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Supplementary Figure from Drug-Induced Epigenomic Plasticity Reprograms Circadian Rhythm Regulation to Drive Prostate Cancer toward Androgen Independence

In prostate cancer, androgen receptor (AR)–targeting agents are very effective in various disease stages. However, therapy resistance inevitably occurs, and little is known about how tumor cells adapt to bypass AR suppression. Here, we performed integrative multiomics analyses on tissues isolated before and after 3 months of AR-targeting enzalutamide monotherapy from patients with high-risk prostate cancer enrolled in a neoadjuvant clinical trial. Transcriptomic analyses demonstrated that AR inhibition drove tumors toward a neuroendocrine-like disease state. Additionally, epigenomic profiling revealed massive enzalutamide-induced reprogramming of pioneer factor FOXA1 from inactive chromatin sites toward active cis-regulatory elements that dictate prosurvival signals. Notably, treatment-induced FOXA1 sites were enriched for the circadian clock component ARNTL. Posttreatment ARNTL levels were associated with patients’ clinical outcomes, and ARNTL knockout strongly decreased prostate cancer cell growth. Our data highlight a remarkable cistromic plasticity of FOXA1 following AR-targeted therapy and revealed an acquired dependency on the circadian regulator ARNTL, a novel candidate therapeutic target.Significance:Understanding how prostate cancers adapt to AR-targeted interventions is critical for identifying novel drug targets to improve the clinical management of treatment-resistant disease. Our study revealed an enzalutamide-induced epigenomic plasticity toward prosurvival signaling and uncovered the circadian regulator ARNTL as an acquired vulnerability after AR inhibition, presenting a novel lead for therapeutic development.See related commentary by Zhang et al., p. 2017.This article is highlighted in the In This Issue feature, p. 2007

Supplementary Data from Drug-Induced Epigenomic Plasticity Reprograms Circadian Rhythm Regulation to Drive Prostate Cancer toward Androgen Independence

Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice we determined that CAFs in both invasive lobular breast cancer and triple negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo tracing and in vitro studies revealed the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. In vitro functional assays showed that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data show that CD26+ and CD26- NFs transform into distinct CAF subpopulations in breast cancer.

Significance Immunoglobulin heavy chain checkpoint (IgHCC) is a critical step during early B cell development. The role of immunoglobulin heavy chain (IgHC) at this step is well established. However, with the expanding knowledge of RNA in regulating central biological processes, there could be a noncoding contribution of IgHC mRNA (IgHR) in controlling the IgHCC. Here, we generated a novel mouse model that enabled us to determine a potential role of IgHR in the IgHCC, independent of IgHC signaling. Our data indicate that IgHR has no role in IgHCC and the latter is predominantly controlled by IgHC, as proposed earlier. Furthermore, this study highlights the sensitivity of progenitor B cells to low amounts of IgHC., Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.

Prostate cancer development and progression is largely dependent on androgen receptor (AR) signaling. AR is a hormone-dependent transcription factor, which binds to thousands of sites throughout the human genome to regulate expression of directly responsive genes, including pro-survival genes that enable tumor cells to cope with increased cellular stress. ERN1 and XBP1 – two key players of the unfolded protein response (UPR) – are among such stress-associated genes. Here, we show that XBP1 levels in primary prostate cancer are associated with biochemical recurrence in five independent cohorts. Patients who received AR-targeted therapies had significantly lower XBP1 expression, whereas expression of the active form of XBP1 (XBP1s) was elevated. In vitro results show that AR-induced ERN1 expression led to increased XBP1s mRNA and protein levels. Furthermore, ChIP-seq analysis revealed that XBP1s binds enhancers upon stress stimuli regulating genes involved in UPR processes, eIF2 signaling and protein ubiquitination. We further demonstrate genomic overlap of AR- and XBP1s-binding sites, suggesting genomic conversion of the two signaling cascades. Transcriptomic effects of XBP1 were further studied by knockdown experiments, which lead to decreased expression of androgen-responsive genes and UPR genes. These results suggest a two-step mechanism of gene regulation, which involves androgen-induced expression of ERN1, thereby enhancing XBP1 splicing and transcriptional activity. This signaling cascade may prepare the cells for the increased protein folding, mRNA decay and translation that accompanies AR-regulated tumor cell proliferation.

