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Deregulation of the Wnt/β-catenin signaling pathway drives the development of colorectal cancer, but understanding of this pathway remains incomplete. Here, we report that the damage-specific DNA-binding protein DDB2 is critical for β-catenin–mediated activation of RNF43, which restricts Wnt signaling by removing Wnt receptors from the cell surface. Reduced expression of DDB2 and RNF43 was observed in human hyperplastic colonic foci. DDB2 recruited EZH2 and β-catenin at an upstream site in the Rnf43 gene, enabling functional interaction with distant TCF4/β-catenin–binding sites in the intron of Rnf43. This novel activity of DDB2 was required for RNF43 function as a negative feedback regulator of Wnt signaling. Mice genetically deficient in DDB2 exhibited increased susceptibility to colon tumor development in a manner associated with higher abundance of the Wnt receptor–expressing cells and greater activation of the downstream Wnt pathway. Our results identify DDB2 as both a partner and regulator of Wnt signaling, with an important role in suppressing colon cancer development. Cancer Res; 77(23); 6562–75. ©2017 AACR.

Table S1: Sequences of DsiRNA; Table S2: Primers for ChIP assays; Table S3: Primers for gene expression assays; Table S4: Correlation analysis of DDB2 and RNF43 expression in patient colonic samples; Table S5:Primers and probes for 3C assay

Figure S6: Association of DDB2, TCF4 and β-catenin on Rnf43 upstream region and intron regions.Figure S7: EZH2 and PAF are required for Rnf43 expression.Figure S8: DDB2 is required for the RNF43-mediated sequestration of TCF4 to the nuclear membrane.Figure S9: WES showed missense mutation in Ctnnb1 gene.Figure S10: DDB2 regulates RNF43 and Wnt target gene expression in normal colonic tissues.Figure S11: Model of how DDB2 regulates Wnt pathway and colon cancer development through RNF43.

Supplementary methods of Protein Extraction, Immunoblotting and Co-immunoprecipitations, Chromatin Conformation Capture Assay, Immunofluorescent Staining and Whole Exome Sequencing (WES)

The long-term survival of patients with advanced urothelial carcinoma (UCa) is limited because of innate resistance to treatment. We identified elevated expression of the histone methyltransferase EZH2 as a hallmark of aggressive UCa and hypothesized that EZH2 inhibition, via a small-molecule catalytic inhibitor, might have antitumor effects in UCa. Here, in a carcinogen-induced mouse bladder cancer model, a reduction in tumor progression and an increase in immune infiltration upon EZH2 inhibition were observed. Treatment of mice with EZH2i causes an increase in MHC class II expression in the urothelium and can activate infiltrating T cells. Unexpectedly, we found that the lack of an intact adaptive immune system completely abolishes the antitumor effects induced by EZH2 catalytic inhibition. These findings show that immune evasion is the only important determinant for the efficacy of EZH2 catalytic inhibition treatment in a UCa model.

Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by inflammation, demyelination, and neurodegeneration. The ideal MS therapy would both specifically inhibit the underlying autoimmune response and promote repair/regeneration of myelin as well as maintenance of axonal integrity. Currently approved MS therapies consist of non-specific immunosuppressive molecules/antibodies which block activation or CNS homing of autoreactive T cells, but there are no approved therapies for stimulation of remyelination nor maintenance of axonal integrity. In an effort to repurpose an FDA-approved medication for myelin repair, we chose to examine the effectiveness of digoxin, a cardiac glycoside (Na

Although PD-1/PD-L1 therapies have shown significant activity across a range of tumor types, only a subset of patients (10-30%) achieve durable responses. With the goal of improving response and application to PD-1/PD-L1 therapies, we have leveraged our proprietary Xilio Advanced Cytokine Therapies (X-ACT) platform to develop PD1/IL2-ACT, a PD-1 blocker enhanced with a tumor-activated, engineered IL-2 agonist. The activity of IL-2 is blocked by a protein domain that prevents IL-2Rβγ binding until activated in the tumor microenvironment by matrix metalloproteinases . Tumor selective activation of IL-2 enables dosing of PD1/IL2-ACT at sufficient exposure levels to block immunosuppressive PD1/PD-L1 signaling as well as mitigating systemic IL-2 driven toxicity. PD1/IL2-ACT is designed to enable in cis engagement of IL-2 receptors on antigen experienced PD-1+ CD8+ T cells in the tumor microenvironment (TME), inducing effector T cell functions while preventing regulatory T cell (Treg) mediated immune suppression. In a preclinical model, PD1/IL2-ACT, compared to anti-PD1 alone, induced significant tumor growth inhibition. Further, PD1/IL2-ACT was better tolerated than a PD-1 directed unmasked IL-2Rβγ biased agonist in the same preclinical model. Pharmacodynamic studies showed that PD1/IL2-ACT did not significantly change immune cell profiles in the periphery but increased antigen specific CD8+ T cells in the TME, consistent with preferential engagement of PD1+ effector T cells at the tumor site. Ex vivo protease cleavage assays performed in human tumors demonstrated significant activation of PD1/IL2-ACT by tumors of multiple indications but minimal activation in human plasma. A pharmacokinetic study in nonhuman primates revealed PD1/IL2-ACT can achieve comparable exposure levels to existing PD-1 blocking immunotherapies. Taken together, these data illustrate that PD1/IL2-ACT has the potential to broaden the activity of existing immunotherapies by inhibiting the PD1/PDL1 axis as well as directing an immunostimulatory cytokine to antigen specific T cells in the TME. Citation Format: Ertan Eryilmaz, Wilson Guzman, Dheeraj S. Tomar, Parker Johnson, Stephanie Hsiao, Lisa Quinn, Nancy Chan, Jimit Lakhani, Brendan Whalen, Rosa Quiroz, Kurt Jenkins, Oleg Yerov, Will Scott, Justin Greene, Zhen Liu, Megan McLaughlin, Sallyann Vu, Chelsea Turcotte, Hanumantha R. Madala, Jacob Taylor, Caitlin O'Toole, Magali Pederzoli-Ribeil, Haley Duprey, Natalia Malkova, Damiano Fantini, David Crowe, Sean Yang, Kyle Smith, Joseph Card, Janice Lee, Kerri Smith, Benjamin Nicholson, Jijun Dong, Jennifer O'Neil, Carl U. Bialucha. Tumor-activated PD1-directed IL-2 increased antigen specific T cells in tumors and demonstrated anti-tumor activity in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 572.

