Interferon alpha (IFNα) is used for the treatment of hepatitis C infection and whilst efficacious it is associated with multiple adverse events including reduced leukocyte, erythrocyte, and platelet counts, fatigue, and depression. These events are most likely caused by systemic exposure to interferon. We therefore hypothesise that targeting the therapeutic directly to the intended site of action in the liver would reduce exposure in blood and peripheral tissue and hence improve the safety and tolerability of IFNα therapy. We genetically fused IFN to a domain antibody (dAb) specific to a hepatocyte restricted antigen, asialoglycoprotein receptor (ASGPR). Our results show that the murine IFNα2 homolog (mIFNα2) fused to an ASGPR specific dAb, termed DOM26h-196-61, could be expressed in mammalian tissue culture systems and retains the desirable biophysical properties and activity of both fusion partners when measured in vitro. Furthermore a clear increase in in vivo targeting of the liver by mIFNα2-ASGPR dAb fusion protein, compared to that observed with either unfused mIFNα2 or mIFNα2 fused to an isotype control dAb VHD2 (which does not bind ASGPR) was demonstrated using microSPECT imaging. We suggest that these findings may be applicable in the development of a liver-targeted human IFN molecule with improved safety and patient compliance in comparison to the current standard of care, which could ultimately be used as a treatment for human hepatitis virus infections.

No abstract

Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAβGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments.

Advances in the development of clinically effective therapeutic strategies for targeting KRAS mutant tumors have been hampered by the lack of suitable in vitro tools and the inability to correlate findings from current in vitro KRAS dependency models with clinical results. Studies to evaluate targeted agents in KRAS dependent and independent cell systems have traditionally utilized panels of cell lines grown in 2D, which differ not only in KRAS status but also exhibit multiple genetic differences. An additional confounding element is that traditional 2D assays do not accurately mimic tumor architecture or growth. Our study focused on addressing two essential questions: (1) Can the consequence of a KRAS mutation be studied without the influence of other genetic factors? (2) Is there a requirement for more in vivo-like conditions to reveal KRAS dependency in vitro? To overcome confounding genetic factors, Horizon Discovery's proprietary gene editing technology GENESIS™ was used to knock-out an endogenous KRAS G13D mutation from DLD-1 colon carcinoma cells in order to create a pair of isogenic cell lines that differ only in terms of their KRAS status. Extensive profiling of the KRAS mutant and wild type cells under a range of growth formats was then performed to identify conditions which reveal dependency on KRAS. In standard 2D formats, cell growth was independent of KRAS with both cell lines demonstrating identical growth rates. However, knock-out of the KRAS mutation severely compromised cell growth under 3D conditions which more closely mimic true tumor biology. Interestingly, growth of the wild type cells was compromised when grown in two distinct 3D formats; soft agar or grown without a supporting matrix under low anchorage conditions. These results demonstrate the essential nature of 3D conditions for revealing KRAS dependency. In order to profile anti-cancer compounds using a truly KRAS dependent system, cellular responses to a series of targeted agents were compared in DLD-1 cells in 2D and 3D assays. Multiple MEK inhibitors demonstrated clear sensitivity under KRAS dependent conditions, with increased potency seen in the 3D assay compared to the 2D assay. Targeted agents unrelated to KRAS such as JAK and SYK inhibitors and non-targeted agents such as Paclitaxel exhibited similar potency in both 2D and 3D assays, further validating this KRAS dependency model. The increasing evidence that 3D conditions are critical for revealing KRAS dependency suggests that previous studies performed under standard 2D conditions may have underestimated levels of dependency. To address this, a study to systematically evaluate KRAS dependency across a panel of more than thirty KRAS mutant and wild type cancer cell lines under 2D and 3D conditions is now underway. Greater knowledge surrounding the biology and an understanding of how best to model KRAS dependency will be key to advancing effective KRAS pathway targeted agents into the clinic. Citation Format: Rebecca Foster, Clare Mudd, Ceri Wiggins, Chris Torrance. 3-Dimensional growth reveals KRAS dependency. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4289. doi:10.1158/1538-7445.AM2013-4289