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Tumor recurrence is a leading cause of death and is thought to arise from a population of residual cells that survive treatment. These residual cancer cells can persist, locally or at distant sites, for years or decades. Therefore, understanding the pathways that regulate residual cancer cell survival may suggest opportunities for targeting these cells to prevent recurrence. Previously, it was observed that the proapoptotic protein (PAWR/Par-4) negatively regulates residual cell survival and recurrence in mice and humans. However, the mechanistic underpinnings on how Par-4 expression is regulated are unclear. Here, it is demonstrated that Par-4 is transcriptionally upregulated following treatment with multiple drugs targeting the PI3K–Akt–mTOR signaling pathway, and identify the Forkhead family of transcription factors as mediators of this upregulation. Mechanistically, Foxo3a directly binds to the Par-4 promoter and activates its transcription following inhibition of the PI3K–Akt pathway. This Foxo-dependent Par-4 upregulation limits the long-term survival of residual cells following treatment with therapeutics that target the PI3K–Akt pathway. Taken together, these results indicate that residual breast cancer tumor cell survival and recurrence requires circumventing Foxo-driven Par-4 upregulation and suggest that approaches to enforce Par-4 expression may prevent residual cell survival and recurrence. Mol Cancer Res; 16(4); 599–609. ©2018 AACR.

Figures S1-3 show regulation of Par-4 by PI3K-Akt-Foxo pathway. Table 1 shows primer sequences.

High-grade urothelial cancer contains intrinsic molecular subtypes that exhibit differences in underlying tumor biology and can be divided into luminal-like and basal-like subtypes. We describe here the first subtype-specific murine models of bladder cancer and show that Upk3a-CreERT2; Trp53L/L; PtenL/L; Rosa26LSL-Luc (UPPL, luminal-like) and BBN (basal-like) tumors are more faithful to human bladder cancer than the widely used MB49 cells. Following engraftment into immunocompetent C57BL/6 mice, BBN tumors were more responsive to PD-1 inhibition than UPPL tumors. Responding tumors within the BBN model showed differences in immune microenvironment composition, including increased ratios of CD8+:CD4+ and memory:regulatory T cells. Finally, we predicted and confirmed immunogenicity of tumor neoantigens in each model. These UPPL and BBN models will be a valuable resource for future studies examining bladder cancer biology and immunotherapy.Significance: This work establishes human-relevant mouse models of bladder cancer. Cancer Res; 78(14); 3954–68. ©2018 AACR.

Supplemental Figure Legends

Supplemental Table 1: Flow cytometry antibody information. Supplemental Table 2: List of genes (top 10% most differentially expressed genes) used to cluster mouse cell lines from Figure 3D. Supplemental Table 3: List of genes (top 10% most differentially expressed genes) used to cluster mouse cell line tumors from Figure 3E. Supplemntal Table 4. Class I predicted neoantigens. Supplemental Table 5. Class II predicted neoantigens

Supplemental Figure 1: Inactivation of Pten and Trp53 in Keratin 5 expressing cells does not result in bladder cancer. Supplemental Figure 2: Histologic features of BBN induced tumors. Supplemental Figure 3: Generation of KT normal urothelial cells and comparison to MB49 and 3T3 cells. Supplemental Figure 4: Hierarchical clustering of MB49, BBN963, and UPPL1541 cell lines and cell line derived tumors. Supplemental Figure 5: IPA Pathways enriched in BBN963 versus UPPL1541 cell line derived tumors. Supplemental Figure 6: MB49 cell line derived tumors are more mesenchymal than BBN963 and UPPL1541 cell line derived tumors. Supplemental Figure 7: Immune Gene Signatures and correlation between tumor-infiltrating lymphocyte phenotype and tumor size. Supplemental Figure 8: TCR clonotype in anti-PD-1 treated BBN963 tumors. Supplemental Figure 9: Memory T cell gene signature expression in TCGA BLCA tumors. Supplemental Figure 10: Oncoprint of whole exome sequencing of UPPL1541, BBN963, and MB49 cell line derived tumors.

Purpose: Improved risk stratification and predictive biomarkers of treatment response are needed for non–muscle-invasive bladder cancer (NMIBC). Here we assessed the clinical utility of targeted RNA and DNA molecular profiling in NMIBC. Experimental Design: Gene expression in NMIBC samples was profiled by NanoString nCounter, an RNA quantification platform, from two independent cohorts (n = 28, n = 50); targeted panel sequencing was performed in a subgroup (n = 50). Gene signatures were externally validated using two RNA sequencing datasets of NMIBC tumors (n = 438, n = 73). Established molecular subtype classifiers and novel gene expression signatures were assessed for associations with clinicopathologic characteristics, somatic tumor mutations, and treatment outcomes. Results: Molecular subtypes distinguished between low-grade Ta tumors with FGFR3 mutations and overexpression (UROMOL-class 1) and tumors with more aggressive clinicopathologic characteristics (UROMOL-classes 2 and 3), which were significantly enriched with TERT promoter mutations. However, UROMOL subclasses were not associated with recurrence after bacillus Calmette-Guérin (BCG) immunotherapy in two independent cohorts. In contrast, a novel expression signature of an inflamed tumor microenvironment (TME) was associated with improved recurrence-free survival after BCG. Expression of immune checkpoint genes (PD-L1/PD-1/CTLA-4) was associated with an inflamed TME, but not with higher recurrence rates after BCG. FGFR3 mutations and overexpression were both associated with low immune signatures. Conclusions: Assessment of the immune TME, rather than molecular subtypes, is a promising predictive biomarker of BCG response. Modulating the TME in an immunologically “cold” tumor warrants further investigation. Integrated transcriptomic and exome sequencing should improve treatment selection in NMIBC.

