Targeting lung cancer through inhibition of checkpoint kinases

Inhibitors of checkpoint kinases ATR, Chk1 and Wee1 are currently being tested in preclinical and clinical trials. Here, we review the basic principles behind the use of such inhibitors as anticancer agents, and particularly discuss their potential for treatment of lung cancer. As lung cancer is one of the most deadly cancers, new treatment strategies are highly needed. We discuss how checkpoint kinase inhibition in principle can lead to selective killing of lung cancer cells while sparing the surrounding normal tissues. Several features of lung cancer may potentially be exploited for targeting through inhibition of checkpoint kinases, including mutated p53, low ERCC1 levels, amplified Myc, tumor hypoxia and presence of lung cancer stem cells. Synergistic effects have also been reported between inhibitors of ATR/Chk1/Wee1 and conventional lung cancer treatments, such as gemcitabine, cisplatin or radiation. Altogether, inhibitors of ATR, Chk1 and Wee1 are emerging as new cancer treatment agents, likely to be useful in lung cancer treatment. However, as lung tumors are very diverse, the inhibitors are unlikely to be effective in all patients, and more work is needed to determine how such inhibitors can be utilized in the most optimal ways

Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption

Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage

TLR9 stimulation of B-cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent of DNA damage responses. Implications for Common variable immunodeficiency

<div><p>In the present study, we address the important issue of whether B-cells protected from irradiation-induced cell death, may survive with elevated levels of DNA damage. If so, such cells would be at higher risk of gaining mutations and undergoing malignant transformation. We show that stimulation of B-cells with the TLR9 ligands CpG-oligodeoxynucleotides (CpG-ODN) prevents spontaneous and irradiation-induced death of normal peripheral blood B-cells, and of B-cells from patients diagnosed with Common variable immunodeficiency (CVID). The TLR9-mediated survival is enhanced by the vitamin A metabolite retinoic acid (RA). Importantly, neither stimulation of B-cells via TLR9 alone or with RA increases irradiation-induced DNA strand breaks and DNA damage responses such as activation of ATM and DNA-PKcs. We prove that elevated levels of γH2AX imposed by irradiation of stimulated B-cells is not due to induction of DNA double strand breaks, but merely reflects increased levels of total H2AX upon stimulation. Interestingly however, we unexpectedly find that TLR9 stimulation of B-cells induces low amounts of inactive p53, explained by transcriptional induction of <i>TP53</i>. Taken together, we show that enhanced survival of irradiated B-cells is not accompanied by elevated levels of DNA damage. Our results imply that TLR9-mediated activation of B-cells not only promotes cell survival, but may via p53 provide cells with a barrier against harmful consequences of enhanced activation and proliferation. As CVID-derived B-cells are more radiosensitive and prone to undergo apoptosis than normal B-cells, our data support treatment of CVID patients with CpG-ODN and RA.</p></div

TLR9 stimulation does not enhance DNA damage-induced DNA strand breaks.

<p>B-cells were treated with CpG-ODN (0.5 μg/ml) in the presence or absence of RA (200 nM) for 24 hours prior to irradiation (IR; 5 Gy). After indicated time points the cells were lysed, and single cell gel electrophoresis was performed. DNA was stained with SYBRgold, and DNA damage was estimated measuring the % DNA in the tail. <b>(A)</b> Pictures of representative comets from non-irradiated and irradiated cells. One representative experiment is shown. <b>(B)</b> The results (% DNA in tails) are presented as histograms of mean values ±SEM (n = 4, *<i>p</i> < 0.05, paired t-test).</p

TLR9 stimulation enhances the levels of H2AX in B-cells.

