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Supplementary Video 1 from Protein Kinase A Effects of an Expressed PRKAR1A Mutation Associated with Aggressive Tumors

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Supplementary Video 2 from Protein Kinase A Effects of an Expressed PRKAR1A Mutation Associated with Aggressive Tumors

Lung cancer remains the second most commonly diagnosed cancer worldwide and the leading cause of cancer-related mortality. The mapping of genomic alterations and their role in lung-cancer progression has been followed by the development of new therapeutic options. Several novel drugs, such as targeted therapy and immunotherapy, have significantly improved outcomes. However, many patients with lung cancer do not benefit from existing therapies or develop progressive disease, leading to increased morbidity and mortality despite initial responses to treatment. Alterations in DNA-damage repair (DDR) genes represent a cancer hallmark that impairs a cell’s ability to prevent deleterious mutation accumulation and repair. These alterations have recently emerged as a therapeutic target in breast, ovarian, prostate, and pancreatic cancers. The role of DDR alterations remains largely unknown in lung cancer. Nevertheless, recent research efforts have highlighted a potential role of some DDR alterations as predictive biomarkers of response to treatment. Despite the failure of PARP inhibitors (main class of DDR targeting agents) to improve outcomes in lung cancer patients, there is some evidence suggesting a role of PARP inhibitors and other DDR targeting agents in benefiting a distinct subset of lung cancer patients. In this review, we will discuss the existing literature on DDR alterations and homologous recombination deficiency (HRD) state as predictive biomarkers and therapeutic targets in both non-small cell lung and small cell lung cancer.

Synthetic lethality occurs between two genes when silencing of either gene alone enables viable outcomes but inhibition of both is lethal. Understanding context-dependent functioning of synthetic lethality allows therapeutic targeting in cancer. Furthermore, the paradigm shift toward precision medicine to treat cancer necessitates the establishment of biomarkers to help determine which patient populations might respond to specific drug combinations. Elucidating synthetically lethal gene combinations in cancer could establish clinically relevant drug combinations as well as biomarkers to better treat patients. Here, we have reviewed the recent synthetically lethal gene combinations in preclinical and clinical settings and discuss how this approach could help reveal potential biomarkers.

Introduction: Allostatic load (AL) is a composite measure of chronic stress including cardiovascular, immune, and metabolic biomarkers. AL has previously been associated with health disparities in cardiovascular disease, metabolic syndrome, and certain malignancies. However, there are no prior publications to the best of our knowledge evaluating the association of AL and clinical outcomes in patients with advanced prostate cancer (PCa). Methods: We identified 139 subjects with metastatic or biochemically recurrent (BCR) PCa treated at the University of Illinois Hospital and Health Sciences System between January 2015 and May 2022. Subjects were assigned AL scores based on retrospective assessment of 9 biomarkers (systolic and diastolic blood pressure, heart rate, alkaline phosphatase, albumin, creatinine, body mass index, glucose, white blood cell count) collected at the time of diagnosis of advanced disease. Subjects were assigned a total AL score whereby each of the individual 9 biomarkers were given a value of 0 or 1 if threshold value was met. Results: Of the 139 subjects, the median age at PCa diagnosis was 63. The majority were African American (65.4%), 14.4% were Hispanic or Latino, 12.2% were white and non-Hispanic or Latino, and race/ethnicity was other or unknown for 7.9%. A majority (62.6%) had localized or regional nodal disease at diagnosis but later developed metastases or BCR, while 37.4% had de novo metastatic disease. Those with de novo metastatic disease had a higher mean AL score compared to those who had localized or regional nodal disease at diagnosis (2.62 vs. 2.08, p=0.03). Nearly all of the subjects (98.6%) received androgen deprivation therapy (ADT) while 1.4% had surgical castration. Most (66.2%) had local therapies such as surgery or radiation prior to or in addition to ADT. A minority (39.6%) developed castrate-resistant disease while 60.4% did not. For subjects who developed castrate-resistant disease, median time to castrate-resistance was 31 months. Those who developed castrate-resistant disease had a higher mean AL score compared to those who did not (2.58 vs. 2.08, p=0.03). In the study population, 25.9% had a low sum Gleason score (≤7) at diagnosis, 46.0% had a high sum Gleason score (≥8) at diagnosis, and 28.1% did not have a Gleason score available at diagnosis. Subjects with a high sum Gleason score had higher mean AL compared to those with low sum Gleason scores, though this finding did not reach statistical significance (2.47 vs. 1.94, p=0.06). In our study, 32 out of 139 subjects had at least one biomarker missing from their records and therefore not included in the subject’s AL score. The most common biomarkers missing were albumin and alkaline phosphatase. Discussion: In our pilot, single-institution study, higher AL may be associated with more aggressive phenotypes of PCa including de novo metastatic disease, earlier progression to castrate-resistance, and higher Gleason scores. Prospective studies are needed to further validate the role AL plays in PCa aggressiveness. Citation Format: Michael Weinfeld, Ryan Nguyen, Frank Weinberg, Vijayakrishna Gadi, Natalie Reizine. Increased allostatic load is associated with castrate-resistance and de novo metastatic disease in prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C032.

