Constitutively activated STAT3 plays a critical role in non-small cell lung carcinoma (NSCLC) progression by mediating proliferation and survival. STAT3 activation in normal cells is transient, making it an attractive target for NSCLC therapy. The therapeutic potential of blocking STAT3 in NSCLC was assessed utilizing a decoy approach by ligating a double-stranded 15-mer oligonucleotide that corresponds to the STAT3 response element of STAT3-target genes, to produce a cyclic STAT3 decoy (CS3D). The decoy was evaluated using NSCLC cells containing either wild-type EGFR (201T) or mutant EGFR with an additional EGFRi resistance mutation (H1975). These cells are resistant to EGFR inhibitors and require an alternate therapeutic approach. CS3D activity was compared with an inactive cyclic control oligonucleotide (CS3M) that differs by a single base pair, rendering it unable to bind to STAT3 protein. Transfection of 0.3 μmol/L of CS3D caused a 50% inhibition in proliferation in 201T and H1975 cells, relative to CS3M, and a 2-fold increase in apoptotic cells. Toxicity was minimal in normal cells. CS3D treatment caused a significant reduction of mRNA and protein expression of the STAT3 target gene c-Myc and inhibited colony formation by 70%. The active decoy decreased the nuclear pool of STAT3 compared with the mutant. In a xenograft model, treatments with CS3D (5 mg/kg) caused a potent 96.5% and 81.7% reduction in tumor growth in 201T ( < 0.007) and H1975 models ( < 0.0001), respectively, and reduced c-Myc and p-STAT3 proteins. Targeting STAT3 with the cyclic decoy could be an effective therapeutic strategy for NSCLC. .
Background Tumor models resistant to EGFR tyrosine kinase inhibitors or cisplatin express higher levels of the immune checkpoint molecule PD‐L1. We sought to determine whether PD‐L1 expression is elevated in head and neck squamous cell carcinoma (HNSCC) models of acquired cetuximab resistance and whether the expression is regulated by bromodomain and extraterminal domain (BET) proteins. Methods Expression of PD‐L1 was assessed in HNSCC cell line models of acquired cetuximab resistance. Proteolysis targeting chimera (PROTAC)‐ and RNAi‐mediated targeting were used to assess the role of BET proteins. Results Cetuximab‐resistant HNSCC cells expressed elevated PD‐L1 compared to cetuximab‐sensitive controls. Treatment with the BET inhibitor JQ1, the BET PROTAC MZ1, or RNAi‐mediated knockdown of BRD2 decreased PD‐L1 expression. Knockdown of BRD2 also reduced the elevated levels of PD‐L1 seen in a model of acquired cisplatin resistance. Conclusions PD‐L1 is significantly elevated in HNSCC models of acquired cetuximab and cisplatin resistance where BRD2 is the primary regulator.
The STAT3 pathway is frequently overactive in non-small cell lung cancer (NSCLC), an often fatal disease with known risk factors including tobacco and chemical exposures. Whether STAT3 can be downmodulated to delay or prevent development of lung cancer resulting from an environmental exposure has not been previously tested. A circular oligonucleotide STAT3 decoy (CS3D) was used to treat mice previously exposed to the tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. CS3D contains a double-stranded STAT3 DNA response element sequence and interrupts STAT3 signaling by binding to STAT3 dimers, rendering them unable to initiate transcription at native STAT3 DNA binding sites. An intermittent course of CS3D decreased the development of airway preneoplasias by 42% at 1 week posttreatment, reduced the progression of preneoplasia to adenomas by 54% at 8 weeks posttreatment, and reduced the size and number of resulting lung tumors by 49.7% and 29.5%, respectively, at 20 weeks posttreatment. No toxicity was detected. A mutant cyclic oligonucleotide with no STAT3 binding ability was used as a control. Chemopreventive effects were independent of the KRAS mutational status of the tumors. In lungs harvested during and after the treatment course with CS3D, airway preneoplasias had reduced STAT3 signaling. Chemopreventive effects were accompanied by decreased VEGFA expression, ablated IL6, COX-2, and p-NF-kB, and decreased pulmonary M2 macrophages and myeloid-derived suppressor cells. Thus, downmodulation of STAT3 activity using a decoy molecule both reduced oncogenic signaling in the airway epithelium and favored a lung microenvironment with reduced immunosuppression.
