Chromosomal translocations are responsible for the appearance of oncogenes encoding fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGF␤R, TEL/TRKC(L), and NPM/ALK (6, 35). BCR/ABL is derived from relocation of the portion of the c-ABL gene from chromosome 9 to the portion of the BCR gene locus on chromosome 22 [t(9;22)] and is present in most chronic myelogenous leukemia (CML) patients and a cohort of acute lymphocytic leukemia (ALL) patients (11,19,64). TEL/ABL results from a t(9;12) translocation reported in ALL, acute myelogenous leukemia (AML), and atypical CML (35) and consists of the amino-terminal fragment of the TEL domain fused in-frame with exon 2 of ABL (24). TEL/JAK2 was characterized as a product of a t(9;12) translocation which includes the TEL oligomerization domain and JAK2 catalytic domain (37) and was found in ALL (37, 51). TEL/PDGF␤R is associated with a t(5;12) translocation which juxtaposes the amino-terminal region of TEL with the transmembrane and tyrosine kinase domains of the platelet-derived growth factor receptor ␤ (23). TEL/PDGF␤R was found in chronic myelomonocytic leukemia (35). The consequence of t(12;15) is expression of the TEL/TRKC fusion tyrosine kinase associated with AML, infantile fibrosarcoma, and congenital mesoblastic nephroma (41). The TEL/TRKC fusion in AML [TEL/TRKC(L)] includes exons 1 to 4 of the TEL gene fused in frame to the tyrosine kinase domain of TRKC lacking a 42-bp exon near the C terminus of the TRKC moiety. NPM/ALK, formed by the t(2;5) translocation, was implicated in the pathogenesis of anaplastic large cell lymphoma (38). The NPM/ALK fusion gene encodes a 75-kDa hybrid protein that contains the aminoterminal portion of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase ALK (anaplastic lymphoma kinase) (44). These FTKs (BCR/ABL-related FTKs) show structural similarities, which include an amino-terminal oligomerization domain responsible for constitutive oligomerization and activation of the associated tyrosine kinase of the carboxy-terminal fusion partner.FTKs and other oncogenic tyrosine kinases such as v-Src and HER-2/neu activate multiple signaling pathways responsible for protection from apoptosis, induction of growth factor-independent proliferation, transformation, and resistance to therapeutic drugs and to ␥-radiation (25,41,43,53,54,61,85). Resistance to DNA-damaging agents is a cause for failure in the therapy of human cancer, including hematological malignancies. Several mechanisms of resistance to DNA damage

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.

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• Imatinib does not prevent accumulation of genomic instability in CML-CP.• Imatinib-refractory leukemia stem cells may be a source of genomic instability in CML-CP.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. ROSinduced 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 TKIrefractory 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. (Blood. 2013;121(20):4175-4183)

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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 lovastatintreated patients. Lovastatin has also been proved to potentiate antitumor effects of both cisplatin and TNF-␣ 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.

IntroductionAn effective immune response to severe bacterial infections requires a robust production of the innate immunity cells from hematopoietic stem and progenitor cells (HSPCs) in a process called emergency myelopoiesis. In sepsis, an altered immune response that leads to a failure of bacterial clearance is often observed. In this study, we aimed to evaluate the impact of sepsis on human HSPCs in the bone marrow (BM) microenvironment of humanized mice subjected to acute endotoxemia and polymicrobial sepsis.MethodsHumanized mice (hu-NSG) were generated by transplanting NOD.Cg-Prkdc/scidIL2rγ (NSG) mice with the human cord blood CD34+ cells. Eight weeks after the transplantation, hu-NSG mice were subjected to sepsis induced by endotoxemia—Escherichia coli lipopolysaccharide (LPS)—or by cecal ligation and puncture (CLP). Twenty-four hours later, HSPCs from BM were analyzed by flow cytometry and colony-forming unit (CFU) assay. CLP after inhibition of Notch signaling was also performed. The effects of LPS on the in vitro proliferation of CD34+ cells from human BM were tested by CellTrace Violet dye staining.ResultsThe expression of Toll-like receptor 4 receptor was present among engrafted human HSPCs. Both CLP and endotoxemia decreased (by 43 % and 37 %) cellularity of the BM. In addition, in both models, accumulation of early CD34+ CD38− HSCs was observed, but the number of CD34+ CD38+ progenitors decreased. After CLP, there was a 1.5-fold increase of proliferating CD34+ CD38−Ki-67+ cells. Moreover, CFU assay revealed a depressed (by 75 % after LPS and by 50 % after CLP) production of human hematopoietic colonies from the BM of septic mice. In contrast, in vitro LPS stimulated differentiation of CD34+ CD38− HSCs but did not induce proliferation of these cells in contrast to the CD34+ CD38+ progenitors. CLP sepsis modulated the BM microenvironment by upregulation of Jagged-1 expression on non-hematopoietic cells, and the proliferation of HSCs was Notch-dependent.ConclusionsCLP sepsis and endotoxemia induced a similar expansion and proliferation of early HSCs in the BM, while committed progenitors decreased. It is suggestive that the Notch pathway contributed to this effect. Targeting early hematopoiesis may be considered as a viable alternative in the existing arsenal of supportive therapies in sepsis.

To investigate the effect of the ceramide moiety of GM1 ganglioside on its association with detergent resistant membrane domains (DRMs) in human leukemia HL-60 cells, [(3)H] labeled GM1 molecular species (GM1s) with ceramides consisting of C18 sphingosine acetylated or acylated with C(8), C(12), C(14), C(16), C(18), C(22), C(24), C(18:1), C(22:1), or C(24:1) fatty acids (FAs), or C20 sphingosine acetylated or acylated with C(8) or C(18) FA were prepared and added to culture media. GM1s uptake by HL-60 cells was affected by the structure of their ceramides. Resistance to removal with trypsin and the stoichiometry of [(125)I] cholera toxin (CT) binding indicated that the added GM1s were incorporated into the membranes of the cells used for the isolation of DRMs in a manner resembling endogenous gangliosides. The ceramide moieties of the GM1s determined their occurrence in DRMs and the dependence of their recovery in this membrane fraction on the amount of Triton X-100 (TX) used for extraction as well as on cholesterol depletion. The GM1s with sphingosine acylated with C(14), C(16), C(18) C(22), or C(24) FAs were similarly abundant in DRMs. GM1s acylated with C(18:1), C(22:1), or C(24:1) were less abundant than those acylated with saturated FA of the same length. GM1s acetylated or acylated with C(8) FA were detected in DRMs in the lowest proportion. Depletion of 73% of cell cholesterol with methyl-beta-cyclodextrin significantly affected the recovery in DRMs of GM1s acetylated or acylated with C(8) or unsaturated FAs but not of GM1 acylated with C(18), C(22), or C(24) FAs. After cross-linking with CT B subunit, all GM1s were recovered in DRMs in a similarly high proportion irrespective of their ceramide structure or cholesterol depletion. DRMs prepared with low TX concentration at the TX/cell protein ratio of 0.3:1 were separated by multistep sucrose density gradient centrifugation into two fractions. The GM1s with sphingosine acetylated or acylated with C(18) or C(18:1) FAs occurred in these fractions in different proportions.