Identification of Colorectal Cancer Cell Stemness from Single-Cell RNA Sequencing.

Cancer stem cells (CSC) play a critical role in metastasis, relapse, and therapy resistance in colorectal cancer. While characterization of the normal lineage of cell development in the intestine has led to the identification of many genes involved in the induction and maintenance of pluripotency, recent studies suggest significant heterogeneity in CSC populations. Moreover, while many canonical colorectal cancer CSC marker genes have been identified, the ability to use these classical markers to annotate stemness at the single-cell level is limited. In this study, we performed single-cell RNA sequencing on a cohort of 6 primary colon, 9 liver metastatic tumors, and 11 normal (nontumor) controls to identify colorectal CSCs at the single-cell level. Finding poor alignment of the 11 genes most used to identify colorectal CSC, we instead extracted a single-cell stemness signature (SCS_sig) that robustly identified "gold-standard" colorectal CSCs that expressed all marker genes. Using this SCS_sig to quantify stemness, we found that while normal epithelial cells show a bimodal distribution, indicating distinct stem and differentiated states, in tumor epithelial cells stemness is a continuum, suggesting greater plasticity in these cells. The SCS_sig score was quite variable between different tumors, reflective of the known transcriptomic heterogeneity of CRC. Notably, patients with higher SCS_sig scores had significantly shorter disease-free survival time after curative intent surgical resection, suggesting stemness is associated with relapse. IMPLICATIONS: This study reveals significant heterogeneity of expression of genes commonly used to identify colorectal CSCs, and identifies a novel stemness signature to identify these cells from scRNA-seq data.

Phase 1 study of the safety, pharmacokinetics, and preliminary efficacy of CA102N as monotherapy and in combination with trifluridine-tipiracil in patients with locally advanced or metastatic solid tumors.

CA102N is a covalently bound conjugate of modified nimesulide (Nim) and NaHA, the sodium salt of hyaluronic acid (HA). HA is a natural ligand of cluster of differentiation 44 (CD44), which is over-expressed in colorectal cancer (CRC). CA102N is designed to deliver nimesulide directly to the tumor via the interaction of HA and CD44. A Phase 1, 2-part (dose escalation, dose expansion), non-randomized, open-label, first-in-human study of CA102N, as monotherapy and in combination with trifluridine-tipiracil, was conducted in patients with locally advanced or metastatic solid tumors. The CA102N doses evaluated were 0.36 mg/kg, 0.54 mg/kg, and 0.72 mg/kg Nim equivalent. The primary endpoints were dose-limiting toxicities (DLTs) in Cycle 1 as well as serious adverse events (SAEs) and treatment-emergent adverse events (TEAEs) throughout the study; secondary endpoints were pharmacodynamics parameters, objective tumor response, and urinary pharmacodynamics markers of target inhibition. Between April 2019 and October 2021, 37 patients were enrolled in 3 US centers. No DLTs were observed in Part 1, and 0.72 mg/kg Nim equivalent was the dose selected for Part 2. In total, 52 TEAEs in 18 patients were CA102N-related; 4 (in 3 patients) were ≥ Grade 3. Exploratory analysis in the dose expansion cohort revealed a median progression-free survival of 3.7 (1.0, 6.77) months. Based on this study, CA102N as monotherapy or in combination with trifluridine-tipiracil, was safe and well-tolerated at the recommended Phase 2 dose of 0.72 mg/kg Nim equivalent in patients with locally advanced or metastatic solid tumors. Preliminary evidence of antitumor activity in CRC warrants further clinical development. (ClinicalTrials.gov registration number: NCT03616574. Registration date: August 6, 2018).

An Aspirin a Day: New Pharmacological Developments and Cancer Chemoprevention.

