DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.

The replication stress response, which serves as an anticancer barrier, is activated not only by DNA damage and replication obstacles but also oncogenes, thus obscuring how cancer evolves. Here, we identify that oncogene expression, similar to other replication stress–inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness. DNA fiber analysis and electron microscopy reveal that activation of translesion synthesis (TLS) polymerases restricts replication fork slowing, reversal, and fork degradation without inducing replication gaps despite the continuation of replication during stress. Consistent with gap suppression (GS) being fundamental to cancer, we demonstrate that a small-molecule inhibitor targeting the TLS factor REV1 not only disrupts DNA replication and cancer cell fitness but also synergizes with gap-inducing therapies such as inhibitors of ATR or Wee1. Our work illuminates that GS during replication is critical for cancer cell fitness and therefore a targetable vulnerability.

The Food and Drug Administration-approved antifungal agent, itraconazole (ITZ), has been increasingly studied for its novel biological properties. In particular, ITZ inhibits the hedgehog (Hh) signaling pathway and has the potential to serve as an anticancer chemotherapeutic against several Hh-dependent malignancies. We have extended our studies on ITZ analogues as Hh pathway inhibitors through the design, synthesis, and evaluation of novel des-triazole ITZ analogues that incorporate modifications to the triazolone/side chain region of the scaffold. Our overall results suggest that the triazolone/side chain region can be replaced with various functionalities (hydrazine carboxamides and meta-substituted amides) resulting in improved potency when compared to ITZ. Our studies also indicate that the stereochemical orientation of the dioxolane ring is important for both potent Hh pathway inhibition and compound stability. Finally, our studies suggest that the ITZ scaffold can be successfully modified in terms of functionality and stereochemistry to further improve its anti-Hh potency and physicochemical properties.

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Rev1 is a protein scaffold of the translesion synthesis (TLS) pathway, which employs low‐fidelity DNA polymerases for replication of damaged DNA. The TLS pathway helps cancers tolerate DNA damage induced by genotoxic chemotherapy, and increases mutagenesis in tumors, thus accelerating the onset of chemoresistance. TLS inhibitors have emerged as potential adjuvant drugs to enhance the efficacy of first‐line chemotherapy, with the majority of reported inhibitors targeting protein‐protein interactions (PPIs) of the Rev1 C‐terminal domain (Rev1‐CT). We previously identified phenazopyridine (PAP) as a scaffold to disrupt Rev1‐CT PPIs with Rev1‐interacting regions (RIRs) of TLS polymerases. To explore the structure‐activity relationships for this scaffold, we developed a protocol for co‐crystallization of compounds that target the RIR binding site on Rev1‐CT with a triple Rev1‐CT/Rev7R124A/Rev3‐RBM1 complex, and solved an X‐ray crystal structure of Rev1‐CT bound to the most potent PAP analogue. The structure revealed an unexpected binding pose of the compound and informed changes to the scaffold to improve its affinity for Rev1‐CT. We synthesized eight additional PAP derivatives, with modifications to the scaffold driven by the structure, and evaluated their binding to Rev1‐CT by microscale thermophoresis (MST). Several second‐generation PAP derivatives showed an affinity for Rev1‐CT that was improved by over an order of magnitude, thereby validating the structure‐based assumptions that went into the compound design.