Cancer-associated systemic inflammation is strongly linked to poor disease outcome in patients with cancer1,2. For most human epithelial tumour types, high systemic neutrophil-to-lymphocyte ratios are associated with poor overall survival3, and experimental studies have demonstrated a causal relationship between neutrophils and metastasis4,5. However, the cancer-cell-intrinsic mechanisms that dictate the substantial heterogeneity in systemic neutrophilic inflammation between tumour-bearing hosts are largely unresolved. Here, using a panel of 16 distinct genetically engineered mouse models for breast cancer, we uncover a role for cancer-cell-intrinsic p53 as a key regulator of pro-metastatic neutrophils. Mechanistically, loss of p53 in cancer cells induced the secretion of WNT ligands that stimulate tumour-associated macrophages to produce IL-1β, thus driving systemic inflammation. Pharmacological and genetic blockade of WNT secretion in p53-null cancer cells reverses macrophage production of IL-1β and subsequent neutrophilic inflammation, resulting in reduced metastasis formation. Collectively, we demonstrate a mechanistic link between the loss of p53 in cancer cells, secretion of WNT ligands and systemic neutrophilia that potentiates metastatic progression. These insights illustrate the importance of the genetic makeup of breast tumours in dictating pro-metastatic systemic inflammation, and set the stage for personalized immune intervention strategies for patients with cancer.

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B‐cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B‐cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro‐proliferative, pro‐GC program. In addition, DOT1L indirectly supports the repression of an anti‐proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B‐cell naivety and GC B‐cell differentiation., The histone H3K79 methyltransferase DOT1L plays a central role in B cell development and differentiation. DOT1L maintains B cells naivety by orchestrating critical transcriptional and epigenetic regulators.

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B cell fate remain unclear. Here we identified a central role for the histone H3K79 methyltransferase DOT1L in controlling B cell differentiation. Murine B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells showed aberrant differentiation and prematurely acquired plasma cell features. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro-proliferative, pro-GC program. In addition, DOT1L supports the repression of an anti-proliferative, plasma cell differentiation program by maintaining expression of the H3K27 methyltransferase Ezh2, the catalytic component of Polycomb Repressor Complex 2 (PRC2). Our findings show that DOT1L is a central modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B cell naivety and GC B cell differentiation.

Although hair bulge stem cells are not the cancer cells-of-origin, they contribute to two-stage DMBA/TPA skin carcinogenesis in an α3β1-dependent manner., Epidermal-specific deletion of integrin α3β1 almost completely prevents the formation of papillomas during 7,12-Dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis. This dramatic decrease in tumorigenesis was thought to be due to an egress and premature differentiation of α3β1-depleted hair bulge (HB) stem cells (SCs), previously considered to be the cancer cells-of-origin in the DMBA/TPA model. Using a reporter mouse line with inducible deletion of α3β1 in HBs, we show that HB SCs remain confined to their niche regardless of the presence of α3β1 and are largely absent from skin tumors. However, tumor formation was significantly decreased in mice deficient for α3β1 in HB SCs. RNA sequencing of HB SCs isolated from short-term DMBA/TPA–treated skin showed α3β1-dependent expression of the matricellular protein connective tissue growth factor (CCN2), which was confirmed in vitro, where CCN2 promoted colony formation and 3D growth of transformed keratinocytes. Together, these findings show that HBs contribute to skin tumorigenesis in an α3β1-dependent manner and suggest a role of HB SCs in creating a permissive environment for tumor growth through the modulation of CCN2 secretion.

