The current integrative pathobiological hypothesis states that pancreatic cancer (PDAC) develops and progresses in response to an interaction between known oncogenes and downstream epigenomic regulators. Congruently, this study tests a new combinatorial therapy based on the inhibition of the Aurora kinase A (AURKA) oncogene and one of its targets, the H3K9 methylation-based epigenetic pathway. This therapeutic combination is effective at inhibiting the in vitro growth of PDAC cells both, in monolayer culture systems, and in 3D spheroids and organoids. The combination also reduces the growth of PDAC xenografts in vivo. Mechanistically, it was found that inhibiting methyltransferases of the H3K9 pathway in cells, which are arrested in G2/M after targeting Aurora kinase A, decreases H3K9 methylation at centromeres, induces mitotic aberrations, triggers an aberrant mitotic check point response, and ultimately leads to mitotic catastrophe. Combined, this data describes for the first time a hypothesis-driven design of an efficient combinatorial treatment that targets a dual oncogenic-epigenomic pathway to inhibit PDAC cell growth via a cytotoxic mechanism that involves perturbation of normal mitotic progression to end in mitotic catastrophe. Therefore, this new knowledge has significant mechanistic value as it relates to the development of new therapies, as well as biomedical relevance.
We have previously shown that amino acid changes in the human Kruppel-Like Factor (KLF) 11 protein is associated with the development of maturity onset diabetes of the young VII, whereas complete inactivation of this pathway by the -331 human insulin mutation causes neonatal diabetes mellitus. Here, we report that Klf11-/- mice have decreased circulating insulin levels, alterations in the control of blood glucose and body weight, as well as serum dyslipidemia, but do not develop diabetes. Functional assays using ex vivo liver tissue sections demonstrate that Klf11-/- mice display increased insulin sensitivity. Genome-wide experiments validated by pathway-specific quantitative PCR arrays reveal that the Klf11-/- phenotype associates to alterations in the regulation of gene networks involved in lipid metabolism, in particular those regulated by peroxisome proliferator-activated receptor-γ. Combined, these results demonstrate that the major phenotype given by the whole-body deletion of Klf11 in mouse is not diabetes but increased insulin sensitivity, likely due to altered transcriptional regulation in target tissues. The absence of diabetes in the Klf11-/- mouse either indicates an interspecies difference for the role of this transcription factor in metabolic homeostasis between mouse and humans, or potentially highlights the fact that other molecular factors can compensate for its absence. Nevertheless, the data of this study, gathered at the whole-organism level, further support a role for KLF11 in metabolic processes like insulin sensitivity, which regulation is critical in several forms of diabetes.
Because of its dismal outcome, pancreatic ductal adenocarcinoma (PDAC) remains a therapeutic challenge making the testing of new pharmacologic tools a goal of paramount importance. Here, we developed a rational approach for inhibiting PDAC growth based on leveraging cell-cycle arrest of malignant cells at a phase that shows increased sensitivity to distinct epigenomic inhibitors. Specifically, we simultaneously inhibited checkpoint kinase 1 (Chk1) by prexasertib and the G9a histone methyltransferase with BRD4770, thereby targeting two key pathways for replication fork stability. Methodologically, the antitumor effects and molecular mechanisms of the combination were assessed by an extensive battery of assays, utilizing cell lines and patient-derived cells as well as 3D spheroids and xenografts. We find that the prexasertib-BRD4770 combination displays a synergistic effect on replication-associated phenomena, including cell growth, DNA synthesis, cell-cycle progression at S phase, and DNA damage signaling, ultimately leading to a highly efficient induction of cell death. Moreover, cellular and molecular data reveal that the synergistic effect of these pathways can be explained, at least in large part, by the convergence of both Chk1 and G9a functions at the level of the ATR-RPA-checkpoint pathway, which is operational during replication stress. Thus, targeting the epigenetic regulator G9a, which is necessary for replication fork stability, combined with inhibition of the DNA damage checkpoint, offers a novel approach for controlling PDAC growth through replication catastrophe. Implications: This study offers an improved, context-dependent, paradigm for the use of epigenomic inhibitors and provides mechanistic insight into their potential therapeutic use against PDAC.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine N-Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes KrasG12D-mediated acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN) formation in both the Pdx1-Cre and P48Cre/+KrasG12D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway-mediated ADM upon EHMT2 deletion. Notably, KrasG12D increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from KrasG12D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during KrasG12D-mediated initiation. The inhibitory effect on KrasG12D-induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis. This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo, extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC.
Pancreatic ductular adenocarcinoma (PDAC) ranks fourth as a cause of cancer death in the USA and is almost universally fatal, with the annual number of deaths equivalent to the number of newly diagnosed cases. Valuable research in the field has revealed genetic aberrations that contribute to PDAC development and progression, with KRAS being one of the most frequent mutations in more than 90% of patient samples. However, to date, any efforts to directly target KRAS have failed in the clinic. Thus, there is indisputably an urgent need to further improve our understanding of molecular mechanisms underlying PDAC development as to identify novel therapeutic targets, including druggable important downstream targets and nodes orchestrated by oncogenic KRAS. In particular, we are interested in epigenetic pathways involved in PDAC development and progression due to the potential reversibility of any alteration, unlike genetic mutation. In the current study, using a cell model that allows inducible expression of mutant KRASG12D, we find that protein levels of the dimethyl-K9H3 histone methyl transferase (HMT), G9a, and its complex partners are increased in response to activation of the oncogenic Kras pathway. Furthermore, the activation of this oncogenic pathway results in the formation of the G9a-GLP-Wiz trimer complex, as determined by affinity protein purification, combined with mass spectrometry. In vivo experiments involving the cross of the Pdx1-CRE/LSL-KRASG12D mice with G9afl/fl animals demonstrate that a loss of the H3K9Me2 mark in the nucleus of exocrine cells is accompanied by a significantly reduced number of PanIN lesions. RNA-Seq experiments from these animals reveal that these mice have reduced levels of typical molecular markers of PanINs. In addition, these experiments show changes in the levels in several genes, which have been previously been shown to synergize with Kras to mediate pancreatic cancer initiation. Congruently, pharmacological inhibition of G9a using BRD4770 displays an inhibitory effect on KRASG12D-induced cell proliferation. Combined, these data provide evidence for a key role of the meK9H3-G9a pathway as a mediator of the oncogenic Kras response and defines a novel point of potential therapeutic intervention for PDAC. Citation Format: Angela Mathison, Ann Salmonson, Brooke Paradise, Mckenna Missfeldt, Juan Iovanna, Daniel Billadeau, Raul Urrutia, Gwen Lomberk. The epigenetic regulator, G9a, is a KRAS-inducible protein and its inactivation inhibits PanIN formation by this oncogene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1391. doi:10.1158/1538-7445.AM2017-1391
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