Enhanced Radiosensitivity in Solid Tumors using a Tumor-selective Alkyl Phospholipid Ether Analog

Abstract

Antitumor alkyl phospholipid (APL) analogs comprise a group of structurally related molecules with remarkable tumor selectivity. Some of these compounds have shown radiosensitizing capabilities. CLR127 is a novel, clinical-grade antitumor APL ether analog, a subtype of synthetic APL broadly targeting cancer cells with limited uptake in normal tissues. The purpose of this study was to investigate the effect of CLR127 to modulate radiation response across several adult and pediatric cancer types in vitro as well as in murine xenograft models of human prostate adenocarcinoma, neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma. In vitro, CLR127 demonstrated selective uptake in cancer cells compared to normal cells. In cancer cells, CLR127 treatment prior to radiation significantly decreased clonogenic survival in vitro, and led to increased radiation-induced double-stranded DNA (dsDNA) breakage compared with radiation alone, which was not observed in normal controls. In animal models, CLR127 effectively increased the antitumor response to fractionated radiotherapy and led to delayed tumor regrowth at potentially clinically achievable doses. In conclusion, our study highlights the ability of CLR127 to increase radiation response in several cancer types. Given almost universal uptake of CLR127 in malignant cells, future research should test whether the observed effects can be extended to other tumor types. Our data provide a strong rationale for clinical testing of CLR127 as a tumor-targeted radiosensitizing agent. Mol Cancer Ther; 17(11); 2320-8. ©2018 AACR.

Figure 1.

Uptake of CLR1501 in cancer cells compared to normal cells. Flow cytometry of the uptake of CLR1501 (a CLR127 fluorescent analog) by normal cells (HUFI, human skin fibroblasts; MCF10A, human mammary gland epithelial cells) and tumor cell lines. Averages ± standard error (SE) from three repeats per cell type. *p ≤ 0.05, ** p ≤ 0.01 tumor cells versus normal cells. RU=relative units.

Figure 2.

Clonogenic survival of cancer (CHLA-20, Rh30, PC-3) and normal cells (MCF10A) exposed to various XRT doses, with or without CLR127 pretreatment in increasing concentrations as indicated. Radiosensitization of tumor cells by CLR127. Data points represent mean values, bars indicate standard deviation. Enhancement ratios (ER₁₀) as shown. * p ≤ 0.05; ** p ≤ 0.001.

Figure 3.

Immunofluorescence microscopy was performed with anti-γH2AX antibody to visualize the DNA repair process (Fig. 3A, upper panel, scale bar: 10 μm). The data points (Fig. 3B, lower panel) represent mean values of γH2AX foci per nucleus, while the error bars show 95% confidence intervals. With the combined treatment of IR and CLR127, quantification of γH2AX foci revealed there are significant increase of mean foci count in cancer cell lines Rh30 and PC-3 at 24 hours compared to the samples treated with IR only (** p<0.01, *** p<0.001). Normal cell MCF10A did not show significant changes in foci number across all time points. NR: no irradiation; IR: irradiated.

Figure 4.

Effect of CLR127 (CLR) and radiation on DNA damage repair proteins pBRCA1 and XLF in vitro. Cells were treated with or without 7.5 μM CLR for 16 hours and exposed to 5 Gy radiation. Cells were collected directly before radiation (0 h), and at 1, 3, and 6 hours (h) and subjected to western blotting. The obtained bands were quantified relative to β-actin (XLF) or vinculin (pBRCA1) loading control using ImageJ software. Left row: Representative western blots shown. Right: Wide bars show mean relative pBRCA1 and XLF expression, respectively. Bars show mean values of three independent experiments, small bars depict standard error. Statistical differences were calculated with Student’s two-tailed t-test (*p≤0.05; **p≤0.01).

Figure 5.

CLR127 augments radiation response in xenografted pediatric cancers and adult prostate adenocarcinoma. Nude mice bearing human xenografts were treated with XRT (dashed line with triangles), CLR127 (solid line with diamonds), or both modalities (dotted line with solid circles); control/untreated mice: solid line with squares. Symbols denote mean tumor volume ± SE. Gray arrowheads indicate fractionated radiation. Black arrowheads indicate intravenous injection of CLR127. Drug (CLR127) and XRT doses are indicated in the figures. n=10–12 xenografts per group. Combination treatment vs. XRT alone p ≤ 0.001 in all tumors except SK-N-AS (not significant).

Figure 6.

Effect of in vivo CLR127 and radiation on DNA damage repair. Mice bearing human prostate carcinoma PC-3 xenografts were treated with CLR127 (30 mg/kg/dose x 1 dose), XRT (8 Gy x 1 fraction) or the combination of CLR127 and XRT. CLR127 was injected intravenously 72 hours prior to radiation administration to allow for maximal uptake. Tumors were harvested 24 hours after radiation. γH2AX expression in the tumor xenograft was examined by immunohistochemistry. Shown is the percentage of γH2AX positive cells obtained from average of twelve visual fields randomly selected from three tumors per each treatment condition: excipient control/no treatment; CLR127 only; radiation only (XRT); and radiation and CLR127 treatment. Bars represent standard error. Panels: examples of visual fields under microscopic examination (20x magnification). Upper: CLR127 (CLR) and XRT, lower: XRT only. ** p ≤ 0.001.