The Loss of ATRX Increases Susceptibility to Pancreatic Injury and Oncogenic KRAS in Female But Not Male Mice

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ORIGINAL RESEARCH

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59 60 61 62 63 64 65 Claire C. Young,1,2,3,7 Ryan J. Baker,1,7 Christopher J. Howlett,4 Todd Hryciw,5,6 66 Joshua E. Herman,6 Douglas Higgs,8 Richard Gibbons,8 Howard Crawford,9 Arthur Brown,5,6 67 68 and Christopher L. Pin1,2,3,7 69 70 1 Department of Paediatrics, 2Department of Physiology and Pharmacology, 3Department of Oncology, 4Department of 5 Pathology and Laboratory Medicine, and Department of Anatomy and Cell Biology, Schulich School of Medicine & Dentistry, 71 72 University of Western Ontario, 6Robarts Research Institute, and 7Children’s Health Research Institute, London, Ontario, Canada; 8MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom; 73 and 9Molecular & Integrative Physiology and Internal Medicine, University of Michigan, Ann Arbor, Michigan 74 75 76 77 Experimentally-induced Chronic pancreatitis 78 Female 79 Regeneration Acinar cell Absence of ATRX 80 Fibrosis Injury 81 Centroacinar cell Male Duct cell 82 Adult induction of oncogenic KRAS Fibroblast 83 Activated 84 KRASG12D Stellate cell 85 Inflammatory cell 86 No ATRX 87 Male Female 88 Graphical Abstract Young et al (2018). Induc on of chronic pancrea s leads to stellate cell ac va on, inflamma on, and de-differen a on of acinar cells that is normally resolved over the period of several days. In 89 the absence of ATRX, there is increased injury, acinar to duct cell metaplasia (ADM) and, specifically in female ) female mice lacking mice, increased fibrosis. When combined with oncogenic KRAS (KRAS 90 ATRX display significantly more ADM, fibrosis and inflamma on, and increased progression of pancrea c 91 intraepithelial neoplasia (PanIN). However, male mice lacking ATRX show reduced progression through to PanINs and less ADM. 92 93 94 METHODS: Mice allowing conditional loss of Atrx within pancre- 95 SUMMARY atic acinar cells were examined after induction of recurrent 96 cerulein-induced pancreatitis or oncogenic KRAS (KRASG12D). HisFemale mice lacking ATRX in the pancreas have increased 97 tologic, biochemical, and molecular analysis examined pancreatic sensitivity to pancreatic cancer, whereas male mice without 98 pathologies up to 2 months after induction of Atrx deletion. ATRX are protected. This study identifies such susceptibility 99 in pancreatic cancer and highlights the need for sex-specific RESULTS: Mice lacking Atrx showed more progressive damage, 100 approaches in cancer treatment. inflammation, and acinar-to-duct cell metaplasia in response to 101 injury relative to wild-type mice. In combination with KRASG12D, 102 Atrx-deficient acinar cells showed increased fibrosis, inflammation, 103 BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is progression to acinar-to-duct cell metaplasia, and pre-cancerous 104 the third leading cause of cancer death in North America, lesions relative to mice expressing only KRASG12D. This sensi- 105 accounting for >30,000 deaths annually. Although somatic tivity appears only in female mice, mimicking a significant preva106 activating mutations in KRAS appear in 97% of PDAC patients, lence of ATRX mutations in human female PDAC patients. 107 additional factors are required to initiate PDAC. Because 108 mutations in genes encoding chromatin remodelling proteins CONCLUSIONS: Our results indicate the absence of ATRX 109 have been implicated in KRAS-mediated PDAC, we investi- increases sensitivity to injury and oncogenic KRAS only in Q7110 female mice. This is an instance of a sex-specific mutation that gated whether loss of chromatin remodeler ɑ-thalassemia, mental-retardation, X-linked (ATRX) affects oncogenic KRAS’s enhances oncogenic KRAS’s ability to promote pancreatic 111 ability to promote PDAC. ATRX affects DNA replication, repair, intraepithelial lesion formation. (Cell Mol Gastroenterol Hepatol 112 113 and gene expression and is implicated in other cancers 2018;-:-–-; https://doi.org/10.1016/j.jcmgh.2018.09.004) 114 including glioblastomas and pancreatic neuroendocrine tumors. The hypothesis was that deletion of Atrx in pancreatic Keywords: Epigenetics; Pancreatic Ductal Adenocarcinoma; 115 116 acinar cells will increase susceptibility to injury and oncogenic MIST1; SOX9.

The Loss of ATRX Increases Susceptibility to Pancreatic Injury and Oncogenic KRAS in Female But Not Male Mice

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ancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in North America, with a 5-year survival rate of w9% (Pancreatic Cancer Facts, PANCAN). PDAC is characterized by increased genomic instability,1 a poor response to chemotherapeutic intervention, and often diagnosed at later stages because of a lack of clinical symptoms and poor diagnostic markers even for susceptible populations. During the last 10 years, elegant lineage tracing studies identified acinar cells as the cell of origin in many PDAC cases.2–4 In these cases, the initiating events include acinar-to-duct cell metaplasia (ADM), in which mature acinar cells transiently revert to a pancreatic progenitor-like state, increasing the potential for progression to neoplastic lesions termed pancreatic intraepithelial lesions (PanINs) and PDAC.2,5 Mutations in KRAS that lead to a constitutively active form of the protein are a hallmark of PDAC, present in 97% of cases.3 However, the constitutive activation of KRAS alone appears to be insufficient to drive PDAC progression, and additional acquired mutations and/or pancreatic injury are required.1,6,7 Recent molecular characterization of PDAC tumors has identified 4 subtypes for PDAC and mutations that define 10 pathways commonly affected in these tumors. In addition to identifying RAS and NOTCH signaling as key oncogenic pathways, somatic mutations in genes involved in chromatin remodeling and SWI/SNF function were identified.8 Studies using genetically modified mouse strains harboring null for the chromatin remodeling proteins brahma-related gene 19 or B-cell specific Moloney virus insertion site 110 increased and decreased, respectively, the ability for oncogenic KRAS to promote PDAC progression. These studies confirm the importance of maintaining chromosome organization/integrity in preventing KRASmediated oncogenic progression. Our goal was to explore the idea that other components that contribute to SWI/SNF function and genome integrity may be functionally linked to oncogenic KRAS activity in promoting PDAC. ATRX (ɑ-thalassemia, mental-retardation, X-linked) is a member of the SWI/SNF family of proteins and interacts with death associated protein 6 (DAXX) to maintain or remodel appropriate nucleosome organization within the genome.11,12 In addition to its role in chromatin remodeling, studies have identified roles for ATRX in maintaining genomic stability,13 maintaining proper DNA replication and repair,14 and affecting gene expression.15 It has been proposed that ATRX-dependent deposition of histone variant H3.3 prevents the formation of alternative DNA structures during replication, allowing for proper facilitation of the replicative process.15,16 Complete loss of ATRX function during development is lethal,17 but hypomorphic ATRX mutations are the underlying cause for ATRX syndrome, a developmental disorder in males involving significant cognitive impairment, facial abnormalities, and development of ɑ-thalassemia.18 More recently, somatic mutations in ATRX have been identified in a number of cancers including glioblastomas and pancreatic neuroendocrine tumors.19–22 To date, the role of ATRX in the adult pancreas or PDAC has not been examined. Therefore, we investigated the effect

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of ATRX deletion on pancreatic injury and oncogenic KRASmediated PDAC progression. We generated mouse lines in which exon 18 of the mouse ATRX gene could be conditionally deleted in pancreatic acinar cells on its own or in combination with activation of oncogenic KRAS. Our results showed that loss of ATRX alters the response to recurrent pancreatic injury, suggesting a role for ATRX in the repair and regeneration of acinar tissue after pancreatic insult. Furthermore, combination of Atrx deletion with oncogenic KRAS activation significantly enhanced pancreatic damage within 2 months relative to oncogenic KRAS alone. Surprisingly, this ability to sensitize the pancreatic acinar cells to the oncogenic action of mutated KRAS was observed exclusively in female mice. These results indicate that ATRX loss cooperates with activated KRAS to promote pancreatic disease in a sex-specific manner.