Interleukin-12 (IL-12) is a proinflammatory cytokine, which bridges innate and adaptive immunity via induction of T helper 1 differentiation and promotes cytolytic activity of natural killer and T cells. IL-12 has demonstrated potent antitumor activity in syngeneic mouse models and promising anti-tumor efficacy in humans. However, development of IL-12-based treatments has been limited by severe systemic toxicities in the clinical setting. To overcome toxicity and potentially improve the therapeutic index of IL-12 in a clinical setting, XTX301 was engineered as a half-life extended and masked IL-12. The masking domain of XTX301 is designed to pharmacologically inactivate IL-12 in circulation and non-tumor tissue, while enabling generation of an active IL-12 moiety upon cleavage of a linker sequence by matrix metalloproteinases that are enriched in the tumor microenvironment. To confirm the ability of human tumor associated proteases to activate XTX301, cleavage was assessed in primary human tumor samples and in plasma from cancer patients. We observed cleavage of XTX301 in the majority of human tumors samples tested, but no activation was observed in plasma from cancer patients. Human IL-12 does not bind and signal through the mouse IL-12 receptors, hence three murine surrogates were created for in vivo studies: mXTX301, a non-cleavable control, and an unmasked control molecule. Tumor growth inhibition was observed after a single dose of mXTX301 as low as 0.039 mg/kg. The non-cleavable form of mXTX301 was less potent than mXTX301, demonstrating that the anti-tumor activity of mXTX301 is dependent on protease activation. The unmasked control was not well tolerated, with a > 20% body weight loss observed by Day 6, resulting in 75% of animals being euthanized by Day 11. Unlike the unmasked control, mXTX301 was well-tolerated at all tested doses, as evidenced by no loss in animals’ body weights, and demonstrated minimal pharmacodynamic activity in non-tumor tissues. In tumors, mXTX301 stimulated the infiltration of cytotoxic CD8+ T cells and induced the expression of several immune-related genes including those associated with IFN-γ cell signaling, antigen processing and presentation, and defense response as well as enrichment of gene signatures for T cells, natural killer (NK) cells, macrophages and dendritic cells as determined by RNA sequencing. In non-human primates, the highest non-severely toxic dose of XTX301 was 2mg/kg dosed weekly for a total of 4 doses. In summary, a half-life extended tumor-activated IL-12 molecule, mXTX301, demonstrated anti-tumor activity in preclinical mouse models with improved tolerability compared to a systemically active IL-12 molecule suggesting that XTX301 has potential for exerting potent anti-tumor activity while widening the therapeutic index of IL-12 treatment. Citation Format: Natalia Malkova, Ekta Patel, Sallyann Vu, Damiano Fantini, Rebekah O'Donnell, Manoussa Fanny, Justin Greene, Wilson Guzman, David Crowe, Stephanie Hsiao, Parker Johnson, Megan McLaughlin, Oleg Yerov, Kurt Jenkins, Katarina Halpin-Veszeleiova, Hanumantha Rao Madala, Caitlin O'Toole, Jake Taylor, Magali Pederzoli-Ribeil, Benjamin Nicholson, Carl Uli Bialucha, Jennifer E. O'Neil. A half-life extended, tumor-activated IL-12 increased the infiltration of effector immune cells into the tumor microenvironment and demonstrated anti-tumor activity in syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 587.

Cancer cells accumulate somatic mutations as result of DNA damage, inaccurate repair and other mechanisms. Different genetic instability processes result in characteristic non-random patterns of DNA mutations, also known as mutational signatures. We developed mutSignatures, an integrated R-based computational framework aimed at deciphering DNA mutational signatures. Our software provides advanced functions for importing DNA variants, computing mutation types, and extracting mutational signatures via non-negative matrix factorization. Specifically, mutSignatures accepts multiple types of input data, is compatible with non-human genomes, and supports the analysis of non-standard mutation types, such as tetra-nucleotide mutation types. We applied mutSignatures to analyze somatic mutations found in smoking-related cancer datasets. We characterized mutational signatures that were consistent with those reported before in independent investigations. Our work demonstrates that selected mutational signatures correlated with specific clinical and molecular features across different cancer types, and revealed complementarity of specific mutational patterns that has not previously been identified. In conclusion, we propose mutSignatures as a powerful open-source tool for detecting the molecular determinants of cancer and gathering insights into cancer biology and treatment.

Bladder cancer is the fourth most common cancer in men and fifth most common overall. The use of next-generation sequencing (NGS) approaches is crucial to precisely characterize the molecular defects of tumors, and this information could be combined with other clinical data, such as tumor histology and TNM staging, with the goal of precise tumor classification. In many settings, targeted NGS is evaluated in patients with first- and second-line metastatic cancer. Yet, in the decade to come we anticipate increased application of precision oncology at all stages of bladder cancer with the aim of customizing cancer treatment. Here, we review the genomic and transcriptomic features associated with risk stratification in bladder cancer and summarize the current efforts for precision oncology in localized urothelial carcinomas.

The growing attention toward the benefits of single-cell RNA sequencing (scRNA-seq) is leading to a myriad of computational packages for the analysis of different aspects of scRNA-seq data. For researchers without advanced programing skills, it is very challenging to combine several packages in order to perform the desired analysis in a simple and reproducible way. Here we present DIscBIO, an open-source, multi-algorithmic pipeline for easy, efficient and reproducible analysis of cellular sub-populations at the transcriptomic level. The pipeline integrates multiple scRNA-seq packages and allows biomarker discovery with decision trees and gene enrichment analysis in a network context using single-cell sequencing read counts through clustering and differential analysis. DIscBIO is freely available as an R package. It can be run either in command-line mode or through a user-friendly computational pipeline using Jupyter notebooks. We showcase all pipeline features using two scRNA-seq datasets. The first dataset consists of circulating tumor cells from patients with breast cancer. The second one is a cell cycle regulation dataset in myxoid liposarcoma. All analyses are available as notebooks that integrate in a sequential narrative R code with explanatory text and output data and images. R users can use the notebooks to understand the different steps of the pipeline and will guide them to explore their scRNA-seq data. We also provide a cloud version using Binder that allows the execution of the pipeline without the need of downloading R, Jupyter or any of the packages used by the pipeline. The cloud version can serve as a tutorial for training purposes, especially for those that are not R users or have limited programing skills. However, in order to do meaningful scRNA-seq analyses, all users will need to understand the implemented methods and their possible options and limitations.

Primary cutaneous follicle center lymphomas (PCFCLs) are indolent B-cell lymphomas that predominantly remain skin restricted and manageable with skin-directed therapy. Conversely, secondary cutaneous involvement by usual systemic follicular lymphoma (secondary cutaneous follicular lymphoma [SCFL]) has a worse prognosis and often necessitates systemic therapy. Unfortunately, no histopathologic or genetic features reliably differentiate PCFCL from SCFL at diagnosis. Imaging may miss low-burden internal disease in some cases of SCFLs, leading to misclassification as PCFCL. Whereas usual systemic FL is well characterized genetically, the genomic landscapes of PCFCL and SCFL are unknown. Herein, we analyzed clinicopathologic and immunophenotypic data from 30 cases of PCFCL and 10 of SCFL and performed whole-exome sequencing on 18 specimens of PCFCL and 6 of SCFL. During a median follow-up of 7 years, 26 (87%) of the PCFCLs remained skin restricted. In the remaining 4 cases, systemic disease developed within 3 years of diagnosis. Although the SCFLs universally expressed BCL2 and had BCL2 rearrangements, 73% of the PCFCLs lacked BCL2 expression, and only 8% of skin-restricted PCFCLs had BCL2 rearrangements. SCFLs showed low proliferation fractions, whereas 75% of PCFCLs had proliferation fractions >30%. Of the SCFLs, 67% had characteristic loss-of-function CREBBP or KMT2D mutations vs none in skin-restricted PCFCL. Both SCFL and skin-restricted PCFCL showed frequent TNFRSF14 loss-of-function mutations and copy number loss at chromosome 1p36. These data together establish PCFCL as a unique entity with biological features distinct from usual systemic FL and SCFL. We propose 3 criteria based on BCL2 rearrangement, chromatin-modifying gene mutations (CREBBP, KMT2D, EZH2, and EP300), and proliferation index to classify cutaneous FL specimens based on the likelihood of concurrent or future systemic spread.

