Functional screening of amplification outlier oncogenes in organoid models of early tumorigenesis.

Somatic copy number gains are pervasive across cancer types, yet their roles in oncogenesis are insufficiently evaluated. This inadequacy is partly due to copy gains spanning large chromosomal regions, obscuring causal loci. Here, we employed organoid modeling to evaluate candidate oncogenic loci identified via integrative computational analysis of extreme copy gains overlapping with extreme expression dysregulation in The Cancer Genome Atlas. Subsets of "outlier" candidates were contextually screened as tissue-specific cDNA lentiviral libraries within cognate esophagus, oral cavity, colon, stomach, pancreas, and lung organoids bearing initial oncogenic mutations. Iterative analysis nominated the kinase DYRK2 at 12q15 as an amplified head and neck squamous carcinoma oncogene in p53-/- oral mucosal organoids. Similarly, FGF3, amplified at 11q13 in 41% of esophageal squamous carcinomas, promoted p53-/- esophageal organoid growth reversible by small molecule and soluble receptor antagonism of FGFRs. Our studies establish organoid-based contextual screening of candidate genomic drivers, enabling functional evaluation during early tumorigenesis.

Real-world survival analysis by tumor mutational burden in non-small cell lung cancer: a multisite U.S. study.

BACKGROUND: Tumor mutational burden (TMB) is a potential biomarker to predict tumor response to immuno-oncology agents in patients with metastatic non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: A multi-site cohort study evaluated patients diagnosed with stage IV NSCLC between 2012 and 2019 who had received comprehensive genomic profiling (CGP) and any NSCLC-related treatment at 9 U.S. cancer centers. Baseline characteristics and clinical outcomes were compared between patients with TMB <10 and TMB ≥10. RESULTS: Among the 667 patients with CGP results, most patients received CGP from Foundation Medicine (64%) or Caris (20%). Patients with TMB ≥10 (vs. TMB <10) were associated with a positive smoking history. TMB was associated with ALK (p = 0.01), EGFR (p < 0.01), and TP53 (p < 0.05) alterations. TMB >10 showed a significant association towards longer overall survival (OS) (HR: 0.43, 95% CI: 0.21-0.88, p = 0.02) and progression-free survival (PFS) (HR: 0.43, 95% CI: 0.21-0.85, p = 0.02) in patients treated with first-line immunotherapy and tested by Foundation Medicine or Caris at treatment initiation. CONCLUSIONS: TMB levels greater than or equal to 10 mut/Mb, when tested by Foundation Medicine or Caris at treatment initiation, were significantly associated with improved OS and PFS among patients treated with first-line immunotherapy-containing regimens. Additional prospective research is warranted to validate this biomarker along with PD-L1 expression.

Chemodetection and Destruction of Host Urea Allows Helicobacter pylori to Locate the Epithelium.

The gastric pathogen Helicobacter pylori interacts intimately with the gastric mucosa to avoid the microbicidal acid in the stomach lumen. The cues H. pylori senses to locate and colonize the gastric epithelium have not been well defined. We show that metabolites emanating from human gastric organoids rapidly attract H. pylori. This response is largely controlled by the bacterial chemoreceptor TlpB, and the main attractant emanating from epithelia is urea. Our previous structural analyses show that TlpB binds urea with high affinity. Here we demonstrate that this tight binding controls highly sensitive responses, allowing detection of urea concentrations as low as 50 nM. Attraction to urea requires that H. pylori urease simultaneously destroys the signal. We propose that H. pylori has evolved a sensitive urea chemodetection and destruction system that allows the bacterium to dynamically and locally modify the host environment to locate the epithelium.

c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression.

Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis.

Organoid modeling for cancer precision medicine.

Three-dimensional organotypic culture models show great promise as a tool for cancer precision medicine, with potential applications for oncogene modeling, gene discovery and chemosensitivity studies.

Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

c-Jun N-Terminal Kinases Mediate a Wide Range of Targets in the Metastatic Cascade.

