Transcription factor expression repertoire basis for epigenetic and transcriptional subtypes of colorectal cancers.

Colorectal cancers (CRCs) form a heterogenous group classified into epigenetic and transcriptional subtypes. The basis for the epigenetic subtypes, exemplified by varying degrees of promoter DNA hypermethylation, and its relation to the transcriptional subtypes is not well understood. We link cancer-specific transcription factor (TF) expression alterations to methylation alterations near TF-binding sites at promoter and enhancer regions in CRCs and their premalignant precursor lesions to provide mechanistic insights into the origins and evolution of the CRC molecular subtypes. A gradient of TF expression changes forms a basis for the subtypes of abnormal DNA methylation, termed CpG-island promoter DNA methylation phenotypes (CIMPs), in CRCs and other cancers. CIMP is tightly correlated with cancer-specific hypermethylation at enhancers, which we term CpG-enhancer methylation phenotype (CEMP). Coordinated promoter and enhancer methylation appears to be driven by downregulation of TFs with common binding sites at the hypermethylated enhancers and promoters. The altered expression of TFs related to hypermethylator subtypes occurs early during CRC development, detectable in premalignant adenomas. TF-based profiling further identifies patients with worse overall survival. Importantly, altered expression of these TFs discriminates the transcriptome-based consensus molecular subtypes (CMS), thus providing a common basis for CIMP and CMS subtypes.

A Rare Red Herring: Intrahepatic Splenosis Masquerading as Hepatocellular Carcinoma.

Splenosis is defined as viable splenic tissue that is autotransplanted into other compartments in the body. Intrahepatic splenosis is a rare diagnosis that can be difficult for clinicians to identify. The most common causes of splenosis include abdominal trauma and splenectomy. While most patients with intrahepatic splenosis are asymptomatic, in the presence of risk factors of hepatocellular carcinoma, it is paramount to rule out malignancy. In this report, we present a patient with imaging findings concerning for hepatocellular carcinoma, ultimately diagnosed with percutaneous biopsy and technetium-99m-tagged heat-damaged red blood cell scintigraphy-proven intrahepatic splenosis.

Pancreatoblastoma in Elderly Adults: Report of Two Patients.

Introduction. Pancreatoblastoma is a rare malignant epithelial neoplasm of the pancreas, which often shows multiple lines of differentiation, but is defined by neoplastic cells with acinar differentiation and characteristic squamoid nests. Pediatric patients are most commonly affected, and although a subset is known to occur in adults, the diagnosis is rarely considered in elderly adults. Methods. The clinicopathologic features of two cases of pancreatoblastoma in elderly patients were examined. Results. Two patients (age 80 and 81 years) presented with pancreatoblastoma, including one with early-stage pancreatic disease and one with liver metastasis. Biopsies and one pancreatic resection specimen showed characteristic histomorphologic features, including prominent acinar differentiation and abundant squamoid nests. Both cases had complete loss of SMAD4 (DPC4) immunolabeling. Next generation sequencing was performed on one case and revealed copy number loss of chromosome 11p and 9p21 (CDKN2A/B) and pathogenic or likely pathogenic variants in APC, SMAD4, and PIK3CA. The APC and SMAD4 variants occurred at allele frequencies suggestive of germline mutations, raising the possibility that this patient may have an inherited cancer predisposition syndrome. Conclusions. We present two cases which extend the upper age limit for reported pancreatoblastoma, including one with genetic findings suggestive of an inherited cancer predisposition syndrome.

Pediatric autoimmune gastritis: clinical correlates and histologic features.

Autoimmune gastritis is a well-known pathologic entity, but there are few studies that examine its clinical and histologic presentation in children. This is a single institution, retrospective study performed on patients diagnosed from 2011 through 2019. Patients were identified by their pathologic diagnosis within the laboratory information system. The electronic medical record and archived slides were reviewed. Twenty-two children (3 months to 18 years; median, 10.9 years) with autoimmune gastritis were diagnosed of a total of 14,257 nonconsultation gastric biopsies from unique patients (0.15% prevalence). Patients with autoimmune gastritis were diagnosed at an average age of 10.9 years and were mostly female (68.2% women, 31.8% men). The majority had extragastric immune disorders (13/22; 59.1%). All patients in the study had gastric body mucosa with enterochromaffin-like cell hyperplasia, atrophy, and histologic features of chronic injury. Most biopsies showed gastric body metaplasia (n = 19) or active gastric inflammation. However, antral atrophy was also observed in 12 patients, and antral metaplasia was identified in one patient; four patients had active chronic antral gastritis. All biopsies were negative for Helicobacter pylori. Pediatric autoimmune gastritis is a rare disorder that should be recognized because of its systemic effects with long-term morbidity. In addition, the possibility of tandem extragastric immune disorders should be considered when a diagnosis of pediatric autoimmune gastritis is established.