DNA repair pathways have been associated with variability in hepatocellular carcinoma (HCC) clinical outcomes, but the mechanism through which DNA repair varies as a function of liver regeneration and other HCC characteristics is poorly understood. We curated a panel of 199 genes representing 15 DNA repair pathways to identify DNA repair expression classes and evaluate their associations with liver features and clinicopathologic variables in The Cancer Genome Atlas (TCGA) HCC study. We identified two groups in HCC, defined by low or high expression across all DNA repair pathways. The low-repair group had lower grade and retained the expression of classical liver markers, whereas the high-repair group had more clinically aggressive features, increased p53 mutant-like gene expression, and high liver regenerative gene expression. These pronounced features overshadowed the variation in the low-repair subset, but when considered separately, the low-repair samples included three subgroups: L1, L2, and L3. L3 had high DNA repair expression with worse progression-free (HR 1.24, 95% CI 0.81–1.91) and overall (HR 1.63, 95% CI 0.98–2.71) survival. High-repair outcomes were also significantly worse compared with the L1 and L2 groups. HCCs vary in DNA repair expression, and a subset of tumors with high regeneration profoundly disrupts liver biology and poor prognosis.

Background: ARID1A mutations (ARID1Amut) occur in up to 30% of metastatic bladder cancers (mBC) and have been associated with poorer response to standard treatments and shorter overall survival. We previously identified bromodomain and extraterminal (BET) protein inhibition as a potential treatment paradigm for mBC, with particular potency of BET inhibitors (BETi) in cells lines containing ARID1A inactivating mutations (ARID1Amut). BETi prevents BET protein bromodomains from interacting with acetylated histone lysine tails and promoting transcription of multiple oncogenes. Our previous data shows that treatment with the pan-BETi OTX-015 reduces ARID1B expression, and significantly reduces both gene and protein expression of RAD51/RAD51 in ARID1Amut cells compared to ARID1A wild type (ARID1AWT), suggesting impaired DNA damage repair function. Here we evaluate the phenotypic effects of BETi on proliferation and migration in preclinical models of BC. Methods: To evaluate OTX-015-induced changes to gene expression, 5637 (ARID1AWT) and HT1197 (ARID1Amut) cells were treated with 1 μM OTX-015 for 48-h. RNA was extracted and sent to Novogene for bulk RNA-seq. Differential expression was quantified using DESeq2, and gene enrichment was analyzed using GSEA. 5637 and HT1197 cells were treated with 1 or 5 μM OTX-015 for 48-h, and colony forming potential was assessed by crystal violet staining after 10 days. Cells were treated with the same treatment schema as above, and a wound-healing assay evaluated wound closure over 48-h. Differences in the percent area of wells covered with colonies and relative wound closure were assessed by FIJI v.2.9.1. Results: BET inhibition of ARID1Amut HT1197 cells lead to a significant decrease in the pathway enrichment for G2/M checkpoint (NES=-2.78, q Conclusions: These preliminary data highlight how OTX-015 potently inhibits proliferation and migration in ARID1Amut cells. These data will be confirmed in isogenic cell lines harboring inactivating ARID1A mutations, and support future mechanistic exploration (e.g., BETi-mediated cell cycle dysregulation) in ARID1Amut bladder cancer. Citation Format: Ryan M. Kemper, Manfred Meng, Jeffrey S. Damrauer, William Y. Kim, Daniel J. Crona. OTX-015 inhibits proliferation and migration in ARID1A-mutated bladder cancer. [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 6281.

Primary liver cancer, consisting of both cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC), is the second leading cause of cancer deaths worldwide. Our goal is to genomically characterize rare HCC subclasses to provide insight into disease biology. Leveraging The Cancer Genome Atlas (TCGA) to perform a combined analysis of CCA (n = 36) and HCC (n = 275), we integrated multiple genomic platforms, to assess transcriptional profiles, mutational signatures, and copy number patterns to uncover underlying etiology and linage specific patterns. We identified two molecular classes distinct from prototypical HCC tumors. The first, CCA-Like, although histologically indistinguishable from HCC, had enrichment of CCA mutations (IDH1, BAP1), mutational signatures, and transcriptional patterns (SOX9, KRT19). CCA-Like, however, retained a copy number landscape similar to HCC, suggesting a hepatocellular linage. The second, Blast-Like, is enriched in TP53 mutations, HBV infection, exposure related mutational signatures and transcriptionally similar to hepatoblasts. Although these subclasses are molecularly distinct, they both have a worse progression-free survival compared to classical HCC tumors, yet are clinically treated the same. The identification of and characterization of CCA-Like and Blast-Like subclasses advance our knowledge of HCC as well as represents an urgent need for the identification of class specific biomarkers and targeted therapy., Jeffrey Damrauer, Markia Smith et al. used existing datasets from cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) to characterize two subsets of HCC distinct from prototypical HCC tumors, based on comprehensive analysis of molecular data. The two classes differed from HCC by their copy number, gene expression and mutational signature and exhibited worse progression free survival, highlighting the need to identify class-specific biomarkers and develop targeted therapies for these forms of cancer.

