Targeted Inhibition of DNA-PKcs, ATM, ATR, PARP, and Rad51 Modulate Response to X Rays and Protons.

Small molecule inhibitors are currently in preclinical and clinical development for the treatment of selected cancers, particularly those with existing genetic alterations in DNA repair and DNA damage response (DDR) pathways. Keen interest has also been expressed in combining such agents with other targeted antitumor strategies such as radiotherapy. Radiotherapy exerts its cytotoxic effects primarily through DNA damage-induced cell death; therefore, inhibiting DNA repair and the DDR should lead to additive and/or synergistic radiosensitizing effects. In this study we screened the response to X-ray or proton radiation in cell lines treated with DDR inhibitors (DDRis) targeting ATM, ATR, DNA-PKcs, Rad51, and PARP, with survival metrics established using clonogenic assays. We observed that DDRis generate significant radiosensitization in cancer and primary cells derived from normal tissue. Existing genetic defects in cancer cells appear to be an important consideration when determining the optimal inhibitor to use for synergistic combination with radiation. We also show that while greater radiosensitization can be achieved with protons (9.9 keV/µm) combined with DDRis, the relative biological effectiveness is unchanged or in some cases reduced. Our results indicate that while targeting the DDR can significantly radiosensitize cancer cells to such combinations, normal cells may also be equally or more severely affected, depending on the DDRi used. These data highlight the importance of identifying genetic defects as predictive biomarkers of response for combination treatment.

An empirical model of proton RBE based on the linear correlation between x-ray and proton radiosensitivity.

BACKGROUND: Proton relative biological effectiveness (RBE) is known to depend on physical factors of the proton beam, such as its linear energy transfer (LET), as well as on cell-line specific biological factors, such as their ability to repair DNA damage. However, in a clinical setting, proton RBE is still considered to have a fixed value of 1.1 despite the existence of several empirical models that can predict proton RBE based on how a cell's survival curve (linear-quadratic model [LQM]) parameters α and ß vary with the LET of the proton beam. Part of the hesitation to incorporate variable RBE models in the clinic is due to the great noise in the biological datasets on which these models are trained, often making it unclear which model, if any, provides sufficiently accurate RBE predictions to warrant a departure from RBE = 1.1. PURPOSE: Here, we introduce a novel model of proton RBE based on how a cell's intrinsic radiosensitivity varies with LET, rather than its LQM parameters. METHODS AND MATERIALS: We performed clonogenic cell survival assays for eight cell lines exposed to 6 MV x-rays and 1.2, 2.6, or 9.9 keV/µm protons, and combined our measurements with published survival data (n = 397 total cell line/LET combinations). We characterized how radiosensitivity metrics of the form DSF% , (the dose required to achieve survival fraction [SF], e.g., D10% ) varied with proton LET, and calculated the Bayesian information criteria associated with different LET-dependent functions to determine which functions best described the underlying trends. This allowed us to construct a six-parameter model that predicts cells' proton survival curves based on the LET dependence of their radiosensitivity, rather than the LET dependence of the LQM parameters themselves. We compared the accuracy of our model to previously established empirical proton RBE models, and implemented our model within a clinical treatment plan evaluation workflow to demonstrate its feasibility in a clinical setting. RESULTS: Our analyses of the trends in the data show that DSF% is linearly correlated between x-rays and protons, regardless of the choice of the survival level (e.g., D10% , D37% , or D50% are similarly correlated), and that the slope and intercept of these correlations vary with proton LET. The model we constructed based on these trends predicts proton RBE within 15%-30% at the 68.3% confidence level and offers a more accurate general description of the experimental data than previously published empirical models. In the context of a clinical treatment plan, our model generally predicted higher RBE-weighted doses than the other empirical models, with RBE-weighted doses in the distal portion of the field being up to 50.7% higher than the planned RBE-weighted doses (RBE = 1.1) to the tumor. CONCLUSIONS: We established a new empirical proton RBE model that is more accurate than previous empirical models, and that predicts much higher RBE values in the distal edge of clinical proton beams.

Effect of boron compounds on the biological effectiveness of proton therapy.