Stage T1 bladder cancers have the highest progression and recurrence rates of all non-muscle-invasive bladder cancers (NMIBCs). Most T1 cancers are treated with bacillus Calmette-Guerin (BCG), but many will progress or recur, and some T1 patients will die from bladder cancer. Particularly aggressive tumors could be treated with early cystectomy. To better understand the molecular heterogeneity of T1 cancers, we performed transcriptome profiling and unsupervised clustering, and identified five consensus subtypes of T1 tumors treated with repeat transurethral resection (reTUR) and induction and maintenance BCG. The T1-LumGU subtype was associated with carcinoma in situ (CIS; six/13, 46% of all CIS), had high E2F1 and EZH2 expression, and was enriched in E2F target and G2M checkpoint hallmarks. The T1-Inflam subtype was inflamed and infiltrated with immune cells. While most T1 tumors were classified as luminal papillary, the T1-TLum subtype had the highest median luminal papillary score and FGFR3 expression, no recurrence events, and the fewest copy number gains. T1-Myc and T1-Early subtypes had the most recurrences (14/30 within 24 mo), the highest median MYC expression, and, when combined, had significantly worse recurrence-free survival than the other three subtypes. T1-Early had five (38%) recurrences within the first 6 mo of BCG, and repressed IFN-α and IFN-γ hallmarks and inflammation. We developed a single-patient T1 classifier and validated our subtype biology in a second cohort of T1 tumors. Future research will be necessary to validate the proposed T1 subtypes and to determine if therapies can be individualized for each subtype. PATIENT SUMMARY: We identified and characterized expression subtypes of high-grade stage T1 bladder cancer that are biologically heterogeneous and have variable responses to bacillus Calmette-Guerin treatment. We validated the subtypes and describe a single-patient classifier.

Background Baricitinib, an oral selective Janus kinase (JAK)1 and 2 inhibitor, resulted in significant clinical improvements in patients with active systemic lupus erythematosus (SLE) receiving standard background therapy in the phase 2 study JAHH (NCT02708095). Baricitinib may impact key cytokines implicated in the pathogenesis of SLE through effects on the JAK/signal transducer and activator of transcription (STAT) signaling pathway. The impact of baricitinib on STAT-related gene expression and associations with clinical response in SLE were evaluated. Methods 314 patients were randomized 1:1:1 to receive once-daily placebo, baricitinib 2-mg, or baricitinib 4-mg for 24 weeks in JAHH. Patients were ≥18 years of age, had a diagnosis of SLE, and had active disease involving skin or joints. RNA isolated from whole blood at baseline and weeks 2, 4, 12, and 24 was analyzed using Affymetrix HTA2.0 array. Results Gene expression profiling demonstrated a statistically significant elevation of STAT1 and STAT2 gene expression at baseline in SLE patients. There was a significant association between the overexpression of STAT1 and STAT2 at baseline. Baricitinib 4-mg treatment resulted in modest reduction in STAT1, STAT2, and STAT4 expression, and a statistically significant reduction in multiple genes downstream of STAT1, STAT2, and STAT4. The reduction in expression of STAT-associated genes with baricitinib treatment correlated with clinical improvement in SLE patients using SLEDAI-2K measurements (table 1). Conclusions Baricitinib may partially mediate its effect in SLE through changes in STAT-related gene expression, with changes associated with clinical improvement in SLE. Acknowledgements Funded by Eli Lilly and Company.

Patients with locally advanced and metastatic urothelial carcinoma have a low survival rate (median 15.7 months, 13.1–17.8), with only a 23% response rate to monotherapy treatment with anti-PDL1 checkpoint immunotherapy. To identify new therapeutic targets, we profiled the immune regulatory signatures during murine cancer development using the BBN carcinogen and identified an increase in the expression of the T cell inhibitory protein B7-H4 (VTCN1, B7S1, B7X). B7-H4 expression temporally correlated with decreased lymphocyte infiltration. While the increase in B7-H4 expression within the bladder by CD11b+ monocytes is shared with human cancers, B7-H4 expression has not been previously identified in other murine cancer models. Higher expression of B7-H4 was associated with worse survival in muscle-invasive bladder cancer in humans, and increased B7-H4 expression was identified in luminal and luminal-papillary subtypes of bladder cancer. Evaluation of B7-H4 by single-cell RNA-Seq and immune mass cytometry of human bladder tumors found that B7-H4 is expressed in both the epithelium of urothelial carcinoma and CD68+ macrophages within the tumor. To investigate the function of B7-H4, treatment of human monocyte and T cell co-cultures with a B7-H4 blocking antibody resulted in enhanced IFN-γ secretion by CD4+ and CD8+ T cells. Additionally, anti-B7-H4 antibody treatment of BBN-carcinogen bladder cancers resulted in decreased tumor size, increased CD8+ T cell infiltration within the bladder, and a complimentary decrease in tumor-infiltrating T regulatory cells (Tregs). Furthermore, treatment with a combination of anti-PD-1 and anti-B7-H4 antibodies resulted in a significant reduction in tumor stage, a reduction in tumor size, and an increased level of tumor necrosis. These findings suggest that antibodies targeting B7-H4 may be a viable strategy for bladder cancers unresponsive to PD-1 checkpoint inhibitors.

The N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model is an attractive model system of muscle-invasive bladder cancer (MIBC) as it recapitulates the histology of human tumors in a background with intact immune system. However, it was unknown whether this carcinogen-induced model also mimicked human MIBC at the molecular and mutational level. In our study, we analyzed gene expression and mutational landscape of the BBN model by next-generation sequencing followed by a bioinformatic comparison to human MIBC using data from The Cancer Genome Atlas and other repositories. BBN tumors showed overexpression of markers of basal cancer subtype, and had a high mutation burden with frequent Trp53 (80%), Kmt2d (70%), and Kmt2c (90%) mutations by exome sequencing, similar to human MIBC. Many variants corresponded to human cancer hotspot mutations, supporting their role as driver mutations. We extracted two novel mutational signatures from the BBN mouse genomes. The integrated analysis of mutation frequencies and signatures highlighted the contribution of aberrations to chromatin regulators and genetic instability in the BBN tumors. Together, our study revealed several similarities between human MIBC and the BBN mouse model, providing a strong rationale for its use in molecular and drug discovery studies.

