CRISPR/Cas9-Mediated Point Mutation in Nkx3.1 Prolongs Protein Half-Life and Reverses Effects Nkx3.1 Allelic Loss.

NKX3.1 is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. NKX3.1 is haploinsufficient, and pathogenic reduction in protein levels may result from genetic loss, decreased transcription, and increased protein degradation caused by inflammation or PTEN loss. NKX3.1 acts by retarding proliferation, activating antioxidants, and enhancing DNA repair. DYRK1B-mediated phosphorylation at serine 185 of NKX3.1 leads to its polyubiquitination and proteasomal degradation. Because NKX3.1 protein levels are reduced, but never entirely lost, in prostate adenocarcinoma, enhancement of NKX3.1 protein levels represents a potential therapeutic strategy. As a proof of principle, we used CRISPR/Cas9-mediated editing to engineer in vivo a point mutation in murine Nkx3.1 to code for a serine to alanine missense at amino acid 186, the target for Dyrk1b phosphorylation. Nkx3.1S186A/-, Nkx3.1+/- , and Nkx3.1+/+ mice were analyzed over one year to determine the levels of Nkx3.1 expression and effects of the mutant protein on the prostate. Allelic loss of Nkx3.1 caused reduced levels of Nkx3.1 protein, increased proliferation, and prostate hyperplasia and dysplasia, whereas Nkx3.1S186A/- mouse prostates had increased levels of Nkx3.1 protein, reduced prostate size, normal histology, reduced proliferation, and increased DNA end labeling. At 2 months of age, when all mice had normal prostate histology, Nkx3.1+/- mice demonstrated indices of metabolic activation, DNA damage response, and stress response. These data suggest that modulation of Nkx3.1 levels alone can exert long-term control over premalignant changes and susceptibility to DNA damage in the prostate. SIGNIFICANCE: These findings show that prolonging the half-life of Nkx3.1 reduces proliferation, enhances DNA end-labeling, and protects from DNA damage, ultimately blocking the proneoplastic effects of Nkx3.1 allelic loss.

Nestin+NG2+ Cells Form a Reserve Stem Cell Population in the Mouse Prostate.

In the prostate, stem and progenitor cell regenerative capacities have been ascribed to both basal and luminal epithelial cells. Here, we show that a rare subset of mesenchymal cells in the prostate are epithelial-primed Nestin-expressing cells (EPNECs) that can generate self-renewing prostate organoids with bipotential capacity. Upon transplantation, these EPNECs can form prostate gland tissue grafts at the clonal level. Lineage-tracing analyses show that cells marked by Nestin or NG2 transgenic mice contribute to prostate epithelium during organogenesis. In the adult, modest contributions in repeated rounds of regression and regeneration are observed, whereas prostate epithelial cells derived from Nestin/NG2-marked cells are dramatically increased after severe irradiation-induced organ damage. These results indicate that Nestin/NG2 expression marks a novel radioresistant prostate stem cell that is active during development and displays reserve stem cell activity for tissue maintenance.

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.

Differential requirements of androgen receptor in luminal progenitors during prostate regeneration and tumor initiation.

Master regulatory genes of tissue specification play key roles in stem/progenitor cells and are often important in cancer. In the prostate, androgen receptor (AR) is a master regulator essential for development and tumorigenesis, but its specific functions in prostate stem/progenitor cells have not been elucidated. We have investigated AR function in CARNs (CAstration-Resistant Nkx3.1-expressing cells), a luminal stem/progenitor cell that functions in prostate regeneration. Using genetically--engineered mouse models and novel prostate epithelial cell lines, we find that progenitor properties of CARNs are largely unaffected by AR deletion, apart from decreased proliferation in vivo. Furthermore, AR loss suppresses tumor formation after deletion of the Pten tumor suppressor in CARNs; however, combined Pten deletion and activation of oncogenic Kras in AR-deleted CARNs result in tumors with focal neuroendocrine differentiation. Our findings show that AR modulates specific progenitor properties of CARNs, including their ability to serve as a cell of origin for prostate cancer.

A computational systems approach identifies synergistic specification genes that facilitate lineage conversion to prostate tissue.

To date, reprogramming strategies for generating cell types of interest have been facilitated by detailed understanding of relevant developmental regulatory factors. However, identification of such regulatory drivers often represents a major challenge, as specific gene combinations may be required for reprogramming. Here we show that a computational systems approach can identify cell type specification genes (master regulators) that act synergistically, and demonstrate its application for reprogramming of fibroblasts to prostate tissue. We use three such master regulators (FOXA1, NKX3.1 and androgen receptor, AR) in a primed conversion strategy starting from mouse fibroblasts, resulting in prostate tissue grafts with appropriate histological and molecular properties that respond to androgen-deprivation. Moreover, generation of reprogrammed prostate does not require traversal of a pluripotent state. Thus, we describe a general strategy by which cell types and tissues can be generated even with limited knowledge of the developmental pathways required for their specification in vivo.

