3.1、The NOTCH1 signal was gradually activated as the disease progressed
Lung squamous cell carcinoma was offen caused by the chronic inflammatory lung injury. The GSE136831 contained single cell data set of chronic obstructive pulmonary disease(COPD) and pulmonary fibrosis (IPF) (20). Cell communication analysis of single cell data found IPF was more serious than COPD (Fig. 1A), (Supplementary Fig. 1). As the severity of the disease progressed, the NOTCH1 signals between cells communication became more and more complex that illustrated the NOTCH1 signaling pathway was more deeply activated with the deepening of pathological degree (Fig. 1B), (Supplementary Fig. 3). The results of single cell analysis indicated that NOTCH1 was mainly activated in basal stem cells and aberrant_basaloid cells (Abnormal basal stem cells) of the airway epithelium (Fig. 1C), (Supplementary Fig. 2). Basal stem cells can differentiate into other epithelium cells (Fig. 1D).
The expression of NOTCH1 showed a decreasing trend in the twisted death of airway tissue cells of the LPS injuried lung of mice. While, the expression of NOTCH1 increased in the hyperplastic epithelium after airway repair. This suggested that NOTCH1 was activated along with airway epithelial repair(Fig. 1E,F).
The excessive growth might cause tumors. Therefore, we analyzed NOTCH1 expression in the pathological tissue of lung squamous cell carcinoma. The expression of NOTCH1 in tumor tissues was higher than that in adjacent tissues and was also observed in poorly differentiated carcinomas. Interestingly, A high expression of NOTCH1 was found in the airway epithelium with paracancer hyperplasia(Fig. 1G). This indicated that NOTCH1 played an important role in the occurrence and development of tumors.
3.2、The function of NOTCH1 was related to epigenetic enzymes, while it was unfavorable to tumor innate immunity and immune infiltration
However, in the TCGA database for lung squamous cell carcinoma, NOTCH1 was not highly correlated with key genes of innate immunity (Fig. 2A). The expression of NOTCH1 in LUSC also showed a negative correlation with the target of immune cell infiltration, which showed cold tumor characteristics (Fig. 2B). Only in the single-cell data of pulmonary fibrosis, the cCAS/STING signaling pathway were activatied. The aberrant_basaloid cells closely communicated with other cells with the cCAS/STING signaling (Fig. 2C). It proved that there was an important link between the pathologic deterioration and innate immune activation in the aberrant_basaloid cells. NOTCH1 might play an important role in immunotherapy of lung squamous cell carcinoma. Only targeted at NOTCH1 may not be a good strategy for transfering cold tumors to hot tumors.
We analyzed the transcriptome of LUSC to further explore the function of NOTCH1. GO analysis indicated the NOTCH1 functional enriched in protein dealkylation and alkylation and NADH (Fig. 2D).The dealkylation and alkylation of proteins was associated with epigenetic enzymes KDM4A and SETD2 (Fig. 2E).
We detected the change in NAD+/NADH, KDM4A and SETD2 after NOTCH1 was knocked down. After inhibition of NOTCH1, NAD + content decreased significantly and NADH content increased (Fig. 2F).This suggested NOTCH1 inhibited the action of NADH dehydrogenase. The NAD+/NADH ratio regulates the activity of many enzymes in cell.The expression of KDM4A increased significantly after NOTCH1 knockdown, while SETD2 decreased (Fig. 2G,H).
Epigenetic enzymes KDM4A and SETD2 were frequently mutated in cancers that may cause innate cellular immunity, inflammation, and genomic instability. Stable cell lines with KDM4A and SETD2 knockouts were constructed by Crisper Cas9 technology. Thus formed two tumor microenvironments that complement each other for methylation loss, which were used to explore the effect of NOTCH1 inhibitory therapy(Fig. 2I,J). The immunofluorescence test showed that H3K36me3 was almost disappear after the elimination of SETD2, while H3K9me3 increased significantly. The fluorescence intensity of H3K36me3 and H3K9me3 increased slightly after KDM4A was knocked out (Fig. 2K,M). This indicated that NOTCH1 may interact with KDM4A through metabolic activity associated with NADH dehydrogenase. H3K36me3 and H3K9me3 was showed differently in KDM4A and SETD2 knockouts cells.
