Molecular Basis of XRN2-Deficient Cancer Cell Sensitivity to Poly(ADP-ribose) Polymerase Inhibition.

R-loops (RNA-DNA hybrids with displaced single-stranded DNA) have emerged as a potent source of DNA damage and genomic instability. The termination of defective RNA polymerase II (RNAPII) is one of the major sources of R-loop formation. 5'-3'-exoribonuclease 2 (XRN2) promotes genome-wide efficient RNAPII termination, and XRN2-deficient cells exhibit increased DNA damage emanating from elevated R-loops. Recently, we showed that DNA damage instigated by XRN2 depletion in human fibroblast cells resulted in enhanced poly(ADP-ribose) polymerase 1 (PARP1) activity. Additionally, we established a synthetic lethal relationship between XRN2 and PARP1. However, the underlying cellular stress response promoting this synthetic lethality remains elusive. Here, we delineate the molecular consequences leading to the synthetic lethality of XRN2-deficient cancer cells induced by PARP inhibition. We found that XRN2-deficient lung and breast cancer cells display sensitivity to two clinically relevant PARP inhibitors, Rucaparib and Olaparib. At a mechanistic level, PARP inhibition combined with XRN2 deficiency exacerbates R-loop and DNA double-strand break formation in cancer cells. Consistent with our previous findings using several different siRNAs, we also show that XRN2 deficiency in cancer cells hyperactivates PARP1. Furthermore, we observed enhanced replication stress in XRN2-deficient cancer cells treated with PARP inhibitors. Finally, the enhanced stress response instigated by compromised PARP1 catalytic function in XRN2-deficient cells activates caspase-3 to initiate cell death. Collectively, these findings provide mechanistic insights into the sensitivity of XRN2-deficient cancer cells to PARP inhibition and strengthen the underlying translational implications for targeted therapy.

Fate, transformation and toxicological implications of environmental diclofenac: Role of mineralogy and solar flux.

Diclofenac is an emerging surface water contaminant, yet the environmental impact of its degradation products remains elusive. The current study focuses on mineralogy-controlled diclofenac photo-degradation and its potential health impacts. Under irradiated conditions, we studied the effects of kaolinite, hematite, and anatase on diclofenac degradation. Our results showed that kaolinite doubled the diclofenac degradation rate, which can be attributed to the high catalytic effect, mediated via increased surface area and pore size of mineral surface in the low pH. Conversely, anatase, a crystal phase of titanium dioxide (TiO2), diminished the diclofenac degradation compared to treatments without TiO2. Hematite, on the other hand, showed no effect on diclofenac degradation. Photo-degradation products also varied with the mineral surface. We further assessed in vitro toxicological effects of photo-degraded products on two human cell lines, HEK293T and HepG2. Biological assays confirmed that photo-degraded compound 6 (1-(2,6-dichlorophenyl)indolin-2-one) decreased HEK293T cell survival significantly (p < 0.05) when compared to diclofenac in all concentrations. At lower concentrations, inhibition of HEK293T cells caused by compounds 4 (2-(8-chloro-9H-carbazol-1-yl)acetic acid), and 5 (2-(9H-carbazol-1-yl)acetic acid) was greater than diclofenac. Compound 7 (1-phenylindolin-2-one) was toxic only at 250 µM. Additionally, compound 6 decreased HepG2 cell viability significantly when compared to diclofenac. Overall, our data highlighted that mineralogy plays a vital role in environmental diclofenac transformation and its photo-degraded products. Some photo-degraded compounds can be more cytotoxic than the parent compound, diclofenac.

Cystargolide-based amide and ester Pz analogues as proteasome inhibitors and anti-cancer agents.

A series of cystargolide-based ß-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 (IC50 = 21 nM) and to be less cytotoxic toward the normal tissue model MCF10A cells than carfilzomib.

DNA damage induced by KP372-1 hyperactivates PARP1 and enhances lethality of pancreatic cancer cells with PARP inhibition.

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 ß-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.

XRN2 interactome reveals its synthetic lethal relationship with PARP1 inhibition.

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.