Background Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analyses of their RNA and DNA profiles are challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin result in false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. However, currently available algorithms are limited and improvements in accuracy and ease of use are necessary. Results We developed the R-package XenofilteR, which separates mouse from human sequence reads based on the edit-distance between a sequence read and reference genome. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human DNA sequence data. These analyses revealed that XenofilteR removes > 99.9% of sequence reads of mouse origin while retaining human sequences. This allowed for mutation analysis of xenograft samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic tumor mutations. Conclusions XenofilteR accurately dissects RNA and DNA sequences from mouse and human origin, thereby outperforming currently available tools. XenofilteR is open source and available at https://github.com/PeeperLab/XenofilteR. Electronic supplementary material The online version of this article (10.1186/s12859-018-2353-5) contains supplementary material, which is available to authorized users.

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor suppressor, may also be exploited therapeutically., Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is driven by SMARCA4 loss. Here the authors demonstrate that SCCOHT cells are highly sensitive to CDK4/6 inhibition and provide mechanistic insights, whereby this druggable vulnerability is driven by cyclin D1 deficiency induced by SMARCA4 loss.

Monocytes/macrophages (MΦs), osteoclasts (OCs), and dendritic cells (DCs) are closely related cell types of high clinical significance, but the exact steps in their lineage commitment are unclear. In studies on MΦ and DC development, OC development is generally not addressed. Furthermore, findings on DC development are confusing, because monocytes can also differentiate into DC-like cells. To resolve these issues, we have examined the development of monocytes/MΦs, OCs, and DCs from common progenitors, using the homeostatic driver cytokines macrophage colony-stimulating factor, RANK ligand (L), and Flt3L. In mouse bone marrow, B220-CD11blow/-c-Kit+c-Fms+ cells could be dissected into a CD27+Flt3+ population that proved oligopotent for MΦ/OC/DC development (MODP) and a CD27low/-Flt3- population that proved bipotent for MΦ/OC development (MOP). Developmental potential and relationship of MODP and downstream MOP populations are demonstrated by differentiation cultures, functional analysis of MΦ/OC/DC offspring, and genome-wide messenger RNA expression analysis. A common DC progenitor (CDP) has been described as committed to plasmacytoid and conventional DC development. However, the human CDP proved identical to the MODP population, whereas the mouse CDP largely overlapped with the MODP population and was accordingly oligopotent for MΦ, OC, and DC development. The CX3CR1+ MΦ/DC progenitor (MDP) population described in the mouse generated MΦs and OCs but not DCs. Thus, monocytes/MΦs, OCs, and DCs share a common progenitor that gives rise to a bipotent MΦ/OC progenitor, but a dedicated DC progenitor is currently undefined. The definition of these progenitor populations may serve diagnostics and interventions in diseases with pathogenic activity of MΦs, OCs, or DCs.

Arp2/3 complex assembles branched actin filaments key to many cellular processes, but its organismal roles remain poorly understood. Here we employed conditional arpc4 knockout mice to study the function of the Arp2/3 complex in the epidermis. We found that depletion of the Arp2/3 complex by knockout of arpc4 results in skin abnormalities at birth that evolve into a severe psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2. Knockout of arpc4 in cultured keratinocytes was sufficient to induce nuclear accumulation of Nrf2, upregulation of Nrf2-target genes and decreased filamentous actin levels. Furthermore, pharmacological inhibition of the Arp2/3 complex unmasked the role of branched actin filaments in Nrf2 regulation. Consistently, we unveiled that Nrf2 associates with the actin cytoskeleton in cells and binds to filamentous actin in vitro. Finally, we discovered that Arpc4 is downregulated in both human and mouse psoriatic epidermis. Thus, the Arp2/3 complex affects keratinocytes’ shape and transcriptome through an actin-based cell-autonomous mechanism that influences epidermal morphogenesis and homeostasis.