3.3、 The inhibition of NOTCH1 in KDM4A and SETD2 knockouts cells caused the opposite effect on the cGAS/STING signal pathway.
The therapeutic effect of NOTCH1 inhibition under the two different genomic instability microenvironments showed the the opposite effect on the cGAS/STING signal pathway. In the KDM4A -/- cells, cGAS/STING signal pathway was activited, it was aggravated that lead to apoptosis when treated with NOTCH1 inhibition. While in the SETD2-/- cells, NOTCH1 inhibitory therapy cGAS/STING signal pathway went down and encouraged the cell proliferation.
IRF3 (Interferon regulatory factor 3) as a member of interferon regulatory factors, was a core transcription factor for IFN and IFN-inducible gene (ISG) expression. cGAS-STING activation was monitored by examining phosphorylation levels of STING (pSTING) and TBK1 (pTBK1), as well as IRF3 (pIRF3)(21).In KDM4A-/- cells, IRF3, STING and TBK1 form trimer complexes, IRF3-p was activated by TBK1 phosphorylation. IRF3-p and IFNB1 were further activated in NOTCH1 and KDM4A double-knock cells (Fig. 3A,B,C,D). The expression of CTLA4 was most significant in the only KDM4A knockout cells (Fig. 3A).IRF3 could also be transported to mitochondria, interacted with Bax and than induced apoptosis(22). In NOTCH1 and KDM4A double-knock cells, anti-apoptosis protein BCL-xl and BCL-2 were significantly decreased (Fig. 3A,B), that suggested that apoptosis may be induced. However, in the cell lines that SETD2 knocked out with the NOTCH1 knocked down or not, IRF3-p was not significant (Fig. 3C,D).
In CCK8 assay, the cell proliferation all accelerated in KDM4A or SETD2 knocked out cells. NOTCH1 inhibitor DAPT further promoted SETD2 knocked out cell proliferation(Fig. 3E). The apoptosis test was confirmed in the NOTCH1 and KDM4A double-knock cells, causing severe apoptosis (Fig. 3F). In clone formation assay, wild-type cells stopped forming obvious clones with DAPT administered. With DAPT administered in the KDM4A knockout cells caused severe apoptosis in cells. While in the SETD2 knockout cells, DAPT had not affected the clone formation (Fig. 3G).
3.4、 NOTCH1 significantly affected NAD+/NADH metabolic pathways and thus affected DNA repair.
The difference of DNA damage in cells were detected by immunofluorescence assay. DNA damage showed the opposite trend in KDM4A and SETD2 knockouts cells with the NOTCH1 inhibition respectively. A large amount of dsDNA appeared in the cytoplasm of KDM4A knockout cells, little dsDNA was found in SETD2 knockout cells or NOTCH1 inhibited cells. The mitochondrial staining showed that mitochondrial aggregation occurred in NOTCH1 knockdown cells (Fig. 4A). NAD+/NADH test also showed the opposite trend in the two type cells (Fig. 4B).
Inhibited NOTCH1 slightly lower the gamma γH2AX in nomal cells. But in KDM4A knocked out cells, Inhibited NOTCH1 Significantly increased γH2AX. Compared SETD2 knockout cells, Inhibited NOTCH1 slightly increased γH2AX. This suggested that NOTCH1 exacerbates cell damage in genomically unstable cells (Fig. 4C,D). While, ATM-p showed the opposite trend in the two different genomically unstable cells (Fig. 4E,F). The above experiments showed that NOTCH1 affected DNA repair was different in the different genomically unstable cells, might promote DNA repair in KDM4A-/- cells, but block DNA repair in SETD2-/- cells.
3.5、The NOTCH1 significantly affected the nucleotide synthesis pathway in KDM4A-/- cells.
