Genetic Testing for Hypertriglyceridemia in Academic Lipid Clinics: Implications for Precision Medicine-Brief Report.

BACKGROUND: Severe hypertriglyceridemia is often caused by variants in genes of triglyceride metabolism. These variants include rare, heterozygous pathogenic variants (PVs), or multiple common, small-effect single nucleotide polymorphisms that can be quantified using a polygenic risk score (PRS). The role of genetic testing to examine PVs and PRS in predicting risk for pancreatitis and severity of hypertriglyceridemia is unknown. METHODS: We examined the relationship of PVs and PRSs associated with hypertriglyceridemia with the highest recorded plasma triglyceride level and risk for acute pancreatitis in 363 patients from 3 academic lipid clinics who underwent genetic testing (GBinsight's Dyslipidemia Comprehensive Panel). Categories of hypertriglyceridemia included: normal triglyceride (<200 mg/dL), moderate (200-499 mg/dL), severe (500-999 mg/dL), or very severe (≥1000 mg/dL). RESULTS: PVs and high PRSs were identified in 37 (10%) and 59 (16%) individuals, respectively. Patients with both had increased risk for very severe hypertriglyceridemia compared with those with neither genetic risk factor. Risk for acute pancreatitis was also increased in individuals with both genetic risk factors (odds ratio, 5.1 [P=0.02] after controlling for age, race, sex, body mass index, and highest triglyceride level), but not in individuals with PV or high PRS alone. CONCLUSIONS: The presence of both PV and high PRS significantly increased risk for very severe hypertriglyceridemia and acute pancreatitis, whereas PV or PRS alone only modestly increased risk. Genetic testing may help identify patients with hypertriglyceridemia who have the greatest risk for developing pancreatitis and may derive the greatest benefit from novel triglyceride-lowering therapies.

Atorvastatin-associated rhabdomyolysis in a patient with a novel variant of the SLCO1B1 gene: A case report.

We report the case of an individual with severe hypercholesterolemia who experienced rhabdomyolysis with high dose atorvastatin. Genetic testing was undertaken to evaluate for suspected familial hypercholesterolemia (FH) and for the presence of gene variants associated with susceptibility to statin associated muscle disease. Genetic testing identified the presence of a potentially damaging variant of the hepatic xenobiotic transporter pump SLCO1B1, a single nucleotide variant (SNV) (rs77271279, c.481+1G>T) that disrupts the canonical donor splice motif. Although this variant has not previously been reported as associated with rhabdomyolysis and thus requires validation in population studies, it likely played a role in this patient's susceptibility to rhabdomyolysis based on functional assessment of the effect of this variant on SLCO1B1 protein function and given the known role of this transporter in statin uptake by the liver. The presence of this gene variant reinforced our decision to treat the patient's hypercholesterolemia with non-statin alternatives (PCSK9 inhibitor and ezetimibe). Genetic testing also identified the presence of a second SLCO1B1 gene variant, c.1200C>G (p.Phe400Leu, rs59113707) and homozygosity for an intron variant of the apolipoprotein(a) (LPA) gene (c.2604.138G>A intron variant, rs9457951) associated with increased Lp(a), a risk factor for atherosclerotic cardiovascular disease. Notably, all three variants are rare in persons of European descent but more frequent in African-Americans. These findings underscore the role of disabling mutations of the SLCO1B1 gene in statin myopathy and the need to validate these and other gene variants associated with statin myopathy in a population of patients with statin-associated muscle disease.

The Role of Genetics in Cardiovascular Risk Reduction: Findings From a Single Lipid Clinic and Review of the Literature.

