Kyung Yong Lee

Background/Objectives: Irinotecan is a component of standard first-line regimens for advanced pancreatic ductal adenocarcinoma (PDAC), yet cells that survive irinotecan treatment may retain the capacity to re-establish growth. Most anticancer drug screens use short-term growth inhibition as an initial criterion for compound selection, potentially excluding compounds with limited acute activity. We asked whether some of these excluded candidates might nevertheless show substantial long-term activity and enhance irinotecan responses in combination. Methods: We first screened 3524 compounds for short-term growth inhibition and advanced compounds with limited acute activity to a 10-day colony-growth screen. Long-term hits were narrowed to clinically relevant anticancer agents and then screened with irinotecan. Talazoparib showed the lowest combination-to-single-agent score and was selected for further evaluation. The combination was evaluated across multiple BRCA1/2-wild-type PDAC cell lines and further validated using additional PARP inhibitors and a xenograft model. RNA sequencing and γH2AX analysis were used to examine associated DNA damage-response features. Results: Of the 1877 compounds advanced from the initial 3-day screen, 334 (17.8%) met the long-term hit criterion. In short- versus long-term comparisons, irinotecan plus talazoparib produced greater growth suppression and higher Bliss synergy scores in 10-day than in 72 h assays. Enhanced long-term responses were observed across five BRCA1/2-wild-type PDAC cell lines, and similar long-term effects were observed with three additional PARP inhibitors. The combination also showed synergistic antitumor activity in an MIA PaCa-2 subcutaneous xenograft model. Transcriptomic and γH2AX analyses provided findings consistent with replication-stress and DNA-damage-response processes. Conclusions: A substantial subset of compounds with limited acute activity showed substantial long-term activity. PARP inhibition enhanced irinotecan responses and emerged as a candidate combination strategy in BRCA1/2-wild-type PDAC.

Background: Double-strand breaks (DSBs) are primarily repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). Given that DSBs are highly cytotoxic, PARP inhibitors (PARPi), a prominent class of anticancer drugs, are designed to target tumors with HR deficiency (HRD), such as those harboring BRCA mutations. However, many tumor cells acquire resistance to PARPi, often by restoring HR in HRD cells through the inactivation of NHEJ. Therefore, identifying novel regulators of NHEJ could provide valuable insights into the mechanisms underlying PARPi resistance. Methods: Cellular DSBs were assessed using neutral comet assays and phospho-H2AX immunoblotting. Fluorescence-based reporter assays quantified repair via NHEJ or HR. The recruitment of proteins that promote NHEJ and HR to DSBs was analyzed using immunostaining, live-cell imaging following laser-induced microirradiation, and FokI-inducible single DSB generation. Loss-of-function experiments were performed in multiple human cancer cell lines using siRNA-mediated knockdown or CRISPR-Cas9 gene knockout. Cell viability assays were conducted to evaluate resistance to PARP inhibitors. Additionally, bioinformatic analyses of public databases were performed to investigate the association between TLK expression and BRCA1 status. Results: We demonstrate that human tousled-like kinase (TLK) orthologs are essential for NHEJ-mediated repair of DSBs and for PARPi sensitivity in cells with BRCA1 mutation. TLK1 and TLK2 exhibit redundant roles in promoting NHEJ, and their deficiency results in a significant accumulation of DSBs. TLKs are required for the proper localization of 53BP1, a key factor in promoting the NHEJ pathway. Consequently, TLK deficiency induces PARPi resistance in triple-negative breast cancer (TNBC) and ovarian cancer (OVCA) cell lines with BRCA1 deficiency, as TLK deficiency in BRCA1-depleted cells, impairs 53BP1 recruitment to DSBs and reduces NHEJ efficiency, while restoring HR. Conclusions: We have identified TLK proteins as novel regulators of NHEJ repair and PARPi sensitivity in BRCA1-depleted cells, suggesting that TLK repression may represent a previously unrecognized mechanism by which BRCA1 mutant cancers acquire PARPi resistance.

Effectively targeting cancer stemness is essential for successful cancer therapy. Recent studies have revealed that SOX2, a pluripotent stem cell factor, significantly contributes to cancer stem cell (CSC)-like characteristics closely associated with cancer malignancy. However, its contradictory impact on patient survival in specific cancer types, including lung adenocarcinoma (LUAD), underscores the need for more comprehensive research to clarify its functional effect on cancer stemness. In this study, we demonstrate that SOX2 is not universally required for the regulation of CSC-like properties in LUAD. We generated SOX2 knockouts in A549, H358, and HCC827 LUAD cells using the CRISPR/Cas9 system. Our results reveal unchanged CSC characteristics, including sustained proliferation, tumor sphere formation, invasion, migration, and therapy resistance, compared to normal cells. Conversely, SOX2 knockdown using conditional shRNA targeting SOX2, significantly reduced CSC traits. However, these loss-of-function effects were not rescued by SOX2 resistant to shRNA, underscoring the potential for SOX2 protein level-independent results in prior siRNA- or shRNA-based research. Ultimately, our findings demonstrate that SOX2 is not absolutely essential in LUAD cancer cells. This emphasizes the necessity of considering cancer subtype-dependent and context-dependent factors when targeting SOX2 overexpression as a potential therapeutic vulnerability in diverse cancers.

Significance: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers to treat. Existing drugs rarely work as a single agent. Therefore, identifying novel drug combinations that will synergistically and selectively kill PDAC cells is a significant unmet need. Here, we used CRISPR gene knockout s creening to identify combinatorial targets of gemcitabine (Gem) in PDAC. We discovered that PRMT5 depletion makes PDAC cells significantly sensitive to Gem. Our mechanistic findings show that PRMT5 inhibition impairs DNA repair mechanism. Hence the combination results in synergistic accumulation of Gem-induced DNA damage. We show that the combination leads to synergistic cell death in PDAC cells in vitro and in vivo.