Despite remarkable success of immune checkpoint inhibitors, the majority of cancer patients have yet to receive durable benefits. Here, in order to investigate the metabolic alterations in response ...to immune checkpoint blockade, we comprehensively profile serum metabolites in advanced melanoma and renal cell carcinoma patients treated with nivolumab, an antibody against programmed cell death protein 1 (PD1). We identify serum kynurenine/tryptophan ratio increases as an adaptive resistance mechanism associated with worse overall survival. This advocates for patient stratification and metabolic monitoring in immunotherapy clinical trials including those combining PD1 blockade with indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase (IDO/TDO) inhibitors.
The RNF43_p.G659fs mutation occurs frequently in colorectal cancer, but its function remains poorly understood and there are no specific therapies directed against this alteration. In this study, we ...find that RNF43_p.G659fs promotes cell growth independent of Wnt signaling. We perform a drug repurposing library screen and discover that cells with RNF43_p.G659 mutations are selectively killed by inhibition of PI3K signaling. PI3K/mTOR inhibitors yield promising antitumor activity in RNF43
isogenic cell lines and xenograft models, as well as in patient-derived organoids harboring RNF43_p.G659fs mutations. We find that RNF43
binds p85 leading to increased PI3K signaling through p85 ubiquitination and degradation. Additionally, RNA-sequencing of RNF43
isogenic cells reveals decreased interferon response gene expression, that is reversed by PI3K/mTOR inhibition, suggesting that RNF43
may alter tumor immunity. Our findings suggest a therapeutic application for PI3K/mTOR inhibitors in treating RNF43_p.G659fs mutant cancers.
Immunotherapy with checkpoint inhibitors, such as the programmed death-1 (PD-1) antibodies pembrolizumab and nivolumab, are effective in a variety of tumors, yet not all patients respond. Tumor ...microsatellite instability-high (MSI-H) has emerged as a biomarker of response to checkpoint blockade, leading to the tissue agnostic approval of pembrolizumab in MSI-H cancers. Here we describe a patient with MSI-H colorectal cancer that was treated with this immune checkpoint inhibitor and exhibited progression of disease. We examined this intrinsic resistance through genomic, transcriptional, and pathologic characterization of the patient's tumor and the associated immune microenvironment. The tumor had typical MSI-H molecular features, including a high neoantigen load. We also identified biallelic loss of the gene for β
-microglobulin (
), whose product is critical for antigen presentation. Immune infiltration deconvolution analysis of bulk transcriptome data from this anti-PD-1-resistant tumor and hundreds of other colorectal cancer specimens revealed a high natural killer cell and M2 macrophage infiltration in the patient's cancer. This was confirmed by single-cell transcriptome analysis and multiplex immunofluorescence. Our study provides insight into resistance in MSI-H tumors and suggests immunotherapeutic strategies in additional genomic contexts of colorectal cancer.
Routine tumor-node-metastasis (TNM) staging of colorectal cancer is imperfect in predicting survival due to tumor pathobiological heterogeneity and imprecise assessment of tumor spread. We leveraged ...Bayesian additive regression trees (BART), a statistical learning technique, to comprehensively analyze patient-specific tumor characteristics for the improvement of prognostic prediction. Of 75 clinicopathologic, immune, microbial, and genomic variables in 815 stage II-III patients within two U.S.-wide prospective cohort studies, the BART risk model identified seven stable survival predictors. Risk stratifications (low risk, intermediate risk, and high risk) based on model-predicted survival were statistically significant (hazard ratios 0.19-0.45, vs. higher risk; P < 0.0001) and could be externally validated using The Cancer Genome Atlas (TCGA) data (P = 0.0004). BART demonstrated model flexibility, interpretability, and comparable or superior performance to other machine-learning models. Integrated bioinformatic analyses using BART with tumor-specific factors can robustly stratify colorectal cancer patients into prognostic groups and be readily applied to clinical oncology practice.
We examined whether the association between alcohol consumption and CRC incidence was stronger for tumors with higher contributions of defective MMR (dMMR)-related tumor mutational signatures (TMSs).
...We used data from 227,916 men and women who participated in the Nurses' Health Study (1980-2016), the Nurses' Health Study II (1991-2017), and the Health Professionals Follow-up Study (1986-2016). Dietary data was collected every 4 years through validated food frequency questionnaires. Relative contributions of two dMMR-related TMSs (c-dMMRa/SBS15 and c-dMMRb/SBS26) were quantified using whole-exome sequencing data in a subset of incident CRC cases. Duplication-method Cox proportional hazards regression models were used to assess the association between alcohol consumption and the risk of CRC subtypes according to different contributions of the TMSs. All statistical tests were 2-sided.