Disseminated cancer cells rely on intricate interactions among diverse cell types in the tumor-associated stroma, vasculature, and immune system for survival and growth. Ubiquitous expression of c-Jun N-terminal kinase (jnk) genes in various cell types permits their control of metastasis. In early stages of metastasis, JNKs affect tumor-associated inflammation and angiogenesis as well as tumor cell migration and intravasation. Within the tumor stroma, JNKs are essential for the release of growth factors that promote epithelial-to-mesenchymal transition (EMT) in tumor cells. JNK3, the least ubiquitous isoform, facilitates angiogenesis by increasing endothelial cell migration. Importantly, JNK expression in tumor cells integrates stromal signals to promote tumor cell invasion. However, JNK isoforms differentially regulate migration toward the endothelial barrier. Once tumor cells enter the bloodstream, JNKs increase circulating tumor cell (CTC) survival and homing to tissues. By promoting fibrosis, JNKs improve CTC attachment to the endothelium. Once anchored, JNKs stimulate EMT to facilitate tumor cell extravasation and enhance the secretion of endothelial barrier disrupters. Tumor cells attract barrier-disrupting macrophages by JNK-dependent transcription of macrophage chemoattractant molecules. In the secondary tissue, JNKs are instrumental in the premetastatic niche and stimulate tumor cell proliferation. JNK expression in cancer cells stimulates tissue-remodeling macrophages to improve tumor colonization. However, in T-cells, JNKs alter cytokine production that increases tumor surveillance and inhibits the recruitment of tissue-remodeling macrophages. Therapeutically targeting JNKs for metastatic disease is attractive considering their promotion of metastasis; however, specific JNK tools are needed to determine their definitive actions within the context of the entire metastatic cascade.

Retrofitting the genome: L1 extinction follows endogenous retroviral expansion in a group of muroid rodents.

Long interspersed nuclear element 1 (LINE-1; L1) retrotransposons are the most common retroelements in mammalian genomes. Unlike individual families of endogenous retroviruses (ERVs), they have remained active throughout the mammalian radiation and are responsible for most of the retroelement movement and much genome rearrangement within mammals. They can be viewed as occupying a substantial niche within mammalian genomes. Our previous demonstration that L1s and B1 short interspersed nuclear elements (SINEs) are inactive in a group of South American rodents led us to ask if other elements have amplified to fill the empty niche. We identified a novel and highly active family of ERVs (mysTR). To determine whether loss of L1 activity was correlated with expansion of mysTR, we examined mysTR activity in four South American rodent species that have lost L1 and B1 activity and four sister species with active L1s. The copy number of recent mysTR insertions was extremely high, with an average of 4,200 copies per genome. High copy numbers exist in both L1-active and L1-extinct species, so the mysTR expansion appears to have preceded the loss of both SINE and L1 activity rather than to have filled an empty niche created by their loss. It may be coincidental that two unusual genomic events--loss of L1 activity and massive expansion of an ERV family--occur in the same group of mammals. Alternatively, it is possible that this large ERV expansion set the stage for L1 extinction.

Jnk2 effects on tumor development, genetic instability and replicative stress in an oncogene-driven mouse mammary tumor model.

Oncogenes induce cell proliferation leading to replicative stress, DNA damage and genomic instability. A wide variety of cellular stresses activate c-Jun N-terminal kinase (JNK) proteins, but few studies have directly addressed the roles of JNK isoforms in tumor development. Herein, we show that jnk2 knockout mice expressing the Polyoma Middle T Antigen transgene developed mammary tumors earlier and experienced higher tumor multiplicity compared to jnk2 wildtype mice. Lack of jnk2 expression was associated with higher tumor aneuploidy and reduced DNA damage response, as marked by fewer pH2AX and 53BP1 nuclear foci. Comparative genomic hybridization further confirmed increased genomic instability in PyV MT/jnk2-/- tumors. In vitro, PyV MT/jnk2-/- cells underwent replicative stress and cell death as evidenced by lower BrdU incorporation, and sustained chromatin licensing and DNA replication factor 1 (CDT1) and p21(Waf1) protein expression, and phosphorylation of Chk1 after serum stimulation, but this response was not associated with phosphorylation of p53 Ser15. Adenoviral overexpression of CDT1 led to similar differences between jnk2 wildtype and knockout cells. In normal mammary cells undergoing UV induced single stranded DNA breaks, JNK2 localized to RPA (Replication Protein A) coated strands indicating that JNK2 responds early to single stranded DNA damage and is critical for subsequent recruitment of DNA repair proteins. Together, these data support that JNK2 prevents replicative stress by coordinating cell cycle progression and DNA damage repair mechanisms.

