Giacomo G. Rossetti

An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers^1–6. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair⁷. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription–replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription–replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.

An important advance in cancer therapy in the last decade has been the development of poly ADP-ribose polymerase (PARP) inhibitors for the treatment of select ovarian and breast cancers. The clinical benefit stems from the synthetic lethality of PARP inhibitors with homologous recombination (HR) deficiency, which deficiency is prevalent in the cancer types listed above. The current model to explain the synthetic lethality is based on the observation that PARP inhibitors trap PARPs on DNA: the trapped PARPs block progression of the replisome, leading to the formation of DNA double-strand breaks (DSBs), which require HR for repair. Here, we propose a novel mechanism to explain the synthetic lethality between PARP inhibitors and HR deficiencies. We show that PARP1 functions together with the proteins TIMELESS and TIPIN to protect the replisome from transcription-replication conflicts (TRCs). In the absence of any one of these proteins, TRCs evolved into DNA DSBs that required HR for repair, explaining the observed synthetic lethality. In further support of this model, when we inhibited transcription elongation, which prevents the emergence of transcription-replication conflicts, then the HR-deficient cancer cells became resistant to PARP inhibitors. We further observed that trapping of PARPs on DNA was not required for the synthetic lethality with HR deficiency, since we could observe strong synthetic lethality simply by depleting PARP1 and PARP2 by siRNA. Rather, trapping of PARPs on DNA correlated with the ability of the various PARP inhibitors to inhibit PARP enzymatic activity in cells; the strongest trappers were also the most potent inhibitors of PARP1 in cells. In vitro, all PARP inhibitors tested were almost equipotent in their ability to inhibit the enzymatic activity of PARP1, meaning that the potency of PARP1 inhibitors in vitro did not reflect their inhibitory potency in cells. Our model provides a new framework for understanding the mechanism of action of PARP inhibitors in the clinic and the mechanisms by which resistance can emerge. Citation Format: Thanos D. Halazonetis, Michalis Petropoulos, Giacomo G. Rossetti, Angeliki Karamichali, Alena Freudenmann, Luca Iacovino, Vasilis Dionellis, Sotirios K. Sotiriou. DNA damage generated by transcription-replication conflicts explains the synthetic lethality of PARP inhibitors with homologous recombination deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1566.

BRCA1orBRCA2germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification andBRCA1/2mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of β-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed β-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression ofCDKN1Agene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, β-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that β-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.

The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with Apc^lox/lox, LSL-Kras^G12D, and Tp53^lox/lox targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.