Chemoprevention refers to the use of natural or synthetic agents to reverse, suppress, or prevent the progression or recurrence of cancer. A large body of preclinical and clinical data suggest the ability of aspirin to prevent precursor lesions and cancers, but much of the clinical data are inferential and based on descriptive epidemiology, case control, and cohort studies or studies designed to answer other questions (e.g., cardiovascular mortality). Multiple pharmacological, clinical, and epidemiologic studies suggest that aspirin can prevent certain cancers but may also cause other effects depending on the tissue or disease and organ site in question. The best-known biological targets of aspirin are cyclooxygenases, which drive a wide variety of functions, including hemostasis, inflammation, and immune modulation. Newly recognized molecular and cellular interactions suggest additional modifiable functional targets, and the existence of consensus molecular cancer subtypes suggests that aspirin may have differential effects based on tumor heterogeneity. This review focuses on new pharmacological developments and innovations in biopharmacology that clarify the potential role of aspirin in cancer chemoprevention.

Association of Prediagnosis Obesity and Postdiagnosis Aspirin With Survival From Stage IV Colorectal Cancer.

Importance: The potential relationship between obesity and colorectal cancer (CRC) outcome is poorly understood in patients with late-stage disease. Increased body mass index may negate aspirin use for cancer prevention, but its role as a factor on the effectiveness of postdiagnosis aspirin use is unclear. Objective: To evaluate how prediagnosis obesity and postdiagnosis aspirin use may be associated with overall survival in patients with late-stage colorectal cancer. Design, Setting, and Participants: This cross-sectional study used self-reported data from patients with metastatic or treatment-refractory disease who consented to a clinical protocol at MD Anderson Cancer Center, a large US cancer treatment center. Patients were enrolled between 2010 and 2018 and followed up for mortality through July 2020. Analyses were conducted through March 2022. Exposures: Body mass index in the decade prior to initial diagnosis and regular aspirin use at survey completion. Main Outcomes and Measures: Overall survival was measured from stage IV diagnosis until death or last follow-up. Cox proportional hazards models were constructed to estimate associations of prediagnosis obesity and postdiagnosis aspirin use with overall survival. Results: Of 656 patients included in this analysis, 280 (42.7%) were women, 135 (20.6%) were diagnosed with CRC before age 45 years, 414 (63.1%) were diagnosed between ages 45 and 65 years, and 107 (16.3%) were diagnosed at 65 years or older; 105 patients (16.0%) were Black or Hispanic, and 501 (76.4%) were non-Hispanic White. Controlling for age, sex, race, stage at initial diagnosis, and weight change between prediagnosis and survey date, patients with obesity in the decade prior to CRC diagnosis had significantly higher likelihood of death (hazard ratio, 1.45; 95% CI, 1.11-1.91) compared with those with normal prediagnosis body mass index. Furthermore, only patients with normal prediagnosis body mass index experienced significant survival benefit with postdiagnosis aspirin use (hazard ratio, 0.59; 95% CI, 0.39-0.90). Conclusions and Relevance: In this cross-sectional study, our findings suggest potentially differential tumor development in the long-term physiologic host environment of obesity. Confirmation and further evaluation are needed to determine whether prediagnosis body mass index may be used to estimate the benefit from postdiagnosis aspirin use.

Autologous humanized mouse models to study combination and single-agent immunotherapy for colorectal cancer patient-derived xenografts.

Designing studies of immunotherapy is limited due to a lack of pre-clinical models that reliably predict effective immunotherapy responses. To address this gap, we developed humanized mouse models of colorectal cancer (CRC) incorporating patient-derived xenografts (PDX) with human peripheral blood mononuclear cells (PBMC). Humanized mice with CRC PDXs were generated via engraftment of autologous (isolated from the same patients as the PDXs) or allogeneic (isolated from healthy donors) PBMCs. Human T cells were detected in mouse blood, tissues, and infiltrated the implanted PDXs. The inclusion of anti-PD-1 therapy revealed that tumor responses in autologous but not allogeneic models were more comparable to that of patients. An overall non-specific graft-vs-tumor effect occurred in allogeneic models and negatively correlated with that seen in patients. In contrast, autologous humanized mice more accurately correlated with treatment outcomes by engaging pre-existing tumor specific T-cell populations. As autologous T cells appear to be the major drivers of tumor response thus, autologous humanized mice may serve as models at predicting treatment outcomes in pre-clinical settings for therapies reliant on pre-existing tumor specific T-cell populations.

