High-throughput screening to identify synthetic lethal compounds in RCC

Synthetic lethal compounds selectively kill cancer cells by targeting tumour cell-essential processes, but leave healthy cells unharmed. Research is ongoing to identify such compounds in a range of cancers (reviewed in McLornan et al., 2014) including renal cell carcinoma (RCC). New research from Wolff et al., (2015) has identified homoharringtonine (HHT) as synthetically lethal for a subset of clear cell RCC (ccRCC) tumours associated with VHL mutations.

The high-throughput screen completed by Wolff et al. used the 786-O VHL-deficient cell line alongside a VHL-reconstituted isogenic control line. The cell lines were differentially labelled with GFP and mCherry fluorochromes respectively, enabling imaging-based assessment of cell survival in mixed cultures. Mixed cultures better recapitulate the in vivo setting of tumour and healthy cells side-by-side and reduce the variation caused by sequential screening thereby increasing sensitivity.

Approximately 12,800 compounds – including all FDA approved and NIH experimental drugs (UT Southwestern High-Throughput Screening Core) – were screened for synthetic lethality and synergistic activity with mTOR inhibitor sirolimus. 139 compounds significantly reduced the ratio of VHL-deficient to VHL- reconstituted cells with 15 showing reproducible results at concentrations below 5μM. None of the compounds were significantly more effective in combination with sirolimus than without.

The plant alkaloid HHT was chosen for further investigation due to existing FDA approval for treatment of chronic myeloid leukaemia (CML) thereby representing a drug repurposing opportunity. CML studies have identified that HHT most likely inhibits protein synthesis and increases turnover of the anti-apoptotic factor MCL-1 resulting in apoptosis.

HHT at 50nM, a clinically relevant concentration, induced cell death in 30-40% of VHL-defective cells – but under 10% of the VHL-reconstituted cells – within 36 hours, with evidence of apoptosis within 12 hours. Wolff et al. report an upregulation of anti-apoptotic factor Bcl-xL in the VHL-reconstituted cells that was absent from the VHL-deficient cells as previously reported (Devarajan et al., 2001) and would reduce protection from pro-apoptotic signals.

To confirm lethality in vivo Wolff et al. grafted six independent ccRCC cell lines into nude mice treated with either HHT, sirolimus or an empty vesicle control.  The tumour graft lines were derived from individual ccRCC patient samples (Sivanand et al., 2012) and confirmed to carry VHL mutations, however detailed characterisation of the lines is unavailable. Two of the six tumour graft lines responded to HTT showing an observable inhibition of tumour growth (40.3-63.7%) with evidence of necrosis on examination. In comparison the majority of tumours responded to treatment with rapamycin, showing similar signs of necrosis, but not to empty vehicle treatment. As predicted from the cellular screen there was no synergistic action between HHT and sirolimus on tumour growth.

These results indicate that HHT could be repurposed for some cases of VHL-mutated ccRCC.  It is unclear why the 786-O cell line, plus only two of the graft cell lines, were susceptible to HHT, when the other four VHL-mutation tumour graft cell lines were not. HHT’s effectiveness in CML, which is not associated with VHL mutations, suggests that additional modifiers are involved.  Identifying biomarkers in these lines would enable selection of the patients most likely to respond. This also highlights the need to assess multiple cancer-specific cell lines in synthetic lethality screens to compensate for the heterogenetic nature of cancer and identify compounds for other subtypes.

As previously described on the blog high-throughput screens have identified synthetic lethal compounds for VHL, HLRCC and BHD RCC lines. BHD tumourigenesis is reliant on a second FLCN mutation (Vocke et al., 2005) making tumour cells genetically distinct from surrounding tissue. Previous studies have identified treatment with mithramycin or paclitaxel, and the inhibition of Slingshot2 as capable of selectively killing FLCN-null cells. These compounds act in a variety of mechanisms increasing the understanding of FLCN function and tumourigenic pathways. Further screens may identify additional, potentially synergistic, compounds including other pre-approved compounds for potential repurposing.

  • Devarajan P, De Leon M, Talasazan F, Schoenfeld AR, Davidowitz EJ, Burk RD (2001). The von Hippel-Lindau gene product inhibits renal cell apoptosis via Bcl-2-dependent pathways. J Biol Chem. Nov 2;276(44):40599-605. PMID: 11514546.
  • McLornan DP, List A, Mufti GJ (2014). Applying synthetic lethality for the selective targeting of cancer. N Engl J Med. Oct 30;371(18):1725-35. Review. PMID: 25354106.
  • Sivanand S, Peña-Llopis S, Zhao H, Kucejova B, Spence P, Pavia-Jimenez A,Yamasaki T, McBride DJ, Gillen J, Wolff NC, Morlock L, Lotan Y, Raj GV, Sagalowsky A, Margulis V, Cadeddu JA, Ross MT, Bentley DR, Kabbani W, Xie XJ, Kapur P, Williams NS, Brugarolas J (2012). A validated tumorgraft model reveals activity of dovitinib against renal cell carcinoma. Sci Transl Med. Jun 6;4(137):137ra75. PMID: 22674553.
  • Vocke CD, Yang Y, Pavlovich CP, Schmidt LS, Nickerson ML, Torres-Cabala CA, Merino MJ, Walther MM, Zbar B, Linehan WM (2015). High frequency of somatic frameshift BHD gene mutations in Birt-Hogg-Dubé-associated renal tumors. J Natl Cancer Inst. Jun 15;97(12):931-5. PMID: 15956655.
  • Wolff NC, Pavía-Jiménez A, Tcheuyap VT, Alexander S, Vishwanath M, Christie A, Xie XJ, Williams NS, Kapur P, Posner B, McKay RM, & Brugarolas J (2015). High-throughput simultaneous screen and counterscreen identifies homoharringtonine as synthetic lethal with von Hippel-Lindau loss in renal cell carcinoma. Oncotarget, 6 (19), 16951-62 PMID: 26219258.
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