We documented 825 incident CRC cases with available TMS data over 26-36 years of follow-up. The association between alcohol consumption and CRC incidence was stronger for tumors with higher contributions of c-dMMRb/SBS26 (P-heterogeneitytrend = 0.02) compared to tumors with lower contributions of this TMS. Compared with nondrinkers, drinkers with ≥15 g/d of alcohol had a high risk of c-dMMRb/SBS26-high CRC multivariable-adjusted HR: 2.43 (95% CI: 1.55-3.82), but not c-dMMRb/SBS26-low CRC 0.86 (95% CI: 0.57-1.28) or c-dMMRb/SBS26-moderate CRC 1.14 (95% CI: 0.76-1.71). No significant differential associations were observed for c-dMMRa/SBS15 (P-heterogeneitytrend = 0.41).
High alcohol consumption was associated with an increased incidence of CRC containing higher contributions of c-dMMRb/SBS26, suggesting that alcohol consumption may be involved in colorectal carcinogenesis through the DNA mismatch repair pathway.
Various species of the intestinal microbiota have been associated with the development of colorectal cancer.sup.1,2, but it has not been demonstrated that bacteria have a direct role in the ...occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin.sup.3. This compound is believed to alkylate DNA on adenine residues.sup.4,5 and induces double-strand breaks in cultured cells.sup.3. Here we expose human intestinal organoids to genotoxic pks.sup.+E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract
Introduction: RNF43 is a transmembrane E3 ubiquitin ligase and WNT signaling suppressor that is commonly mutated in colorectal cancer (CRC). A C-terminal RNF43 hotspot mutation, ...RNF43_G659fs, occurs in approximately 36% (55/151) of microsatellite-instability (MSI)-high CRCs, but its underlying mechanism and function remain poorly understood. This study investigated the functional role of RNF43_G659fs in order to evaluate potential novel therapeutic approaches for tumors harboring this mutation.
Methods: Isogenic RNF43117mut and RNF43659mut cell line models were generated using the CRISPR/Cas9 system to evaluate CRC tumorigenesis and WNT dependency. RNF43659mut was screened with a novel high-throughput drug repurposing library that employed a set of 5363 small molecules to identify compounds capable of selectively inhibiting RNF43659mut cell growth. Small molecules that selectively killed the RNF43659mut cells were validated in organoid models. Proteomic analysis, RNA-Seq and gene set enrichment analysis (GSEA) were performed to characterize mechanistic interactions and related signaling pathways of RNF43659mut in CRC.
Results: Unlike N-terminal RNF43 frameshift mutations, we observed that RNF43659mut conferred a growth advantage over RNF43WT cells independent of WNT signaling. Furthermore, RNF43659mut and RNF43WT exhibited differential drug responses in the high-throughput drug repurposing screen which revealed that RNF43659mut cells were vulnerable to PI3K/AKT/mTOR inhibitors, including BYL-719 (Alpelisib). Enhanced AKT and mTOR activation was observed in RNF43659mut cell and attenuated by BYL-719 treatment in a dose-dependent manner. These results were subsequently validated in patient-derived organoid models. Furthermore, immunoprecipitation and proteomic analysis revealed interactions between RNF43_G659fs and p85, a negative regulator of PI3K. We also demonstrated that the RNF43_G659fs mutant activated PI3K/AKT/mTOR signaling through binding and degradation of p85. Consistent with the role of PI3K in immunomodulation, our RNA-Seq results showed that the RNF43_G659fs mutation was positively related to NF-kB activation (Normalized Enrichment Score=1.842, p<0.01) and inversely related to Interferon-alpha/Interferon-gamma response pathways (Normalized Enrichment Score= -1.992, p<0.01; Normalized Enrichment Score= -1.577, p<0.01, respectively), indicating its role in tumor microenvironment remodeling.
Conclusion: This study confirms that RNF43659mut is an essential driver mutation in CRC and provides evidence that patients harboring RNF43_G659fs-mutant tumors may respond favorably to PI3K inhibition.
Citation Format: Lishan Fang, Dane Ford-Roshon, Max Russo, Casey O'Brien, Carino Gurjao, Maximilien Grandclaudon, Steven M. Corsello, Srivatsan Raghavan, Namrata Udeshi, James Berstler, Ewa Sicinska, Kimmie Ng, Marios Giannakis. RNF43 G659fs is an oncogenic mutation in colorectal cancer and sensitizes tumor cells to PI3K/mTOR inhibition abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 960.
