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Epithelial metaplasia, or the histologically evident replacement of one differentiated somatic cell type with another, offers insights into mucosal homeostasis as an adaptation to diverse inflammatory pathogenic stimuli. The cellular framework for metaplasia can be created by coordination of tissue-specific transcription factors (TFs) with epigenetic regulators of chromatin remodeling, allowing altered TF binding of inflammation-associated exposed genomic loci. For example, intestinal metaplasia (aberrant goblet cell differentiation and CDX2 expression, a pan-intestinal TF) is a common metaplastic response to chronic injury in the upper gastrointestinal tract, associated with increased risk of epithelial dysplasia and cancer. Inflammatory bowel disease (IBD) is an etiologically complex chronic inflammatory disease characterized by relapsing cycles of intestinal injury and healing. IBD patients carry increased risk of colitis-associated dysplasia and colorectal cancer (CAC) proportional to disease duration, extent, and severity. SATB2, or special AT-rich binding protein 2, is a chromatin organizing protein with homeostatic roles in osteoblastic, neural, and colonic differentiation. Intriguingly, others have recently shown that SATB2 is uniquely lost in human IBD dysplasia and CAC, in contrast to sporadic CRC where its expression is typically retained. (By contrast, CDX2 shows retained expression in both CAC and CRC.) Other recent work has shown that colon-specific Satb2 knockout in mice results in conversion of colon to small intestine, including emergence of villi and Paneth cells. Colonic mucosal biopsies from chronic IBD patients show histologic features reminiscent of small intestine with so-called Paneth cell metaplasia (PCM) and villiform, distorted growth. The molecular mediators of this epithelial remodeling in IBD are unknown. We hypothesized that SATB2 loss could result in a form of “small intestinal metaplasia” in human IBD colon. To investigate this, we selected a cohort of 20 IBD distal colon mucosal biopsies with marked crypt architectural changes and PCM and performed SATB2 immunostaining (n=7 Crohn’s disease, n=13 ulcerative colitis). Indeed, 19 of 20 such samples showed striking evidence of SATB2 loss. The pattern of SATB2 loss was heterogeneous; some tissue fragments showed complete epithelial loss as expected in small intestine, whereas others showed patchy loss in alternating crypts, even some showing heterogeneous expression within the same crypt. As positive and negative controls, we included normal distal colon and terminal ileum samples, observing expected intact and negative SATB2 expression, respectively. Of note, CDX2 was strongly expressed across the entire cohort, even in IBD tissue with loss of SATB2 expression. We propose that SATB2 loss is a pre-neoplastic metaplastic response to chronic injury in human colon. Future work will investigate how SATB2 loss enables a plasticity state permissive for emergence of TP53 mutations, dysplasia, and CAC. Citation Format: Maged Zeineldin, Reem Abu-Shamma, Tatianna Larman. Implicating SATB2 loss in a metaplasia-dysplasia sequence in human inflammatory bowel disease [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B026.

Adenomatous polyposis coli (APC) is best known for its crucial role in colorectal cancer suppression. Rodent models with various Apc mutations have enabled experimental validation of different Apc functions in tumors and normal tissues. Since the development of the first mouse model with a germline Apc mutation in the early 1990s, 20 other Apc mouse and rat models have been generated. This article compares and contrasts currently available Apc rodent models with particular emphasis on providing potential explanations for their reported variation in three areas: (i) intestinal polyp multiplicity, (ii) intestinal polyp distribution, and (iii) extraintestinal phenotypes. Cancer Res; 73(8); 2389–99. ©2013 AACR.

