Spatial mapping of the DNA adducts in cancer.

DNA adducts and strand breaks are induced by various exogenous and endogenous agents. Accumulation of DNA damage is implicated in many disease processes, including cancer, aging, and neurodegeneration. The continuous acquisition of DNA damage from exogenous and endogenous stressors coupled with defects in DNA repair pathways contribute to the accumulation of DNA damage within the genome and genomic instability. While mutational burden offers some insight into the level of DNA damage a cell may have experienced and subsequently repaired, it does not quantify DNA adducts and strand breaks. Mutational burden also infers the identity of the DNA damage. With advances in DNA adduct detection and quantification methods, there is an opportunity to identify DNA adducts driving mutagenesis and correlate with a known exposome. However, most DNA adduct detection methods require isolation or separation of the DNA and its adducts from the context of the nuclei. Mass spectrometry, comet assays, and other techniques precisely quantify lesion types but lose the nuclear context and even tissue context of the DNA damage. The growth in spatial analysis technologies offers a novel opportunity to leverage DNA damage detection with nuclear and tissue context. However, we lack a wealth of techniques capable of detecting DNA damage in situ. Here, we review the limited existing in situ DNA damage detection methods and examine their potential to offer spatial analysis of DNA adducts in tumors or other tissues. We also offer a perspective on the need for spatial analysis of DNA damage in situ and highlight Repair Assisted Damage Detection (RADD) as an in situ DNA adduct technique with the potential to integrate with spatial analysis and the challenges to be addressed.

Phosphodiesterase 10A (PDE10A) as a novel target to suppress ß-catenin and RAS signaling in epithelial ovarian cancer.

A leading theory for ovarian carcinogenesis proposes that inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, nonsteroidal anti-inflammatory drugs (NSAIDs) exert promising chemopreventive activity for ovarian cancer. Unfortunately, toxicity is associated with long-term use of NSAIDs due to their cyclooxygenase (COX) inhibitory activity. Previous studies suggest the antineoplastic activity of NSAIDs is COX independent, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. Here we evaluate PDE10A as a novel therapeutic target for ovarian cancer. Analysis of The Cancer Genome Atlas (TCGA) ovarian tumors revealed PDE10A overexpression was associated with significantly worse overall survival for patients. PDE10A expression also positively correlated with the upregulation of oncogenic and inflammatory signaling pathways. Using small molecule inhibitors, Pf-2545920 and a novel NSAID-derived PDE10A inhibitor, MCI-030, we show that PDE10A inhibition leads to decreased ovarian cancer cell growth and induces cell cycle arrest and apoptosis. We demonstrate these pro-apoptotic properties occur through PKA and PKG signaling by using specific inhibitors to block their activity. PDE10A genetic knockout in ovarian cancer cells through CRISP/Cas9 editing lead to decreased cell proliferation, colony formation, migration and invasion, and in vivo tumor growth. We also demonstrate that PDE10A inhibition leads to decreased Wnt-induced ß-catenin nuclear translocation, as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. These findings implicate PDE10A as novel target for ovarian cancer chemoprevention and treatment.

An in vivo model of glioblastoma radiation resistance identifies long noncoding RNAs and targetable kinases.

Key molecular regulators of acquired radiation resistance in recurrent glioblastoma (GBM) are largely unknown, with a dearth of accurate preclinical models. To address this, we generated 8 GBM patient-derived xenograft (PDX) models of acquired radiation therapy-selected (RTS) resistance compared with same-patient, treatment-naive (radiation-sensitive, unselected; RTU) PDXs. These likely unique models mimic the longitudinal evolution of patient recurrent tumors following serial radiation therapy. Indeed, while whole-exome sequencing showed retention of major genomic alterations in the RTS lines, we did detect a chromosome 12q14 amplification that was associated with clinical GBM recurrence in 2 RTS models. A potentially novel bioinformatics pipeline was applied to analyze phenotypic, transcriptomic, and kinomic alterations, which identified long noncoding RNAs (lncRNAs) and targetable, PDX-specific kinases. We observed differential transcriptional enrichment of DNA damage repair pathways in our RTS models, which correlated with several lncRNAs. Global kinomic profiling separated RTU and RTS models, but pairwise analyses indicated that there are multiple molecular routes to acquired radiation resistance. RTS model-specific kinases were identified and targeted with clinically relevant small molecule inhibitors. This cohort of in vivo RTS patient-derived models will enable future preclinical therapeutic testing to help overcome the treatment resistance seen in patients with GBM.