Targeting RAS Mutant Colorectal Cancer with Dual Inhibition of MEK and CDK4/6.

KRAS and NRAS mutations occur in 45% of colorectal cancers, with combined MAPK pathway and CDK4/6 inhibition identified as a potential therapeutic strategy. In the current study, this combinatorial treatment approach was evaluated in a co-clinical trial in patient-derived xenografts (PDX), and safety was established in a clinical trial of binimetinib and palbociclib in patients with metastatic colorectal cancer with RAS mutations. Across 18 PDX models undergoing dual inhibition of MEK and CDK4/6, 60% of tumors regressed, meeting the co-clinical trial primary endpoint. Prolonged duration of response occurred predominantly in TP53 wild-type models. Clinical evaluation of binimetinib and palbociclib in a safety lead-in confirmed safety and provided preliminary evidence of activity. Prolonged treatment in PDX models resulted in feedback activation of receptor tyrosine kinases and acquired resistance, which was reversed with a SHP2 inhibitor. These results highlight the clinical potential of this combination in colorectal cancer, along with the utility of PDX-based co-clinical trial platforms for drug development. SIGNIFICANCE: This co-clinical trial of combined MEK-CDK4/6 inhibition in RAS mutant colorectal cancer demonstrates therapeutic efficacy in patient-derived xenografts and safety in patients, identifies biomarkers of response, and uncovers targetable mechanisms of resistance.

Novel Markers for Liquid Biopsies in Cancer Management: Circulating Platelets and Extracellular Vesicles.

Although radiologic imaging and histologic assessment of tumor tissues are classic approaches for diagnosis and monitoring of treatment response, they have many limitations. These include challenges in distinguishing benign from malignant masses, difficult access to the tumor, high cost of the procedures, and tumor heterogeneity. In this setting, liquid biopsy has emerged as a potential alternative for both diagnostic and monitoring purposes. The approaches to liquid biopsy include cell-free DNA/circulating tumor DNA, long and micro noncoding RNAs, proteins/peptides, carbohydrates/lectins, lipids, and metabolites. Other approaches include detection and analysis of circulating tumor cells, extracellular vesicles, and tumor-activated platelets. Ultimately, reliable use of liquid biopsies requires bioinformatics and statistical integration of multiple datasets to achieve approval in a Clinical Laboratory Improvement Amendments setting. This review provides a balanced and critical assessment of recent discoveries regarding tumor-derived biomarkers in liquid biopsies along with the potential and pitfalls for cancer detection and longitudinal monitoring.

Precision Combination Therapies Based on Recurrent Oncogenic Coalterations.

Cancer cells depend on multiple driver alterations whose oncogenic effects can be suppressed by drug combinations. Here, we provide a comprehensive resource of precision combination therapies tailored to oncogenic coalterations that are recurrent across patient cohorts. To generate the resource, we developed Recurrent Features Leveraged for Combination Therapy (REFLECT), which integrates machine learning and cancer informatics algorithms. Using multiomic data, the method maps recurrent coalteration signatures in patient cohorts to combination therapies. We validated the REFLECT pipeline using data from patient-derived xenografts, in vitro drug screens, and a combination therapy clinical trial. These validations demonstrate that REFLECT-selected combination therapies have significantly improved efficacy, synergy, and survival outcomes. In patient cohorts with immunotherapy response markers, DNA repair aberrations, and HER2 activation, we have identified therapeutically actionable and recurrent coalteration signatures. REFLECT provides a resource and framework to design combination therapies tailored to tumor cohorts in data-driven clinical trials and preclinical studies. SIGNIFICANCE: We developed the predictive bioinformatics platform REFLECT and a multiomics- based precision combination therapy resource. The REFLECT-selected therapies lead to significant improvements in efficacy and patient survival in preclinical and clinical settings. Use of REFLECT can optimize therapeutic benefit through selection of drug combinations tailored to molecular signatures of tumors. See related commentary by Pugh and Haibe-Kains, p. 1416. This article is highlighted in the In This Issue feature, p. 1397.

