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MT-EGFR expression in HBEC cells is associated with dramatically elevated levels of replication factors.

Supplemental Figure Legends

HPLC chromatogram of ADT 061

HPLC chromatogram of compound 4.

AST and ALT activity of male APC+/Min-FCCC mice administered varying doses of ADT 061 in the diet. Enzyme activity was measured in plasma samples. Results are presented as mean {plus minus} SEM.

Mass spectroscopy of compound 5.

Reaction steps for synthesis of ADT 061 (a) 3-(4-methoxyphenyl)-2-methylacrylic acid (compound 1): 4-Methoxybenzaldehyde (219 g, 1.61 mole), propionic anhydride (315g, 2.42 mole), and sodium propionate (155g, 1.61 mole) were stirred at 140 ºC until a clear solution was achieved (~48 h). The solution was cooled to room temperature and poured into 8L of ice water. The precipitation formed was collected by filtration, transferred into a 2L round-bottom flask, and refluxed in 1.5 L of ethanol for 3h. The flask was stored at -20 ºC overnight. Compound 1 was obtained as a colorless crystal (213g) after filtration. Compound 1 is also commercially available from AstraTech (# W18287,95% purity). (b) 3-(4-Methoxyphenyl)-2-methylpropanoic acid (compound 2): p-Methoxy-α-methylcinnamic acid (213g) and palladium on active charcoal (Pd-C, 10%, 2g) were suspended in 1.5 L of 95% ethanol and warmed to 60 ºC in a water bath. The warm suspension was immediately put on a catalytic hydrogenator, treated with hydrogen (40 psi). The reaction was completed within 45 min, as indicated by the complete dissolving of the solid starting material. The catalyst was removed by filtration, and the filtrate was concentrated to give compound 2 as a colorless oil (215g), which was used for the next reaction step without further purification and characterization. Compound 2 is also commercially available from PharmaBlocks (# PBTQ6955, 97% purity). (c) 6-Methoxy-2-methyl-2,3-dihydro-1H-inden-1-one (compound 3): Phosphoryl acid (98%, 500g) and polyphosphoryl acid (PPA, 450g) were pre-warmed separately to 70{degree sign}C in a water bath before being transferred to a 2L, three-necked flask equipped with a thermometer, a mechanical stirrer, and a dropping funnel. The flask was kept in an oil bath at 55-60 {degree sign}C for 1 h until smooth stirring was achieved. Compound 2 (95 g) was added dropwise over a period of 5 min. The temperature was carefully raised to 70-75{degree sign}C for 15 min. The reaction solution was immediately transferred to 6L of ice water and stirred until the PPA was completely dissolved. The mixture was extracted with ethyl ether (1L Ã- 3). The organic layer was dried with sodium sulfate and concentrated. The residue was purified by a silica gel column and eluted with hexane and acetone. Purity was monitored by TLC. Compound 3 was obtained as a clear, colorless oil (76g) that was used for the next reaction step without further purification and characterization. Compound 3 is also commercially available from AstraTech (# 96721, 95% purity). (d) 2-(5-Methoxy-2-methyl-1H-inden-3-yl) acetic acid (compound 4): A mixture of compound 3 (76g, 0.39 mole), cyanoacetic acid (36.6g, 0.43 mole), acetic acid (40 mL) and ammonium acetate (9 g) in 500 mL of toluene was refluxed with stirring for 48h. The liberated water was collected by a Dean-Stark trap. The reaction mixture was cooled and filtered, and the filtrate was concentrated. The residue was dissolved in 250 mL of ethanol. A solution of potassium hydroxide (90 g) in 360 mL of water was added to the solution. The mixture was refluxed overnight under argon. The organic solvents were removed under vacuum, and the remaining aqueous solution was diluted with 500 mL of water, extracted with ethyl ether, boiled with active charcoal for 1h, then filtered. The filtrate was acidified with 6N HCl and sonicated for 2h. The precipitate was collected by filtration, washed with water, refluxed in 180 mL of acetone for 2h, and stored at -20 ºC overnight. The precipitate was collected by filtration and dried under vacuum to give compound 4 as a colorless solid (42g). The mother liquor was concentrated, and a second crop (3.5g) was obtained after repeating the recrystallization procedure. The structure was confirmed by mass spectrometry (M+H: 219.04) and 1H-NMR (DMSO-d6) Î'(ppm): 7.240(d, 1H); 6.804(d, 1H); 6.658(m, 1H); 3.781 (s, 3H); 3.741(s, 2H); 3.250(s, 2H); 2.050(s, 3H). Purity was determined by HPLC (98.7%) (Suppl. Fig. 2a-c). (e) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid (compound 5): Compound 4 (32.7g, 0.150 mole), 3, 4, 5-trimethoxybenzaldehyde (35.3g, 0.180 mole) and sodium methoxide (21g) in 300 mL of anhydrous methanol were refluxed in a 500 mL round-bottomed flask overnight. After cooling, the reaction mixture was diluted with 150 mL of acetone and sonicated for 30 min. The precipitate was collected by filtration and washed twice with acetone, then dissolved in 150 mL of water. The aqueous solution was acidified with 6N HCl and sonicated for 30 min. The precipitate was collected by filtration, washed with water, recrystallized from methanol, and dried under vacuum to afford compound 5 as a yellow crystal (51.7g). The structure was confirmed by mass spectrometry (M+H: 397.11) and 1H-NMR (DMSO-d6) Î'(ppm): 12.377 (s, 1H); 7.429 (d, 1H); 7.139 (s, 1H); 6.877(s, 2H); 6.669 (d, 1H); 6.546(dd, 1H); 3.780 (s, 3H); 3.780(s, 3H); 3.737(s, 6H); 3.545(s, 2H); 21.121(s, 3H). Purity was determined by HPLC (99.64%) (Suppl. Fig. 3a-c). (f) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)-N-(pyridin-3-yl) acetamide (ADT 061): To a 500 mL round-bottomed flask containing compound 5 (39.6g, 0.100 mole) and 300 mL of anhydrous dichloromethane, 1,1'-carbonyldiimidazole (CDI, 20.0g, 0.115 mole) was added in portions and stirred for 15 min at room temperature. 3-Amino-pyridine (10.6g, 0.115 mole) was added, followed by anhydrous pyridine (80 mL). The solution was stirred at 40{degree sign}C overnight, treated with 5g of sodium hydroxide in 20 mL of water for 10 min, then diluted with 300 mL of dichloromethane, and washed with water (250 mL x 3). The solution was concentrated, and the residue was purified with a silica gel column. Recrystallization from methanol and then from ethyl acetate afforded ADT 061 as a yellow crystal (32.7g). The structure of ADT 061 was confirmed by mass spectrometry (M+H: 473.2) and 1H-NMR (DMSO-d6) Î'(ppm): 10.453(sb, 1H); 8.765(d, 1H); 8.276(dd, 1H); 8.044(m, 1H); 7.431(d, 1H); 7.351(dd, 1H); 7.142(s,1H); 6.914(s, 1H); 6.880(s, 2H); 6.542(m, 1H); 3.781 (s, 6H); 3.731(s, 3H); 3.726(s, 3H); 3.696(s, 2H); 2.194(s, 3H). Purity was determined by HPLC (>99.7%) (Suppl. Fig. 4a-c).