Repair-Assisted Damage Detection Reveals Biological Disparities in Prostate Cancer between African Americans and European Americans.

African Americans (AA) are two times more likely to be diagnosed with and succumb to prostate cancer (PCa) compared to European Americans (EA). There is mounting evidence that biological differences in these tumors contribute to disparities in patient outcomes. Our goal was to examine the differences in DNA damage in AA and EA prostate tissues. Tissue microarrays with matched tumor-benign adjacent pairs from 77 AA and EA PCa patients were analyzed for abasic sites, oxidative lesions, crosslinks, and uracil content using the Repair Assisted Damage Detection (RADD) assay. Our analysis revealed that AA PCa, overall, have more DNA damage than EA PCa. Increased uracil and pyrimidine lesions occurred in AA tumors, while EA tumors had more oxidative lesions. AA PCa have higher levels of UMP and folate cycle metabolites than their EA counterparts. AA PCa showed higher levels of UNG, the uracil-specific glycosylase, than EA, despite uracil lesions being retained within the genome. AA patients also had lower levels of the base excision repair protein XRCC1. These results indicate dysfunction in the base excision repair pathway in AA tumors. Further, these findings reveal how metabolic rewiring in AA PCa drives biological disparities and identifies a targetable axis for cancer therapeutics.

Suppression of Colon Tumorigenesis in Mutant Apc Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic ß-Catenin.

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 ß-catenin, inhibited Wnt-induced nuclear translocation of ß-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/ß-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.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Cytoprotective Effect of Vitamin D on Doxorubicin-Induced Cardiac Toxicity in Triple Negative Breast Cancer.

BACKGROUND: Doxorubicin (Dox) is a first-line treatment for triple negative breast cancer (TNBC), but its use may be limited by its cardiotoxicity mediated by the production of reactive oxygen species. We evaluated whether vitamin D may prevent Dox-induced cardiotoxicity in a mouse TNBC model. METHODS: Female Balb/c mice received rodent chow with vitamin D3 (1500 IU/kg; vehicle) or chow supplemented with additional vitamin D3 (total, 11,500 IU/kg). the mice were inoculated with TNBC tumors and treated with intraperitoneal Dox (6 or 10 mg/kg). Cardiac function was evaluated with transthoracic echocardiography. The cardiac tissue was evaluated with immunohistochemistry and immunoblot for levels of 4-hydroxynonenal, NAD(P)H quinone oxidoreductase (NQO1), C-MYC, and dynamin-related protein 1 (DRP1) phosphorylation. RESULTS: At 15 to 18 days, the mean ejection fraction, stroke volume, and fractional shortening were similar between the mice treated with vitamin D + Dox (10 mg/kg) vs. vehicle but significantly greater in mice treated with vitamin D + Dox (10 mg/kg) vs. Dox (10 mg/kg). Dox (10 mg/kg) increased the cardiac tissue levels of 4-hydroxynonenal, NQO1, C-MYC, and DRP1 phosphorylation at serine 616, but these increases were not observed with vitamin D + Dox (10 mg/kg). A decreased tumor volume was observed with Dox (10 mg/kg) and vitamin D + Dox (10 mg/kg). CONCLUSIONS: Vitamin D supplementation decreased Dox-induced cardiotoxicity by decreasing the reactive oxygen species and mitochondrial damage, and did not decrease the anticancer efficacy of Dox against TNBC.

