Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer.

Bladder cancer is the fifth most prevalent cancer in the U.S., yet is understudied, and few laboratory models exist that reflect the biology of the human disease. Here, we describe a biobank of patient-derived organoid lines that recapitulates the histopathological and molecular diversity of human bladder cancer. Organoid lines can be established efficiently from patient biopsies acquired before and after disease recurrence and are interconvertible with orthotopic xenografts. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Analyses of drug response using bladder tumor organoids show partial correlations with mutational profiles, as well as changes associated with treatment resistance, and specific responses can be validated using xenografts in vivo. Our studies indicate that patient-derived bladder tumor organoids represent a faithful model system for studying tumor evolution and treatment response in the context of precision cancer medicine.

Manipulating the 3D organization of the largest synthetic yeast chromosome.

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

Identification of causal genetic drivers of human disease through systems-level analysis of regulatory networks.

Identification of driver mutations in human diseases is often limited by cohort size and availability of appropriate statistical models. We propose a framework for the systematic discovery of genetic alterations that are causal determinants of disease, by prioritizing genes upstream of functional disease drivers, within regulatory networks inferred de novo from experimental data. We tested this framework by identifying the genetic determinants of the mesenchymal subtype of glioblastoma. Our analysis uncovered KLHL9 deletions as upstream activators of two previously established master regulators of the subtype, C/EBPß and C/EBPδ. Rescue of KLHL9 expression induced proteasomal degradation of C/EBP proteins, abrogated the mesenchymal signature, and reduced tumor viability in vitro and in vivo. Deletions of KLHL9 were confirmed in > 50% of mesenchymal cases in an independent cohort, thus representing the most frequent genetic determinant of the subtype. The method generalized to study other human diseases, including breast cancer and Alzheimer's disease.

Alternative splicing is a developmental switch for hTERT expression.

Telomere length control is critical for cellular lifespan and tumor suppression. Telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere reserves. Its silencing upon differentiation leads to gradual telomere shortening in somatic cells. Here, we report that transcriptional regulation through cis-regulatory elements only partially accounts for telomerase activation in pluripotent cells. Instead, developmental control of telomerase is primarily driven by an alternative splicing event, centered around hTERT exon 2. Skipping of exon 2 triggers hTERT mRNA decay in differentiated cells, and conversely, its retention promotes telomerase accumulation in pluripotent cells. We identify SON as a regulator of exon 2 alternative splicing and report a patient carrying a SON mutation and suffering from insufficient telomerase and short telomeres. In summary, our study highlights a critical role for hTERT alternative splicing in the developmental regulation of telomerase and implicates defective splicing in telomere biology disorders.

Inactive PARP1 causes embryonic lethality and genome instability in a dominant-negative manner.

PARP1 (poly-ADP ribose polymerase 1) is recruited and activated by DNA strand breaks, catalyzing the generation of poly-ADP-ribose (PAR) chains from NAD+. PAR relaxes chromatin and recruits other DNA repair factors, including XRCC1 and DNA Ligase 3, to maintain genomic stability. Here we show that, in contrast to the normal development of Parp1-null mice, heterozygous expression of catalytically inactive Parp1 (E988A, Parp1+/A) acts in a dominant-negative manner to disrupt murine embryogenesis. As such, all the surviving F1 Parp1+/A mice are chimeras with mixed Parp1+/AN (neoR retention) cells that act similarly to Parp1+/-. Pure F2 Parp1+/A embryos were found at Mendelian ratios at the E3.5 blastocyst stage but died before E9.5. Compared to Parp1-/- cells, genotype and expression-validated pure Parp1+/A cells retain significant ADP-ribosylation and PARylation activities but accumulate markedly higher levels of sister chromatid exchange and mitotic bridges. Despite proficiency for homologous recombination and nonhomologous end-joining measured by reporter assays and supported by normal lymphocyte and germ cell development, Parp1+/A cells are hypersensitive to base damages, radiation, and Topoisomerase I and II inhibition. The sensitivity of Parp1+/A cells to base damages and Topo inhibitors exceed Parp1-/- controls. The findings show that the enzymatically inactive PARP1 dominant negatively blocks DNA repair in selective pathways beyond wild-type PARP1 and establishes a crucial physiological difference between PARP1 inactivation vs. deletion. As a result, the expression of enzymatically inactive PARP1 from one allele is sufficient to abrogate murine embryonic development, providing a mechanism for the on-target side effect of PARP inhibitors used for cancer therapy.