APOBEC enzymes are responsible for a mutation signature (TCW>T/G) implicated in a wide variety of tumors. We explore the APOBEC mutational signature in bladder cancer and the relationship with specific mutations, molecular subtype, gene expression, and survival using sequencing data from The Cancer Genome Atlas (n = 395), Beijing Genomics Institute (n = 99), and Cancer Cell Line Encyclopedia. Tumors were split into “APOBEC-high” and “APOBEC-low” based on APOBEC enrichment. Patients with APOBEC-high tumors have better overall survival compared to those with APOBEC-low tumors (38.2 vs. 18.5 months, p = 0.005). APOBEC-high tumors are more likely to have mutations in DNA damage response genes (TP53, ATR, BRCA2) and chromatin regulatory genes (ARID1A, MLL, MLL3), while APOBEC-low tumors are more likely to have mutations in FGFR3 and KRAS. APOBEC3A and APOBEC3B expression correlates with mutation burden, regardless of bladder tumor molecular subtype. APOBEC mutagenesis is associated with increased expression of immune signatures, including interferon signaling, and expression of APOBEC3B is increased after stimulation of APOBEC-high bladder cancer cell lines with IFNγ. In summary, APOBEC-high tumors are more likely to have mutations in DNA damage response and chromatin regulatory genes, potentially providing more substrate for APOBEC enzymes, leading to a hypermutational phenotype and the subsequent enhanced immune response.

Deregulation of the Wnt/β-catenin signaling pathway drives the development of colorectal cancer, but understanding of this pathway remains incomplete. Here, we report that the damage-specific DNA-binding protein DDB2 is critical for β-catenin–mediated activation of RNF43, which restricts Wnt signaling by removing Wnt receptors from the cell surface. Reduced expression of DDB2 and RNF43 was observed in human hyperplastic colonic foci. DDB2 recruited EZH2 and β-catenin at an upstream site in the Rnf43 gene, enabling functional interaction with distant TCF4/β-catenin–binding sites in the intron of Rnf43. This novel activity of DDB2 was required for RNF43 function as a negative feedback regulator of Wnt signaling. Mice genetically deficient in DDB2 exhibited increased susceptibility to colon tumor development in a manner associated with higher abundance of the Wnt receptor–expressing cells and greater activation of the downstream Wnt pathway. Our results identify DDB2 as both a partner and regulator of Wnt signaling, with an important role in suppressing colon cancer development. Cancer Res; 77(23); 6562–75. ©2017 AACR.

Background:Baricitinib (BARI), an oral, selective Janus kinase (JAK)1 and JAK2 inhibitor, improved disease severity in adults with systemic lupus erythematosus (SLE) receiving standard background therapy in a phase 2 trial1. There were no meaningful reductions in least squares mean change from baseline (BL) in levels of serologic biomarkers for SLE with BARI treatment, including anti-double-stranded deoxyribonucleic acid (anti-dsDNA) antibodies and complement component (C)3 and C41.Objectives:Evaluate the median change from BL in serologic biomarkers in subgroups and the overall population of BARI-treated SLE patients, in addition to the Systemic Lupus Erythematosus Responder Index-4 (SRI-4) response by normalization of anti-dsDNA.Methods:Data were assessed from the phase 2 trial JAHH (NCT02708095). The median change from BL in anti-dsDNA, IgG, C3, and C4 was evaluated over time among the following populations at BL: anti-dsDNA positive (≥30 IU/mL), low C3 (Results:Among patients who were anti-dsDNA positive at BL, significant decreases of anti-dsDNA antibodies were observed for BARI 2-mg and 4-mg compared to PBO beginning at Wks 2 and 4, respectively, and continuing through Wk 24 (Figure 1 and Table 1). Moreover, reductions of IgG levels were found for BARI-treated patients including significant decreases for BARI 4-mg compared to PBO at Wks 12 and 24 (Table 1). Among patients who had low levels of C3 and C4 at BL, no significant differences in median change from BL were observed over time with BARI compared to PBO. For patients who were anti-dsDNA positive at BL, no relationship in SRI-4 responder rate was observed for those who stayed positive or achieved normal levels by Wk 24, possibly due to the limited sample size.Conclusion:BARI treatment resulted in a rapid and sustained significant decrease in anti-dsDNA antibodies compared to PBO among anti-dsDNA positive SLE patients at BL, as well as a significant decrease in IgG levels in the 4-mg group at Wks 12 and 24. These data suggest that BARI may have an effect on B cell activity in SLE.References:[1]Wallace D et al. Lancet. 2018;392:222-231.Table 1.PBOBARI 2-mgBARI 4-mgWeek412244122441224Anti-dsDNA (IU/mL)a0.2 (-17.2, 17.3)2.6 (-14.8, 18.4)3.0 (-14.9, 28.3)-15.4** (-31.4, 1.9)-18.1* (-42.0, 4.1)-29.6** (-55.1, 10.3)-17.9** (-42.7, 1.8)-23.3*** (-50.9, -5.9)-15.1** (-71.9, -4.6)IgG (g/L)b-0.31 (-1.1, 0.4)0.09 (-1.1, 0.7)-0.04 (-0.9, 0.9)-0.60 (-1.3, 0.6)-0.30 (-1.3, 0.4)-0.51 (-1.7, 0.6)-0.56 (-1.2, 0.2)-0.65** (-1.3, 0.2)-0.60** (-1.7, 0.2)Data are median change from baseline (25th, 75th percentiles). aData were assessed for patients that were anti-dsDNA positive (≥30 IU/mL) at baseline (PBO N=51, BARI 2-mg N=56, BARI 4-mg N=53). bData were assessed for all patients (PBO N=105, BARI 2-mg N=105, BARI 4-mg N=104). *p≤0.05, **p≤0.01, ***p≤0.001 for BARI vs. PBO.Acknowledgements:The authors would like to acknowledge Nicole L. Byers, of Eli Lilly and Company, for medical writing and process support.Disclosure of Interests:Thomas Dörner Speakers bureau: Eli Lilly and Company, Roche, and Samsung, Consultant of: AbbVie, Celgene, Eli Lilly and Company, Janssen, Novartis, Roche, Samsung, and UCB, Grant/research support from: Janssen, Novartis, Roche, Sanofi, and UCB, Ronald van Vollenhoven Consultant of: Abbvie, Biotest, BMS, Celgene, Crescendo, Eli Lilly and Company, GSK, Janssen, Merck, Novartis, Pfizer, Roche, UCB, and Vertex, Grant/research support from: Abbvie, Amgen, BMS, GSK, Pfizer, Roche, and UCB, Andrea Doria Speakers bureau: GSK, Janssen, Pfizer, and Roche, Consultant of: Celgene, Eli Lilly and Company, and GSK, Bochao Jia Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Damiano Fantini Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Jorge Ross Terres Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Maria Silk Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Stephanie de Bono Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Peter Fischer Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Daniel J. Wallace Consultant of: Amgen, Aurunia, Eli Lilly and Company, EMD Merck Serono, GSK, and Pfizer

Survival of patients with urothelial carcinoma (including bladder cancer and upper tract urothelial carcinoma) is limited by our current approaches to staging, surgery, and chemotherapy. Large-scale, next-generation sequencing collaborations, such as The Cancer Genome Atlas, have already identified drivers and vulnerabilities of urothelial carcinoma. This disease has a high degree of mutational heterogeneity and a high frequency of somatic mutations compared with other solid tumours, potentially resulting in an increased neoantigen burden. Mutational heterogeneity is mediated by multiple factors including the apolipoprotein B mRNA editing enzyme catalytic polypeptide family of enzymes, smoking exposure, viral integrations, and intragene and intergene fusion proteins. The mutational landscape of urothelial carcinoma, including specific mutations in pathways and driver genes, such as FGFR3, ERBB2, PIK3CA, TP53, and STAG2, affects tumour aggressiveness and response to therapy. The next generation of therapies for urothelial carcinoma will be based on patient-specific targetable mutations found in individual tumours. This personalized-medicine approach to urothelial carcinoma has already resulted in unique clinical trial design and has the potential to improve patient outcomes and survival.

