Key Points• Del(18p), together with del(17p)/TP53 mutations, is present at a high frequency before ibrutinib treatment.• BTK mutations drive ibrutinib relapse, but del(17p)/TP53 mutations may be dispensable.Ibrutinib has generated remarkable responses in patients with chronic lymphocytic leukemia (CLL), including those with an unfavorable cytogenetic profile. However, patients develop resistance, with poor outcomes and no established treatment options. Mutations in BTK and PLCG2 have emerged as main mechanisms of drug resistance, but not all patients carry these mutations. Further understanding of mechanisms of resistance is urgently needed and will support rational development of new therapeutic strategies. To that end, we characterized the genomic profiles of serial samples from 9 patients with ibrutinib-relapsed disease, including 6 who had Richter transformation. Mutations, indels, copy-number aberrations, and loss of heterozygosity were assessed using next-generation sequencing and single-nucleotide polymorphism array. We found that 18p deletion (del(18p)), together with del(17p)/TP53 mutations, was present in 5 of 9 patients before ibrutinib therapy. In addition to BTK C481 , we identified BTK T316A, a structurally novel mutation located in the SH2 domain of BTK. Minor BTK clones with low allele frequencies were captured in addition to major BTK clones. Although TP53 loss predisposes patients for relapse, clone size of TP53 loss may diminish during disease progression while mutant BTK clone expands. In patients who had Richter transformation, we found that the transformed cells were clonal descendants of circulating leukemia cells but continued to undergo evolution and drifts.Surprisingly, transformed lymphoma cells in tissue may acquire a different BTK mutation from that in the CLL leukemia cells. Collectively, these results provide insights into clonal evolution underlying ibrutinib relapse and prompt further investigation on genomic abnormalities that have clinical application potential.

Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states.

SM. Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 294: L46-L56, 2008. First published November 16, 2007 doi:10.1152/ajplung.00247.2007.-Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P Ͻ 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P Ͻ 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow. carnitine metabolism; oxidative stress THE DEVELOPMENT OF PULMONARY HYPERTENSION and its associated increased vascular reactivity are common accompaniments of congenital heart disease with increased pulmonary blood flow (3). Endothelial dysfunction is thought to be an early hallmark of pulmonary hypertension (4). There is increasing histological and physiological evidence that endothelial injury and the resulting aberration in the balance of its regulatory mechanisms play an important role in the development of pulmonary hypertension (4). Children with pulmonary hypertension have evidence of endothelial dysfunction as indicated by impaired endothelium-dependent relaxation in early disease and decreased endothelial nitric oxide synthase (eNOS) protein levels in late disease (11,23). However, data delineating the role of endotheliumderived NO in the disease have been less clear. For example, we (5) and others (72) have shown that pulmonary expression of NOS can be paradoxically inc...

Carnitines are involved in mitochondrial transport of fatty acids and are of critical importance for maintaining normal mitochondrial function. This review summarizes recent experimental and clinical studies showing that mitochondrial dysfunction secondary to a disruption of carnitine homeostasis may play a role in decreased NO signaling and the development of endothelial dysfunction. Future challenges include development of agents that can positively modulate Lcarnitine homeostasis which may have high therapeutic potential.

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The pathogenesis of pulmonary hypertension is a complex multifactorial process that involves the remodeling of pulmonary arteries. This remodeling process encompasses concentric medial thickening of small arterioles, neomuscularization of previously nonmuscular capillary-like vessels, and structural wall changes in larger pulmonary arteries. The pulmonary arterial muscularization is characterized by vascular smooth muscle cell (SMC) hyperplasia and hypertrophy. In addition, in uncontrolled pulmonary hypertension, the clonal expansion of apoptosis-resistant endothelial cells leads to the formation of plexiform lesions. Based upon a large number of studies in animal models, the three major stimuli that drive the vascular remodeling process are inflammation, shear stress and hypoxia. Although, the precise mechanisms by which these stimuli impair pulmonary vascular function and structure are unknown, reactive oxygen species (ROS)-mediated oxidative damage appears to play an important role. ROS are highly reactive due to their unpaired valence shell electron. Oxidative damage occurs when the production of ROS exceeds the quenching capacity of the anti-oxidant mechanisms of the cell. ROS can be produced from complexes in the cell membrane (nicotinamide adenine dinucleotide phosphate-oxidase), cellular organelles (peroxisomes and mitochondria), and in the cytoplasm (xanthine oxidase). Furthermore, low levels of tetrahydrobiopterin (BH4) and L-arginine the rate limiting co-factor and substrate for endothelial nitric oxide synthase (eNOS), can cause the uncoupling of eNOS, resulting in decreased NO production and increased ROS production. This review will focus on the ROS generation systems, scavenger antioxidants, and oxidative stress associated alterations in vascular remodeling in pulmonary hypertension.