Urothelial Cancer - Genomic Analysis to Improve Patient Outcomes and Research (NCT02643043), UC-GENOME, is a genomic analysis and biospecimen repository study in 218 patients with metastatic urothelial carcinoma. Here we report on the primary outcome of the UC-GENOME—the proportion of subjects who received next generation sequencing (NGS) with treatment options—and present the initial genomic analyses and clinical correlates. 69.3% of subjects had potential treatment options, however only 5.0% received therapy based on NGS. We found an increased frequency ofTP53E285Kmutations as compared to non-metastatic cohorts and identified features associated with benefit to chemotherapy and immune checkpoint inhibition, including: Ba/Sq and Stroma-rich subtypes, APOBEC mutational signature (SBS13), and inflamed tumor immune phenotype. Finally, we derive a computational model incorporating both genomic and clinical features predictive of immune checkpoint inhibitor response. Future work will utilize the biospecimens alongside these foundational analyses toward a better understanding of urothelial carcinoma biology.

INTRODUCTION AND OBJECTIVE:Improved risk stratification and predictive biomarkers of treatment response are needed for non-muscle-invasive bladder cancer (NMIBC). Here we assessed the clinical util...

Summary Cellular and molecular aberrations contribute to the disparity of human cancer incidence and etiology between ancestry groups. Multiomics profiling in The Cancer Genome Atlas (TCGA) allows for querying of the molecular underpinnings of ancestry-specific discrepancies in human cancer. Here, we provide a protocol for integrative associative analysis of ancestry with molecular correlates, including somatic mutations, DNA methylation, mRNA transcription, miRNA transcription, and pathway activity, using TCGA data. This protocol can be generalized to analyze other cancer cohorts and human diseases. For complete details on the use and execution of this protocol, please refer to Carrot-Zhang et al. (2020)., Graphical abstract, Highlights • Protocols for ancestry associations with TCGA molecular data • Protocols for ancestry associations with oncogenic pathways • Statistical and power analysis for determining significant associations • Key considerations of potential confounding factors, Cellular and molecular aberrations contribute to the disparity of human cancer incidence and etiology between ancestry groups. Multiomics profiling in The Cancer Genome Atlas (TCGA) allows for querying of the molecular underpinnings of ancestry-specific discrepancies in human cancer. Here, we provide a protocol for integrative associative analysis of ancestry with molecular correlates, including somatic mutations, DNA methylation, mRNA transcription, miRNA transcription, and pathway activity, using TCGA data. This protocol can be generalized to analyze other cancer cohorts and human diseases.

Background The dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM. Methods We compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models. Findings B7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines. Interpretation We demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM. Fund Alex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.

Background: Bladder cancer (BC) is a common and deadly disease. Inactivating ARID1A mutations occur in up to 30% of metastatic BC tumors, making it the most commonly mutated epigenetic gene and the 4th most commonly mutated gene overall in BC. ARID1A mutations are associated with decreased response to therapy and poor prognosis; thus, there is a need to develop therapies specifically targeting ARID1A mutant tumors. Using the UNC EpiG Diamond compound library, a set of well-annotated small molecule probes targeting chromatin regulatory proteins, we determined that inhibitors of bromodomain and extraterminal (BET) proteins potently inhibit the viability of BC cells. Here, we tested the hypothesis that ARID1Amut cells are particularly sensitized to the pan-BET inhibitor OTX-015. Methods: AR1D1A-competent (ARID1AWT) 5637 and ARID1A-mutated (ARID1Amut) HT1197 cells were treated with eight ascending concentrations of OTX-015 (0.1 nM-100 µM), and cell viability was measured using CellTiter-Glo™ after 72-120-hour incubations. IC50 values were calculated using a four-parameter non-linear regression model. Gene expression of BET inhibitor target genes, MYC, ARID1B, and RAD51, were evaluated by RT-PCR after a 48-hour incubation, normalized to an SDHA housekeeper and compared to a 0.1% DMSO control. CellTiter-Glo™ and RT-PCR experiments were conducted in technical and biologic triplicates. Western blotting evaluated OTX-015 effects on c-MYC, BAF250B (encoded by ARID1B), and RAD51 protein expression after 72-hour incubation. Results: OTX-015 was 8-times more potent in ARID1Amut HT1197 cells at 120 h than in ARID1AWT 5637 cells (IC50: 0.12 μM vs. 1.0 μM). OTX-015 treatment (1 µM) significantly reduced ARID1B and RAD51 mRNA expression versus 0.1% DMSO control (83% and 86% reduction, respectively, both P Conclusions: These preliminary results support future lines of inquiry into the molecular mechanisms that underlie sensitization of ARID1Amut cells to BET protein inhibition. Citation Format: Ryan M. Kemper, Heemaja Mewada, Jeffrey S. Damrauer, Brian Hardy, Stephen F. Frye, Kenneth H. Pearce, Jesse Raab, William Y. Kim, Daniel James Crona. ARID1A mutated bladder cancer cells are sensitized to BET protein inhibition with OTX-015 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3267.

