Different from the chemotherapy of "killing a thousand enemies and losing eight hundred", oncogene targeted therapy is called "biological missile" because of its precise action on cancer-causing targets.
In recent years, a variety of targeted therapy drugs have been successfully applied to the clinic, however, the actual benefits for patients with advanced tumors are not ideal, and one of the important reasons is: drug resistance. And we know very little about the mechanisms by which targeted therapy acquires resistance.
Studies have found that residual cancer cells that survive targeted therapy and subsequent DNA damage repair are important reasons for drug resistance in tumor cells [1]. Therefore, exploring the molecules related to DNA damage repair induced by targeted therapy and clarifying the mechanism of tumor resistance are crucial to the development of targeted drugs and the selection of clinical protocols.
Recently, Dr. Kris C. Wood of Duke University and his team found that dna double-strand breaks (DSB) and DNA double-strand break repair exist in tumor cells that survive targeted therapy, and this repair process relies on ataxia capillary dilation mutation (ATM) enzymes.
They also found that targeted therapy drugs, in combination with ATM inhibitors, eradicated surviving residual tumor cells in vitro cell lines and animal models, resulting in a longer-lasting therapeutic response[2].
This research result has laid a theoretical foundation for the clinical development of ATM inhibitors and the integration of existing targeted therapy protocols, and the relevant research results have recently been published in the prestigious journal Science Translational Medicine.
Thesis homepage
Next, let's look at how this research is conducted.
First, to investigate whether targeted therapy activates the DNA damage response signaling pathway (DDR), the team used a set of tumor cell lines that are sensitive to homologous targeted therapy: epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) cell line (PC9, HCC827), anaplastic lymphoma kinase (ALK) gene rearrangement NSCLC cell line (H3122), BRAF gene mutant melanoma cell line (A375), FLT3 gene-mutant acute myeloid leukemia (AML) cell line (MOLM13), KRAS gene-mutant pancreatic cancer cell line (MIA PaCa-2) and NSCLC cell line (A549).
After treating the above cell lines with different doses of homologous targeted therapy drugs for 24 h, they found a significant increase in the expression of phosphorylated ATMs (p-ATMs) and γH2AX at the S1981 site. It should be noted that ATMs S1981 locus phosphorylation is necessary to activate the downstream DNA damage response signaling pathway, and γH2AX produced by phosphorylation of H2AX (member of the H2A family X) is a biomarker of DNA double-strand breaks.
Subsequent cell cloning experiments showed that a significant increase in gamma H2AX expression was still observed at doses of drugs that did not affect cell viability, a conclusion that was further validated in three Gefitinib-resistant EGFR mutant NSCLC cell lines (PC9R, GR4, WZR12), while annexin V staining (for detecting apoptosis) was observed in PC9 and A549 cells treated with targeted drugs as negative.
Finally, the neutral comet test (a technique for detecting DNA damage at the single-cell level) found that the presence of DNA double-strand breaks was detected 24 hours after targeted drug treatment in PC9 and A549 cells, suggesting that the observed ATM activation was due to DNA damage.
The above results show that tumor cells that survive targeted therapy will experience DNA double strand breaks and subsequent ATM activation.
The DNA damage response signaling pathway is activated in targeted drug-treated tumor cells
So, how is ATM activated? Let's move on.
First, PC9 and HCC827 cells were observed to increase phosphorylation expression at the checkpoint kinase 2 (Chk2) T68 site after 24 h of treatment with gefitinib (EGFR blocker); subsequently, the researchers treated PC9 cells with ATM kinase inhibitors (AZD0156) alone or in combination with gefitinib, and found that the combination of AZD0156 and gefitinib eliminated the expression of gamma H2AX and the activation of DNA double-strand break repair pathways.
In subsequent experiments, the researchers found that as gefitinib dosage and treatment time increased, p-ATM and gamma H2AX expression also gradually increased, while initiator caspase 9 and performer caspase 3 were cleaved.