Next-generation sequencing (NGS) genomic oncology profiling assays have emerged as key drivers of personalized cancer care and translational research. However, validation of these assays to meet strict clinical standards has been historically problematic because of both significant assay complexity and a scarcity of optimal validation samples. Herein, we present the clinical validation of 76 genes from a novel 1212-gene large-scale hybrid capture cancer sequencing assay (University of Chicago Medicine OncoPlus) using full-data comparisons against multiple clinical NGS amplicon-based assays to yield dramatic increases in per-sample data comparison efficiency compared with previously published validations. Using a sample set of 104 normal, solid tumor, and hematopoietic malignancy specimens, head-to-head NGS data analyses allowed for 6.8 million individual clinical base call comparisons, including 2729 previously confirmed variants, with 100% sensitivity and specificity. University of Chicago Medicine OncoPlus showed excellent performance for detection of single-nucleotide variants, insertions/deletions up to 52 bp, and FLT3 internal tandem duplications of up to 102 bp or larger. Highly concordant copy number variant and ALK/RET/ROS1 gene fusion detection were also observed. In addition to underlining the efficiency of NGS validation via full-data benchmarking against existing clinical NGS assays, this study also highlights the degree of performance similarity between hybrid capture and amplicon assays that is attainable with the application of strict quality control parameters and optimized computational analytics.

Rationale Disruption of the circadian clock in mice produces vascular dysfunction as evidenced by impairments in endothelium-dependent signaling, vasomotion, and blood vessel remodeling. Although the altered function of endothelial NO synthase and the overproduction of reactive oxygen species are central to dysfunction of the endothelium, to date, the impact of the circadian clock on endothelial NO synthase coupling and vascular reactive oxygen species production is not known. Objective The goals of the present study were to determine whether deletion of a critical component of the circadian clock, Bmal1, can influence endothelial NO synthase coupling and reactive oxygen species levels in arteries from Bmal1-knockout (KO) mice. Methods and Results Endothelial function was reduced in aortae from Bmal1-KO mice and improved by scavenging reactive oxygen species with polyethylene glycol-superoxide dismutase and nonselectively inhibiting cyclooxygenase isoforms with indomethacin. Aortae from Bmal1-KO mice exhibited enhanced superoxide levels as determined by electron paramagnetic resonance spectroscopy and dihydroethidium fluorescence, an elevation that was abrogated by administration of nitro-L -arginine methyl ester. High-performance liquid chromatography analysis revealed a reduction in tetrahydrobiopterin and an increase in dihydrobiopterin levels in the lung and aorta of Bmal1-KO mice, whereas supplementation with tetrahydrobiopterin improved endothelial function in the circadian clock KO mice. Furthermore, levels of tetrahydrobiopterin, dihydrobiopterin, and the key enzymes that regulate biopterin bioavailability, GTP cyclohydrolase and dihydrofolate reductase exhibited a circadian expression pattern. Conclusions Having an established influence in the metabolic control of glucose and lipids, herein, we describe a novel role for the circadian clock in metabolism of biopterins, with a significant impact in the vasculature, to regulate coupling of endothelial NO synthase, production of superoxide, and maintenance of endothelial function. (Circ Res. 2012; 111:1157–1165.)