BACKGROUND: Genetic information is not routinely obtained in the management of most lipid disorders or in primary or secondary prevention of cardiovascular disease (CVD). We sought to determine the prevalence of pathogenic variants associated with lipoprotein metabolism or coronary artery disease (CAD) in a single lipid clinic and discuss the future use of genetic information in CVD prevention. METHODS: Genetic testing was offered to patients with hypertriglyceridemia (defined as pre-treatment fasting triglycerides ≥150 mg/dL), elevated LDL-C (defined as pre-treatment ≥190 mg/dL), low HDL-C (defined as ≤40 mg/dL), elevated lipoprotein (a) (defined as ≥50 mg/dL or 100 nmol/L) or premature CAD (defined as an acute coronary syndrome or revascularization before age 40 years in men and 50 years in women) using next-generation DNA sequencing of 327 exons and selected variants in 129 genes known or suspected to be associated with lipoprotein metabolism or CAD. RESULTS: 82 of 84 patients (97.6%) were found to have a variant associated with abnormal lipid metabolism or CAD. The most common pathogenic or likely pathogenic variants included those of the LDL receptor (15 patients) and lipoprotein lipase (9 patients). Other common variants included those of apolipoprotein A5 (14 patients) and variants associated with elevated lipoprotein (a) (25 patients). CONCLUSIONS: The majority of patients presenting to a single lipid clinic were found to have at least one variant associated with abnormal lipoprotein metabolism or CAD. Incorporating genetic information, including the use of genetic risk scores, is anticipated in the future care of lipid disorders and CVD prevention.

Transcription factor HBP1 is a direct anti-cancer target of transcription factor FOXO1 in invasive oral cancer.

Either FOXO1 or HBP1 transcription factor is a downstream effector of the PI3K/Akt pathway and associated with tumorigenesis. However, the relationship between FOXO1 and HBP1 in oral cancer remains unclear. Analysis of 30 oral tumor specimens revealed that mean mRNA levels of both FOXO1 and HBP1 in non-invasive and invasive oral tumors were found to be significantly lower than that of the control tissues, and the status of low FOXO1 and HBP1 (< 0.3 fold of the control) was associated with invasiveness of oral tumors. To investigate if HBP1 is a direct transcription target of FOXO1, we searched potential FOXO1 binding sites in the HBP1 promoter using the MAPPER Search Engine, and two putative FOXO1 binding sites located in the HBP1 promoter -132 to -125 bp and -343 to -336 bp were predicted. These binding sites were then confirmed by both reporter gene assays and the in cellulo ChIP assay. In addition, Akt activity manipulated by PI3K inhibitor LY294002 or Akt mutants was shown to negatively affect FOXO1-mediated HBP1 promoter activation and gene expression. Last, the biological significance of the FOXO1-HBP1 axis in oral cancer malignancy was evaluated in cell growth, colony formation, and invasiveness. The results indicated that HBP1 knockdown potently promoted malignant phenotypes of oral cancer and the suppressive effect of FOXO1 on cell growth, colony formation, and invasion was alleviated upon HBP1 knockdown in invasive oral cancer cells. Taken together, our data provide evidence for HBP1 as a direct downstream target of FOXO1 in oral cancer malignancy.

SIRT1 and LSD1 competitively regulate KU70 functions in DNA repair and mutation acquisition in cancer cells.

Acquisition of BCR-ABL mutations underlies drug resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors, but the molecular mechanisms of mutation acquisition are poorly understood. We previously showed that lysine deacetylase sirtuin 1, SIRT1, promotes acquisition of BCR-ABL mutations in association with enhancing KU70 mediated non-homologous end joining DNA repair. In this study, we demonstrate that lysine specific demethylase 1 (LSD1) plays an opposite role to SIRT1 in regulating DNA repair and mutation acquisition. In response to therapeutic stress and DNA damage, LSD1 and SIRT1 compete for binding to KU70 on DNA damage foci globally and on the ABL locus. The recruitment of SIRT1 or LSD1 to KU70 impacts chromatin structure but does not correlate well with their direct histone modification functions, and SIRT1 helps maintain histone H4K16 acetylation and open chromatin for repair. The competitive KU70 binding by these proteins affects cancer cells' ability to repair broken DNA and acquire resistant genetic mutations in CML and prostate cancer cells. We identify that the core domain of KU70 binds both LSD1 and SIRT1, forming a molecular basis for the competition. The C-terminal SAP motif of KU70 mediates LSD1/SIRT1 competitive interaction by suppressing LSD1 binding to KU70 and ectopic expression of SAP-deleted KU70 to CML cells compromises their ability to acquire BCR-ABL mutations. Our study reveals a novel cellular stress response mechanism in cancer cells and a key role of LSD1/SIRT1/KU70 dynamic interaction in regulating DNA repair and mutation acquisition.

ATRA-induced cellular differentiation and CD38 expression inhibits acquisition of BCR-ABL mutations for CML acquired resistance.