Of vascular defense, hemostasis, cancer, and platelet biology: an evolutionary perspective.

We have established considerable expertise in studying the role of platelets in cancer biology. From this expertise, we were keen to recognize the numerous venous-, arterial-, microvascular-, and macrovascular thrombotic events and immunologic disorders are caused by severe, acute-respiratory-syndrome coronavirus 2 (SARS-CoV-2) infections. With this offering, we explore the evolutionary connections that place platelets at the center of hemostasis, immunity, and adaptive phylogeny. Coevolutionary changes have also occurred in vertebrate viruses and their vertebrate hosts that reflect their respective evolutionary interactions. As mammals adapted from aquatic to terrestrial life and the heavy blood loss associated with placentalization-based live birth, platelets evolved phylogenetically from thrombocytes toward higher megakaryocyte-blebbing-based production rates and the lack of nuclei. With no nuclei and robust RNA synthesis, this adaptation may have influenced viral replication to become less efficient after virus particles are engulfed. Human platelets express numerous receptors that bind viral particles, which developed from archetypal origins to initiate aggregation and exocytic-release of thrombo-, immuno-, angiogenic-, growth-, and repair-stimulatory granule contents. Whether by direct, evolutionary, selective pressure, or not, these responses may help to contain virus spread, attract immune cells for eradication, and stimulate angiogenesis, growth, and wound repair after viral damage. Because mammalian and marsupial platelets became smaller and more plate-like their biophysical properties improved in function, which facilitated distribution near vessel walls in fluid-shear fields. This adaptation increased the probability that platelets could then interact with and engulf shedding virus particles. Platelets also generate circulating microvesicles that increase membrane surface-area encounters and mark viral targets. In order to match virus-production rates, billions of platelets are generated and turned over per day to continually provide active defenses and adaptation to suppress the spectrum of evolving threats like SARS-CoV-2.

The Provocative Roles of Platelets in Liver Disease and Cancer.

Both platelets and the liver play important roles in the processes of coagulation and innate immunity. Platelet responses at the site of an injury are rapid; their immediate activation and structural changes minimize the loss of blood. The majority of coagulation proteins are produced by the liver-a multifunctional organ that also plays a critical role in many processes: removal of toxins and metabolism of fats, proteins, carbohydrates, and drugs. Chronic inflammation, trauma, or other causes of irreversible damage to the liver can dysregulate these pathways leading to organ and systemic abnormalities. In some cases, platelet-to-lymphocyte ratios can also be a predictor of disease outcome. An example is cirrhosis, which increases the risk of bleeding and prothrombotic events followed by activation of platelets. Along with a triggered coagulation cascade, the platelets increase the risk of pro-thrombotic events and contribute to cancer progression and metastasis. This progression and the resulting tissue destruction is physiologically comparable to a persistent, chronic wound. Various cancers, including colorectal cancer, have been associated with increased thrombocytosis, platelet activation, platelet-storage granule release, and thrombosis; anti-platelet agents can reduce cancer risk and progression. However, in cancer patients with pre-existing liver disease who are undergoing chemotherapy, the risk of thrombotic events becomes challenging to manage due to their inherent risk for bleeding. Chemotherapy, also known to induce damage to the liver, further increases the frequency of thrombotic events. Depending on individual patient risks, these factors acting together can disrupt the fragile balance between pro- and anti-coagulant processes, heightening liver thrombogenesis, and possibly providing a niche for circulating tumor cells to adhere to-thus promoting both liver metastasis and cancer-cell survival following treatment (that is, with minimal residual disease in the liver).

