In vivo modeling of metastatic human high-grade serous ovarian cancer in mice.

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.

Clonal Dynamics In Vivo of Virus Integration Sites of T Cells Expressing a Safety Switch.

Safety switches are becoming relevant for the clinical translation of T-cell-based immunotherapies. In patients receiving an allogeneic hematopoietic stem cell transplant, the inducible caspase-9 gene (iC9) safety switch expressed by donor-derived T lymphocytes efficiently controls acute graft versus host disease (GvHD). However, in vivo elimination of iC9-T cells by the chemical inducer of dimerization (CID) that activates the iC9 protein is incomplete. To study this effect, we characterized the clonal diversity and dynamics of vector insertion sites (VIS) in iC9-T cells pre- and post-CID administration in four patients who developed GvHD. We identified 3,203 VIS among four patients and followed their in vivo clonal dynamics up to 161 days post-CID. VIS were categorized by their proximity to host genome elements, gene associations, and cis-modulatory relationship to mapped promoters. We found that VIS are preferentially located near open chromatin and promoter regions; furthermore, there was no evidence for selection bias among VIS surviving the CID treatment. The majority of iC9-T cells with high normalized VIS copy number at the time of GvHD onset were eliminated by CID, while iC9-T cells detectable post-CID generally have low normalized VIS copy number. We propose that suboptimal iC9 transgene expression is responsible for the incomplete elimination of iC9-T cells and illustrate here by simple model how cis-modulatory influences of local genome context and T-cell receptor activation status at time of CID treatment contribute to stochastic sparing of iC9-T cells.

Tumor immune contexture is a determinant of anti-CD19 CAR T cell efficacy in large B cell lymphoma.

Axicabtagene ciloleucel (axi-cel) is an anti-CD19 chimeric antigen receptor (CAR) T cell therapy approved for relapsed/refractory large B cell lymphoma (LBCL) and has treatment with similar efficacy across conventional LBCL subtypes. Toward patient stratification, we assessed whether tumor immune contexture influenced clinical outcomes after axi-cel. We evaluated the tumor microenvironment (TME) of 135 pre-treatment and post-treatment tumor biopsies taken from 51 patients in the ZUMA-1 phase 2 trial. We uncovered dynamic patterns that occurred within 2 weeks after axi-cel. The biological associations among Immunoscore (quantification of tumor-infiltrating T cell density), Immunosign 21 (expression of pre-defined immune gene panel) and cell subsets were validated in three independent LBCL datasets. In the ZUMA-1 trial samples, clinical response and overall survival were associated with pre-treatment immune contexture as characterized by Immunoscore and Immunosign 21. Circulating CAR T cell levels were associated with post-treatment TME T cell exhaustion. TME enriched for chemokines (CCL5 and CCL22), γ-chain receptor cytokines (IL-15, IL-7 and IL-21) and interferon-regulated molecules were associated with T cell infiltration and markers of activity. Finally, high density of regulatory T cells in pre-treatment TME associated with reduced axi-cel-related neurologic toxicity. These findings advance the understanding of LBCL TME characteristics associated with clinical responses to anti-CD19 CAR T cell therapy and could foster biomarker development and treatment optimization for patients with LBCL.

Estrogen Receptors alpha and beta as determinants of gene expression: influence of ligand, dose, and chromatin binding.

Estrogen receptors alpha and beta (ERalpha and ERbeta) mediate the actions of estrogens in a variety of normal and cancer target cells. Estrogens differ in their preference for these ERs, and many phytoestrogens bind preferentially to ERbeta. To investigate how phytoestrogens such as genistein impact ER-regulated gene expression, we used adenoviral gene delivery of ERbeta coupled with ERalpha depletion with small interfering RNA to generate human breast cancer (MCF-7) cells expressing four complements of ERalpha and ERbeta. We examined the dose-dependent effects of genistein on genome-wide gene expression by DNA microarrays and monitored the recruitment of ERs and coregulators to responsive regions of estrogen-regulated genes. At a low (6 nm) concentration, genistein regulated gene expression much more effectively in cells coexpressing ERalpha and ERbeta than in cells expressing ERalpha alone, whereas at high concentration (300 nm), genistein induced transcriptome changes very similar to that of 17beta-estradiol. We demonstrate that ERbeta is preferentially activated by genistein and is recruited to estrogen-responsive genomic sites and that differential occupancy of ERalpha and ERbeta by genistein and 17beta-estradiol in turn influences the recruitment patterns of coregulators such as steroid receptor coactivator 3 (SRC3) and receptor-interacting protein 140 (RIP140). Our observations indicate that genistein is a potency-selective ligand for gene expression regulation by ERalpha and ERbeta and that the ability of ERalpha and ERbeta to serve as determinants of gene expression is greatly influenced by the nature of the ligand, by ligand dose, and by the differential abilities of ligand-ER complexes to recruit different coregulators at ER binding sites of hormone-regulated genes.

