Poster Presentation 27th Lorne Cancer Conference 2015

Investigating acquired resistance to novel Pol I transcription inhibitors for the treatment of hematologic malignancies (#133)

Don Cameron 1 , Gretchen Poortinga 1 , Grant McArthur 1 , Ross Hannan 1
  1. Peter MacCallum Cancer Institute, East Melbourne, Vic, Australia

RNA Polymerase I (Pol I) uniquely transcribes the 47S pre-ribosomal RNA (rRNA) molecule, which is then processed into three of the four rRNA molecules required for ribosome synthesis. Pol I transcribes the ribosomal DNA (rDNA) in discrete regions within the nuclei, called nucleoli. Cancer cells have long been reported to have larger nucleoli and higher basal levels of Pol I transcription than normal cells to accommodate for their greater rate of protein production and cell growth1,2. Considering the reliance of malignant cells on rDNA transcription, along with the role of Pol I in controlling cell growth, an emerging therapeutic consideration is that targeting Pol I directly represents a viable option for treating cancer2 .

This hypothesis led to the development of CX-5461, a first-in-class small molecule inhibitor of Pol I transcription3. Using the Eμ-Myc lymphoma mouse model, the Hannan lab demonstrated that inhibition of Pol I transcription by CX-5461 can selectively activate the p53 pathway in tumour cells, while leaving non-malignant cells unharmed4. These promising pre-clinical results led to the commencement of the CX-5461 Phase I clinical trial in haematological malignancies at Peter Mac in June 2013.

To investigate methods of resistance to CX-5461, mice injected with Eμ-Myc lymphoma cells were treated with CX-5461 continuously until relapse. The relapse tumours were harvested at sacrifice and sequenced by whole exome capture.

In each relapsed tumour, we identified multiple mutations in Top2a, a Type II Topoisomerase able to reduce torsional stress in supercoiled DNA by enabling the passage of one DNA strand through a temporary double strand break in another5.In addition, Top2a has been found to have other functions in a range of other processes including chromosomal crossover6, telomere protection7, and chromosomal condensation8, suggesting a wider role for Top2a in maintenance of the cell’s genomic integrity. Interestingly, Top2a was recently shown to directly bind to Pol I transcription initiation factor RRN3, and to create transient double-strand DNA breaks at the rDNA promoter region9.

We have shown that clones containing heterozygous mutations in Top2a are resistant to CX-5461 treatment in vitro and in vivo. However, the Top2a +/mut clones are not resistant to Pol I elongation inhibition by Dactinomycin, suggesting that the resistance to CX-5461 is mediated specifically at the Pol I initiation level. These clones do not express the mutant allele and as a result have lower basal levels of wild-type Top2a in comparison to drug-naïve Eμ-Myc cells. Also, they have lower basal rates of Pol I transcription and are smaller in size, though their proliferation rate remains unchanged. Our model suggests that CX-5461 resistance in the Top2a +/mut cells is due to a muted drug response owing to reduced basal Top2a and Pol I loading at the rDNA repeats.

These data suggest that cells with low Top2a and low basal Pol I transcription are resistant to CX-5461 treatment, and these phenotypes might provide valuable biomarkers for current and future CX-5461 trials.

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  8. Farr, C. J., Antoniou-Kourounioti, M., Mimmack, M. L., Volkov, A. & Porter, A. C. G. The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells. Nucleic Acids Res. 42, 4414–4426 (2014).
  9. Ray, S. et al. Topoisomerase IIα promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation. Nat Commun 4, 1598 (2013).