Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1− DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure-and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1−DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.

Ewing’s sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing’s sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing’s sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing’s sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing’s sarcoma patients with PARP inhibitors.

Purpose: We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve anti-tumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to a play role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim to achieve improved clinical efficacy and wider therapeutic window. This next generation PARPi could provide a paradigm shift in clinical outcomes achieved by first generation PARPi, particularly in combination. Patients and Methods: AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematological toxicity was evaluated in rat models as monotherapy and combination. Results: AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first generation PARPi, AZD5305 has minimal effects on hematological parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1mg/kg QD achieved greater depth of tumor regression compared to olaparib 100mg/kg QD, and longer duration of response. Conclusions: AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain therapeutic benefits of non-selective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in Ph1 trials (NCT04644068).

Current clinical poly (ADP-ribose) polymerase (PARP) inhibitors target both PARP1 and PARP2 and they all cause clinical cytopenias with varying severity. Understanding the mechanism underlying the hematological toxicity of these agents is key for the rational design of a best-in-class molecule with greater therapeutic potential, both as monotherapy and in combination with chemotherapy. We validated the rat as a physiologically competent translational model to investigate PARP inhibitor-driven hematological toxicity. Here we demonstrate that in comparison with a representative PARP1/2 inhibitor, olaparib, the novel highly potent PARP1-selective inhibitor and trapper, AZD5305, does not cause hematological toxicity as a monotherapy in pre-clinical rat models at predicted clinically efficacious exposures. Thus, monotherapy toxicity of PARP1/2 inhibitors likely depends on PARP2 inhibition. Next, we proceeded to investigate whether PARP1-selective inhibition would be better tolerated in chemotherapy combinations than PARP1/2 inhibition. We performed a rat in vivo study comparing daily olaparib or daily AZD5305 at matched exposures in combination with one cycle of intravenous carboplatin for 14 days. We show that olaparib and PARP1-selective AZD5305 cause comparable exacerbation of carboplatin-induced peripheral blood effects implicating PARP1 inhibition in combination-driven hematological toxicity. Importantly however, AZD5305+carboplatin showed improved hematological tolerability over olaparib+carboplatin because peripheral reticulocytes and bone marrow erythroid precursor cells recover in the presence of continuous AZD5305 but not in the presence of continuous olaparib. Importantly, this differentiation was maintained in a subsequent rat in vivo study, where daily olaparib or daily AZD5305 were combined with two three-weekly cycles of a higher dose of carboplatin to more closely mimic clinical protocol. AZD5305+carboplatin was associated with a more rapid recovery of reticulocytes, red blood cells and hemoglobin following both cycles of carboplatin. In contrast, olaparib+carboplatin was associated with a slower recovery resulting in a more sustained reduction in red cells and hemoglobin during both the first and second cycle of carboplatin. Thus, in rodents the novel potent PARP1-selective inhibitor AZD5305 has improved hematological tolerability over dual PARP1/2 inhibitors, both as a monotherapy and in carboplatin combinations. Citation Format: Sonja J. Gill, Ruth Macdonald, Carmen Pin, Rob Collins, Emilyanne Leonard, Gareth Maglennon, Andy Pike, Peter Cotton, Glen Hawthorne, Jordan Pugh, Rebecca Sargeant, Daniel Sutton, James Atkinson, Stewart Jones, Sarah Chinery, Mark Anderton. The novel PARP1-selective inhibitor AZD5305 has reduced hematological toxicity when compared to PARP1/2 inhibitors in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1374.

Radiotherapy is an effective anticancer treatment, but combinations with targeted agents that maximize efficacy while sparing normal tissue are needed. Here, we assess the radiopotentiation profiles of DNA damage response inhibitors (DDRi) olaparib (PARP1/2), ceralasertib (ATR), adavosertib (WEE1), AZD0156 (ATM), and KU-60648 (DNA-PK). We performed a radiotherapy combination screen and assessed how drug concentration and cellular DDR deficiencies influence the radiopotentiation ability of DDRi. We pre-selected six lung cancer cell lines with different genetic/signaling aberrations (including mutations in TP53 and ATM) and assessed multiple concentrations of DDRi in combination with a fixed radiotherapy dose by clonogenic assay. The effective concentration of DDRi in radiotherapy combinations is lower than that required for single-agent efficacy. This has the potential to be exploited further in the context of DDR deficiencies to increase therapeutic index and we demonstrate that low concentrations of AZD0156 preferentially sensitized p53-deficient cells. Moreover, testing multiple concentrations of DDRi in radiotherapy combinations indicated that olaparib, ceralasertib, and adavosertib have a desirable safety profile showing moderate increases in radiotherapy dose enhancement with increasing inhibitor concentration. Small increases in concentration of AZD0156 and particularly KU-60648, however, result in steep increases in dose enhancement. Radiopotentiation profiling can inform on effective drug doses required for radiosensitization in relation to biomarkers, providing an opportunity to increase therapeutic index. Moreover, multiple concentration testing demonstrates a relationship between drug concentration and radiotherapy effect that provides valuable insights that, with future in vivo validation, can guide doseescalation strategies in clinical trials.