Chie Onishi

Survivin Selectively Modulates Genes Deregulated in Human Leukemia Stem Cells

ITD-Flt3 mutations are detected in leukemia stem cells (LSCs) in acute myeloid leukemia (AML) patients. While antagonizing Survivin normalizes ITD-Flt3-induced acute leukemia, it also impairs hematopoietic stem cell (HSC) function, indicating that identification of differences in signaling pathways downstream of Survivin between LSC and HSC are crucial to develop selective Survivin-based therapeutic strategies for AML. Using a Survivin-deletion model, we identified 1,096 genes regulated by Survivin in ITD-Flt3-transformed c-kit+, Sca-1+, and lineageneg (KSL) cells, of which 137 are deregulated in human LSC. Of the 137, 124 genes were regulated by Survivin exclusively in ITD-Flt3+ KSL cells but not in normal CD34neg KSL cells. Survivin-regulated genes in LSC connect through a network associated with the epidermal growth factor receptor signaling pathway and falls into various functional categories independent of effects on apoptosis. Pathways downstream of Survivin in LSC that are distinct from HSC can be potentially targeted for selective anti-LSC therapy.

Internal tandem duplication of FLT3 deregulates proliferation and differentiation and confers resistance to the FLT3 inhibitor AC220 by Up-regulating RUNX1 expression in hematopoietic cells

Internal tandem duplication in the FLT3 gene (FLT3/ITD), which is found in patients with acute myeloid leukemia (AML), causes resistance to FLT3 inhibitors. We found that RUNX1, a transcription factor that regulates normal hematopoiesis, is up-regulated in patients with FLT3/ITD+ AML. While RUNX1 can function as a tumor suppressor, recent data have shown that RUNX1 is required for AML cell survival. In the present study, we investigated the functional role of RUNX1 in FLT3/ITD signaling. FLT3/ITD induced growth factor-independent proliferation and impaired G-CSF mediated myeloid differentiation in 32D hematopoietic cells, coincident with up-regulation of RUNX1 expression. Silencing of RUNX1 expression significantly decreased proliferation and secondary colony formation, and partially abrogated the impaired myeloid differentiation of FLT3/ITD+ 32D cells. Although the number of FLT3/ITD+ 32D cells declined after incubation with the FLT3/ITD inhibitor AC220, the cells became refractory to AC220, concomitant with up-regulation of RUNX1. Silencing of RUNX1 abrogated the emergence and proliferation of AC220-resistant FLT3/ITD+ 32D cells in the presence of AC220. Our data indicate that FLT3/ITD deregulates cell proliferation and differentiation and confers resistance to AC220 by up-regulating RUNX1 expression. These findings suggest an oncogenic role for RUNX1 in FLT3/ITD+ cells and that inhibition of RUNX1 function represents a potential therapeutic strategy in patients with refractory FLT3/ITD+ AML.

Internal tandem duplication mutations in FLT3 gene augment chemotaxis to Cxcl12 protein by blocking the down-regulation of Rho-associated kinase via the Cxcl12/Cxcr4 signaling axis.