Opportunity to increase deceased donation for United States veterans.

Recent changes to organ procurement organization (OPO) performance metrics have highlighted the need to identify opportunities to increase organ donation in the United States. Using data from the Organ Procurement and Transplantation Network (OPTN), Scientific Registry of Transplant Recipients (SRTR), and Veteran Health Administration Informatics and Computing Infrastructure Clinical Data Warehouse (VINCI CDW), we sought to describe historical donation performance at Veteran Administration Medical Centers (VAMCs). We found that over the period 2010-2019, there were only 33 donors recovered from the 115 VAMCs with donor potential nationwide. VA donors had similar age-matched organ transplant yields to non-VA donors. Review of VAMC records showed a total of 8474 decedents with causes of death compatible with donation, of whom 5281 had no infectious or neoplastic comorbidities preclusive to donation. Relative to a single state comparison of adult non-VA inpatient deaths, VAMC deaths were 20 times less likely to be characterized as an eligible death by SRTR. The rate of conversion of inpatient donation-consistent deaths without preclusive comorbidities to actual donors at VAMCs was 5.9% that of adult inpatients at non-VA hospitals. Overall, these findings suggest significant opportunities for growth in donation at VAMCs.

Associations between DNA Damage and PD-L1 Expression in Ovarian Cancer, a Potential Biomarker for Clinical Response.

Programmed death ligand-1 (PD-L1) inhibitors are currently under investigation as a potential treatment option for ovarian cancer. Although this therapy has shown promise, its efficacy is highly variable among patients. Evidence suggests that genomic instability influences the expression of PD-L1, but little is known about this relationship in ovarian cancer. To examine the relationship between PD-L1 expression and genomic instability, we measured DNA damage using Repair Assisted Damage Detection (RADD). We then correlated the presence of persistent DNA damage in the ovarian tumor with protein expression of PD-L1 using immunohistochemistry. Ovarian tumors showed a high prevalence of oxidative DNA damage. As the level of oxidative DNA damage increased, we saw a significant correlation with PD-L1 expression. The highest correlation between DNA damage and PD-L1 expression was observed for mucinous ovarian tumors (r = 0.82), but a strong correlation was also observed for high grade serous and endometrioid tumors (r = 0.67 and 0.69, respectively). These findings link genomic instability to PD-L1 protein expression in ovarian cancer and suggest that persistent DNA damage can be used as a potential biomarker for patient selection for immunotherapy treatment.

Exploiting DNA repair defects in triple negative breast cancer to improve cell killing.

BACKGROUND: The lack of molecular targets for triple negative breast cancer (TNBC) has limited treatment options and reduced survivorship. Identifying new molecular targets may help improve patient survival and decrease recurrence and metastasis. As DNA repair defects are prevalent in breast cancer, we evaluated the expression and repair capacities of DNA repair proteins in preclinical models. METHODS: DNA repair capacity was analyzed in four TNBC cell lines, MDA-MB-157 (MDA-157), MDA-MB-231 (MDA-231), MDA-MB-468 (MDA-468), and HCC1806, using fluorescence multiplex host cell reactivation (FM-HCR) assays. Expression of DNA repair genes was analyzed with RNA-seq, and protein expression was evaluated with immunoblot. Responses to the combination of DNA damage response inhibitors and primary chemotherapy drugs doxorubicin or carboplatin were evaluated in the cell lines. RESULTS: Defects in base excision and nucleotide excision repair were observed in preclinical TNBC models. Gene expression analysis showed a limited correlation between these defects. Loss in protein expression was a better indicator of these DNA repair defects. Over-expression of PARP1, XRCC1, RPA, DDB1, and ERCC1 was observed in TNBC preclinical models, and likely contributed to altered sensitivity to chemotherapy and DNA damage response (DDR) inhibitors. Improved cell killing was achieved when primary therapy was combined with DDR inhibitors for ATM, ATR, or CHK1. CONCLUSION: Base excision and nucleotide excision repair pathways may offer new molecular targets for TNBC. The functional status of DNA repair pathways should be considered when evaluating new therapies and may improve the targeting for primary and combination therapies with DDR inhibitors.

