SMAD4 Mutations Research Articles

Suppressive cancer nonstop extension mutations increase C-terminal hydrophobicity and disrupt evolutionarily conserved amino acid patterns

Nonstop extension mutations, a.k.a. stop-lost or stop-loss mutations, convert a stop codon into a sense codon resulting in translation into the 3’ untranslated region until the next in-frame stop codon, thereby extending the C-terminus of a protein. In cancer, only nonstop mutations in SMAD4 have been functionally characterized, while the impact of other nonstop mutations remain unknown. Here, we exploit our pan-cancer NonStopDB dataset and test all 2335 C-terminal extensions arising from somatic nonstop mutations in cancer for their impact on protein expression. In a high-throughput screen, 56.1% of the extensions effectively reduce protein abundance. Extensions of multiple tumor suppressor genes like PTEN, APC, B2M, CASP8, CDKN1B and MLH1 are effective and validated for their suppressive impact. Importantly, the effective extensions possess a higher hydrophobicity than the neutral extensions linking C-terminal hydrophobicity with protein destabilization. Analyzing the proteomes of eleven different species reveals conserved patterns of amino acid distribution in the C-terminal regions of all proteins compared to the proteomes like an enrichment of lysine and arginine and a depletion of glycine, leucine, valine and isoleucine across species and kingdoms. These evolutionary selection patterns are disrupted in the cancer-derived effective nonstop extensions.

Molecular Profiling of Low-Grade Appendiceal Mucinous Neoplasms (LAMN).

Low-grade appendiceal mucinous neoplasia (LAMN) represents a relatively rare tumor of the appendix typically diagnosed incidentally through appendectomy for acute appendicitis. In cases where perforation occurs, mucinous content may disseminate into the abdominal cavity, leading to the development of pseudomyxoma peritonei (PMP). The primary objective of this study was to elucidate the molecular characteristics associated with various stages of LAMN and PMP. DNA was extracted from LAMN, primary PMPs, recurrent PMPs, and adenocarcinomas originating from LAMN. The subsequent analysis involved the examination of mutational hotspot regions within 50 cancer-related genes, covering over 2800 COSMIC mutations, utilizing amplicon-based next-generation sequencing (NGS). Our findings revealed activating somatic mutations within the MAPK-signaling pathway across all tumors examined. Specifically, 98.1% of cases showed mutations in KRAS, while one tumor harbored a BRAF mutation. Additionally, GNAS mutations were identified in 55.8% of tumors, with no significant difference observed between LAMN and PMP. While LAMN rarely displayed additional mutations, 42% of primary PMPs and 60% of recurrent PMPs showed additional mutations. Notably, both adenocarcinomas originating from LAMN showed mutations within TP53. Furthermore, 7.7% (4/52) of cases exhibited a potentially targetable KRAS G12C mutation. In four patients, NGS analysis was performed on both primary PMP and recurrent PMP/adenocarcinoma samples. While mutations in KRAS and GNAS were detected in almost all samples, 50% of recurrent cases displayed an additional SMAD4 mutation, suggesting a notable alteration during disease progression. Our findings indicate two key points: First, mutations within the MAPK pathway, particularly in KRAS, are evident across all tumors, along with a high frequency of GNAS mutations. Second, progression toward PMP or adenocarcinoma is associated with an accumulation of additional mutations within common oncogenic pathways.

Molecular genetic analysis of colorectal carcinoma with an aggressive extraintestinal immunohistochemical phenotype

Colorectal cancer (CRC) is a leading global cause of illness and death. There is a need for identification of better prognostic markers beyond traditional clinical variables like grade and stage. Previous research revealed that abnormal expression of cytokeratin 7 (CK7) and loss of the intestinal-specific Special AT-rich sequence-binding protein 2 (SATB2) are linked to poor CRC prognosis. This study aimed to explore these markers’ prognostic significance alongside two extraintestinal mucins (MUC5AC, MUC6), claudin 18, and MUC4 in 285 CRC cases using immunohistochemistry on tissue microarrays (TMAs). CK7 expression and SATB2-loss were associated with MUC5AC, MUC6, and claudin 18 positivity. These findings suggest a distinct "non-intestinal" immunohistochemical profile in CRC, often right-sided, SATB2-low, with atypical expression of CK7 and non-colorectal mucins (MUC5AC, MUC6). Strong MUC4 expression negatively impacted cancer-specific survival (hazard ratio = 2.7, p = 0.044). Genetic analysis via next-generation sequencing (NGS) in CK7 + CRCs and those with high MUC4 expression revealed prevalent mutations in TP53, APC, BRAF, KRAS, PIK3CA, FBXW7, and SMAD4, consistent with known CRC mutation patterns. NGS also identified druggable variants in BRAF, PIK3CA, and KRAS. CK7 + tumors showed intriguingly common (31.6%) BRAF V600E mutations corelating with poor prognosis, compared to the frequency described in the literature and databases. Further research on larger cohorts with a non-colorectal immunophenotype and high MUC4 expression is needed.

Clinical and genomic features of classical and basal transcriptional subtypes in pancreatic cancer.

