Poly(ADP-ribose) polymerase inhibitors (PARPi) are selectively active in cells with homologous recombination (HR) deficiency (HRD) caused by mutations in BRCA1, BRCA2, and other pathway members. We sought small molecules that induce HRD in HR-competent cells to induce synthetic lethality with PARPi and extend the utility of PARPi. We demonstrated that inhibition of bromodomain containing 4 (BRD4) induced HRD and sensitized cells across multiple tumor lineages to PARPi regardless of BRCA1/2, TP53, RAS, or BRAF mutation status through depletion of the DNA double-stand break resection protein CtIP (C-terminal binding protein interacting protein). Importantly, BRD4 inhibitor (BRD4i) treatment reversed multiple mechanisms of resistance to PARPi. Furthermore, PARPi and BRD4i are synergistic in multiple in vivo models.

Mutant RAS has remained recalcitrant to targeted therapy efforts. Here we demonstrate that combined treatment with poly ADP ribose polymerase (PARP) inhibitors and MEK inhibitors evokes unanticipated, synergistic cytotoxic effects in vitro and in vivo in multiple RAS mutant tumor models across tumor lineages where RAS mutations are prevalent. The effects of PARP and MEK inhibitor combinations are independent of BRCA1/2 and p53 mutation status suggesting that the synergistic activity is likely to be generalizable. Synergistic activity of PARP and MEK inhibitor combinations in RAS mutant tumors is associated with: 1) induction of BIM-mediated apoptosis, 2) decrease in expression of components of the homologous recombination DNA repair pathway, 3) decrease in homologous recombination DNA damage repair capacity, 4) decrease in DNA damage checkpoint activity, 5) increase in PARP inhibitor-induced DNA damage, 6) decrease in vascularity which could increase PARP inhibitor efficacy by inducing hypoxia, and 7) elevated PARP1 protein, which increases trapping activity of PARP inhibitors. Mechanistically, enforced expression of FOXO3a, which is a target of the RAS/MAPK pathway, was sufficient to recapitulate the functional consequences of MEK inhibitors including synergy with PARP inhibitors. Thus the ability of mutant RAS to suppress FOXO3a and its reversal by MEK inhibitors accounts, at least in part, for the synergy of PARP and MEK inhibitors in RAS mutant tumors. The rational combination of PARP and MEK inhibitors warrants clinical investigation in patients with RAS mutant tumors where there are few effective therapeutic options.

Highlights d We characterize effects of PARP and WEE1 inhibitors on functional proteomics d Concurrent PARP and WEE1 blockade effectively inhibits tumors but is poorly tolerated d Sequential PARP and WEE1 inhibition minimizes toxicity while maintaining efficacy d Basal replication stress influences the therapeutic index of sequential therapy