Until recently, the treatment of patients with advanced non-small-cell lung cancer (NSCLC) whose tumors did not have a targetable genetic alteration was cytotoxic chemotherapy alone. This treatment provided only modest survival benefit. The introduction of immune checkpoint inhibitors targeting programmed cell death 1 protein (PD-1) signaling pathway in the treatment of patients with NSCLC has had significant effect on patient survival. Atezolizumab, nivolumab and pembrolizumab have been shown to be superior to chemotherapy in patients with recurrent NSCLC. Recently, pembrolizumab has been combined with chemotherapy in the front-line setting and has demonstrated an improvement in overall survival in NSCLC patients as compared to chemotherapy alone. In this review we will focus on the clinical trials that led to approval of combination pembrolizumab and chemotherapy as first-line treatment for patients with advanced NSCLC as well as discuss other combinations of immunotherapy and chemotherapy that have also been evaluated.

BACKGROUND: Lung cancer death rates and incidence in both men and women have decreased over the past two decades. However, certain subsets of non-small cell lung cancer (NSCLC) have arisen with poor outcomes. Identifying factors which contribute to poorer outcomes as well as those that inform early detection strategies remain unmet needs. We present data from a contemporaneous group of NSCLC patients that received care at a single University teaching hospital to understand clinical and pathological factors influencing outcomes in the past decade. METHODS: A cohort of 2,289 patients with NSCLC who established care at the Rogel Cancer Center, University of Michigan between January 2011 and April 2019 were identified. Patient characteristics and clinical outcomes were recorded using electronic health records. The Kaplan-Meier method and the Cox proportional model were used to assess relationship between clinic-pathological factors and survival. RESULTS: Of the 2,289 patients, 92% were >50 years of age while 8% were

Introduction: Allostatic load (AL) is a multi-system measure of chronic stress shown to be an emerging framework for understanding disparities in cancer outcomes, but there have been no AL studies in lung cancer. Methods: We conducted a retrospective review of patients diagnosed with stage IV lung adenocarcinoma from 2016 - 2020 at the University of Illinois Hospital and Health System. An AL index included 9 biomarkers (systolic blood pressure, diastolic blood pressure, resting heart rate, BMI, albumin, eGFR, creatinine, glucose, and use of medications for hypertension, diabetes, or hypocholesteremia) with 1 point assigned for each biomarker meeting a pre-determined cutoff. AL was dichotomized between AL-high (5-9) and AL-low (0-4) with median progression-free survival of first-line therapy (mPFS1) and median overall survival (mOS) estimated for both groups with Kaplan-Meier curves. Rates of clinically actionable driver mutations (driver-POS), including EGFR, ALK, ROS1, BRAF V600e, RET, MET, NTRK, and KRAS G12C, were compared for both groups using chi-squared test. Mean AL was compared between those who were driver-POS and those who did not have a clinically actionable driver mutation (driver-NEG) using independent samples t-test. Results: A total of 61 patients were identified with 56% female and 74% former or current tobacco users. The cohort was 49% Black, 21% White, and 13% Hispanic or Latino. Mean AL at diagnosis was 3.3 (+/- 1.4). AL-high patients comprised 20% (12) of the cohort compared with 80% (49) AL-low. Between AL-high and AL-low cohorts, there was no statistically significant difference in mPFS1 (15 vs. 13 months, P = 0.98) and mOS (34 vs. 18 months, P = 0.33). Rates of driver-POS were lower in AL-high compared with AL-low (25% vs 69%, P Conclusions: Patients whose tumors did not have a clinically actionable driver mutation were associated with a higher AL, suggestive of higher chronic stress playing a role in more aggressive tumor biology. No statistically significant survival difference was observed in AL-high versus AL-low populations for mPFS1 and mOS. Larger, prospective trials are planned to correlate AL at diagnosis with clinical and biomarker data. AL, which serves as a measure of physiological dysfunction due to chronic stress associated with social disadvantage, may have implications for tumor biology and clinical outcomes in lung cancer. Citation Format: Ryan H. Nguyen, Michael Weinfeld, Mary Pasquinelli, Frank Weinberg, Natalie Reizine, Lawrence Feldman, Vijayakrishna K. Gadi. Allostatic load, tumor genomics, and outcomes in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3663.