Single luminal epithelial progenitors can generate prostate organoids in culture.

The intrinsic ability to exhibit self-organizing morphogenetic properties in ex vivo culture may represent a general property of tissue stem cells. Here we show that single luminal stem/progenitor cells can generate prostate organoids in a three-dimensional culture system in the absence of stroma. Organoids generated from CARNs (castration-resistant Nkx3.1-expressing cells) or normal prostate epithelia exhibit tissue architecture containing luminal and basal cells, undergo long-term expansion in culture and exhibit functional androgen receptor signalling. Lineage-tracing demonstrates that luminal cells are favoured for organoid formation and generate basal cells in culture. Furthermore, tumour organoids can initiate from CARNs after oncogenic transformation and from mouse models of prostate cancer, and can facilitate analyses of drug response. Finally, we provide evidence supporting the feasibility of organoid studies of human prostate tissue. Our studies underscore the progenitor properties of luminal cells, and identify in vitro approaches for studying prostate biology.

Luminal cells are favored as the cell of origin for prostate cancer.

The identification of cell types of origin for cancer has important implications for tumor stratification and personalized treatment. For prostate cancer, the cell of origin has been intensively studied, but it has remained unclear whether basal or luminal epithelial cells, or both, represent cells of origin under physiological conditions in vivo. Here, we use a novel lineage-tracing strategy to assess the cell of origin in a diverse range of mouse models, including Nkx3.1(+/-); Pten(+/-), Pten(+/-), Hi-Myc, and TRAMP mice, as well as a hormonal carcinogenesis model. Our results show that luminal cells are consistently the observed cell of origin for each model in situ; however, explanted basal cells from these mice can generate tumors in grafts. Consequently, we propose that luminal cells are favored as cells of origin in many contexts, whereas basal cells only give rise to tumors after differentiation into luminal cells.

Lineage analysis of basal epithelial cells reveals their unexpected plasticity and supports a cell-of-origin model for prostate cancer heterogeneity.

A key issue in cancer biology is whether oncogenic transformation of different cell types of origin within an adult tissue gives rise to distinct tumour subtypes that differ in their prognosis and/or treatment response. We now show that initiation of prostate tumours in basal or luminal epithelial cells in mouse models results in tumours with distinct molecular signatures that are predictive of human patient outcomes. Furthermore, our analysis of untransformed basal cells reveals an unexpected assay dependence of their stem cell properties in sphere formation and transplantation assays versus genetic lineage tracing during prostate regeneration and adult tissue homeostasis. Although oncogenic transformation of basal cells gives rise to tumours with luminal phenotypes, cross-species bioinformatic analyses indicate that tumours of luminal origin are more aggressive than tumours of basal origin, and identify a molecular signature associated with patient outcome. Our results reveal the inherent plasticity of basal cells, and support a model in which different cells of origin generate distinct molecular subtypes of prostate cancer.

Fine mapping of an epilepsy modifier gene on mouse Chromosome 19.

Mutations in voltage-gated sodium channels are associated with several types of human epilepsy. Variable expressivity and penetrance are common features of inherited epilepsy caused by sodium channel mutations, suggesting that genetic modifiers may influence clinical severity. The mouse model Scn2a(Q54) has an epilepsy phenotype due to a mutation in Scn2a that results in elevated persistent sodium current. Phenotype severity in Scn2a(Q54) mice is dependent on the genetic background. Congenic C57BL/6J.Q54 mice have delayed onset and low seizure frequency compared to (C57BL/6J x SJL/J)F1.Q54 mice. Previously, we identified two modifier loci that influence the Scn2a(Q54) epilepsy phenotype: Moe1 (modifier of epilepsy 1) on Chromosome 11 and Moe2 on Chromosome 19. We have constructed interval-specific congenic strains to further refine the position of Moe2 on Chromosome 19 to a 5-Mb region. Sequencing and expression analyses of genes in the critical interval suggested two potential modifier candidates: (1) voltage-gated potassium channel subunit subfamily V, member 2 (Kcnv2), and (2) SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 2 (Smarca2). Based on its biological role in regulating membrane excitability and the association between ion channel variants and seizures, Kcnv2 is a strong functional candidate for Moe2. Modifier genes affecting the epilepsy phenotype of Scn2a(Q54) mice may contribute to variable expressivity and penetrance in human epilepsy patients with sodium channel mutations.

Deleterious variants of FIG4, a phosphoinositide phosphatase, in patients with ALS.