Results To determine whether ATRX deletion affected the phenotype of mature acinar cells, AtrxflD18 mice23 were mated to mice expressing creERT from the Mist1 locus (Mist1creERT; Figure 1A), allowing for acinar-specific deletion in the pancreas.24,25 Deletion of exon 18 results in complete loss of ATRX, likely because of mRNA instability.23 A shorter isoform of ATRX may still be expressed, but this truncated form lacks the SWI/SNF domain.26 Tamoxifen was administered to 2- to 4-month-old mice, and ATRX accumulation was assessed 7, 35, or 60 days after dosing (Figure 1B and C). Immunofluorescence analysis confirmed 98% of acinar cells were ATRX-negative at all time points, demonstrating efficient Atrx deletion and indicating mature acinar cells do not require ATRX for maintained viability (Figure 1C). Co-immunofluorescence for ATRX and insulin and identification of duct nuclei based on morphology confirmed Atrx deletion specifically in acinar cells (Figure 1D). Histologic analysis showed no obvious phenotypes regarding disorganization of acinar cells or injury/ inflammation (Figure 2A), although intralobular adipocytes were observed at a higher frequency in Mist1creERT/þ AtrxflD18 mice. Because loss of ATRX showed limited effects on overall pancreatic morphology, we focused specifically on the 60-day time point to determine whether any phenotypes occurred in response to loss of ATRX. Immunofluorescence analysis for proliferative markers Ki67 (Figure 2B) and pH3 (data not shown) or TUNEL analysis (Figure 2C) revealed increased numbers of proliferating and apoptotic cells after ATRX deletion. Because ATRX is Abbreviations used in this paper: ADM, acinar-to-duct cell metaplasia; ANOVA, analysis of variance; ATRX, ɑ-thalassemia, mentalretardation, X-linked; CIP, cerulein induced pancreatitis; CPA, carboxypeptidase; DAXX, death associated protein 6; ds, double stranded; PanIN, pancreatic intraepithelial lesion; PDAC, pancreatic ductal adenocarcinoma; WT, wild-type.

© 2018 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X https://doi.org/10.1016/j.jcmgh.2018.09.004

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involved in double stranded (ds) DNA repair,14,27 we examined gH2AX accumulation, a marker for unresolved dsDNA breaks. Mist1creERT/þAtrxflD18 pancreatic tissue showed an increase in the number of cells accumulating gH2AX (Figure 2D), suggesting loss of ATRX leads to an

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inability to resolve dsDNA breaks. These results suggest that short-term loss of ATRX in pancreatic acini has no overt consequences on pancreatic morphology but may increase susceptibility to events that require intact DNA repair pathways, such as pancreatic injury.

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To examine this possibility, Mist1creERT/þ and Mist1creERT/þ AtrxflD18 mice were subjected to recurrent pancreatic injury for 11 days and allowed to recover for 3 days. We chose a mild dosing regimen so damage in control mice would be limited. No significant differences were observed between Mist1creERT/þ and Mist1creERT/þAtrxflD18 mice based on body weight, behavior, or gross tissue morphology 3 days after cessation of cerulein (data not shown). However, histologic analysis showed marked differences between the 2 genotypes in response to cerulein treatment. As expected, cerulein-treated Mist1creERT/þ mice show intra-acinar edema but no evidence of inflammation or fibrosis (Figure 3A), likely because of the mild nature of the cerulein treatment. Conversely, cerulein-treated Mist1creERT/þAtrxflD18 mice showed increased damage (Figure 3A) and fibrosis in female Mist1creERT/þAtrxflD18 mice relative to controls (Figure 3B and C), as indicated by H&E and trichrome histology, respectively. This enhanced cellular damage was further confirmed by the strong immunofluorescence signal for F4/ 80 antigen that was indicative of extensive macrophage infiltration in Mist1creERT/þAtrxflD18 mice relative to controls (Figure 3D). Quantification of the tissue damage confirmed increased sensitivity to recurrent cerulein induced pancreatitis (CIP) and indicated that female Mist1creERT/þAtrxflD18 mice are clearly more sensitive than male mice to these effects (Figure 3C, Table 2). Whereas analysis of acinar cell death by cleaved caspase-3 showed no difference between genotypes (Figure 4A), notably enhanced cell turnover based on cleaved caspase-3 (Figure 4B) or proliferative capacity by Ki67 staining (Figure 5E) was observed in Mist1creERT/þAtrxflD18 pancreata in areas showing classic features of ADM after CIP. No such areas of ADM were observed in Mist1creERT/þ mice (Table 2). Tissue histology also revealed a notable increase in ADM in Mist1creERT/þAtrxflD18 tissue based on the appearance of tubular complexes (Figure 3B). Western blot analysis (Figure 5A) for mature acinar cell markers amylase and pro-carboxypeptidase (CPA) confirmed a different response to recurrent CIP in Mist1creERT/þAtrxflD18 mice. Ceruleintreated Mist1creERT/þ mice exhibited increased accumulation of amylase and CPA compared with saline-treated controls, which was indicative of a regenerative response to injury. However, Mist1creERT/þAtrxflD18 mice did not show this recovery from injury (Figure 5A). This difference in enzyme accumulation was not due to increased release of enzymes in response to injury because circulating levels of amylase were not significantly different between genotypes (Figure 5B). Immunohistochemical analysis confirmed

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decreased CPA accumulation in both putative ADM as well as surrounding acinar cells (Figure 5C). Surprisingly, this decrease in CPA accumulation appeared to be more dramatic in cerulein-treated female Mist1creERT/þAtrxflD18 mice. Acini (delineated by a dotted line) show limited CPA accumulation in tissue sections from female mice (Figure 5C). We next examined expression of the progenitor/duct cell marker SOX9, a transcription factor that increases during regeneration and is required for ADM.4 In adult pancreatic tissue, SOX9 is expressed only in a subset of duct and centroacinar cells,4,28 which we confirmed by immunofluorescence on sections from saline-treated and ceruleintreated Mist1creERT/þ mice (Figure 5D and E). Conversely, increased SOX9 nuclear accumulation was observed in female and male Mist1creERT/þAtrxflD18 pancreatic tissue specifically after CIP treatment (Figure 5D), accumulating in ADM and some acinar cells (Figure 5D and E). Taken together, these data suggest loss of ATRX increased the sensitivity of acinar cells to recurrent cerulein exposure. These findings suggest that Mist1creERT/þAtrxflD18 mice will have increased susceptibility to oncogenic properties of mutated KRAS, because maintenance of the acinar phenotype constrains KRAS-induced transformation.5,9,29 Therefore, we next introduced an inducible form of oncogenic KRAS (KrasLSL-G12D/þ) into the Mist1creERT/þAtrxflD18 genotype (Mist1creERT/þKrasLSL-G12D/þAtrxflD18 hereafter referred to as MKA; Figure 6A). Cre-mediated induction of KRASG12D þ/deletion of Atrx was initiated in 2- to 4-month-old congenic Mist1creERT/þ, Mist1creERT/þAtrxflD18, Mist1creERT/þKrasLSL-G12D/þ, and MKA mice (Figure 6B), and acinar cell–specific loss of ATRX was confirmed by immunohistochemistry (Figure 6C). No significant differences were observed in body weight between groups during the course of the experiment (Figure 6D), and assessment of serum amylase levels at the time of death revealed no differences between genotypes (Figure 6E). Because of the presence of oral squamous papilloma tumors in KRASG12D-expressing mice, the experiment was terminated at 60 days after tamoxifen administration (data not shown). Gross morphologic examination revealed enlarged spleens in female Mist1creERT/þKrasLSL-G12D/þ and MKA mice (Figure 7). Histologic examination revealed normal pancreatic morphology in Mist1creERT/þ and Mist1creERT/þAtrxflD18 mice (Figure 8A). Mist1creERT/þKrasLSL-G12D/þ mice also showed typical pancreatic morphology for the most part, with a few instances of ADM or PanINs (Figure 8A). This is consistent with previous reports that activation of oncogenic KRASG12D in mature acinar cells was insufficient on its own to cause

Figure 1. (See previous page). Loss of ATRX in the pancreas is specific to acinar cells. (A) Schematic of Atrx deficient model. AtrxflD18 mice carrying loxP sites that flank exon 18. CreERT is expressed from the Mist1 promoter. On tamoxifen administration to Mist1creERT/þAtrxflD18 mice, cre recombinase becomes localized to the nucleus and produces deletion of exon 18 of the Atrx gene. This leads to degradation of full-length Atrx mRNA. (B) Immunofluorescence for ATRX in pancreatic tissue from Mist1creERT/þ (WT) mice 7, 35, or 60 days after tamoxifen gavage in Mist1creERT/þAtrxflD18 mice (AtrxflD18). White arrows indicate residual ATRX expression. Magnification bars ¼ 25 mm. (C) Atrx deletion efficiency was quantified as the percentage of acinar cells lacking ATRX (n ¼ 3 for all groups). (D) Localization of insulin (green) and ATRX (red) by co-immunofluorescence (IF) demonstrates acinar-specific knockout of ATRX expression in Mist1creERT/þAtrxflD18 mice 20 days after tamoxifen treatment. White arrows indicate ATRX expression in pancreatic duct cells. Sections were counterstained with DAPI to reveal nuclei. Magnification bar ¼ 50 mm.