Retinoids play key roles in development, differentiation, and homeostasis through regulation of specific target genes by the retinoic acid receptor/retinoid X receptor (RAR/RXR) nuclear receptor complex. Corepressors and coactivators contribute to its transcriptional control by creating the appropriate chromatin environment, but the precise composition of these nuclear receptor complexes remains to be elucidated. Using an RNA interference-based genetic screen in mouse F9 cells, we identified the transcriptional corepressor CTBP2 (C-terminal binding protein 2) as a coactivator critically required for retinoic acid (RA)-induced transcription. CTBP2 suppression by RNA interference confers resistance to RA-induced differentiation in diverse murine and human cells. Mechanistically, we find that CTBP2 associates with RAR/RXR at RA target gene promoters and is essential for their transactivation in response to RA. We show that CTBP2 is indispensable to create a chromatin environment conducive for RAR/RXR-mediated transcription by recruiting the histone acetyltransferase p300. Our data reveal an unexpected function of the corepressor CTBP2 as a coactivator for RAR/RXR in RA signaling.

Oncogenic fusion genes that involve kinases have proven to be effective targets for therapy in a wide range of cancers. Unfortunately, the diagnostic approaches required to identify these events are struggling to keep pace with the diverse array of genetic alterations that occur in cancer. Diagnostic screening in solid tumours is particularly challenging, as many fusion genes occur with a low frequency. To overcome these limitations, we developed a capture enrichment strategy to enable high-throughput transcript sequencing of the human kinome. This approach provides a global overview of kinase fusion events, irrespective of the identity of the fusion partner. To demonstrate the utility of this system, we profiled 100 non-small cell lung cancers and identified numerous genetic alterations impacting fibroblast growth factor receptor 3 (FGFR3) in lung squamous cell carcinoma and a novel ALK fusion partner in lung adenocarcinoma. (c) 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Chromosomal translocations are a hallmark of cancer. Unraveling the molecular mechanism of these rare genetic events requires a clear distinction between correlative and causative risk-determinants, where technical and analytical issues can be excluded. To meet this goal, we performed in-depth analyses of publicly available genome-wide datasets. In contrast to several recent reports, we demonstrate that chromosomal translocation risk is causally unrelated to promoter stalling (Spt5), transcriptional activity, or off-targeting activity of the activation-induced cytidine deaminase. Rather, an open chromatin configuration, which is not promoter-specific, explained the elevated translocation risk of promoter regions. Furthermore, the fact that gene size directly correlates with the translocation risk in mice and human cancers further demonstrated the general irrelevance of promoter-specific activities. Interestingly, a subset of translocations observed in cancer patients likely initiates from double-strand breaks induced by an access-independent process. Together, these unexpected and novel insights are fundamental in understanding the origin of chromosome translocations and, consequently, cancer.

Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analysis of their RNA and DNA profiles is challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin can result both in the generation of false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. Therefore, we developed the open-source R-package XenofilteR, which separates mouse from human sequence reads based on the number of discordant base pairs between each read and the reference genomes. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human whole genome and whole exome DNA sequence data. This analysis revealed that XenofilteR removes >99.9% of sequence reads of mouse origin while retaining sequence reads of human origin. The filtering allowed for mutation analysis of PDX samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic mutations present in the original tumor. These findings were further validated in breast cancer and melanoma PDX samples, confirming the retrieval of accurate variant allele frequencies and somatic mutations. In conclusion, XenofilteR accurately dissects sequence reads from mouse and human origin in PDX sequence data, thereby outperforming currently available tools. Citation Format: Oscar Krijgsman, Roelof JC Kluin, Kristel Kemper, Thomas Kuilman, Julian R. de Ruiter, Vivek Iyer, Josep V. Forment, Paulien Cornelissen-Steijger, Iris de Rink, Petra ter Brugge, Ji-Ying Song, Sjoerd Klarenbeek, Ultan McDermott, Jos Jonkers, Arno Velds, David J. Adams, Daniel S. Peeper. XenofilteR: Computational dissection of mouse and human reads in PDX and xenograft sequence data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1041.