ADT 061 pharmacokinetics and tissue distribution. ADT 061 was detected in plasma at a concentration of 2.8 µM 0.5 hr after a single oral administration of a dose of 100 mg/kg in 0.5% CMC/0.25% T80 in water to female C57BL/6 mice and reached plasma Cmax of 4.9 µM 4 hr after administration, which is >10 times higher than human colon cancer cell growth inhibition IC50 values. ADT 061 reached a Cmax in colon mucosa within 2 hr after oral administration (62.6 nmol/g) and was still present at concentrations exceeding IC50 values 8 hr after administration (28.9 nmol/g). ADT 061 concentrations in lungs, ovaries, and uterus (1-2 nmol/g) were appreciably lower than colon mucosa levels, while the ADT 061 concentration in the brain was nearly undetectable 2 and 8 hr post treatment (0.1 and 0.2 nmol/g, respectively) (n=4, mean {plus minus} SD).

Supplementary Figure Legends.

Eadie-Hofstee plot of data from Fig. 2C. Error bars = SEM

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic β-catenin, inhibited Wnt-induced nuclear translocation of β-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer.Prevention Relevance:PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/β-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASC) to long-lived plasma cells (LLPC). We provide single cell transcriptional resolution of 17,347 BM ASC from 5 healthy adults. Fifteen clusters were identified ranging from newly minted ASC (cluster 1) expressing MKI67 and high MHC Class II that progressed to late clusters 5-8 through intermediate clusters 2-4. Additional clusters included early and late IgM-predominant ASC of likely extra-follicular origin; IFN-responsive; and high mitochondrial activity ASC. Late ASCs were distinguished by differences in G2M checkpoints, MTOR signaling, distinct metabolic pathways, CD38 expression, and utilization of TNF-receptor superfamily members. They mature through two distinct paths differentiated by the degree of TNF signaling through NFKB. This study provides the first single cell resolution atlas and molecular roadmap of LLPC maturation, thereby providing insight into differentiation trajectories and molecular regulation of these essential processes in the human BM microniche. This information enables investigation of the origin of protective and pathogenic antibodies in multiple diseases and development of new strategies targeted to the enhancement or depletion of the corresponding ASC.One Sentence Summary:The single cell transcriptomic atlas of human bone marrow plasma cell heterogeneity shows maturation of class-switched early and late subsets, specific IgM and Interferon-driven clusters, and unique heterogeneity of the late subsets which encompass the long-lived plasma cells.

Background Aberrant activation of androgen receptor signalling following castration therapy is a common clinical observation in prostate cancer (PCa). Earlier, we demonstrated the role of MYB overexpression in androgen-depletion resistance and PCa aggressiveness. Here, we investigated MYB-androgen receptor (AR) crosstalk and its functional significance. Methods Interaction and co-localization of MYB and AR were examined by co-immunoprecipitation and immunofluorescence analyses, respectively. Protein levels were measured by immunoblot analysis and enzyme-linked immunosorbent assay. The role of MYB in ligand-independent AR transcriptional activity and combinatorial gene regulation was studied by promoter-reporter and chromatin immunoprecipitation assays. The functional significance of MYB in castration resistance was determined using an orthotopic mouse model. Results MYB and AR interact and co-localize in the PCa cells. MYB-overexpressing PCa cells retain AR in the nucleus even when cultured under androgen-deprived conditions. AR transcriptional activity is also sustained in MYB-overexpressing cells in the absence of androgens. MYB binds and promotes AR occupancy to the KLK3 promoter. MYB-overexpressing PCa cells exhibit greater tumorigenicity when implanted orthotopically and quickly regain growth following castration leading to shorter mice survival, compared to those carrying low-MYB-expressing prostate tumours. Conclusions Our findings reveal a novel MYB-AR crosstalk in PCa and establish its role in castration resistance.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic β-catenin, inhibited Wnt-induced nuclear translocation of β-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer. Prevention Relevance: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/β-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes nosocomial pneumonia, urinary tract infections, and bacteremia. A hallmark of P. aeruginosa pathogenesis is disruption of host cell function by the type III secretion system (T3SS) and its cognate exoenzyme effectors.