Acquired resistance through genetic mutations is a major obstacle in targeted cancer therapy, but the underlying mechanisms are poorly understood. Here we studied mechanisms of acquired resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) by examining genome-wide gene expression changes in KCL-22 CML cells versus their resistant KCL-22M cells that acquire T315I BCR-ABL mutation following TKI exposure. Although T315I BCR-ABL is sufficient to confer resistance to TKIs in CML cells, surprisingly we found that multiple drug resistance pathways were activated in KCL-22M cells along with reduced expression of a set of myeloid differentiation genes. Forced myeloid differentiation by all-trans-retinoic acid (ATRA) effectively blocked acquisition of BCR-ABL mutations and resistance to the TKIs imatinib, nilotinib or dasatinib in our previously described in vitro models of acquired TKI resistance. ATRA induced robust expression of CD38, a cell surface marker and cellular NADase. High levels of CD38 reduced intracellular nicotinamide adenine dinucleotide (NAD+) levels and blocked acquired resistance by inhibiting the activity of the NAD+-dependent SIRT1 deacetylase that we have previously shown to promote resistance in CML cells by facilitating error-prone DNA damage repair. Consequently, ATRA treatment decreased DNA damage repair and suppressed acquisition of BCR-ABL mutations. This study sheds novel insight into mechanisms underlying acquired resistance in CML, and suggests potential benefit of combining ATRA with TKIs in treating CML, particularly in advanced phases.

HBP1-mediated transcriptional regulation of DNA methyltransferase 1 and its impact on cell senescence.

The activity of DNA methyltransferase 1 (DNMT1) is associated with diverse biological activities, including cell proliferation, senescence, and cancer development. In this study, we demonstrated that the HMG box-containing protein 1 (HBP1) transcription factor is a new repressor of DNMT1 in a complex mechanism during senescence. The DNMT1 gene contains an HBP1-binding site at bp -115 to -134 from the transcriptional start site. HBP1 repressed the endogenous DNMT1 gene through sequence-specific binding, resulting in both gene-specific (e.g., p16(INK4)) and global DNA hypomethylation changes. The HBP1-mediated repression by DNMT1 contributed to replicative and premature senescence, the latter of which could be induced by Ras and HBP1 itself. A detailed investigation unexpectedly revealed that HBP1 has dual and complex transcriptional functions, both of which contribute to premature senescence. HBP1 both repressed the DNMT1 gene and activated the p16 gene in premature senescence. The opposite transcriptional functions proceeded through different DNA sequences and differential protein acetylation. While intricate, the reciprocal partnership between HBP1 and DNMT1 has exceptional importance, since its abrogation compromises senescence and promotes tumorigenesis. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence, with an impact on overall DNA methylation state.

Sirtuins in hematological aging and malignancy.

Aging of the hematological system causes anemia, reduced immunity, and increased incidence of hematological malignancies. Hematopoietic stem cells (HSCs) play a crucial role in this process as their functions decline during aging. Sirtuins are a family of protein lysine modifying enzymes that have diverse roles in regulating metabolism, genome stability, cell proliferation, and survival, and have been implicated in mammalian aging and longevity. Here we provide an updated overview of sirtuins in aging research; particularly, how increased activity of SIRT1, SIRT3, or SIRT6 improves several aging parameters, and may possibly increase lifespan in mice. We review the literature on how sirtuins may play a role in HSC aging and hematological malignancies, and how key signaling pathways of HSCs may be affected by sirtuins. Among them, SIRT1 plays a critical role in chronic myelogenous leukemia, an age-dependent malignancy, and inhibition of SIRT1 sensitizes leukemic stem cells to tyrosine kinase inhibitor treatment and blocks acquisition of resistant oncogene mutations. In-depth understanding of sirtuins in HSC aging and malignancy may help design novel strategies to deter hematological aging and improve treatment of hematological malignancies.

Overcoming CML acquired resistance by specific inhibition of Aurora A kinase in the KCL-22 cell model.