Introduction: Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Recently, MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, was successfully translated to FFPE tissue showing to be substantially equivalent to fresh tissue. In recent years, RNA-sequencing (RNA-Seq) became the standard method for transcriptome analysis, because of its low background signal and its ability of quantifying a large dynamic range of expression levels. Here we report a preliminary analysis of the FFPE MammaPrint 70-gene profile using RNA-Seq technology and the comparison with the MammaPrint® microarray diagnostic test in a series of FFPE samples. Methods: RNA-Seq was carried out using a strand-specific RNA library preparation followed by target enrichment of the coding region of the human transcriptome without relying on the presence of poly-A tail. RNA sequencing libraries were prepared starting from a minimal amount of 20 ng of total RNA based on the DV200 metric assessment. The library pools were single-end sequenced on the Illumina HiSeq 2500 instrument at the length of 65bp. The resulting sequences were mapped to the human reference genome (build 38) using TopHat v2.1. Tophat was guided by using a transcriptome index from Ensembl (version 77). The HTSeq-count tool was used to generate the total number of uniquely mapped reads for each gene. Gene expressions were normalized with Count Per Million (CPM) normalization and log2 transformed afterwards. Microarray data of the sample were available for analysis comparison. Results: On average, we obtained 22 million reads assigned to gene per sample (min=15M, max=28M). The number of reads assigned to genes vary from 61% to 70% of the total number of reads. Between 80% and 90% of the reads assigned to genes mapped to protein coding genes which is comparable to fresh frozen material. The 70-gene signature was successfully mapped to the RNA-Seq genes. A median raw read-count of 384 was observed for the 70-gene profile among the samples. Importantly, we observed a high concordance (R2 Pearson correlation=0.97) between the MammaPrint index calculated using the RNA-Seq data and the correspondent Microarray MammaPrint index. Additionally, the BluePrint profile, a microarray diagnostic test for breast cancer molecular subtyping, was successfully translated to the RNA-Seq platform. As with the MammaPrint profile, BluePrint showed high concordance between the two technologies with high correlation values for each of the subtypes (Luminal R2 Pearson correlation=0.98, Basal R2 Pearson correlation=0.97, HER2 R2 Pearson correlation=0.77). Conclusions: Next Generation RNA-sequencing is a feasible technology to assess diagnostic signatures, such as the 70 gene MammaPrint and BluePrint profiles. Citation Format: Lorenza Mittempergher, Jacob B. Spangler, Mireille H. Snel, Leonie J. Delahaye, Iris de Rink, Sun Tian, Annuska M. Glas, Rene Bernards. Assessment of the MammaPrint 70-gene profile using RNA sequencing technology [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 5409. doi:10.1158/1538-7445.AM2017-5409

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway(1). In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers(2,3). Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration(4). Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases(5). In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent endresection of DSBs in BRCA1 deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance(6). Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Urothelial carcinoma (UC) of the bladder is one of the most common cancers worldwide. The treatment of patients with metastatic or locally advanced UC consists of platinum-based chemotherapy. Unfortunately, a large number of patients will ultimately develop resistance. Therapeutic options at that point are very limited: the last FDA drug approval for the treatment of bladder cancer dates back more than 2 decades. Identification of activated signaling pathways in UC can provide new targets for treatment. Recent DNA and RNA sequencing projects in invasive UC have revealed somatic mutations in several cancer genes. Some of these mutated genes, such as FGFR3 and PIK3CA, could potentially guide therapy. However, our knowledge of gene rearrangements in bladder cancer remains limited. These fusion genes often involve multiple fusion partners, as was reported for FGFR3 fusions in bladder cancer, which represents a significant challenge for discovery and for subsequent diagnostic screening. Therefore, a global detection method is needed to fully understand the diversity of alterations driving this disease. We used a high-throughput platform to systematically profile kinase fusions through specific enrichment of kinase transcripts. Using this approach, we screened 80 muscle invasive UC specimens and identified a number of activating mutations and novel fusion transcripts. The fusion genes identified in this discovery set will be validated in a second cohort of UC specimens to determine their frequency. Functional validation will be presented for some of these fusion genes. These genetic alterations may provide new avenues for individualized molecular treatment of UC patients. Citation Format: Floris Groenendijk, Iris de Rink, Laura Mertens, Annegien Broeks, Yann Neuzillet, Jeroen de Jong, Bas van Rhijn, Rene Bernards, Michiel van der Heijden. Identification of kinase fusion genes in bladder cancer through kinome-centered RNA sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2767. doi:10.1158/1538-7445.AM2014-2767