This study shows that the generation of human long-lived plasma cells requires blood antibody secreting cells to undergo epigenetic and transcriptional reprogramming in response to the bone marrow microniche to become apoptosis resistant and survive to secrete antibodies for a lifetime., Antibody secreting cells (ASCs) circulate after vaccination and infection and migrate to the BM where a subset known as long-lived plasma cells (LLPCs) persists and secrete antibodies for a lifetime. The mechanisms by which circulating ASCs become LLPCs are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared with BM LLPCs. Compared with blood ASCs, BM LLPCs have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. LLPCs up-regulate pro-survival genes MCL1, BCL2, and BCL-XL while simultaneously down-regulating pro-apoptotic genes HRK1, CASP3, and CASP8. Consistent with reduced gene expression, the pro-apoptotic gene loci are less accessible in LLPCs. Of the pro-survival genes, only BCL2 is concordant in gene up-regulation and loci accessibility. Using a novel in vitro human BM mimetic, we show that blood ASCs undergo similar morphological and molecular changes that resemble ex vivo BM LLPCs. Overall, our study demonstrates that early-minted blood ASCs in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPCs.

Phosphofurin acidic cluster sorting protein-1 (PACS-1) is a multifunctional membrane traffic regulator that plays important roles in organ homeostasis and disease. In this study, we elucidate a novel nuclear function for PACS-1 in maintaining chromosomal integrity. PACS-1 progressively accumulates in the nucleus during cell cycle progression, where it interacts with class I histone deacetylases 2 and 3 (HDAC2 and HDAC3) to regulate chromatin dynamics by maintaining the acetylation status of histones. PACS-1 knockdown results in the proteasome-mediated degradation of HDAC2 and HDAC3, compromised chromatin maturation, as indicated by elevated levels of histones H3K9 and H4K16 acetylation, and, consequently, increased replication stress-induced DNA damage and genomic instability.

Dihydroxyacetone phosphate (DHAP) is the endogenous by-product of fructose metabolism. Excess DHAP in cells can induce advanced glycation end products and oxidative stress. Dihydroxyacetone (DHA) is the triose precursor to DHAP. DHA is used as the active ingredient in sunless tanning products, including aerosolized spray tans, and is formed by the combustion of solvents found in electronic cigarettes. Human exposure to DHA has been increasing as the popularity of sunless tanning products and electronic cigarettes have grown. Topically applied DHA is absorbed through the viable layers of the skin and into the bloodstream. Exogenous exposure to DHA is cytotoxic in immortalized keratinocytes and melanoma cells with cell cycle arrest induced within 24 h and cell death occurring by apoptosis at consumer relevant concentrations of DHA within 72 h. Less is known about systemic exposures to DHA that occur following absorption through skin, and now through inhalation of the aerosolized DHA used in spray tanning. In the present study, HEK 293T cells were exposed to consumer-relevant concentrations of DHA to examine the cytotoxicity of systemic exposures. HEK 293T cells were sensitive to consumer-relevant doses of DHA with an IC(50) value of 2.4 ± 0.3 mM. However, cell cycle arrest did not begin until 48 h after DHA exposure. DHA exposed cells showed altered metabolic activity with decreased mitochondrial function and decreased lactate and ATP production observed within 24 h of exposure. Autofluorescent imaging and NAD+ sensors also revealed an imbalance in the redox cofactors NAD+/NADH within 24 h of exposure. Cell death occurred by autophagy indicated by increases in LC3B and SIRT1. Despite DHA’s ability to be converted to DHAP and integrated into metabolic pathways, the metabolic dysfunction and starvation responses observed in the HEK 293T cells indicate that DHA does not readily contribute to the energetic pool in these cells.

Protein–protein interactions regulate many essential enzymatic processes in the cell. Somatic mutations outside of an enzyme active site can therefore impact cellular function by disruption of critical protein–protein interactions. In our investigation of the cellular impact of the T304I cancer mutation of DNA Polymerase β (Polβ), we find that mutation of this surface threonine residue impacts critical Polβ protein–protein interactions. We show that proteasome-mediated degradation of Polβ is regulated by both ubiquitin-dependent and ubiquitin-independent processes via unique protein–protein interactions. The ubiquitin-independent proteasome pathway regulates the stability of Polβ in the cytosol via interaction between Polβ and NAD(P)H quinone dehydrogenase 1 (NQO1) in an NADH-dependent manner. Conversely, the interaction of Polβ with the scaffold protein X-ray repair cross complementing 1 (XRCC1) plays a role in the localization of Polβ to the nuclear compartment and regulates the stability of Polβ via a ubiquitin-dependent pathway. Further, we find that oxidative stress promotes the dissociation of the Polβ/NQO1 complex, enhancing the interaction of Polβ with XRCC1. Our results reveal that somatic mutations such as T304I in Polβ impact critical protein–protein interactions, altering the stability and sub-cellular localization of Polβ and providing mechanistic insight into how key protein–protein interactions regulate cellular responses to stress.

Antibody secreting cells (ASC) circulate after vaccination and migrate to the bone marrow (BM) where a subset known as long-lived plasma cells (LLPC) persist and secrete antibodies for a lifetime. The mechanisms of how circulating ASC become LLPC are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared to BM LLPC. LLPC acquire transcriptional and epigenetic changes in the apoptosis pathway to support their survival. Upregulation of pro-survival gene expression accompanies downregulation of pro-apoptotic gene expression in LLPC. While pro-apoptotic gene loci are less accessible, pro-survival gene loci are not always accompanied by accessibility changes. Importantly, we show similar LLPC morphological and transcriptional maturation of blood ASC in response to the novel in vitro BM mimetic. In all, our study demonstrates that blood ASC in the BM microniche must undergo morphological and molecular changes to mature into apoptotic-resistant LLPC.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-cyclooxygenase inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic β-catenin, inhibited Wnt-induced nuclear translocation of β-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc(+/min-FCCC) mouse model of colorectal cancer (CRC) without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing CRC.

