Project Portugal 2030

Summary

Considering previous results obtained by team members (PSoares) and others it became clear that an effective therapeutic strategy should target promoter mutation-driven TERT gene expression, and not the telomerase complex, since i) TERT exerts other effects in cancer cells beyond immortalization, (i.e. beyond telomere length maintenance) which are associated to mechanisms of tumorigenesis; and, ii) telomerase activity is required in normal cells for critical biological processes (e.g. lymphocytes and tissue stem cells). Thus, we want to address the following PROBLEM: Re-activated telomerase mechanisms act as drivers of tumorigenesis. So far there is no therapeutic molecule under clinical development targeted towards the specific mechanism of re-activation derived from TERT promoter mutations. Some strategies under clinical development have concentrated on taregeting TERT enzymatic activity (e.g. Imetelstat, a 13-mer oligonucleotide blocking the catalytic site), and vaccines against TERT positive cells (e.g. Tertomotide, a TERT peptide vaccine), but have been hampered by toxicity issues and lack of effectiveness. Our HYPOTHESIS is that we can achieve suppression of TERTp transcriptional re-activation in a mutation specific way through novel gene and RNA-targeted oligonucleotide (ON) tools. SPECIFIC OBJECTIVES consist of: A) Assessment of anti-gene oligonucleotide (agONs) design features and nucleotide modifications that allow stable and efficient hybridization with chromosomal DNA, at regions of short homopurine/homopyrimidine stretches, for mutant promoter targeting. The strategy will consist of creating agONs capable of forming a triple helix in the supercoiled dsDNA (Hoogsteen binding) followed by enhanced dsDNA strand invasion, to increase binding stability. Ultimately this will allow to refine the design rules for the development of agONs as tools for other gene/promoter targeting applications (AIM 1). B) Study of mutTERTp anti-gene ONs biological activity. By physically interfering with the de novo transcription factors binding site, created by the mutations, as well as altering the local DNA structure, we will avoid TERTp transcriptional reactivation (AIM 2). C) Develop cancer targeted bi-functional ONs. Design antisense ONs (ASO) sequences that specifically inhibit the GABPß1L subunit of the GBAP tetramer transcription factor (targeting exon 9 that encodes the ß1L(long) isoform and is absent in the ß1S(short) isoform). The GABPß1L subunit of the GBAP tetramer has been linked to specific mutant promoter binding and re-activation. Subsequently we will link both ASO and agON to assemble a multi-modal oligonucleotide therapeutic for increased efficacy which goes further beyond any state-of-the-art. This is also further supported by a cancer targeting approach that will be achieved by folate modification of the bi-specific oligo structure, in a ONdrug-conjugate type approach(AIM 3). D) Both agONs and ASOs (collectively defined as ONs) will be studied independently and in combination (in the bi-specific ON strategy - bisON) to assess if SYNERGISTIC effects can be observed (AIM 4). The creation of this targeted therapy would be able to cover a large burden of malignant tumors that frequently harbor TERTp genetic alterations (thyroid, liver, skin, CNS cancers), and where few options remain once resistance to first line treatment is acquired.