PURPOSE: We assessed whether adding sodium borocaptate (BSH) or 4-borono-l-phenylalanine (BPA) to cells irradiated with proton beams influenced the biological effectiveness of those beams against prostate cancer cells to investigate if the alpha particles generated through proton-boron nuclear reactions would be sufficient to enhance the biological effectiveness of the proton beams. METHODS: We measured clonogenic survival in DU145 cells treated with 80.4-ppm BSH or 86.9-ppm BPA, or their respective vehicles, after irradiation with 6-MV X-rays, 1.2-keV/µm (low linear energy transfer [LET]) protons, or 9.9-keV/µm (high-LET) protons. We also measured γH2AX and 53BP1 foci in treated cells at 1 and 24 h after irradiation with the same conditions. RESULTS: We found that BSH radiosensitized DU145 cells across all radiation types. However, no difference was found in relative radiosensitization, characterized by the sensitization enhancement ratio or the relative biological effectiveness, for vehicle- versus BSH-treated cells. No differences were found in numbers of γH2AX or 53BP1 foci or γH2AX/53BP1 colocalized foci for vehicle- versus BSH-treated cells across radiation types. BPA did not radiosensitize DU145 cells nor induced any significant differences when comparing vehicle- versus BPA-treated cells for clonogenic cell survival or γH2AX and 53BP1 foci or γH2AX/53BP1 colocalized foci. CONCLUSIONS: Treatment with 11 B, at concentrations of 80.4 ppm from BSH or 86.9 ppm from BPA, had no effect on the biological effectiveness of proton beams in DU145 prostate cancer cells. Our results agree with published theoretical calculations indicating that the contribution of alpha particles from such reactions to the total absorbed dose and biological effectiveness is negligible. We also found that BSH radiosensitized DU145 cells to X-rays, low-LET protons, and high-LET protons but that the radiosensitization was not related to DNA damage.

Transcriptional consequences of XPA disruption in human cell lines.

Nucleotide excision repair (NER) in mammalian cells requires the xeroderma pigmentosum group A protein (XPA) as a core factor. Remarkably, XPA and other NER proteins have been detected by chromatin immunoprecipitation at some active promoters, and NER deficiency is reported to influence the activated transcription of selected genes. However, the global influence of XPA on transcription in human cells has not been determined. We analyzed the human transcriptome by RNA sequencing (RNA-Seq). We first confirmed that XPA is confined to the cell nucleus even in the absence of external DNA damage, in contrast to previous reports that XPA is normally resident in the cytoplasm and is imported following DNA damage. We then analyzed four genetically matched human cell line pairs deficient or proficient in XPA. Of the ∼14,000 genes transcribed in each cell line, 325 genes (2%) had a significant XPA-dependent directional change in gene expression that was common to all four pairs (with a false discovery rate of 0.05). These genes were enriched in pathways for the maintenance of mitochondria. Only 27 common genes were different by more than 1.5-fold. The most significant hits were AKR1C1 and AKR1C2, involved in steroid hormone metabolism. AKR1C2 protein was lower in all of the immortalized XPA-deficient cells. Retinoic acid treatment led to modest XPA-dependent activation of some genes with transcription-related functions. We conclude that XPA status does not globally influence human gene transcription. However, XPA significantly influences expression of a small subset of genes important for mitochondrial functions and steroid hormone metabolism. The results may help explain defects in neurological function and sterility in individuals with xeroderma pigmentosum.

The ERCC1 and ERCC4 (XPF) genes and gene products.

The ERCC1 and ERCC4 genes encode the two subunits of the ERCC1-XPF nuclease. This enzyme plays an important role in repair of DNA damage and in maintaining genomic stability. ERCC1-XPF nuclease nicks DNA specifically at junctions between double-stranded and single-stranded DNA, when the single-strand is oriented 5' to 3' away from a junction. ERCC1-XPF is a core component of nucleotide excision repair and also plays a role in interstrand crosslink repair, some pathways of double-strand break repair by homologous recombination and end-joining, as a backup enzyme in base excision repair, and in telomere length regulation. In many of these activities, ERCC1-XPF complex cleaves the 3' tails of DNA intermediates in preparation for further processing. ERCC1-XPF interacts with other proteins including XPA, RPA, SLX4 and TRF2 to perform its functions. Disruption of these interactions or direct targeting of ERCC1-XPF to decrease its DNA repair function might be a useful strategy to increase the sensitivity of cancer cells to some DNA damaging agents. Complete deletion of either ERCC1 or ERCC4 is not compatible with viability in mice or humans. However, mutations in the ERCC1 or ERCC4 genes cause a remarkable array of rare inherited human disorders. These include specific forms of xeroderma pigmentosum, Cockayne syndrome, Fanconi anemia, XFE progeria and cerebro-oculo-facio-skeletal syndrome.