Transcriptional profiling of pancreatic cancers (PC) has defined two main transcriptional subtypes, classical and basal. Initial data suggest shorter survival for patients with basal tumors and differing treatment sensitivity to FOLFIRINOX (FFX) and gemcitabine nab-Paclitaxel (GnP) by transcriptional subtype. We examined 8,743 patients with RNA sequencing from PCs performed at Caris Life Sciences (Phoenix, AZ). Classical and basal subtypes were identified using PurIST algorithm on RNA-sequencing and two cohorts were analyzed: (1) Biomarker cohort included patients with complete molecular profiling data (n = 7,250); (2) Outcomes cohort included patients with metastatic disease with available survival outcomes (n=5,335). In the biomarker cohort, 3,063 tumors (42.2%) were strongly classical (SC), and 2,015 tumors (27.8%) were strongly basal (SB). SC and SB tumors showed strong associations with histologic phenotypes and biopsy site. SB tumors had higher rates of KRAS, TP53, and ARID1A mutations, lower rates of SMAD4 mutation, and transcriptional evidence of epithelial mesenchymal transition. Sixty of 77 cases (78%) maintained their transcriptional subtype between temporally and/or spatially disparate lesions. In the outcomes cohort, SB subtype was associated with shorter overall survival time, regardless of whether they received FFX or GnP as first line chemotherapy. Mutant KRAS allele type was prognostic of outcomes, however this impact was restricted to SC tumors, whereas all mutant KRAS alleles had similarly poor outcomes in SB tumors. SB subtype is a strong independent predictor of worse outcomes, irrespective of upfront chemotherapy regimen. Clinical trials should investigate PC transcriptional subtypes as a biomarker.

The Role of TGF-β1 and Mutant SMAD4 on Epithelial-Mesenchymal Transition Features in Head and Neck Squamous Cell Carcinoma Cell Lines.

The aim of the present study was to investigate possible differences in the sensitivity of HNSCC cells to known EMT regulators. Three HNSCC cell lines (UM-SCC-1, -3, -22B) and the HaCaT control keratinocyte cell line were exposed to transforming growth factor beta 1 (TGF-β1), a known EMT master regulator, and the cellular response was evaluated by real-time cell analysis (RTCA), Western blot, quantitative PCR, flow cytometry, immunocytochemistry, and the wound closure (scratch) assay. Targeted sequencing on 50 cancer-related genes was performed using the Cancer Hotspot Panel v2. Mutant, and wild type SMAD4 cDNA was used to generate recombinant SMAD4 constructs for expression in mammalian cell lines. The most extensive response to TGF-β1, such as cell growth and migration, β-actin expression, or E-cadherin (CDH1) downregulation, was seen in cells with a more epithelial phenotype. Lower response correlated with higher basal p-TGFβ RII (Tyr424) levels, pointing to a possible autocrine pre-activation of these cell lines. Targeted sequencing revealed a homozygous SMAD4 mutation in the UM-SCC-22B cell line. Furthermore, PCR cloning of SMAD4 cDNA from the same cell line revealed an additional SMAD4 transcript with a 14 bp insertion mutation, which gives rise to a truncated SMAD4 protein. Overexpression of this mutant SMAD4 protein in the highly epithelial control cell line HaCaT resulted in upregulation of TGF-β1 and vimentin. Consistent with previous reports, the invasive and metastatic potential of HNSCC tumor cells appears associated with the level of autocrine secretion of EMT regulators such as TGF-β1, and it could be influenced by exogenous EMT cytokines such as those derived from immune cells of the tumor microenvironment. Furthermore, mutant SMAD4 appears to be a significant contributor to the mesenchymal transformation of HNSCC cells.

Abstract C015: Selective and pairwise epistatic effects of somatic mutations in KRAS wild-type pancreatic cancer