Targeting oncogenic KRAS with molecular brush-conjugated antisense oligonucleotides.

The mutant form of the guanosine triphosphatase (GTPase) KRAS is a key driver in human tumors but remains a challenging therapeutic target, making KRASMUT cancers a highly unmet clinical need. Here, we report a class of bottlebrush polyethylene glycol (PEG)-conjugated antisense oligonucleotides (ASOs) for potent in vivo KRAS depletion. Owing to their highly branched architecture, these molecular nanoconstructs suppress nearly all side effects associated with DNA-protein interactions and substantially enhance the pharmacological properties of the ASO, such as plasma pharmacokinetics and tumor uptake. Systemic delivery to mice bearing human non-small-cell lung carcinoma xenografts results in a significant reduction in both KRAS levels and tumor growth, and the antitumor performance well exceeds that of current popular ASO paradigms, such as chemically modified oligonucleotides and PEGylation using linear or slightly branched PEG. Importantly, these conjugates relax the requirement on the ASO chemistry, allowing unmodified, natural phosphodiester ASOs to achieve efficacy comparable to that of chemically modified ones. Both the bottlebrush polymer and its ASO conjugates appear to be safe and well tolerated in mice. Together, these data indicate that the molecular brush-ASO conjugate is a promising therapeutic platform for the treatment of KRAS-driven human cancers and warrant further preclinical and clinical development.

Intra-epithelial non-canonical Activin A signaling safeguards prostate progenitor quiescence.

The healthy prostate is a relatively quiescent tissue. Yet, prostate epithelium overgrowth is a common condition during aging, associated with urinary dysfunction and tumorigenesis. For over thirty years, TGF-ß ligands have been known to induce cytostasis in a variety of epithelia, but the intracellular pathway mediating this signal in the prostate, and its relevance for quiescence, have remained elusive. Here, using mouse prostate organoids to model epithelial progenitors, we find that intra-epithelial non-canonical Activin A signaling inhibits cell proliferation in a Smad-independent manner. Mechanistically, Activin A triggers Tak1 and p38 ΜAPK activity, leading to p16 and p21 nuclear import. Spontaneous evasion from this quiescent state occurs upon prolonged culture, due to reduced Activin A secretion, a condition associated with DNA replication stress and aneuploidy. Organoids capable to escape quiescence in vitro are also able to implant with increased frequency into immunocompetent mice. This study demonstrates that non-canonical Activin A signaling safeguards epithelial quiescence in the healthy prostate, with potential implications for the understanding of cancer initiation, and the development of therapies targeting quiescent tumor progenitors.

Atg7 cooperates with Pten loss to drive prostate cancer tumor growth.

Understanding new therapeutic paradigms for both castrate-sensitive and more aggressive castrate-resistant prostate cancer is essential to improve clinical outcomes. As a critically important cellular process, autophagy promotes stress tolerance by recycling intracellular components to sustain metabolism important for tumor survival. To assess the importance of autophagy in prostate cancer, we generated a new autochthonous genetically engineered mouse model (GEMM) with inducible prostate-specific deficiency in the Pten tumor suppressor and autophagy-related-7 (Atg7) genes. Atg7 deficiency produced an autophagy-deficient phenotype and delayed Pten-deficient prostate tumor progression in both castrate-naïve and castrate-resistant cancers. Atg7-deficient tumors display evidence of endoplasmic reticulum (ER) stress, suggesting that autophagy may promote prostate tumorigenesis through management of protein homeostasis. Taken together, these data support the importance of autophagy for both castrate-naïve and castrate-resistant growth in a newly developed GEMM, suggesting a new paradigm and model to study approaches to inhibit autophagy in combination with known and new therapies for advanced prostate cancer.