Despite high remission rates after chemotherapy, only 30-40% of acute myeloid leukemia (AML) patients survive five years after diagnosis. The main cause for this treatment failure is insufficient eradication of a subpopulation of chemotherapy resistant leukemic cells with a stem cell-like character. These so-called leukemic stem cells (LSC) are thought to be responsible for relapse. We hypothesized that success of novel anti-AML therapies relies on functional manipulation of genes including microRNAs, resulting in elimination of leukemic (stem) cells while sparing residual co-existing normal hematopoietic stem cells (HSC). We aimed at identification of microRNAs differentially expressed between HSC, LSC and the AML bulk obtained from the same AML bone marrow, taking into account the effects of the leukemic microenvironment. To that end, we described immunophenotypic markers that can distinguish LSC from HSC. Moreover, we identified that HSC have higher aldehyde dehydrogenase activity than LSC (Schuurhuis et al. Plos One 2013). Comparing the microRNA expression profile of LSC with that of HSC showed that microRNA-551b (miR-551b) is highly expressed in residual HSC in the AML bone marrow. To determine whether miR-551b is a HSC specific microRNA we purified stem and progenitor cell subsets from normal bone marrow and showed that miR-551b is the highest expressed in the two most primitive CD34+CD38- populations i.e. CD90+CD45RA- HSC and CD90-CD45RA- multipotent progenitors. To investigate whether the expression of miR-551b is of clinical importance in AML we determined its expression in AML bone marrow samples (n=154) and showed that high miR-551b is associated with lower complete remission (CR) rates after the first cycle of induction chemotherapy, shorter relapse free survival and a worse overall survival. In line with miR-551b being a stem cell miRNA, high expression in AML was associated with an undifferentiated morphology (FAB M0). To shed more light on the functional role of miR-551b in AML we correlated the expression of miR-551b with overall gene expression in a large panel of AML patients. Many of the genes that highly correlated with miR-551b like; MLLT3, INPP4B, HTR1F, HOPX, PROM1 and others, are also present in published HSC signatures. In conclusion, miR-551b is specifically expressed in normal stem and multipotent progenitor cells and high expression in AML is associated with poor prognosis. Currently, our research focuses on the function of miR-551b in AML. Citation Format: David C. de leeuw, Fedor Denkers, Peter Valk, Iris de Rink, Ron Kerkhoven, Gerrit Jan Schuurhuis, Gert J. Ossenkoppele, Linda Smit. MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2014-3890

3065 Background: The development of new biomarkers often requires fresh frozen (FF) samples. Recently we showed that microarray gene expression data generated from FFPE material are comparable to data extracted from the FF counterpart, including known signatures such as the 70-gene prognosis signature (Mittempergher L et al., 2011). As described by Luo et al (2010) RNA profiling using next generation sequencing (RNA-Seq) is now applicable to archival FFPE specimens. Methods: Technical performance and the comparison between the RNA-Seq 70-gene read-out and the MammaPrint test (Glas et al., 2006) is evaluated in a series of 15 patients (11/15 with matched FFPE/FF material). RNA-Seq was carried out using minor adjustments of the Illumina TruSeq RNA preparation method. RNA sequencing libraries were prepared starting from 100ng of total RNA. Next, the DSN (Duplex-Specific Nuclease) normalization process was used to remove ribosomal RNA and other abundant transcripts (Luo et al, 2010). The libraries were paired-end sequenced on the Illumina HiSeq 2000 instrument with multiplexing of 4 libraries per lane. The resulting sequences were mapped to the human reference genome (build 37) using TopHat 1.3.1(Trapnell et al., 2009). The HTSeq-count tool was used to generate the total number of uniquely mapped reads for each gene. Results: Between 14% and 45% of the total number of reads were assigned to protein-coding genes. The minimum coverage per 1000bp of CDS was 38 reads. The 70 MammaPrint genes were successfully mapped to the RNA-Seq transcripts. We calculated the Pearson correlation coefficient between the centroids of the original good prognosis template (van’t Veer et al., 2002) and the 70-gene read count determined by RNA-Seq of each sample. Predictions based on the 70-gene RNA-Seq data showed a high agreement with the actual MammaPrint test predictions (>90%), irrespective of whether the RNA-seq was performed on FF or FFPE tissue. Conclusions: New generation RNA-sequencing is a feasible technology to assess diagnostic signatures.