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widespread pancreatic damage.3 Conversely, MKA mice demonstrated a variable phenotype based on sex (Figure 8A). Male MKA mice (n ¼ 10) appeared phenotypically normal with negligible PanIN formation and few pockets of fibrosis relative to Mist1creERT/þKrasLSL-G12D/þ mice. Conversely, all female MKA mice (n ¼ 6) developed PanINs and fibrosis, with some mice displaying extensive inflammation and fibrosis, along with disruptions in acinar cell organization consistent with a chronic pancreatitis phenotype (Figure 8A). Trichrome staining confirmed increased fibrosis only in female MKA pancreatic tissue relative to Mist1creERT/þKrasLSL-G12D/þ mice (Figure 9A), and Alcian blue histology confirmed increases in neoplastic PanIN lesions (Figure 9B). The tissue showed significant variability in fibrosis between mice in both MKA and Mist1creERT/þKrasLSL-G12D/þ cohorts (Figure 9C), although female MKA mice in general had increased damage (MKA ¼ 15.2% ± 9.2% damaged area vs Mist1creERT/þ KrasLSL-G12D/þ ¼ 5.0% ± 1.6%), whereas male MKA mice had decreased damage (MKA ¼ 0.2% ± 0.2% vs Mist1creERT/þ KrasLSL-G12D/þ ¼ 5.8% ± 3.8%). On the basis of a 2-way ANOVA, no significant differences were observed between any group regarding damaged (ie, lesion) area. However, quantification of lesion type (Figure 9D) indicated significantly more pre-cancerous lesions develop in female MKA mice relative to all groups except Mist1creERT/þKrasLSL-G12D/þ male mice. Quantification of overall pancreatic fibrosis, inflammation, and ADM, as described earlier, confirmed increased injury in MKA females relative to Mist1creERT/þ KrasLSL-G12D/þ counterparts (Table 3). To assess PanIN formation within Mist1creERT/þKrasLSLG12D/þ and MKA pancreatic tissue, the percentage of pancreatic lobules containing at least one instance of each lesion type (ranging from ADM to PanIN3) was quantified on H&E stained sections (Figure 8B) and statistically compared by 2-way ANOVAs (Table 4). MKA female mice had significantly fewer lobules consisting only of normal acini relative to all other groups (Table 4, P < .05). Whereas both Mist1creERT/þKrasLSL-G12D/þ and MKA mice exhibited ADM, the incidence of PanIN1 in female MKA mice was 2.5-fold higher (16.21% ± 8.3% of lobules; n ¼ 10) relative to female Mist1creERT/þKrasLSL-G12D/þ mice (6.52% ± 3.5%; n ¼ 6), and female MKA mice contained significantly more lobules with PanIN2 lesions than all other genotypes and sexes (Figure 8B, Table 4; P < .01). Interestingly, histologic analysis of male MKA mice revealed no PanIN1 or PanIN2 lesions (Figure 8B, Table 4). To determine whether presumptive ADM and PanINs were arising from ATRX null acinar cells, we examined the expression of transcription factors involved in ADM. SOX9 (Figure 10A and B) and PDX1 (data not shown)

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accumulation was assessed by immunofluorescence and immunohistochemistry, respectively. As observed earlier, no SOX9þ acinar cells were observed in Mist1creERT/þ and Mist1creERT/þAtrxflD18 mice. Similarly, Mist1creERT/þ KrasLSL-G12D/þ tissue was devoid of SOX9þ acinar cells, although pockets of putative SOX9 ADM were observed (Figure 10A). Whereas male MKA mice showed few SOX9expressing cells, SOX9þ cells and ADM were readily apparent in female MKA mice (Figure 10A and B). This widespread SOX9 accumulation in both ADM and acinar cells adjacent to areas of damage suggested SOX9 expression precedes ADM, which is in support of previous studies indicating SOX9 expression is required for ADM. Similar increases for PDX1 were observed in female MKA tissue, with PDX1þ cells readily observed in ADM and PanINs (data not shown) compared with all other genotypes. In many cases, cells within ADMs and PanIN lesions also stained for Ki67 (Figure 10B), indicating an increase in proliferation in these areas. Quantification of Ki67þ acinar cells showed no significant difference between genotypes, suggesting that proliferation likely occurs after ADM (Figure 10C). This expression pattern of SOX9 suggests normal progression through ADM in MKA mice but does not indicate whether ADM arises from ATRX-positive cells. Therefore, ATRX accumulation was examined to confirm an acinar cell origin for ADM and PanINs. All presumptive ADM observed in male Mist1creERT/þKrasLSL-G12D/þ mice tissue was ATRX positive. Similarly, all ADM and PanINs in female Mist1creERT/þKrasLSL-G12D/þ tissue contained exclusively ATRX-positive cells. In Mist1creERT/þKrasLSL-G12D/þAtrxflD18/x female mice, which harbor one transcriptionally active copy of the Atrx gene, ADM and PanINs contained mixed populations of ATRXþ and ATRX– cells. Fifty-nine percent ± 17% of lesions contained at least 1 ATRXþ cell, with the other 41% ± 17% lesions containing only ATRX-negative cells (Figure 10D and E). The ATRX– lesions likely arise from cells in which the non-targeted Atrx gene has been silenced as part of X chromosome inactivation. Although some ATRXþ cells were observed in PanINs and ADMs of MKA mice, the majority of lesions were completely devoid of ATRX expression in male (82% ± 7%) and female (73% ± 10%) mice. Because ATRX is ubiquitously expressed, the absence of ATRX in PanINs and ADM suggests the origin of these lesions in MKA mice as acinar cells. Finally, to determine whether sex-specific susceptibility conferred by the absence of ATRX on KRAS mice translates to humans, we queried the International Cancer Genome Consortium (dcc.icgc.org) database for ATRX mutations (Figure 11). The International Cancer Genome Consortium database includes whole genome sequence analysis for 729 patients from Australian and Canadian tumor sequencing

Figure 2. (See previous page). Mature acinar cells do not require ATRX for maintenance, but ATRX loss induces low levels of pancreatic damage. (A) Representative H&E staining of Mist1creERT/þ (WT) and Mist1creERT/þAtrxflD18 (AtrxflD18) pancreatic tissue 60 days after last tamoxifen gavage. Intralobular adipocytes are indicated by *. (B–D) Representative images and quantification for (B) Ki67 immunofluorescence (Mist1creERT/þ [WT; n ¼ 4]; Mist1creERT/þAtrxflD18 [ATRX–; n ¼ 5]), (C) TUNEL (Mist1creERT/þ, n ¼ 9; Mist1creERT/þAtrxflD18, n ¼ 10), or (D) gH2AX IF (Mist1creERT/þ [n ¼ 7]; Mist1creERT/þAtrxflD18 [n ¼ 11]) 60 days after tamoxifen treatment. White arrows indicate positive cells. Magnification bar ¼ 50 mm. Data were assessed by using two-tailed t test with Tukey post hoc test. Error bars represent means ± standard error; *P < .005.