SUMMARYAssembly and disassembly of DNA repair protein complexes at sites of DNA damage is essential to maintain genomic integrity. We investigated factors coordinating assembly of the base excision repair (BER) proteins, DNA polymerase β (Polβ) and XRCC1, to DNA lesion sites, identifying a new role for Polβ in regulating XRCC1 disassembly from DNA repair complexes and conversely, demonstrating Polβ’s dependence on XRCC1 for complex assembly. RealPAR, a genetically-encoded probe for live cell imaging of poly(ADP-ribose) (PAR), reveals that Polβ and XRCC1 require PAR for repair complex assembly and PAR degradation for disassembly. We find that BER complex assembly is further modulated by attenuation / augmentation of NAD+ biosynthesis. Finally, SIRT6 does not regulate PARP1 activation but impairs XRCC1 recruitment, leading to diminished Polβ abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for both PARP1 and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.

International audience; changes in nicotinamide adenine dinucleotide (nAD +) levels that compromise mitochondrial function trigger release of DNA damaging reactive oxygen species. NAD + levels also affect DNA repair capacity as nAD + is a substrate for pARp-enzymes (mono/poly-ADp-ribosylation) and sirtuins (deacetylation). The ecto-5′-nucleotidase CD73, an ectoenzyme highly expressed in cancer, is suggested to regulate intracellular nAD + levels by processing NAD + and its bio-precursor, nicotinamide mononucleotide (NMN), from tumor microenvironments, thereby enhancing tumor DNA repair capacity and chemotherapy resistance. We therefore investigated whether expression of CD73 impacts intracellular nAD + content and nAD +-dependent DNA repair capacity. Reduced intracellular NAD + levels suppressed recruitment of the DNA repair protein XRCC1 to sites of genomic DNA damage and impacted the amount of accumulated DNA damage. Further, decreased NAD + reduced the capacity to repair DNA damage induced by DNA alkylating agents. Overall, reversal of these outcomes through nAD + or NMN supplementation was independent of CD73. In opposition to its proposed role in extracellular nAD + bioprocessing, we found that recombinant human CD73 only poorly processes NMN but not NAD +. A positive correlation between CD73 expression and intracellular NAD + content could not be made as CD73 knockout human cells were efficient in generating intracellular NAD + when supplemented with nAD + or NMN. The tumor microenvironment has the potential to provide critical metabolites to promote tumor cell growth and immune modulation 1 as well as support cellular metabolism via metabolic coupling 2 or metabolic plasticity 3,4. A key energy metabolite in the tumor microenvironment is the hydride exchanger nicotinamide adenine dinu-cleotide (NAD + /NADH) which plays an important role in redox reactions for a number of energy-related and regulatory processes 5-8. Mammalian cells, in order to sustain intracellular NAD + homeostasis, can preferentially utilize a de novo synthesis pathway from L-tryptophan (Trp) or the Preiss-Handler pathway from nicotinic acid (NA), or employ the more effective salvage pathway 9 , which initiates from nicotinamide (NAM), or the nicotina-mide riboside (NR) kinase pathway. It is suggested that a source of NAD + and related NAD + metabolites arises from cell lysis at sites of inflammation or tumor cell necrosis 10 , providing substrates for NAD +-consuming gly-cohydrolase ectoenzymes such as CD38 in concert with connexin 43 11 or NAD +-consuming pyrophosphatases such as NPP5 12. NAD + is also an essential substrate for signaling and protein modification factors that impact cell death, stress responses and genome stability via mono-or poly-ADP-ribosylation (PARP family proteins) 13 , chromatin status via deacetylation (sirtuins) 14 and overall functional capacity of mitochondria 15. Importantly, nuclear/mitochon-drial crosstalk is mediated in part by NAD + and NAD + precursors to facilitate genome stability and the cellular response to genotoxic and cytostatic insults 16,17. The last few years have opened a new chapter in NAD + biology since a decrease in the cellular NAD + level has been associated with aging and a variety of pathological syndromes including obesity, neurodegenerative diseases

Bisphenol A (BPA) is used heavily in the production of polycarbonate plastics, thermal receipt paper, and epoxies. Ubiquitous exposure to BPA has been linked to obesity, diabetes, and breast and reproductive system cancers. Resistance to chemotherapeutic agents has also been shown in cancer cell models. Here, we investigated BPA's ability to confer resistance to camptothecin (CPT) in mouse embryonic fibroblasts (MEFs). MEFs are sensitive to CPT; however, co-exposure of BPA with CPT improved cell survival. Co-exposure significantly reduced Top1-DNA adducts, decreasing chromosomal aberrations and DNA strand break formation. This decrease occurs despite BPA treatment increasing the protein levels of Top1. By examining chromatin structure after BPA exposure, we determined that widespread compaction and loss of nuclear volume occurs. Therefore, BPA reduced CPT activity by reducing the accessibility of DNA to Top1, inhibiting DNA adduct formation, the generation of toxic DNA strand breaks, and improving cell survival.