EGFR signaling promotes resistance to CHK1 inhibitor prexasertib in triple negative breast cancer.

Aim: Innate resistance to the CHK1 inhibitor prexasertib has been described, but resistance mechanisms are not understood. We aimed to determine the role epidermal growth factor receptor (EGFR) plays in innate resistance to prexasertib in triple negative breast cancer (TNBC). Methods: Using a panel of pre-clinical TNBC cell lines, we measured the sensitivity to prexasertib. We examined the effect activation of EGFR had on prexasertib sensitivity. We measured the synergy of dual blockade of EGFR with erlotinib and CHK1 with prexasertib in TNBC cell lines and xenografts. Results: EGFR overexpression and activation increased resistance to CHK1 inhibition by prexasertib. EGFR promoted the phosphorylation of BCL2-associated agonist of cell death (BAD), inactivating its pro-apoptotic functions. Inhibition of EGFR reversed BAD phosphorylation, increasing sensitivity to prexasertib. Conclusion: The use of prexasertib as a monotherapy in TNBC has been limited due to modest clinical responses. We demonstrated that EGFR activation contributes to innate resistance to prexasertib in TNBC and potentially other cancers. EGFR expression status should be considered in clinical trials examining prexasertib's use as a monotherapy or combination therapy.

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer.

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 ß (POL ß), 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 cancers (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 in the TCGA database and its expression and localization in TNBC cell lines. The TCGA database revealed high XRCC1 expression in TNBC tumors and TNBC cell lines show variable, but mostly high expression of XRCC1. XRCC1 localized outside of the nucleus in some TNBC cell lines, altering their ability to repair base lesions and single-strand breaks. Subcellular localization of POL ß also varied and did not correlate with XRCC1 localization. Basal levels of DNA damage correlated with observed changes in XRCC1 expression, localization, and measure repair capacity. The results confirmed that XRCC1 expression changes indicate DNA repair capacity changes but emphasize that basal DNA damage levels along with protein localization are better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in TNBC preclinical models and tumors, XRCC1 expression levels should be assessed when evaluating treatment responses of TNBC preclinical model cells.

DNA damage measurements within tissue samples with Repair Assisted Damage Detection (RADD).

Exposures to genotoxic carcinogens and reactive species result in strand breaks and a spectrum of covalent modifications to DNA that can induce mutations and contribute to the initiation and progression of cancer. Measurements of DNA damage within tissue or tumor samples can serve as a biomarker for exposures or assess changes in DNA repair capacity relevant in cancer development and treatment. Numerous methods to characterize DNA damage exist. However, these methods are primarily applicable to isolated DNA or cultured cells, often require a substantial amount of material, and may be limited to the detection and quantification of only a handful of DNA adducts. Here, we used the Repair Assisted Damage Detection (RADD) assay to detect and excise DNA adducts using a cocktail of DNA repair enzymes, then the damage site within the genome are tagged for detection using a modified nucleotide. We previously demonstrated the RADD assay can detect lesions within isolated DNA and fixed cells, and now RADD can be used to detect DNA adducts and DNA strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue samples. We verified the ability of the RADD assay to detect DNA damage in tissue by exogenously inducing DNA damage with X-rays and restriction enzymes. We also showed that RADD can be multiplexed with antibodies to detect cell cycle markers or other proteins of interest. Finally, we showed that RADD can detect DNA damage within clinically relevant ovarian tumor samples. RADD is a flexible and easy-to-use assay that allows relative damage levels to be determined within FFPE samples and allows the heterogeneity of DNA adducts and strand breaks within clinically relevant samples to be measured.

Management and Survival of Adult Patients with Pilocytic Astrocytoma in the National Cancer Database.