Insulin Conformation Changes in Hybrid Alginate-Gelatin Hydrogel Particles.

There is a strong need to develop an insulin delivery system suitable for oral administration and preserving natural (α-helix) insulin conformation. In this work, we fabricated alginate-gelatin hydrogel beads for insulin encapsulation. Altering matrix composition and crosslinking agents has resulted in various surface morphologies and internal spatial organization. The structures of the insulin-loaded matrices were studied using optical and field emission electronic microscopy. We use FTIR spectroscopy to identify insulin conformation changes as affected by the hydrogel matrices. It was found that blended alginate-gelatin matrices demonstrate better encapsulation efficiency and stronger swelling resistance to a simulated gastric environment than sodium alginate beads crosslinked with the CaCl2. FTIR measurements reveal conformation changes in insulin. It is also confirmed that in the presence of gelatin, the process of insulin fibrinogenesis ceases due to intermolecular interaction with the gelatin. Performed molecular modeling shows that dipole-dipole interactions are the dominating mechanism that determines insulin behavior within the fabricated matrix.

Prostate cancer cell heterogeneity and plasticity: Insights from studies of genetically-engineered mouse models.

Although prostate adenocarcinoma lacks distinguishable histopathological subtypes, prostate cancer displays significant inter- and intratumor heterogeneity at the molecular level and with respect to disease prognosis and treatment response. In principle, understanding the basis for prostate cancer heterogeneity can help distinguish aggressive from indolent disease, and help overcome castration-resistance in advanced prostate cancer. In this review, we will discuss recent advances in understanding the cell types of origin, putative cancer stem cells, and tumor plasticity in prostate cancer, focusing on insights from studies of genetically engineered mouse models (GEMMs). We will also outline future directions for investigating tumor heterogeneity using mouse models of prostate cancer.

Prevalence of oral and blood oncogenic human papillomavirus biomarkers among an enriched screening population: Baseline results of the MOUTH study.

BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal cancer screening is being explored in research studies, but strategies to identify an appropriate population are not established. The authors evaluated whether a screening population could be enriched for participants with oncogenic HPV biomarkers using risk factors for oral HPV. METHODS: Participants were enrolled at Johns Hopkins Hospitals and Mount Sinai Icahn School of Medicine. Eligible participants were either men aged 30 years or older who had two or more lifetime oral sex partners and a personal history of anogenital dysplasia/cancer or partners of patients who had HPV-related cancer. Oral rinse and serum samples were tested for oncogenic HPV DNA, RNA, and E6 or E7 antibodies, respectively. Participants with any biomarker were considered at-risk. RESULTS: Of 1108 individuals, 7.3% had any oncogenic oral HPV DNA, and 22.9% had serum antibodies for oncogenic HPV E6 or E7. Seventeen participants (1.5%) had both oral and blood biomarkers. HPV type 16 (HPV16) biomarkers were rarer, detected in 3.7% of participants, including 20 with oral HPV16 DNA and 22 with HPV16 E6 serum antibodies (n = 1 had both). In adjusted analysis, living with HIV (adjusted odds ratio, 2.65; 95% CI, 1.60-4.40) and older age (66-86 vs. 24-45 years; adjusted odds ratio, 1.70; 95% CI, 1.07-2.70) were significant predictors of being at risk. Compared with the general population, the prevalence of oral HPV16 (1.8% vs. 0.9%), any oncogenic oral HPV DNA (7.3% vs. 3.5%), and HPV16 E6 antibodies (2.2% vs. 0.3%) was significantly elevated. CONCLUSIONS: Enrichment by the eligibility criteria successfully identified a population with higher biomarker prevalence, including HPV16 biomarkers, that may be considered for screening trials. Most in this group are still expected to have a low risk of oropharyngeal cancer.

Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A.

Myostatin (MSTN) is a transforming growth factor-ß (TGF-ß) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-ß family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Test Characteristics of Cross-sectional Imaging and Concordance With Endoscopy in Postoperative Crohn's Disease.

BACKGROUND & AIMS: Postoperative Crohn's disease (CD) surveillance relies on endoscopic monitoring. The role of cross-sectional imaging is less clear. We evaluated the concordance of cross-sectional enterography with endoscopic recurrence and the predictive ability of radiography for future CD postoperative recurrence. METHODS: We performed a multi-institution retrospective cohort study of postoperative adult patients with CD who underwent ileocolonoscopy and cross-sectional enterography within 90 days of each other following ileocecal resection. Imaging studies were interpreted by blinded, expert CD radiologists. Patients were categorized by presence of endoscopic postoperative recurrence (E+) (modified Rutgeerts' score ≥i2b) or radiographic disease activity (R+) and grouped by concordance status. RESULTS: A total of 216 patients with CD with paired ileocolonoscopy and imaging were included. A majority (54.2%) exhibited concordance (34.7% E+/R+; 19.4% E-/R-) between studies. The plurality (41.7%; n = 90) were E-/R+ discordant. Imaging was highly sensitive (89.3%), with low specificity (31.8%), in detecting endoscopic postoperative recurrence. Intestinal wall thickening, luminal narrowing, mural hyper-enhancement, and length of disease on imaging were associated with endoscopic recurrence (all P < .01). Radiographic disease severity was associated with increasing Rutgeerts' score (P < .001). E-/R+ patients experienced more rapid subsequent endoscopic recurrence (hazard ratio, 4.16; P = .033) and increased rates of subsequent endoscopic (43.8% vs 22.7%) and surgical recurrence (20% vs 9.5%) than E-/R- patients (median follow-up, 4.5 years). CONCLUSIONS: Cross-sectional imaging is highly sensitive, but poorly specific, in detecting endoscopic disease activity and postoperative recurrence. Advanced radiographic disease correlates with endoscopic severity. Patients with radiographic activity in the absence of endoscopic recurrence may be at increased risk for future recurrence, and closer monitoring should be considered.

Prostate organogenesis: tissue induction, hormonal regulation and cell type specification.

Prostate organogenesis is a complex process that is primarily mediated by the presence of androgens and subsequent mesenchyme-epithelial interactions. The investigation of prostate development is partly driven by its potential relevance to prostate cancer, in particular the apparent re-awakening of key developmental programs that occur during tumorigenesis. However, our current knowledge of the mechanisms that drive prostate organogenesis is far from complete. Here, we provide a comprehensive overview of prostate development, focusing on recent findings regarding sexual dimorphism, bud induction, branching morphogenesis and cellular differentiation.

Obesity-associated variants within FTO form long-range functional connections with IRX3.

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.

ProNodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm.

The secreted glycoprotein sonic hedgehog (Shh) is expressed in the prechordal mesoderm, where it plays a crucial role in induction and patterning of the ventral forebrain. Currently little is known about how Shh is regulated in prechordal tissue. Here we show that in the embryonic chick, Shh is expressed transiently in prechordal mesoderm, and is governed by unprocessed Nodal. Exposure of prechordal mesoderm microcultures to Nodal-conditioned medium, the Nodal inhibitor CerS, or to an ALK4/5/7 inhibitor reveals that Nodal is required to maintain both Shh and Gsc expression, but whereas Gsc is largely maintained through canonical signalling, Nodal signals through a non-canonical route to maintain Shh. Further, Shh expression can be maintained by a recombinant Nodal cleavage mutant, proNodal, but not by purified mature Nodal. A number of lines of evidence suggest that proNodal acts via FGFR3. ProNodal and FGFR3 co-immunoprecipitate and proNodal increases FGFR3 tyrosine phosphorylation. In microcultures, soluble FGFR3 abolishes Shh without affecting Gsc expression. Further, prechordal mesoderm cells in which Fgfr3 expression is reduced by Fgfr3 siRNA fail to bind to proNodal. Finally, targeted electroporation of Fgfr3 siRNA to prechordal mesoderm in vivo results in premature Shh downregulation without affecting Gsc. We report an inverse correlation between proNodal-FGFR3 signalling and pSmad1/5/8, and show that proNodal-FGFR3 signalling antagonises BMP-mediated pSmad1/5/8 signalling, which is poised to downregulate Shh. Our studies suggest that proNodal/FGFR3 signalling governs Shh duration by repressing canonical BMP signalling, and that local BMPs rapidly silence Shh once endogenous Nodal-FGFR3 signalling is downregulated.

