Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin.

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1-UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state.

Significant changes in macrophage and CD8 T cell densities in primary prostate tumors 2 weeks after SBRT.

BACKGROUND: Radiotherapy impacts the local immune response to cancers. Prostate Stereotactic Body Radiotherapy (SBRT) is a highly focused method to deliver radiotherapy often used to treat prostate cancer. This is the first direct comparison of immune cells within prostate cancers before and after SBRT in patients. METHODS: Prostate cancers before and 2 weeks after SBRT are interrogated by multiplex immune fluorescence targeting various T cells and macrophages markers and analyzed by cell and pixel density, as part of a clinical trial of SBRT neoadjuvant to radical prostatectomy. RESULTS: Two weeks after SBRT, CD68, and CD163 macrophages are significantly increased while CD8 T cells are decreased. SBRT markedly alters the immune environment within prostate cancers.

The intraprostatic immune environment after stereotactic body radiotherapy is dominated by myeloid cells.

BACKGROUND: Hundreds of ongoing clinical trials combine radiation therapy, mostly delivered as stereotactic body radiotherapy (SBRT), with immune checkpoint blockade. However, our understanding of the effect of radiotherapy on the intratumoral immune balance is inadequate, hindering the optimal design of trials that combine radiation therapy with immunotherapy. Our objective was to characterize the intratumoral immune balance of the malignant prostate after SBRT in patients. METHODS: Sixteen patients with high-risk, non-metastatic prostate cancer at comparable Gleason Grade disease underwent radical prostatectomy with (n = 9) or without (n = 7) neoadjuvant SBRT delivered in three fractions of 8 Gy over 5 days completed 2 weeks before surgery. Freshly resected prostate specimens were processed to obtain single-cell suspensions, and immune-phenotyped for major lymphoid and myeloid cell subsets by staining with two separate 14-antibody panels and multicolor flow cytometry analysis. RESULTS: Malignant prostates 2 weeks after SBRT had an immune infiltrate dominated by myeloid cells, whereas malignant prostates without preoperative treatment were more lymphoid-biased (myeloid CD45+ cells 48.4 ± 19.7% vs. 25.4 ± 7.0%; adjusted p-value = 0.11; and CD45+ lymphocytes 51.6 ± 19.7% vs. 74.5 ± 7.0%; p = 0.11; CD3+ T cells 35.2 ± 23.8% vs. 60.9 ± 9.7%; p = 0.12; mean ± SD). CONCLUSION: SBRT drives a significant lymphoid to myeloid shift in the prostate-tumor immune infiltrate. This may be of interest when combining SBRT with immunotherapies, particularly in prostate cancer.

Phase 1 Trial of Stereotactic Body Radiation Therapy Neoadjuvant to Radical Prostatectomy for Patients With High-Risk Prostate Cancer.

PURPOSE: This study aimed to evaluate the feasibility and safety of prostate stereotactic body radiation therapy (SBRT) neoadjuvant to radical prostatectomy (RP) in a phase 1 trial. The primary endpoint was treatment completion rate without severe acute surgical complications. Secondary endpoints included patient-reported quality of life and physician-reported toxicities. METHODS AND MATERIALS: Patients with nonmetastatic high-risk or locally advanced prostate cancer received 24 Gy in 3 fractions to the prostate and seminal vesicles over 5 days, completed 2 weeks before RP. Patients with pN1 disease were treated after multidisciplinary discussion and shared decision making. Patient-reported quality of life (International Prostate Symptom Score and Expanded Prostate Cancer Index Composite 26-item version questionnaires) and physician-reported toxicity (Common Terminology Criteria for Adverse Events, version 4.03) were assessed before SBRT, immediately before surgery, and at 3-month intervals for 1 year. RESULTS: Twelve patients were enrolled, and 11 completed treatment (1 patient had advanced disease on prostate-specific membrane antigen positron emission tomography after enrollment but before treatment). There were no significant surgical complications. After RP, 2 patients underwent additional radiation therapy to nodes with androgen suppression for pN1 disease. Median follow-up after completion of treatment was 20.1 months, with 9 of 11 patients having a follow-up period of >12 months. Two patients had biochemical recurrence (prostate-specific antigen ≥0.05) within the first 12 months, with an additional 2 patients found to have biochemical recurrence after the 12-month period. The highest Common Terminology Criteria for Adverse Events genitourinary grades were 0, 1, 2, and 3 (n = 1, 4, 4, and 2, respectively), and the highest gastrointestinal grades were 0, 1, and 2 (n = 9, 1, and 1, respectively). At 12 months, incontinence was the only grade ≥2 toxicity. One and 2 of 9 patients had grade 2 and 3 incontinence, respectively. On the Expanded Prostate Cancer Index Composite (26-item version), the mean/median changes in scores from baseline to 12 months were -32.8/-31.1 for urinary incontinence, -1.6/-6.2 for urinary irritative/obstructive, -2.1/0 for bowel, -34.4/-37.5 for sexual function, and -10.6/-2.5 for hormonal. The mean/median change in International Prostate Symptom Score from baseline to 12 months was 0.5/0.5. CONCLUSIONS: RP after neoadjuvant SBRT appears to be feasible and safe at the dose tested. The severity of urinary incontinence may be higher than RP alone.

