Grazyna Hoser

BCR/ABL Regulates Mammalian RecA Homologs, Resulting in Drug Resistance

RAD51 is one of six mitotic human homologs of the E. coli RecA protein (RAD51-Paralogs) that play a central role in homologous recombination and repair of DNA double-strand breaks (DSBs). Here we demonstrate that RAD51 is important for resistance to cisplatin and mitomycin C in cells expressing the BCR/ABL oncogenic tyrosine kinase. BCR/ABL significantly enhances the expression of RAD51 and several RAD51-Paralogs. RAD51 overexpression is mediated by a STAT5-dependent transcription as well as by inhibition of caspase-3-dependent cleavage. Phosphorylation of the RAD51 Tyr-315 residue by BCR/ABL appears essential for enhanced DSB repair and drug resistance. Induction of the mammalian RecA homologs establishes a unique mechanism for DNA damage resistance in mammalian cells transformed by an oncogenic tyrosine kinase.

Fusion Tyrosine Kinases Induce Drug Resistance by Stimulation of Homology-Dependent Recombination Repair, Prolongation of G 2 /M Phase, and Protection from Apoptosis

ABSTRACT Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC(L), and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic leukemias and non-Hodgkins lymphoma. FTK-transformed cells displayed drug resistance against the cytostatic drugs cisplatin and mitomycin C. These cells were not protected from drug-mediated DNA damage, implicating activation of the mechanisms preventing DNA damage-induced apoptosis. Various FTKs, except TEL/TRKC(L), can activate STAT5, which may be required to induce drug resistance. We show that STAT5 is essential for FTK-dependent upregulation of RAD51, which plays a central role in homology-dependent recombinational repair (HRR) of DNA double-strand breaks (DSBs). Elevated levels of Rad51 contributed to the induction of drug resistance and facilitation of the HRR in FTK-transformed cells. In addition, expression of antiapoptotic protein Bcl-xL was enhanced in cells transformed by the FTKs able to activate STAT5. Moreover, cells transformed by all examined FTKs displayed G2/M delay upon drug treatment. Individually, elevated levels of Rad51, Bcl-xL, or G2/M delay were responsible for induction of a modest drug resistance. Interestingly, combination of these three factors in nontransformed cells induced drug resistance of a magnitude similar to that observed in cells expressing FTKs activating STAT5. Thus, we postulate that RAD51-dependent facilitation of DSB repair, antiapoptotic activity of Bcl-xL, and delay in progression through the G2/M phase work in concert to induce drug resistance in FTK-positive leukemias and lymphomas.

Rac2-MRC-cIII-generated ROS cause genomic instability in chronic myeloid leukemia stem cells and primitive progenitors

Chronic myeloid leukemia in chronic phase (CML-CP) is induced by BCR-ABL1 oncogenic tyrosine kinase. Tyrosine kinase inhibitors eliminate the bulk of CML-CP cells, but fail to eradicate leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) displaying innate and acquired resistance, respectively. These cells may accumulate genomic instability, leading to disease relapse and/or malignant progression to a fatal blast phase. In the present study, we show that Rac2 GTPase alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III (MRC-cIII), thereby generating high levels of reactive oxygen species (ROS) in CML-CP LSCs and primitive LPCs. MRC-cIII-generated ROS promote oxidative DNA damage to trigger genomic instability, resulting in an accumulation of chromosomal aberrations and tyrosine kinase inhibitor-resistant BCR-ABL1 mutants. JAK2(V617F) and FLT3(ITD)-positive polycythemia vera cells and acute myeloid leukemia cells also produce ROS via MRC-cIII. In the present study, inhibition of Rac2 by genetic deletion or a small-molecule inhibitor and down-regulation of mitochondrial ROS by disruption of MRC-cIII, expression of mitochondria-targeted catalase, or addition of ROS-scavenging mitochondria-targeted peptide aptamer reduced genomic instability. We postulate that the Rac2-MRC-cIII pathway triggers ROS-mediated genomic instability in LSCs and primitive LPCs, which could be targeted to prevent the relapse and malignant progression of CML.