Neuroblastoma is a clinically heterogeneous tumor responsible for 15% of cancer-related deaths in children. Older age at diagnosis and MYCN amplification are associated with poor prognosis in neuroblastoma. Previously, sequencing stage 4 neuroblastoma tumors revealed recurrent mutations in the chromatin remodeler ATRX that are significantly associated with older age and mutually exclusive with MYCN amplification. Here, we expanded our genetic analysis of neuroblastoma across all ages and stages. We sequenced 475 neuroblastoma tumors and found that the incidence of ATRX mutations is correlated with older age at diagnosis and advanced stages of the disease. We also directly studied the mutual exclusivity between ATRX mutations and MYCN amplification using both cultured cells and orthotopic mouse models. We found that targeting ATRX using shRNA or CRISPR-Cas-9 reduces cell viability in MYCN-amplified cells. Additionally, induction of MYCN overexpression results in cell death in ATRX-mutant neuroblastoma cells. To characterize the molecular basis of the ATRX-mutations/MYCN-amplification incompatibility, we studied epigenetic, metabolic, and replicative changes associated with MYCN amplification in these cells. We discovered that induction of MYCN overexpression provokes mitochondrial abnormalities, metabolic reprograming, and replicative stress in ATRX mutant cells. Collectively, these data show synthetic lethality between ATRX mutations and MYCN amplification and suggest that the chromatin remodeler ATRX is important in reducing replicative stress associated with MYCN amplification in neuroblastoma. These data will also help in identifying therapeutic targets, which will reduce the morbidity and mortality associated with high-risk neuroblastoma. Citation Format: Maged Zeineldin, Sara M. Federico, Lyra Griffith, Jongyre Jeon, Xiang Chen, John Easton, Jianrong Wu, Shenghua Mao, Yanling Liu, Arlene Naranjo, Alberto S. Pappo, Michael D. Hogarty, Michael A. Dyer. Synthetic lethality between ATRX mutations and MYCN amplification in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR11.

Age and stage of disease at diagnosis are associated with outcome in neuroblastoma. A previous study identified ATRX point mutations and in-frame deletions in older patients with stage 4 disease. To validate the frequency of ATRX mutations and assess the relation of mutations and known prognostic variables, we collaborated with the Children’s Oncology Group and sequenced the entire ATRX genomic locus in 475 neuroblastoma tumor samples to validate the frequency of ATRX mutations and assess the relation of mutations and known prognostic variables. Older age at diagnosis, stage 4 disease, and unfavorable histology were significantly associated with mutations in the ATRX gene; MYCN amplification was mutually exclusive from ATRX mutations. To directly test if ATRX mutations and MYCN amplification are incompatible in neuroblastoma, we mutated the ATRX gene in MYCN amplified neuroblastoma cell lines and ectopically expressed MYCN in ATRX mutant neuroblastoma cell lines. Both approaches showed that ATRX and MYCN amplification are incompatible in neuroblastoma in culture and in vivo. To gain a better understanding of the underlying molecular and cellular mechanisms of this incompatibility, we performed a series of experiments including telomere analysis, epigenetic profiling, electron microscopic analysis of dying cells, and metabolic profiling. We discovered that induction of MYCN expression in ATRX mutant neuroblastoma cells leads to metabolic reprogramming, mitochondrial dysfunction, increased reactive oxygen species, and replicative stress. We propose that this leads to synthetic lethality in ATRX mutant neuroblastomas because of the underlying replicative stress in those cells as a result of dysfunction of this essential histone chaperone complex. Citation Format: Maged Zeineldin, Sara Federico, Xiang Chen, Beisi Xu, Elizabeth Stewart, Arlene Naranjo, Michael D. Hogarty, Michael A. Dyer. MYCN amplification and ATRX mutations are incompatible in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA18.

Mutation of the tumor suppressor gene Adenomatous Polyposis Coli (APC) is considered an initiating event in the development of most intestinal tumors. Although much effort has been spent determining functions of the APC protein, to date little is known about the mechanisms that regulate cellular APC levels. Here we report that in cultured cells, induction of a heat-shock response, via heat or compounds such as the HSP90 inhibitor 17-AAG, resulted in increased levels of APC. A novel non-toxic small molecule that we developed, KN1, also induced a heat-shock response and led to increased APC levels in both cultured cells and mice. To investigate the effect of heat-shock response induction and elevation of Apc level in intestinal tumorigenesis, we performed a series of experiments treating mice with either KN1 or 17-AAG. We tested these compounds on two mouse models with different germline Apc mutations, ApcMin/+ and Apc1322T/+. In both cases, a moderate dose of either drug did not change tumor burden, but surprisingly altered the distribution of intestinal polyps. In a third mouse model, colonic tumors were induced via administration of the mutagen azoxymethane (AOM) and colon irritant dextran sodium sulfate (DSS) rather than by germline Apc mutation. In AOM-DSS-treated mice, KN1 reduced tumor incidence, multiplicity, and size. Moreover, KN1-treated mice lost significantly less weight and had smaller spleens than vehicle-treated mice, suggesting KN1 may also suppress colitis. We conclude that induction of the heat-shock response affects intestinal tumorigenesis in germline Apc-mutant and colitis-induced tumor models. Citation Format: Maged Zeineldin, William McGuinness, Brian Blagg, Roger Rajewski, Kristi L. Neufeld. Induction of the heat-shock response upregulates the tumor suppressor APC and alters intestinal tumorigenesis in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2728. doi:10.1158/1538-7445.AM2013-2728