BACKGROUND: Adult pilocytic astrocytomas (PAs) are relatively rare central nervous system (CNS) tumors with a favorable prognosis. We sought to investigate existing clinical management strategies and overall survival (OS) as a function of various clinical characteristics in a cohort of adult patients with PA. METHODS: The study cohort comprised all patients age >18 years diagnosed with a CNS PA diagnosed between 2004 and 2014 and included in the National Cancer Database. Clinical and treatment-related characteristics were recorded and analyzed for associations with OS following diagnosis using univariate and multivariate analyses. RESULTS: A total of 3057 adult patients, with a median age of 32 years, met the inclusion criteria. At diagnosis, 1138 patients (41%) had cerebral tumors, 832 (30%) had cerebellar tumors, 252 (9%) had tumors of the spinal cord, and 534 (19%) had tumors of unspecified location. More than three-quarters (77%) of the patients underwent surgery alone as local therapy, with the remainder split among surgery plus radiation (11.9%), radiation alone (4.5%), and biopsy alone (6.9%). On multivariate analysis, factors associated with inferior OS included older age (hazard ratio [HR], 1.05; P < 0.001), lower income (P < 0.001), higher Charlson/Deyo score (P = 0.023), larger tumor size (P = 0.023), and radiation therapy technique (P < 0.001; HR, 3.37 for external beam radiation therapy [EBRT]). CONCLUSIONS: Our data provide large-scale prognostic information from a contemporary cohort of patients with PA, confirming that age, median income, Charlson/Deyo Score, and tumor size have significant effects on OS. Although resection status, tumor size, and location likely bias against EBRT, novel therapeutics are clearly needed in patients with tumors not amenable to resection or radiosurgery.

Mining ZINC Database to Discover Potential Phosphodiesterase 9 Inhibitors Using Structure-Based Drug Design Approach.

In view of the emerging clinical indications for Phosphodiesterase 9 inhibitors e.g. treatment of Alzheimer, diabetes, cancer, and the limited number of its selective inhibitors which possess a single chemical scaffolds, a structure-based approach was undertaken to mine the ZINC database by virtual screening to identify novel PDE9 inhibitors. The database, which was never reported to have been used before for discovery of PDE9 inhibitors, was screened against the ligand binding pocket of the PDE9 complex (PDB:4GH6) using molecular docking programs, MOE and AutoDock Vina in PyRx. Three different scoring functions were used to evaluate the docking poses and scores of the compounds, and the compounds were selected through consensus selection, thus reducing the margin of error in docking. The highest scoring compounds were then selected and purchased for in vitro testing as PDE9 inhibitors and cancer growth inhibitory agents. This led to the discovery of three previously unreported potent PDE 9 inhibitory compounds with two unique chemical scaffolds. Consistent with the role of PDE9 in cancer cell growth, the compounds also inhibited the growth of breast tumor cell lines, MCF-7 and MDA-468 at concentrations comparable to those that inhibited PDE9.

A molecular program for contralateral trajectory: Rig-1 control by LIM homeodomain transcription factors.

Despite increasing evidence for transcriptional control of neural connectivity, how transcription factors regulate discrete steps in axon guidance remains obscure. Projection neurons in the dorsal spinal cord relay sensory signals to higher brain centers. Some projection neurons send their axons ipsilaterally, whereas others, commissural neurons, send axons contralaterally. We show that two closely related LIM homeodomain proteins, Lhx2 and Lhx9, are expressed by a set of commissural relay neurons (dI1c neurons) and are required for the dI1c axon projection. Midline crossing by dI1c axons is lost in Lhx2/9 double mutants, a defect that results from loss of expression of Rig-1 from dI1c axons. Lhx2 binds to a conserved motif in the Rig-1 gene, suggesting that Lhx2/9 regulate directly the expression of Rig-1. Our findings reveal a link between the transcriptional programs that define neuronal subtype identity and the expression of receptors that guide distinctive aspects of their trajectory.