Lovastatin potentiates antitumor activity of doxorubicin in murine melanoma via an apoptosis-dependent mechanism

Lovastatin, a drug successfully used in the clinic to prevent and to treat coronary heart disease, has recently been reported to decrease the incidence of melanoma in lovastatin‐treated patients. Lovastatin has also been proved to potentiate antitumor effects of both cisplatin and TNF‐α in murine melanoma models. Recently, an augmented therapeutic effect of lovastatin and doxorubicin has been reported in 3 tumor models in mice. In our preliminary study lovastatin caused retardation of melanoma growth in mice treated with doxorubicin (Feleszko et al. J Natl Cancer Inst 1998;90:247–8). In the present report, we supplement our preliminary observations and demonstrate in 2 murine and 2 human melanoma cell lines that lovastatin effectively potentiates the cytostatic/cytotoxic activity of doxorubicin in vitro via an augmentation of apoptosis (estimated with PARP‐cleavage assay, annexin V assay and TUNEL). The combined antiproliferative activity of lovastatin and doxorubicin was evaluated using the combination index (CI) method of Chou and Talalay, revealing synergistic interactions in melanoma cells exposed to lovastatin and doxorubicin. In B16F10 murine melanoma model in vivo, we have demonstrated significantly increased sensitivity to the combined treatment with both lovastatin (5 mg/kg for 14 days) and doxorubicin (4 × 1 mg/kg) as compared with either agent acting alone. Lovastatin treatment resulted also in significant reduction of the number of experimental metastasis in doxorubicin‐treated mice. The results of our studies suggest that lovastatin may enhance the effectiveness of chemotherapeutic agents in the treatment of malignant melanomas.

Genomic instability may originate from imatinib-refractory chronic myeloid leukemia stem cells.

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.

Does the bcr/abl-mediated increase in the efficacy of DNA repair play a role in the drug resistance of cancer cells?

BCR/ABL oncogenic tyrosine kinase is responsible for the pathogenesis of Philadelphia chromosome‐positive human leukemia and is generated by a specific reciprocal chromosome translocation, t(9;22)(q34−;q11+). We examined the role of DNA repair in therapeutic drug resistance to idarubicin in the murine pro‐B lymphoid cell line BaF3 and its BCR/ABL ‐transformed clone. These cells can be used as models of human leukemias. The MTT assay revealed that BCR/ABL ‐transformed cells displayed resistance to idarubicin in the range 0.3–0.5μm, compared with the control BaF3 cells. Idarubicin at 0.3 and 1μm induced DNA damage in the form of strand‐breaks and/or alkali labile sites in both transformed and control cells in comet assays. TheBCR/ABL ‐transformed cells needed only 60min to remove damage to their DNA, whereas controls took 120min. We hypothesize that this observed increase in the efficacy of repair in BCR/ABL‐ positive cells is involved in their resistance to idarubicin.

Id1 Transcription Inhibitor-Matrix Metalloproteinase 9 Axis Enhances Invasiveness of the Breakpoint Cluster Region/Abelson Tyrosine Kinase-Transformed Leukemia Cells

Breakpoint cluster region/Abelson (BCR/ABL) tyrosine kinase enhances the ability of leukemia cells to infiltrate various organs. We show here that expression of the helix-loop-helix transcription factor Id1 is enhanced by BCR/ABL in a signal transducer and activator of transcription 5 (STAT5)-dependent manner. Enhanced expression of Id1 plays a key role in BCR/ABL-mediated cell invasion. Down-regulation of Id1 in BCR/ABL leukemia cells by the antisense cDNA significantly reduced their invasive capability through the Matrigel membrane and their ability to infiltrate hematopoietic and nonhematopoietic organs resulting in delayed leukemogenesis in mice. The Id1-promoted cell invasiveness was seemingly mediated by matrix metalloproteinase 9 (MMP9). Transactivation of MMP9 promoter in BCR/ABL cells was dependent on Id1 and abrogation of the MMP9 catalytic activity by a metalloproteinase inhibitor or blocking antibody decreased invasive capacity of leukemia cells. These data suggest that BCR/ABL-STAT5-Id1-MMP9 pathway may play a critical role in BCR/ABL-mediated leukemogenesis by enhancing invasiveness of leukemia cells.