Objectives: The egg-laying hen is an ideal model to study chemoprevention of epithelial ovarian cancer (EOC) as aging hens have a high rate of spontaneous EOC and similar clinicopathological characteristics of the disease to women. Our previous research revealed that the novel non-COX inhibitory NSAID-derivative, MCI-030, decreased ovarian cancer incidence in the hen. MCI-030 inhibits PDE10A, which was shown to be an effective therapeutic target in colon and lung cancers, reducing beta-catenin and MAPK signaling. Molecular analysis of MCI-030 treated hens was performed to study the mechanism by which PDE10A inhibition can reduce ovarian cancer risk. Methods: A total of 225 White leghorn hens aged 2.5 to 3 years old were treated through their feed with MCI-030 or its parent compound, sulindac, for 6- and 12-months and compared to a control group (Figure 1A). Necropsy was performed at each time point to formally assess for tumors with 60 hens evaluated at 6-months and 85 at 12-months. Logistic regression with Firth correction to estimate effect size for this pilot study was performed. Ovary and oviduct tissues were harvested and formalin-fixed or flash-frozen then paraffin-embedded or homogenized, respectively. Molecular analyses were performed using fluorescent immunohistochemistry, TUNEL assays, and western blotting. Results: A trend of decreased risk of malignancy was observed in the MCI-030 group at 6- and 12-months versus control with odds ratios (OR) of 0.56 and 0.75, respectively (95% CI 0.121-2.633, 0.22-2.58). In contrast, this trend was not observed for sulindac (6-month OR=1.56, 95% CI 0.405-6.06, 12-month OR=0.94, 95% CI 0.29-3.03). Assessment of apoptosis in hen ovaries by TUNEL assay demonstrated that MCI-030 significantly increased apoptosis both overall and in ovarian tumors (Figure 1B). Western blotting revealed that pERK was significantly decreased in ovaries of sulindac and MCI-030 hens (Figure 1C). Immunohistochemistry showed that MCI-030 significantly decreased nuclear translocation of beta-catenin and levels of active beta-catenin in ovarian tumors (Figure 1D). Download : Download high-res image (224KB) Download : Download full-size image Conclusions: MCI-030 appears to be a promising agent for the chemoprevention and treatment of ovarian cancer. Molecular analysis of its efficacy in hens revealed that MCI-030 increases tumor apoptosis and decreases pERK and beta-catenin oncogenic signaling. Further investigation of MCI-030 in additional model systems and in-human trials is warranted.

Although survival outcomes have improved dramatically over the last few decades in newly diagnosed myeloma patients, elderly patients have not yielded the same magnitude of benefit as evidenced by higher rates of reported myeloma-related deaths in patients over the age of 75. This is of particular importance given this cohort comprises a large proportion of myeloma patients with the median age of diagnosis being 70 years. One contributor to this discrepancy is reduced use of high-dose therapy and autologous stem cell transplantation (HDT/ASCT) in this population because of concerns for increased toxicity and safety. The objective of this retrospective analysis is to evaluate survival and safety outcomes in 53 newly diagnosed patients ≥74 years of age who underwent HDT/ASCT at our institution in comparison to 122 control patients in the same age bracket who did not undergo stem cell transplantation during this same time period. Patients treated at our institution were identified in our institutional myeloma database by age. They were all treated between November 2006 and October 2016 at the Winship Cancer Institute of Emory University. Fifty-three patients were identified who had undergone HDT/ASCT, and, to assess the relative benefit of ASCT, 122 control patients in the same age range were also identified who did not undergo HDT/ASCT during the same time period. The median age for the entire cohort was 77 years (74 years in the ASCT group versus 78 in the non-ASCT group). Median time to ASCT was 6 months (range 2-57 months). There were no gender or race differences between the 2 groups, although a higher proportion of high-risk patients underwent HDT/ASCT. Ninety-three percent of ASCT patients received triplet induction therapy with a proteasome inhibitor and immunomodulatory agent backbone in comparison to only 55% of patients the non-ASCT group. The median progression-free survival (PFS) for the ASCT group was 50 months versus 30 months in the non-ASCT group. The median overall survival (OS) was 80 months versus 40 months, respectively. In high-risk patients, the median PFS was 60.8 months, and the median OS was 77.8 months in the ASCT group compared to 26 months and 38 months in the non-ASCT group, respectively. There were no transplant-related deaths within the first 100 days in the ASCT group. This study offers real-world perspective and data on the safety and survival benefit of ASCT in the elderly population with a near doubling of OS when compared to those treated with similar regimens and modern agents without ASCT. These data provide a rationale for offering ASCT in elderly patients pending a thorough pretransplantation evaluation.

Background Melphalan as high dose therapy (HDT) is a proven effective conditioning regimen for patients undergoing myeloma autotransplants and is associated with known peri-transplant toxicities that warrant a discussion with patients. Efforts to optimize supportive care have resulted in significant decline in transplant related morbidities and mortality. In this context, we have reviewed the observed toxicities among patients receiving autotransplants in the recent years with an intent to deliver consistency in informing patients of the known toxicities. Methods We conducted a retrospective analysis of myeloma patients undergoing autologous stem cell transplant over a two-year period. Demographic and outcomes data for the patients were obtained from our IRB approved myeloma database and responses were evaluated per IMWG Uniform Response Criteria. Fisher’s exact and Cochran-Mante-Haenszel tests were used when groups were compared. Results Of the 395 patients that underwent autotransplants 308 patients received a dose of 200 mg/m2 (Mel200) and 87 received 140 mg/m2 (Mel140). Median age of the patients was 62 years (range, 17-78). Impressively, African American patients comprised of 41.5% of the entire cohort. Relevant comorbidities prior to transplant include hypertension (62%), renal failure (5%), coronary artery disease (5%) and h/o atrial fibrillation (4%). 11% of patients had KPS ≤70, median pretransplant EF was 55% (30-86%), and DLCO corrected was 87% (30-156). Median time for neutrophil engraftment, platelet engraftment and LOS were 14 (12-24), 14 (11-21), 15 (7-53) days respectively. While peri-transplant, neutropenic fevers were seen in 67% of the patients, majority were of non-infectious nature. Documented bacteremia was seen in 13% of patients, clostridium difficile infection in 8%, respiratory infections in 5%, and other infections were seen in 2% (cellulitis- 1% and UTI -1%). Any grade mucositis was seen in 31% and grade 3 mucositis in 7%. Atrial fibrillation was seen in 5% and renal failure in 4%. Readmission rates were 5%, median time to readmission was 4 days (range, 1-20). Transplant related mortality at the 30-day mark was 0.25% (1 incident due to a intracranial aneurysm rupture) and at 100-day mark was 0.75% (1 death due to septic shock and 1 due to cardiac failure). Unfortunately, 2 patients succumbed to aggressive myeloma during the same period. Conclusion While we are able to offer HDT to patients with advanced age, cardiac toxicities and renal insufficiency, an explicit conversation regarding known peri-transplant toxicities is necessary to make an informed decision. Very interestingly, 93% of this cohort received risk-adapted maintenance initiating at a median time of 105 days from autotransplants, a surrogate to suggest adequate recovery from transplant related morbidities. Outcomes regarding AA patients and additional progression free and overall survival data by cytogenetic risk will be presented at the meeting.