Abstract About 8 to 10% of all pancreatic adenocarcinomas (PAAD) do not possess canonical mutations in KRAS. A sizable proportion of these KRAS wild-type (WT) pancreatic cancers harbor mutations in alternative MAPK-pathway driver genes. These alternative MAPK-pathway genes represent a set of therapeutic targets that might be exploited to treat individuals in this PAAD subset. Previous studies have prioritized the mutational- target landscape of KRAS WT PAAD by ranking genes determined by significance thresholds to be drivers by the prevalence of their somatic mutations in tumors. These prevalences usefully inform the size of the patient population that would be candidates for targeted therapeutics. However, prevalence is markedly influenced by underlying mutation rate. It is not a metric of cancer effect; it does not directly quantify the degree to which a driver mutation increases cancer cell survival and proliferation, nor do observations of co-occurrence or mutual exclusivity quantify epistasis. Therefore, to better guide the development of targeted therapeutics, we assembled a rich dataset of 6,391 PAAD tumor sequences from TCGA, UTSW, ICGC, QCMG, Yale-Gilead, and the AACR Project GENIE.We analyzed the cancer effect sizes of somatic variants in PAADs. Furthermore, we determined the selective epistatic interactions between KRAS and other PAAD driver genes. Nine of the 20 highest-effect variants were nonsynonymous substitutions in KRAS, with the prevalence of these mutations ranging from 18 to 2390 (median 93). This high frequency of highly oncogenic KRAS mutants supports the longstanding conception that KRAS mutations dominate the oncogenic landscape of PAAD. However, of the 6,391 cases analyzed, 627 (9.8%) were KRAS WT. To further investigate the oncogenic landscape of these samples, we employed a pairwise epistasis model of selection. We discovered that not only are alternative MAPK drivers (BRAF and NRAS) present in KRAS WT PAAD, but that they show antagonistic epistasis with KRAS mutation. Moreover, we found that non-MAPK drivers CTNNB1 and IDH1 also exhibit substantial antagonistic epistasis with KRAS. Also, in a KRAS-substituted background, selection for mutations in tumor suppressor genes TP53, CDKN2A, and SMAD4 was increased. Furthermore, mutations in BRAF, NRAS, CTNNB1, TP53, CDKN2A, and SMAD4, genes commonly considered to be drivers of PAAD, in addition to a previously unrecognized driver IDH1, suppressed selection for KRAS mutations. Not only MAPK drivers, but also other oncogenic drivers of PAAD at both high and low prevalence exhibit antagonistic somatic selective epistasis. Treatments targeting these variants that are antagonistically epistatic with KRAS may rescue the full strength of selection on KRAS variants, warranting continued monitoring for KRAS variants in resurgent cancer. Moreover, as resistance to KRAS inhibitors is likely caused by gained alterations in alternative MAPK drivers, exploration of treatments targeting these mutations may additionally provide an avenue to overcome therapeutic resistance in PAADs. Citation Format: Rishi M Shah, Can M Ersahin, Jeffrey D Mandell, Vincent L Cannataro, Jeffrey P Townsend. Selective and pairwise epistatic effects of somatic mutations in KRAS wild-type pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C015.

Abstract C030: Evolutionary and epistatic analyses reveal genic interactions with KRAS during malignant progression of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) patients face poor prognoses attributable to delayed disease detection, resulting in distant metastases and thereby reducing treatment efficacy. The challenge of detecting PDAC lesions during early stages emanates from an inauspicious trifecta of clinical factors: early-stage malignancies are often asymptomatic, existing biomarkers are insufficiently sensitive, and the physical location of the pancreas stymies reliable screening. A promising strategy towards improving outcomes is the resection of high-risk, pre-malignant lesions deemed likely to progress into carcinomas. Intraductal papillary mucinous neoplasms (IPMNs) are both large enough to be detected during abdominal imaging and can originate PDAC lesions. However, a patient may have many IPMN cysts, many of which will never progress. As pancreatic resection carries with it a high risk of morbidity, distinguishing the the evolutionary trajectories of IPMNs that will and will not progress could inform treatment strategies. Additionally, these trajectories may serve as disruptable targets to manage pre- neoplastic lesions, preventing their progression to PDAC. The vast majority PDAC cases contain mutations in KRAS and GNAS with secondary driver mutations in TP53, SMAD4, CDKN2A, and RNF43. Recent work has compared the frequency of these mutations in PDAC with low-grade and high-grade IPMN lesions, finding that they more common in high than low- grade lesions. Interestingly, unlike KRAS or GNAS, KLF4 mutations are prevalent in low-grade IPMNs, rare in high-grade IPMNs, and almost never observed in full-fledged carcinoma, suggesting distinct evolutionary trajectories possibly mediated by epistatic interactions between KLF4 and KRAS. However, the prevalence of a mutation is not dispositive as to its importance in tumorgenesis. Mutations in TTN, for instance, are frequently observed in PDAC and other cancers, but are widely considered to play a modest role, if any, in these cancers. To understand the contribution of a mutation to the evolution of cancer, we must instead deconvolve selection from baseline mutation rate across genomic sites and among patients to distinguish truly oncogenic mutations from mutations that occur frequently merely due to high underlying mutational rates. Here, we quantify the evolutionary and epistatic interplay between KRAS, KLF4, and other frequent mutations in IPMNs and PDAC during malignant transition. First, we report the cancer effect sizes of single-nucleotide variants in IPMNs at varying dysplastic stages. Then, we infer the synergistic and antagonistic interactions between driver mutations, providing insight into the order in which driver mutations occur. We then corroborate this epistasis-inferred ordering with the ordering of mutations recovered from ancestral state reconstruction derived from time-calibrated phylogenies. Lastly, we probe the mutational processes most responsible for conferring highly oncogenic mutations in IPMNs and PDACs to identify potentially actionable or preventable pathways of disease progression. Metabolism Citation Format: Nic Fisk, Derek Song, Margaret Moore, Cole G Jensen, Vincent L. Cannataro, Mofeed Nagib, Jeffrey D Mandell, Luisa Escobar-Hoyos, John W Kunstman, Jeffrey P Townsend. Evolutionary and epistatic analyses reveal genic interactions with KRAS during malignant progression of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C030.