Mutations of the lipid phosphatase FIG4 that regulates PI(3,5)P(2) are responsible for the recessive peripheral-nerve disorder CMT4J. We now describe nonsynonymous variants of FIG4 in 2% (9/473) of patients with amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS). Heterozygosity for a deleterious allele of FIG4 appears to be a risk factor for ALS and PLS, extending the list of known ALS genes and increasing the clinical spectrum of FIG4-related diseases.

Evidence for a direct role of the disease modifier SCNM1 in splicing.

We originally isolated Scnm1 as a disease modifier gene that is required for efficient in vivo splicing of a mutant splice donor site in the sodium channel Scn8a. It was previously unclear whether the modifier effect on splicing was direct or indirect. We now report evidence that sodium channel modifier 1 (SCNM1) has a direct role in splicing. SCNM1 protein interacts with the spliceosome protein U1-70K in the yeast two-hybrid system, and is co-localized with U1-70K in nuclear speckles in mammalian cells. SCNM1 is also co-immunoprecipitated with the spliceosomal core Smith (Sm) proteins and demonstrates functional activity in a minigene splicing assay. In a yeast two-hybrid screen, SCNM1 interacted with LUC7L2, a mammalian homolog of a yeast protein involved in recognition of non-consensus splice donor sites. This interaction requires the acidic C-terminal domain of SCNM1 which is truncated by the disease susceptibility variant Scnm1(R187X) in mouse strain C57BL/6J. Luc7L2 transcripts are widely distributed in mammalian tissues, and undergo alternative splicing and polyadenylation. LUC7L2 is also co-localized with U1-70K and may function with SCNM1 in recognition of weak splice donor sites. In summary, Scnm1 is the first example of a modifier gene which influences disease severity through a trans-effect on splicing of the disease gene transcript.

Severe epilepsy resulting from genetic interaction between Scn2a and Kcnq2.

A mutation in the voltage-gated sodium-channel Scn2a results in moderate epilepsy in transgenic Scn2a(Q54) mice maintained on a C57BL/6J strain background. The onset of progressive epilepsy begins in adults with short-duration partial seizures that originate in the hippocampus. The underlying abnormality is an increase in persistent sodium current in hippocampal neurons. The voltage-gated potassium channel Kcnq2 is responsible for generating M current (I(KM)) that is thought to control excitability and limit repetitive firing of hippocampal neurons. To determine whether impaired M current would exacerbate the seizure phenotype of Scn2a(Q54) mice, we carried out genetic crosses with two mutant alleles of Kcnq2. Szt1 mice carry a spontaneous deletion that removes the C-terminal domain of Kcnq2. A novel Kcnq2 missense mutation V182M was identified by screening the offspring of ENU-treated males for reduced threshold to electrically evoked minimal clonic seizures. Double mutant mice carrying the Scn2a(Q54) transgene together with either of the Kcnq2 mutations exhibited severe epilepsy with early onset, generalized tonic-clonic seizures and juvenile lethality by 3 weeks of age. This dramatic exacerbation of the sodium-channel mutant phenotype indicates that M current plays a critical role in preventing seizure initiation and spreading in this animal model. The genetic interaction between Scn2a and Kcnq2 demonstrates that combinations of mild alleles of monogenic epilepsy genes can result in severe disease and provides a model for complex inheritance of human epilepsy. The data suggest that interaction between these genes might contribute to the variable expressivity observed in human families with sodium-channel mutations. In a screen of 23 SMEI patients with missense mutations of SCN1A, no second-site mutations in KCNQ2 were identified.

Genetic modifiers affecting severity of epilepsy caused by mutation of sodium channel Scn2a.

Mutations in the voltage-gated sodium channels SCN 1 A and SCN 2 A are responsible for several types of human epilepsy. Variable expressivity among family members is a common feature of these inherited epilepsies, suggesting that genetic modifiers may influence the clinical manifestation of epilepsy. The transgenic mouse model Scn 2 a(Q 54) has an epilepsy phenotype as a result of a mutation in Scn 2 a that slows channel inactivation. The mice display progressive epilepsy that begins with short-duration partial seizures that appear to originate in the hippocampus. The partial seizures become more frequent and of longer duration with age and often induce secondary generalized seizures. Clinical severity of the Scn 2 a(Q 54) phenotype is influenced by genetic background. Congenic C57BL/6J.Q 54 mice exhibit decreased incidence of spontaneous seizures, delayed seizure onset, and longer survival in comparison with [C57BL/6J x SJL/J]F(1).Q 54 mice. This observation indicates that strain SJL/J carries dominant modifier alleles at one or more loci that determine the severity of the epilepsy phenotype. Genome-wide interval mapping in an N(2) backcross revealed two modifier loci on Chromosomes 11 and 19 that influence the clinical severity of of this sodium channel-induced epilepsy. Modifier genes affecting clinical severity in the Scn 2 a(Q 54) mouse model may contribute to the variable expressivity seen in epilepsy patients with sodium channel mutations.