SUMMARY RTK/RAS/RAF pathway alterations (RPAs) are a hallmark of lung adenocarcinoma (LUAD). In this study, we use whole-genome sequencing (WGS) of 85 cases found to be RPA(–) by previous studies from The Cancer Genome Atlas (TCGA) to characterize the minority of LUADs lacking apparent alterations in this pathway. We show that WGS analysis uncovers RPA(+) in 28 (33%) of the 85 samples. Among the remaining 57 cases, we observe focal deletions targeting the promoter or transcription start site of STK11 (n = 7) or KEAP1 (n = 3), and promoter mutations associated with the increased expression of ILF2 (n = 6). We also identify complex structural variations associated with high-level copy number amplifications. Moreover, an enrichment of focal deletions is found in TP53 mutant cases. Our results indicate that RPA(–) cases demonstrate tumor suppressor deletions and genome instability, but lack unique or recurrent genetic lesions compensating for the lack of RPAs. Larger WGS studies of RPA(–) cases are required to understand this important LUAD subset., In Brief Carrot-Zhang et al. perform whole-genome characterization of lung adenocarcinomas (LUADs) lacking RTK/RAS/RAF pathway alterations (RPAs) and identify mutations or structural variants in both coding and non-coding spaces that define a unique entity of RPA(–) LUADs and potentially explain the underlying biology of this disease., Graphical Abstract

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease., This study shows that the cell cycle E3 ubiquitin ligase APC/C is a regulator of several chromatin regulatory proteins, including the multivalent epigenetic reader and writer UHRF1. Perturbing UHRF1 ubiquitylation and degradation alters cell cycle and DNA methylation patterning, pointing to a key role for cell cycle degradation in shaping chromatin environments.

Background Recent molecular characterization of urothelial cancer (UC) has suggested potential pathways in which to direct treatment, leading to a host of targeted therapies in development for UC. In parallel, gene expression profiling has demonstrated that high-grade UC is a heterogeneous disease. Prognostic basal-like and luminal-like subtypes have been identified and an accurate transcriptome BASE47 classifier has been developed. However, these phenotypes cannot be broadly investigated due to the lack of a clinically viable diagnostic assay. We sought to develop and evaluate a diagnostic classifier of UC subtype with the goal of accurate classification from clinically available specimens. Methods Tumor samples from 52 patients with high-grade UC were profiled for BASE47 genes concurrently by RNAseq as well as NanoString. After design and technical validation of a BASE47 NanoString probeset, results from the RNAseq and NanoString were used to translate diagnostic criteria to the Nanostring platform. Evaluation of repeatability and accuracy was performed to derive a final Nanostring based classifier. Diagnostic classification resulting from the NanoString BASE47 classifier was validated on an independent dataset (n = 30). The training and validation datasets accurately classified 87% and 93% of samples, respectively. Results Here we have derived a NanoString-platform BASE47 classifier that accurately predicts basal-like and luminal-like subtypes in high grade urothelial cancer. We have further validated our new NanoString BASE47 classifier on an independent dataset and confirmed high accuracy when compared with our original Transcriptome BASE47 classifier. Conclusions The NanoString BASE47 classifier provides a faster turnaround time, a lower cost per sample to process, and maintains the accuracy of the original subtype classifier for better clinical implementation.

We evaluated ancestry effects on mutation rates, DNA methylation, and mRNA and miRNA expression among 10,678 patients across 33 cancer types from The Cancer Genome Atlas. We demonstrated that cancer subtypes and ancestry-related technical artifacts are important confounders that have been insufficiently accounted for. Once accounted for, ancestry-associated differences spanned all molecular features and hundreds of genes. Biologically significant differences were usually tissue-specific but not specific to cancer. However, admixture and pathway analyses suggested some of these differences are causally related to cancer. Specific findings included an increased FBXW7 mutations in patients of African origin, decreased VHL and PBRM1 mutations in renal cancer patients of African origin, and decreased immune activity in bladder cancer patients of East Asian origin.

SummaryThe survival of residual tumor cells following therapy and their eventual recurrence constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during tumor relapse. We used cellular barcoding to directly monitor clonal dynamics during tumor recurrence in a genetically engineered mouse model. We found that the clonal diversity of tumors progressively decreased during tumor regression, residual disease, and recurrence. Only a fraction of subclones survived oncogene withdrawal and persisted in residual tumors. The minimal residual disease phase itself was accompanied by a continued attrition of clones, suggesting an ongoing process of selection during dormancy. The reactivation of dormant residual cells into recurrent tumors followed several distinct evolutionary routes. Approximately half of the recurrent tumors exhibited a striking clonal dominance in which one or two subclones comprised the vast majority of the tumor. The majority of these clonal recurrent tumors exhibited evidence of de novo acquisition of Met amplification, and were sensitive to small-molecule Met inhibitors. A second group of recurrent tumors exhibited marked polyclonality, with thousands of subclones and a clonal architecture very similar to primary tumors. These polyclonal recurrent tumors were not sensitive to Met inhibitors, but were instead dependent upon an autocrine IL-6 – Stat3 pathway. These results suggest that the survival and reactivation of dormant tumors can proceed via multiple independent routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substratesin silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates and we show that several chromatin proteins bind APC/C, oscillate during the cell cycle and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

High-grade urothelial cancer contains intrinsic molecular subtypes that exhibit differences in underlying tumor biology and can be divided into luminal-like and basal-like subtypes. We describe here the first subtype-specific murine models of bladder cancer and show that Upk3a-CreERT2; Trp53L/L; PtenL/L; Rosa26LSL-Luc (UPPL, luminal-like) and BBN (basal-like) tumors are more faithful to human bladder cancer than the widely used MB49 cells. Following engraftment into immunocompetent C57BL/6 mice, BBN tumors were more responsive to PD-1 inhibition than UPPL tumors. Responding tumors within the BBN model showed differences in immune microenvironment composition, including increased ratios of CD8+:CD4+ and memory:regulatory T cells. Finally, we predicted and confirmed immunogenicity of tumor neoantigens in each model. These UPPL and BBN models will be a valuable resource for future studies examining bladder cancer biology and immunotherapy. Significance: This work establishes human-relevant mouse models of bladder cancer. Cancer Res; 78(14); 3954–68. ©2018 AACR.

