Correlating ROS1 Protein Expression With ROS1 Fusions, Amplifications, and Mutations.

INTRODUCTION: In this study, we sought to further characterize ROS1 protein expression in solid tumors with the complete spectrum of ROS1 genomic alterations. METHODS: ROS1 immunohistochemistry (IHC) was performed using the ROS1 (SP384) class I assay per manufacturer's instructions on a variety of solid tumors (n = 32) with known ROS1 genomic alterations. Genomic alterations included fusions (n = 17), gene amplifications (n = 10), and short-variant mutations (n = 11). RESULTS: Of the 32 cases with ROS1 IHC results, 100% (11 of 11) with canonical ROS1 fusions were positive for ROS1 IHC. Among noncanonical ROS1 fusions, only two (of five) cases with SQSTM1-ROS1 and RDX-ROS1 fusions were positive for ROS1 IHC whereas PTPRK-ROS1 (two) and TTC28-ROS1 fusions were negative for ROS1 IHC. One sample with a canonical ROS1 fusion and co-occurring ROS1 resistance mutation (6094G>A, p.G2032R) was positive for ROS1 IHC. A total of 10% (one of 10) of ROS1 amplified tumors were positive for ROS1 IHC. None of the cases (zero of five) with ROS1 short-variant mutations were positive for ROS1 protein expression. CONCLUSIONS: These findings suggest that if ROS1 IHC was used as a screening tool for ROS1 fusion, a subset of fusion-negative tumors will reveal positive IHC staining highlighting the value of reflexing to genomic profiling to confirm the presence of a targetable fusion-driver before the initiation of therapy. In addition, the ability of comprehensive genomic profiling to detect ROS1 resistance mutations will be important for clinical decision making.

Aerosolized 3-bromopyruvate inhibits lung tumorigenesis without causing liver toxicity.

3-Bromopyruvate, an alkylating agent and a well-known inhibitor of energy metabolism, has been proposed as a specific anticancer agent. However, the chemopreventive effect of 3-bromopyruvate in lung tumorigenesis has not been tested. In this study, we investigated the chemopreventive activity of 3-bromopyruvate in a mouse lung tumor model. Benzo(a)pyrene was used to induce lung tumors, and 3-bromopyruvate was administered by oral gavage to female A/J mice. We found that 3-bromopyruvate significantly decreased tumor multiplicity and tumor load by 58% and 83%, respectively, at a dose of 20 mg/kg body weight by gavage. Due to the known liver toxicity of 3-bromopyruvate in animal models given large doses of 3-bromopyruvate, confirmed in this study, we decided to test the chemopreventive activity of aerosolized 3-bromopyruvate in the same lung tumor model. As expected, aerosolized 3-bromopyruvate similarly significantly decreased tumor multiplicity and tumor load by 49% and 80%, respectively, at a dose of 10 mg/mL by inhalation. Interestingly, the efficacy of aerosolized 3-bromopyruvate did not accompany any liver toxicity indicating that it is a safer route of administering this compound. Treatment with 3-bromopyruvate increased immunohistochemical staining for cleaved caspase-3, suggesting that the lung tumor inhibitory effects of 3-bromopyruvate were through induction of apoptosis. 3-Bromopyruvate also dissociated hexokinase II from mitochondria, reduced hexokinase activity, and blocked energy metabolism in cancer cells, finally triggered cancer cell death and induced apoptosis through caspase-3, and PARP in human lung cancer cell line. The ability of 3-bromopyruvate to inhibit mouse lung tumorigenesis, in part through induction of apoptosis, merits further investigation of this compound as a chemopreventive agent for human lung cancer.

Elicitation of reactive oxygen species in Chlamydia pneumoniae-stimulated macrophages: a Ca2+-dependent process involving simultaneous activation of NADPH oxidase and cytochrome oxidase genes.