Because NOTCH1 may have an effect on DNA by regulating metabolism, we examined the metabolomics in NOTCH1 knockdown cells. Metabolomics showed that NOTCH1 was enriched in the folate synthesis pathway (Fig. 5A). Folic acid was key to cellular carbon metabolism and nucleotide synthesis. D-Ribulose-1,5-bisphosphate and 2-Deoxyinosine-5-monophosphate that constituted the building blocks of DNA was abnormally changed (Fig. 5B). We also specifically analyzed the metabolic pathways of nucleotide synthesis, 5-methyl THF, dIMP, 5-methylcytidine, D-Ribulose-5-phosphate, D-Ribulose-1,5-bisphosphate, PRPP, uridine, dUMP were All showed a decrease trend, while 7,8-Dihydrofolate, N-Acetyl-L-methionine showed a rise trend (Fig. 5C). It illustrated that NOTCH1 inhibition interfered with folate metabolism and nucleotide synthesis.
One carbon unit produced purine and thymidylate through the folic acid cycle. S-adenosylmethionine (SAM) was mainly derived from one-carbon metabolism, was a methyl donor, and was involved in histone and DNA methylation. Deoxyribonucleotides, they were the building blocks of DNA elongation during DNA repair. We mapped the metabolic pathway from folate to nucleotide synthesis, noticed the DHFR,PRPS1/2,PRPS1L1,PRPS1/3,PPAT were rate-limiting enzymes for nucleotide synthesis (Fig. 5D). The mRNA changed of key rate-limiting enzymes were detected by QPCR assay and found them changed in cells under different conditions and showed a consistent change rule. These rate-limiting enzymes were all significantly increased in KDM4A and NOTCH1 knockout cells, but in SETD2 and NOTCH1 knockout cells were not significant (Fig. 5E). The rapid increased of PRPS1/2 can also cause cell apoptosis(23). Cellular levels of deoxyribonucleotides varied greatly in response to cell proliferation, and an imbalance in a single dNTP content can affect DNA repair errors or mitochondrial DNA depletion(24). The cGAS/STING axis activation induced ATM-mediated Phosphorylation of PRPS1/2 T228 to response to DNA damage(25). This suggested that cGAS/STING axis may coordinate with NOTCH1 to regulate rate-limiting enzymes.
3.6、The synergistic action of NOTCH1 and TBK1 regulated the changes of rate-limiting enzymes in nucleotide synthesis
TBK1 was a TRAF-associated NF-kB activator(TANK)Binding kinase 1 and the core kinase of the antiviral innate immune response, and its overactivation can lead to inflammation or tissue damage, so its activity is strictly regulated. TBK1 could be activated by a variety of factors, such as pathogen-associated molecular patterns (PAMP), inflammatory cytokines, and oncogenic kinases. TBK1 mainly mediated IRF3/7 activation and NF-kB signaling, and can also activate STAT3 and STAT6 to regulate inflammatory cytokine production and innate immune activation(26). We found that NOTCH1 knockdown caused significant upregulation of TBK1-p protein, even in KDM4A or SETD2 knockout cells (Fig. 6A,B). This indicated that the NOTCH1 pathway may directly regulate the activation of t TBK1-p.
Ionizing radiation induced to activate TBK1 and phosphorylated PRPS1/2, Further promoted deoxyribonucleotide synthesis, DNA repair and cell viability(25). Elevated TBK1-p was not the only factor for the expression of Nucleotide rate-limiting enzyme. While, according to (Fig. 4E,F), ATM-p was also significantly activated in the NOTCH1 and KDM4A double-knock cells, this suggests that the stimulation of TBK1 on deoxynucleotide rate-limiting enzymes depends on cGAS/STING signal and ATM-p activation(Fig. 5E). This is similar to the reference file that TBK1 need to depend on cGAS/STING axis and ATM-p to phosphorylate PRPS1/2(25).
