Gavande, N. S. | Search bioRxiv

bioRxiv2020

Chemical exhaustion of RPA in cancer treatment

Replication protein A (RPA) plays essential roles in DNA replication, repair, recombination and the DNA-damage response (DDR). We have developed second generation RPA inhibitors (RPAis) that block the RPA-DNA interaction. These DNA-binding inhibitors (DBis) can elicit a state of cellular RPA exhaustion resulting in single agent in vitro anticancer activity across a broad spectrum of cancers and in vivo activity in two non-small cell lung cancer models. The cellular response to RPAi treatment suggests a threshold exists before RPA inhibition induces cell death. Chemical RPA exhaustion potentiates the anticancer activity of other DDR inhibitors as well as traditional DNA damaging cancer therapeutics. Consistent with the chemical RPA exhaustion model, we demonstrate that the effects of RPAi on replication fork dynamics and DNA damage signaling are similar to other known DDR inhibitors. In accordance with the RPA threshold model, retrospective analysis of lung cancer patient data demonstrates high RPA expression as a negative prognostic biomarker for overall survival in smoking-related lung cancers. Similarly, relative expression of RPA is a predictive marker for response to chemotherapy. These observations are consistent with the increase in RPA expression serving as an adaptive mechanism that allows tolerance of the genotoxic stress resulting from carcinogen exposure. These data demonstrate a unique mechanism of action of RPAis eliciting a state of RPA exhaustion that impacts the DDR and may provide an effective therapeutic option for difficult to treat lung cancers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/404640v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@d662e4org.highwire.dtl.DTLVardef@f68b11org.highwire.dtl.DTLVardef@241c0corg.highwire.dtl.DTLVardef@ad7398_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Discovery and Development of Novel DNA-PK Inhibitors by Targeting the unique Ku-DNA Interaction

DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBis) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBis directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the activity of IR and radiomimetics. Analysis of Ku-null cells demonstrates that Ku-DBis cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBis. The utility of Ku-DBis was also demonstrated in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBis, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.

biochemistry↗