Chlamydia pneumoniae, a respiratory pathogen implicated in the development and progress of atherosclerosis, is known to infect and survive in macrophages, despite macrophage producing reactive oxygen species (ROS). To gain insight into ROS generation in macrophages infected with C. pneumoniae and to explore factors accounting for their final levels and effect, we investigated the role of NADPH oxidase and cytochrome oxidase pathways in the production and modulation of ROS. We also determined the operational role of Ca2+ signaling in the process. Macrophages stimulated with C. pneumoniae exhibit early release of ROS via up-regulation of NADPH oxidase and cytochrome c oxidase activities. Increasing the dose of C. pneumoniae led to an increase in the expression of these enzymes gene production, which was accompanied by a significant up-regulation of their gene products, implying a probable activation of transcriptional and translational processes, respectively. The change in levels of free Ca2+, influx across plasma membrane and efflux from intracellular store into cytosol all exhibited a significant regulatory role on the ROS generation pathways in macrophages. The observed events were shown to be dependent on binding of C. pneumoniae to CD14 receptors of macrophages. The data reported here imply that macrophages infected with C. pneumoniae produce ROS through membrane-associated NADPH oxidase with oxidative phosphorylation levels depending on Ca2+ influx signals.

An important role of phospholipase Cgamma1 in pre-B-cell development and allelic exclusion.

Phospholipase Cgamma1 (PLCgamma1) has been reported to be expressed predominantly in T cells and to play an important role in T-cell receptor signaling. Here we show that PLCgamma1 is expressed throughout B-cell development, with high expression in B-cell progenitors, and is involved in pre-B-cell receptor (pre-BCR) signaling. Reduced expression of PLCgamma1, in the absence of PLCgamma2 (PLCgamma1+/-PLCgamma2-/-), impedes early B-cell development at the pro-B- to pre-B-cell transition and impairs immunoglobulin heavy chain allelic exclusion, hallmarks of defective pre-BCR signaling. In contrast, early B-cell development is largely normal, whereas late B-cell maturation is impaired in the absence of PLCgamma2 alone (PLCgamma2-/-) and overexpression of PLCgamma1 in PLCgamma2-/- mice fails to restore BCR-mediated B-cell proliferation and maturation. These studies reveal an essential role of PLCgamma1, distinct from that of PLCgamma2, in B-cell development.

Induction of lipoprotein lipase gene expression in Chlamydia pneumoniae-infected macrophages is dependent on Ca2+ signaling events.

Unregulated uptake of low density lipoprotein (LDL) in macrophages is the hallmark of early atherogenic lesions, and Chlamydia pneumoniae infection of macrophages induces this process by an unknown mechanism. It was therefore aimed in this study to investigate (i) the role of C. pneumoniae in macrophage expression of the lipoprotein lipase (LpL) gene, (ii) the probable role of Ca2+ influx signals and (iii) the effect of the process on LDL uptake. Lipoprotein lipase mRNA expression and LpL activity in infected RAW-264.7 cells were significantly upregulated. A biphasic Ca2+ influx signal was observed in infected cells with a moderate influx (303 nM Ca2+) favoring optimal LpL gene expression. Also, the antagonists of L-type Ca2+ channel in macrophages significantly down-regulated LpL gene expression and the biomolecular content of C. pneumoniae responsible for the observed events was in part found to be Chlamydia lipopolysaccharide (cLPS). Investigations aimed at determining the specific relevance of Ca(2+)-dependent lipoprotein lipase gene expression in C. pneumoniae-infected macrophages showed that the condition caused enhanced uptake of LDL which was abrogated by Calphostin-C-mediated down-regulation of LpL. This discovery of a specialized Ca2+ influx signal-mediated LpL upregulation in C. pneumoniae-infected macrophages provides a mechanistic insight into early events involving C. pneumoniae in macrophage foam cell formation resulting from LDL uptake.

