Christensen, Trace

Abstract By regulating gene expression networks that mediate neoplastic behavior, epigenetic protein complexes are the ultimate effectors of oncogenic pathways. This hierarchical position of epigenetic pathways within the mechanisms that regulate cancer makes them ideal candidates for therapeutic targets. The current work was designed to characterize the role of the K9H3 Histone Methyltransferase (HMT) pathway in mediating oncogenic signals downstream of Aurora Kinase A (AurkA), with the goal of designing efficient combinatorial therapies against PDAC. Affinity protein purification, combined with mass spectrometry, demonstrates that HP1γ isolated from mitotic cells interacts with AurkA and the HMTs, G9a and GLP. We also find that this complex is critical for mitotic progression and cell proliferation. Congruently, treatment of PDAC cells with individual drugs against AurkA or HP1-HMT inhibits cell growth by inducing senescence, a cytostatic response. However, the combination of these agents has a synergistic effect of reducing cell growth in both, monolayer and spheroid cultures to result in a cytotoxic effect. Confocal and electron microscopy, along with cell cycle analysis, demonstrate that the cytotoxic effect of this combination is due to induction of mitotic catastrophe, a distinct form of cell death that occurs during mitosis. Molecularly, the combination of AurkA and HP1-HMT inhibition bypasses G2/M arrest with downregulation of the Chk1-Cdc25c pathway. In vivo treatment of PDAC orthotopic xenografts with the HP1-HMT inhibitor alone demonstrated reduced PDAC growth, and increased efficacy in PDAC growth reduction was observed in combination with the AurkA inhibitor over either individual treatment. Thus, our data demonstrate a novel AurkA-HP1-HMT mitotic pathway that holds promise for potential pharmacological targeting in combinatorial therapy for this malignancy. Citation Format: Angela Mathison, Ann Salmonson, Mckenna Missfeldt, Thiago de Assuncao, Jennifer Bintz, Trace Christensen, Sarah Kossak, Asha Nair, Juan Iovanna, Robert Huebert, Gwen Lomberk. Pharmacological targeting of the Aurora A and Histone 3 lysine 9 methyltransferase pathways in pancreatic cancer induces mitotic catastrophe [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 4060. doi:10.1158/1538-7445.AM2017-4060

Abstract The current integrative pathobiologic 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 three-dimensional 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 AURKA, decreases H3K9 methylation at centromeres, induces mitotic aberrations, triggers an aberrant mitotic check point response, and ultimately leads to mitotic catastrophe. Combined, these data describe 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. Implications: These results outline a model for the combined inhibition of a genetic-to-epigenetic pathway to inhibit cell growth and suggest an important and provocative consideration for harnessing the capacity of cell-cycle inhibitors to enhance the future use of epigenetic inhibitors. Mol Cancer Res; 15(8); 984–97. ©2017 AACR.

Abstract A circulating isoform of EGFR has been identified as an emerging serum biomarker in cancer patients. Serum concentrations of EGFR have been evaluated as screening, diagnostic, prognostic, and theragnostic markers for diverse human malignancies, including breast, ovarian, endometrial, colorectal, and lung cancer. Yet full-length EGFR is rarely shed from normal or malignant cells. Here we demonstrate that an alternately spliced EGFR isoform, termed ‘sEGFR,’ gives rise to the circulating sEGFR protein through proteolytic cleavage of a cell surface precursor. This cleavage event is inhibited by fibronectin and stimulated by both an anti-Δ5/α1 integrin antibody and the metalloprotease activator 4-aminophenylmercuric acetate. Preliminary results identify ADAM17/TACE as at least one mediator of sEGFR shedding. Shed sEGFR binds to EGF with high affinity. Two FDA-approved therapeutic anti-EGFR antibodies, cetuximab (Erbitux) and panitumumab (Vectibix), inhibit shedding of sEGFR, implicating sEGFR as a potential alternative target for these antibodies, consistent with previous studies indicating that baseline serum sEGFR concentrations may predict responsiveness to anti-EGFR-targeted therapy. These observations bear striking similarity to aspects of trastuzumab regulation of HER2, shedding, and, by analogy, may have implications for the design of future clinical trials using EGFR-directed therapeutics, including cetuximab and panitumimab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3624. doi:1538-7445.AM2012-3624