Periodic Reporting for period 2 - PARPinhibit (A systems biology approach to tackle PARP-inhibitors resistance and identify novel therapeutic targets to overcome it)
Reporting period: 2022-12-31 to 2023-12-30
The Poly(ADP-ribose) polymerase 1 (PARP1) gene encodes a nuclear protein that transfers ADP-ribose residues (PAR) onto target substrates; this post-translational modification is called PARylation. Inhibition of PARP1 is widely used in cancer therapy to treat cancers where DNA repair is defective, particularly through homologous recombination (HR), such as breast, prostate, pancreatic, and ovarian cancers caused by inactivation of BRCA1 or BRCA2; however, despite their demonstrated efficacy, development of resistance to PARP inhibitors (PARPi) is a wide diffuse problem and still little is known about how to tackle this significant challenge. Therefore, the overall objectives of the project are to identify targets that can be modulated as single agents or in the context of a combined approach to overcome development of PARPi resistance; we planned to do that by identification of genes whose inhibition influence PARylation activity and PARP1 retention to DNA and that also influence sensitivity to PARP inhibitors. We can conclude that we discovered and studied in depth an interesting novel target important for PARP1 activity, able to regulate its function and DNA binding in cells. Moreover, we evaluate how modulation of this gene influences sensitivity of ovarian cancer cells to treatment with PARPi, to DNA damage induced with ionizing radiation exposition and to a combination of both treatments. In conclusion, with further analysis, treatment of this target could become relevant in cancer therapy, particularly in those cancers where PARP inhibitors are currently administered such as ovarian cancer.
To search for novel modulators of PARylation activity and PARPi sensitivity, we harnessed three different datasets each one analyzing PARP1 interaction network from different point of view: (i) The first one analyzes genes that show a genetic interaction with PARP1, (ii) the second one contains genes coding for proteins that biochemically interact with PARP inhibitors directly or indirectly, (iii) the third one contains genes coding for proteins that directly or indirectly physically interact with PARP1. This analysis led to the discovery of SART1 as the most interesting target by being present in all datasets, therefore we decided to study it further. To inform the choice of cancer models in which to explore the role of the identified target in PARPi sensitivity we first examined publicly available information from normal tissue databases (https://gtexportal.org/home/) and cancer data (https://www.cbioportal.org/). Through this preliminary analysis we decided to perform our analysis in ovarian cancer cell lines, since SART1 expression in this tissue is one of the highest among all normal tissues and copy number alterations (CNA) of this gene, such as amplification, are frequent in ovarian cancer patients. Thus, we created different stably silenced cell lines based on the previous results and we studied how this downregulation influences PARP1 activity and its retention to DNA. Particularly, SART1 silencing determines an increase in PARylation activity and steady binding of PARP1 to DNA, even in absence of DNA damage. Next, to narrow down the interaction and to assess which domain of the proteins was the most important to regulate PARP1 activity, we created several deletion and mutagenized constructs, and we expressed them in silenced cells. This precise analysis showed that SART1 n-terminal domain is important to regulate PARP1 activity and particularly a RGG/RG repeat present in this part of the protein. Finally, to study if targeting of SART1 led to an increased sensitivity to PARPi, we treated different ovarian cancer cells silenced or proficient for SART1 with three different PARP inhibitors (Olaparib, Rucaparib and Talazoparib) and we did this analysis in context where BRCA1 is present or absent, since in cancer therapy these drugs are particularly efficient where there is deficiency in BRCA1 activity. Moreover, to have a broader view, we did the latter analysis in silenced cells treated with ionizing radiation (IR), rather than PARP inhibitors. This preliminary analysis shows that ovarian cancer cell lines deficient for BRCA1 becomes more sensible to all PARPi tested when SART1 is downregulated, while the same cell line proficient for BRCA1 did not. Conversely, silencing of SART1 determines an increase to DNA damage induced with IR irrespective of BRCA1 status. Finally, we studied if combination of IR and PARPi could improve efficiency of PARP inhibition. Interestingly, we discovered that by first inducing DNA damage with a small dose of IR, cell lines in which SART1 silencing had no influence in PARPi sensitivity become more sensible to all drugs tested when SART1 is downregulated. In the end, this project led to the discovery and analysis of a novel regulator of PARP1 activity. This protein is important to modulate PARylation and its silencing determines an increased retention of PARP1 to DNA, a process dependent on a few aminoacids present in the n-terminal domain. Moreover, by modulating its expression, we were able to make ovarian cancer cells more sensible to PARP inhibitors, an effect even more prominent when combined with other sources of DNA damage such as IR. During the lifetime of the project, these results have been disseminated to the scientific audience on different occasions. The most prominent ones are the Moffitt Scientific Symposium that took place in the host institution on 11 May 2022 and the “Recombination Mechanism” conference that took place in Lisbon from 10 Jul 2023 to 13 Jul 2023. Lastly, a paper based on these results has been submitted, reviewed and provisionally accepted by iScience journal (cell press). Shortly, it will be available to the whole scientific community to be read and discussed.
This project greatly improved the state of the art, by enlarging the knowledge about PARP1 regulation and discovering a new important protein that seems to be essential for PARP1 to properly function in cells. Results obtained show an unprecedent activity of SART1 within DNA repair, PARP activity regulation, sensitivity to PARPi and to DNA damage induced by ionizing radiation. This opens the way to further analysis that in the future could lead to the development of drugs targeting this protein enhancing PARP1 inhibitors by either overcoming resistance development or improving their efficiency in patients nonresponsive to these drugs. The impact of these results could be indeed relevant, since development of resistance to PARPi is a wide diffuse problem and the possibility to make them functional in those patients that are or become irresponsive is something really important in cancer therapy. Furthermore, by generally increasing cell’s sensitivity to DNA damage, SART1 downregulation is something to take in consideration in combination with radio or chemotherapy treatment and its expression level in patients should be analyzed to modulate efficacy of these treatments. In the end, we expect that the paper based on the results from this project that will soon be published will have a great impact in the scientific community and it will lead the way to further analysis that potentially will have a great impact from a socio-economic point of view by promoting development of a new kind of drug useful in ovarian cancer therapy and potentially even in other kind of cancers.