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884 885 886 ADM (based on worst Fibrosis (based on 887 pancreatic lobule) trichrome stain) 888 0 None present 0 None present 889 1 >10% of lobule 1 <5% of tissue area 890 2 10%–30% of lobule 2 5%–10% of tissue area 891 3 30%–50% of lobule 3 10%–20% of tissue area 892 4 >50% of lobule 4 >20% of tissue area 893 Inflammation 894 0 None present 895 1 Focal: small, contained areas 896 897 2 Mild: small, slightly diffuse areas 898 3 Moderate: diffuse areas Discussion 899 4 Severe: diffuse areas, significant presence throughout Pancreatic ductal adenocarcinoma is currently the third 900 the tissue leading cause of cancer-related deaths in North America 901 NOTE. Grading of pancreatic lesions: pancreatic lesions (www.pancan.org). Five-year survival rates have increased 902 (ranging from ADM to PanIN grade 3) were quantified and only marginally in the last 30 years because of late stage of 903 classified into the following categories: ADM, PanIN grade 1, diagnosis and insensitivity to conventional chemotheraPanIN grade 2, PanIN grade 3, or PDAC based on morpho- peutics. Therefore, detecting factors that increase sensitivity 904 logic characteristics including cell shape (cuboidal or of pancreatic tissue to the oncogenic properties of mutated 905 906 columnar), presence of mucin accumulation, nuclear atypia, KRAS is important in identifying alternative therapeutic and pseudostratification, and papillary or cribriform structure. 907 diagnostic options. In this study, we have shown that acinar908 specific loss of ATRX, a chromatin remodelling protein, 909 studies and consists of 324 female patients (42%) and 405 affects the tissue’s response to injury and constitutive 910 male patients (53%). Gender was not identified in 5% of the mutant KRAS activity. Using a novel mouse line that allows 911 patients. Therefore, the proportion of PDAC patients re- for acinar-specific ablation of Atrx, we show loss of ATRX 912 ported as female was 0.44. KRAS mutations were identified increased the sensitivity for pancreatic injury. In addition, 913 in 591 patients, with a similar ratio of male (55%) to female we showed loss of Atrx dramatically enhanced the ability of 914 (45%) patients. Two hundred sixty-eight mutations were oncogenic KRAS to promote precancerous lesions in the 915 observed within the ATRX gene, encompassing 145 (w19%) pancreas. Importantly, these effects were observed in a sex916 PDAC patients, with 68% of ATRX mutations in female specific fashion, with only female mice displaying sensitivity 917 patients. Therefore, the proportion of PDAC patients to loss of ATRX. Our results also suggest that loss of ATRX 918 carrying ATRX mutations that were female was 0.68. The may reduce the sensitivity to oncogenic KRAS in male mice. 919 2 Q12 difference in proportions is significant, c (1, N ¼ 729) ¼ This is evidence of a gender-specific susceptibility factor 920 41.633; P < .00001 (Figure 11B), suggesting ATRX muta- and suggests stratification of PDAC based on their molecular 921 tions are related to the sex of the patient. profile may identify new targets for therapy and diagnosis. 922 Most ATRX mutations in both sexes are found in nonAlthough we have not defined a role for ATRX in normal 923 coding regions, and the impact on expression is unknown. acinar cell physiology, it appears ATRX is dispensable for 924 However, 8 patients harbor ATRX mutations with a pre- maintaining the acinar cell phenotype in the adult. This is 925 dicted impact on protein function. All but one of these consistent with previous studies that identified roles for 926 mutations occur in female patients, suggesting a sex-specific ATRX only in mitotically active tissue, where loss of ATRX 927 928 Figure 3. (See previous page). Loss of ATRX sensitizes acinar tissue to recurrent pancreatic injury 3 days after 929 cessation of pancreatic injury. (A) H&E staining of saline or cerulein (CIP) treated Mist1creERT/þ (WT) and Mist1creERT/þAtrxflD18 930 (AtrxflD18) mice. CIP-treated Mist1creERT/þ mice show vacuolation and intra-acinar edema relative to saline-treated mice. CIP- 931 treated Mist1creERT/þAtrxflD18 mice demonstrated increased damage (black arrows) and putative ADM (white arrows) relative to 932 CIP-treated Mist1creERT/þ mice. Magnification bar ¼ 50 mm. (B) Trichrome stain of Mist1creERT/þ and Mist1creERT/þAtrxflD18 tissue indicating fibrosis. Mist1creERT/þAtrxflD18 mice exhibit increased fibrosis (black arrow) and to a greater extent in female 933 Mist1creERT/þAtrxflD18 mice. Magnification bar ¼ 100 mm. (C) Quantification of fibrosis in pancreatic tissue from Mist1creERT/þ 934 (WT) mice treated with saline (n ¼ 4 female or 3 male) or cerulein (n ¼ 4 female or 5 male) and Mist1creERT/þAtrxflD18 (AtrxflD18) 935 mice treated with saline (n ¼ 5 female or 3 male) or cerulein (n ¼ 5 female and male). Male mice are denoted as black symbols 936 and female mice as red symbols. Data were assessed using 2-way ANOVA with Tukey post hoc test. Error bars represent 937 means ± standard error. When not considering sex (upper graph), no significant differences exist between groups. When sex of 938 the mouse is considered (lower graph), female CIP-treated Mist1creERT/þAtrxflD18 mice are significantly different than all groups 939 (*P < .05 vs all male mouse groups; **P < .01 vs female Mist1creERT/þ and Mist1creERT/þAtrxflD18 saline-treated and female Mist1creERT/þ CIP-treated groups). Individual animals are denoted with black (male) or red (female) markers. (D) Representative 940 immunofluorescent images of F4/80 accumulation in female Mist1creERT/þ and Mist1creERT/þAtrxflD18 tissue (repeated 3 times). 941 Arrows indicate macrophages. Sections were counterstained with DAPI. Magnification bar ¼ 70 mm. 942 Table 1.Histology Grading Criteria

susceptibility in the human patient population. Pancreatic neuroendocrine and glioblastoma patient populations show 46% and 42% of the identified ATRX mutations are in female populations. Conversely, 60% of the ATRX mutations found in the pediatric brain tumor population occur in female patients. However, c2 analysis indicates the proportions of ATRX mutations are independent of sex (Figure 11C and data not shown). Interestingly, mutations in DAXX, the partner for ATRX in depositing H3.3 variant histones into chromatin,11,12 are rare in PDAC and show no sex bias. Other common mutations linked to PDAC, including P16/CDNK2 (Figure 11A) and SMAD4 (data not shown), showed no sex bias.

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943 Table 2.Morphometric Analysis of Pancreatic Tissue After Recurrent Pancreatic Damage 944 945 Male Female 946 Mist1creERT/þAtrxflD18(5) Mist1creERT/þ (4) Mist1creERT/þAtrxflD18 (5) Mist1creERT/þ (5) 947 948 Fibrosis 0±0 0±0 0±0 1.4 ± 0.51 949 Inflammation 0.2 ± 0.20 1.4 ± 0.25 0.75 ± 0.25 2.4 ± 0.40 950 ADM 0±0 0.6 ± 0.25 0±0 1.2 ± 0.37 951 Total 0.2 ± 0.2 2.0 ± 0.45 0.75 ± 0.25 5.0 ± 1.1a 952 NOTE. (#) indicates n value; see methodology for scoring. Histopathologic assessment of pancreatic damage, as indicated by 953 3 factors: fibrosis, inflammation, and presence of ADM. Scores are represented on a grading scale from 0 to 4. Superscript 954 letter “a” indicates groups that are statistically different (P < .01). Data were assessed using 2-way ANOVA and Tukey post 955 hoc test. 956 957 958 maintains genomic stability and regulates cell cycle pro- the female MKA mice suggests that chronic pancreatitis 959 cesses including proper chromosome segregation during occurs in these mice, and the inflammation observed may 960 mitosis.14,27 We did observe small, yet significant increases contribute to increased susceptibility to active KRAS. Loss of 961 962 in acinar cell apoptosis, dsDNA damage, and proliferation in function mutations in ATRX have been identified in other 963 Mist1creERT/þAtrxflD18 mice, suggesting during a longer cancer types, most notably those involving up-regulation 964 period of time (>2 months), the absence of ATRX may lead of the alternative lengthening of telomeres pathway.13,35 965 to more overt damage. Acinar cell division in the adult Mutations in ATRX or binding partner DAXX are often 966 rodent pancreas is <2%,30 which is similar to the observed observed in pancreatic neuroendocrine tumors and glio967 rates of apoptosis and dsDNA damage. However, targeted blastomas, but neither cancer shows a gender preference ablation of other factors, including Xbp131 and Pdx1,32 with or without ATRX mutation. Studies examining PDAC 968 results in rapid loss of acinar tissue, so acinar cell division tumors confirmed an absence of alternative lengthening of 969 is clearly not a prerequisite for development of overt telomeres in every case,36 and PDAC is typically character970 pancreatic pathologies. Mild damage in acinar tissue with ized by telomere attrition.37 Therefore, we suggest that 971 ATRX loss suggested acinar cells may be sensitive to other ATRX is affecting an alternative pathway in PDAC, possibly 972 973 in a DAXX-independent manner. ATRX interacts with factors known to promote pancreatic pathologies. 974 When exposed to recurrent cerulein treatment, only enhancer of zeste homologue 2, a member of polycomb 975 Mist1creERT/þAtrxflD18 mice showed fibrosis, inflammatory repressor complex 2, leading to altered gene expression,38 976 cell infiltration, and regions of ADM, with the effects more and loss of EZH2 also increases sensitivity to oncogenic 977 dramatic in female mice. It is unclear whether loss of ATRX KRAS.33 978 The incidence of human PDAC between sexes is relaleads to increased damage or if regeneration is impaired in Mist1creERT/þAtrxflD18 mice. Unpublished work from our tively equal, with approximately the same number of cases 979 laboratory using an acute pancreatitis regimen indicated occurring in men and women (Canadian Cancer Statistics, 980 similar amounts of damage in control and Mist1creERT/þ 2016). Assessment of International Cancer Genomic Con981 AtrxflD18 mice immediately after injury, suggesting loss of sortium database revealed ATRX single nucleotide poly982 ATRX impairs the regenerative process after injury, and is morphisms in almost 20% of PDAC cases (145/729), 983 984 consistent with studies in which other chromatin remodel- although most were in non-coding regions of the gene. 985 ling proteins (EZH233 and BMI110) are required for proper However, whether in the coding or non-coding regions, 986 pancreatic regeneration. It is possible that defects from Atrx ATRX mutations had a higher than expected frequency in 987 loss become more widespread once injury is induced, and female patients, even when taking into consideration that it 988 increased DNA damage and/or replicative defects provide a is an X-linked gene. Therefore, it is possible that ATRX loss 989 barrier to acinar tissue regeneration. However, we found no defines a unique subtype of PDAC, in which female patients differences in apoptosis and proliferation in cerulein-treated are more susceptible, or that loss of ATRX function in male 990 mice, suggesting these are not contributing factors through patients does not allow progression through to a PDAC 991 which ATRX loss promotes damage. phenotype. Although we have observed decreased acinar 992 The combination of Atrx deletion with oncogenic KRAS cell sensitivity to oncogenic KRAS in male mice, female MKA 993 activation produced extensive fibrosis and inflammation, mice show significantly increased progression to PanIN1 994 995 pancreatic damage indicative of chronic pancreatitis, as well and PanIN2 lesions, and the mechanisms underlying the 996 as PanIN lesions up to grade 2. In this instance, damage was extensive pancreatic damage specifically in female MKA 997 exclusive to female mice. Consistent with previous studies,34 mice are unclear. It is possible that loss of ATRX enhances 998 we observed minimal fibrosis and PanIN lesions in KRAS KRAS activity and leads to altered hormonal signalling. 999 mice that indicate oncogenic KRAS activation in adult mice Sex hormone receptors, including estrogen receptors, play a 1000 Q13 required another stimulus, such as chronic pancreatitis, to role in the progression of other cancers such as colorectal 1001 produce invasive PDAC. The pancreatic injury observed in cancer.39 However, the Atrx gene is located on the X