Methylation-eQTL analysis in cancer research.

MOTIVATION: DNA methylation is a key epigenetic factor regulating gene expression. While promoter methylation has been well studied, recent publications have revealed that functionally important methylation also occurs in intergenic and distal regions, and varies across genes and tissue types. Given the growing importance of inter-platform integrative genomic analyses, there is an urgent need to develop methods to discover and characterize gene-level relationships between methylation and expression. RESULTS: We introduce a novel sequential penalized regression approach to identify methylation-expression quantitative trait loci (methyl-eQTLs), a term that we have coined to represent, for each gene and tissue type, a sparse set of CpG loci best explaining gene expression and accompanying weights indicating direction and strength of association. Using TCGA and MD Anderson colorectal cohorts to build and validate our models, we demonstrate our strategy better explains expression variability than current commonly used gene-level methylation summaries. The methyl-eQTLs identified by our approach can be used to construct gene-level methylation summaries that are maximally correlated with gene expression for use in integrative models, and produce a tissue-specific summary of which genes appear to be strongly regulated by methylation. Our results introduce an important resource to the biomedical community for integrative genomics analyses involving DNA methylation. AVAILABILITY AND IMPLEMENTATION: We produce an R Shiny app (https://rstudio-prd-c1.pmacs.upenn.edu/methyl-eQTL/) that interactively presents methyl-eQTL results for colorectal, breast and pancreatic cancer. The source R code for this work is provided in the Supplementary Material. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pilot Clinical Trial of Perioperative Durvalumab and Tremelimumab in the Treatment of Resectable Colorectal Cancer Liver Metastases.

PURPOSE: Despite the prognostic importance of immune infiltrate in colorectal cancer, immunotherapy has demonstrated limited clinical activity in refractory metastatic proficient mismatch-repair (pMMR) colorectal cancer. This study explores combining anti-CTLA-4 and an anti-PD-L1 therapy in the preoperative management of resectable colorectal cancer liver metastases with the intent to improve immune responses in this disease setting. PATIENTS AND METHODS: Patients with resectable colorectal cancer liver-only metastases received one dose of tremelimumab and durvalumab preoperatively followed by single-agent durvalumab postoperatively. Primary objectives were to determine feasibility and safety. RESULTS: A total of 24 patients were enrolled between November 2016 and November 2019. Twenty-three patients received treatment [21 pMMR and 2 deficient mismatch-repair (dMMR)] and subsequently 17 (74%; 95% CI: 53%-88%) underwent surgical resection. Grade 3/4 treatment-related immune toxicity and postoperative grade 3/4 toxicity were seen in 5/23 (22%; 95% CI: 10%-44%) and 2/17 (12%; 95% CI: 2%-38%) patients. The median relapse-free survival (RFS) was 9.7 (95% CI: 8.1-17.8) months, and overall survival was 24.5 (95% CI: 16.5-28.4) months. Four patients demonstrated complete pathologic response, two dMMR patients and two POLE mutation patients. Pre- and post-tumor tissue analysis by flow cytometry, immunofluorescence, and RNA sequencing revealed similar levels of T-cell infiltration, but did demonstrate evidence of CD8+ and CD4+ activation posttreatment. An increase in B-cell transcriptome signature and B-cell density was present in posttreatment samples from patients with prolonged RFS. CONCLUSIONS: This study demonstrates the safety of neoadjuvant combination tremelimumab and durvalumab prior to colorectal cancer liver resection. Evidence for T- and B-cell activation following this therapy was seen in pMMR metastatic colorectal cancer.

Sequential Administration of XPO1 and ATR Inhibitors Enhances Therapeutic Response in TP53-mutated Colorectal Cancer.

BACKGROUND & AIMS: Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. METHODS: To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. RESULTS: Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. CONCLUSIONS: Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.

Development and Validation of a Gene Signature Classifier for Consensus Molecular Subtyping of Colorectal Carcinoma in a CLIA-Certified Setting.

