Assessing the safety, tolerability and efficacy of PLGA-based immunomodulatory nanoparticles in patients with advanced NY-ESO-1-positive cancers: a first-in-human phase I open-label dose-escalation study protocol.

INTRODUCTION: The undiminished need for more effective cancer treatments stimulates the development of novel cancer immunotherapy candidates. The archetypical cancer immunotherapy would induce robust, targeted and long-lasting immune responses while simultaneously circumventing immunosuppression in the tumour microenvironment. For this purpose, we developed a novel immunomodulatory nanomedicine: PRECIOUS-01. As a PLGA-based nanocarrier, PRECIOUS-01 encapsulates a tumour antigen (NY-ESO-1) and an invariant natural killer T cell activator to target and augment specific antitumour immune responses in patients with NY-ESO-1-expressing advanced cancers. METHODS AND ANALYSIS: This open-label, first-in-human, phase I dose-escalation trial investigates the safety, tolerability and immune-modulatory activity of increasing doses of PRECIOUS-01 administered intravenously in subjects with advanced NY-ESO-1-expressing solid tumours. A total of 15 subjects will receive three intravenous infusions of PRECIOUS-01 at a 3-weekly interval in three dose-finding cohorts. The trial follows a 3+3 design for the dose-escalation steps to establish a maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D). Depending on the toxicity, the two highest dosing cohorts will be extended to delineate the immune-related parameters as a readout for pharmacodynamics. Subjects will be monitored for safety and the occurrence of dose-limiting toxicities. If the MTD is not reached in the planned dose-escalation cohorts, the RP2D will be based on the observed safety and immune-modulatory activity as a pharmacodynamic parameter supporting the RP2D. The preliminary efficacy will be evaluated as an exploratory endpoint using the best overall response rate, according to Response Evaluation Criteria in Solid Tumors V.1.1. ETHICS AND DISSEMINATION: The Dutch competent authority (CCMO) reviewed the trial application and the medical research ethics committee (CMO Arnhem-Nijmegen) approved the trial under registration number NL72876.000.20. The results will be disseminated via (inter)national conferences and submitted for publication to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04751786.

TRIM28 is an Epigenetic Barrier to Induced Pluripotent Stem Cell Reprogramming.

Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large-scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses (ERVs) silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of ERVs, thus facilitating the transition through reprogramming. Stem Cells 2017;35:147-157.

Polycomb group gene Ezh2 regulates mammary gland morphogenesis and maintains the luminal progenitor pool.

Specification of the cellular hierarchy in the mammary gland involves complex signaling that remains poorly defined. Polycomb group proteins are known to contribute to the maintenance of stem cell identity through epigenetic modifications, leading to stable alterations in gene expression. The polycomb protein family member EZH2 is known to be important for stem cell maintenance in multiple tissues, but its role in mammary gland development and differentiation remains unknown. Our analyses show that EZH2 is predominantly expressed in luminal cells of the mouse mammary epithelium. As mammary gland development occurs mostly after birth, the analysis of EZH2 gene function in postnatal development is precluded by embryonic lethality of conventional EZH2 knockout mice. To investigate the role of EZH2 in normal mammary gland epithelium, we have generated novel transgenic mice that express doxycycline-regulatable short hairpin (sh) RNAs directed against Ezh2. Knockdown of EZH2 results in delayed outgrowth of the mammary epithelium during puberty, due to impaired terminal end bud formation and ductal elongation. Furthermore, our results demonstrate that EZH2 is required to maintain the luminal cell pool and may limit differentiation of luminal progenitors into CD61(+) differentiated luminal cells, suggesting a role for EZH2 in mammary luminal cell fate determination. Consistent with this, EZH2 knockdown reduced lobuloalveolar expansion during pregnancy, suggesting EZH2 is required for the differentiation of luminal progenitors to alveolar cells.

Analysis of mice lacking DNaseI hypersensitive sites at the 5' end of the IgH locus.

The 5' end of the IgH locus contains a cluster of DNaseI hypersensitive sites, one of which (HS1) was shown to be pro-B cell specific and to contain binding sites for the transcription factors PU.1, E2A, and Pax5. These data as well as the location of the hypersensitive sites at the 5' border of the IgH locus suggested a possible regulatory function for these elements with respect to the IgH locus. To test this notion, we generated mice carrying targeted deletions of either the pro-B cell specific site HS1 or the whole cluster of DNaseI hypersensitive sites. Lymphocytes carrying these deletions appear to undergo normal development, and mutant B cells do not exhibit any obvious defects in V(D)J recombination, allelic exclusion, or class switch recombination. We conclude that deletion of these DNaseI hypersensitive sites does not have an obvious impact on the IgH locus or B cell development.

Identification of a candidate regulatory element within the 5' flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A.

The Igh locus is controlled by cis-acting elements, including Emu and the 3' IgH regulatory region which flank the C region genes within the well-studied 3' part of the locus. Although the presence of additional control elements has been postulated to regulate rearrangements of the VH gene array that extends to the 5' end of the locus, the 5' border of Igh and its flanking region have not been characterized. To facilitate the analysis of this unexplored region and to identify potential novel control elements, we physically mapped the most D-distal VH segments and scanned 46 kb of the immediate 5' flanking region for DNase I hypersensitive sites. Our studies revealed a cluster of hypersensitive sites 30 kb upstream of the most 5' VH gene. Detection of one site, HS1, is restricted to pro-B cell lines and HS1 is accessible to restriction enzyme digestion exclusively in normal pro-B cells, the stage defined by actively rearranging Igh-V loci. Sequence motifs within HS1 for PU.1, Pax5, and E2A bind these proteins in vitro and these factors are recruited to HS1 sequence only in pro-B cells. Transient transfection assays indicate that the Pax5 binding site is required for the repression of transcriptional activity of HS1-containing constructs. Thus, our characterization of the region 5' of the VH gene cluster demonstrated the presence of a single cluster of DNase I hypersensitive sites within the 5' flanking region, and identified a candidate Igh regulatory region defined by pro-B cell-specific hypersensitivity and interaction with factors implicated in regulating VDJ recombination.