Abstract PR-21: Temporal stability and chemotherapy responsiveness of classical and basal transcriptional subtypes of pancreatic cancer

Abstract Background: Transcriptional profiling of pancreatic cancers (PC) has defined classical and basal subtypes; basal tumors have worse outcomes. Initial data suggest worse outcomes to FOLFIRINOX (FFX) compared with gemcitabine nab-paclitaxel (GnP) in basal tumors. In addition, it is hypothesized that classical to basal conversion may lead to chemotherapy resistance, although clinical evidence for subtype conversion and subtype specific chemotherapy sensitivity are sparse. Here, we examined the temporal stability and clinical implications of PC transcriptional subtypes in a large, real-world dataset. Methods: We examined patients with metastatic PC, who had outcomes data and RNA sequencing (n=5,335) performed at Caris Life Sciences (Phoenix, AZ). Classical and basal subtypes were identified along a six subtype continuum from strong classical (SC) to strong basal (SB) on either pole using PurIST algorithm. Overall survival (OS) was defined from date of biopsy of metastatic site to date of death or last known contact and estimated using Kaplan-Meier method. Survival was compared between groups using log-rank test and cox proportional hazards regression. Predictors of subtype discordance were assessed in patients who underwent RNA-sequencing of two tumor biopsies using multivariable logistic regression. Results: In patients with metastatic PC, SB tumors (n = 1,579) had significantly worse OS compared to SC tumors (n = 2,118), with a median OS of 5.7 vs. 10.0 months, respectively (hazard ratio [HR] 1.49, 95% confidence interval [CI], 1.39-1.60, p = 2.77e-27) independent of impact of age, gender and mutations in KRAS, TP53, CDKN2A or SMAD4 in cox proportional hazard models. OS was worse in patients with SB tumors who received either 1L GnP [SB (n = 112) vs. SC (n = 178), median OS: 6.0 vs. 10.4 months (HR 1.35 [95% CI, 1.05-1.75], p = 0.02)], or 1L FFX [SB (n = 155) vs. SC (n = 215), median OS: 8.9 vs. 12.5 months (HR 1.35 (95% CI, 1.06-1.72), p = 0.013)]. The prognostic impact of transcriptional subtype within different KRAS-mutant alleles is not well defined. OS was short and not significantly different between the three most frequent KRAS alleles (G12 V/D/R) in SB tumors (median OS, KRAS G12R/V/D: 5.4 vs. 5.4 vs. 5.5 months). In SC tumors, KRAS G12D was associated with a significantly worse OS compared to KRAS G12R (median OS: 8.6 vs. 11.9 months, HR 1.23 (95% CI, 1.04-1.47), p = 0.018). KRAS G12V had a numerically worse survival compared with KRAS G12R in SC tumors (median OS: 9.4 vs. 11.9 months, HR 1.15 (95% CI, 0.96-1.37), p = 0.12). Sixty of 77 cases (78%) maintained their transcriptional subtype between two temporally &/or spatially disparate lesions. The likelihood of transcriptional subtype change was independent of the duration between (p = 0.79) or site of (p = 0.37) the two biopsies. Conclusions: SB subtype is a strong independent predictor of worse outcomes, irrespective of mutant KRAS allele or upfront chemotherapy regimen, and tends to remain stable between biopsies in individual patients Citation Format: Harshabad Singh, Joanne Xiu, Kevin Kapner, Chen Yuan, Raja R Narayan, Matthew Oberley, Alex Farrell, Rishi Surana, Brandon M Huffmann, Kimberly J Perez, James M Cleary, Alexander C Jordan, Andressa Dias Costa, Hannah L Williams, Srivatsan Raghavan, Benjamin Weinberg, Michael J Pishvaian, Rachna T Shroff, Sanjay Goel, Stephanie K Dougan, Jonathan Nowak, David Spetzler, George Sledge, Brian M Wolpin, Andrew J Aguirre. Temporal stability and chemotherapy responsiveness of classical and basal transcriptional subtypes of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-21.

Identification of TPI1 As a potential therapeutic target in pancreatic cancer with dependency of TP53 mutation using multi-omics analysis.

Mutations of KRAS, CDKN2A, TP53, and SMAD4 are the four major driver genes for pancreatic ductal adenocarcinoma (PDAC), of which mutations of KRAS and TP53 are the most frequently recognized. However, molecular-targeted therapies for mutations of KRAS and TP53 have not yet been developed. To identify novel molecular targets, we newly established organoids with the Kras mutation (KrasmuOR) and Trp53 loss of function using Cre transduction and CRISPR/Cas9 (Krasmu/p53muOR) from murine epithelia of the pancreatic duct in KrasLSL-G12D mice, and then analyzed the proteomic and metabolomic profiles in both organoids by mass spectrometry. Hyperfunction of the glycolysis pathway was recognized in Krasmu/p53muOR compared with KrasmuOR. Loss of function of triosephosphate isomerase (TPI1), which is involved in glycolysis, induced a reduction of cell proliferation in human PDAC cell lines with the TP53 mutation, but not in PDAC or in human fibroblasts without TP53 mutation. The TP53 mutation is clinically recognized in 70% of patients with PDAC. In the present study, protein expression of TPI1 and nuclear accumulation of p53 were recognized in the same patients with PDAC. TPI1 is a potential candidate therapeutic target for PDAC with the TP53 mutation.