Smart Glasses as a Surgical Pathology Grossing Tool.

CONTEXT.­: Smart glasses are a wearable technology that enable hands-free data acquisition and entry. OBJECTIVE.­: To develop a surgical pathology grossing application on a smart glass platform. DESIGN.­: An existing logistics software for the Google Glass Enterprise smart glass platform was used to create surgical pathology grossing protocols. The 2 grossing protocols were developed to simulate grossing a complex (heart) and a simple (kidney) specimen. For both protocols, users were visually prompted by the smart glass device to perform each task, record measurements, or document the field of view. In addition to measuring the total time of the protocol performance, each substep within the protocol was automatically recorded. Subsequently, a report was generated that contained the dictation, images, voice recordings, and the timing of each step. The application was tested by 3 users using the 2 grossing protocols. The users were tracked across 3 grossing procedures for each protocol. RESULTS.­: For the complex specimen grossing the average time across repeated procedures was not significantly different between users (P > .99). However, when grossing times of the complex specimen were compared for repeated performances of the same user, a significant reduction in grossing times was observed with each repetition (P = .002). For the simple specimen, the average grossing time across multiple attempts was different among users (P = .03); however, no improvement in grossing time was observed with repeated performance (P = .499). CONCLUSIONS.­: Augmented reality based grossing applications can provide automated data collection to track the changes in grossing performance over time.

Lenalidomide-Associated Secondary B-Lymphoblastic Leukemia/Lymphoma-A Unique Entity.

OBJECTIVES: Autologous stem cell transplant with lenalidomide maintenance therapy has greatly improved the relapse-free and overall survival rates of patients with multiple myeloma but also has been associated with an increased risk of secondary B-lymphoblastic leukemia/lymphoma (B-ALL). METHODS: We report a comprehensive review of the clinicopathologic features of 2 patients with multiple myeloma who developed secondary B-ALL during lenalidomide maintenance. RESULTS: Our observations showed that the disease may initially present with subtle clinical, morphologic, and flow-cytometric findings. The flow cytometry findings in such cases may initially mimic an expansion of hematogones with minimal immunophenotypic variation. Both patients achieved complete remission of secondary B-ALL after standard chemotherapy; however, one patient continues to have minimal residual disease, and the other experienced relapse. Next-generation sequencing of the relapse specimen showed numerous, complex abnormalities, suggesting clonal evolution. CONCLUSIONS: Our findings suggest the need for increased awareness and further study of this unique form of secondary B-ALL.

Tissue-Specific Regulation of the Wnt/ß-Catenin Pathway by PAGE4 Inhibition of Tankyrase.

Spatiotemporal control of Wnt/ß-catenin signaling is critical for organism development and homeostasis. The poly-(ADP)-ribose polymerase Tankyrase (TNKS1) promotes Wnt/ß-catenin signaling through PARylation-mediated degradation of AXIN1, a component of the ß-catenin destruction complex. Although Wnt/ß-catenin is a niche-restricted signaling program, tissue-specific factors that regulate TNKS1 are not known. Here, we report prostate-associated gene 4 (PAGE4) as a tissue-specific TNKS1 inhibitor that robustly represses canonical Wnt/ß-catenin signaling in human cells, zebrafish, and mice. Structural and biochemical studies reveal that PAGE4 acts as an optimal substrate decoy that potently hijacks substrate binding sites on TNKS1 to prevent AXIN1 PARylation and degradation. Consistently, transgenic expression of PAGE4 in mice phenocopies TNKS1 knockout. Physiologically, PAGE4 is selectively expressed in stromal prostate fibroblasts and functions to establish a proper Wnt/ß-catenin signaling niche through suppression of autocrine signaling. Our findings reveal a non-canonical mechanism for TNKS1 inhibition that functions to establish tissue-specific control of the Wnt/ß-catenin pathway.

Regulation of tankyrase activity by a catalytic domain dimer interface.

Tankyrases (TNKS and TNKS2) are enzymes that catalyze poly-ADP-ribosylation (PARsylation) of their target proteins. Tankyrase-mediated PARsylation plays critical regulatory roles in cell signaling, particularly in the Wnt/ß-catenin pathway. The sterile alpha motif (SAM) domain in tankyrases mediates their oligomerization, which is essential for tankyrase function. The oligomerization regulates the catalytic activity of tankyrases, but the underlying mechanism is unclear. Our analyses of crystal structures of the tankyrase catalytic domain suggest that formation of a head-to-head dimer regulates the catalytic activity. Our activity assays show that residues in the catalytic domain dimer interface are important for the PARsylation activity of tankyrases both in solution and cells. The dimer is weak and may only form in the context of the SAM domain-mediated oligomers of tankyrases, consistent with the dependence of the tankyrase activity on the SAM domain.

