In vitro and in vivo biology characterization of two novel inhibitors targeting the different domains of DNA polymerase theta (Polθ)

DNA polymerase theta (Polθ) is a 290 kD specialized and error-prone Polymerase A family enzyme that executes microhomology-mediated end joining (MMEJ), possessing an N-terminal helicase-like domain and a C-terminal DNA polymerase domain, which are linked by a large, unstructured central region. Polθ is normally expressed at low levels but is frequently overexpressed in tumours and is critical for the survival of homologous recombination (HR)-deficient cancer cells. Accordingly, Polθ has been proposed as an attractive therapeutic target for the treatment of BRCA deficient and DNA repair pathway defective cancers. Several inhibitors targeting either the polymerase or the helicase domain of Polθ have been described and progressed to the clinic. However, it is not clear which axis of intervention is the most beneficial even though inhibition of either domain elicits synthetic lethality with BRCA genes preclinically. Here we compare the in vitro biological profile of two novel Polθ inhibitors, one targeting the C-terminal DNA polymerase (Pol) domain and the other targeting the N-terminal helicase-like domain (Hel). Both inhibitors exhibit similar potency in their respective biochemical assays (Hel inhibitor IC 50 = 2.1 nM, Pol inhibitor IC 50 = 4.2 nM). However, in a colony formation cellular assay the Hel inhibitor elicited significantly stronger synthetic lethality in BRCA2-mutant tumour cells (Hel inhibitor IC 50 = 2.1 nM; Pol inhibitor IC 50 125.6 nM). Furthermore, this differentiation was evident mechanistically, where the Hel inhibitor inhibited MMEJ-mediated repair with a 3-fold higher potency and increased micronuclei formation in cells with a 200-fold higher potency when compared to the Pol inhibitor. When tested in vitro , in combination studies with the PARP inhibitor Niraparib, Hel showed a synergistic effect even at the lowest combination of concentrations tested (4 nM Hel, 15 nM Niraparib), whereas a 10-fold higher concentration of Pol inhibitor was needed to achieve the same synergistic effect. Based on its superior in vitro profile, the helicase inhibitor was progressed to BRCA1- and BRCA2-deficient xenograft models, where it evoked DNA damage and showed a dose-dependent sustained regression of tumour volume, in combination with a PARP inhibitor, even at the lowest dose tested of 3 mg/kg PO QD. The dose selection was based on the unbound exposure of the Hel inhibitor being in excess of the unbound cellular IC50 during the treatment period. Given the tumour regressive effects in vivo and favourable ADME profile in preclinical species and early human dose predictions, the helicase compound is currently undergoing evaluation in non-GLP toxicology studies.