Interference with DNA repair after ionizing radiation by a pyrrole-imidazole polyamide.

Pyrrole-imidazole (Py-Im) polyamides are synthetic non-genotoxic minor groove-binding small molecules. We hypothesized that Py-Im polyamides can modulate the cellular response to ionizing radiation. Pre-treatment of cells with a Py-Im polyamide prior to exposure to ionizing radiation resulted in a delay in resolution of phosphorylated γ-H2AX foci, increase in XRCC1 foci, and reduced cellular replication potential. RNA-sequencing of cell lines exposed to the polyamide showed induction of genes related to the ultraviolet radiation response. We observed that the polyamide is almost 10-fold more toxic to a cell line deficient in DNA ligase 3 as compared to the parental cell line. Alkaline single cell gel electrophoresis reveals that the polyamide induces genomic fragmentation in the ligase 3 deficient cell line but not the corresponding parental line. The polyamide interferes directly with DNA ligation in vitro. We conclude that Py-Im polyamides may be further explored as sensitizers to genotoxic therapies.

A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes.

The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of gamma-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.

The element(s) at the nontranscribed Xist locus of the active X chromosome controls chromosomal replication timing in the mouse.

In female mammalian cells, the inactive X chromosome is replicated late in S phase while the active X chromosome is replicated earlier. The replication times of the X chromosomes reflect a general trend in which late replication is associated with gene repression and earlier replication with transcriptional competence. The X-linked Xist gene is expressed exclusively from the inactive X chromosome where it is involved in the initiation and maintenance of X-inactivation. In contrast, no biological activity has been assigned to the Xist locus of the active X chromosome where the Xist gene is transcriptionally silenced. Here, we provide evidence that the element(s) at the nontranscribed Xist locus of the active X chromosome controls chromosomal replication timing in cis.

Loss of Ubr2, an E3 ubiquitin ligase, leads to chromosome fragility and impaired homologous recombinational repair.

The N-end rule pathway of protein degradation targets proteins with destabilizing N-terminal residues. Ubr2 is one of the E3 ubiquitin ligases of the mouse N-end rule pathway. We have previously shown that Ubr2-/- male mice are infertile, owing to the arrest of spermatocytes between the leptotene/zygotene and pachytene of meiosis I, the failure of chromosome pairing, and subsequent apoptosis. Here, we report that mouse fibroblast cells derived from Ubr2-/- embryos display genome instability. The frequency of chromosomal bridges and micronuclei were much higher in Ubr2-/- fibroblasts than in +/+ controls. Metaphase chromosome spreads from Ubr2-/- cells revealed a high incidence of spontaneous chromosomal gaps, indicating chromosomal fragility. These fragile sites were generally replicated late in S phase. Ubr2-/- cells were hypersensitive to mitomycin C, a DNA cross-linking agent, but displayed normal sensitivity to gamma-irradiation. A reporter assay showed that Ubr2-/- cells are significantly impaired in the homologous recombination repair of a double strand break. In contrast, Ubr2-/- cells appeared normal in an assay for non-homologous end joining. Our results therefore unveil the role of the ubiquitin ligase Ubr2 in maintaining genome integrity and in homologous recombination repair.

Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells.

The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright) cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright) iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright) iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

Dexamethasone induces dysferlin in myoblasts and enhances their myogenic differentiation.

Glucocorticoids are beneficial in many muscular dystrophies but they are ineffective in treating dysferlinopathy, a rare muscular dystrophy caused by loss of dysferlin. We sought to understand the molecular basis for this disparity by studying the effects of a glucocorticoid on differentiation of the myoblast cell line, C2C12, and dysferlin-deficient C2C12s. We found that pharmacologic doses of dexamethasone enhanced the myogenic fusion efficiency of C2C12s and increased the induction of dysferlin, along with specific myogenic transcription factors, sarcolemmal and structural proteins. In contrast, the dysferlin-deficient C2C12 cell line demonstrated a reduction in long myotubes and early induction of particular muscle differentiation proteins, most notably, myosin heavy chain. Dexamethasone partially reversed the defect in myogenic fusion in the dysferlin-deficient C2C12 cells. We hypothesize that a key therapeutic benefit of glucocorticoids may be the up-regulation of dysferlin as an important component of glucocorticoid-enhanced myogenic differentiation.

Inhibition of Atm and/or Atr disrupts gene silencing on the inactive X chromosome.

ATM and ATR are well documented for their roles in maintaining the integrity of genomic DNA by responding to DNA damage and preparing the cell for repair. Since ATM and ATR have been reported to exist in complexes with histone deacetylases, we asked whether Atm and Atr might also uphold gene silencing by heterochromatin. We show that the Atm/Atr inhibitor 2-aminopurine causes the inactive X chromosome to accumulate abnormal chromatin and undergo unwanted gene reactivation. We provide evidence that this gene expression from the inactive X chromosome is not a byproduct of the accumulation of DNA breaks. Individually inhibiting Atm and Atr by either small interfering RNA or the expression of dominant-negative ATM and ATR constructs also compromised X-inactivation. Atm and Atr, therefore, not only function in responding to DNA damage but perhaps also are involved in gene silencing via the maintenance of heterochromatin.