Role of the Exocyst Complex Component Sec6/8 in Genomic Stability.

The exocyst is a heterooctomeric complex well appreciated for its role in the dynamic assembly of specialized membrane domains. Accumulating evidence indicates that this macromolecular machine also serves as a physical platform that coordinates regulatory cascades supporting biological systems such as host defense signaling, cell fate, and energy homeostasis. The isolation of multiple components of the DNA damage response (DDR) as exocyst-interacting proteins, together with the identification of Sec8 as a suppressor of the p53 response, suggested functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in resistance to ionizing radiation (IR) and accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Sec8 perturbation resulted in the accumulation of ATF2 and RNF20 and the promiscuous accumulation of DDR-associated chromatin marks and Rad51 repairosomes. Thus, the exocyst supports DNA repair fidelity by limiting the formation of repair chromatin in the absence of DNA damage.

Disruption of Wnt/ß-Catenin Signaling and Telomeric Shortening Are Inextricable Consequences of Tankyrase Inhibition in Human Cells.

Maintenance of chromosomal ends (telomeres) directly contributes to cancer cell immortalization. The telomere protection enzymes belonging to the tankyrase (Tnks) subfamily of poly(ADP-ribose) polymerases (PARPs) have recently been shown to also control transcriptional response to secreted Wnt signaling molecules. Whereas Tnks inhibitors are currently being developed as therapeutic agents for targeting Wnt-related cancers and as modulators of Wnt signaling in tissue-engineering agendas, their impact on telomere length maintenance remains unclear. Here, we leveraged a collection of Wnt pathway inhibitors with previously unassigned mechanisms of action to identify novel pharmacophores supporting Tnks inhibition. A multifaceted experimental approach that included structural, biochemical, and cell biological analyses revealed two distinct chemotypes with selectivity for Tnks enzymes. Using these reagents, we revealed that Tnks inhibition rapidly induces DNA damage at telomeres and telomeric shortening upon long-term chemical exposure in cultured cells. On the other hand, inhibitors of the Wnt acyltransferase Porcupine (Porcn) elicited neither effect. Thus, Tnks inhibitors impact telomere length maintenance independently of their affects on Wnt/ß-catenin signaling. We discuss the implications of these findings for anticancer and regenerative medicine agendas dependent upon chemical inhibitors of Wnt/ß-catenin signaling.

Identification of therapeutic small-molecule leads in cultured cells using multiplexed pathway reporter readouts.

The rapid expansion of molecular screening libraries in size and complexity in the last decade has outpaced the discovery rate of cost-effective strategies to single out reagents with sought-after cellular activities. In addition to representing high-priority therapeutic targets, intensely studied cell signaling systems encapsulate robust reference points for mapping novel chemical activities given our deep understanding of the molecular mechanisms that support their activity. In this chapter, we describe strategies for using transcriptional reporters of several well-interrogated signal transduction pathways coupled with high-throughput biochemical assays to fingerprint novel compounds for drug target identification agendas.

A multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells.

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Genome-wide RNAi screen reveals disease-associated genes that are common to Hedgehog and Wnt signaling.

The Hedgehog (Hh) and Wnt signal transduction pathways are master regulators of embryogenesis and tissue renewal and represent anticancer therapeutic targets. Using genome-wide RNA interference screening in murine cultured cells, we established previously unknown associations between these signaling pathways and genes linked to developmental malformations, diseases of premature tissue degeneration, and cancer. We identified functions in both pathways for the multitasking kinase Stk11 (also known as Lkb1), a tumor suppressor implicated in lung and cervical cancers. We found that Stk11 loss resulted in disassembly of the primary cilium, a cellular organizing center for Hh pathway components, thus dampening Hh signaling. Loss of Stk11 also induced aberrant signaling through the Wnt pathway. Chemicals that targeted the Wnt acyltransferase Porcupine or that restored primary cilia length by inhibiting the tubulin deacetylase HDAC6 (histone deacetylase 6) countered deviant pathway activities driven by Stk11 loss. Our study demonstrates that Stk11 is a critical mediator in both the Hh and the Wnt pathways, and our approach provides a platform to support the development of targeted therapeutic strategies.