e18731 Background: Immune checkpoint inhibitors (ICI) constitute the mainstay of treatment in several unresectable locally advanced and metastatic solid cancers. mRNA COVID-19 vaccines are immunogenic and can modulate intrinsic host immunostimulatory properties however the effect of COVID-19 mRNA vaccination on outcomes in patients receiving ICI is not well understood. This study examines the outcomes in cancer patients receiving ICI according to their vaccination status. Methods: From January 2021 to December 2021, we identified adult patients with locally advanced and metastatic solid tumors at the University of Illinois Hospital & Health Sciences System who had received at least one dose of ICI, either as monotherapy or in combination with chemotherapy or targeted therapy, in any line of cancer treatment. Patients were stratified by COVID-19 vaccination status and treatment type (monotherapy versus combined chemoimmunotherapy). Endpoints included immune-related adverse events (IRAEs), progression-free survival (PFS) from ICI initiation and overall survival (OS). Results: Among 89 patients meeting these inclusion criteria, the mean age at diagnosis was 66 years, patient sex was about equally split (female 50.5% to male 49.5%), most patients were minorities (including 58.4% African American), vaccinated (78.7%), had lung cancer (57.3%), were stage IV (71.1%), and had received ICI monotherapy (67.4%). There were no significant differences in the rate of IRAEs between vaccinated and non-vaccinated patients ( p= 0.53). Patients who received ICI monotherapy had higher rates of IRAEs ( p< 0.001). There was no difference in PFS between vaccinated and non-vaccinated patients ( p= 0.7) and no difference in OS between vaccinated and non-vaccinated patients ( p= 0.59). Conclusions: In this real-world sample of patients with advanced solid cancers who received ICI there were no significant differences in IRAEs, PFS or OS between vaccinated and non-vaccinated patients, which may be due to the relatively small sample size. Larger real-world datasets with long-term follow-up are needed to study the effect of mRNA COVID-19 vaccination on outcomes in advanced cancer patients receiving ICI.

3138 Background: TMB is routinely reported in cancer patients tested with broad-panel next generation sequencing and has become a predictive biomarker associated with response to checkpoint inhibitor (CPI) therapy. Sequencing of paired tumor and normal specimens allows correction of TMB estimates with patient-specific germline variants. When a paired normal specimen is unavailable, TMB estimates are corrected using germline variant annotations derived from population-scale germline variant surveys. Germline variants do not generate neoantigens, which is the putative target of the immune response in CPI treated patients. To evaluate TMB differences in paired sequencing (PS) and tumor-only sequencing (TOS), we compared TMB assessments—stratified by race—in two common malignancies. Methods: Using de identified records from the Tempus clinico-genomic database, cohorts of patients with non-small cell lung cancer (NSCLC) and breast cancer sequenced using the Tempus xT NGS platform (DNA-seq of 595-648 genes at 500x coverage, whole exome capture RNA-seq) and noted to not have microsatellite instability, were identified for analyses. The Kruskall-Wallis test was used to compare TMB distributions. Results: Among 4,817 NSCLC patients with race information (13% Black (B), 5% Asian (A), 82% White (W), 3,052 had PS, and 1,765 had TOS performed. Median TMB for B, A, and W patients was 5.8, 2.6, and 4.7 (within group p < 0.0001), respectively in patients with PS, and 9.5, 6, and 7.4 (within group p < 0.0001), in patients with TOS. Comparisons across PS and TOS were highly significant (p < 0.0001). The absolute difference in median TMB was 3.7, 3.4, and 2.5, respectively. Among 3,191 patients with breast cancer (17% B, 4% A, 78% W), 2,220 had PS, and 971 had TOS. Median TMB for B, A, and W patients was 2.6, 2.1, and 2.6 (within group p = 0.11), respectively, in patients with PS, and 6.3, 5.8, and 4.7 (within group p < 0.0001) in patients with TOS. Comparisons across PS and TOS were highly significant (p < 0.0001). The absolute difference in median TMB was 3.7, 3.7, and 2.1, respectively. Conclusions: PS reduces estimated TMB compared to TOS across all racial groups with a pronounced difference in Black and Asian racial groups. This is expected as population databases of germline variation are based on cohorts predominantly from individuals of European ancestry, leading to artifactually high TMB in minorities tested with TOS. As a result, artifactually elevated TMB estimates from TOS may promote treatment with CPI in patients with a low probability of response which could exacerbate known race-based outcome disparities. PS provides a more accurate estimate of TMB regardless of race and could reduce the use of CPI in patients with a low likelihood of response.