Macrophage L-type Ca2+ channel antagonists alter Chlamydia pneumoniae MOMP and HSP-60 mRNA gene expression, and improve antibiotic susceptibility.

Recent data have shown a unique relationship between Ca2+ signaling in macrophages through L-type channels and the outcome of C. pneumoniae infection of such cells. The present investigation seeks to provide insights into the manner in which macrophage L-type Ca2+ channel operation affects major outer membrane protein (MOMP) and heat shock protein-60 (HSP-60) mRNA gene expression (factors associated with Chlamydia chronicity), and the possible effect of this on antibiotic susceptibility. Intracellular calcium ([Ca2+]i) chelation using varying doses of 1,2-bis (o-aminophenoxy) ethane-N,N,N'N'--tetra acetic acid (acetoxymethyl) ester (BAPTA-AM) induced an increase in MOMP and a decrease in HSP-60 mRNA gene expression. L-type Ca2+ channel antagonists produced an identical but enhanced effect. Since these findings associate specialized Ca2+ channels to Chlamydia chronicity, it was important to determine Ca2+ channel effect on the usual antibiotic refractory form of C. pneumoniae in macrophages. Inhibition of macrophage L-type Ca2+ channel operation improved C. pneumoniae antibiotic susceptibility assessed by decreased inclusion counts or down-regulated MOMP and HSP-60 mRNA gene expression. These findings provide molecular insights into how specialized Ca2+ channels influence Chlamydia chronic course in macrophages and demonstrates a role for L-type Ca2+ channel inhibitors in enhanced C. pneumoniae susceptibility to antibiotic therapy.

Phospholipase Cgamma2 provides survival signals via Bcl2 and A1 in different subpopulations of B cells.

PLCgamma2 plays a critical role in B cell receptor (BCR) signaling and its targeted deletion results in defective B cell development and function. Here, we show that PLCgamma2 deficiency specifically blocks B cell maturation at the transitional type 2 (T2) to follicular (FO) B cell transition and the PLCgamma2 pathway regulates survival of B cells. BCR-induced apoptosis is dramatically enhanced in all subsets of splenic PLCgamma2-deficient B cells, especially in T2 and FO B cell subpopulations. We also find that all splenic PLCgamma2-deficient B cell subpopulations express abnormally low levels of Bcl-2 protein. In addition, PLCgamma2 deficiency disrupts BCR-mediated induction of A1 expression. Enforced expression of Bcl-2 prevents BCR-induced apoptosis in all splenic PLCgamma2-deficient B cell subpopulations and partially restores the numbers of PLCgamma2-deficient FO B cells. In contrast to Bcl-2, enforced expression of A1 preferentially prevents BCR-induced apoptosis in PLCgamma2-deficient FO B cells and partially restores the numbers of these B cells. Therefore, the PLCgamma2 pathway provides a survival signal via regulation of Bcl-2 in all splenic B cell subpopulations and via additional induction of A1 in mature FO B cells.

Identification of Shp-2 as a Stat5A phosphatase.

Stat5A, a member of the signal transducers and activators of transcription (Stat) family, is activated upon a single tyrosine phosphorylation. Although much is known about the activation process, the mechanism by which the tyrosine-phosphorylated Stat5A proteins are inactivated is largely unknown. In this report, we demonstrate that down-regulation of the tyrosine-phosphorylated Stat5A was via dephosphorylation. Using tyrosine-phosphorylated peptides derived from Stat5A, we were able to purify protein-tyrosine phosphatase Shp-2 from cell lysates. Shp-2, but not Shp-1, specifically interacted with Stat5A in vivo, and the interaction was tyrosine phosphorylation-dependent. Moreover, Shp-2 was able to accelerate Stat5A dephosphorylation, and dephosphorylation of Stat5A was dramatically delayed in Shp-2-deficient cells. Therefore, we conclude that Shp-2 is a Stat5A phosphatase, which down-regulates the active Stat5A in vivo.