A recent study suggests that activation of the endogenous pathway caspase can lead to the formation of DNA double-strand breaks and subsequent ATM activation[3]. Therefore, the researchers hypothesized that Bcl-2 anti-apoptotic family members BIM and BAK/BAX activated downstream caspase, and the resulting increase in mitochondrial outer membrane permeability (MOMP), may be responsible for DNA double-strand breaks as well as ATM activation.
To test this hypothesis, they knocked out the expression of BIM and knockdown BAX in PC9 cells, respectively, and found that BIM and BAX low expression eliminated activation of ATMs and gamma H2AX induced by targeted therapy. To further validate this hypothesis, the researchers treated PC9 cells using a pancysteine inhibitor (Q-VD-OPh) alone or in combination with gefitinib, and found that the combination of the two eliminated ATM activation observed when treated with gefitinib alone.
Simultaneous knocking out of caspase 3 and 7 in PC9 cells can similarly eliminate activation of ATM and gamma H2AX in GEFITINIB-treated PC9 cells. How is deoxyribonuclease expression in PC9 cells treated with gefitinib, a key enzyme activated by caspase 3 and 7 that mediates DNA double-strand breaks?
The results showed that deoxyribonuclease expression was relatively unchanged, but the expression of caspase-activated DNA inhibitory factor (ICAD) was significantly reduced; the researchers further knocked out DNA expression in PC9 cells and found that no activation of gamma H2AX was observed in GEFIT-treated PC9 cells, but caspase 3 cleavage and loss of DNA inhibitory factors were still observed.
The above results show that the low expression of DNA inhibitors leads to DNA activation, and consequently induces DNA double-strand breaks and ATM activation, and the activation of caspase and the deletion of DNA inhibitory factors play a role upstream of DNA activation.
EGFR inhibitors induce ATM signaling pathway activation
The above results suggest that cells surviving targeted therapy may require ATM activation to repair DNA double-strand breaks caused by targeted therapy exposure. So can ATM inhibitors improve the intensity and duration of targeted therapy responses?
First, previous experiments have confirmed that AZD0156 can block gefitinib-induced ATM activation, and then researchers using AZD0156 alone or in combination with gefitinib pc9, PC9R, GR4 cells found that AZD0156 can simultaneously increase the sensitivity of both targeted drug-sensitive cell lines (PC9) and targeted drug-resistant cell lines (PC9R, GR4) to gefitinib.
This suggests that tumor cells remaining after treatment with EGFR inhibitors are still sensitive to the combined use of EGFR inhibitors and ATM inhibitors, and this combination treatment regimen can clear tumor cells that survive single treatment with EGFR inhibitors. Next, the research team further verified this conclusion.
A small effect on cell growth was observed in long-term qualitative time-of-progression (TTP) analysis, while gefitinib monotherapy led to an increase in tumor cell resistance at about 40 days, while the combination of gefitinib and AZD0156 was effective in removing residual tumor cells, thereby avoiding the growth of drug-resistant tumor cells.
Finally, the research team assessed the effect of DNA on cellular responses in the combined use of EGFR inhibitors and ATM inhibitors. The study found that after knocking out the expression of DNA in PC9 and GR4 cells, the inhibiting effect of combination therapy on tumor cell growth could be reversed. Next, after inhibiting the expression of THE ATM, it was found that the deletion of the ATM eliminated the activation of gefitinib-induced gamma-H2AX, indicating that AZD0156 played a role by targeting ATM inhibition.
Taken together, these results suggest that dna-mediated DNA double-strand break formation is synthetically dependent on ATMs in tumor cells that survive EGFR inhibitor therapy, and THAT is a key kinase to address this DNA damage. Therefore, the combination of EGFR inhibitors and ATM inhibitors can remove tumor cells that survive under EGFR inhibitor monotherapy, thereby inhibiting the growth of drug-resistant tumor cells.
Schematic diagram of the mechanism
In vitro cell experiments have confirmed that the combination of EGFR inhibitors and ATM inhibitors can inhibit the growth of drug-resistant tumor cells, so will this combination of treatment prevent the growth of tumors in vivo? Next, the research team conducted an in vivo xenotransplantation experiment, and together we saw how the results were.