Fusion oncogenic tyrosine kinases alter DNA damage and repair after genotoxic treatment: role in drug resistance?

Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGF beta R and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic myelogenous leukemias and non-Hodgkins lymphoma. Murine hematopoietic growth factor dependent BaF3 cells and cells transformed by FTK (BaF3-FTK) were used to investigate the role of FTKs in response to DNA damage. FTK-transformed cells displayed resistance to genotoxic treatment including gamma-radiation and cytostatic agents such as idarubicin and MNNG. More FTK-transformed cells survived genotoxic treatment and were able to proliferate in comparison to parental non-transformed cells. Similar or higher levels of DNA damage was detected in gamma-irradiated in BaF3-FTK cells in comparison to BaF3 parental cells. Idarubicin induced different amounts of DNA damage in various BaF3-FTK cells. All BaF3-FTK cells treated with MNNG displayed significantly more DNA damage in comparison to BaF3 cells. Despite the extent of genotoxic effect BaF3-FTK cells were often able to repair damaged DNA more efficiently that the non-transformed counterparts. Inhibition of BCR/ABL kinase activity by STI571 (Gleevec, inatinib mesylate) abrogated the resistance to genotoxic treatment and inhibited DNA repair mechanisms. We hypothesize that facilitation of the DNA repair in FTK-positive cells may contribute to their resistance to genotoxic treatment.

Mechanisms of immune response regulation in lung cancer.

Lung cancer is a leading cause of cancer deaths. As a solid tumor with low antigenicity and heterogenic phenotype lung cancer evades host immune defense. The cytotoxic anticancer effect is suppressed by a complex mechanism in tumor microenvironment. The population of regulatory T cells (Tregs) plays a crucial role in this inhibition of immune response. Tregs are defined by presence of forkhead box P3 (Foxp3) molecule. The high expression of Foxp3 was found in lung cancer cells and in tumor infiltrating lymphocytes (TIL). Cytotoxic T-lymphocyte antigen 4 (CTLA4) is constitutively expressed on Tregs and suppresses T cell activation. The elevated CTLA4 expression in lymphocytes in patients with lung cancer was found. Recently the antibodies blocking CTLA4 showed some clinical efficacy in patients with lung cancer. Cancer cells and immune cells release many cytokines capable to show suppressive immune effect in cancer microenvironment. The most active are transforming growth factorβ (TGFβ) and IL-10. The pleiotropic function of Th17 population is TGFβ related. The myeloid lineage of suppressor cells in lung cancer is represented by tumor associated macrophages (TAM) with phenotype of M2 macrophages and some regulatory properties with releasing amounts of IL-10 and TGFβ. The myeloid derived suppressor cells (MDSCs) control cytotoxic T cell activity in mechanisms which are highly dependent on the context of tumor environment. The mechanisms of anticancer immune response regulation need further investigation as an important target to new way of treatment.

Sepsis immunopathology: perspectives of monitoring and modulation of the immune disturbances

Septic syndromes are the main cause of death in the intensive care units and although the mortality rates is slowly decreasing, the occurrence of the disease has been increasing. The pathogenesis of sepsis includes countless disturbances of the host immune system starting with a harmful, infection-triggered exaggerated inflammatory cascade, followed by the development of an immunoparalysis state. The latter contributes to the failure in pathogen eradication and leads to secondary infections, which are often the cause of fatal complications. In this review, we consider different novel therapeutic strategies for restoration of immune function. The use of glucocorticoids, intravenous immunoglobulins, heparin, recombinant human activated protein C, granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interferon-γ, statins, macrolides and high-volume hemofiltration are discussed. Even though some clinical trials of these regimens are promising, the key to their successful application seems to be the precise monitoring of the status of immune system followed by implementation of the adequate therapy. Thus, in this paper we present disturbances in the immune system in the course of human sepsis, with special attention to the parameters that could be monitored and serve as markers for immunomodulatory therapies. We conclude by briefly presenting the current sepsis treatment strategy.