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  • 1
    Online Resource
    Online Resource
    Identification of Heat Shock Protein 32 (Hsp32) as a Novel Target in Acute Lymphoblastic Leukemia (ALL).
    American Society of Hematology ; 2008
    In:  Blood Vol. 112, No. 11 ( 2008-11-16), p. 1616-1616
    Details
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 1616-1616
    Abstract: Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in various solid tumors and leukemias. We have recently shown that Hsp32 is expressed in leukemic cells and serves as a survival-factor and molecular target in Ph+ chronic myeloid leukemia. In the present study, we examined the expression and functional role of Hsp32 in acute lymphoblastic leukemia (ALL). Leukemic cells were obtained from patients with Ph+ ALL (n=5) and Ph− ALL (n=5). In addition, Ph+ ALL cell lines (Z-119, BV-173, TOM-1, NALM-1) and Ph− ALL cell lines (RAJI, RAMOS, REH, BL-41) were used. As assessed by immunocytochemistry and qRT-PCR, leukemic cells were found to express the Hsp32 protein as well as Hsp32 mRNA in all patients and in all cell lines examined. The Hsp32-inductor hemin was found to promote the expression of Hsp32 in leukemic cells. To determine the functional role of Hsp32 in lymphoblasts, an siRNA against Hsp32 was applied. The siRNA-induced knock down of Hsp32 in RAJI cells was found to be associated with reduced growth and with an increase in apoptotic cells compared to a control siRNA against luciferase (p & lt;0.05). In a next step, two pharmacologic inhibitors of Hsp32, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), were applied. As assessed by 3H-thymidine uptake experiments, both drugs were found to inhibit proliferation in the BCR/ABL+ cell lines Z-119, BV-173, and TOM-1, and in the BCR/ABL-negative ALL cell lines RAJI, RAMOS, REH, and BL-41. The effects of PEG-ZnPP and SMA-ZnPP were dose-dependent with IC50 values ranging between 1 and 10 μM, and were found to be associated with apoptosis as determined by microscopy as well as by flow cytometry and AnnexinV-staining. In NALM-1 cells, PEG-ZnPP and SMA-ZnPP also produced apoptosis and growth arrest, but the IC50 for SMA-ZnPP was slightly higher compared to other cell lines (20 μM). Effects of Hsp32-targeting drugs were also observed in primary leukemic cells obtained from patients with Ph+ ALL and Ph− ALL, with IC50 values ranging between 1 and 10 μM. No major differences were found when comparing results in imatinib-sensitive and imatinib-resistant patients. In drug combination experiments, Hsp32-targeting drugs were found to cooperate with imatinib and with AMN107 (nilotinib) in producing growth-inhibition and apoptosis in all Ph+ ALL cell lines tested. Furthermore, we were able to demonstrate strong cooperative antileukemic effects when applying Hsp32-targeting drugs in combination with bendamustine. Overall, these results suggest that Hsp32 may be a novel molecular target in ALL.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V112.11.1616.1616
    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
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  • 2
    Online Resource
    Online Resource
    Bosutinib Blocks Lyn and Btk Activation and Synergizes with the KIT D816V-Targeting Drug Midostaurin in Inducing Apoptosis in Neoplastic Human Mast Cells.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 1717-1717
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 1717-1717
    Abstract: Abstract 1717 Poster Board I-743 Advanced systemic mastocytosis (SM) is a malignant hematopoietic neoplasm characterized by destructive growth of neoplastic mast cells (MC) in various organ systems. In these patients, the response to conventional cytoreductive therapy is poor and the prognosis is grave. The D816V-mutated variant of c-KIT is found in most patients and is considered to be a major transforming oncoprotein in SM that leads to abnormal survival and growth of neoplastic MC. Therefore, agents interfering with the kinase activity of KIT D816V have been developed. One promising agent is midostaurin (PKC412). However, in most patients with advanced SM, therapy with midostaurin is not sufficient to induce long term remissions. In addition, midostaurin is unable to block all pro-oncogenic signaling molecules, such as Lyn and Btk, in neoplastic MC, suggesting that additional oncoproteins and survival factors may play a role in malignant transformation in SM, and that novel therapeutic strategies are required to block such KIT-independent oncogenic pathways. Especially Lyn and Btk have attracted attention as potential new targets in neoplastic MC. Bosutinib (SKI-606) is a novel multikinase inhibitor that targets a broad spectrum of kinases including Lyn and Btk. The aim of the current study was to evaluate the effect of bosutinib on neoplastic MC, and potential cooperative drug interactions between bosutinib and midostaurin. As assessed by 3H-thymidine uptake, bosutinib was found to inhibit the growth of the MC leukemia cell line HMC-1, including the HMC-1.1 subclone that lacks KIT D816V and HMC-1.2 cells expressing KIT D816V, with similar IC50 values (1-5 μM). Furthermore, bosutinib was found to induce apoptosis in both HMC-1 subclones. Growth-inhibitory and apoptosis-inducing effects of bosutinib were also seen in primary neoplastic MC obtained from