Gene expression preferentially regulated by tamoxifen in breast cancer cells and correlations with clinical outcome.

The beneficial effect of the selective estrogen receptor (ER) modulator tamoxifen in the treatment and prevention of breast cancer is assumed to be through its ability to antagonize the stimulatory actions of estrogen, although tamoxifen can also have some estrogen-like agonist effects. Here, we report that, in addition to these mixed agonist/antagonist actions, tamoxifen can also selectively regulate a unique set of >60 genes, which are minimally regulated by estradiol (E2) or raloxifene in ERalpha-positive MCF-7 human breast cancer cells. This gene regulation by tamoxifen is mediated by ERalpha and reversed by E2 or ICI 182,780. Introduction of ERbeta into MCF-7 cells reverses tamoxifen action on approximately 75% of these genes. To examine whether these genes might serve as markers of tamoxifen sensitivity and/or the development of resistance, their expression level was examined in breast cancers of women who had received adjuvant therapy with tamoxifen. High expression of two of the tamoxifen-stimulated genes, YWHAZ/14-3-3z and LOC441453, was found to correlate significantly with disease recurrence following tamoxifen treatment in women with ER-positive cancers and hence seem to be markers of a poor prognosis. Our data indicate a new dimension in tamoxifen action, involving gene expression regulation that is tamoxifen preferential, and identify genes that might serve as markers of tumor responsiveness or resistance to tamoxifen therapy. This may have a potential effect on the choice of tamoxifen versus aromatase inhibitors as adjuvant endocrine therapy.

Impact of estrogen receptor beta on gene networks regulated by estrogen receptor alpha in breast cancer cells.

Two subtypes of the estrogen receptor (ER), ERalpha and ERbeta, mediate the actions of estrogens, and although 70% of human breast cancers express ERbeta along with ERalpha, little is known about the possible comodulatory effects of these two ERs. To investigate this, we have used adenoviral gene delivery to produce human breast cancer (MCF-7) cells expressing different levels of ERbeta, along with their endogenous ERalpha, and have examined the effects of ERbeta and receptor occupancy, using ER subtype selective ligands, on genome-wide gene expression by microarray and pathway network analysis. ERbeta had diverse effects on gene expression, enhancing or counteracting ERalpha regulation for distinct subsets of estrogen target genes. Strikingly, ERbeta in the absence of estradiol (E2), elicited the stimulation or suppression of many genes that were normally only regulated by ERalpha with E2. In addition, ERbeta plus E2 elicited the expression of a unique group of genes that were not regulated by ERalpha plus E2 alone. The expression of genes in many functional categories were modulated by ERbeta, with the greatest numbers associated with transcription factors and signal transduction pathways. Regulation of multiple components in the TGFbeta and semaphorin pathways, and of genes controlling cell cycle progression and apoptosis, may contribute to the suppression of cell proliferation observed with ERbeta. Our observations suggest that the relative levels of ERbeta and ERalpha in breast cancers are likely to impact cell proliferation and the activities of diverse signaling pathways and their response to ER ligands and endocrine therapies.

Genome-wide dynamics of chromatin binding of estrogen receptors alpha and beta: mutual restriction and competitive site selection.

Estrogen receptors ERalpha and ERbeta, members of the nuclear receptor superfamily, exert profound effects on the gene expression and biological response programs of their target cells. Herein, we explore the dynamic interplay between these two receptors in their selection of chromatin binding sites when present separately or together in MCF-7 breast cancer cells. Treatment of cells (containing ERalpha only, ERbeta only, or ERalpha and ERbeta) with estradiol or ER subtype-selective ligands was followed by chromatin immunoprecipitation analysis with a custom-designed tiling array for ER binding sites across the genome to examine the effects of ligand-occupied and unoccupied ERalpha and ERbeta on chromatin binding. There was substantial overlap in binding sites for these estradiol-liganded nuclear receptors when present alone, but many fewer sites were shared when both ERs were present. Each ER restricted the binding site occupancy of the other, with ERalpha generally being dominant. Binding sites of both receptors were highly enriched in estrogen response element motifs, but when both ERs were present, ERalpha displaced ERbeta, shifting it into new sites less enriched in estrogen response elements. Binding regions of the two ERs also showed differences in their enrichments for other transcription factor binding motifs. Studies with ER subtype-specific ligands revealed that it was the liganded subtype that principally determined the spectrum of chromatin binding. These findings highlight the dynamic interplay between the two ERs in their selection of chromatin binding sites, with competition, restriction, and site shifting having important implications for the regulation of gene expression by these two nuclear receptors.