The study found that THE USE OF ATM inhibitors alone had little effect on tumor growth, with EGFR inhibitors inhibiting tumor growth before tumors developed resistance, and mice treated with a combination of ATM inhibitors and EGFR inhibitors showed sustained tumor regression throughout the study period.
Subsequently, the research team further validated it using three cell models extracted from tumor tissues in lung cancer patients, MGH134 (from patients with EGFR mutation NSCLC who became resistant to first-line erlotinib treatment), MGH1109 (from patients with EGFR mutation NSCLC initial treatment), and MGH006 (from patients with EML4-ALK variant 1 mutation NSCLC).
The results showed that ATM inhibitors could inhibit the growth of resistant tumors in targeted therapy; consistent with this finding, in MGH134 xenograft mice, osimertinib treatment produced initial growth inhibition, followed by tumor progression, while osimertinib's combination treatment with AZD0156 produced sustained tumor regression.
Targeted therapy induces ATM activation in mouse xenograft models
Finally, to explore the possible relevance of these experimental results to the clinic, the research team analyzed ATM activation in tumor tissue in patients treated with EGFR inhibitors.
First, the researchers performed immunohistochemical (IHC) staining of tumor tissues in 5 patients with EGFR mutation NSCLC, and the results showed a significant increase in p-ATM expression in tumor tissues during treatment compared with before receiving the EGFR inhibitor erlotinib; further comprehensive analysis of tumor samples from all patients found a significant increase in p-ATM expression in tumor tissues of progressive patients treated with erlotinib.
Next, the researchers analyzed time-of-clinic progression data from the Sloan-Kettering Actionable Cancer Target (MSK-IMPACT) clinical sequencing cohort database of 11 patients who received first-line erlotinib treatment, who developed both EGFR mutations/erlotinib-sensitized mutations and ATM mutations.
The results of the analysis showed that patients with EGFR mutation NSCLC with ATM deletion mutation had a longer progression-free survival compared with patients with EGFR mutation NSCLC without ATM mutations. The time to disease progression (TTP) of erlotinib treatment was 17.8 ± 10.9 months in tumor patients with ATM loss mutations, compared with 9.0 ± 1.9 months in tumor patients with those with non-functional ATM mutations, which was consistent with the 8 to 12 months clinical progression time observed in multiple studies of patients with unselected EGFR mutation NSCLC receiving first-line erlotinib therapy [4].
These data suggest that ATM activation occurs in tumor cells that survive after treatment with EGFR inhibitors and may play a tumor-protecting role.
Targeted therapy induces ATM activation in the patient's tumor tissue
Overall, this study explored the molecular mechanism of resistance from targeted therapy, further validated the clearance effect of combination therapy of ATM inhibitors and targeted drugs on drug-resistant tumors through in vitro and in vitro experiments, and finally returned to the clinic to detect the expression of NSCLC patients in the targeted anterior and posterior tumor tissues, and the effect of ATM-deficit mutations on the efficacy of targeted drugs.
This study has laid a theoretical foundation for the integration of clinical research and development of ATM inhibitors with existing targeted therapy protocols, as well as the design of clinical trials, and also provided new diagnostic and therapeutic ideas for clinical practice.
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1.Sun C, Fang Y, Yin J, et al. Rational combination therapy with PARP and MEK inhibitors capitalizes on therapeutic liabilities in RAS mutant cancers. Sci Transl Med. 2017;9(392):eaal5148. doi:10.1126/scitranslmed.aal5148
2.Ali M, Lu M, Ang HX, et al. Small-molecule targeted therapies induce dependence on DNA double-strand break repair in residual tumor cells. Sci Transl Med. 2022;14(638):eabc7480. doi:10.1126/scitranslmed.abc7480
3.Liu X, Li F, Huang Q, et al. Self-inflicted DNA double-strand breaks sustain tumorigenicity and stemness of cancer cells. Cell Res. 2017;27(6):764-783. doi:10.1038/cr.2017.41
4.Yu HA, Arcila ME, Rekhtman N, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin Cancer Res. 2013;19(8):2240-2247. doi:10.1158/1078-0432.CCR-12-2246
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