e14595 Background: Management of cancer-related pain with opioids is hypothesized to decrease efficacy of immune checkpoint inhibitors (ICIs) through immunosuppressive effects of opioids, such as down-regulation of major histocompatibility complex class II. There is limited evidence on how opioids affect outcomes in patients receiving ICIs. Methods: We identified 212 patients at the University of Illinois Hospital and Health Sciences System who received an ICI between January 1, 2015 and July 31, 2021. Overall survival (OS) and progression-free survival (PFS) were compared in patients who were not treated with opioids, patients treated with low-dose opioids (defined as less than 60 morphine milliequivalents per day), and patients treated with high-dose opioids (defined as at least 60 morphine milliequivalents per day). Results: Out of 212 patients who received an ICI, 98 (46.2%) received no opioids, 69 (32.6%) received low-dose opioids, and 45 (21.2%) received high-dose opioids. Among all patients, 105 (49.5%) died during the study duration. The overall median survival time was 23 (95% CI = 16, 38) months for the entire sample. Patients who received opioids at any dose had a median OS of 17 months compared to 37 months in those who received no opioids (HR = 1.53, p = 0.0385). When patients receiving opioids were further divided by dose, survival time was highest in those receiving no opioids, next highest in those receiving low-dose opioids, and lowest in those receiving high-dose opioids (37 months vs 18 months vs 10 months, p = 0.0515). Among all 212 patients, progression of disease occurred in 84 (39.6%) patients. The overall median time-to-progression was 24 months. There was no significant difference in median PFS between those treated with opioids (36 months) versus not (23 months, p = 0.156). Conclusions: We observed an association between opioid therapy, especially at higher doses, and decreased median OS in patients receiving ICIs. On the other hand, there was no statistical association between opioid therapy and median PFS. These data highlight a potential drug interaction in oncology care, and further analysis looking at duration of opioid use and ECOG PS is planned.[Table: see text]

Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras–induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4(+) T-helper cell response. IL22 is an effector molecule secreted by CD4(+) and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on non-hematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of pro-tumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8(+) T cell responses, while suppressing the pro-tumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC(+)CCSP(+) stem cells. Thus, we conclude that IL22 promotes Kras mutant lung tumorigenesis by driving a pro-tumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells.

Small-cell lung cancer (SCLC) is an aggressive tumor that accounts for 13–15% of all lung cancer cases and is the seventh most common cause of cancer-related death in the U.S. with a 5-year overall survival rate of 6.3% (1). At initial diagnosis, about 2/3 of patients have extensive-stage disease (ES-SCLC), for which all treatment is given with palliative intent. Platinum-based, two-drug chemotherapy has been the standard first-line treatment for patients with ES-SCLC for over 20 years. In the U.S., the most common regimens utilized are carboplatin plus etoposide (CE) or cisplatin plus etoposide (PE), which yield a response rate of 50–70% and a median overall survival time of 8–11 months (2). Despite the remarkable activity of initial chemotherapy, nearly all patients relapse within months and the benefits of second-line therapy are limited. Thus, there is a desperate need for more effective first-line therapy.

The mechanisms by which steatosis of the liver progresses to non-alcoholic steatohepatitis, and end-stage liver disease remain elusive. Metabolic derangements in hepatocytes controlled by SIRT1 plays a role in the development of fatty liver in inbred-animals. The ability to perform similar studies using human tissue has been limited by the genetically variability in man. We generated human induced pluripotent stem (iPS)-cells with controllable expression of SIRT1. By differentiating edited-iPS cells into hepatocytes and knocking down SIRT1, we found increased fatty acid biosynthesis that exacerbates fat accumulation. To model human fatty livers, we repopulated decellularized rat livers with human mesenchymal cells, fibroblasts, macrophages, and human SIRT1-knockdown iPS-derived hepatocytes and found that the human iPS-derived liver tissue developed macrosteatosis, acquired proinflammatory phenotype and shared a similar lipid and metabolic profiling to human fatty livers. Biofabrication of genetically-edited human liver tissue may become an important tool for investigating human liver biology and disease.

Reactive oxygen species (ROS) are important signaling molecules in cancer. The level of ROS will determine physiological effects. While high levels of ROS can cause damage to tissues and cell death, low levels of ROS can have a proliferative effect. ROS are produced by tumor cells but also cellular components that make up the tumor microenvironment (TME). In this review, we discuss the mechanisms by which ROS can affect the TME with particular emphasis on tumor-infiltrating leukocytes. Greater insight into ROS biology in this setting may allow for therapeutic manipulation of ROS levels in order to remodel the tumor microenvironment and increase anti-tumor activity.

