms, splenomegaly, and anemia/thrombocytopenia lacking hemophagocytosis in the BM. Five of the seven patients had suffered from immunological diseases or cancers. The tumor cells were divided into those with a lymphoplasmacytoid or blastoid appearance. Six cases were confirmed to express CD5 antigen on tumor cells. Three cases presented a chromosomal translocation between CCND3 and the immunoglobulin heavy chain (IGH) loci, t(6;14)(p21;q32). Three and two cases showed unmutated and mutated sequences of the variable region of IGH (VH), respectively, and one case showed deletion of an entire segment of VH. Two cases with t(6;14)(p21;q32) showed an unmutated VH sequence and chromosomal translocation within the switch region of IGH. Further studies are required to determine whether CCND3 expression is associated with a unique subset of diffuse large B cell lymphoma.

Successful treatment with liposomal doxorubicin for widespread Kaposi’s sarcoma and human herpesvirus-8 related severe hemophagocytic syndrome in a patient with acquired immunodeficiency syndrome

Hemophagocytic syndrome (HPS) sometimes occurres in patients with acquired immunodeficiency syndrome (AIDS). Human herpesvirus-8 (HHV-8)/Kaposi’s sarcoma (KS)-associated herpesvirus has so far been recognized as a trigger of HPS in immunosuppressed subject. We describe a 39-year-old man with AIDS who had widespread mucocutaneous and pulmonary KS and severe HPS. No opportunistic infections or neoplasias were detected except for KS. HHV-8-DNA could be detected in this patient by polymerase chain reaction (PCR) in the serum. Clinical symptoms and cytopenia originating from HPS were reduced by pulse therapy of corticosteroid, antibiotics, and virucides, but recurred with dose reduction of the steroid. Mucocutaneous tumors, edema, and dyspnea had progressed rapidly at this time. Liposomal doxorubicin was given and showed marked effects on both mucocutaneous and plural tumors. HPS also subsided and the serum HHV-8 DNA level markedly decreased after initial treatment with liposomal doxorubicin. HHV-8 clearance with liposomal doxorubicin has recently been reported. Liposomal doxorubicin suppressed not only the widespread KS tumors, but also HHV-8 viremia resulting in decreased HPS in this patient.

Capnocytophaga canimorsus sepsis in a methotrexate-treated patient with rheumatoid arthritis

Capnocytophaga canimorsus is a gram-negative rod that can be transmitted primarily by dog bites. This life-threatening organism commonly causes sepsis in patients with splenectomy or alcoholism. A 53-year-old rheumatoid arthritis male treated with methotrexate (MTX) for 5 years was admitted for a 4-day history of fever and dyspnea. He had been bitten on a finger by the family dog 4 days before onset. Laboratory tests revealed pancytopenia, acute renal failure, and evidence of disseminated intravascular coagulation, and he subsequently developed acute respiratory distress syndrome. Furthermore, blood cultures grew gram-negative bacilli and despite intensive treatment, he died 5 days after admission. Later, C. canimorsus was identified from his culture samples using a species-specific polymerase chain reaction. C. canimorsus infections should be considered in the differential diagnosis of sepsis for immunocompromised hosts following animal bites.