DDB2 is a multifunctional protein that participates in both nucleotide excision repair and regulation of gene transcription. In colon cancer cells, chromatin association of XRCC5/6, in the absence of DNA damage, depends on DDB2, and the DDB2–XRCC5/6 interaction promotes the transcription of the antiangiogenic gene SEMA3A., Damaged DNA-binding protein 2 (DDB2), a nuclear protein, participates in both nucleotide excision repair and mRNA transcription. The transcriptional regulatory function of DDB2 is significant in colon cancer, as it regulates metastasis. To characterize the mechanism by which DDB2 participates in transcription, we investigated the protein partners in colon cancer cells. Here we show that DDB2 abundantly associates with XRCC5/6, not involving CUL4 and DNA-PKcs. A DNA-damaging agent that induces DNA double-stranded breaks (DSBs) does not affect the interaction between DDB2 and XRCC5. In addition, DSB-induced nuclear enrichment or chromatin association of XRCC5 does not involve DDB2, suggesting that the DDB2/XRCC5/6 complex represents a distinct pool of XRCC5/6 that is not directly involved in DNA break repair (NHEJ). In the absence of DNA damage, on the other hand, chromatin association of XRCC5 requires DDB2. We show that DDB2 recruits XRCC5 onto the promoter of SEMA3A, a DDB2-stimulated gene. Moreover, depletion of XRCC5 inhibits SEMA3A expression without affecting expression of VEGFA, a repression target of DDB2. Together our results show that DDB2 is critical for chromatin association of XRCC5/6 in the absence of DNA damage and provide evidence that XRCC5/6 are functional partners of DDB2 in its transcriptional stimulatory activity.

Background:Given the unmet clinical needs in systemic lupus erythematosus (SLE), including poor disease control and drug toxicities, new therapies are needed. In a phase 2, randomized, placebo-controlled, double-blind study (JAHH), once-daily baricitinib (bari) resulted in significant clinical improvement in patients (pts) with active SLE versus PBO. Bari inhibits JAK1 and JAK2 signalling, and in turn may affect STAT1, STAT2, STAT4 pathways. Therefore, bari has the potential to simultaneously impact several pro-inflammatory immune cytokines implicated in the pathogenesis of SLE, including IFN-α, IFN-γ, IL-6, IL-12, and IL-23.Objectives:The objectives of the current study were: 1) to examine baseline serum cytokines in the JAHH phase 2 clinical trial for correlations with clinical or immunologic assessments; 2) to determine if changes in serum cytokine levels were associated with bari treatment.Methods:Pts enrolled in the JAHH phase 2 trial received daily treatment with PBO, bari 2 mg, or bari 4 mg through week 24. Serum samples were collected at baseline (week [wk] 0), wk 12, and wk 24) from SLE pts (n=270) and 50 sex- and age-matched controls. Samples were analyzed for: IL-2, IL-3, IL-5, IL-6, IL-10, IL-17A, IL-21, IL-12/23p40, IL-12p70, GM-CSF, IFN-α and IFN-γ using ultrasensitive quantitative assays. IFN gene signature, autoantibodies, C3 and C4 were measured as previously described [1].Results:At wk 0, serum IL-17A, IL-12/23p40, IL-6, IFN-γ and IFN-α were readily detectable. IL-12/23p40 was detectable in 100% of pts vs. 100% of controls, IFN-γ in 89% of pts vs. 66% of controls, IL-6 in 53% of pts vs. 12% of controls and in IFN-α 41% of pts vs. 2% of controls; detection of serum IL-2, GM-CSF, IL-5, IL-10 and IL-17A was variable (Fig 1). At baseline (wk 0), IL-12/23p40 was positively correlated with SLEDAI and IFN gene signature and negatively correlated with serum C4. IL-6 was positively correlated with joint swelling, joint tenderness, IFN-γ and C3. Serum IFN-α was positively correlated with serum IFN-γ, anti-Sm and anti-RNP, and the IFN gene signature (Fig 2). Treatment with bari 4 mg (Fig 1B) significantly decreased serum IL12/23p40 and IL-6 cytokine levels at wk 12 (pFigure 1.* p = 0.015; ** p = 0.001; Abbreviations: LLOQ, Lower limit of quantification.Figure 2.Abbreviations: Anti-dsDNA, Anti-double stranded DNA; Anti-RNP, Anti-ribonucleoprotein; CLASI, Cutaneous lupus erythematosus disease area and severity index; SLEDAI, SLE disease activity index.Conclusion:Bari 4 mg treatment was associated with statistically significant decreases of serum IL-12/23p40 and IL-6 at week 12 which continued through week 24. Serum IFN-α or IFN-γ were not reduced with bari treatment. Thus, bari 4 mg simultaneously impacted multiple pro-inflammatory cytokines implicated in the pathogenesis of SLE.References:[1]Hoffman RW, et al.Arthritis Rheumatol.2017;69(3):643-654.Disclosure of Interests:Thomas Dörner Grant/research support from: Janssen, Novartis, Roche, UCB, Consultant of: Abbvie, Celgene, Eli Lilly, Roche, Janssen, EMD, Speakers bureau: Eli Lilly, Roche, Samsung, Janssen, Yoshiya Tanaka Grant/research support from: Asahi-kasei, Astellas, Mitsubishi-Tanabe, Chugai, Takeda, Sanofi, Bristol-Myers, UCB, Daiichi-Sankyo, Eisai, Pfizer, and Ono, Consultant of: Abbvie, Astellas, Bristol-Myers Squibb, Eli Lilly, Pfizer, Speakers bureau: Daiichi-Sankyo, Astellas, Chugai, Eli Lilly, Pfizer, AbbVie, YL Biologics, Bristol-Myers, Takeda, Mitsubishi-Tanabe, Novartis, Eisai, Janssen, Sanofi, UCB, and Teijin, Michelle A Petri Grant/research support from: GSK, Eli Lilly and Company, Consultant of: Eli Lilly and Company, Josef S. Smolen Grant/research support from: AbbVie, AstraZeneca, Celgene, Celltrion, Chugai, Eli Lilly, Gilead, ILTOO, Janssen, Novartis-Sandoz, Pfizer Inc, Samsung, Sanofi, Consultant of: AbbVie, AstraZeneca, Celgene, Celltrion, Chugai, Eli Lilly, Gilead, ILTOO, Janssen, Novartis-Sandoz, Pfizer Inc, Samsung, Sanofi, Daniel Wallace Consultant of: Amgen, Eli Lilly and Company, EMD Merck Serono, and Pfizer, Brenda Crowe Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Ernst Dow Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Richard E Higgs Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Guilherme Rocha Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Robert Benschop Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Maria Silk Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Stephanie de Bono Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Robert Hoffman Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company, Damiano Fantini Shareholder of: Eli Lilly and Company, Employee of: Eli Lilly and Company