Otto Warburg's theory on the origins of cancer postulates that tumor cells have defects in mitochondrial oxidative phosphorylation and therefore rely on high levels of aerobic glycolysis as the major source of ATP to fuel cellular proliferation (the Warburg effect). This is in contrast to normal cells, which primarily utilize oxidative phosphorylation for growth and survival. Here we report that the major function of glucose metabolism for Kras-induced anchorage-independent growth, a hallmark of transformed cells, is to support the pentose phosphate pathway. The major function of glycolytic ATP is to support growth under hypoxic conditions. Glutamine conversion into the tricarboxylic acid cycle intermediate alpha-ketoglutarate through glutaminase and alanine aminotransferase is essential for Kras-induced anchorage-independent growth. Mitochondrial metabolism allows for the generation of reactive oxygen species (ROS) which are required for Kras-induced anchorage-independent growth through regulation of the ERK MAPK signaling pathway. We show that the major source of ROS generation required for anchorage-independent growth is the Q o site of mitochondrial complex III. Furthermore, disruption of mitochondrial function by loss of the mitochondrial transcription factor A (TFAM) gene reduced tumorigenesis in an oncogenic Kras-driven mouse model of lung cancer. These results demonstrate that mitochondrial metabolism and mitochondrial ROS generation are essential for Kras-induced cell proliferation and tumorigenesis.

Historically, it has been assumed that glycolytic metabolism, not mitochondrial metabolism, is essential for tumor cell proliferation. However, most tumor cells have functional mitochondria, and recent studies suggest that the citric acid cycle (TCA) cycle intermediates are precursors for synthesis of nucleotides, lipids, and amino acids. Here we review the accumulating evidence that mitochondrial metabolism plays an essential role in tumor cell proliferation.

Senescence is a potential tumor-suppressing mechanism and a commonly used model of cellular aging. One current hypothesis to explain senescence, based in part on the correlation of oxygen with senescence, postulates that it is caused by oxidative damage from reactive oxygen species (ROS). Here, we further test this theory by determining the mechanisms of hyperoxia-induced senescence. Exposure to 70% O2 led to stress-induced, telomere-independent senescence. Although hyperoxia elevated mitochondrial ROS production, overexpression of antioxidant proteins was not sufficient to prevent hyperoxia-induced senescence. Hyperoxia activated AMPK; however, overexpression of a kinase-dead mutant of LKB1, which prevented AMPK activation, did not prevent hyperoxia-induced senescence. Knocking down p21 via shRNA, or suppression of the p16/pRb pathway by either BMI1 or HPV16-E7 overexpression, was also insufficient to prevent hyperoxia-induced senescence. However, suppressing p53 function resulted in partial rescue from senescence, suggesting that hyperoxia-induced senescence involves p53. Suppressing both the p53 and pRb pathways resulted in almost complete protection, indicating that both pathways cooperate in hyperoxia-induced senescence. Collectively, these results indicate a ROS-independent but p53/pRb-dependent senescence mechanism during hyperoxia.—Klimova, T. A., Bell, E. L., Shroff, E. H., Weinberg, F. D., Snyder, C. M., Dimri, G. P., Schumacker, P. T., Budinger, G. R. S., Chandel, N. S. Hyperoxia-induced premature senescence requires p53 and pRb, but not mitochondrial matrix ROS.

Purpose: Since the identification of PRKAR1A mutations in Carney complex, substitutions and small insertions/deletions have been found in ∼70% of the patients. To date, no germ-line PRKAR1A deletion and/or insertion exceeded a few base pairs (up to 15). Although a few families map to chromosome 2, it is possible that current sequencing techniques do not detect larger gene changes in PRKAR1A–mutation-negative individuals with Carney complex. Experimental Design: To screen for gross alterations of the PRKAR1A gene, we applied Southern hybridization analysis on 36 unrelated Carney complex patients who did not have small intragenic mutations or large aberrations in PRKAR1A, including the probands from two kindreds mapping to chromosome 2. Results: We found large PRKAR1A deletions in the germ-line of two patients with Carney complex, both sporadic cases; no changes were identified in the remaining patients, including the two chromosome-2-mapping families. In the first patient, the deletion is expected to lead to decreased PRKAR1A mRNA levels but no other effects on the protein; the molecular phenotype is predicted to be PRKAR1A haploinsufficiency, consistent with the majority of PRKAR1A mutations causing Carney complex. In the second patient, the deletion led to in-frame elimination of exon 3 and the expression of a shorter protein, lacking the primary site for interaction with the catalytic protein kinase A subunit. In vitro transfection studies of the mutant PRKAR1A showed impaired ability to bind cyclic AMP and activation of the protein kinase A enzyme. The patient bearing this mutation had a more-severe-than-average Carney complex phenotype that included the relatively rare psammomatous melanotic schwannoma. Conclusions: Large PRKAR1A deletions may be responsible for Carney complex in patients that do not have PRKAR1A gene defects identifiable by sequencing. Preliminary data indicate that these patients may have a different phenotype especially if their defect results in an expressed, abnormal version of the PRKAR1A protein.

Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development and progression by regulating genes that are vital for proliferation, glycolysis, angiogenesis, and metastasis. To identify strategies of targeting the HIF-1 pathway, we screened a siRNA library against the entire druggable genome and a small-molecule library consisting of 691,200 compounds using a HIF-1 reporter cell line. Although the siRNA library screen failed to reveal any druggable targets, the small-molecule library screen identified a class of alkyliminophenylacetate compounds that inhibit hypoxia-induced HIF-1 reporter activity at single-digit nanomolar concentrations. These compounds were found to inhibit hypoxia but not deferoxamine-induced HIF-1α protein stabilization. Further analysis indicated that the alkyliminophenylacetate compounds likely inhibit the HIF-1 pathway through blocking the hypoxia-induced mitochondrial reactive oxygen species (ROS) production. Strikingly, all of the nonalkyliminophenylacetate HIF-1 inhibitors identified from the small-molecule library screen were also found to target mitochondria like the alkyliminophenylacetate compounds. The exclusive enrichment of mitochondria inhibitors from a library of >600,000 diverse compounds by using the HIF-1 reporter assay highlights the essential role of mitochondria in HIF-1 regulation. These results also suggest that targeting mitochondrial ROS production might be a highly effective way of blocking HIF-1 activity in tumors.

Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of the protein kinase Cβ isozyme. The objective of this study was to assess the efficacy of enzastaurin in inducing apoptosis in multiple myeloma (MM) cell lines and to investigate possible mechanisms of apoptosis. Cell proliferation assays were done on a variety of MM cell lines with unique characteristics (dexamethasone sensitive, dexamethasone resistant, chemotherapy sensitive, and melphalan resistant). The dexamethasone-sensitive MM.1S cell line was used to further assess the effect of enzastaurin in the presence of dexamethasone, insulin-like growth factor-I (IGF-I), interleukin-6, and the pan-specific caspase inhibitor ZVAD-fmk. Enzastaurin increased cell death in all cell lines at clinically significant low micromolar concentrations (1–3 μmol/L) after 72 hours of treatment. Dexamethasone and enzastaurin were shown to have an additive effect on MM.1S cell death. Although IGF-I blocked the effect of 1 μmol/L enzastaurin, IGF-I did not abrogate cell death induced with 3 μmol/L enzastaurin. Moreover, enzastaurin-induced cell death was not affected by interleukin-6 or ZVAD-fmk. GSK3β phosphorylation, a reliable pharmacodynamic marker for enzastaurin activity, and AKT phosphorylation were both decreased with enzastaurin treatment. These data indicate that enzastaurin induces apoptosis in MM cell lines in a caspase-independent manner and that enzastaurin exerts its antimyeloma effect by inhibiting signaling through the AKT pathway. [Mol Cancer Ther 2006;5(7):1783–9]

Previous studies have shown that insulin augments GnRH-stimulated LH synthesis and release from primary gonadotrophs. In this study, regulation of LHbeta gene expression by GnRH and insulin was examined in LbetaT2 cells. Endogenous LHbeta mRNA is stimulated 2.4-fold by insulin alone, 2.6-fold by GnRH alone, and 4.7-fold by insulin together with GnRH. This effect of insulin, like GnRH, mapped to sequences -140 to +1 in the mouse LHbeta gene. Insulin together with GnRH stimulates activity of an LHbeta-reporter gene 7.1-fold; whereas, GnRH alone or insulin alone stimulates the reporter activity 2.8- and 3.1-fold, respectively. Blocking the binding of Egr-1 to sequences -51 to -42 in the LHbeta gene inhibits effects of insulin and GnRH. Insulin together with GnRH increases Egr-1 mRNA levels and total Egr-1 binding to LHbeta DNA. These findings indicate that insulin may impact regulation of the reproductive axis at the level of the pituitary.

Background: Inactivation of the human type Ia regulatory subunit (RIa) of cyclic AMP dependent protein kinase (PKA) (PRKAR1A) leads to altered kinase activity, primary pigmented nodular adrenocortical disease (PPNAD), and sporadic adrenal and other tumours. Methods and results: A transgenic mouse carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline responsive promoter (the Tg(Prkar1a*x2as)1Stra, Tg(tTAhCMV)3Uh or tTA/X2AS line) developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia and other features reminiscent of PPNAD, including late onset weight gain, visceral adiposity, and non- dexamethasone suppressible hypercorticosteronaemia, with histiocytic, epithelial hyperplasias, lympho- mas, and other mesenchymal tumours. These lesions were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIb protein levels; the latter biochemical and protein changes were also documented in Carney complex tumours associated with PRKAR1A inactivating mutations and chromosome 17 PRKAR1A locus changes. Conclusion: We conclude that the tTA/X2AS mouse line with a downregulated Prkar1a gene replicates several of the findings in Carney complex patients and their affected tissues, supporting the role of RIa as a candidate tumour suppressor gene.

