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  • American Society of Hematology  (6)
  • Kuwatsuka, Yachiyo  (6)
  • 1
    Online Resource
    Online Resource
    Biomarkers In Cell Death of Imatinib-Resistant Ph-Leukemia Cells During Treatment with mTOR Inhibitor, Everolimus
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 3988-3988
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 3988-3988
    Abstract: Abstract 3988 Aberrant activation of mammalian target of rapamycin (mTOR) signaling pathway has been reported in hematological malignancies including leukemia initiating cells. Although rapamycin and its analogs have proven effective as anticancer agents, the mechanism of action and the solid biomarkers of response have not been fully elucidated. We investigated detailed biomarkers during the cell death of imatinib (IM)-resistant Ph-positive (Ph+) leukemia cells due to quiescence or mutations at the ABL-kinase domain after treatment with mTOR inhibitor, everolimus (Eve, RAD001). Ph+ leukemic NOD/SCID/IL2rγnull (NOG) mice cells were long co-cultured with S17 stromal cells and treated with IM and Eve. While slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ cells were insensitive to IM in spite of BCR-ABL-dephosphorylation, combination treatment with IM and Eve induced substantial cell death including the CD34+ population. In Baf3/p210T315I cells, IM-resistant Ph+ leukemia cell line harboring T315I-mutation, Eve also induced cell death with low IC50 values in PI-exclusion assays. In murine model cutaneously injected with Baf3/p210T315I cells, in vivo-treatment with Eve decreased tumor formation. In these systems during treatment with Eve, we did not observe evident dephosphorylations of BCR-ABL, mTOR itself and 4EBP1, but rapid S6K-dephosphorylation with lower doses and decreased expression of MCL-1. Furthermore, the feedback-loop effects such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a were also detected during the cell death. We are now investigating more efficient strategies using inhibitors screening kit and also planning to examine new generation of mTOR inhibitors to overcome the IM-resistance due to quiescence or T315I-mutation. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Janssen: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.3988.3988
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
    Online Resource
    Online Resource
    Treatment with Bortezomib Overcomes Resistance to Imatinib in Ph-Leukemia Quiescent Cells.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 1426-1426
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 1426-1426
    Abstract: Abstract 1426 Poster Board I-449 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. In order to examine mechanisms of drug resistance in LSCs and to seek strategies to overcome the resistance, we used Ph-positive acute lymphoblastic leukemia patient cells serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Recently several publications have suggested that proteasome inhibitors can induce selective cell death in LSCs. Spleen cells derived from leukemic NOG mice were treated ex vivo with imatinib and the proteasome inhibitor, bortezomib and cell viablility (PI-/Annexin-V-) was compared between treated and non-treated cells. After treatment with imatinib, significantly more residual cells were observed in the CD34+CD38- population compared to the CD34+CD38+ or CD34-CD38+ populations. With nM level of bortezomib, substantial cell death was induced in all populations with up-regulation of phospho-p53 (Ser15). Phosphorylation of BCR-ABL and CrkL was completely inhibited in all populations with imatinib treatment, but not with bortezomib treatment. Regarding cell cycle states, a higher percentage of Hoechst-33342low/Pyronin-Ylow cells was observed in the CD34+CD38- population relative to the other populations, suggesting more cells in the G0 state among the CD34+CD38- population. In co-culturing with S17 stromal cells, quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib, while substantial cell death including CD34+ population was induced with nM level of bortezomib. We are also investigating more detailed biomarkers in the cell death and effects of these drugs both on the primitive leukemia cells and normal hematopoietic cells using the in vivo leukemic NOG mice systems. These results imply that resistance to imatinib in Ph-positive leukemia quiescent cells is independent of BCR-ABL phosphorylation and that treatment with bortezomib can overcome the resistance of Ph-positive LSCs. Disclosures Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe: Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.1426.1426
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2009
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