The UC-GENOME study was designed to capitalize on The Cancer Genome Atlas (TCGA) findings in real world patients with metastatic urothelial carcinoma (UC) with co-equal aims: 1) to provide targeted DNA sequencing for potential clinical decision making at no cost to patients; and 2) to create a clinically annotated biorepository (including tissue, plasma, and PBMCs) for collaborative research. Patients with metastatic UC (n=209, median age 68 y, 74% male) were accrued at 8 academic medical centers between 2016-2019. We report on the first analysis of the targeted DNAseq (n=191) and total RNAseq (n=176) from FFPE specimens (n=169 overlap). Recurrently mutated genes were observed in a similar frequency to TCGA, including TP53 (54%), KMT2D (30%), ARID1A (26%) and FGFR3 (18%). Somatic variant patterns were correlated with previously annotated mutational signatures (COSMICv3), revealing a subset of tumors enriched for APOBEC signatures. Molecular subtypes were defined using the Bladder Cancer Molecular Taxonomy Group's consensus subtyping schema (Ba_Sq=54 [31%], Stroma-rich=64 [36%], LumP=26 [15%], LumU=24 [14%], LumNS=4 [2%], NE=4 [2%]). To further understand the tumor immune features and whether they correlate with durable clinical response, tumor microenvironment deconvolution was performed by applying MiXCR and immune gene signatures. An inflamed phenotype and enhanced T cell receptor richness but not clonality was observed within Ba_Sq and Stroma-rich subtypes, which also had the highest disease control rate (CR/PR + SD ~75%) to immunotherapy. The Stroma-rich subtype had the highest disease control rate (~88%) to chemotherapy. Future efforts will leverage the clinical, DNA and RNAseq data along with other biobanked specimens for collaborative research initiatives. Citation Format: Jeffrey S. Damrauer, Jeff Klomp, Wolfgang Beck, Mi Zhou, Elizabeth Plimack, Matthew Galsky, Petros Grivas, Noah Hahn, Peter O'Donnell, Gopa Iyer, David I. Quinn, Katherine A. Hoadley, William Y. Kim, Matthew I. Milowsky. Urothelial cancer-GENOmic analysis to improve patient outcomes and research (UC-GENOME): a bladder cancer advocacy network (BCAN) led collaborative research study [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 2188.

Summary CRISPR-Cas9 and shRNA high-throughput sequencing screens have abundant applications for basic and translational research. Methods and tools for the analysis of these screens must properly account for sequencing error, resolve ambiguous mappings among similar sequences in the barcode library in a statistically principled manner, and be computationally efficient. Herein we present bcSeq, an open source R package that implements a fast and parallelized algorithm for mapping high-throughput sequencing reads to a barcode library while tolerating sequencing error. The algorithm uses a Trie data structure for speed and resolves ambiguous mappings by using a statistical sequencing error model based on Phred scores for each read. Availability and implementation The package source code and an accompanying tutorial are available at http://bioconductor.org/packages/bcSeq/. Supplementary information Supplementary data are available at Bioinformatics online.

The survival and recurrence of residual tumor cells following therapy constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during relapse. We use cellular barcoding to monitor clonal dynamics during tumor recurrence in vivo. We find that clonal diversity decreases during tumor regression, residual disease, and recurrence. The recurrence of dormant residual cells follows several distinct routes. Approximately half of the recurrent tumors exhibit clonal dominance with a small number of subclones comprising the vast majority of the tumor; these clonal recurrences are frequently dependent upon Met gene amplification. A second group of recurrent tumors comprises thousands of subclones, has a clonal architecture similar to primary tumors, and is dependent upon the Jak/Stat pathway. Thus the regrowth of dormant tumors proceeds via multiple routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities., The cellular composition of recurrent tumors can provide insight into resistance to therapy and inform on second line therapies. Here, using a genetically modified mouse, the authors perform barcoding experiments of the primary tumors to allow them to study the clonal dynamics of tumor recurrence.

