General processing of DNA mismatches can result in the formation of the ATR-activating DNA substrate. In addition, inhibition of ATR in the presence of replication stress can cause lethal premature condensation of chromosomes due to checkpoint loss. A fraction of the cells was harvested for PCR genotyping and the remainder plated for colonies.
S1333 is not in a region of ATR previously known to be involved in the regulation of the kinase. We completed two additional replicates of the whole genome screen in the presence of the CHKi, HU and the ATRi (Figure 4.2A). Summary of the synthetic lethal relationships overlapped in the whole genome screen treated with ATRi, CHKi and HU (A) or with ATRi, cisplatin, or ATRi and cisplatin (B) using a robust Z-score cut off of -2.0.
The list of top fifteen synthetic lethal genes with ATRi or combined ATRi/cisplatin treatment contains a striking number of genes in the DNA replication and ATR pathways (Figures 4.4C and D). In the nucleotide biosynthesis pathway, knockdown of genes involved in the addition of phosphates to nucleotides was identified by ATRi as synthetically lethal (Figure 4.5E). A subset of the cell lines tested were highly responsive and ATR knockdown remarkably sensitized these cells to the inhibitor (Figure 4.6A).
We next wanted to test whether the activity level of the ATR pathway reflects sensitivity to the ATRi. In a study of the ATR frameshift, cell lines containing the frameshift have reduced phosphorylated CHK1 (pCHK1) following DNA damage. The CCLE data of copy number variation and mRNA levels of the ATR pathway genes identify cell lines with ATR signaling defects.
Acquired outlier cell lines were examined by western blot for pS317 CHK1 levels before and after treatment with 2 mM HU for 1 hr compared to the control cell line, U2OS. Cell
Compared to the control cell lines (orange lines), the ATR frameshift (green lines), CHK1 frameshift (blue line), and ATR pathway-deficient cell lines (black lines) showed a wide range of sensitivities to the ATRi. These data indicate that there is no clearly observable relationship between mutations in the ATR pathway and sensitivity to the ATRi. We initially hypothesized that knockdown of ATR sensitizes cells to the ATRi because there was less total protein in the cell.
Our screen and their work identified loss of the polymerase as synthetically lethal with ATRi not loss of proofreading. HU and the clinical compound gemcitabine synergize with ATRi to kill cells (data not shown and [184]). In the primary screen, 71 genes with synthetic lethal ratios are enriched for ATRi at replication forks of iPOND [103].
In the secondary screens, RNF208 knockdown sensitized cells to low dose HU, the CHKi and the ATRi. In the secondary screen, knockdown of BRD3-sensitized cells to the ATRi as well as minor sensitization to HU and CHKi. We tested two clones for increased sensitivity to the ATRi compared to wild type U2OS cells (Figure 5.11B).
However, the BRD3 null cells were as sensitive to ATRi as wild-type U2OS cells. All four siRNAs in the secondary screen resulted in increased sensitivity to ATRi to a similar extent. One objective was to identify a clinically actionable patient population in the cancer clinic to treat with ATRi.
However, this is consistent with our overall findings in the genome-wide screen, downregulation of the ATR signaling pathway sensitizes cells to ATRi. However, there are conflicting reports if defects in HR are synthetically lethal in ATRi. The first two enriched pathways identified as synthetically lethal with ATRi alone and in combination with cisplatin were DNA replication and the ATR pathway.
During my work, I noticed that the doubling time of the cells changes the observed sensitivity to ATRi. From my experiments and others published in the literature, many cell lines have already been identified as resistant to ATRi.
Cancers with reduced ATR functionality should be treated with the ATRi. Reduced ATR function results in increased replication stress and increased dependence on the
Therefore, patients should be treated first with DNA-damaging chemotherapy followed by ATRi to most effectively kill the cancer cells. This appendix provides Table A.2 with the top synthetic lethal genes by ATRi, correlation plots, and Z-score plots for each drug condition. The ATRi overview table includes the percent control for each gene after ATRi treatment and a calculated robust Z-score.
The table also includes the percent viability after gene knockdown and the corresponding robust Z-score. All four replicates of the whole-genome screening with ATRi are listed as well as the calculated average. A gene was considered synthetically lethal and included in this table if the mean robust Z-score was −2.0 or less.
The alamarBlue values for each gene, under each drug condition, for each replicate are graphed. Overall, the replicates were highly correlative, but Arm 1 of the screen with CHKi, HU, and ATRi was more correlative than Arm2 (ATRi, cisplatin, ATRi/cisplatin). The ATR pathway was synthetically lethal, with every drug condition screened for in the whole-genome screens.
ATR pathway and other genes with known functions synthetic lethal with each drug are indicated on the graphs. The top synthetic lethal genes with the ATRi (robust Z-score less than -2.0) were analyzed by ToppGene to identify enriched pathways.