EGFR signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non–small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild-type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells. Gene expression profiling in human bronchial epithelial cells (HBEC) revealed that MT-EGFR expression elevated transcripts related to cell cycle and replication in aerobic and hypoxic conditions and downregulated RAD50, a critical component of nonhomologous end joining and homologous recombination DNA repair pathways. NSCLCs and HBEC with MT-EGFR revealed elevated basal and hypoxia-induced γ-H2AX–associated DNA lesions that were coincident with replication protein A in the S-phase nuclei. DNA fiber analysis showed that, relative to WT-EGFR, MT-EGFR NSCLCs harbored significantly higher levels of stalled replication forks and decreased fork velocities in aerobic and hypoxic conditions. EGFR blockade by cetuximab significantly increased radiosensitivity in hypoxic cells, recapitulating MT-EGFR expression and closely resembling synthetic lethality of PARP inhibition. Implications: This study demonstrates that within an altered DNA damage response of hypoxic NSCLC cells, mutant EGFR expression, or EGFR blockade by cetuximab exerts a synthetic lethality effect and significantly compromises radiation resistance in hypoxic tumor cells. Mol Cancer Res; 15(11); 1503–16. ©2017 AACR.

The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, present a challenge for genome integrity maintenance as this requires the engagement of different repair pathways. Specific ‘repair controllers’ that facilitate repair pathway crosstalk at lesion sites, by regulating base excision repair (BER) protein trafficking, have yet to be identified. We find that DNA polymerase β (Polβ), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partnered with UBR5 restrains double-strand break (DSB) signaling. TRIP12 but not UBR5 controls cellular levels and chromatin loading of Polβ. Required for Polβ foci formation, TRIP12 regulates Polβ involvement after DNA damage. Excessive TRIP12-mediated shuttling of Polβ affects DSB formation and radiation sensitivity underscoring its precedence for BER. The herein discovered trafficking function at the nexus of repair signaling pathways towards Polβ-directed BER optimizes DNA repair pathway choice at complex lesion sites.

Changes in nicotinamide adenine dinucleotide (NAD+) levels that compromise mitochondrial function trigger release of DNA damaging reactive oxygen species. NAD+ levels also affect DNA repair capacity as NAD+ is a substrate for PARP-enzymes (mono/poly-ADP-ribosylation) and sirtuins (deacetylation). The ecto-5′-nucleotidase CD73, an ectoenzyme highly expressed in cancer, is suggested to regulate intracellular NAD+ levels by processing NAD+ and its bio-precursor, nicotinamide mononucleotide (NMN), from tumor microenvironments, thereby enhancing tumor DNA repair capacity and chemotherapy resistance. We therefore investigated whether expression of CD73 impacts intracellular NAD+ content and NAD+-dependent DNA repair capacity. Reduced intracellular NAD+ levels suppressed recruitment of the DNA repair protein XRCC1 to sites of genomic DNA damage and impacted the amount of accumulated DNA damage. Further, decreased NAD+ reduced the capacity to repair DNA damage induced by DNA alkylating agents. Overall, reversal of these outcomes through NAD+ or NMN supplementation was independent of CD73. In opposition to its proposed role in extracellular NAD+ bioprocessing, we found that recombinant human CD73 only poorly processes NMN but not NAD+. A positive correlation between CD73 expression and intracellular NAD+ content could not be made as CD73 knockout human cells were efficient in generating intracellular NAD+ when supplemented with NAD+ or NMN.

DNA repair defects have been increasingly focused on as therapeutic targets. In hormone positive breast cancer, XRCC1-deficient tumors have been identified and proposed as targets for combination therapies that damage DNA and inhibit DNA repair pathways. XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) polymerase 1, DNA polymerase β (POL β), and DNA ligases. Loss of XRCC1 confers BER defects and hypersensitivity to DNA damaging agents. BER defects have not been evaluated in triple negative breast cancer (TNBC), for which new therapeutic targets and therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects in TNBC, we examined XRCC1 expression and localization in the TCGA database and in TNBC cell lines. High XRCC1 expression was observed for TNBC tumors in the TCGA database and expression of XRCC1 varied between TNBC cell lines. We also observed changes in XRCC1 subcellular localization in TNBCs that alter the ability to repair base lesions and single-strand breaks. Subcellular localization changes were also observed for POL β that did not correlate with XRCC1 localization. Basal levels of DNA damage were also measured in the TNBC cell lines, and damage levels correlated with observed changes in XRCC1 expression, localization, and repair functions. The results confirmed that XRCC1 expression changes may indicate DNA repair capacity changes but emphasize that basal DNA damage levels along with expression and localization are better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in TNBC preclinical models and the TCGA database, XRCC1 expression levels should be considered when evaluating treatment responses of TNBC preclinical model cells.

