In this study, we defined critical roles for KDM2B in PDAC pathogenesis and provided insights into the transcriptional functions of this chromatin-modifying enzyme. Examination of human tumor specimens coupled with genome-wide analyses revealed that KDM2B is a central driver of an epigenetic program critical for tumorigenicity of poorly differentiated (quasimesenchymal) PDAC. KDM2B is part of distinct transcriptional modules that have different functions in gene regulation: occupancy of TSSs together with PcG proteins suppresses lineage-specific genes, whereas cobinding with MYC and/or KDM5A positively regulates transcription of sets of genes involved broadly in metabolic homeostasis (Figure C). Loss of these transcriptional programs upon KDM2B silencing in PDAC cell lines resulted in upregulation of pancreatic differentiation genes, sensitization to apoptosis in response to energy stress, and pronounced reduction in tumorigenicity.
The important functional contributions of PcG proteins to KDM2B-regulated tumor maintenance that we defined in PDAC extend prior results demonstrating the interplay of KDM2B and EZH2 (11
). By mapping the genome-wide chromatin binding sites of these enzymes and integrating the data with transcriptional changes caused by KDM2B modulation, we found that PRC2-mediated repression represents a major component of the KDM2B-regulated transcriptome, with 31% of EZH2 target genes cobound by KDM2B, and approximately 45% of these KDM2B-EZH2 targets showing differential expression (predominantly upregulation) upon KDM2B knockdown (Figure , E and F). KDM2B knockdown interfered with the binding of EZH2 — and of PRC1 components at certain loci, a function that correlates with H3K36me2 HDM activity. Given that PRC2 binding is inhibited by active chromatin marks, including di- and trimethylated H3K36 (39
), KDM2B may facilitate the recruitment of PRC2 to target loci through demethylation of H3K36. KDM2B also physically interacts with PcG proteins, both in mammalian cells and in flies, which may contribute to the targeting of PRC1 and PRC2 (12
). Moreover, we observed that KDM2B positively regulated EZH2 levels in a subset of PDAC cell lines via epigenetic silencing of the let-7b
miRNAs, which target EZH2 (Supplemental Figure 7, A–C), as reported in other cell types (11
). Importantly, bioinformatic analysis of 2,158 human tumor specimens showed that the expression levels of EZH2 and KDM2B were closely correlated across tumor types (Supplemental Figure 7D) and in human PDAC specimens (Supplemental Figure 7E), and KDM2B showed the highest degree of coordinate regulation with EZH2 compared with all HDM family members (Supplemental Figure 7F). As silencing of PRC2 targets and induction of EZH2 are associated with poor differentiation and shortened survival across multiple cancer types, including PDAC (22
), these data establish the widespread importance of cooperation between KDM2B and PcG proteins in antagonizing developmental decisions in cancer.
Our ChIP-seq studies showed that greater than 25% of the KDM2B target loci were co-occupied by KDM5A in PDAC cells (Figure A). Likewise, integration of these data with publicly available datasets suggested that there was also considerable overlap in the binding of these 2 factors with c-MYC (Figure , B and C). This latter set of targets was greatly enriched for actively transcribed genes and genes that were downregulated upon KDM2B silencing (Figure , D–F). KDM2B did not appear to mediate demethylation of H3K36 or H3K4 at these loci; rather, KDM2B inactivation reduced H3K4me3 levels in accordance with the observed downregulation of gene expression. These observations implicate KDM2B in the positive regulation of transcription, which was unexpected based on prior functional studies. However, crosstalk between KDM2B and KDM5A is consistent with recent findings in Drosophila
showing genetic interactions between dKDM2 and the KDM5A ortholog Lid (31
). Moreover, while the best-characterized functions of KDM5A/Lid are in transcriptional repression through its H3K4 HDM activity, KDM5A/Lid has been shown to directly activate transcription by binding to MYC or to circadian transcription factors at target loci, a function independent of its demethylase activity (31
). In addition, published KDM5A genome-wide chromatin binding profiles in lymphoma and ES cells (35
) show considerable overlap with our present observations in PDAC cells, connecting KDM5A to active transcription.
The set of genes positively regulated by KDM2B was highly enriched for components of the translational machinery and for mitochondrial function. These profiles are in line with the well-characterized role for MYC in metabolic regulation, including the requirement for induction of ribosomal genes for MYC oncogenic activity (44
). In addition, KDM5A has been shown to bind comparable sets of targets in multiple cell types and appears to be required for maintenance of mitochondrial function (35
). While the magnitude of change in the expression of these KDM2B-KDM5A-MYC–bound genes upon KDM2B knockdown was less pronounced compared with the observed upregulation of the KDM2B-PcG targets, these profiles were remarkable in showing decreases in multiple components of key metabolic pathways (Figure E and Figure , B and C). KDM2B knockdown was associated with an energy stress response, as reflected by activation of AMPK signaling and downregulation of mTOR activity (Figure E, Figure , F and G, and Supplemental Figure 6, E–G). Moreover, these cells were highly sensitized to apoptosis upon energy depletion induced by glucose removal, which suggests that KDM2B maintains tumorigenesis by coordinating both metabolic and cell differentiation decisions (Figure C).
KDM5A is overexpressed in human PDAC specimens, and our data indicated that KDM2B contributes to this induction (Figure , C and D). Although further studies are needed to delineate the associated molecular mechanisms, it is noteworthy that miR-101
, which is directly repressed by KDM2B (11
), is predicted to target KDM5A in addition to EZH2. KDM5A is also amplified in some PDAC specimens (2
) and cell lines (e.g., TU-8902), and the c-MYC locus frequently shows copy number increases in these tumors (2
). Thus, genomic gains, upregulated expression, and functional synergy among these factors are together likely to contribute to the high activity of this transcriptional module in poorly differentiated PDAC.
Our observations linking KDM2B to the tumorigenicity of an aggressive subset of PDAC through integral roles in both PcG- and MYC-mediated transcriptional regulation are notable in light of the emerging relationship between cancers and pluripotent stem cells. Cancer cells and ES cells share the properties of indefinite self-renewal and blocked differentiation and have overlapping gene expression signatures, which supports the view that cancers are transcriptionally reprogrammed toward an ES cell–like state (22
). Recent studies have suggested a refined model, showing that the ES cell self-renewal transcriptional program involves separate regulatory modules centering on PcG, MYC, and the core pluripotency factors (e.g., OCT4, KLF4, and NANOG); in cancers, the partial reactivation of this ES cell signature consisting of the PcG and MYC modules correlates with advanced stage and poor outcome (26
). Thus, the dual transcriptional functions of KDM2B, involving PcG and MYC activity, that cooperate to drive the growth of poorly differentiated PDAC cells impinge directly on 2 distinct pluripotency networks. These findings, together with the previous demonstration that KDM2B enhances somatic cell reprogramming by OCT4 (15
), are consistent with an ability of KDM2B to subvert differentiation in primary cells and to sustain undifferentiated states in advanced cancers.
In addition to predicting poor outcomes, the quasimesenchymal subtype of PDAC also appears to have signaling dependencies different from those of more epithelial (classical) tumors, as reflected by previous studies in cell lines showing that the former subtype has reduced sensitivity to knockdown of the KRAS oncogene and altered responsiveness to anticancer drugs (3
). Our studies suggest that KDM2B regulates a critical epigenetic switch that balances developmental and metabolic decisions in these poorly differentiated PDAC cells. Since KDM2B catalytic activity appears to be required for tumorigenesis, small-molecule inhibitors may provide therapeutic benefit in this chemoresistant PDAC subtype.