Integrated multi-omics characterization of SMAD4 mutant colorectal cancer

Colorectal cancer is one of the most common cancers around the world, which is a severe threat to people’s health. SMAD4 belongs to the dwarfin/SMAD family, which plays a crucial role in TGF-β and BMP signal pathways. As the molecular characterization of colon cancer patients following SMAD4 mutations remains unclear, we integrated multi-omics data of SMAD4 mutant patients to reveal the profile of molecular characterization of SMAD4 mutation. A missense mutation is the most common mutant type of SMAD4. Patients with SMAD4 mutation had worse survival. Tumor tissues from patients carrying the SMAD4 mutation showed a reduction in various immune cells, such as CD4 + memory T cells and memory B cells. Many differential genes were identified compared to the SMAD4 mutation-free group and could be significantly enriched for tumor- and immune-related signaling pathways. In addition, the mutant group had different drug sensitivities than the non-mutant group.

SMAD4 mutations causing Myhre syndrome are under positive selection in the male germline

While it is widely thought that de novo mutations (DNMs) occur randomly, we previously showed that some DNMs are enriched because they are positively selected in the testes of aging men. These "selfish" mutations cause disorders with a shared presentation of features, including exclusive paternal origin, significant increase of the father's age, and high apparent germline mutation rate. To date, all known selfish mutations cluster within the components of the RTK-RAS-MAPK signaling pathway, a critical modulator of testicular homeostasis. Here, we demonstrate the selfish nature of the SMAD4 DNMs causing Myhre syndrome (MYHRS). By analyzing 16 informative trios, we show that MYHRS-causing DNMs originated on the paternally derived allele in all cases. We document a statistically significant epidemiological paternal age effect of 6.3 years excess for fathers of MYHRS probands. We developed an ultra-sensitive assay to quantify spontaneous MYHRS-causing SMAD4 variants in sperm and show that pathogenic variants at codon 500 are found at elevated level in sperm of most men and exhibit a strong positive correlation with donor's age, indicative of a high apparent germline mutation rate. Finally, we performed invitro assays to validate the peculiar functional behavior of the clonally selected DNMs and explored the basis of the pathophysiology of the different SMAD4 sperm-enriched variants. Taken together, these data provide compelling evidence that SMAD4, a gene operating outside the canonical RAS-MAPK signaling pathway, is associated with selfish spermatogonial selection and raises the possibility that other genes/pathways are under positive selection in the aging human testis.

Abstract PR007: Delineating functional drivers of esophageal adenocarcinoma to identify synthetic lethal interactions

Abstract Application of molecular targeted therapies for esophageal adenocarcinoma (EAC) has been limited by a lack of druggable oncogenic drivers. We propose that synthetic lethal interactions may provide new opportunities for targeted therapies in EAC. We have taken an integrated multi-omics approach incorporating Perturb-Seq (CRISPR editing combined with single cell RNA sequencing) and in vivo tumorigenesis assays to perform high-throughput characterisation of >70 high-confidence EAC driver genes, and genome-wide CRISPR-Cas9 knockout screens in isogenic models of EAC tumorigenesis to identify disease relevant synthetic lethal genetic interactions. MS-based proteomics, reverse phase protein arrays, polysome profiling and bulk RNA-sequencing of isogenic models were utilised to interrogate the biology of bona fide EAC drivers and associated driver specific gene dependencies. The overall goals were to (i) enhance our understanding of EAC tumorigenesis, (ii) identify potential opportunities for therapeutic interventions targeting EAC drivers via synthetic lethal-like approaches, and (iii) reduce the complexity of genetic heterogeneity by categorising EAC drivers with similar phenotypic outcomes. Through our approach we have identified complex crosstalk between the tumor suppressor SMAD4 and regulation of mTOR signaling, with specific downstream effects on translational reprogramming in EAC. Mutation or loss of SMAD4 occurs in up to 20% of EAC, but not pre-malignant tissue (Barrett’s esophagus), and is sufficient to promote transformation of pre-malignant cells in our in vivo tumorigenesis model. In this model, xenotransplanted SMAD4-deficient (via CRISPR-Cas9 knockout or shRNA knockdown) Barrett’s metaplasia cells formed invasive, metastatic tumors after a period of latency. SMAD4 deficient cells had downregulated expression of 4E-BP1, which inhibits EIF4E, the cap-dependent translation initiation factor. This was accompanied by increased mTOR activity, including phosphorylation and inactivation of 4EBP1. Moreover, we found that SMAD4-deficient cells preferentially upregulate cap-dependent translation at the expense of IRES mediated translation. Furthermore, perturbation of additional negative regulators of mTOR signaling in combination with SMAD4 knockout exacerbated these effects and accelerated tumorigenesis in vivo. We have extended these findings to a model of Barrett’s esophagus patient-derived organoids (PDOs) and observed increased proliferative potential of our genetically modified PDOs. Finally, analysing gene ontologies for differentially expressed genes from Perturb-seq revealed that driver-dependent transcriptional changes can be categorized into a smaller number of functional pathways allowing us to potentially consider groups of drivers as functional units. This work advances our understanding of EAC tumorigenesis, provides new mechanistic insights into SMAD4-driven transformation as well as novel potential therapeutic avenues for SMAD4-deficient EAC. Citation Format: Julia V. Milne, Ebtihal Mustafa, Kenji Fujihara, Eric Kusnadi, Anna Trigos, Niko Thio, Maree Pechlivanis, Carlos Cabalag, Twishi Gulati, Kaylene Simpson, Cuong Duong, Luc Furic, Wayne Phillips, Nicholas Clemons. Delineating functional drivers of esophageal adenocarcinoma to identify synthetic lethal interactions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR007.

