JARID1 proteins are histone demethylases that both regulate normal cell fates during development and contribute to the epigenetic plasticity that underlies malignant transformation. This H3K4 demethylase family participates in multiple repressive transcriptional complexes at promoters and has broader regulatory effects on chromatin that remain ill-defined. There is growing understanding of the oncogenic and tumor suppressive functions of JARID1 proteins, which are contingent on cell context and the protein isoform. Their contributions to stem cell-like de-differentiation, tumor aggressiveness, and therapy resistance in cancer have sustained interest in the development of JARID1 inhibitors. Here we review the diverse and context-specific functions of the JARID1 proteins that may impact the utilization of emerging targeted inhibitors of this histone demethylase family in cancer therapy.

Objectives/Hypothesis Delineate factors impacting creation and use of patient-derived xenografts (PDXs) of human papilloma virus-related (HPV+) head and neck squamous cell carcinomas (HNSCCs). Study Design Laboratory-based translational study. Methods 51 surgically resected HNSCCs, including 31 HPV+ cancers, were implanted into NOD/SCID/IL-2Rγ−/− (NSG) mice using standardized methodology. Clinical and pathologic factors were tested for association with engraftment. The gross, histologic, and molecular features of established HPV+ PDXs were analyzed in comparison to their tumors of origin. Results Negative HPV status and perineural invasion (PNI) were independent, additive factors associated with increased PDX formation. Epstein-Barr Virus-positive (EBV+) human large B-cell lymphomas grew from 32% of HPV+ HNSCC cases that failed to engraft. Successfully established HPV+ PDXs retained basaloid histology and often developed cystic growth patterns typical of HPV+ nodal metastases. They also maintained elevated p16INK4A levels and expression of E6/E7 viral oncogene transcripts. Conclusion Reduced engraftment by HPV+ tumors lacking PNI likely results in selection biases in HNSCC PDX models. Formation of EBV+ lymphomas in NSG mice further reduces generation of HPV+ models and must be ruled out before long-term use of PDXs. Nevertheless, retention of distinctive pathologic traits and viral oncogene expression by HPV+ PDXs provides a viable in vivo platform for basic and translational studies as well as a resource for generating advanced in vitro models.

The degree of heterogeneity among cancer stem cells (CSC) remains ill-defined and may hinder effective anti-CSC therapy. Evaluation of oral cancers for such heterogeneity identified two compartments within the CSC pool. One compartment was detected using a reporter for expression of the H3K4me3 demethylase JARID1B to isolate a JARID1Bhigh fraction of cells with stem cell-like function. JARID1Bhigh cells expressed oral CSC markers including CD44 and ALDH1 and showed increased PI3-kinase (PI3K) pathway activation. They were distinguished from a fraction in a G0-like cell cycle state characterized by low reactive oxygen species and suppressed PI3K/AKT signaling. G0-like cells lacked conventional CSC markers but were primed to acquire stem cell-like function by upregulating JARID1B, which directly mediated transition to a state expressing known oral CSC markers. The transition was regulated by PI3K signals acting upstream of JARID1B expression, resulting in PI3K inhibition depleting JARID1Bhigh cells but expanding the G0-like subset. These findings define a novel developmental relationship between two cell phenotypes that may jointly contribute to CSC maintenance. Expansion of the G0-like subset during targeted depletion of JARID1Bhigh cells implicates it as a candidate therapeutic target within the oral CSC pool.

Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug Brefeldin A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1Δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1Δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity.chromatin | gene regulation | histone methylation | epigenetics M odifications on histones, such as phosphorylation, acetylation, methylation, and ubiquitination, have been implicated in gene expression and silencing (1, 2). One modification on histone H3 that is associated with active gene expression is the methylation of lysine 4 (H3K4) (3, 4). H3K4 methylation is present concurrently in mono-, di-, and trimethyl forms in the cell (1, 2). Primarily, H3K4 methylation is maintained in a pattern that has trimethylation enriched at the 5′ end of ORFs, dimethylation throughout the gene, and monomethylation enriched near the 3′ end (3, 4). In humans, H3K4 methylation is mediated by methyltransferases MLL1-4 and Set1A and -B complexes, which are homologous to the yeast Set1 H3K4 methyltransferase complex (5-12). In addition, Set1 and the human homologs are known to interact with the Ser5-phosphorylated C-terminal tail domain of RNA polymerase II (3). More recently, di-and trimethylated H3K4 have been shown to be docking sites for chromodomain and plant homeodomain (PHD) fingercontaining proteins (1,13,14). Taken together, this and other studies suggest that the methylation of histone H3K4 plays a role in mediating gene expression by recruiting effector proteins.In Saccharomyces cerevisiae, the Set1 complex (Set1C or COMPASS) is responsible for the methylation of histone H3K4 and the kinetochore protein Dam1 (5,[15][16][17][18][19]. Loss of the catalytic protei...

Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.

Upregulation of the H3K4me3 demethylase JARID1B is linked to acquisition of aggressive, stem cell-like features by many cancer types. However, the utility of emerging JARID1 family inhibitors remains uncertain, in part because JARID1B’s functions in normal development and malignancy are diverse and highly context-specific. In this study, responses of oral squamous cell carcinomas (OSCCs) to catalytic inhibition of JARID1B were assessed using CPI-455, the first tool compound with true JARID1 family selectivity. CPI-455 attenuated clonal sphere and tumor formation by stem-like cells that highly express JARID1B while also depleting the CD44-positive and Aldefluor-high fractions conventionally used to designate OSCC stem cells. Silencing JARID1B abrogated CPI-455’s effects on sphere formation, supporting that the drug acted through this isoform. To further delineate CPI-455’s capacity to block JARID1B’s functions, its biologic effects were compared against those indicated by pathway analysis of the transcriptional profile produced by JARID1B knockdown. Downregulation of multiple gene sets related to stem cell function was consistent with the drug’s observed actions. However, strong E-Cadherin upregulation seen upon silencing JARID1B surprisingly could not be reproduced using CPI-455. Expressing a demethylase-inactive mutant of JARID1B demonstrated suppression of this transcript to be demethylase-independent, and the capacity of mutant JARID1B but not CPI-455 to modulate invasion provided a functional correlate of this finding. These results show that JARID1B catalytic inhibition effectively targets some stem cell-like features of malignancy but also reveal demethylase-independent actions refractory to inhibition. Future application of JARID1 inhibitors in combinatorial use for cancer therapy may be guided by these findings.

No abstract

<p>Differentially expressed genes in JARID1Bhigh vs. bulk LNT14 cells.</p>