Background: ITD-FLT3 mutations induce the refractory phenotype in patients with AML and enhance cell migration toward Cxcl12. Results: The enhanced cell migration toward Cxcl12 by ITD-FLT3 is mediated by inhibiting the down-regulation of Rho-associated kinase. Conclusion: ITD-FLT3 enhances Cxcl12-induced cell migration by modulating the expression of Rho-associated kinase. Significance: Rho-associated kinase may be therapeutically manipulated for the treatment of ITD-FLT3+ AML patients. Internal tandem duplication mutations in the Flt3 gene (ITD-FLT3) enhance cell migration toward the chemokine Cxcl12, which is highly expressed in the therapy-protective bone marrow niche, providing a potential mechanism underlying the poor prognosis of ITD-FLT3+ acute myeloid leukemia. We aimed to investigate the mechanisms linking ITD-FLT3 to increased cell migration toward Cxcl12. Classification of the expression of Cxcl12-regulated genes in ITD-FLT3+ cells demonstrated that the enhanced migration of ITD-FLT3+ cells toward Cxcl12 was associated with the differential expression of genes downstream of Cxcl12/Cxcr4, which are functionally distinct from those expressed in ITD-FLT3− cells but are independent of the Cxcr4 expression levels. Among these differentially regulated genes, the expression of Rock1 in the ITD-FLT3+ cells that migrated toward Cxcl12 was significantly higher than in ITD-FLT3− cells that migrated toward Cxcl12. In ITD-FLT3− cells, Rock1 expression and Mypt1 phosphorylation were transiently up-regulated but were subsequently down-regulated by Cxcl12. In contrast, the presence of ITD-FLT3 blocked the Cxcl12-induced down-regulation of Rock1 and early Mypt1 dephosphorylation. Likewise, the FLT3 ligand counteracted the Cxcl12-induced down-regulation of Rock1 in ITD-FLT3− cells, which coincided with enhanced cell migration toward Cxcl12. Rock1 antagonists or Rock1 shRNA abolished the enhanced migration of ITD-FLT3+ cells toward Cxcl12. Our findings demonstrate that ITD-FLT3 increases cell migration toward Cxcl12 by antagonizing the down-regulation of Rock1 expression. These findings suggest that the aberrant modulation of Rock1 expression and activity induced by ITD-FLT3 may enhance acute myeloid leukemia cell chemotaxis to the therapy-protective bone marrow niche, where Cxcl12 is abundantly expressed.

Asian variant of intravascular large B cell lymphoma causes patients to frequently develop the syndrome of inappropriate antidiuretic hormone secretion

The Asian variant of intravascular large B cell lymphoma is a special type of intravascular lymphoma with hemophagocytic syndrome and hypercytokinemia including interleukin-6, which stimulates antidiuretic hormone synthesis in the hypothalamus. We present here that the syndrome of inappropriate antidiuretic hormone secretion frequently occurs in patients with the Asian variant of intravascular large B cell lymphoma. The syndrome of inappropriate antidiuretic hormone secretion was found in eight of 118 (6.8%) lymphoma patients at the first diagnosis. Although there were six (5.1%) among 118 lymphoma patients with the Asian variant of intravascular large B cell lymphoma, four of the six patients (66.7%) developed the syndrome of inappropriate antidiuretic hormone secretion. In four patients with the Asian variant of intravascular large B cell lymphoma with the syndrome of inappropriate antidiuretic hormone secretion, elevated serum interleukin-6 and low sodium levels were almost normalized after chemotherapy. The Asian variant of intravascular large B cell lymphoma patients frequently develop the syndrome of inappropriate antidiuretic hormone secretion, and interleukin-6 might play a role in the occurrence of this disease. We should pay attention to hyponatremia caused by the syndrome of inappropriate antidiuretic hormone secretion in patients with the Asian variant of intravascular large B cell lymphoma.

Spontaneous Regression of Intravascular Large B-Cell Lymphoma and Apoptosis of Lymphoma Cells: A Case Report

A 61-year-old Japanese woman presented with hemophagocytic syndrome (HPS) and suffered from intravascular large B-cell lymphoma (IVLBCL). After a few days of supportive care, her condition improved without any anti-cancer drugs or steroids. She experienced recurrences of HPS at 15 mon and 21 mon after first presentation, but lymphoma cells were not observed. Relapse of IVLBCL with pulmonary involvement occurred 27 mon after first presentation. She underwent R-CHOP therapy followed by autologous stem cell transplantation. She is currently alive and without lymphoma. Immunostaining by anti-ssDNA suggested that spontaneous regression may have been due to apoptosis of the lymphoma cells.

Erratum: Internal tandem duplication mutations in FLT3 gene augment chemotaxis to Cxcl12 protein by blocking the down-regulation of rho-associated kinase via the Cxcl12/Cxcr4 signaling axis (Journal of Biological Chemistry)

Chie Onishi, Satomi Mori-Kimachi, Tomohiro Hirade, Mariko Abe, Takeshi Taketani, Junji Suzumiya, Toshitsugu Sugimoto, Seiji Yamaguchi, Reuben Kapur, and Seiji Fukuda Dr. Onishi’s name was misspelled. The correct spelling is Chie Onishi, as shown in the author line. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 47, p. 28356, November 20, 2015

Synergistic combination therapy with cotylenin A and vincristine in multiple myeloma models