Colon cancer is the second leading cause of cancer-related mortality in the United States, resulting in over 50,000 deaths annually. Approximately 80% of colon cancers begin with a mutation in the gene coding for the tumor suppressor protein Adenomatous Polyposis Coli, APC. APC protein can shuttle between the cytoplasm and the nucleus of cells, facilitated by two nuclear localization signals (NLS) and multiple nuclear export signals. To better understand functions of nuclear APC, our lab generated a “knock-in” mouse model with mutations introduced that inactivate both Apc NLS. These ApcmNLS/mNLS mice show a dramatic decrease in nuclear Apc. We previously showed increased proliferation and Wnt target gene expression in intestinal epithelial cells from ApcmNLS/mNLS mice, suggesting a role for nuclear APC in inhibition of proliferation and Wnt signaling. We also showed increased tumor number and size in ApcMin mice if they also harbor the ApcmNLS allele. Here we examine the role of nuclear Apc in intestinal epithelial differentiation and stem cell homeostasis. Paraffin-embedded tissue from the intestines of Apc+/+ and ApcmNLS/mNLS mice was sectioned and stained for markers of enterocytes (alkaline phosphatase) and goblet cells (alcian blue). Quantification of the positive cells indicated that ApcmNLS/mNLS mice had fewer differentiated enterocytes and goblet cells than their wild-type littermates. Intestines from ApcmNLS/mNLS mice were also stained for markers of quiescent and active stem cells, DCAMKL-1 and Lgr5 respectively. Quantification of the various stem cell populations will be described. Together, our data supports a role for nuclear Apc in promoting enterocyte and goblet cell differentiation, in suppression of tumors, and in inhibiting intestinal cell proliferation and Wnt signaling. Information gathered from analysis of ApcmNLS/mNLS mice will contribute to our understanding of the functions of nuclear APC in tumor suppression and ultimately the mechanism of intestinal tumorigenesis. Citation Format: Matthew A. Miller, Maged Zeineldin, Kristi L. Neufeld. Demonstrating a role for nuclear Adenomatous polyposis coli in intestinal cell differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1978. doi:10.1158/1538-7445.AM2013-1978

Because mutation of tumor suppressor gene APC is the initiating step in most colorectal cancers (CRC), understanding the full spectrum of APC functions will illuminate better diagnostic, preventive and therapeutic strategies for the disease. Although APC shuttles between the cytoplasm and nucleus, testing proposed roles for nuclear APC in the context of a whole organism was only recently made possible using a mouse model compromised for nuclear Apc which we generated by introducing germline mutations that inactivate the Apc nuclear localization signals (ApcmNLS). Our previous analysis of ApcmNLS mice revealed a role for nuclear Apc in regulation of Wnt signal transduction and intestinal cell proliferation as well as in tumor suppression. In humans, chronic colitis significantly increases CRC risk and APC mutations occur late in this cancer progression. In the current study, we show increased expression of inflammatory mediators cyclo-oxygenase-2 (Cox-2) and macrophage-inflammatory-protein-2 (MIP-2) in colon epithelial cells from ApcmNLS/mNLS mice, suggesting a role for nuclear Apc in suppressing colitis-mediated colon cancer. To test this hypothesis, we initiated colon tumors with a single injection of the mutagen, azoxymethane (AOM) and promoted the tumors with repeated oral administration of dextran sodium sulfate (DSS) to induce colonic inflammation. When treated with AOM-DSS, ApcmNLS/mNLS mice developed more colonic tumors than treated wildtype mice. Tumors from treated ApcmNLS/mNLS and wildtype mice had the same spectrum of β-catenin mutations, proliferation rates and histopathological features, consistent with the ApcmNLS allele enhancing colitis-associated tumor initiation rather than progression. ApcmNLS/mNLS mice had increased weight loss and colonic lymphoid follicles implicating nuclear Apc in suppression of AOM-DSS-induced colitis. These findings reveal novel functions for nuclear Apc and also indicate a critical protective role for Apc early in inflammation-induced colon tumorigenesis. 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 1342. doi:1538-7445.AM2012-1342