Serine/threonine kinase Aurora A is essential for regulating mammalian cell division and is overexpressed in many types of human cancer. However, the role of Aurora A in chemoresistance of chronic myelogenous leukemia (CML) is not well understood. Using the KCL-22 cell culture model we have recently developed for studying mechanisms of CML acquired resistance, we found that Aurora A expression was partially reduced in these cells upon treatment with the tyrosine kinase inhibitor imatinib, which accompanied the acquisition of BCR-ABL mutation for imatinib resistance. Gene knockdown of BCR-ABL also reduced Aurora A expression, and conversely, Aurora A expression increased in hematopoietic progenitor cells after BCR-ABL expression. Inhibition of Aurora A induced apoptosis of CML cells with or without T315I BCR-ABL mutation and suppressed CML cell growth. Inhibition of Aurora A by gene knockdown or a highly specific small molecule inhibitor sensitized CML cells to imatinib treatment and effectively blocked acquisition of BCR-ABL mutations and KCL-22 cell relapse on imatinib, nilotinib or dasatinib. Our results show that Aurora A plays an important role for facilitating acquisition of BCR-ABL mutation and acquired resistance to tyrosine kinase inhibitors in the culture model and suggest that inhibition of Aurora A may provide an alternative strategy to improve CML treatment to overcome resistance.

The green tea compound, (-)-epigallocatechin-3-gallate downregulates N-cadherin and suppresses migration of bladder carcinoma cells.

Green tea has been reported as potential dietary protection against numerous cancers and has been shown to have activity in bladder tumor inhibition in different animal models. The goal of this study was to examine the effects of (-)-epigallocatechin gallate (EGCG-the major phytochemical in green tea) on growth inhibition and behavior of human bladder carcinoma cells and to identify the altered signaling pathway(s) underlying the response to EGCG exposure. EGCG inhibited the in vitro growth of invasive bladder carcinoma cells with an IC(50) range of 70-87 microM. At a concentration of 20 microM, EGCG decreased the migratory potential of bladder carcinoma cells with concomitant activation of p42/44 MAPK and STAT3 and inactivation of Akt. Using biochemical inhibitors of MAPK/ERK, and siRNA to knockdown STAT3 and Akt, inhibition of migration was recorded associated with Akt but not MAPK/ERK or STAT3 signaling in bladder cells. In addition, EGCG downregulated N-cadherin in a dose-dependent manner where reduction in N-cadherin expression paralleled declining migratory potential. Continuous feeding of EGCG to mice prior to and during the establishment of bladder carcinoma xenografts in vivo revealed >50% reduction in mean final tumor volume (P

Target therapy using a small molecule inhibitor against angiogenic receptors in pancreatic cancer.

PURPOSE: PD173074, a small molecule inhibitor of VEGF-RII and FGF-RI, targets neoangiogenesis and mitogenesis. This study aimed to analyze a single-compound-driven inhibition of FGF and VEGF receptors in pancreatic cancer. EXPERIMENTAL DESIGN: RT-PCR and Western blots were performed to quantify protein expression and phosphorylation. Anchorage dependent and independent growth assays were used to study cell growth. With flow cytometry, cell cycle analysis and apoptosis were studied. In vivo HPAF-II and MIA PaCa-2 cells were xenografted. Animals were treated daily for 10 weeks. Immunohistochemistry was used to quantify microvessel density and apoptosis. RESULTS: Highest levels of FGF-RI were detectable in MIA PaCa-2 cells, lowest in HPAF-II cells. PD173074 inhibited cell growth most prominently in cells expressing high levels of FGF-RI. Cell cycle progression was inhibited by blocking transition in the G(0)/G(1) phase, and consequently, apoptosis was increased. In vivo significant inhibition of orthotopic tumor growth was achieved by a combination effect of inhibition of mitogenesis, induction of apoptosis, and reduction of angiogenesis in PD173074-treated animals. CONCLUSIONS: These data highlight VEGF-RII and FGF-RI as therapeutic targets and suggest a potential role for the combined use of tyrosine kinase inhibitors in the management of inoperable pancreatic cancer patients.

Combination therapy for advanced pancreatic cancer using Herceptin plus chemotherapy.

The HER2/neu oncogene is overexpressed in up to 70% of human pancreatic cancer specimens when compared to normal pancreatic tissue. This cell surface receptor can be targeted specifically by the neutralizing antibody Herceptin. Herceptin has been successfully used in combination with other chemotherapeutic agents in breast cancer, a cancer in which only 30% of patients harbor elevated HER2/neu levels. In the present study, we investigated the therapeutic efficacy of Herceptin in combination with gemcitabine and docetaxel. Gemcitabine is currently the standard chemotherapeutic agent used to treat pancreatic cancer. In contrast, docetaxel, a taxane, is only just being investigated in pancreatic cancer. Tumor cell resistance to taxanes is at least in part mediated by the HER2/NEU oncogene. We have previously characterized HER2/NEU expression in human pancreatic cancer cell lines and studied the anti-tumor activity of Herceptin monotherapy in vitro and in vivo. In the present study, combination therapy resulted in a dramatic improvement of animals bearing human pancreatic cancer xenografts. Furthermore, metastasis and production of ascites was lower when a combination of these three agents was used. We conclude that, as with breast cancer, the anti-tumor activity of Herceptin may be improved by combination with taxanes or gemcitabine.