Multiple myeloma is a malignant proliferative disease of plasma cells in the bone marrow and remains largely incurable. Cotylenin A, a fusicoccane diterpene glycoside with a complex sugar moiety, was isolated as a plant-growth regulator. Cotylenin A has been shown to inhibit the growth of various cancer cells. Herein, we examined the anti-myeloma effects of cotylenin A using five human myeloma cell lines (RPMI-8226, KMS-11, KMS-26, KMS-12 PE and KMS-12 BM) and xenografts in immunodeficient mice. Cotylenin A and vincristine synergistically inhibited the growth and induced apoptosis in myeloma cells. While other microtubule-disturbing agents also showed co-operative effects with cotylenin A, other anticancer agents, such as doxorubicin, cisplatin, camptothecin, methotrexate, gemcitabine and 5-fluorouracil, did not show such co-operation with cotylenin A. These differences might be attributed to the effects on autophagic responses. Combined treatment with cotylenin A and vincristine induced autophagy (formation of LC3-II and degradation of p62 protein). However, doxorubicin did not enhance the autophagy induced by cotylenin A. A colony-forming assay indicated that the combined treatment with cotylenin A and vincristine more effectively suppressed the formation of large colonies, which have higher self-renewal activity than vincristine alone. Expression of pluripotency-associated transcription factor Sox2 mRNA in RPMI-8226 myeloma cells was significantly suppressed by treatment with cotylenin A. Combined treatment with cotylenin A and vincristine significantly inhibited the growth of KMS-26 myeloma cells as xenografts. Our results suggest that the combination of cotylenin A and vincristine may have therapeutic value. Recently, it was reported that cotylenin A modulates the 14-3-3 intracellular signaling pathway. The 14-3-3 proteins may be novel targets in treating myeloma. However, our study could not explain how the sensitization to vincristine is related to the effects of cotylenin A on the 14-3-3 signaling pathway and further studies will be needed.

Trafficking of Acute Leukemia Cells Chemokine Receptor Pathways that Modulate Leukemia Cell Dissemination

Recent advances in therapeutic regimens targeting aberrant proliferation of leukemia cells have led to a decline in the mortality of patients with acute leukemia. Nevertheless, a number of patients still suffer from refractory disease or relapse, indicating that more innovative and effective therapeutic strategies are required to achieve superior outcomes. One of the major indicators of poor prognosis in leukemia patients is extramedullary infiltration or dissemination of leukemia cells. In particular, leukemia cell infiltration into the central nervous system is one of the major complications negatively influencing prognosis. However, little is known about the mechanisms responsible for extramedullary dissemination of leukemia cells compared to those responsible for solid tumor metastasis. The chemokine SDF1 (stromal derived factor-1) and its receptor CXCR4 regulate trafficking of normal hematopoietic stem cells (HSC) as well as metastasis of solid tumor cells. Similarly, the majority of acute myeloid leukemia (AML) cells express CXCR4 and migrate in response to SDF1, suggesting that the SDF1/CXCR4 axis may be involved in the dissemination of AML cells to various organs. A recent study also suggests that chemokine/chemokine receptor interactions orchestrate extramedullary dissemination in childhood AML. Moreover, signaling through the chemokine receptor CCR7 is crucial for infiltration of T-ALL cells in the central nervous system. Our recent studies indicate that internal tandem duplication mutations of the Flt3 gene (ITD-Flt3), found in patients with AML, significantly augments migration of hematopoietic cells by deregulating CXCR4 signaling that are qualitatively distinct from cells lacking ITD-Flt3 and facilitate their infiltration to visceral organs while decreasing their homing to the bone marrow. ITD-Flt3 regulates overlapping as well as functionally distinct signaling pathways down-stream of SDF1/CXCR4 compared to cells that do not harbor ITD-Flt3 mutations. The data suggest that ITD-Flt3 may facilitate dissemination of leukemia cells by modulating SDF1/CXCR4 signaling and that blocking this functional cross-talk between ITD-Flt3 and CXCR4 pathways may have therapeutic benefit. Therefore, genes differentially regulated by SDF1 specifically in ITD-Flt3 cells may represent key targets regulating aberrant migration by