Many Tumor Suppressor Genes (TSGs) are silenced through synergistic layers of epigenetic regulation including abnormal DNA hypermethylation of promoter CpG islands, repressive chromatin modifications and enhanced nucleosome deposition over transcription start sites. Many of the protein complexes responsible for silencing of such TSGs remain to be identified. A subset of silenced TSGs controlling key regulatory signaling pathways in colorectal cancer cells can be partially reactivated by in cells disrupted for the DNA methyltransferase 1 and 3B (DNMT1 and 3B) or by DNMT inhibitors (DNMTi). Herein, we used proteomic and functional genomic approaches to identify additional proteins that cooperate with DNMTs in silencing these key silenced TSGs in colon cancer cells. We discovered that DNMTs and the core components of the NuRD nucleosome remodeling complex, chromo domain helicase DNA-binding protein 4 (CHD4), histone deacetylases 1 and 2 (HDAC1 and 2), occupy the promoters of several of these key hypermethylated TSGs and physically and functionally interact to maintain their silencing. Consistent with this, we find an inverse relationship between expression of HDAC1 and 2 and these TSGs in a large panel of primary colorectal tumors. We demonstrate that this DNMT-NuRD complex maintains the silencing of several negative regulators of the WNT signaling pathway. We find that depletion of CHD4 is synthetic lethal with DNMT inhibition in correlation with reactivation of TSGs, suggesting that their combined inhibition may be beneficial for the treatment of colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-386. doi:1538-7445.AM2012-LB-386

Neuroblastoma is a pediatric embryonal malignancy characterized by impaired neuronal differentiation. A better understanding of neuroblastoma differentiation is essential for developing new therapeutic approaches. GDE2 (encoded by GDPD5) is a six-transmembrane-domain glycerophosphodiesterase that promotes embryonic neurogenesis. We find that high GDPD5 expression is strongly associated with favorable outcome in neuroblastoma. GDE2 induces differentiation of neuroblastoma cells, suppresses cell motility, and opposes RhoA-driven neurite retraction. GDE2 alters the Rac-RhoA activity balance and the expression of multiple differentiation-associated genes. Mechanistically, GDE2 acts by cleaving (in cis) and releasing glycosylphosphatidylinositol-anchored glypican-6, a putative co-receptor. A single point mutation in the ectodomain abolishes GDE2 function. Our results reveal GDE2 as a cell-autonomous inducer of neuroblastoma differentiation with prognostic significance and potential therapeutic value.

E-cadherin is a key regulator of epithelial cell–cell adhesion, the loss of which accelerates tumor growth and invasion. E-cadherin is also expressed in hematopoietic cells as well as epithelia. The function of hematopoietic E-cadherin is, however, mostly elusive. In this study, we explored the validity of mouse models to functionally investigate the role of hematopoietic E-cadherin in human hematopoiesis. We generated a hematopoietic-specific E-cadherin knockout mouse model. In mice, hematopoietic E-cadherin is predominantly expressed within the basophil lineage, the expression of which is dispensable for the generation of basophils. However, neither E-cadherin mRNA nor protein were detected in human basophils. In contrast, human hematopoietic E-cadherin marks the erythroid lineage. E-cadherin expression in hematopoiesis thereby revealed striking evolutionary differences between the basophil and erythroid cell lineage in humans and mice. This is remarkable as E-cadherin expression in epithelia is highly conserved among vertebrates including humans and mice. Our study therefore revealed that the mouse does not represent a suitable model to study the function of E-cadherin in human hematopoiesis and an alternative means to study the role of E-cadherin in human erythropoiesis needs to be developed.