EGFR (Epidermal Growth Factor Receptor) is a major target for the treatment of Non-Small Cell Lung Cancer (NSCLC), but patients lacking EGFR mutation are insensitive to EGFR inhibition. Patients with EGFR mutations initially respond to EGFR tyrosine kinase inhibitors (TKIs), but eventually develop acquired resistance as a result of secondary mutations in the EGFR tyrosine kinase domain. Upregulation of Signal Transducer and Activator of Transcription (STAT3), a key oncogenic molecule in NSCLC that is downstream of EGFR signaling, contributes to intrinsic and acquired resistance to EGFR TKIs. In the clinic, agents targeting STAT3 have not been satisfactory to date. To address the limitations of available STAT3 inhibitors, we are currently testing a cyclic oligonucleotide molecule with a novel mechanism that acts as a STAT3 “decoy” (Cyclic STAT3 decoy, CS3D). CS3D mimics the DNA consensus sequence in the promoter region of STAT3-responsive genes, causing the binding of STAT3 dimers and preventing transcriptional regulation of STAT3-target genes. The effects of CS3D are compared to mutant cyclic STAT3 decoy (CS3M), which differs from CS3D by one base-pair. We have tested the ability of CS3D to produce anti-tumor effects in NSCLC cells that are EGFR WT (201T) and in EGFR mutant cells that also carry the T790M resistant mutation (H1975). Initial in vitro studies showed that CS3D caused a 50% inhibition in cell proliferation in 201T and H1975 relative to CS3M using MTS assays. Flow cytometry studies also demonstrated that CS3D caused a 2-fold increase in the percent of apoptotic cells as compared to CS3M. CS3D also caused a 2-fold reduction in expression of STAT3-target genes c-Myc, Bcl-xL and IL-6. Compared to CS3M, CS3D inhibited colony formation by 70%. Using an in vivo mouse xenograft model of 201T and H1975, CS3D caused a 96.5% reduction in tumor growth in 201T (P<0.007) compared to CS3M, while an 81.7% inhibition was seen in H1975 (P<.0001). Utilizing IHC, analysis of residual tumors also illustrated that CS3D induced more caspase3 cleavage relative to CS3M. Additionally, western blot analysis showed 70% reduction in c-Myc protein level in response to CS3D. These results suggest CS3D can be effective as a single therapeutic agent. Combining CS3D with EGFR inhibitors such as afatinib in vitro significantly suppressed cell viability by 85.2% and 80% in 201T and H1975 respectively, as compared to single treatment (CS3D alone reduced cell viability by 53%, and 51.5%, and afatinib alone reduced viability by 31.4% and 34.03% in 201T and H1975, respectively). The combination of CS3D and afatinib warrants further testing in vivo. These data suggest that CS3D alone or in combination with EGFR tyrosine kinase inhibitors produces anti-tumor effects in NSCLC with intrinsic and acquired resistance to EGFR TKIs. Supported by funding to the Masonic Cancer Center from the Minnesota 5th Order of Eagles. Citation Format: Christian Njatcha, Mariya Farooqui, Jennifer R. Grandis, Jill M. Siegfried. Targeting the EGFR/STAT3 axis in NSCLC with resistance to EGFR tyrosine kinase inhibitors using an oligonucleotide-based decoy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4101. doi:10.1158/1538-7445.AM2017-4101
Exposure to tobacco carcinogens such as NNK leads to mutations in genes like K-RAS in premalignant lesions, which progress to adenocarcinoma. Early events in carcinogenesis are also characterized by inflammation. Central to these processes, STAT3 activation has pleotropic pro-tumor effects on proliferation, angiogenesis, and immune evasion. We previously showed that a “decoy” cyclic STAT3 oligonucleotide (CS3D) produced robust antitumor effects in human lung adenocarcinoma models. CS3D mimics the STAT3 DNA response element and acts as a “decoy” to which STAT3 binds, blocking its transcriptional activity. Effects of CS3D were compared to an inactive mutant construct unable to bind to STAT3 (CS3M). Therapeutic effects in human lung cancer xenografts suggested that CS3D might also impair lung cancer development. To test this, mice were exposed to NNK (3mg/kg) for 4 weeks (wks) followed by a 1-wk washout phase to model ex-smokers. Mice were then treated 3 times per wk with CS3D or CS3M (5mg/kg, IV) for 8 wks. Tumor burden and size were then assessed at 8 and 20 wks after the end of treatment. To evaluate effect of CS3D on the development of tumors with K-RAS activating mutations (since STAT3 knockout has been reported to promote KRAS mutant lung cancer), we isolated DNA from tumors by laser capture microdissection and assessed the incidence of G12D activating mutations. CS3D caused a reduction in preneoplasias (P<0.001), tumor size (P<.05), and tumor number (P<.05) by 30%, 50%, and 40% respectively, compared to CS3M. Immunohistochemistry (IHC) showed a tumor microenvironment (TME) that is less primed for tumor progression. Markers of angiogenesis (VEGF) and cell cycle progression (MYC) were downregulated by CS3D (P<.05). Strikingly, IHC analysis at 20 wks after the final treatment revealed a substantial decrease in STAT3 and NF-kB activation (P<.05) suggesting a long-lasting effect of CS3D. However, we detected higher levels of IL-6 at 8 wks and 20 wks after the end of treatment which could indicate a rescuing feedback loop. IHC also showed a 2.6 fold increase in macrophage infiltration at 8 wks. Sequencing of DNA from residual tumors collected at 20 wks (n = 10) showed no significant difference in tumors with K-RAS mutation between CS3D (33.3%) and CS3M (27.3%), showing that pharmacological inhibition of STAT3 with CS3D doesn’t select for K-RAS mutant tumors. To examine immunomodulation, mice were exposed to 4 wks of NNK with a 1-wk washout phase and 1 wk of CS3D or CS3M treatment. Lungs were collected for flow cytometry analysis to define the immune cell profile. CS3D favored an antitumor immune response over an immunosuppressive TME by increasing the number of lung M1 macrophages while decreasing M2 macrophages and MDSCs. Ratio of M1 to M2 was 4:1 with CS3D and 1:3 with CS3M. This is the first evidence that CS3D has chemopreventive properties in lung cancer without promoting KRAS-driven tumors, and modulating the TME is involved in the preventive effect. Citation Format: Christian Njatcha, Jiayi Wang, Huiyu Li, Mariya Farooqui, Jill M. Siegfried. A novel STAT3 decoy inhibitor prevents lung cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3875.
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