In Vivo Targeting Replication Protein A for Cancer Therapy

Abstract

Replication protein A (RPA) plays essential roles in DNA replication, repair, recombination, and the DNA damage response (DDR). 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. We have developed second-generation RPA inhibitors (RPAis) that block the RPA-DNA interaction and optimized formulation for in vivo analyses. Data demonstrate that unlike first-generation RPAis, second-generation molecules show increased cellular permeability and induce cell death via apoptosis. Second-generation RPAis elicit single-agent in vitro anticancer activity across a broad spectrum of cancers, and the cellular response suggests existence of a threshold before chemical RPA exhaustion induces cell death. Chemical RPA inhibition potentiates the anticancer activity of a series of DDR inhibitors and traditional DNA-damaging cancer therapeutics. Consistent with chemical RPA exhaustion, we demonstrate that the effects of RPAi on replication fork dynamics are similar to other known DDR inhibitors. An optimized formulation of RPAi NERx 329 was developed that resulted in single-agent anticancer activity in two non-small cell lung cancer models. These data demonstrate a unique mechanism of action of RPAis eliciting a state of chemical RPA exhaustion and suggest they will provide an effective therapeutic option for difficult-to-treat lung cancers.

Keywords: DNA damage response; DNA repair and cancer; DNA repair inhibitors; Replication Protein A; Replication Stress Response.

Figure 1

Kaplan–Meyer retrospective analysis of overall survival as a function of replication protein A (RPA) gene expression in non-small cell lung cancer (NSCLC). Blue numbering indicates patients with low RPA expression, red numbering indicates patients with high RPA expression. Analysis represents a 500-patient cohort from the caARRAY, with optimized cutoff. (A) Former and current smokers. HR = 1.63 (1.17–2.28) log-rank: p = 0.0035. (B) Former and current smokers who received chemotherapy. HR = 1.69 (1–2.86) log-rank: p = 0.049. (C) Never smokers.

Figure 2

Replication protein A inhibitor (RPAi) inhibitory activity. (A) Chemical structure of RPAi’s 329 and 2004. (B) EMSA analysis of RPA–DNA interaction inhibition by 329 and 2004. Lanes 3–6 in each panel contain 6.25, 12.5, 25, and 50 µM of the indicated RPAi, respectively. The * indicates the position of the Escherichia coli SSB–single-stranded DNA (ssDNA) complex that serves as an internal specificity control. (C) Cell viability of H460 NSCLC cells in response to 329 and 2004.

Figure 3

The 329 induction of apoptotic cell death. (A) Analysis of caspase 3/7 activity in H460 cells following 24 h of treatment with 1% DMSO or the indicated concentrations of 329. Fluorescence images were captured as described in the Materials and Methods. (B) Quantification of caspase 3/7 activity. Fluorescence was measured in 96-well plates using a Biotek Synergy H1 plate reader following 24-h incubation with the indicated drugs and concentrations.

Figure 4

Cellular activity of 329 in 60 cancer cell lines. Cell lines were treated with a 4-log range of replication protein A inhibitor (RPAi) 329 for 72 h. Cell viability was assessed using CellTiter-Glo luminescent viability assay. The data represent the average of triplicate treatments, and the data were fit using non-linear regression analysis to calculate cellular IC₅₀s. (A) IC₅₀ results from each cell line grouped by tumor type. (B) Hill coefficients for individual cell lines. The horizontal lines above cell line names indicated the tumor sites in the order depicted in panel (A).

Figure 5

Replication protein A inhibitor (RPAi) impact on replication fork dynamics. (A) Schematic depiction of experimental design. DNA was pulse-labeled with IdU for 20 min. After IdU removal, replication forks were stalled by the addition of HU or left to replicate with vehicle treatment. HU was removed, and replication was labeled with CldU. Following CldU, cells were treated with the DDRi or vehicle. (B) Quantification of results from DNA fiber analysis in H460 cells treated with the indicated agents. HU was used at a final concentration of 2.5 mM, the ATRi VE-822 at 2 µM, and the RPAi 329 at 50 µM. Data presented are combined from three individual experiments (100 fibers analyzed per experiment; 300 fibers total). Red bar indicates the median value of CldU/IdU. Data were analyzed by ANOVA with Bonferroni test for multiple comparisons (****p < 0.0001).

Figure 6

Analysis of replication protein A inhibitor (RPAi) 329 combination treatment. (A) Chou-Talalay analysis of combination with chemotherapeutics. The combination index (CI) is plotted as a function of the fraction of cell affected (Fa) for each treatment combination of the 329. (B) Chou-Talalay analysis of combination with DDR-targeted agents as described in panel (A).

Figure 7

The 329 in vivo analysis. (A) Stability analysis. Compound stability was assessed over a 14-day time period at varying temperatures as indicated. (B) Pharmacokinetic analysis. Time course of drug plasma concentration over 24 h following drug administration as indicated in legend.

Figure 8

In vivo analysis of anticancer activity of 329. (A) Anticancer activity was assessed in human H460 NCSLC tumor xenografts in NOD/SCID mice. Mice were implanted subcutaneously on day 1 with H460 NSCLC cells, tumors were measured by calipers, and mice assigned randomly to treatment arms. Treatment with 329 was initialized at day 6 and administered via intraperitoneal injection (IP) once daily (20 mg/kg), as indicated (|). Tumor volumes were completed with caliper measurement biweekly. (B) A549 cells were implanted subcutaneously, mice were randomized, and treatment with 329 was initiated at day 11 via IP (40 mg/kg) and treated once daily as indicated (|). (C) Tumor weight from A549 cells was determined on day 32. Statistically significance differences from vehicle-treated tumors are indicated by the asterisk *p < 0.05; ***p < 0.01.