PURPOSE: Consensus molecular subtyping (CMS) of colorectal cancer has potential to reshape the colorectal cancer landscape. We developed and validated an assay that is applicable on formalin-fixed, paraffin-embedded (FFPE) samples of colorectal cancer and implemented the assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. EXPERIMENTAL DESIGN: We performed an in silico experiment to build an optimal CMS classifier using a training set of 1,329 samples from 12 studies and validation set of 1,329 samples from 14 studies. We constructed an assay on the basis of NanoString CodeSets for the top 472 genes, and performed analyses on paired flash-frozen (FF)/FFPE samples from 175 colorectal cancers to adapt the classifier to FFPE samples using a subset of genes found to be concordant between FF and FFPE, tested the classifier's reproducibility and repeatability, and validated in a CLIA-certified laboratory. We assessed prognostic significance of CMS in 345 patients pooled across three clinical trials. RESULTS: The best classifier was weighted support vector machine with high accuracy across platforms and gene lists (>0.95), and the 472-gene model outperforming existing classifiers. We constructed subsets of 99 and 200 genes with high FF/FFPE concordance, and adapted FFPE-based classifier that had strong classification accuracy (>80%) relative to "gold standard" CMS. The classifier was reproducible to sample type and RNA quality, and demonstrated poor prognosis for CMS1-3 and good prognosis for CMS2 in metastatic colorectal cancer (P < 0.001). CONCLUSIONS: We developed and validated a colorectal cancer CMS assay that is ready for use in clinical trials, to assess prognosis in standard-of-care settings and explore as predictor of therapy response.

Sulindac plus a phospholipid is effective for polyp reduction and safer than sulindac alone in a mouse model of colorectal cancer development.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and sulindac are effective for colorectal cancer prevention in humans and some animal models, but concerns over gastro-intestinal (GI) ulceration and bleeding limit their potential for chemopreventive use in broader populations. Recently, the combination of aspirin with a phospholipid, packaged as PL-ASA, was shown to reduce GI toxicity in a small clinical trial. However, these studies were done for relatively short periods of time. Since prolonged, regular use is needed for chemopreventive benefit, it is important to know whether GI safety is maintained over longer use periods and whether cancer prevention efficacy is preserved when an NSAID is combined with a phospholipid. METHODS: As a first step to answering these questions, we treated seven to eight-week-old, male and female C57B/6 Apcmin/+ mice with the NSAID sulindac, with and without phosphatidylcholine (PC) for 3-weeks. At the end of the treatment period, we evaluated polyp burden, gastric toxicity, urinary prostaglandins (as a marker of sulindac target engagement), and blood chemistries. RESULTS: Both sulindac and sulindac-PC treatments resulted in significantly reduced polyp burden, and decreased urinary prostaglandins, but sulindac-PC treatment also resulted in the reduction of gastric lesions compared to sulindac alone. CONCLUSIONS: Together these data provide pre-clinical support for combining NSAIDs with a phospholipid, such as phosphatidylcholine to reduce GI toxicity while maintaining chemopreventive efficacy.

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

Proteome Instability Is a Therapeutic Vulnerability in Mismatch Repair-Deficient Cancer.

Deficient DNA mismatch repair (dMMR) induces a hypermutator phenotype that can lead to tumorigenesis; however, the functional impact of the high mutation burden resulting from this phenotype remains poorly explored. Here, we demonstrate that dMMR-induced destabilizing mutations lead to proteome instability in dMMR tumors, resulting in an abundance of misfolded protein aggregates. To compensate, dMMR cells utilize a Nedd8-mediated degradation pathway to facilitate clearance of misfolded proteins. Blockade of this Nedd8 clearance pathway with MLN4924 causes accumulation of misfolded protein aggregates, ultimately inducing immunogenic cell death in dMMR cancer cells. To leverage this immunogenic cell death, we combined MLN4924 treatment with PD1 inhibition and found the combination was synergistic, significantly improving efficacy over either treatment alone.

