Background We have previously shown that systemic infusion of insulin-like growth factor-1 (IGF-1) exerts anti-inflammatory and anti-oxidant effects and reduces atherosclerotic burden in apolipoprotein E (Apoe) deficient mice. Monocytes/macrophages express high levels of IGF-1 receptor (IGF1R) and play a pivotal role in atherogenesis but the potential effects of IGF-1 on their function are unknown. Methods and Results To determine mechanisms whereby IGF-1 reduces atherosclerosis and to explore the potential involvement of monocytes/macrophages, we created monocyte/ macrophage specific IGF1R knockout (MΦ-IGF1R-KO) mice on Apoe−/− background. We assessed atherosclerotic burden, plaque features of stability, and monocyte recruitment to atherosclerotic lesions. Phenotypic changes of IGF1R-deficient macrophages were investigated in culture. MΦ-IGF1R-KO significantly increased atherosclerotic lesion formation, as assessed by Oil-red-O staining of en face aortae and aortic root cross-sections, and changed plaque composition to a less stable phenotype, characterized by increased macrophage and decreased α-smooth muscle actin-positive cell population, fibrous cap thinning, and decreased collagen content. Brachiocephalic artery lesions of MΦ-IGF1R-KO mice had histological features implying plaque vulnerability. Macrophages isolated from MΦ-IGF1R-KO mice showed enhanced proinflammatory responses upon stimulation by IFNγ and oxidized LDL and elevated antioxidant gene expression levels. Moreover, IGF1R deficient macrophages had decreased expression of ABCA1 and ABCG1 and reduced lipid efflux. Conclusions Our data indicate that macrophage IGF1R signaling suppresses macrophage and foam cell accumulation in lesions and reduces plaque vulnerability, providing a novel mechanism whereby IGF-1 exerts anti-atherogenic effects.

Three discrete forms of feline leukemia virus (FeLV)-associated lymphoma have been described clinically: (1) thymic, (2) alimentary, and (3) multicentric. The most common and best-characterized lymphomas are of T-cell origin, generally occurring in the thymus. These tumors typically contain mature T-cells, involve the activation of a distinctive set of proto-oncogenes, and contain FeLV proviruses whose long terminal repeat (LTR) sequences contain tandemly repeated enhancers. Previous studies of a small group of extrathymic, multicentric lymphomas implicated a different set of genetic determinants. The present study expands those observations by examining the lineage of origin, the involvement of proto-oncogenes, and the structure of LTR and env gene sequences in a set of 11 natural, extrathymic lymphomas of the multicentric type. A pattern of genetic events associated with FeLV-positive multicentric lymphomas emerges from this analysis that is clearly distinct from the pattern associated with thymic lymphomas. The tumors do not contain T-cells or B-cells, as evidenced by the germ line organization of TCR beta and IgH loci. Proto-oncogenes strongly implicated in T-cell lymphomagenesis are not involved in these tumors. Rather, a distinct set of proto-oncogenes may be involved. Most striking is the repeated occurrence of an FeLV isolate whose LTR and env gene bear unique sequence elements.

Feline leukaemia virus (FeLV)-945 was previously identified in natural multicentric lymphomas and contains a 21 bp tandem triplication in the LTR. In the present study, FeLV LTR variation was examined in the cohort from which FeLV-945 was identified. The objectives of the study were to evaluate FeLV LTR variation within the cohort, to determine whether the FeLV-945 LTR was associated uniquely with multicentric lymphoma and to evaluate functional attributes that may have contributed selective advantage to the predominant LTR variants observed. T-cell tumours uniformly contained LTRs with duplicated enhancer sequences, although enhancer duplications conferred little transcriptional advantage. Non-T-cell malignant, proliferative and degenerative diseases contained LTRs with two, three or four tandemly repeated copies of the 21 bp sequence originally identified in FeLV-945. While the length and termini of enhancer duplications were variable, the 21 bp repeat unit was invariant. Triplication of the 21 bp repeat conferred the optimal replicative advantage in feline cells.

Feline leukemia virus (FeLV), like other naturally occurring retroviruses, is characterized by a high degree of genetic diversity. FeLV-945 is a natural isolate derived from non-B-cell non-T-cell lymphomas classified anatomically as multicentric. FeLV-945 exhibits a unique structural motif in the LTR composed of a 21-bp tandem triplication downstream of a single copy of enhancer. The unique FeLV-945 LTR is precisely conserved among eight independent multicentric lymphomas collected in a geographic cluster. Previous studies using reporter gene constructs predict that the FeLV-945 LTR would confer a replicative advantage on the virus that contains it, particularly in primitive hematopoietic cells. Such an advantage may account for the precise conservation of the unique LTR sequence. To test that prediction, a set of recombinant, infectious FeLVs was developed that are isogenic other than the presence of the FeLV-945 LTR or mutations of it. Replication assays show that the FeLV-945 LTR confers a distinct growth advantage in K-562, FEA, and 3201 cells and implicate the 21-bp triplication in that function. Replacement of two copies of the triplicated element with random sequence greatly diminished the replicative capacity, thus implicating the triplicated sequence itself in LTR function. The 21-bp triplication was shown to contain specific nuclear protein binding sites, which may account for the selective pressure to conserve the sequence.