Several risk factors have been established for colorectal cancer, yet their direct mutagenic effects in patients' tumors remain to be elucidated. Here, we leveraged whole-exome sequencing data from ...900 colorectal cancer cases that had occurred in three U.S.-wide prospective studies with extensive dietary and lifestyle information. We found an alkylating signature that was previously undescribed in colorectal cancer and then showed the existence of a similar mutational process in normal colonic crypts. This alkylating signature is associated with high intakes of processed and unprocessed red meat prior to diagnosis. In addition, this signature was more abundant in the distal colorectum, predicted to target cancer driver mutations
p.G12D,
p.G13D, and
p.E545K, and associated with poor survival. Together, these results link for the first time a colorectal mutational signature to a component of diet and further implicate the role of red meat in colorectal cancer initiation and progression. SIGNIFICANCE: Colorectal cancer has several lifestyle risk factors, but the underlying mutations for most have not been observed directly in tumors. Analysis of 900 colorectal cancers with whole-exome sequencing and epidemiologic annotations revealed an alkylating mutational signature that was associated with red meat consumption and distal tumor location, as well as predicted to target
p.G12D/p.G13D.
.
While evidence indicates that
(
) may promote colorectal carcinogenesis through its suppressive effect on T-cell-mediated antitumor immunity, the specific T-cell subsets involved remain uncertain.
We ...measured
DNA within tumor tissue by quantitative PCR on 933 cases (including 128
-positive cases) among 4,465 incident colorectal carcinoma cases in two prospective cohorts. Multiplex immunofluorescence combined with digital image analysis and machine learning algorithms for CD3, CD4, CD8, CD45RO (PTPRC isoform), and FOXP3 measured various T-cell subsets. We leveraged data on
, microsatellite instability (MSI), tumor whole-exome sequencing, and M1/M2-type tumor-associated macrophages TAM; by CD68, CD86, IRF5, MAF, and MRC1 (CD206) multimarker assay. Using the 4,465 cancer cases and inverse probability weighting method to control for selection bias due to tissue availability, multivariable-adjusted logistic regression analysis assessed the association between
and T-cell subsets.
The amount of
was inversely associated with tumor stromal CD3
lymphocytes multivariable OR, 0.47; 95% confidence interval (CI), 0.28-0.79, for
-high vs. -negative category;
= 0.0004 and specifically stromal CD3
CD4
CD45RO
cells (corresponding multivariable OR, 0.52; 95% CI, 0.32-0.85;
= 0.003). These relationships did not substantially differ by MSI status, neoantigen load, or exome-wide tumor mutational burden.
was not significantly associated with tumor intraepithelial T cells or with M1 or M2 TAMs.
The amount of tissue
is associated with lower density of stromal memory helper T cells. Our findings provide evidence for the interactive pathogenic roles of microbiota and specific immune cells.
Interval colorectal cancers (CRCs), cancers diagnosed after a screening/surveillance examination in which no cancer is detected, and before the date of next recommended examination, reflect an ...unprecedented challenge in CRC detection and prevention. To better understand this poorly characterized CRC variant, we examined the clinical and mutational characteristics of interval CRCs in comparison with screen detected CRCs.
We included 1175 CRCs documented in the Prostate, Lung, Colorectal, and Ovarian (PLCO) cancer screening trial and 3661 CRCs in the Nurses’ Health Study (NHS) and Health Professionals Follow-up Study (HPFS). Multivariable Cox models were performed to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) of death risk. Whole exome sequencing was conducted in 147 PLCO cases and 796 NHS/HPFS cases.
A total of 619 deaths (312 CRC-specific) and 2404 deaths (1904 CRC-specific) were confirmed during follow-up of PLCO and NHS/HPFS, respectively. Compared with screen detected CRCs, interval CRCs had a multivariate-adjusted HR (95% CI) of 1.47 (1.21–1.78) for CRC-specific mortality and 1.27 (1.09–1.47) for overall mortality (meta-analysis combining all 3 cohorts). However, we did not observe significant differences in mutational features between interval and screen detected CRCs (false discovery rate adjusted P > .05).
Interval CRCs had a significantly increased risk of death compared with screen detected CRCs that were not explained by established clinical prognostic factors, including stage at diagnosis. The survival disadvantage of interval CRCs did not appear to be explained by differences in the genomic landscape of tumors characterized by whole exome sequencing.
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Colorectal cancers diagnosed between screening examinations (interval cancers) had significantly increased risk of death than screen detected cancers, although no significant molecular differences were observed between them at a whole exome sequencing level.
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