Urothelial cancer (UC) is the fifth most common cancer in the US. While cancer cells may be recognized initially by the immune system, this response is reduced as the tumor escapes immune surveillance over time. An understanding of the immune responses early in cancer progression could identify new therapies that can suppress tumor progression. To that end, we used a murine model of bladder cancer induced by the carcinogen BBN, which replicates the clinical presentation of UC in humans. RNAseq analysis of bladder tumors from mice exposed to BBN revealed an increase in immune regulatory signatures as early as 2 – 4 weeks post-initiation of exposure. These findings were confirmed by FACS analysis of spleen and bladders of mice at 0.5, 1, 2, 3 and 5 months post-initiation of exposure to BBN. The data show a bi-phasic pattern in the immune response, where there is an early increase in the number of Treg and Teff (CD4+ and CD8+) cells within the bladder at 0.5 – 1 months post-initiation of BBN exposure. The Treg and CD8+ T cells populations appear to decrease at 2 months and then increase at 3 and 5 months (corresponding to development of detectible tumor development). To determine if early suppression of Tregs affects tumor development, we depleted Tregs in wildtype C57BL/6 mice with anti-CD25 or diphtheria toxin treatment of Foxp3-DTR mice during the first month of BBN exposure. The data show that the absence of functional Tregs during the first month results in sustained Teff activity at 4 months. The increased Teff cells were found to induce a decrease in overall bladder weight, tumor development, and consequently contained fewer infiltrating immune cells. These findings suggest that early inhibition of Tregs allows for long-lived killing of tumor cells.

Background Bladder cancer is the fifth most common cancer in the United States and smoking is the largest known risk factor. Tobacco-derived carcinogens may induce the accumulation of somatic mutations in urothelial cells, and likely promote tumorigenesis. However, it is still unknown whether smoking-induced bladder carcinogenesis results in tumors with distinctive molecular features that can be therapeutically exploited. Methods We investigated the genomic alterations of human bladder cancer and examined their association with patient smoking history. We performed bioinformatic analyses and looked at differences in gene expression, somatic mutations, and DNA mutational signatures comparing nonsmokers, reformed smokers, and current smokers. Results We detected a limited set of gene expression and gene mutation differences between smokers and nonsmokers. We also identified a specific mutational signature that is enriched in tumors from smokers. This mutational signature was described before and has been linked to specific DNA repair defects in human bladder tumors, as well as to the direct effect of nitrosamine carcinogens in the BBN murine model of bladder cancer. Conclusion We showed associations between smoking status and selected mutational signatures, which could provide insights in the biology of bladder carcinogenesis and tumor progression.

The lysine methyltransferase KMT2C (also known as MLL3), a subunit of the COMPASS complex, implements monomethylation of Lys4 on histone H3 (H3K4) at gene enhancers. KMT2C (hereafter referred to as MLL3) frequently incurs point mutations across a range of human tumor types, but precisely how these lesions alter MLL3 function and contribute to oncogenesis is unclear. Here we report a cancer mutational hotspot in MLL3 within the region encoding its plant homeodomain (PHD) repeats and demonstrate that this domain mediates association of MLL3 with the histone H2A deubiquitinase and tumor suppressor BAP1. Cancer-associated mutations in the sequence encoding the MLL3 PHD repeats disrupt the interaction between MLL3 and BAP1 and correlate with poor patient survival. Cancer cells that had PHD-associated MLL3 mutations or lacked BAP1 showed reduced recruitment of MLL3 and the H3K27 demethylase KDM6A (also known as UTX) to gene enhancers. As a result, inhibition of the H3K27 methyltransferase activity of the Polycomb repressive complex 2 (PRC2) in tumor cells harboring BAP1 or MLL3 mutations restored normal gene expression patterns and impaired cell proliferation in vivo. This study provides mechanistic insight into the oncogenic effects of PHD-associated mutations in MLL3 and suggests that restoration of a balanced state of Polycomb-COMPASS activity may have therapeutic efficacy in tumors that bear mutations in the genes encoding these epigenetic factors.

BackgroundThe APOBEC family of enzymes is responsible for a mutation signature characterized by a TCW>T/G mutation. APOBEC-mediated mutagenesis is implicated in a wide variety of tumors, including bladder cancer. In this study, we explore the APOBEC mutational signature in bladder cancer and the relationship with specific mutations, molecular subtype, gene expression, and survival. We hypothesized that tumors with high levels of APOBEC-mediated mutagenesis would be enriched for mutations in DNA damage response genes and associated with higher expression of genes related to activation of the immune system.MethodsGene expression (n=408) and mutational (n=395) data from the Cancer Genome Atlas (TCGA) bladder urothelial carcinoma provisional dataset was utilized for analysis. Tumors were split into “APOBEC-high” and “APOBEC-low” tumors based on APOBEC enrichment score. Analysis was performed with R.FindingsPatients with APOBEC-high tumors have better overall survival compared to those with APOBEC-low tumors (38.2 vs 18.5 months, p=0.005). Tumors enriched for APOBEC mutagenesis are more likely to have mutations in DNA damage response genes (TP53, ATR, BRCA2), and chromatin regulatory genes (MLL, MLL3), while APOBEC-low tumors are more likely to have mutations inFGFR3andKRAS. APOBEC3AandAPOBEC3Bexpression correlates with total mutational burden, regardless of bladder tumor molecular subtype. APOBEC mutagenesis and enrichment is associated with increased expression of immune-related genes, including interferon signaling.InterpretationTumors enriched for APOBEC mutagenesis are more likely to have mutations in DNA damage response genes and chromatin regulatory genes, potentially providing more single-strand DNA substrate forAPOBEC3AandAPOBEC3B, leading to a hypermutational phenotype and the subsequent immune response.HighlightsABPOEC enzymes, particularlyAPOBEC3AandAPOBEC3B, are responsible for the predominant pattern of mutagenesis in bladder cancerTumors enriched for APOBEC-mediated mutagenesis are more likely to have mutations in DNA damage response genes and chromatin regulatory genes, while tumors not enriched for APOBEC-mediated mutagenesis are more likely to have mutations inKRASandFGFR3APOBEC enrichment is associated with upregulation of genes involved in the immune response