Molecular genetics of prostate cancer: new prospects for old challenges.

Despite much recent progress, prostate cancer continues to represent a major cause of cancer-related mortality and morbidity in men. Since early studies on the role of the androgen receptor that led to the advent of androgen deprivation therapy in the 1940s, there has long been intensive interest in the basic mechanisms underlying prostate cancer initiation and progression, as well as the potential to target these processes for therapeutic intervention. Here, we present an overview of major themes in prostate cancer research, focusing on current knowledge of principal events in cancer initiation and progression. We discuss recent advances, including new insights into the mechanisms of castration resistance, identification of stem cells and tumor-initiating cells, and development of mouse models for preclinical evaluation of novel therapuetics. Overall, we highlight the tremendous research progress made in recent years, and underscore the challenges that lie ahead.

Low birth weight is associated with impaired murine kidney development and function.

BackgroundLow birth weight (LBW) neonates have impaired kidney development that leaves them susceptible to kidney disease and hypertension during adulthood. The study here identifies events that blunt nephrogenesis and kidney development in the murine LBW neonate.MethodsWe examined survival, kidney development, GFR, gene expression, and cyto-/chemokines in the LBW offspring of malnourished (caloric and protein-restricted) pregnant mice.ResultsMalnourished pregnant mothers gave birth to LBW neonates that had 40% reduced body weight and 54% decreased survival. Renal blood perfusion was reduced by 37%, whereas kidney volume and GFR were diminished in the LBW neonate. During gestation, the LBW neonatal kidney had 2.2-fold increased apoptosis, 76% decreased SIX2+ progenitor cells, downregulation of mesenchymal-to-epithelial signaling factors Wnt9b and Fgf8, 64% less renal vesicle formation, and 32% fewer nephrons than controls. At birth, increased plasma levels of IL-1ß, IL-6, IL-12(p70), and granulocyte-macrophage colony-stimulating factor in the LBW neonate reduced SIX2+ progenitor cells.ConclusionIncreased pro-inflammatory cytokines in the LBW neonate decrease SIX2+ stem cells in the developing kidney. Reduced renal stem cells (along with the decreased mesenchymal-to-epithelial signaling) blunt renal vesicle generation, nephron formation, and kidney development. Subsequently, the mouse LBW neonate has reduced glomeruli volume, renal perfusion, and GFR.

Inactivation of p53 and Pten promotes invasive bladder cancer.

Although bladder cancer represents a serious health problem worldwide, relevant mouse models for investigating disease progression or therapeutic targets have been lacking. We show that combined deletion of p53 and Pten in bladder epithelium leads to invasive cancer in a novel mouse model. Inactivation of p53 and PTEN promotes tumorigenesis in human bladder cells and is correlated with poor survival in human tumors. Furthermore, the synergistic effects of p53 and Pten deletion are mediated by deregulation of mammalian target of rapamycin (mTOR) signaling, consistent with the ability of rapamycin to block bladder tumorigenesis in preclinical studies. Our integrated analyses of mouse and human bladder cancer provide a rationale for investigating mTOR inhibition for treatment of patients with invasive disease.