(P 300) ANTI-TUMOR ACTIVITY OF PARP7 INHIBITOR RBN-2397 IN SOFT-TISSUE SARCOMAS

Objective: Sarcomas are a heterogeneous group of mesenchymal malignancies that lead to death in about 50% of patients. The prognosis of patients with advanced disease is still poor with a median overall survival of only 18 months. Thus, there is an imperious need to find new therapeutic alternatives in order to improve sarcoma patient’s outcome. A recent study investigating the transcriptomic 608 tumors across STS subtypes, established an immune-based classification based on the composition of the tumor microenvironment. The immune-high group characterized by the presence of B cell rich tertiary lymphoid structures (TLS) demonstrated improved survival and a high response rate to PD-1 blockade by pembrolizumab. This work provides novel evidence regarding the presence of immune subtypes in STS patients.
Ribon has developed a first-in-class PARP7 inhibitor RBN-2397 that potently inhibit the growth of cancer cells and restore interferon signaling, effectively releasing the “brake” cancer uses to hide from the immune system and suppress both innate and adaptive immune mechanisms. In several cancer models, RBN-2397 demonstrated durable tumor growth inhibition, potent antiproliferative activity and restoration of interferon signaling. Therefore, PARP7 may represent a new promising therapeutic target that can help to improve the outcome of patients with STS.

Methods: The anti-proliferative and cytotoxic effects of RBN-2397 alone or in combination with cytotoxic agents were first investigated in a panel of 14 STS (soft-tissue sarcoma) cell lines established at Institut Bergonié (Bordeaux, France) using a cell imaging multimode microplate reader, the Cytation 3 technology. Cell cycle phases and apoptosis were then analyzed by flow cytometry and clonogenicity by crystal violet staining. We also assessed the amount of double-strand DNA breaks by STRIDE (SensiTive Recognition of Individual DNA Ends), a highly sensitive technique for the detection of DNA damage in situ in the nucleus and cytosol. Finally, to identify the pathways involved in PARP7 inhibition, we performed a transcriptomic analysis by RNA sequencing in 7 STS cell lines.

Results: We found that all the 14 STS cell lines were sensitive to PARP7 inhibition with RBN-2397 with IC50 range between 60 nM and 12 µM. This sensitivity was characterized by a cytostatic effect with a cell accumulation in G1 phase of cell cycle and a decrease in colony formation without any increase in apoptosis. To assess whether PARP7 inhibition can influence the efficacy of chemotherapy in STS cells, we performed viability assays in combination with either doxorubicin or trabectedin in 10 STS cell lines. Combinations of RBN-2397/doxorubicin or RBN2397/trabectedin were synergistic in 90% and 80% of cell lines respectively, the remaining ones being additive. This synergy is characterized by an increased rate of apoptosis or cell cycle arrest depending on the cell line. To deepen the mechanism of action, we also analyzed the DNA damages in situ by STRIDE technique and found that PARP7 inhibition induces dsDNA breaks in nucleus but also in cytoplasm in some cell lines and that the combination with doxorubicin or trabectedin increased the DNA damages content (Figure 1). Finally, a gene set enrichment analysis (GSEA) after RNA sequencing of 7 STS cell lines revealed an upregulation of several cytokine and TNF signaling pathway as well as an enhanced inflammatory response after treatment with RBN-2397 alone or in combination with chemotherapy.

Conclusion: PARP7 inhibition has anti-tumor activity in soft-tissue sarcomas through cell cycle inhibition and induction of DNA damage. Interestingly, the combination with PARP7 inhibition increases the efficacy of chemotherapy already used in sarcoma management providing a clear rationale for its clinical investigation in this indication.