Abstract 4506: CL67: A novel G-quadruplex stabilising agent inhibits the HIF-1 pathway
Authors: Welsh Sarah J. , Lombardo Caterina M. , Valentine Helen , Neidle Stephen 
Published: June 2012

Hypoxic cancer cells are found in all solid tumours and are the most difficult cancer cells to kill. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that plays a critical role in mediating the growing tumours response to hypoxia. Downstream genes activated by HIF-1 lead to increased glycolysis, resistance to apoptosis and increased tumour angiogenesis. HIF-1 over-expression has been associated with increased patient mortality in several tumour types. HIF-1, a heterodimer, consists of a HIF-1α subunit whose rapid breakdown under aerobic conditions is mediated by the von Hippel Lindau protein (pVHL), and an oxygen-insensitive HIF-1ß (ARNT) subunit. HIF-1α protein is elevated many fold in cells under conditions of hypoxia (&lt; 1- 5% O2), and has been found to be increased in many human tumours but is absent in most normal tissues. HIF-1α is, thus, an attractive molecular target for the development of novel cancer drugs with the potential to decrease cancer cell survival and inhibit angiogenesis.

The promoter of HIF-1α contains a guanine-rich sequence capable of forming a G-quadruplex structure which modulates the activity of the HIF-1α promoter. We have identified a number of compounds which bind to G-quadruplex structures and preferentially inhibit the growth of renal cancer cells in vitro compared to other tumour cell lines (IC50 of 1.4 – 2.6 µM in the human renal cancer cell lines RCC4, RCC4VHL, 786-0 and A498 compared to IC50 of 8 – 63 µM in MCF-7 human breast, A549 human lung, MiaPaCa and Panc-1 human pancreatic, and PC-3 human prostate cancer cell lines). The lead compound (CL67) shows 2-fold increased sensitivity in RCC4 cells lacking the VHL gene compared to RCC4 cells transfected with the VHL gene (RCC4VHL) (IC50 of 1.3 µM and 2.6 µM respectively). Cell viability is also preferentially reduced in RCC4 cells compared with RCC4VHL cells, particularly under hypoxic conditions (1% O2; 2.1-fold decreased cell viability in RCC4 compared to RCC4VHL cells).

CL67 inhibits HIF-1α protein under normoxia (20% O2) and hypoxia (1% O2) measured using Western blotting. However, HIF-1α protein is inhibited in both RCC4 and RCC4VHL cells suggesting that CL67 inhibits HIF-1α using a VHL-independent mechanism. HIF-1α protein is reduced in a dose-dependent manner within 2 hours of treatment of RCC4 cells with 5 × IC50. Maximal inhibition of HIF-1α protein (to 5% of pre-treatment levels) is achieved after 6 hours. HIF-1 transactivation, measured using transient transfection of cells with a plasmid expressing luciferase under the control of multiple copies of the hypoxia-response element (Professor G Melillo) is also reduced in a dose-dependent manner to 8% of pre-treatment activity after 4 hours. Xenograft studies are on-going.

CL67 therefore represents a potent novel inhibitor of the HIF-1 pathway which shows preferential activity in renal cancer cell lines, likely by interfering with G-quadruplex structures within promoter sequences.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4506. doi:10.1158/1538-7445.AM2011-4506