Mice with complete inactivation of the type Ialpha regulatory subunit (RIalpha) of cyclic (c) AMP-dependent protein kinase (PKA) (coded by the Prkar1a gene) die early in embryonic life. To bypass the early embryonic lethality of Prkar1a-/- mice, we established transgenic mice carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline-responsive promoter. Mice developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia, and other features reminiscent of PPNAD, and histiocytic and epithelial hyperplasias, lymphomas, and other mesenchymal tumors. This mouse provides a useful tool for the investigation of cAMP, RIalpha, and PKA functions and confirms Prkar1a's critical role in tumorigenesis in endocrine and other tissues.

Glutathione peroxidase 1 (GPx-1) has been implicated in the etiology of several common diseases due to the association between specific allelic variations and cancer risk. The most common among these variations are the codon 198 polymorphism that results in either a leucine or proline and the number of alanine repeat codons in the coding sequence. The molecular and biologic consequences of these variations remain to be characterized. Toward achieving this goal, we have examined the cellular location of GPx-1 encoded by allelic variants by ectopically expressing these genes in MCF-7 human breast carcinoma cells that produce undetectable levels of GPx-1, thus achieving exclusive expression in the same cellular environment. A differential distribution between the cytoplasm and mitochondria was observed, with the allele expressing the leucine-198 polymorphism and 7 alanine repeats being more cytoplasmically located than the other alleles examined. To assess whether the distribution of GPx-1 between the cytoplasm and mitochondria had a biologic consequence, we engineered derivative GPx-1 proteins that were targeted to the mitochondria by the addition of a mitochondria targeting sequence and expressed these proteins in MCF-7 cells. These cells were examined for their response to oxidative stress, energy metabolism, and impact on cancer-associated signaling molecules. The results obtained indicated that both primary GPx-1 sequence and cellular location have a profound impact on cellular biology and offer feasible hypotheses about how expression of distinct GPx-1 alleles can affect cancer risk. Cancer Res; 74(18); 5118–26. ©2014 AACR.

Predicting RNA structures with pseudoknots in general is an NP-complete problem. Accordingly, several authors have suggested subclasses that provide polynomial time prediction algorithms by allowing (respectively, disallowing) certain structural motives. In this article, we introduce a unifying algebraic view on most of these classes. That way it becomes possible to find linear time recognition algorithms that decide whether or not a given structure is member of a class (we offer these algorithms as a web service to the scientific community). Furthermore, by presenting a general translation scheme of our algebraic descriptions into multiple context-free grammars, and proving a new correspondence of multiple context-free grammars and generating functions, it becomes possible to derive the precise asymptotic size of all the classes, solving some open problems such as enumerating the Rivas & Eddy class of pseudoknots.

Background Random biological sequences are a topic of great interest in genome analysis since, according to a powerful paradigm, they represent the background noise from which the actual biological information must differentiate. Accordingly, the generation of random sequences has been investigated for a long time. Similarly, random object of a more complicated structure like RNA molecules or proteins are of interest. Results In this article, we present a new general framework for deriving algorithms for the non-uniform random generation of combinatorial objects according to the encoding and probability distribution implied by a stochastic context-free grammar. Briefly, the framework extends on the well-known recursive method for (uniform) random generation and uses the popular framework of admissible specifications of combinatorial classes, introducing weighted combinatorial classes to allow for the non-uniform generation by means of unranking. This framework is used to derive an algorithm for the generation of RNA secondary structures of a given fixed size. We address the random generation of these structures according to a realistic distribution obtained from real-life data by using a very detailed context-free grammar (that models the class of RNA secondary structures by distinguishing between all known motifs in RNA structure). Compared to well-known sampling approaches used in several structure prediction tools (such as SFold) ours has two major advantages: Firstly, after a preprocessing step in time O ( n 2 ) for the computation of all weighted class sizes needed, with our approach a set of m random secondary structures of a given structure size n can be computed in worst-case time complexity O m ⋅ n ⋅ log ( n ) while other algorithms typically have a runtime in O ( m ⋅ n 2 ) . Secondly, our approach works with integer arithmetic only which is faster and saves us from all the discomforting details of using floating point arithmetic with logarithmized probabilities. Conclusion A number of experimental results shows that our random generation method produces realistic output, at least with respect to the appearance of the different structural motifs. The algorithm is available as a webservice at http://wwwagak.cs.uni-kl.de/NonUniRandGen and can be used for generating random secondary structures of any specified RNA type. A link to download an implementation of our method (in Wolfram Mathematica) can be found there, too.

Historically, it has been assumed that oxidative stress contributes to tumor initiation and progression solely by inducing genomic instability. Recent studies indicate that reactive oxygen species are upregulated in tumors and can lead to aberrant induction of signaling networks that cause tumorigenesis and metastasis. Here we review the role of redox-dependent signaling pathways and transcription factors that regulate tumorigenesis.