Despite a high number of neoantigens and T cell infiltration, most locally advanced and metastatic bladder cancers (BC) (>70%) do not respond to anti-PD1 treatment. Thus, targeting additional checkpoints may play an important role in treatment of BC. We sought to identify negative regulators of T cell activity and evaluate their function using a previously validated pre-clinical carcinogen-induced mouse model of BC. To identify regulators of T cell activity, we first investigated the timing and infiltration of T cells during tumor development in the BBN mouse BC model. We found that numbers of CD4+ and CD8+ T cell increased dramatically within the first 1-2 weeks, but then decreased over the next six weeks decreasing to the lowest levels at 8 weeks before increasing again coordinating with increasing tumor volume. During the decrease in T cell numbers, macrophages increased and by transcriptome profiling we identified increased levels of the T cell inhibitory protein B7-H4. B7-H4+CD11b+ macrophages increased over 4 weeks with opposite expression of CD4+ and CD8+ T cells by FACS. By IHC we localized B7-H4 expression in macrophages adjacent to tumor. A bioinformatic analysis of the human TCGA identified highest B7-H4 expression in luminal infiltrated subtypes of MIBCs, but did not correlate with CD4, CD8 or overall tumor mutation burden. MIBCs with elevated B7-H4 expression had significantly worse survival. In vitro, addition of anti-B7-H4 significantly increased proliferation and IFN-γ production by human T cells co-cultured with B7-H4 expressing APCs. To determine the role of B7-H4 in BC, we treated mice with overt BC (beginning at 4 months of carcinogen administration) with vehicle, anti-PD1 or anti-B7-H4 antibody. Compared to vehicle treated animals, anti-B7-H4 treated mice had a reduction in the number of pT3 tumors (50% vs. 73%), but less than anti-PD1 treated tumors (27%). However, anti-B7-H4 treated mice had significantly increased tumor infiltrating CD8+ T cells per histological field (77.1+34.3 vs. 11.5+7.8; p=0.0121), as well as a significant increase splenic CD8+IFN-γ+ T cells and levels of secreted IFN-γ upon anti-CD3 stimulation. Transcriptome profiling of tumors that responded to anti-B7-H4 antibody by RNA-Seq identified a decrease in mitotic cell cycle, cell cycle checkpoints, RHO GTPases and mitotic spindle checkpoint pathways in responsive tumors. In summary, we confirm the expression of B7-H4 in macrophages found in a murine model of BC and correlate expression to poor survival in the TCGA of MIBC. Inhibition of B7-H4 results in lymphocyte proliferation in vitro and inhibition of B7-H4 in mice resulted in decreased tumor stage and increased CD8+ TILs. This data suggests that B7-H4 may be a candidate alternative checkpoint that can be targeted in humans with BC unresponsive to PD1 therapy. Citation Format: Joshua Meeks, Alexander P. Glaser, Damiano Fantini, Yanni Yu, Valerie Eaton, Joseph R. Podojil, Stephen D. Miller. B7H4 as a T cell inhibitory regulator in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2405.

Bladder cancer is the fourth most common cancer in men and fifth most common malignancy in the United States. Survival of patients with muscle-invasive or metastatic bladder cancer is poor, with only 35% survival at 5 years. Developing innovative anticancer drugs relies on the availability of model systems that closely recapitulate the corresponding human disease at both the histologic and molecular levels. One of the major limitations in the field of bladder cancer research is the limited availability of such models. The N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model is an attractive model system of muscle-invasive bladder cancer (MIBC) since it recapitulates the complex histology of human tumors in a background with intact immune system. However, it was unknown whether this carcinogen-induced model also mimicked human MIBC at the molecular and mutational level. In our study, we analyzed the mutational landscape of the BBN model by whole-exome sequencing followed by a bioinformatic comparison to human MIBC using genomic data from The Cancer Genome Atlas and other repositories. Similar to human MIBC, BBN tumors had a high mutation burden. Next, we extracted mouse-specific mutational signatures that correspond to different genetic instability processes operating in BBN tumors. Interestingly, one of these de novo extracted signatures aligned to a human mutational signature previously associated to DNA-repair deficiencies in human bladder cancers. We further analyzed the most commonly mutated genes and their intratumor mutation frequencies with the aim of identifying potential driver mutations. Our analyses converged on three genes that seem crucial in the process of tumorigenesis in both mouse and human bladder cancers: the tumor suppressor Trp53 as well as the genes Kmt2c and Kmt2d. These encode for methyltransferases involved in the methylation of histone H3K4 and play a key role in the epigenetic regulation of enhancer activity. Also, many of the putative driver mutations found in the BBN genomes corresponded to human cancer hotspot mutations, supporting their role as driver mutations. Together, our study revealed several similarities between human MIBC and the BBN mouse model. Our results highlighted the contribution of aberrations to chromatin regulators and genetic instability in both human and BBN bladder tumors, thus providing a strong rationale for the use of this mouse model in molecular and drug discovery studies. Citation Format: Damiano Fantini, Alexander P. Glaser, Rimar J. Kalen, Wang Yiduo, Yanni Yu, Joshua J. Meeks. A carcinogen-induced mouse model recapitulates the molecular alterations of human muscle-invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B29.

The prion protein (PrP) is highly conserved and ubiquitously expressed, suggesting that it plays an important physiological function. However, despite decades of investigation, this role remains elusive. Here, by using animal and cellular models, we unveil a key role of PrP in the DNA damage response. Exposure of neurons to a genotoxic stress activates PRNP transcription leading to an increased amount of PrP in the nucleus where it interacts with APE1, the major mammalian endonuclease essential for base excision repair, and stimulates its activity. Preventing the induction of PRNP results in accumulation of abasic sites in DNA and impairs cell survival after genotoxic treatment. Brains from Prnp(-/-) mice display a reduced APE1 activity and a defect in the repair of induced DNA damage in vivo. Thus, PrP is required to maintain genomic stability in response to genotoxic stresses.

Inhibition of DNA repair has been proposed as a mechanism underlying heat-induced sensitization of tumour cells to some anticancer treatments. Base excision repair (BER) constitutes the main pathway for the repair of DNA lesions induced by oxidizing or alkylating agents. Here, we report that mild hyperthermia, without toxic consequences per se, affects cellular DNA glycosylase activities, thus impairing BER. Exposure of cells to mild hyperthermia leads to a rapid and selective inactivation of OGG1 (8-oxoguanine DNA glycosylase) associated with the relocalisation of the protein into a detergent-resistant cellular fraction. Following its inactivation, OGG1 is ubiquitinated and directed to proteasome-mediated degradation, through a CHIP (C-terminus of HSC70-interacting protein) E3 ligase-mediated process. Moreover, the residual OGG1 accumulates in the perinuclear region leading to further depletion from the nucleus. As a consequence, HeLa cells subjected to hyperthermia and exposed to a genotoxic treatment have a reduced capacity to repair OGG1 cognate base lesions and an enhanced cell growth defect. The partial alleviation of this response by OGG1 overexpression indicates that heat-induced glycosylase inactivation contributes to the synergistic effect of hyperthermia on genotoxic treatments. Taken together, our results suggest that OGG1 inhibition contributes to heat-induced chemosensitisation of cells and could lay the basis for new anticancer therapeutic protocols that include hyperthermia.