Characteristics of patients (pts) with disease relapse despite absence of molecular residual disease (MRD) after resection of colorectal oligometastases (CRM): Implications from PRECISION study.

3602 Background: Detection of MRD after curative-intent resection of CRM is prognostic for disease relapse and may guide decisions regarding adjuvant chemotherapy (ACT). However, some pts without MRD detected immediately after resection may also experience disease relapse. We explored the features of pts enrolled in a prospective multicenter study with disease relapse who did and did not have post-resection MRD. Methods: Pts with previously untreated resectable CRM scheduled for surgery were prospectively enrolled between March 2021 and October 2022. Pretreatment genomic alterations were assessed in circulating tumor DNA (ctDNA) by Guardant360, while MRD was assessed 4 weeks after resection with a methylation-based ctDNA MRD assay (Guardant Reveal). Characteristics of pts who were MRD negative but relapsed later were analyzed. Results: Of 118 eligible pts enrolled, pretreatment ctDNA was detected in 99 (84%), 62 (53%) of whom had poor genomic profile, defined as a mutation in BRAF, RAS, PIK3CA, and/or SMAD4. Of 110 pts who underwent surgery, postoperative MRD was detected in 35 (32%). With a median follow-up time of 11.0 months (m), 37 pts recurred, of whom 14 were MRD negative and 23 MRD positive. MRD negative pts were more likely to develop lung metastases (64.3% vs. 30.4%, p=0.09) but less likely to have liver metastases (21.4% vs. 60.9%, p=0.03). Median recurrence-free survival (RFS) of MRD-negative relapsed pts was longer than that of MRD positive pts (8.6 m vs. 3.5 m; hazard ratio [HR], 0.28 [95% CI, 0.13-0.58]). Among MRD-negative pts with recurrence, risk factors most associated with recurrence were a history of lymph node metastases (LNM) (odds ratio [OR], 6.20 [95% CI, 1.28-30.1]) and poor pre-operative genomic profile (OR, 3.85 [95% CI, 1.08-13.70]). Among all resected pts, LNM (HR, 2.55 [95% CI, 1.10–5.89]), poor genomic profile (HR, 2.97 [95% CI, 1.29–6.85]), and MRD positivity (HR, 6.36 [95% CI, 2.96–13.64]) were significantly associated with shorter RFS in multivariate analyses. Conclusions: Pts with a history of LNM or poor pre-operative genomic profile were at risk of recurrence despite the absence of MRD after curative-intent resection of CRM. Clinical trial information: UMIN000042490.

Liquid biopsy multiomics enable clinical triage for early colorectal cancer diagnosis.

e15599 Background: Colorectal cancer (CRC) is the second most common cause of cancer death in the United State. Despite advancements in screening, diagnosis, and treatment, the disease remains a formidable public health issue. In 2023, approximately 153020 individuals were diagnosed with CRC and 52550 will die from the disease. In order to meet early diagnostic, we developed this multiomics assay to enable clinical triage for liquid biopsy screening. Methods: Xenonucleic acid (XNAs) are artificial genetic polymers that retain the Watson-Crick base-pairing capability and exhibit high chemical and biological stability. They can be employed as molecular clamps in RT-qPCR due to the stronger hybridizing/binding capability seen in XNA/DNA duplexes than DNA/DNA duplexes. We are developing an XNA-based multiplex RT-qPCR assay for APC, BRAF, CTNNB1, KRAS, NRAS, PIK3CA, SMAD4 and TP53 gene mutation panel and CRC 9 genes methylation panel combined with multiplex protein panel, called ColoscapeTM Plus. We enrolled in FIT positive subjects of both genders, aged 50-74, attending the CRC screening program, we tested 152 plasma samples, 75 health, 50 AA and 27 early CRC with this multiomics assay. Results: The assay results showed that its sensitivity was about 44% (95% CI: 0.302- 0.587) and specificity about 80% (95%CI: 0.689- 0.880) for advanced adenoma (AA). For CRC stage I and II, its sensitivity is about 63% and 78% with 80% specificity separately. Conclusions: The primary data has shown that this low cost and rapid turnaround time multiomics assay has a comparable sensitivity for early colorectal cancer diagnostic.

An overview of chemically induced rodent models for sporadic colorectal cancer: Histopathological and translational perspectives.