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Figure 4. Mist1creERT/DAtrxflD18 pancreatic tissue shows increased apoptosis after recurrent CIP treatment. (A) Acinar cell–specific cleaved caspase-3 comparing Mist1creERT/þ (WT) and Mist1creERT/þAtrxflD18 (AtrxflD18) pancreatic tissue after saline (SAL) or cerulein (CIP) treatment. Quantification in pancreatic tissue from Mist1creERT/þ (WT) mice treated with saline (n ¼ 4 female or 3 male) or cerulein (n ¼ 4 female or 5 male) and Mist1creERT/þAtrxflD18 (AtrxflD18) mice treated with saline (n ¼ 5 female or 4 male) or cerulein (n ¼ 5 female and male). Data were assessed using 2-way ANOVA with Tukey post hoc test. Error bars represent means ± standard error. (B) Immunohistochemistry for cleaved caspase-3 in male and female Mist1creERT/þ (WT) or Mist1creERT/þAtrxflD18 pancreatic tissue 3 days after cessation of cerulein treatment. Arrows indicate apoptotic cells. Magnification bar ¼ 50 mm.

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Figure 5. Mist1creERT/ D AtrxflD18 pancreatic tissue shows reduced regeneration after recurrent CIP treatment. Tissue was examined 3 days after cessation of recurrent CIP treatment. (A) Western blot analysis for CPA, amylase, SOX9, and total ERK1/2 (tERK1/2; loading control). Increased accumulation of digestive enzymes was observed specifically in Mist1creERT/þ (WT) mice, whereas Mist1creERT/þAtrxflD18 (AtrxflD18) mice did not demonstrate similar accumulation. (B) Serum amylase levels in mice (n ¼ 3–4 for each group). Data were assessed by using 2way ANOVA with Tukey post hoc test. Bars represent mean ± standard error. No significant difference in body weight or amylase levels was observed between genotypes. (C) Immunohistochemistry for CPA in Mist1creERT/þ or Mist1creERT/þAtrxflD18 mice. CPA accumulation was decreased in female Mist1creERT/þAtrxflD18 mice (acinus is indicated by dotted line). (D) Representative immunofluorescence for SOX9 (green) expression was increased in male Mist1creERT/ þ flD18 Atrx and female Mist1creERT/þAtrxflD18 tissue (white arrows). Magnification bars ¼ 50 mm. (E) Higher magnification images show SOX9 expression in duct, putative ADM (white arrows), and acinar cell (yellow arrowheads). Sections were co-stained for Ki-67 (red arrowheads) and counterstained with DAPI.

chromosome and is a target of X inactivation. Therefore, it would be expected that female mice heterozygous (AtrxflD18/x) for the mutant Atrx allele would also show similar effects because approximately half of the acinar cells lose ATRX expression. Immunohistochemistry for ATRX confirmed that at least a portion of acinar cells in heterozygous AtrxflD18/x

mice did lose ATRX expression (data not shown), but these mice did not demonstrate increased damage or susceptibility to oncogenic KRAS. These results suggest complete loss of ATRX is required for enhanced KRAS activity and pancreatic damage to occur in female mice. However, such a model also does not account for decreased sensitivity in male MKA mice.

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Figure 6. Induction of oncogenic KRAS with Atrx deletion. (A) Schematic of Atrx-deficient mouse model with oncogenic KRAS activation. (B) Experimental timeline for tamoxifen gavage in congenic Mist1creERT/þ, Mist1creERT/þAtrxflD18, Mist1creERT/ þ KrasLSL-G12D/þ, and MKA mice. Names used for each line within the figures are depicted in bold. (C) Immunohistochemistry for ATRX in acinar (black arrow) and duct (red arrow) cells of Mist1creERT/þ and Mist1creERT/þAtrxflD18 mice, ATRX expression is limited to islet (open arrow; delineated by dotted line) and duct cells (red arrow). (D) Change in body weight (%) of mice after Atrx deletion ± oncogenic KRAS activation. Mouse genotypes include Mist1creERT/þ (WT; n ¼ 13), Mist1creERT/þAtrxflD18 (AtrxflD18; n ¼ 12), Mist1creERT/þKrasLSL-G12D/þ (KrasLSL-G12D/þ; n ¼ 17), and MKA (n ¼ 16). Points are represented as mean weight ± standard error. (E) Serum amylase levels in WT (n ¼ 7 male or 3 female), AtrxflD18 (n ¼ 7 male or 5 female), KrasLSLG12D/þ (n ¼ 6 male or 5 female), and MKA (n ¼ 8 male or 6 female) mice 60 days after Atrx deletion ± oncogenic KRAS activation. Data were assessed using 2-way ANOVA with Tukey post hoc test. Bars represent mean ± standard error.

Sex-specific mechanisms could also be explained by a difference in inflammatory response. It is possible that female AtrxflD18/ flD18 mice are more susceptible to factors promoting inflammation. During recurrent pancreatic injury, AtrxflD18/ flD18 mice showed increased inflammation in comparison with male counterparts, resulting in higher levels of damage. In combination with oncogenic KRAS, increased inflammation in AtrxflD18/ flD18 mice may amplify KRAS activity and activation of downstream pathways, including MAPK and PI3K-PDK1-Akt signaling, leading to

increased cell survival and proliferation. Accordingly, increased KRASG12D activity by inflammatory cytokines (nuclear factor kappa B, interleukin 6) has been demonstrated previously.40,41 The presence of an inflammatory response in female AtrxflD18/ flD18 mice, which is not observed in male AtrxflD18/y mice, could lead to increased damage. It is also possible that sex-specific differences exist regarding the function of SOX9. Previous work suggests SOX9 is required for initiation of ADM,4 and we show