Objective: A leading theory for ovarian carcinogenesis proposes that the inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, epidemiological studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease ovarian cancer risk. Previous studies suggest, however, that the antineoplastic activity of NSAIDs does not require the traditional cyclooxygenase (COX) enzyme inhibition, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a potentially unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. This study evaluates the effects of phosphodiesterase 10A (PDE10A) inhibition as a novel chemopreventive and therapeutic approach for ovarian cancer. Methods: We investigated the effects of PDE10A small molecule inhibitors, Pf-2545920 and MCI-030 (a novel non-COX inhibitory NSAID-derivative), and PDE10A knockout by CRISPR/Cas9 gene editing in various ovarian cancer cell lines using in vitro assays that measured cell proliferation, cell viability, cell cycle arrest, apoptosis, cell migration and invasion. Downstream signaling pathways affected by PDE10A inhibition and gene knockout (KO) were assessed by western-blotting, confocal microscopy, and RNA sequencing. Results: Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer database and an institutional cohort of ovarian cancer patients revealed that high PDE10A mRNA expression was associated with significantly worse overall survival. PDE10A expression was also positively correlated with oncogenic and inflammatory signaling pathways in the TCGA dataset. PDE10A inhibition with Pf-2545920 or MCI-030 decreased ovarian cancer cell proliferation, while inducing cell cycle arrest and apoptosis. Using inhibitors to block PKA (H89) and PKG (KT5823) kinase activity, we demonstrated that the pro-apoptotic effects of PDE10A inhibition were mediated by activation of cGMP/PKG and cAMP/PKA signaling. SKOV3 and OV-90 PDE10A KO cells showed decreased colony formation, cell proliferation, and migration and invasion properties compared to their wild-type (WT) counterparts. Moreover, PDE10A inhibition decreased Wnt-induced β-catenin nuclear translocation as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. RNA sequencing of SKOV3 PDE10A KO clones revealed that pathways associated with cancer, Wnt and TGF-β signaling were downregulated compared to WT control cells. Conclusions: Our data demonstrate that PDE10A has pro-tumorigenic effects in ovarian cancer cells and its high expression in ovarian cancer patients is associated with poor prognosis. Altogether, our results warrant future studies of PDE10A as a novel target for ovarian cancer chemoprevention and/or treatment. Citation Format: Rebecca M. Barber, Elaine Gavin, Alla Musiyenko, Wito Richter, Kevin J. Lee, Annelise Wilhite, Joel F. Andrews, Steve McClellan, Ileana Aragon, Antonio Ward, Xi Chen, Adam Keeton, Kristy Berry, Gary A. Piazza, Jennifer M. Scalici, Luciana Madeira da Silva. PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1213.

The enzymes of the base excision repair (BER) pathway form DNA lesion-dependent, transient complexes that vary in composition based on the type of DNA damage [1]. These protein sub-complexes facilitate substrate/product handoff to ensure reaction completion so as to avoid accumulation of potentially toxic DNA repair intermediates [2]. However, in the mammalian cell, additional signaling molecules are required to fine-tune the activity of the BER pathway enzymes and to facilitate chromatin/histone reorganization for access to the DNA lesion for repair. These signaling enzymes include nicotinamide adenine dinucleotide (NAD(+)) dependent poly(ADP-ribose) polymerases (PARP1, PARP2) and class III deacetylases (SIRT1, SIRT6) that comprise a key PARP-NAD-SIRT axis to facilitate the regulation and coordination of BER in the mammalian cell. Here, we briefly describe the key nodes in the BER pathway that are regulated by this axis and highlight the cellular and organismal variation in NAD(+) bioavailability that can impact BER signaling potential. We discuss how cellular NAD(+) is required for BER to maintain genome stability and to mount a robust cellular response to DNA damage. Finally, we consider the dependence of BER on the PARP-NAD-SIRT axis for BER protein complex assembly.

// Kevin Lee 1 , Ashley S. Lindsey 1 , Nan Li 2 , Bernard Gary 1 , Joel Andrews 3 , Adam B. Keeton 1 , Gary A. Piazza 1 1 Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA 2 Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, Birmingham, Alabama, USA 3 Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA Correspondence to: Kevin Lee, e-mail: kjlee@health.southalabama.edu Keywords: β-catenin, PKG, PKA, PDE10A, colon cancer Received: September 09, 2015 Accepted: December 07, 2015 Published: December 21, 2015 ABSTRACT Phosphodiesterase 10A (PDE10) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington’s disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibits colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activates PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduces the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibits the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, resulting in caspase activation and apoptosis. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells.

OBJECTIVES Although highly active antiretroviral therapy has led to improved survival in HIV-infected individuals, outcomes for HIV-infected patients with sepsis in the post-highly active antiretroviral therapy era are conflicting. Access to highly active antiretroviral therapy and healthcare disparities continue to affect outcomes. We hypothesized that HIV-infected patients with severe sepsis would have worse outcomes compared with their HIV-uninfected counterparts in a large safety-net hospital where access to healthcare is low and delivery of critical care is delayed. DESIGN Secondary analysis of an ongoing prospective observational study between 2006 and 2010. SETTING Three adult ICUs (medical ICU, surgical ICU, and neurologic ICU) at Grady Memorial Hospital, Atlanta, GA. PATIENTS Adult patients with severe sepsis in the ICU. INTERVENTIONS Baseline patient characteristics and clinical outcomes were collected. HIV-infected and HIV-uninfected patients with sepsis were compared using t tests, chi-square tests, and logistic regression; p values less than 0.05 indicated significance. MEASUREMENTS AND MAIN RESULTS Of 1,095 patients with severe sepsis enrolled, 165 (15%) were positive for HIV, with a median CD4 count of 41 (8-167). Twenty-two percent of HIV-infected patients were on highly active antiretroviral therapy prior to admission, and 80% had a CD4 count less than 200. HIV-infected patients had a greater hospital mortality (50% vs 38%; p < 0.01). HIV infection (odds ratio = 1.78; p = 0.005) was an independent predictor of mortality by multivariate regression modeling after adjusting for age, history of pneumonia, history of hospital-acquired infection, and history of sepsis. CONCLUSIONS HIV-infected patients with severe sepsis continue to suffer worse outcomes compared with HIV-uninfected patients in a large urban safety-net hospital caring for patients with limited access to medical care. Further studies need to be done to investigate the effect of socioeconomic status and mitigate healthcare disparities among critically ill HIV-infected patients.