Globally, colorectal cancer (CRC) is one of the most frequently diagnosed human gastrointestinal neoplasia and the second leading cause of cancer-related death in both men and women. Despite considerable efforts currently devoted to the study of the biology and treatment of CRC, patient prognosis and survival are still poor. Sporadic CRC is a complex multistep disease and usually emerges in the setting of lifestyle and dietary changes mainly observed in industrialized countries with high human development index (HDI) (westernized style). The molecular pathogenesis of sporadic CRC presents genetic heterogeneity with APC, RAS, PIK3CA, TGFBR, SMAD4, and TP53 mutations usually detected during the progression of this malignancy. The establishment of sporadic CRC models has become essential for both basic and translational research to improve our understanding of the pathophysiology, unravel new molecular drivers, and preventive/therapeutic improvement of this malignancy. Chemically induced rodent models of sporadic CRC recapitulate most key morphological and genetic/epigenetic events observed during the promotion and progression of this malignancy, establishing effective diagnostic and prevention strategies to be translated into clinical practice. The present review gathers the main features of the state-of-the-art evidence on chemically induced rodent models, widely applied for translational modelling of sporadic CRC with a specific focus on histopathology and prevention perspectives. Our narrative review reinforces the persistent value of these bioassays and encourages the use of multimodel strategies for further investigations.

Characterization of necroptosis activators in colorectal cancer.

3522 Background: Necroptosis is a pro-inflammatory form of programmed cell death triggered by cellular stresses and extrinsic cytokines. It is a potential therapeutic target for immunotherapy (IO) in colorectal cancer (CRC). Here, we present comprehensive clinical and molecular characterization of key necroptosis regulators in CRC. Methods: 24,257 samples of CRC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq), NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES), and PD-L1 expression (22C3 clone; TPS ≥1%) were analyzed. Expression of activators in the necroptosis pathway ( RIPK3, MLKL, CYLD, and ZBP1) was categorized as either high (H) or low (L) using median expression as the cutoff. CRCs with high expression of all four activators were considered to have high propensity for necroptosis activity (NA-H, N=4595) compared to tumors with low expression in all activators (NA-L, N = 4946). RNA deconvolution analysis with QuantiSEQ estimated immune cell infiltration in the tumor microenvironment (TME). Differences in overall survival (OS) were analyzed from insurance claims data and calculated from treatment initiation using Kaplan-Meier estimates. Statistical significance was set as a P-value adjusted for multiple comparisons ( q<0.05). Results: NA-H was associated with increased PD-L1 positivity (4.0% vs 2.3%), immune cell infiltration, and T-cell inflamed score (all q<0.001). TP53 mutations were more frequent in NA-H, while SMAD2, SMAD4, KRAS, and NRAS mutations were associated with NA-L (all q<0.01). NA-H was associated with activation of KRAS signaling, allograft rejection, inflammatory/interferon gamma response, and pancreatic beta cell function pathways (all q<0.05). NA-H showed significantly improved OS in FOLFOX/FOLFIRI (F/F) (NA-L: 34.3 vs NA-H: 39.6 months [mo], P < 0.001; HR = 0.83, 95% CI 0.77 – 0.90) and those treated with IO (15.3 vs 22.4 mo, P = 0.018; HR = 0.77, 95% CI 0.62– 0.96) treated CRC patients. The benefit remained in TP53-mutated CRC treated with F/F (34.6 vs 41.0 mo, P < 0.001; HR = 0.82, 95% CI 0.74-0.90) and IO (10.0 vs 21.8 mo, P = 0.027; HR = 0.68, 95% CI 0.48-0.96) but not in the TP53-wild-type subgroup. Improved OS was observed with increased expression of CYLD and ZBP1(Table). The benefit remained in TP53-mutated CRC but not in the TP53 wildtype subgroup. Conclusions: This is the largest molecular and clinical characterization of necroptotic genes in CRC. Our data shows that activation of the necroptosis pathway is associated with immune cell infiltration/pathway activation, PD-L1 expression, and improved OS on F/F and IO. This benefit is more pronounced in TP53-mutated CRC, suggesting possible novel therapeutic targets.[Table: see text]

Juvenile polyposis syndrome in children: The impact of SMAD4 and BMPR1A mutations on clinical phenotype and polyp burden.

A constitutional disease-causing variant (DCV) in the SMAD4 or BMPR1A genes is present in 40%-60% of patients with juvenile polyposis syndrome (JPS). The aim of this study was to characterize the clinical course and polyp burden in children with DCV-positive JPS compared to DCV-negative JPS. Demographic, clinical, genetic, and endoscopic data of children with JPS were compiled from eight international centers in the ESPHGAN/NASPGHAN polyposis working group. A total of 124 children with JPS were included: 69 (56%) DCV-negative and 55 (44%) DCV-positive (53% SMAD4 and 47% BMPR1A) with a median (interquartile range) follow-up of 4 (2.8-6.4) years. DCV-positive children were diagnosed at an older age compared to DCV-negative children [12 (8-15.7) years vs. 5 (4-7) years, respectively, p < 0.001], had a higher frequency of family history of polyposis syndromes (50.9% vs. 1.4%, p < 0.001), experienced a greater frequency of extraintestinal manifestations (27.3% vs. 5.8%, p < 0.001), and underwent more gastrointestinal surgeries (16.4% vs. 1.4%, p = 0.002). The incidence rate ratio for the development of new colonic polyps was 6.15 (95% confidence interval 3.93-9.63, p < 0.001) in the DCV-positive group compared to the DCV-negative group, with an average of 12.2 versus 2 new polyps for every year of follow-up. There was no difference in the burden of polyps between patients with SMAD4 and BMPR1A mutations. This largest international cohort of pediatric JPS revealed that DCV-positive and DCV-negative children exhibit distinct clinical phenotype. These findings suggest a potential need of differentiated surveillance strategies based upon mutation status.