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status found in metazoans that is known to be activated by stimuli that increase the cellular AMP/ATP ratio. Full activation of AMPK requires specific phosphorylation within the activation loop of the catalytic domain of the alpha-subunit by upstream kinases such as the serine/threonine protein kinase LKB1. Here we show that hypoxia activates AMPK through LKB1 without an increase in the AMP/ATP ratio. Hypoxia increased reactive oxygen species (ROS) levels and the antioxidant EUK-134 abolished the hypoxic activation of AMPK. Cells deficient in mitochondrial DNA (rho(0) cells) failed to activate AMPK during hypoxia but are able to in the presence of exogenous H(2)O(2). Furthermore, we provide genetic evidence that ROS generated within the mitochondrial electron transport chain and not oxidative phosphorylation is required for hypoxic activation of AMPK. Collectively, these data indicate that oxidative stress and not an increase in the AMP/ATP ratio is required for hypoxic activation of AMPK.

Most PRKAR1A tumorigenic mutations lead to nonsense mRNA that is decayed; tumor formation has been associated with an increase in type II protein kinase A (PKA) subunits. The IVS6+1G>T PRKAR1A mutation leads to a protein lacking exon 6 sequences [R1 alpha Delta 184-236 (R1 alpha Delta 6)]. We compared in vitro R1 alpha Delta 6 with wild-type (wt) R1 alpha. We assessed PKA activity and subunit expression, phosphorylation of target molecules, and properties of wt-R1 alpha and mutant (mt) R1 alpha; we observed by confocal microscopy R1 alpha tagged with green fluorescent protein and its interactions with Cerulean-tagged catalytic subunit (C alpha). Introduction of the R1 alpha Delta 6 led to aberrant cellular morphology and higher PKA activity but no increase in type II PKA subunits. There was diffuse, cytoplasmic localization of R1 alpha protein in wt-R1 alpha- and R1 alpha Delta 6-transfected cells but the former also exhibited discrete aggregates of R1 alpha that bound C alpha; these were absent in R1 alpha Delta 6-transfected cells and did not bind C alpha at baseline or in response to cyclic AMP. Other changes induced by R1 alpha Delta 6 included decreased nuclear C alpha. We conclude that R1 alpha Delta 6 leads to increased PKA activity through the mt-R1 alpha decreased binding to C alpha and does not involve changes in other PKA subunits, suggesting that a switch to type II PKA activity is not necessary for increased kinase activity or tumorigenesis.

The tumor suppressor PTEN is mutated or deleted in many tumors, causing the activation of the PI3K pathway. Here, we show that the loss of PTEN increases the transcriptional activity of hypoxia-inducible factor 1 (HIF-1) through the inactivation of Forkhead transcription factors (FOXO) in PTEN-null cells. Reintroduction of PTEN into the nucleus, overexpression of a nonphosphorylatable FOXO3a, which accumulates in the nucleus, or inhibition of nuclear export of FOXO3a by leptomycin B represses HIF-1 transcriptional activity in PTEN-null cells. HIF-1 transcriptional activity increases in PTEN-positive cells depleted of FOXO3a with siRNA. PTEN and FOXO3a regulate the transactivation domain of HIF-1α. Chromatin immunoprecipitation indicates that FOXO3a complexes with HIF-1α and p300 on the Glut-1 promoter, a HIF-1 target gene. Overexpression of p300 reverses FOXO3a-mediated repression of HIF-1 transcriptional activity. Coimmunoprecipitation and GAL4-HIF-1α transactivation assays reveal that FOXO3a interferes with p300-dependent HIF-1 transcriptional activity. Thus, FOXO3a negatively regulates HIF-1 transcriptional activity.

Mutations of the human type Iα regulatory subunit (RIα) of cyclic AMP-dependent protein kinase (PKA; PRKAR1A) lead to altered kinase activity, primary pigmented nodular adrenocortical disease, and tumors of the thyroid and other tissues. To bypass the early embryonic lethality of Prkar1a−/− mice, we established transgenic mice carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline-responsive promoter. Down-regulation of Prkar1a by up to 70% was achieved in transgenic mouse tissues and embryonic fibroblasts, with concomitant changes in kinase activity and increased cell proliferation, respectively. Mice developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia, and other features reminiscent of primary pigmented nodular adrenocortical disease, histiocytic and epithelial hyperplasias, lymphomas, and other mesenchymal tumors. These were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIβ protein levels. This mouse provides a novel, useful tool for the investigation of cyclic AMP, RIα, and PKA functions and confirms the critical role of Prkar1a in tumorigenesis in endocrine and other tissues.