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1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 Figure 7. Gross morphology does not reveal significant differences on dissection. Mist1creERT/þ (WT), Mist1creERT/ 1493 þ AtrxflD18 (AtrxflD18), Mist1creERT/þKrasLSL-G12D/þ (KrasLSL-G12D/þ), or MKA mice. Although female MKA mice may show some 1494 edema, the only easily identified difference is splenomegaly (*), which consistently appeared in Mist1creERT/þKrasLSL-G12D/þ and 1495 MKA mice. 1496 1497 1498 increased SOX9 accumulation in female MKA acinar cells inflammation) provide an additional driving factor for KRAS 1499 surrounding damage. Increased susceptibility in female activity and pancreatic damage, leading to a female-specific 1500 MKA mice could include sex-specific hormonal or inflam- phenotype. 1501 matory pathways that provoke increased SOX9 expression. 1502 As mentioned, female AtrxflD18/ flD18 mice have an ampli1503 1504 fied inflammatory response, and inflammatory signaling Materials and Methods 1505 pathways can influence SOX9 expression during develop- Animal Generation and Cre Induction Mouse experiments were approved by the Animal Care 1506 ment.42 Alternatively, hormonal factors could play a role in sex-specific Sox9 regulation. Recently, up-regulation of and Use Committee at Western University (Protocol #2017- 1507 estrogen receptor a-receptor activity in breast cancer cells 001). All mice used in this study were maintained in a C57Bl6 1508 has been associated with increased SOX9 expression, background. Mice expressing creERT from the Mist1 locus 1509 although this study occurs in the context of estrogen (Mist1creERT/þ)5 were crossed with mice harboring an Atrx 1510 allele with exon 18 flanked by loxP sites,23 producing 1511 deprivation.43 It would be interesting to observe the long-term effects male (Mist1creERT/þAtrxflD18/y) and female (Mist1creERT/þ 1512 of ATRX deletion on oncogenic KRAS-mediated PDAC for- AtrxflD18/fl D18) mice, collectively referred to as Mist1creERT/þ 1513 mation. Because of the prevalence of tumors developing in AtrxflD18. Mist1creERT/þAtrxflD18 mice were crossed to mice 1514 the oral mucosa, we were forced to kill MKA mice before containing an inducible oncogenic KRAS (loxP-STOP-loxP 1515 overt PDAC development. These tumors likely arise because (LSL)-KRASG12D)45 to produce Mist1creERT/þKrasLSL-G12D/þ Q81516 of the expression of Mist1creERT in other tissues, and we AtrxflD18 mice (referred to as MKA). Furthermore, female 1517 are currently generating AtrxflD18/ flD18 mice with a mice containing one (Mist1creERT/þAtrxflD18/x) or two (Mis- 1518 pancreas-specific inducible cre-recombinase, which will t1creERT/þAtrxx/x) copies of the Atrx allele showed no obvious 1519 allow for longer-term analysis. It is possible that having only phenotypic differences and were combined as a single 1520 a single copy of Mist1 contributes to the Mist1creERT/þ Mist1creERT/þ control group. Female mice expressing 1521 AtrxflD18 and MKA phenotypes. Although previous studies44 KRASG12D that were heterozygous (Mist1creERT/þKrasLSL-G12D/þ 1522 and unpublished work from our laboratory demonstrated AtrxflD18/x) or homozygous for Atrx (Mist1creERT/þKrasLSL-G12D/þ 1523 no difference in the phenotype between mice heterozygous Atrxx/x) also showed no obvious morphologic differences 1524 or wild-type (WT) for MIST1 expression, using a different and were collectively referred to as Mist1creERT/þ 1525 1526 cre-recombinase (such as Ptf1acreERT) would rule out any KrasLSL-G12D/þ mice. contribution of Mist1 haploinsufficiency to the results Tamoxifen (Sigma-Aldrich, St Louis, MO; cat. #T5648) 1527 observed in this study. was administered through oral gavage (2 mg/mouse) 3 1528 In summary, we identified that loss of ATRX enhanced times over 5 days. This resulted in >95% recombination in 1529 pancreatic injury and susceptibility to KRAS-mediated acinar cells and no recombination in duct cells.24 Mice were 1530 pancreatic damage. Potential gender-specific factors within monitored for 60 days from first tamoxifen gavage, and 1531 AtrxflD18/ flD18 mice (including hormonal factors or increased body weight was measured weekly. 1532

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Cerulein Induced Pancreatitis To induce recurrent pancreatic injury, control (Mist1creERT/þ) and Mist1creERT/þAtrxflD18 mice were given intraperitoneal injections of saline or cerulein (75 mg/kg body weight; Sigma-Aldrich; cat. #C9026) twice daily for 11 days, followed by a 3-day recovery period. Mice were weighed daily throughout the injury protocol. Mice

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were killed and processed for histologic, molecular, biochemical, and blood serum analysis.46 Serum amylase was quantified by using Phadebas tablets (Magle Life Sciences, Lund, Sweden; cat. #1302) following manufacturer’s instructions.

Histology, Immunohistochemistry, and Immunofluorescent Analysis The head of the pancreas was used for paraffin sections, and cryostat sections were obtained from the middle portion of pancreas. Tissue was washed 2 times in phosphate-buffered saline and then dehydrated through a series of alcohol washes for embedding into paraffin. Paraffin sections (5 mm) were stained by using standard H&E, Alcian blue (Mucin Stain; Abcam Inc, Cambridge, MA; cat. #ab150662) or Masson’s trichrome stain (Abcam Inc; cat. #ab150686) protocols. Sections were imaged by using the Aperio CS2 Digital Scanner and Aperio ImageScope software (Leica Biosystems Imaging Inc, San Diego, CA). Total tissue area was quantified by using the Fiji software,47 and area of damage was quantified as a percentage of total area. Levels of pancreatic damage were assessed by using a grading scale based on 3 factors: fibrosis, inflammation, and presence of acinar to ductal metaplasia. Tissue sections were scored by multiple individuals blinded to mouse genotypes on a scale from 0 to 4. Descriptions of each score can be found in Table 1 along with a description of scoring PanIN lesions. For immunohistochemical analysis, paraffin tissue sections were stained by using the ABC staining system (Santa Cruz Biotechnology Inc, Dallas, TX) or the VectaStain ABC HRP kit with ImmPACT DAB Peroxidase (HRP) Substrate (Vector Laboratories, Brockville, ON, Canada; cat. #SK-4105) according to kit instructions. Cleaved caspase-3 (rabbit 1:100; Cell Signaling Technology, Danvers, MA; cat. #966455) staining was completed by using the Ventana Discovery Ultra XT autostainer (Ventana Medical Systems Inc, Tucson, AZ). Primary antibodies used are specific to ATRX (rabbit; diluted 1:100 in 1.5% mouse blocking serum in phosphate-buffered saline; Santa Cruz Biotechnology Inc; cat. #sc15408), PDX1 (rabbit; 1:1000; Abcam Inc; cat. #ab47267), Ki67 (rabbit 1:500; Abcam Inc; cat. #ab15580). For immunofluorescence, cryostat sections were processed Figure 8. Loss of ATRX enhances KRASG12D’s ability to promote pre-cancerous lesions in female mice. (A) H&E images of pancreatic tissue from male and female Mist1creERT/þ (WT), Mist1creERT/þAtrxflD18 (AtrxflD18), Mist1creERT/ þ KrasLSL-G12D/þ (KrasLSL-G12D/þ), and MKA mice 60 days after tamoxifen treatment. Arrows indicate focal ADM or PanIN lesions. Dotted white line delineates significant lesion area from acinar tissue. Islets are indicated by I. Magnification bar ¼ 200 mm. (B) Average percentage of lobules containing at least one instance of ADM, PanIN1, or PanIN2 in KrasLSLG12D/þ (n ¼ 7 male or 10 female) and MKA (n ¼ 10 male or 6 female) mice. Data were assessed by using 2-way ANOVA with Tukey post hoc test. Representative examples of ADM, PanIN1, and PanIN2 lesions from female MKA mice. Magnification bars ¼ 50 mm.

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Figure 9. Histology of pancreatic tissue after Atrx deletion. Representative (A) trichrome or (B) Alcian blue images of pancreatic tissue from male and female Mist1creERT/þ (WT), Mist1creERT/þAtrxflD18 (AtrxflD18), Mist1creERT/þKrasLSL-G12D/þ (KrasLSL-G12D/þ), and MKA mice 60 days after tamoxifen treatment. Increased tissue fibrosis was routinely observed in MKA females (arrows). Magnification bar ¼ 200 mm. Increased fibrosis (open arrow) and duct metaplasia (closed arrow) are observed in female MKA tissue. Quantification of (C) damaged area as percentage of total pancreatic area based on appearance of PanINs or fibrosis, or (D) number of ADM and PanINs (lesions) observed within Mist1creERT/þ (WT; n ¼ 8 male or 5 female), Mist1creERT/þAtrxflD18 (AtrxflD18; n ¼ 8 male or 5 female), Mist1creERT/þKrasLSL-G12D/þ (KrasLSL-G12D/þ; n ¼ 7 male or 10 female), and MKA (n ¼ 10 male or 6 female) mice 60 days after tamoxifen treatment. Number of ADM and PanINs (lesions) was significantly increased in MKA females compared with all other groups except male Mist1creERT/þKrasLSL-G12D/þ (KrasLSL-G12D/þ) mice. Data were assessed by using 2-way ANOVA with Tukey post hoc test. Bars represent mean ± standard error; *P < .05.