The clinical efficacy displayed by ibrutinib in chronic lymphocytic leukemia (CLL) has been challenged by the frequent emergence of resistant clones. The ibrutinib target, Bruton's tyrosine kinase (BTK), is essential for B cell receptor signaling, and most resistant cases carry mutations in BTK or PLCG2, a downstream effector target of BTK. Recent findings show that MI-2, a small molecule inhibitor of the para-caspase MALT1, is effective in preclinical models of another type of BCR pathway-dependent lymphoma. We therefore studied the activity of MI-2 against CLL and ibrutinib-resistant CLL. Treatment of CLL cells in vitro with MI-2 inhibited MALT1 proteolytic activity, reduced BCR and NF-κB signaling, inhibited nuclear translocation of RelB and p50, and decreased Bcl-xL levels. MI-2 selectively induced dose and time-dependent apoptosis in CLL cells, sparing normal B lymphocytes. Furthermore, MI-2 abrogated survival signals provided by stromal cells and BCR cross-linking and was effective against CLL cells harboring features associated with poor outcomes, including 17p deletion and unmutated IGHV. Notably, MI-2 was effective against CLL cells collected from patients harboring mutations conferring resistance to ibrutinib. Overall, our findings provide a preclinical rationale for the clinical development of MALT1 inhibitors in CLL, in particular for ibrutinib-resistant forms of this disease.

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Objective We have shown that insulin-like growth factor I (IGF-1) infusion in Apoe−/− mice decreased atherosclerotic plaque size and plaque macrophage and lipid content suggesting that IGF-1 suppressed formation of macrophage-derived foam cells. Since 12/15-lipoxygenase (12/15-LOX) plays an important role in OxLDL and foam cell formation, we hypothesized that IGF-1 downregulates 12/15-LOX, thereby suppressing lipid oxidation and foam cell formation. Approach and Results We found that IGF-1 decreased 12/15-LOX plaque immunopositivity and serum OxLDL levels in Apoe−/− mice. IGF-1 reduced 12/15-LOX protein and mRNA levels in cultured THP-1 macrophages and IGF-1 also decreased expression of STAT6 transcription factor. IGF-1 reduction in macrophage 12/15-LOX was mediated in part via a PI3 kinase- and STAT6-dependent transcriptional mechanism. IGF-1 suppressed THP-1 macrophage ability to oxidize lipids and form foam cells. IGF-1 downregulated 12/15-LOX in human blood-derived primary macrophages and IGF-1 decreased LDL oxidation induced by these cells. IGF-1 reduced LDL oxidation and formation of foam cells by wild type murine peritoneal macrophages, however these effects were completely blocked in 12/15-LOX-null macrophages suggesting that the ability of IGF-1 to reduce LDL oxidation and foam cells formation is dependent on its ability to downregulate 12/15-LOX. Conclusions Overall our data demonstrate that IGF-1 reduces lipid oxidation and foam cell formation via downregulation of 12/15-LOX and this mechanism may play a major role in the anti-atherosclerotic effects of IGF-1.

Feline leukemia virus (FeLV) proviruses have been characterized from naturally occurring non-B-cell, non-T-cell tumors occurring in the spleens of infected cats. These proviruses exhibit a unique sequence motif in the long terminal repeat (LTR), namely, a 21-bp tandem triplication beginning 25 bp downstream of the enhancer. The repeated finding of the triplication-containing LTR in non-B-cell, non-T-cell lymphomas of the spleen suggests that the unique LTR is an essential participant in the development of tumors of this particular phenotype. The nucleotide sequence of the triplication-containing LTR most closely resembles that of FeLV subgroup C. Studies performed to measure the ability of the triplication-containing LTR to modulate gene expression indicate that the 21-bp triplication provides transcriptional enhancer function to the LTR that contains it and that it substitutes at least in part for the duplication of the enhancer. The 21-bp triplication confers a bona fide enhancer function upon LTR-directed reporter gene expression; however, the possibility of a spacer function was not eliminated. The studies demonstrate further that the triplication-containing LTRacts preferentially in a cell-type-specific manner, i.e., it is 12-fold more active in K-562 cells than is an LTR lacking the triplication. A recombinant, infectious FeLV bearing the 21-bp triplication in U3 was constructed. Cells infected with the recombinant were shown to accumulate higher levels of viral RNA transcripts and virus particles in culture supernatants than did cells infected with the parental type. The triplication-containing LTR is implicated in the induction of tumors of a particular phenotype, perhaps through transcriptional regulation of the virus and/or adjacent cellular genes, in the appropriate target cell.