The overall prognosis for pancreatic cancer remains dismal and potent chemotherapeutic agents that selectively target this cancer are critically needed. Elevated expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is frequent in pancreatic cancer, and it offers promising tumor-selective targeting. Recently, KP372-1 was identified as a novel NQO1 redox cycling agent that induces cytotoxicity in cancer cells by creating redox imbalance; however, the mechanistic basis of KP372-1-induced cytotoxicity remains elusive. Here, we show that KP372-1 sensitizes NQO1-expressing pancreatic cancer cells and spares immortalized normal pancreatic duct cells, hTERT-HPNE. Notably, we found that KP372-1 is ~ 10- to 20-fold more potent than β-lapachone, another NQO1 substrate, against pancreatic cancer cells. Mechanistically, our data strongly suggest that reactive oxygen species produced by NQO1-dependent redox cycling of KP372-1 cause robust DNA damage, including DNA breaks. Furthermore, we found that KP372-1-induced DNA damage hyperactivates the central DNA damage sensor protein poly(ADP-ribose) polymerase 1 (PARP1) and activates caspase-3 to initiate cell death. Our data also show that the combination of KP372-1 with PARP inhibition creates enhanced cytotoxicity in pancreatic cancer cells. Collectively, our study provides mechanistic insights into the cytotoxicity instigated by KP372-1 and lays an essential foundation to establish it as a promising chemotherapeutic agent against cancer.

Persistent R-loops (RNA-DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5′-3′-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition. Abbreviations BER Base excision repair Co-IP Co-immunoprecipitation DAVID Database for Annotation, Visualization and Integrated Discovery DSBs DNA double strand breaks FDR False discovery rate HR Homologous recombination IPA Ingenuity Pathway Analysis MS Mass spectrometry NHEJ Non-homologous end joining PAR Poly(ADP-ribose) PMSF Phenylmethylsulfonyl fluoride STRING Search Tool for the Retrieval of INteracting Genes/proteins TAP Tandem affinity purification Preserving genomic integrity is critical for ensuring survival and faithful transmission of genetic information. Compromised genomic integrity results in several debilitating diseases such as neurodegenerative disorders and cancer 1,2. The nucleic acid structures containing RNA-DNA hybrids with a displaced single-stranded DNA (R-loops) are generated in cells during transcription and their persistence leads to DNA damage and genomic

A series of cystargolide-based β-lactone analogues containing nitrogen atoms at the Pz portion of the scaffold were prepared and evaluated as proteasome inhibitors, and for their cytotoxicity profile toward several cancer cell lines. Inclusion of one, two or even three nitrogen atoms at the Pz portion of the cystargolide scaffold is well tolerated, producing analogues with low nanomolar proteasome inhibition activity, in many cases superior to carfilzomib. Additionally, analogue 8g , containing an ester and pyrazine group at Pz, was shown to possess significant activity toward RPMI 8226 cells (IC 50 = 21 nM) and to be less cytotoxic toward the normal tissue model MCF10A cells than carfilzomib.

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