Immunofluorescence of TBK1-p showed that most TBK1-p was in the cytoplasm. When the cell inhibited NOTCH1, part of TBK1-p was more inclined to enter the nucleus. In KDM4A-knocked out cell lines, TBK1-p was remarkably concentrated in the nucleus (Fig. 6C). Analysis of the data from the database found a significant correlation between TBK1 and PRPS1/2 in lung cancer (Fig. 6D) .
NF-kB/RelA activation was important for the complete induction of type I IFN, RelA was required for virus-induced type I IFN production. Activated IKKβwas required for NF-kB and TBK1-IRF3 activation. TBK1 was also essential for the full activation of IKKβ(27). p50-p65 was a dimer in NF-kB activation that increases cytokine expression(28).We analyzed changes in P65 and P65-P expression, P65 expression showed a significant gradient reduction in the conditioned cells, while P65-P was not significantly activated (Fig. 6E,F). The inflammatory factor NF-kB/RelA was reduced in the regulated cells.
In order to further explored the interaction of TBK1 and NOTCH1 on the activation of PRPS1/2 enzyme, We used an inhibitor of TBK1(GSK8612) to detect changes in the expression of rate-limiting enzymes for nucleotide synthesis. Interestingly, single inhibition of TBK1 in wild-type cell lines also caused significant increases in rate-limiting enzymes for nucleotide synthesis. This indicated that inhibition of TBK1 itself had the function of affecting the rate-limiting enzyme of nucleotide synthesis. Single activation of TBK1 phosphorylation could not significantly affect the rate-limiting enzyme expression, but inhibition of TBK1 expression could significantly affect the rate-limiting enzyme expression. Interestingly, after TBK1 inhibition, inhibition of NOTCH1 again reduced the elevated rate-limiting enzyme (Fig. 6G). The same effect was observed in KDM4A-/- cells (Fig. 6H). This also indicated NOTCH1 and TBK1 synergistically regulated the changes of rate-limiting enzymes in nucleotide synthesis.
3.7、TBK1 activated ATM-p depend on cGAS/STING axis, while the double deletion of NOTCH1 and TBK would cause increased DNA damage.
Inhibition of TBK1 alone did not significantly increaseγH2AX, but dual inhibition of TBK1 and NOTCH1 did significantly increasedγH2AX. These results indicated that although the dual inhibition of TBK1 and NOTCH1 regulated the expression of PRPS1/2, it also increased DNA damage (Fig. 7A,B,C) .
Through immunofluorescence, we observed the cell localization and fluorescence intensity changes of IRF3-p, and the fluorescence intensity changes of IRF3-P were consistent with western blot. IRF3-p seems to have been concentrated in the nucleus all along. When TBK1 inhibitor was re-applied to these types of cells, it was interesting to note that the fluorescence intensity of IRF3-p was enhanced(Fig. 7F), and western blot showed that IRF3-p was not completely inhibited after TBK1 inhibitor(Fig. 7D,E). This suggests that TBK1 was not the only factor contributing to IRF3-p. The activation of IRF3-p may also be caused by DNA damage. After the double knockdown of KDM4A and NOTCH1, the addition of TBK1 inhibitor resulted in the decrease of rate-limiting enzymes for nucleotide synthesis. Therefore, the sharp increase of rate-limiting enzymes expression were not the reason cause of IRF3-p.
ATM-p andγH2AX were all significantly increased in cells with double knockdown KDM4A and NOTCH1(Fig. 7G). In order to observe the effect of TBK1 on ATM-p, after adding TBK1 inhibitor to cells, ATM-p decreased significantly only in KDM4A and KDM4A, NOTCH1 double-knocked cells (Fig. 7H,I). The activation of ATM-p was dependent on the activation of TBK1 in the cGAS/STING pathway. We used ATM inhibitors(KU-55933 )to explore whether the inhibition of ATM-P could also affect the expression of TBK1-p. Western Blot experiment showed that ATM inhibition did not significantly affect the activation of TBK1, and the expression trend of TBK1 was consistent with that before the addition of inhibitor(Fig. 7J,K,M). These results indicated that DNA damage and repair might not directly stimulate the activation of TBK1-p.