Pain scores improve analgesic administration patterns for trauma patients in the emergency department.

OBJECTIVE: To determine the efficacy of pain scores in improving pain management practices for trauma patients in the emergency department (ED). METHODS: A prospective, observational study of analgesic administration to trauma patients was conducted over a nine-week period following educational intervention and introduction of verbal pain scores (VPSs). All ED nursing and physician staff in an urban Level I trauma center were trained to use the 0-10 VPS. Patients younger than 12 years old, having a Glasgow Coma Scale score (GCS) <8, or requiring intubation were excluded from analysis. Demographics, mechanism of injury, vital signs, pain scores, and analgesic data were extracted from a computerized ED database and patients' records. The staff was blinded to the ongoing study. RESULTS: There were 150 patients studied (183 consecutive trauma patients seen; 33 patients excluded per criteria). Pain scores were documented for 73% of the patients. Overall, 53% (95% confidence interval [CI] = 45% to 61%) of the patients received analgesics in the ED. Of the patients who had pain scores documented, 60% (95% CI = 51% to 69%) received analgesics, whereas 33% (95% CI = 18% to 47%) of the patients without pain scores received analgesics. No patient with a VPS < 4 received analgesics, whereas 72% of patients with a VPS > 4 and 82% with a VPS > 7 received analgesics. Mean time to analgesic administration was 68 minutes (95% CI = 49 to 87). CONCLUSIONS: Pain assessment using VPS increased the likelihood of analgesic administration to trauma patients with higher pain scores in the ED.

Pancreatic cancer growth is inhibited by blockade of VEGF-RII.

BACKGROUND: Angiogenesis is important in the development and progression of pancreatic cancer. Therefore antiangiogenic therapy targeting endothelial cells may represent a promising therapeutic option. The aim of the study was to evaluate antiangiogenic therapy as a potential therapeutic option in pancreatic cancer. METHODS: Replication-deficient retroviruses encoding truncated VEGF-RII were used to block vascular endothelial growth factor (VEGF) signaling. Tumor growth of 3 pancreatic cancer cell lines was assayed in a nude mouse model in which each pancreatic cancer cell line was subcutaneously inoculated together with retrovirus-producing cells. Expression of VEGF was assayed by RT-PCR and by enzyme-linked immunosorbent assay. Oxygen tension in tumors was determined polarographically. RESULTS: All 3 pancreatic cancer cell lines expressed VEGF mRNA, with the highest VEGF secretion seen in MIA PaCa-2 cells. In vivo therapeutic intervention through dominant negative inhibition of VEGF-RII significantly reduced the growth rate of subcutaneous tumors and inhibited tumor neoangiogenesis. Tumor oxygenation, however, was not altered in xenograft tumors treated with dominant negative retroviruses. CONCLUSION: The ligand/receptor system consisting of VEGF and VEGF-RII seems to be of biologic significance in the pathogenesis of pancreatic cancer growth. Therefore therapeutic intervention in this angiogenic system by a retroviral-based gene transfer technology represents a rational and feasible new technique to inhibit tumor growth.

Patterns of analgesic use in trauma patients in the ED.

The objective was to describe patterns of analgesic use for trauma patients treated in our emergency department (ED). We reviewed analgesic use in consecutive patients meeting American College of Surgeons (ACS) Trauma Center Guidelines. A comprehensive database was abstracted from this institution's Trauma Registry and medical records of each patient. A total of 38% (95% CI: 31-46%) of patients received analgesics. Time to administration of first dose of analgesia was 109 minutes (95% CI: 85-133). Women, patients with long bone and pelvic fractures, and those with a longer ED stay were most likely to receive analgesics. Patients with head trauma and those admitted to the intensive care unit were least likely to receive analgesics. Morphine was the most frequent analgesic used with an average total dose of 14 milligrams. A majority of patients meeting ACS Trauma Center Guidelines did not receive analgesics in the ED.