<p><b>(A)</b> B-cells were treated with CpG-ODN (0.5 μg/ml) in the presence or absence of RA (200 nM) for 24 hours prior to irradiation (IR; 10 Gy). After indicated time points, ethanol-fixed samples were bar-coded with pacific blue and four samples were mixed into a single tube before staining with antibodies recognizing γH2AX and DNA stain FxCycleTM Far Red. The median level of γH2AX was analyzed using flow cytometry. The histogram represents average of median levels of γH2AX ±SEM at indicated time points after irradiation related to non-irradiated cells (n = 3, *<i>p</i> < 0.05, **<i>p</i> < 0.01, paired t-test). <b>(B)</b> B-cells were treated as indicated in (A) and fixed with ethanol 2 hours after irradiation. The samples were barcoded and three samples were mixed in one tube before staining with antibodies recognizing H2AX and DNA stain FxCycleTM Far Red. The histograms present average of median levels of H2AX related to non-irradiated cells ±SEM (n = 4, *<i>p</i> < 0.05, **<i>p</i> < 0.01, paired t-test).</p

TLR9 stimulation induces p53 expression.

<p><b>(A)</b> Normal B-cells were treated with CpG-ODN (0.5 μg/ml) in the presence or absence of RA (200 nM) for 24 hours prior to irradiation (IR; 10 Gy). After indicated time points B-cells were collected and subjected to western blot analysis with antibodies recognizing calnexin (as loading control), p53, or p53 phosphorylated at serine 15 (S15). The right panel shows a more exposed version of the blot depicting induction of p53 in non-irradiated cells. One representative experiment of three is shown. Original uncropped blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185708#pone.0185708.s005" target="_blank">S5 Fig</a>. <b>(B)</b> Densitometric analysis of the p53 expression (based on low exposure) at 4 hours after irradiation was normalized to calnexin. The results are presented as histograms of mean values ±SEM (n = 6, *<i>p</i> < 0.05, **<i>p</i> < 0.01, paired t-test). <b>(C)</b> B-cells were treated as described in (A), and lysates were subjected to western blot analysis with antibodies recognizing calnexin (as loading control) and p21^Cip. One representative experiment of three is shown. Original uncropped blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185708#pone.0185708.s006" target="_blank">S6 Fig</a>. <b>(D)</b> B-cells were stimulated as described in (A), and the cells were harvested 24 hours after irradiation. The mRNA level of <i>TP53</i> was quantified by RT-qPCR. The amounts of <i>TP53</i> mRNA related to reference genes (<i>TBP</i> and <i>B2M</i>) were quantified using the 2^-ΔCt-method. The results are presented as histograms of mean values ±SEM (n = 4 *<i>p</i> < 0.05, paired t-test).</p

RA reduces spontaneous and irradiation-induced apoptosis of normal and CVID-derived B-cells stimulated via TLR9.

<p>Normal and CVID-derived B-cells were treated with CpG-ODN (0.5 μg/ml) in the presence or absence of RA (200 nM) for 24 hours prior to irradiation (IR; 10 Gy). After additional 24 hours, cell death was measured as the percentage of PI-positive cells. Each symbol represents the data from an individual patient or healthy control, and the horizontal lines represent median values, (n = 7, *<i>p</i> < 0.05, Wilcoxon signed rank test).</p

TLR9 stimulation does not activate DDRs upstream of p53.

<p><b>(A)</b> B-cells were treated with CpG-ODN (0.5 μg/ml) in the presence or absence of RA (200 nM) for 24 hours prior to irradiation (IR; 10 Gy). After indicated time points, B-cells were collected and subjected to western blot analysis with antibodies recognizing calnexin (as loading control) and phosphorylated ATM (S1981). One representative experiment of three is shown. Original uncropped blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185708#pone.0185708.s007" target="_blank">S7 Fig</a>. <b>(B)</b> Densitometric analysis of pATM expression at 0.5 hours after irradiation was normalized to calnexin. The results are presented as histograms of mean values ±SEM (n = 5, *<i>p</i> < 0.05, paired t-test). <b>(C)</b> B-cells were treated as indicated in (A), and the B-cells were harvested one hour after irradiation and subjected to western blot analysis with antibodies recognizing calnexin (as loading control), pDNA-PKcs (S2056, upper panel) and pATR (Thr1989, lower panel). One representative experiment of three is shown. Original uncropped blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185708#pone.0185708.s008" target="_blank">S8 Fig</a>.</p