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Table 3.Morphometric Analysis of Pancreatic Tissue 60 Days After Activation of KRASG12D and Loss of Atrx Mist1creERT/þ

Mist1creERT/þAtrxflD18

Male (8)

Female (5)

Male (8)

Fibrosis

0±0

0±0

Inflammation

0±0

0±0

ADM

0±0

0.2 ± 0.2

0±0

Total

0 ± 0a

0.2 ± 0.2a

0.14 ± 0.14a

Female (5)

0±0

0±0

0.14 ± 0.14

Mist1creERT/þKrasLSL-G12D/þ Male (7)

Female (10)

MKA Male (10)

Female (6)

1 ± 0.66

0.7 ± 0.47

0±0

1.83 ± 0.48

1 ± 0.54

1 ± 0.52

0.2 ± 0.13

2.17 ± 0.70

0±0

1.57 ± 0.65

1.5 ± 0.45

0.4 ± 0.16

3 ± 0.45

0.33 ± 0.21a

3.57 ± 1.8a,b

3.2 ± 1.4a,b

0.6 ± 0.27a

7 ± 1.57b

0.33 ± 0.21

NOTE. (#) indicates n value; see methodology for scoring. Histopathologic assessment of pancreatic damage, as indicated by 3 factors: fibrosis, inflammation, and presence of ADM. Scores are represented on a grading scale from 0 to 4. Superscript letters “a” and “b” indicate groups that are statistically different (P < .01). Data were assessed using 2-way ANOVA and Tukey post hoc test.

as previously described.46 Primary antibodies used were specific to ATRX (rabbit; diluted 1:100 in blocking solution; Santa Cruz Biotechnology Inc), MIST1 (rabbit; 1:500),48 b-catenin (mouse; 1:500; BD Biosciences, Mississauga, ON, Canada; cat. #610153, lot#5121508), SOX9 (rabbit; 1:500; MilliporeSigma, Etobicoke, ON, Canada; cat. #AB5535, lot#3018860), insulin (mouse 1:500; Sigma; cat. #I2018; lot#092K4841), or gH2AX (rabbit; 1:200; Santa Cruz Biotechnology Inc; cat. #sc-101696, lot#12613). Secondary antibodies used include anti-rabbit FITC (cat. #111-545003, lot#125266) and anti-mouse FITC (cat. #115-025-003, lot#125278; 1:250; Jackson ImmunoResearch, West Grove, PA). Sections were counterstained with 4’,6-diamidino-2phenylindole and imaged using a Leica DM5500B microscope with LAS V4.4 software (Leica Microsystems Ltd, Wetzlar, Germany).

Protein Isolation and Western Blot Analyses Protein was isolated from the middle portion of the pancreata and homogenized on ice using a Potter Elvehiem Homogenizer in extraction buffer (50 mmol/L Tris [pH 7.2], 5 mmol/L MgCl2, 1 mmol/L CaCl2, 1% NP-40, 0.5 mmol/L DTT, 0.5 mmol/L PMSF, 10 mmol/L NaF, 2 mmol/L NaVO4, 150 nmol/L aprotinin, 10 mmol/L, pepstatin, 50 mmol/L leupeptin).46 Homogenates were sonicated samples for 20 seconds on ice (level 4 Fisher Sonic Dismemberator) and

centrifuged 10 minutes at 4 C at 14,000g. Supernatants were taken and frozen at –80 C until used. Isolated protein was resolved by sodium dodecylsulfate–gel electrophoresis in 10% acrylamide gels and transferred to a polyvinylidene difluoride membrane (Bio-Rad; cat. #162-0177) for Western blot analyses.48 Primary antibodies were specific for total MAPK (tERK1/2) (rabbit 1:2500 in 5% BSA-0.1% Tween20; Cell Signaling Technology; cat. #9102, lot #26), carboxypeptidase (CPA) (rabbit 1:1000 in 5% NFDM; R&D Systems, Minneapolis, MN; cat. #AF2765, lot #wo00117071), and amylase (rabbit 1:1000 in 5% NFDM; Abcam; cat. #ab21156). After overnight incubation, blots were incubated in secondary antibody (anti-rabbit HRP, 1:10,000; Jackson Labs, Bar Harbor, ME; cat. #111-035144) diluted in 5% NFDM for 1 hour at room temperature. Blots were developed by using Clarity Western blot ECL kit (Biorad; cat. #1705061) and visualized by using the VersaDoc system with Quantity One 1-D analysis software (Bio-Rad).

TUNEL Assay To assess apoptosis, cryostat sections were processed using the In Situ Cell Death detection kit (Roche, Laval, QC, Canada; cat. #11684795910) per manufacturer’s directions. Sections were counterstained with DAPI. The number of TUNEL-positive cells was quantified using 7 random fields

Table 4.Classification of ADM and PanIN Lesions 60 Days After Activation of KRASG12D and Loss of Atrx Percent of lobules determined by highest lesion grade Genotype KRASLSL-G12D MKA

Sex

Normal

ADM

PanIN1

PanIN2

Male (7)

83.1 ± 7.8

10.8 ± 4.2

4.9 ± 3.6

1.3 ± 1.3

Female (10)

60.1 ± 8.2

33.3 ± 6.3

6.5 ± 3.5

Male (10)

81.2 ± 7.8

18.8 ± 7.8

Female (6)

46.9 ± 11.9a

25.2 ± 5.7

0±0 16.2 ± 8.3b

0±0 0±0 11.7 ± 6.3a

NOTE. (#) indicates n values. Data were assessed using 2-way ANOVA and Tukey post hoc test. These data are presented in Figure 8B. a Difference from all other groups (P < .05). b Difference from male MKA mice (P < .05).

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Figure 10. PanIN lesions in Mist1creERT/DAtrxflD18 mice are derived from ATRX– acinar cells undergoing ADM. (A) Immunofluorescent images for SOX9 in male and female Mist1creERT/þ (WT), Mist1creERT/þAtrxflD18 (AtrxflD18), Mist1creERT/ þ KrasLSL-G12D/þ (KrasLSL-G12D/þ), and MKA mice 60 days after tamoxifen treatment. Insets show nuclear localization of SOX9 in putative ADM in MKA mice. (B) Co-immunofluorescence of putative ADM in female MKA pancreatic tissue shows SOX9 nuclear accumulation in proliferating cells based on Ki67 accumulation (red). White arrows indicate cells expressing Ki67 and SOX9. Sections were counterstained for DAPI. Magnification bars ¼ 50 mm. (C) Quantification of Ki67þ acinar cells in Mist1creERT/þ, Mist1creERT/þAtrxflD18, Mist1creERT/þKrasLSL-G12D/þ, and MKA mice. Bars represent mean ± standard error. N ¼ 3 for each group. (D) Immunohistochemistry for ATRX in putative ADM and PanINs in male and female Mist1creERT/þKrasLSL-G12D/þ (KrasLSL-G12D/þ) and MKA mice. ATRXþ lesions are identified in Mist1creERT/þKrasLSL-G12D/þ mice (black arrow), whereas female MKA revealed both ATRXþ (*) and ATRX– (**) cells within the PanINs. ATRXþ cells are found in ducts (D) and islets (I) of MKA males and in interstitial fibroblasts in MKA females. (E) Quantification of lesions with at least 1 ATRXþ cell (black) or no ATRXþ cells (gray) in Mist1creERT/þ (WT; n ¼ 3 for male and female), Mist1creERT/þAtrxflD18 (AtrxflD18; n ¼ 3 for male and female), Mist1creERT/þKrasLSL-G12D/þ (KrasLSL-G12D/þ; n ¼ 3 for male or n ¼ 6 for female), and MKA (n ¼ 4 for male or n ¼ 3 for female) mice. Bars represent mean % ± standard error.