Background: Previously, miR-345 was identified as one of the most significantly downregulated microRNAs in pancreatic cancer (PC); however, its functional significance remained unexplored. Methods: miR-345 was overexpressed in PC cells by stable transfection, and its effect on growth, apoptosis and mitochondrial-membrane potential was examined by WST-1, Hoechst-33342/Annexin-V, and JC-1 staining, respectively. Gene expression was examined by quantitative reverse-transcription-PCR and/or immunoblotting, and subcellular fractions prepared and caspase-3/7 activity determined by commercially available kits. miR-345 target validation was performed by mutational analysis and luciferase-reporter assay. Results: miR-345 is significantly downregulated in PC tissues and cell lines relative to normal pancreatic cells, and its expression decreases gradually in PC progression model cell lines. Forced expression of miR-345 results in reduced growth of PC cells because of the induction of apoptosis, accompanied by a loss in mitochondrial membrane potential, cytochrome-c release, caspases-3/7 activation, and PARP-1 cleavage, as well as mitochondrial-to-nuclear translocation of apoptosis-inducing factor. These effects could be reversed by the treatment of miR-345-overexpressing PC cells with anti-miR-345 oligonucleotides. BCL2 was characterised as a novel target of miR-345 and its forced-expression abrogated the effects of miR-345 in PC cells. Conclusions: miR-345 downregulation confers apoptosis resistance to PC cells, and its restoration could be exploited for therapeutic benefit.

We determined the prevalence of risk factors for the development of acute respiratory distress syndrome (ARDS), outcomes of critical illness, and the impact of highly active antiretroviral therapy in HIV-1-infected patients. We hypothesized that in an urban county hospital, HIV-1-infected patients with ARDS would have a higher mortality than their HIV-1-uninfected counterparts.Subjects were enrolled between 2006 and 2012. Baseline patient demographics, comorbidities, illness severity, causes of ARDS, and clinical outcomes were obtained. The primary end point was hospital mortality.A total of 178 subjects with ARDS were enrolled in the study; 40 (22%) were infected with HIV-1. The median CD4 count was 75 (15.3-198.3), and 25% were on highly active antiretroviral therapy. HIV-1-infected subjects were significantly younger (44 vs 52 years; P.01) and had higher rates of asthma, chronic obstructive pulmonary disease, pneumonia, history of hospital-acquired infections, and prior sepsis. HIV-1-infected subjects had greater illness severity by Acute Physiology and Chronic Health Evaluation II scores (29 [24-31] vs 24 [22-25]; P.01). Hospital mortality was not higher among HIV-1-infected subjects compared with HIV-1-uninfected subjects (50.0% vs 38.4%; P = .19).In patients with ARDS, HIV-1 infection was associated with greater illness severity but was not associated with higher mortality in ARDS. Future studies need to be done to evaluate the factors that contribute to high morbidity and mortality in medically vulnerable populations who develop ARDS.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Objective Bronchoscopy and bronchoalveolar lavage (BAL) are common procedures in intensive care units; however, no contemporaneous safety and outcomes data have been reported, particularly for critically ill patients. Design This is a retrospective analysis of prospectively collected data from teaching hospital adult intensive care units. Interventions One hundred mechanically ventilated patients with severe sepsis, septic shock, acute lung injury (ALI), and/or acute respiratory distress syndrome underwent bronchoscopy with unilateral BAL. Data collected included demographics, presence of sepsis or ALI, Pa o 2 to F io 2 ratio, positive end-expiratory pressure, Acute Physiology and Chronic Health Evaluation score, Sequential Organ Failure Assessment score, and peri- or postprocedural complications. Results Men comprised 51% of the patients; 81% of the patients were black, and 15% were white. The mean age was 52 (SD, ± 16) years. The mean Acute Physiology and Chronic Health Evaluation score was 22 (± 7.5), whereas the median Sequential Organ Failure Assessment score was 9 (interquartile range, 5-12). Ten patients (10%) had complications during or immediately after the procedure. Hypoxemia during or immediately after the BAL was the most common complication. Ninety percent of the complications were related to transient hypoxemia, whereas bradycardia and hypotension each occurred in 1 patient. Age, female sex, and higher positive end-expiratory pressure were associated with complications. Conclusions Bronchoscopy with BAL in critically ill patients with sepsis and ALI is well tolerated with low risk of complications, primarily related to manageable hypoxemia.

Emerging data highlight the significance of chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 (CXCL12/CXCR4) signaling axis in the chemoresistance of several malignancies, including prostate cancer (PCa); however, underlying mechanisms remain largely elusive. Here, we demonstrate that CXCL12 treatment rescues the PCa cells from docetaxel (DTX)-induced toxicity by overriding its effect on cell cycle (G2/M phase arrest). We further demonstrate that the chemoprotective effect of CXCL12 is abolished upon pharmacological inhibition or RNA interference-mediated silencing of CXCR4. Moreover, microtubule stabilization caused by DTX is suppressed in CXCL12-stimulated PCa cells as revealed by immunofluorescence and immunoblot analyses. The effect of CXCL12 on microtubule stabilization is abrogated when PCa cells are pre-treated with a CXCR4 antagonist. In additional studies, we show that the chemoprotective action of CXCL12/CXCR4 signaling is mediated by p21-activated kinase 4 (PAK4)-dependent activation of Lim domain kinase 1 (LIMK1), and inhibition of either PAK4 or LIMK1 leads to re-sensitization of PCa cells to DTX-induced tubulin polymerization and cellular toxicity even in the presence of CXCL12. Altogether, our findings uncover a novel mechanism underlying CXCL12/CXCR4 signaling-induced PCa chemoresistance and suggest that targeting of this signaling axis or its downstream effector pathway could lead to therapeutic enhancement of DTX.