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Colorectal cancer (CRC) is the most common gastrointestinal malignancy in the U.S.A. and approximately 50% of patients develop metastatic disease (mCRC). Despite our understanding of long non-coding RNAs (lncRNAs) in primary colon cancer, their role in mCRC and treatment resistance remains poorly characterized. Therefore, through transcriptome sequencing of normal, primary, and distant mCRC tissues we find 148 differentially expressed RNAs Associated with Metastasis ( RAMS ). We prioritize RAMS11 due to its association with poor disease-free survival and promotion of aggressive phenotypes in vitro and in vivo. A FDA-approved drug high-throughput viability assay shows that elevated RAMS11 expression increases resistance to topoisomerase inhibitors. Subsequent experiments demonstrate RAMS11 -dependent recruitment of Chromobox protein 4 (CBX4) transcriptionally activates Topoisomerase II alpha (TOP2α ) . Overall, recent clinical trials using topoisomerase inhibitors coupled with our findings of RAMS11- dependent regulation of TOP2α supports the potential use of RAMS11 as a biomarker and therapeutic target for mCRC. The role of long non-coding RNAs (lncRNAs) in metastatic colorectal cancer (mCRC) and treatment resistance is unclear. Here, the authors use transcriptome sequencing of matched normal, primary, and metastatic CRC tissues to discover and validate that lncRNA RAMS11 promotes metastasis and resistance to topoisomerase inhibitors in mCRC.

Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients. The MAPK pathway is an important therapeutic target in pancreatic ductal adenocarcinoma (PDAC), but success is limited by pathway reactivation, which drives resistance. Here, the authors investigate the mechanism underlying HER2-reactivation post KRAS-MAPK inhibition, identifying combination of MAPK and HER2 inhibition as a therapeutic strategy.

Background Pancreatic ductal adenocarcinoma (PDAC) has a high fatality rate, with surgery as the only curative treatment. Identification of new biomarkers related to survival may help guide discovery of new pathophysiologic pathways and potential therapeutic targets. As long-chain ceramides have been linked to tumor proliferation, we sought to determine if ceramide levels were prognostic in PDAC. Methods Patients from two phase I studies of PDAC were followed for all-cause mortality. Ceramide levels (C24:0, C22:0, and C16:0) were quantified before treatment and at study intervals. Multivariable Cox regression models assessed the association of ceramide levels and mortality after adjusting for other univariable predictors, including time-dependent tumor resection. The ability of repeated ceramide measures to discriminate patients at risk for mortality was also assessed using multivariable modeling and the c-statistic. Results Higher plasma C16:0 concentration was associated with higher all-cause mortality in univariable and multivariable analysis (adjusted hazard ratio [aHR] 1.41, 95% confidence interval [CI] 1.09–1.82; p  < 0.01). In contrast, a higher plasma C24:0/C16:0 ratio was associated with lower all-cause mortality in multivariable analysis (aHR 0.69, 95% CI 0.49–0.97; p  = 0.032). Discrimination of mortality was significantly improved with the addition of either plasma C16:0 or C24:0/C16:0 levels, with optimal discrimination occurring using repeated measures of the C24:0/C16:0 ratio (c-statistic 0.73 vs. c-statistic 0.66; p  < 0.001). Conclusions Higher plasma C16:0 and lower C24:0/C16:0 ratios are independently associated with mortality in PDAC and show an ability to improve discrimination of mortality in this deadly disease. Further studies are needed to confirm this association and evaluate this novel pathway for potential therapeutic targets.

Therapy targeting the BRAF-MEK cascade created a treatment revolution for patients with BRAF mutant advanced melanoma. Unfortunately, 80% patients treated will progress by 5 years follow-up. Thus, it is imperative we study mechanisms of melanoma progression and therapeutic resistance. We created a scRNA (single cell RNA) atlas of 128,230 cells from 18 tumors across the treatment spectrum, discovering melanoma cells clustered strongly by transcriptome profiles of patients of origins. Our cell-level investigation revealed gains of 1q and 7q as likely early clonal events in metastatic melanomas. By comparing patient tumors and their derivative cell lines, we observed that PD1 responsive tumor fraction disappears when cells are propagated in vitro . We further established three anti-BRAF-MEK treatment resistant cell lines using three BRAF mutant tumors. ALDOA and PGK1 were found to be highly expressed in treatment resistant cell populations and metformin was effective in targeting the resistant cells. Our study suggests that the investigation of patient tumors and their derivative lines is essential for understanding disease progression, treatment response and resistance.

Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs \"captures\" the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically \"fit\" for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0-45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40-60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign.

Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in human A375 melanoma cell lines and observed decreased expression of CDC20 , changes in migration capabilities, increased growth of xenografts, and an altered transcriptional state previously associated with a more proliferative and less migratory state. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20 , led to perturbation of key melanoma phenotypes. A non-coding variant upstream of the CDC20 promoter leads to down-regulation of CDC20 expression and up-regulation of a more proliferative and melanocytic transcriptional program that may aid in accelerating tumor growth.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.

BackgroundInteractions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors.MethodWith patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient’s hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual’s TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor.ResultsAutologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth.ConclusionsHumanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing.

Alport syndrome is a human hereditary glomerulonephritis which results in end-stage renal failure (ESRF) in most cases. It is caused by mutations in any one of the collagen α3(IV), α4(IV), or α5(IV) chain genes ( COL4A3- COL4A5). Patients carrying identical mutations can exhibit very different disease courses, suggesting that other genes or the environment influence disease progression. We previously generated a knockout mouse model of Alport syndrome by mutating Col4a3. Here, we show that genetic background strongly influences the timing of onset of disease and rate of progression to ESRF in these mice. On the 129×1/SvJ background, Col4a3 −/− mice reached ESRF at ∼66 days of age, while on the C57BL/6J background, the mean age at ESRF was 194 days of age. This suggests the existence of modifier genes that influence disease progression. A detailed histopathological analysis revealed that glomerular basement membrane lesions typical of Alport syndrome were significantly more frequent in homozygotes on the 129×1/SvJ background than on the C57BL/6J background as early as two weeks of age, suggesting that modifier genes act by influencing glomerular basement membrane structure. Additional data indicated that differential physiological responses to basement membrane splitting also underlie the differences in disease progression. We attempted to map the modifier genes as quantitative trait loci (QTLs) using age at ESRF as the quantitative trait. Genome scans were performed on mice at the two extremes in a cohort of mutant F1 × C57BL/6J backcross mice. Analysis with Map Manager QT revealed QTLs linked to markers on chromosomes 9 and 16. A more detailed understanding of how these QTLs act could lead to new approaches for therapy in diverse renal diseases.