Over half of breast-cancer-related deaths are due to recurrence 5 or more years after initial diagnosis and treatment. This latency suggests that a population of residual tumor cells can survive treatment and persist in a dormant state for many years. The role of the microenvironment in regulating the survival and proliferation of residual cells following therapy remains unexplored. Using a conditional mouse model for Her2-driven breast cancer, we identify interactions between residual tumor cells and their microenvironment as critical for promoting tumor recurrence. Her2 downregulation leads to an inflammatory program driven by TNFα/NFκB signaling, which promotes immune cell infiltration in regressing and residual tumors. The cytokine CCL5 is elevated following Her2 downregulation and remains high in residual tumors. CCL5 promotes tumor recurrence by recruiting CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors. Blocking this TNFα-CCL5-macrophage axis may be efficacious in preventing breast cancer recurrence., eLife digest Breast cancer is the second-leading cause of cancer-related deaths in women. Recurrence of breast-cancer five or more years after initial diagnosis and treatment causes more than half of these deaths. This suggests that some tumor cells survived treatment and persisted undetected. These residual tumor cells may not grow for years and are often surrounded by other cells, including immune system cells. What role these surrounding immune cells play in triggering future growth of these residual tumor cells is not clear. Many breast cancer patients receive chemotherapy, which kills all quickly dividing cells. Targeted therapies, which block signals necessary for cancer cell growth, are also used often. More recently, scientists have developed treatments that use a patients own immune system to fight off cancer. Scientists are currently studying whether combining these immunotherapies with chemotherapy or targeted therapies increases the likelihood of eliminating cancer. Learning more about the role surrounding immune cells play in allowing residual tumor cells to persist and regrow is important to understanding how to treat cancer more successfully and prevent recurrence. Now, Walens et al. show that immune cells called macrophages supply residual breast cancer cells in mice with a protein called collagen that they need to grow. In the experiments, mice with an aggressive form of breast cancer called Her2 received targeted cancer therapy. After the treatment, tumor cells in the mice released small molecules called cytokines that attract immune system cells. Levels of one cytokine called CCL5 rose after treatment and remained high in residual tumors in the mice. The experiments also revealed that CCL5 levels were high in residual breast cancer tumors collected from women. This shows that high levels of CCL5 appear to shorten the amount of time between tumor treatment and recurrence because CCL5 attracts macrophages that deposit collagen in the residual tumors. Scientists believe collagen promotes tumor growth because recurrent tumors have high levels of collagen and breast cancer patients with high levels of collagen in their tumors often have worse outcomes. Treatments that prevent or block the release of CCL5 or that stop macrophages from supplying the residual tumor cells with collagen may help prevent recurrence.

A compounding feature of greater than 50% of all cancers is the high incidence of the cachexia syndrome, a complex metabolic disorder characterized by extreme weight loss due mainly to the gross depletion of skeletal muscle tissue. Although studies into the cause of cancer cachexia has spanned over multiple decades, little is known about the effects of various cancer treatments themselves on cachexia. For example, chemotherapy agents induce side effects such as nausea and anorexia, but these symptoms do not fully account for the changes seen with cancer cachexia. In this study we examine the effects of chemotherapeutic compounds, specifically, cisplatin in the colon-26 adenocarcinoma model of cancer cachexia. We find that although cisplatin is able to reduce tumor burden as expected, muscle wasting in mice nevertheless persists. Strikingly, cisplatin alone was seen to regulate muscle atrophy, which was independent of the commonly implicated ubiquitin proteasome system. Finally, we show that cisplatin is able to induce NF-κB activity in both mouse muscles and myotube cultures, suggesting that an additional side effect of cancer treatment is the regulation of muscle wasting that may be mediated through activation of the NF-κB signaling pathway.

Tumor recurrence is a leading cause of death and is thought to arise from a population of residual cells that survive treatment. These residual cancer cells can persist, locally or at distant sites, for years or decades. Therefore, understanding the pathways that regulate residual cancer cell survival may suggest opportunities for targeting these cells to prevent recurrence. Previously, it was observed that the proapoptotic protein (PAWR/Par-4) negatively regulates residual cell survival and recurrence in mice and humans. However, the mechanistic underpinnings on how Par-4 expression is regulated are unclear. Here, it is demonstrated that Par-4 is transcriptionally upregulated following treatment with multiple drugs targeting the PI3K–Akt–mTOR signaling pathway, and identify the Forkhead family of transcription factors as mediators of this upregulation. Mechanistically, Foxo3a directly binds to the Par-4 promoter and activates its transcription following inhibition of the PI3K–Akt pathway. This Foxo-dependent Par-4 upregulation limits the long-term survival of residual cells following treatment with therapeutics that target the PI3K–Akt pathway. Taken together, these results indicate that residual breast cancer tumor cell survival and recurrence requires circumventing Foxo-driven Par-4 upregulation and suggest that approaches to enforce Par-4 expression may prevent residual cell survival and recurrence. Mol Cancer Res; 16(4); 599–609. ©2018 AACR.

SUMMARY The transcription factor FOXM1 contributes to cell cycle progression and is significantly upregulated in basal-like breast cancer (BLBC). Despite its importance in normal and cancer cell cycles, we lack a complete understanding of mechanisms that regulate FOXM1. We identified USP21 in an RNAi-based screen for deubiquitinases that control FOXM1 abundance. USP21 increases the stability of FOXM1, and USP21 binds and deubiquitinates FOXM1 in vivo and in vitro, indicating a direct enzyme-substrate relationship. Depleting USP21 downregulates the FOXM1 transcriptional network and causes a signifi-cant delay in cell cycle progression. Significantly, USP21 depletion sensitized BLBC cell lines and mouse xenograft tumors to paclitaxel, an anti-mitotic, frontline therapy in BLBC treatment. USP21 is the most frequently amplified deubiquitinase in BLBC patient tumors, and its amplification co-occurs with the upregulation of FOXM1 protein. Altogether, these data suggest a role for USP21 in the proliferation and potentially treatment of FOXM1-high, USP21-high BLBC., Graphical Abstract, In Brief The cell cycle transcription factor FOXM1 is activated in basal-like breast cancer (BLBC) and associated with therapeutic resistance and poor patient outcomes. Arceci et al. show USP21 antagonizes FOXM1 degradation, thereby promoting proliferation and paclitaxel resistance. USP21 is catalytically active and recurrently overexpressed in BLBC, representing a potential therapeutic target.