X chromosome inactivation and Xist evolution in a rodent lacking LINE-1 activity.

Dosage compensation in eutherian mammals occurs by inactivation of one X chromosome in females. Silencing of that X chromosome is initiated by Xist, a large non-coding RNA, whose coating of the chromosome extends in cis from the X inactivation center. LINE-1 (L1) retrotransposons have been implicated as possible players for propagation of the Xist signal, but it has remained unclear whether they are essential components. We previously identified a group of South American rodents in which L1 retrotransposition ceased over 8 million years ago and have now determined that at least one species of these rodents, Oryzomys palustris, still retains X inactivation. We have also isolated and analyzed the majority of the Xist RNA from O. palustris and a sister species retaining L1 activity, Sigmodon hispidus, to determine if evolution in these sequences has left signatures that might suggest a critical role for L1 elements in Xist function. Comparison of rates of Xist evolution in the two species fails to support L1 involvement, although other explanations are possible. Similarly, comparison of known repeats and potential RNA secondary structures reveals no major differences with the exception of a new repeat in O. palustris that has potential to form new secondary structures.

Loss of LINE-1 activity in the megabats.

LINE-1 (L1) retrotransposons are the most abundant type of mammalian retroelement. They have profound effects on genome plasticity and have been proposed to fulfill essential host functions, yet it remains unclear where they lie on the spectrum from parasitism to mutualism. Their ubiquity makes it difficult to determine the extent of their effects on genome evolution and gene expression because of the relative dearth of animal models lacking L1 activity. We have isolated L1 sequences from 11 megabat species by a method that enriches for recently inserted L1s and have done a bioinformatic examination of L1 sequences from a 12th species whose genome was recently shotgun sequenced. An L1 extinction event appears to have occurred at least 24 million years ago (MYA) in an ancestor of the megabats. The ancestor was unusual in having maintained two highly divergent long-term L1 lineages with different levels of activity, which appear, on an evolutionary scale, to have simultaneously lost that activity. These megabat species can serve as new animal models to ask what effect loss of L1 activity has on mammalian genome evolution and gene expression.

MysTR: an endogenous retrovirus family in mammals that is undergoing recent amplifications to unprecedented copy numbers.

A large percentage of the repetitive elements in mammalian genomes are retroelements, which have been moved primarily by LINE-1 retrotransposons and endogenous retroviruses. Although LINE-1 elements have remained active throughout the mammalian radiation, specific groups of endogenous retroviruses generally remain active for comparatively shorter periods of time. Identification of an unusual extinction of LINE-1 activity in a group of South American rodents has opened a window for examination of the interplay in mammalian genomes between these ubiquitous retroelements. In the course of a search for any type of repetitive sequences whose copy numbers have substantially changed in Oryzomys palustris, a species that has lost LINE-1 activity, versus Sigmodon hispidus, a closely related species retaining LINE-1 activity, we have identified an endogenous retrovirus family differentially amplified in these two species. Analysis of three full-length, recently transposed copies, called mysTR elements, revealed gag, pro, and pol coding regions containing stop codons which may have accumulated either before or after retrotransposition. Isolation of related sequences in S. hispidus and the LINE-1 active outgroup species, Peromyscus maniculatus, by PCR of a pro-pol region has allowed determination of copy numbers in each species. Unusually high copy numbers of approximately 10,000 in O. palustris versus 1,000 in S. hispidus and 4,500 in the more distantly related P. maniculatus leave open the question of whether there is a connection between endogenous retrovirus activity and LINE-1 inactivity. Nevertheless, these independent expansions of mysTR represent recent amplifications of this endogenous retrovirus family to unprecedented levels.