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein-protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

The apurinic endonuclease 1/redox factor-1 (APE1) has a crucial function in DNA repair and in redox signaling in mammals, and recent studies identify it as an excellent target for sensitizing tumor cells to chemotherapy. APE1 is an essential enzyme in the base excision repair pathway of DNA lesions caused by oxidation and alkylation. As importantly, APE1 also functions as a redox agent maintaining transcription factors involved in cancer promotion and progression in an active reduced state. Very recently, a new unsuspected function of APE1 in RNA metabolism was discovered, opening new perspectives for this multifunctional protein. These observations underline the necessity to understand the molecular mechanisms responsible for fine-tuning its different biological functions. This survey intends to give an overview of the multifunctional roles of APE1 and their regulation in the context of considering this protein a promising tool for anticancer therapy.

APE1/Ref-1 (also called HAP1 or APEX, and here referred to as APE1), the mammalian ortholog of Escherichia coli Xth (exonuclease III), is a vital protein (20) that acts as a master regulator of cellular response to oxidative stress conditions and contributes to the maintenance of genome stability (55, 56). APE1 is involved in both the base excision repair (BER) pathways of DNA lesions, acting as the major apurinic/apyrimidinic (AP) endonuclease, and in transcriptional regulation of gene expression as a redox coactivator of different transcription factors, such as early growth response protein 1 (Egr-1), NF-κB, and p53 (55, 56). These two biological activities are located in two functionally distinct protein domains. In fact, the N-terminal region, containing the nuclear localization signal (NLS) sequence, is principally devoted to redox activity through Cys65, while the C-terminal one exerts enzymatic activity on the abasic sites of DNA (56, 63). The protein C terminus is highly conserved during phylogenesis, while the N terminus is not. Except in mammals, which always show a high sequence conservation (more than 90%), and Danio, Drosophila, Xenopus, and Dictyostelium (presenting a sequence identity of less than 40%), the N-terminal region is mostly absent in other organisms. A third APE1 function, which is regulated by Lys6/Lys7 acetylation (7), is indirect binding to the negative calcium response elements (nCaRE) of some promoters (i.e., PTH and APE1 promoters), thus acting as a transcriptional repressor (12, 30). In different mammalian cell types, the APE1 subcellular distribution is mainly nuclear and is critical for controlling cellular proliferative rate (20, 25, 28). However, cytoplasmic, mitochondrial, and endoplasmic reticulum localization has also been reported (11, 22, 33, 50, 54). Interestingly, cytoplasmic expression of APE1 has been correlated with aggressiveness of different tumors (55, 56), although its role in tumorigenic processes is completely unknown. To date, no subnuclear distribution of APE1 has been reported. APE1 is an abundant protein (about 104 to 105 copies/cell) within eukaryotic cells and has a relatively long half-life (about 8 h). Thus, fine-tuning of the multiple APE1 functions may reside on its posttranslational modifications and on the modulation of its interactome under different conditions. While some posttranslational modifications have a functional role (i.e., Lys6/Lys7 acetylation) (7, 17), little information is available on APE1 protein interacting partners, except for those that are involved in BER function (38). Interestingly, proteolysis at residue Lys31 has recently been related to an enhanced immune cell death mediated by granzymes A and K (16, 23). This proteolytic event reduces APE1 accumulation within nuclei (16, 29) and its interaction with XRCC1 (60) and alters APE1 functions (16, 23). Recently, proteolysis occurring at Asn33 (giving rise to a protein form called NΔ33APE1) has also been described (11), suggesting that removal of the NLS may constitute a general mechanism for redirecting APE1 toward noncanonical subcellular compartments, such as mitochondria (11, 33, 54). Unfortunately, neither has the specific protease responsible for this cleavage been identified in nonimmune cells, nor has the mitochondrial localization signal been mapped yet. Mitochondrial localization of APE1 could be associated with a potential role in mtDNA repair of oxidized bases (11, 33, 54). However, since it is not clear whether NΔ33APE1 maintains its DNA repair activity in vivo (16) or acquires an aspecific endonuclease activity for double-stranded DNA (dsDNA) (66), at present it is not possible to drive any definitive conclusion. Moreover, as the truncated NΔ33APE1 form is associated with an apoptotic phenotype (23), it cannot be excluded that its generation may causatively be involved in the cytotoxic effect, driving proapoptotic triggering directly within mitochondria. The first 42 amino acids of APE1 are highly unordered in the protein crystallographic structure (3, 35), while the remainder of the protein has a globular fold (21). It is therefore presumable that the protein's N terminus is used for interacting with other partners, thus modulating the different APE1 functions. Interestingly, a similar bipartite arrangement for Rrp1, the Drosophila homologue of mammalian APE1, has been described, pointing out a functional role of the unstructured N-terminal domain in modulating protein-protein interactions (42, 52). By using an unbiased proteomic approach, in this work we have identified and characterized a novel APE1 complex. We found that APE1 N terminus is essential for binding to a number of proteins involved in ribosome biogenesis and RNA processing. Among the interacting partners, we focused on the nucleophosmin (NPM1)-APE1 interaction. NPM1 is an abundant protein which specifically resides within the granular region of the nucleolus and has been implicated in a variety of cellular processes, including centrosome duplication, maintenance of the genome's integrity, and ribosome biogenesis (19). NPM1 has a chaperone activity regulated by phosphorylation (51) and an endoribonuclease activity at a specific site of the spacer region between the 5.8S and the 28S rRNAs (43). Here, we demonstrate that the NPM1-APE1 interaction is required for APE1 subnuclear localization and for modulating the cleansing process of rRNA. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus possibly affecting gene expression through posttranscriptional mechanisms.

APE1/Ref-1, the mammalian ortholog of E. coli Xth, and a multifunctional protein possessing both DNA repair and transcriptional regulatory activities, has dual role in controlling cellular response to oxidative stress. It is rate-limiting in repair of oxidative DNA damage including strand breaks and also has co-transcriptional activity by modulating genes expression directly regulated by Egr-1 and p53 transcription factors. PTEN, a phosphoinositide phosphatase, acts as an 'off' switch in the PI-3 kinase/Akt signalling pathway and regulates cell growth and survival. It is shown here that transient alteration in the APE1 level in HeLa cells modulates PTEN expression and that acetylatable APE1 is required for the activation of the PTEN gene. Acetylation of APE1 enhances its binding to distinct trans-acting complexes involved in activation or repression. The acetylated protein is deacetylated in vivo by histone deacetylases. It was found that exposure of HeLa cells to H(2)O(2) and to histone deacetylase inhibitors increases acetylation of APE1 and induction of PTEN. The absence of such induction in APE1-downregulated HeLa cells confirmed APE1's role in regulating inducible PTEN expression. That APE1-dependent PTEN expression is mediated by Egr-1 was supported by experiments with cells ectopically expressing Egr-1. Thus, the data open new perspectives in the comprehension of the many functions exerted by APE1 in controlling cell response to oxidative stress.