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1946 1947 1948 1949 1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

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2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 of view from each mouse and calculated as percentage of 2037 TUNEL-positive cells compared with DAPI counts. 2038 2039 2040 Statistical Analysis In all cases, data were analyzed for significance by using 2041 2042 an unpaired, two-tailed t test or 2-way analysis of variance 2043 (ANOVA) with Tukey post hoc test. Values are depicted as 2044 means ± standard error of the mean. Significance is 2045 considered P < .05. 2046 2047 2048 References 1. Hingorani SR, Wang L, Multani AS, et al. Trp53R172H 2049 and KrasG12D cooperate to promote chromosomal 2050 instability and widely metastatic pancreatic ductal 2051 Q14 adenocarcinoma in mice. Cancer Cell 2005;7:469–483. 2052 2. Zhu L, Shi G, Schmidt CM, et al. Acinar cells contribute 2053 to the molecular heterogeneity of pancreatic intra2054 epithelial neoplasia. Am J Pathol 2007;171:263–273. 2055 3. Morris JPt, Cano DA, Sekine S, et al. Beta-catenin blocks 2056 Kras-dependent reprogramming of acini into pancreatic 2057 cancer precursor lesions in mice. J Clin Invest 2010; 2058 120:508–520. 2059 4. Kopp JL, von Figura G, Mayes E, et al. Identification of 2060 Sox9-dependent acinar-to-ductal reprogramming as the 2061 principal mechanism for initiation of pancreatic ductal 2062 adenocarcinoma. Cancer Cell 2012;22:737–750. 2063

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Figure 11. Mutation analysis on PDAC patients based on data from the International Cancer Genome Consortium. (A) Comparison of gender breakdown for all PDAC cases, or for mutations in ATRX, known driver mutations for PDAC (KRAS, CDNK2/P16), or for DAXX. Impact ATRX mutations refer to those mutations that occur with the protein coding region and are predicted to have a negative impact on protein function. (B and C) Chisquared analyses determining if ATRX mutations and sex are independent variables in PDAC (B) or pancreatic neuroendocrine tumors (PNETs; C).

5. Shi G, Direnzo D, Qu C, et al. Maintenance of acinar cell organization is critical to preventing Kras-induced acinar-ductal metaplasia. Oncogene 2013;32: 1950–1958. 6. Bardeesy N, Aguirre AJ, Chu GC, et al. Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse. Proc Natl Acad Sci U S A 2006;103:5947–5952. 7. De La OJ, Emerson LL, Goodman JL, et al. Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia. Proc Natl Acad Sci U S A 2008; 105:18907–18912. 8. Bailey P, Chang DK, Nones K, et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 2016;531:47–52. 9. von Figura G, Fukuda A, Roy N, et al. The chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma. Nat Cell Biol 2014;16:255–267. 10. Bednar F, Schofield HK, Collins MA, et al. Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism. Carcinogenesis 2015; 36:730–738. 11. Lewis PW, Elsaesser SJ, Noh KM, et al. Daxx is an H3.3specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. Proc Natl Acad Sci U S A 2010; 107:14075–14080.

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3 October 2018
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27. Ritchie K, Seah C, Moulin J, et al. Loss of ATRX leads to chromosome cohesion and congression defects. J Cell Biol 2008;180:315–324. 28. Prevot PP, Simion A, Grimont A, et al. Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia. Gut 2012; 61:1723–1732. 29. Martinelli P, Madriles F, Canamero M, et al. The acinar regulator Gata6 suppresses KrasG12V-driven pancreatic tumorigenesis in mice. Gut 2016;65:476–486. 30. Ledda-Columbano GM, Perra A, Pibiri M, et al. Induction of pancreatic acinar cell proliferation by thyroid hormone. J Endocrinol 2005;185:393–399. 31. Hess DA, Humphrey SE, Ishibashi J, et al. Extensive pancreas regeneration following acinar-specific disruption of Xbp1 in mice. Gastroenterology 2011; 141:1463–1472. 32. Roy N, Takeuchi KK, Ruggeri JM, et al. PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance. Genes Dev 2016;30:2669–2683. 33. Mallen-St Clair J, Soydaner-Azeloglu R, Lee KE, et al. EZH2 couples pancreatic regeneration to neoplastic progression. Genes Dev 2012;26:439–444. 34. Guerra C, Schuhmacher AJ, Canamero M, et al. Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice. Cancer Cell 2007;11:291–302. 35. de Wilde RF, Heaphy CM, Maitra A, et al. Loss of ATRX or DAXX expression and concomitant acquisition of the alternative lengthening of telomeres phenotype are late events in a small subset of MEN-1 syndrome pancreatic neuroendocrine tumors. Mod Pathol 2012; 25:1033–1039. 36. Heaphy CM, de Wilde RF, Jiao Y, et al. Altered telomeres in tumors with ATRX and DAXX mutations. Science 2011; 333:425. 37. van Heek T, Rader AE, Offerhaus GJ, et al. K-ras, p53, and DPC4 (MAD4) alterations in fine-needle aspirates of the pancreas: a molecular panel correlates with and supplements cytologic diagnosis. Am J Clin Pathol 2002; 117:755–765. 38. Cardoso C, Timsit S, Villard L, et al. Specific interaction between the XNP/ATR-X gene product and the SET domain of the human EZH2 protein. Hum Mol Genet 1998;7:679–684. 39. Nussler NC, Reinbacher K, Shanny N, et al. Sex-specific differences in the expression levels of estrogen receptor subtypes in colorectal cancer. Gend Med 2008; 5:209–217. 40. Daniluk J, Liu Y, Deng D, et al. An NF-kappaB pathwaymediated positive feedback loop amplifies Ras activity to pathological levels in mice. J Clin Invest 2012; 122:1519–1528. 41. Baumgart S, Chen NM, Siveke JT, et al. Inflammationinduced NFATc1-STAT3 transcription complex promotes pancreatic cancer initiation by KrasG12D. Cancer Discov 2014;4:688–701. 42. Akiyama H, Stadler HS, Martin JF, et al. Misexpression of Sox9 in mouse limb bud mesenchyme induces

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polydactyly and rescues hypodactyly mice. Matrix Biology 2007;26:224–233. Jeselsohn R, Cornwell M, Pun M, et al. Embryonic transcription factor SOX9 drives breast cancer endocrine resistance. PNAS 2017;114:E4482–E4491. Karki A, Humphrey SE, Steele RE, et al. Silencing Mist1 gene expression is essential for recovery from acute pancreatitis. PLoS One 2015;10:e0145724. Jackson EL, Willis N, Mercer K, et al. Analysis of lung tumor initiation and progression using conditional expression of K-ras. Genes Develop 2001;15:3243–3248. Fazio EN, Young CC, Toma J, et al. Activating transcription factor 3 promotes loss of the acinar cell phenotype in response to cerulein-induced pancreatitis in mice. Mol Biol Cell 2017;28:2347–2359. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9:676–682. Pin CL, Bonvissuto AC, Konieczny SF. Mist1 expression is a common link among serous exocrine cells exhibiting regulated exocytosis. Anat Rec 2000;259:157–167.

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2300 2301 Correspondence 2302 Address correspondence to: Christopher Pin, Department of Paediatrics, 2303 University of Western Ontario, Children’s Health Research Institute, 5th Floor, Victoria Research Laboratories, London, Ontario, Canada N6C 2V5. Q22304 Q3 e-mail: [email protected]; fax: 519-685-8186. 2305 Author contributions 2306 CY: acquisition of data, analysis and interpretation of data, drafting of 2307 the manuscript; RB: acquisition of data, analysis and interpretation 2308 of data; CH: analysis and interpretation of data; TH: acquisition of data; JH: acquisition of data; DH: material support; RG: material support; 2309 HC: acquisition of data, analysis and interpretation of data, critical 2310 revision of the manuscript for important intellectual content; AB: critical revision of the manuscript for important intellectual content; CP: study 2311 concept and design, drafting of the manuscript, critical revision of the 2312 manuscript for important intellectual content, obtained funding, study Q42313 supervision. 2314 Conflicts of interest Q52315 The authors disclose no conflicts. 2316 Funding 2317 Funding was provided to C.P. by the Rob Lutterman Foundation and Cancer Research Society of Canada. C.Y. was funded by a Canadian Graduate 2318 Scholarship. R.B. received a Canadian Association of Gastroenterology Q17 Q62319 summer scholarship. 2320 2321 2322 2323 2324 2325 2326 2327 2328 2329 2330 2331 2332 2333 2334 2335 2336 2337 2338 2339 2340 2341 2342 2343 2344 2345 2346 2347 2348 2349 2350 2351 2352 2353 2354 2355 2356 2357 2358 Received February 28, 2018. Accepted September 6, 2018.

FLA 5.5.0 DTD
JCMGH392 proof
3 October 2018
12:18 pm
ce CLR