Tumor progression is the process by which cancer cells acquire increasingly aggressive characteristics, including resistance to therapies and an increased propensity to metastasize. The combination of these two processes results in recurrence of tumors in distant sites, and is chiefly responsible for cancer mortality. For instance, tumor recurrence is the leading cause of death from breast cancer. Recurrent breast cancers are thought to arise from a population of residual cells that survive treatment and persist, often in a dormant state, for years or decades before resuming proliferation. Primary breast tumors are heterogeneous, harboring different subclones of (epi)genetically distinct cells, and tumor recurrence may result from the progressive outgrowth of aggressive subclones. As a consequence, understanding the clonal dynamics of dormancy and recurrence is essential for developing strategies to prevent or treat recurrence. However, studying these processes in patients is challenging, given the difficulty in identifying residual disease and obtaining recurrent tumors. To overcome these obstacles, we have used DNA barcoding to study the clonal dynamics of breast cancer recurrence in inducible genetically engineered mouse models. These models exhibit key features of breast cancer progression as it occurs in women, including the survival of cancer cells following therapy and their eventual spontaneous recurrence. In these models doxycycline (dox) administration induces expression of an oncogene (e.g., HER2/neu, Wnt1, or Myc), leading to mammary tumor formation. Subsequent withdrawal of dox induces oncogene downregulation and complete tumor regression, mimicking targeted therapies. However, a cluster of residual cells survives oncogene downregulation and resides in a dormant, nonproliferative state in the mammary gland, and these cells eventually reinitiate proliferation to form a recurrent tumor. Using these models, we find that breast tumors can follow two distinct evolutionary routes to recurrence. Approximately half of the recurrent tumors exhibit a striking clonal dominance in which one or two subclones comprise the vast majority of the tumor. These clonal recurrent tumors exhibit evidence of de novo acquisition of Met amplification, and are sensitive to small-molecule Met inhibitors. The other half of recurrent tumors exhibit marked polyclonality, with thousands of subclones and a clonal architecture very similar to primary tumors. These polyclonal recurrent tumors are not sensitive to Met inhibitors, but have acquired dependence upon an epigenetic pathway for their growth and survival and are sensitive to drugs targeting this pathway. These results suggest that tumor recurrence can proceed via multiple independent routes, producing recurrent tumors with distinct clonal dynamics, genetic alterations, and drug sensitivities. This abstract is also being presented as Poster B45. Citation Format: Jeffrey Damrauer, James V. Alvarez. Clonal dynamics during breast cancer dormancy and recurrence [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 PR09.

Nuclear factor-κB (NF-κB) signaling is necessary for many types of muscle atrophy, yet only some of the required components have been identified. Gene transfer of a dominant negative (d.n.) IKKβ into rat soleus muscles showed complete inhibition of 7-day disuse-induced activation of a κB reporter gene, while overexpression of wild-type (w.t.) IKKβ did not. Overexpression of a d.n. IKKβ-EGFP fusion protein showed that atrophy was inhibited by 50%, indicating that IKKβ is required for the atrophy process. Overexpression of constitutively active (c.a.) IKKβ-EGFP showed a marked increase in NF-κB activity and a decrease in fiber size of weight-bearing soleus muscles, while muscles overexpressing w.t. IKKβ-HA had no effect. The same results were found for IKKα; overexpression of a d.n. form of the protein decreased unloading-induced NF-κB activation and inhibited atrophy by 50%, while overexpression of the w.t. protein had no effect. Overexpression of a c.a. IKKα–EGFP fusion protein showed that IKKα was suffic...

Ovarian clear-cell carcinoma (OCCC) is an aggressive form of ovarian cancer with high ARID1A mutation rates. Here we present a mutant mouse model of OCCC. We find that ARID1A inactivation is not sufficient for tumour formation, but requires concurrent activation of the phosphoinositide 3-kinase catalytic subunit, PIK3CA. Remarkably, the mice develop highly penetrant tumours with OCCC-like histopathology, culminating in haemorrhagic ascites and a median survival period of 7.5 weeks. Therapeutic treatment with the pan-PI3K inhibitor, BKM120, prolongs mouse survival by inhibiting the tumour cell growth. Cross-species gene expression comparisons support a role for IL-6 inflammatory cytokine signalling in OCCC pathogenesis. We further show that ARID1A and PIK3CA mutations cooperate to promote tumour growth through sustained IL-6 overproduction. Our findings establish an epistatic relationship between SWI/SNF chromatin remodelling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signalling. We propose that ARID1A protects against inflammation-driven tumorigenesis.