Signet ring cell colorectal cancer: genomic insights into a rare subpopulation of colorectal adenocarcinoma.

BACKGROUND: Signet ring cell carcinoma (SRCC) is a rare subtype of colorectal cancer (CRC). The aim of this study was to characterise the genomic alterations and outcomes of SRCC. METHODS: Medical records of metastatic CRC (mCRC) patients whose tumours were evaluated by NGS analysis were reviewed. SC-mCRC were classified into two groups: SRCC (>50% signet ring cells) and adenocarcinoma (AC) with SC component (≤50% signet ring cells). RESULTS: Six hundred and sixty-five mCRC patients were included. Of the 93 mCRC cases with SC features, 63 had slides for review. Of those 63 cases, 35 were confirmed SRCC, and 28 were AC with SC component. Compared with AC group, KRAS and PIK3CA mutations (mts) were found in only 11% (OR: 0.13) and 3% (OR: 0.15) of SRCC cases, respectively. In contrast to the 44% rate of APC mts in AC group, only 3% of SRCC patients had APC mts (OR = 0.04). CONCLUSIONS: SRCC has distinct molecular features, including low rates of KRAS, PIK3CA and APC mts. Further study to identify activation pathways and potential therapeutic targets are needed.

CTC analysis: an update on technological progress.

There is a growing need for a more accurate, real-time assessment of tumor status and the probability of metastasis, relapse, or response to treatment. Conventional means of assessment include imaging and tissue biopsies that can be highly invasive, may not provide complete information of the disease's heterogeneity, and not ideal for repeat analysis. Therefore, a less-invasive means of acquiring similar information at greater time points is necessary. Liquid biopsies are samples of a patients' peripheral blood and hold potential of addressing these criteria. Ongoing research has revealed that a tumor can release circulating cells, genetic materials (DNA or RNA), and exosomes into circulation. These potential biomarkers can be captured in a liquid biopsy and analyzed to determine disease status. To achieve these goals, numerous technologies have been developed. In this review, we discuss both prominent and newly developed technologies for circulating tumor cell capture and analysis and their clinical impact.

Clinical and molecular characterization of early-onset colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) incidence is increasing in adults younger than 50 years. This study evaluated clinical and molecular features to identify those features unique to early-onset CRC that differentiate these patients from patients 50 years old or older. METHODS: Baseline characteristics were evaluated according to the CRC onset age with 3 independent cohorts. A fourth cohort was used to describe the impact of age on the consensus molecular subtype (CMS) prevalence. RESULTS: This retrospective review of more than 36,000 patients with CRC showed that early-onset patients were more likely to have microsatellite instability (P = .038), synchronous metastatic disease (P = .009), primary tumors in the distal colon or rectum (P < .0001), and fewer BRAF V600 mutations (P < .001) in comparison with patients 50 years old or older. Patients aged 18 to 29 years had fewer adenomatous polyposis coli (APC) mutations (odds ratio [OR], 0.56; 95% confidence interval [CI], 0.35-0.90; P = .015) and an increased prevalence of signet ring histology (OR, 4.89; 95% CI, 3.23-7.39; P < .0001) in comparison with other patients younger than 50 years. In patients younger than 40 years, CMS1 was the most common subtype, whereas CMS3 and CMS4 were uncommon (P = .003). CMS2 was relatively stable across age groups. Early-onset patients with inflammatory bowel disease were more likely to have mucinous or signet ring histology (OR, 5.54; 95% CI, 2.24-13.74; P = .0004) and less likely to have APC mutations (OR, 0.24; 95% CI, 0.07-0.75; P = .019) in comparison with early-onset patients without predisposing conditions. CONCLUSIONS: Early-onset CRC is not only distinct from traditional CRC: special consideration should be given to and further investigations should be performed for both very young patients with CRC (18-29 years) and those with predisposing conditions. The etiology of the high rate of CMS1 in patients younger than 40 years deserves further exploration.