    Online Resource
    Online Resource
    Treatment with mTOR Inhibitor, Everolimus (RAD001) Overcomes Resistance to Imatinib in Ph-Leukemia Quiescent or T315I-Mutated Cells.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 3277-3277
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 3277-3277
    Abstract: Abstract 3277 Poster Board III-1 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. Aberrant activation of mTOR signaling has also been reported to be involved in LSCs. In order to examine mechanisms of drug resistance in Ph-positive (Ph+) LSCs and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., Proc Natl Acad Sci USA, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells derived from leukemic NOG mice were co-cultured with S17 stromal cells and treated with imatinib and the mTOR inhibitor, everolimus (RAD001, Novartis Pharmaceuticals). While quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib in spite of BCR-ABL- and CrkL-dephosphorylation, substantial cell death including CD34+ population was induced with nM level of everolimus. In imatinib-resistant Ph+ leukemia cell lines harboring T315I-mutation (Baf3p210/T315I and TCC-Y/T315I), everolimus induced cell death with low IC50 values in PI-exclusion assays. We are also investigating detailed biomarkers in the cell death (such as phosphorylation of 4E-BP1 or p70 S6K) and effects of theses drugs in the leukemic NOG mice systems. These results imply that treatment with everolimus can overcome the resistance to imatinib in Ph+ LSCs or T315I-mutated cells. Disclosures: Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe:Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.3277.3277
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2009
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 4
    Online Resource
    Online Resource
    Rapid Reduction of Chronic Myeloid Leukemia Stem Cells After Treatment with Second-Generation BCR-ABL Kinase Inhibitors, Dasatinib and Nilotinib
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 4457-4457
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 4457-4457
    Abstract: Abstract 4457 Chronic myeloid leukemia (CML) is effectively treated with imatinib (IM), however, several mathematical models and ex vivo-examinations suggested that IM-therapy does not eradicate BCR-ABL-positive hematopoietic stem cells (HSC). We prospectively (0, 3, 6 and 12 months after IM-therapy) investigated 16 newly diagnosed and 22 long-term followed CML-chronic phase (CP) cases using methods previously reported (Jamieson et al., N Engl J Med, 2004. and Abe et al., Int J Hematol, 2008) (Figure 1) with FACSAria™ and quantitative RT-PCR of BCR-ABL among each sorted population; total mononuclear cells, HSC/Thy-1+, HSC/Thy-1–, common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP). In optimal responders to IM-therapy, BCR-ABL transcripts in the HSC populations (HSC/Thy-1+ and HSC/Thy-1–) tended to be more retentive than other populations while gradual reduction was observed during the first 12 months in all populations. And discrepancy of minimum residual diseases (MRD) between the HSC populations and other populations was larger in patients after longer IM-therapy. In evaluating properties of CML stem cells and other markers, we observed irrelevant distribution of side population (SP) and expressions of ABC transporters (ABCB1 and ABCG2) in comparison with CD34/38 expression. We also prospectively investigated BCR-ABL transcripts in each population of 23 IM-resistant or -intolerant CML-CP cases and one newly diagnosed CML-accelerated phase (AP) case during treatment with second-generation tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib. Treatment with each inhibitor induced more rapid reduction of BCR-ABL transcripts even in the HSC population (CD34+CD38–) during the first 6 months and there was no significant difference of MRD among each population in optimal responders to 2nd TKIs-therapy. In the stromal co-culturing system using primary cells and leukemic NOD/SCID/IL2rgnull (NOG) mice xenotransplanted with Ph+ leukemia cells, retention of quiescent slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ population after IM-treatment were observed and cell death mechanisms after treatment with 2nd TKIs are also under investigation. These results imply that therapy with 2nd TKIs could be a promising approach for quick and efficient reduction of the CML stem cells and cure of disease. Figure 1 Figure 1. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.4457.4457
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
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  • 5
    Online Resource
    Online Resource
    Treatment with mTOR Inhibitor, Everolimus (RAD001) Overcomes Resistance to Imatinib In Ph-Leukemia Quiescent Cells.