Introduction: High-grade, muscle-invasive bladder cancer has recently been shown to harbor intrinsic molecular subtypes with distinct biologic features. Current murine models of bladder cancer, including the prominent carcinogen induced model MB49, do not account for subtype specific characteristics, leaving a gap in available tools for understanding subtype specific differences in bladder cancer. We have developed and validated immunocompetent, subtype specific models of bladder cancer, and we have used these models to assess differential responses to immune checkpoint inhibition. Methods: Two distinct models of murine bladder cancer were developed in a C57BL/6 background. The UPPL models were generated through Pten/Trp53 conditional knockout in Uroplakin3a expressing cells. BBN models were generated through exposure of wild-type C57BL/6 mice to the carcinogen N-Butyl-N-(4-hydmoxybutyl)nitrosamine and subsequent generation of cell lines from spontaneous tumors. RNAseq was performed on several BBN and UPPL tumors and cell lines, with findings validated with flow cytometry and T/B cell receptor (TCR/BCR) amplicon sequencing of tumor infiltrating lymphocytes (TILs). Results: BBN and UPPL models reflected characteristics of human basal and luminal bladder cancers, respectively. BBN (basal) models demonstrated higher immune gene signature expression, with concordantly higher numbers of TILs compared to the UPPL (luminal) model (p < 0.0001). Two BBN and two UPPL models were assessed for response to anti-PD-1 therapy in vivo as syngeneic tumors grown in wild type C57BL/6 mice. One of the BBN lines (BBN963) demonstrated robust control of tumor growth in some animals, including multiple complete responses (p = 0.0003), but also tumors that progressed, leading us to characterize BBN963 as a mixed response model. The marked response to PD-1 blockade in BBN963 was associated with significantly higher sharing of TCR CDR3 sequences among TILs compared to sequences of the other tumors (p = 0.003). In addition, analysis of BBN963 tumors by flow cytometry demonstrated naïve and memory T cell phenotypes correlated with increased and decreased tumor sizes, respectively. Closer examination of individual BBN963 tumor responses to PD-1 blockade revealed distinct responder and non-responder infiltrating immune cell phenotypes. Responders demonstrated a less diverse B cell repertoire (p = 0.0043) with increased BCR CDR3 sequence sharing (p < 0.0001). Discussion: We have developed two unique classes of murine bladder cancer lines, UPPL and BBN, with gene expression and TIL profiles that closely correlate with human luminal and basal bladder cancers, respectively. The BBN and UPPL subtype specific models can serve as a tool for elucidating bladder cancer responses to immunotherapy. The mixed response of BBN963 tumors to PD-1 blockade should be an asset for assessing pathways mediating response to checkpoint blockade as well as the value of combination therapy. [C.S., R.S, B.V, W.K contributed equally to this work] Citation Format: Christof C. Smith, Ryoichi Saito, Lisa M. Bixby, Takanobu Utsumi, Jordan Kardos, Shengjie Chai, Sara E. Wobker, Bhavani Krishnan, Jeffrey S. Damrauer, Jonathan S. Serody, David Darr, Benjamin G. Vincent, William Y. Kim. Development of subtype specific mouse models of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1654. doi:10.1158/1538-7445.AM2017-1654

Rapamycin derivatives allosterically targeting mTOR are currently FDA approved to treat advanced renal cell carcinoma (RCC), and catalytic inhibitors of mTOR/PI3K are now in clinical trials for treating various solid tumors. We sought to investigate the relative efficacy of allosteric versus catalytic mTOR inhibition, evaluate the crosstalk between the mTOR and MEK/ERK pathways, as well as the therapeutic potential of dual mTOR and MEK inhibition in RCC. Pharmacologic (rapamycin and BEZ235) and genetic manipulation of the mTOR pathway were evaluated by in vitro assays as monotherapy as well as in combination with MEK inhibition (GSK1120212). Catalytic mTOR inhibition with BEZ235 decreased proliferation and increased apoptosis better than allosteric mTOR inhibition with rapamycin. While mTOR inhibition upregulated MEK/ERK signaling, concurrent inhibition of both pathways had enhanced therapeutic efficacy. Finally, primary RCC tumors could be classified into subgroups [(I) MEK activated, (II) Dual MEK and mTOR activated, (III) Not activated, and (IV) mTOR activated] based on their relative activation of the PI3K/mTOR and MEK pathways. Patients with mTOR only activated tumors had the worst prognosis. In summary, dual targeting of the mTOR and MEK pathways in RCC can enhance therapeutic efficacy and primary RCC can be subclassified based on their relative levels of mTOR and MEK activation with potential therapeutic implications.

We sought to define whether there are intrinsic molecular subtypes of high-grade bladder cancer. Consensus clustering performed on gene expression data from a meta-dataset of high-grade, muscle-invasive bladder tumors identified two intrinsic, molecular subsets of high-grade bladder cancer, termed “luminal” and “basal-like,” which have characteristics of different stages of urothelial differentiation, reflect the luminal and basal-like molecular subtypes of breast cancer, and have clinically meaningful differences in outcome. A gene set predictor, bladder cancer analysis of subtypes by gene expression (BASE47) was defined by prediction analysis of microarrays (PAM) and accurately classifies the subtypes. Our data demonstrate that there are at least two molecularly and clinically distinct subtypes of high-grade bladder cancer and validate the BASE47 as a subtype predictor. Future studies exploring the predictive value of the BASE47 subtypes for standard of care bladder cancer therapies, as well as novel subtype-specific therapies, will be of interest.

Urothelial carcinoma of the bladder is a common malignancy that causes approximately 150,000 deaths per year worldwide. So far, no molecularly targeted agents have been approved for treatment of the disease. As part of The Cancer Genome Atlas project, we report here an integrated analysis of 131 urothelial carcinomas to provide a comprehensive landscape of molecular alterations. There were statistically significant recurrent mutations in 32 genes, including multiple genes involved in cell-cycle regulation, chromatin regulation, and kinase signalling pathways, as well as 9 genes not previously reported as significantly mutated in any cancer. RNA sequencing revealed four expression subtypes, two of which (papillary-like and basal/squamous-like) were also evident in microRNA sequencing and protein data. Whole-genome and RNA sequencing identified recurrent in-frame activating FGFR3-TACC3 fusions and expression or integration of several viruses (including HPV16) that are associated with gene inactivation. Our analyses identified potential therapeutic targets in 69% of the tumours, including 42% with targets in the phosphatidylinositol-3-OH kinase/AKT/mTOR pathway and 45% with targets (including ERBB2) in the RTK/MAPK pathway. Chromatin regulatory genes were more frequently mutated in urothelial carcinoma than in any other common cancer studied so far, indicating the future possibility of targeted therapy for chromatin abnormalities.