the bone marrow of patients with SM (n=3). As assessed by phosphoblotting, bosutinib did not inhibit the autophosphorylation of mutant KIT in HMC-1 cells, but was found to completely inhibit the phosphorylation of Lyn and Btk. To confirm the target-function of Lyn and Btk in neoplastic MC, siRNA experiments were performed. Knockdown of Lyn or Btk resulted in induction of apoptosis and growth-inhibition in HMC-1 cells. We next attempted to exploit target-specific and complementing effects of midostaurin and bosutinib by combining both substances. As expected, combined application of bosutinib and midostaurin resulted in a complete inhibition of phosphorylation of KIT, Lyn, and Btk in HMC-1.1 and HMC-1.2 cells. We were also able to show that bosutinib synergizes with midostaurin in inducing apoptosis in both HMC-1 subclones. Synergistic effects were also observed when combining midostaurin with Lyn- or Btk-siRNA. Together, we have identified Lyn and Btk as novel KIT-independent survival molecules in neoplastic MC. Inhibition of these kinases by siRNA-knockdown or by bosutinib leads to growth-inhibition and apoptosis. Synergistic pro-apoptotic effects were observed with the combination “bosutinib + midostaurin”, suggesting that simultaneous targeting of KIT and Lyn/Btk may be a powerful strategy to counteract the survival of neoplastic MC. This drug combination may therefore be an interesting approach to overcome drug-resistance in advanced forms of SM. Disclosures Valent: Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.1717.1717
    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
    Revealing Six Phases of CML Stem Cell Development to Explain Clinical Phenomena Seen in TKI-Treated Patients.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 4263-4263
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 4263-4263
    Abstract: Abstract 4263 Chronic myeloid leukemia (CML) is a stem cell (SC) disease defined by the BCR/ABL oncoprotein that is considered essential for abnormal growth and accumulation of neoplastic cells. Based on in vitro studies and mathematical models, CML clones are considered to be organized hierarchically similar to normal hematopoiesis. More recent data suggest, that CML cells grow in subclones that usually exhibit SC function but vary in their leukemia-initiating potential. The situation is even more complex in patients treated with TKI. In these patients, intrinsic as well as acquired resistance against TKI have been described and recognized as an emerging problem and challenge in practice and research. Most SC concepts focus on imatinib-resistant mutants of BCR/ABL, that are detectable in subclones. However, several questions and phenomena that occur in TKI-treated patients remain to be solved. Based on laboratory and clinical observations, we propose the existence of 6 distinct phases of CML SC development (SCD): a) a Ph-negative phase, b) an early Ph-positive preleukemic SCD-phase in which subclones are (very) small and usually undetectable, c) a pre/leukemic SCD-phase in which one or more subclones expand(s) and replace(s) normal myelopoiesis but still produce(s) normal WBC, d) chronic phase (CP), e) accelerated phase (AP), and f) a blast phase (BP). The latency period until progression into a next SCD phase is variable and may depend on several different factors including subclone inhibition by chalones like lipocalin and other factors, different growth kinetics produced by various BCR/ABL mutants, and TKI-induced subclone-selection. Phase a) may explain the rare occurrence of Ph-negative subclones (+OCA) during TKI treatment. Phase b) may explain why BCR/ABL mutants are not detectable before TKI therapy is initiated, why mutant- and ACA subclones “appear” in CCyR patients after a certain latency period, and why one patient can develop two or more BCR/ABL mutants in different subclones. Phase b) may also explain the rare detection of very small quantities of BCR/ABL in healthy individuals and constant low level-MRD in a few CML patients in whom therapy was stopped. Phase c) can explain early CML patients in whom WBC are normal in repeated tests; and explain relapsed TKI-resistant patients in whom under TKI therapy normal hematopoiesis is replaced by the mutant subclone but WBC remain normal for weeks to several months. The SCD phases a) through c) are not accessible in any of the conventional xenotransplant models available. Even in SCD phases d) through f), it is quite difficult to demonstrate stable long term engraftment in xenotransplant mouse models, although stem cell subclones obtained from patients in f) may grow in NOG- or NSG mice similar to AML SC. However, even if this may be a reproducible approach, analysis of all relevant subclones in these patients will probably not be achievable unless additional oncogenes are introduced in these subclones. In summary, our new concept extends the hydra model of cancer stem cell development for CML by introducing a step wise progression-model with 6 defined phases of SCD. This model may have theoretical and clinical implications for patients with freshly diagnosed and TKI-resistant CML and for cancer stem cell research in general. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.4263.4263
    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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