Genomic profiling of small bowel adenocarcinoma: a pooled analysis from 3 databases.

Small bowel adenocarcinoma is a rare disease. The genomic profiling tumours according to clinical characteristics and its impact on the prognosis remains unclear. A pooled analysis of clinical data, genomic profiling and MisMatch Repair (MMR) status from three databases was performed. A total of 188 tumour samples were analysed. A predisposing disease was reported in 22.3%, mainly Lynch syndrome and Crohn's disease. The tumours were localized in 80.2% and metastatic in 18.8%. The most frequent mutations were KRAS (42.0%) among them 7/79 are G12C, TP53 (40.4%), APC (19.1%), PIK3CA (18.6%), SMAD4 (12.8%) and ERBB2 (9.6%). Mutation distribution differed according to predisposing disease for TP53, ERBB2, IDH1, FGFR3, FGFR1 and KDR. KRAS and SMAD4 mutations were more frequent in metastatic tumour, whereas ERBB2 mutations were absent in metastatic tumour. For localized tumour, APC mutation was independently associated with a poor overall survival (OS) (p = 0.0254). 31.8% of localized tumours and 11.3% of metastatic tumours were dMMR (29.8% of the entire cohort). A dMMR status was associated with a better OS (HR = 0.61 [0.39-0.96], p = 0.0316). There is a different genomic profile according to the stage and predisposing disease. dMMR and APC mutation in localized tumour predict a better prognosis.

Pancreatic Cancer: A Review on Pathophysiology, Naturopathy, Clinical Treatment and Outcomes

Abstract: The study aimed to comprehend the molecular mechanisms and pathophysiology of pancreatic cancer with an emphasis on the advances in treatment options and the use of natural products as anticancer agents. The study involved a literature survey using PubMed, Web of Science and Google scholar database. The literature search was done using keywords “Pancreatic cancer”, “Chemotherapy”, “Mutations”, and “Natural compounds”. 266 articles were studied of which 201 were taken into consideration based on relevance to the topic. Pancreatic cancer is associated with mutations of CDKN2A (encoding p16), KRAS, TP53 and SMAD4. MAPK, PI3K-AKT, and TGF- β pathway dysfunction also led to pancreatic cancer. Current clinical trial activities in pancreatic cancer target angiogenesis, surface receptors, cell cycle, DNA damage response, etc. Studies have shown that combining surgical resection with adjuvant chemotherapy increases survival rates in patients. New treatment options are on the rise for this cancer type, which is perioperative or neo-adjuvant therapy. Gemcitabine as a single treatment agent in pancreatic cancer has shown promising response with chemotherapy regimens using two combinations- Folfirinox and Gemcitabine/Nab-Paclitaxel giving a better response rate. Numerous natural substances, including curcumin, aloe vera, and taxol, which suppress oxidative stress, angiogenesis, JAK2 STAT3 pathways, and enhanced natural killer cell activity, have been explored as potential treatments for pancreatic cancer. With pancreatic cancer having a poor prognosis, investigations to comprehend its molecular underpinnings and research on natural chemicals could lead to the development of safer treatment alternatives with enhanced survival rates for pancreatic cancer patients.

Longitudinal Comparative Analysis of Circulating Tumor DNA and Matched Tumor Tissue DNA in Patients with Metastatic Colorectal Cancer Receiving Palliative First-Line Systemic Anti-Cancer Therapy.

This study aimed to compare tumor tissue DNA (ttDNA) and circulating tumor DNA (ctDNA) to explore the clinical applicability of ctDNA and to better understand clonal evolution in patients with metastatic colorectal cancer undergoing palliative first-line systemic therapy. We performed targeted sequencing analysis of 88 cancer-associated genes using germline DNA, ctDNA at baseline (baseline-ctDNA), and ctDNA at progressive disease (PD-ctDNA). The results were compared with ttDNA data. Among 208 consecutively enrolled patients, we selected 84 (41 males; median age, 59 years; range, 35 to 90 years) with all four sample types available. A total of 202 driver mutations were found in 34 genes. ttDNA exhibited the highest mutation frequency (n=232), followed by baseline-ctDNA (n=155) and PD-ctDNA (n=117). Sequencing ctDNA alongside ttDNA revealed additional mutations in 40 patients (47.6%). PD-ctDNA detected 13 novel mutations in 10 patients (11.9%) compared to ttDNA and baseline-ctDNA. Notably, seven mutations in five patients (6.0%) were missense or nonsense mutations in APC, TP53, SMAD4, and CDH1 genes. In baseline-ctDNA, higher maximal variant allele frequency (VAF) values (p=0.010) and higher VAF values of APC (p=0.012), TP53 (p=0.012), and KRAS (p=0.005) mutations were significantly associated with worse overall survival. While ttDNA remains more sensitive than ctDNA, our ctDNA platform demonstrated validity and potential value when ttDNA was unavailable. Post-treatment analysis of PD-ctDNA unveiled new pathogenic mutations, signifying cancer's clonal evolution. Additionally, baseline-ctDNA's VAF values were prognostic after treatment.