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 1579-1579
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 1579-1579
    Abstract: Abstract 1579 Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia and eradication of LSCs should be necessary for the cure. In order to examine mechanisms of drug resistance in Ph+ leukemia to imatinib (IM) due to stem cell properties and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., PNAS, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells from leukemic NOG mice were treated ex vivo with IM and growth factors, and cell viablility (PI/AnnexinV-staining) was compared between treated and non-treated cells. After treatment with IM, significantly more residual cells were observed in the CD34+/38- population compared to the CD34+/38+ or CD34-/38+ populations. Phosphorylation of BCR-ABL and CrkL was completely inhibited in all populations with IM treatment. Regarding cell cycle states, a higher percentage of quiescent slow-cycling (Hoechst 33342low/Pyronin Ylow) cells was observed in the CD34+/38- population relative to the other populations. Recently, aberrant activation of mTOR signaling has also been reported to be involved in LSCs. Leukemic spleen cells in longer co-culturing with stromal cells were treated with IM and mTOR inhibitor, everolimus (Eve, RAD001). While slow-cycling CD34high+ cells were insensitive to IM, combination treatment with IM and Eve induced significant cell death also in the quiescent population. In vivo-treatment with Eve decreased tumor cells in leukemic NOD mice inoculated with the leukemic NOG cells under the sub-lethally irradiated condition. Treatment with Eve also reduced long-term colony formation of Ph+ leukemia patient samples including Y253H-mutated Ph-ALL with less toxicity to normal cord blood cells. These results imply that treatment with Eve is promising for overcoming the resistance to IM due to quiescent property in Ph+ LSCs. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Janssen: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.1579.1579
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 6
    Online Resource
    Online Resource
    Treatment with Hsp90 Inhibitor, 17-AAG Overcomes Resistance to Small Molecule FLT3-Inhibitors in FLT3/ITD-Positive Leukemia Cells Harboring N676K-Mutation.
    American Society of Hematology ; 2008
    In:  Blood Vol. 112, No. 11 ( 2008-11-16), p. 1619-1619
    Details
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 1619-1619
    Abstract: The receptor tyrosine kinase FLT3 is mutated in ~30% of acute myeloid leukemia (AML) and small molecules that selectively inhibit FLT3 kinase activity induce apoptosis in blasts from AML patients with FLT3 mutations and prolong survival in animal models of FLT3-induced myeloproliferative disease. Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3-inihibitors have shown measurable clinical responses. However, most of these responses were transient and in some cases an additive single amino acid substitution (such as N676K) within FLT3 kinase domain was reported to be a possible cause for the temporary response. We previously showed a novel FLT3 inhibitor, FI-700, selectivity suppresses the growth of leukemia cells with FLT3 mutations (Kiyoi, et al., Clin Cancer Res 07). In order to examine how we can overcome drug resistance due to additional kinase mutations, we cultured MOLM-13 (FLT3/ITD-positive leukemia cell line) cells with increasing doses of FI-700 to generate several FI-700-resistant sublines. In 6 of 6 clones with resistance to more than 1 μM FI-700, FLT3N676K mutation was detected in one allele, and these were also resistant to small molecule FLT3-inhibitors such as AG1295 and AG1296. In a drug-screening for this kind of subline (MOLM-13/FLT3N676K), sub-molar treatment with molecular chaperon Hsp90 inhibitor, 17-AAG, inhibited growth as revealed by MTT-assays, and a combination of 17-AAG and FI-700 synergistically induced cell death as indicated by DNA-histograms and PI/Annexin-V assays. Expression of FLT3 by FACS and phosphorylation of FLT3 and its substrate molecules (such as STAT5), were significantly inhibited by the combined treatment of FI-700 and 17-AAG. To reveal the detailed mechanisms underlying these synergistic effects, we are currently investigating the binding of FLT3 and Hsp90, the degradation and the localization of FLT3 in these cells. We are also investigating the in vivo-effects of combined treatment in NOD/SCID mice inoculated with MOLM-13/FLT3N676K. These results imply that treatment of FLT3/ITD-positive leukemia cells with 17-AAG is a promising strategy for overcoming the drug resistance of cells with mutations in the FLT3 kinase domain.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V112.11.1619.1619
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
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