The ATLANTIC trial reported that higher PD-L1 expression in tumors was involved in a higher objective response in patients with EGFR
/ALK
non-small cell lung cancer (NSCLC), indicating the ...possibility of anti-PD-1/PD-L1 therapy as a third-line (or later) treatment for advanced NSCLC. Therefore, the determination of status and regulatory mechanisms of PD-L1 in EGFR mutant NSCLC before and after acquired EGFR-TKIs resistance are meaningful.
The correlation among PD-L1, c-MET, and HGF was analyzed based on TCGA datasheets and paired NSCLC specimens before and after acquired EGFR-TKI resistance. EGFR-TKI resistant NSCLC cells with three well-known mechanisms, c-MET amplification, hepatocyte growth factor (HGF), and EGFR-T790M, were investigated to determinate PD-L1 expression status and immune escape ability. PD-L1-deleted EGFR-TKIs sensitive and resistant cells were used to evaluate the immune escape ability of tumors in mice xenograft models.
Positive correlations were found among PD-L1, c-MET, and HGF, based on TCGA datasheets and paired NSCLC specimens. Moreover, the above three resistant mechanisms increased PD-L1 expression and attenuated activation and cytotoxicity of lymphocytes in vitro and in vivo, and downregulation of PD-L1 partially restored the cytotoxicity of lymphocytes. Both MAPK and PI3K pathways were involved in the three types of resistance mechanism-induced PD-L1 overexpression, whereas the NF-kappa B pathway was only involved in T790M-induced PD-L1 expression.
HGF, MET-amplification, and EGFR-T790M upregulate PD-L1 expression in NSCLC and promote the immune escape of tumor cells through different mechanisms.
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Drug tolerance is the basis for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) including osimertinib, through mechanisms that still remain unclear. ...Here, we show that while AXL-low expressing EGFR mutated lung cancer (EGFRmut-LC) cells are more sensitive to osimertinib than AXL-high expressing EGFRmut-LC cells, a small population emerge osimertinib tolerance. The tolerance is mediated by the increased expression and phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), caused by the induction of its transcription factor FOXA1. IGF-1R maintains association with EGFR and adaptor proteins, including Gab1 and IRS1, in the presence of osimertinib and restores the survival signal. In AXL-low-expressing EGFRmut-LC cell-derived xenograft and patient-derived xenograft models, transient IGF-1R inhibition combined with continuous osimertinib treatment could eradicate tumors and prevent regrowth even after the cessation of osimertinib. These results indicate that optimal inhibition of tolerant signals combined with osimertinib may dramatically improve the outcome of EGFRmut-LC.
The
deletion polymorphism is associated with apoptosis resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI), such as gefitinib and erlotinib, in non-small cell lung cancer (NSCLC) harboring
...mutations. Here, we investigated whether the
deletion polymorphism contributes to resistance against osimertinib, a third-generation EGFR-TKI. In addition, we determined the efficacy of a histone deacetylase (HDAC) inhibitor, vorinostat, against this form of resistance and elucidated the underlying mechanism.
We used
-mutated NSCLC cell lines, which were either heterozygous or homozygous for the
deletion polymorphism, to evaluate the effect of osimertinib
and
Protein expression was examined by Western blotting. Alternative splicing of
mRNA was analyzed by RT-PCR.
-mutated NSCLC cell lines with the
deletion polymorphism exhibited apoptosis resistance to osimertinib in a polymorphism dosage-dependent manner, and this resistance was overcome by combined use with vorinostat. Experiments with homozygous
deletion-positive cells revealed that vorinostat affected the alternative splicing of
mRNA in the deletion allele, increased the expression of active BIM protein, and thereby induced apoptosis in osimertinib-treated cells. These effects were mediated predominantly by HDAC3 inhibition. In xenograft models, combined use of vorinostat with osimertinib could regress tumors in
-mutated NSCLC cells homozygous for the
deletion polymorphism. Moreover, this combination could induce apoptosis even when tumor cells acquired
-T790M mutations.
These findings indicate the importance of developing HDAC3-selective inhibitors, and their combined use with osimertinib, for treating
-mutated lung cancers carrying the
deletion polymorphism.
.
Leptomeningeal carcinomatosis (LMC) occurs frequently in non–small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations and is associated with acquired resistance to ...EGFR tyrosine kinase inhibitors (EGFR‐TKIs). However, the mechanism by which LMC acquires resistance to osimertinib, a third‐generation EGFR‐TKI, is unclear. In this study, we elucidated the resistance mechanism and searched for a novel therapeutic strategy. We induced osimertinib resistance in a mouse model of LMC using an EGFR‐mutant NSCLC cell line (PC9) via continuous oral osimertinib treatment and administration of established resistant cells and examined the resistance mechanism using next‐generation sequencing. We detected the Kirsten rat sarcoma (KRAS)‐G12V mutation in resistant cells, which retained the EGFR exon 19 deletion. Experiments involving KRAS knockdown in resistant cells and KRAS‐G12V overexpression in parental cells revealed the involvement of KRAS‐G12V in osimertinib resistance. Cotreatment with trametinib (a MEK inhibitor) and osimertinib resensitized the cells to osimertinib. Furthermore, in the mouse model of LMC with resistant cells, combined osimertinib and trametinib treatment successfully controlled LMC progression. These findings suggest a potential novel therapy against KRAS‐G12V–harboring osimertinib‐resistant LMC in EGFR‐mutant NSCLC.
Kirsten rat sarcoma (KRAS)‐G12V mutation plays a key role in the development of osimertinib resistance in EGFR‐mutant non–small cell lung cancer (NSCLC). Leptomeningeal carcinomatosis progression can be controlled by combinatorial treatment with osimertinib and trametinib.
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A novel epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor, osimertinib, has marked efficacy in patients with EGFR‐mutant lung cancer. While epithelial‐mesenchymal transition (EMT) ...plays a role in the resistance to various targeted drugs, its involvement in EGFR‐inhibitor resistance remains largely unknown. Preclinical experiments with osimertinib‐resistant lung cancer cells showed that EMT was associated with decreased microRNA‐200c and increased ZEB1 expression. In several resistant clone cells, pretreatment with the histone deacetylase inhibitor quisinostat helped overcome the resistance by reverting EMT. Furthermore, drug screening from a library of 100 kinase inhibitors indicated that Glycogen synthase kinase‐3 (GSK‐3) inhibitors, such as LY2090314, markedly inhibited the growth and induced apoptosis of resistant cells, specifically those with a mesenchymal phenotype. These results suggest that GSK‐3 inhibition could be useful to circumvent EMT‐associated resistance to osimertinib in EGFR‐mutant lung cancer.
Summary scheme of the role of microRNA (miR)‐200c in the activation of the glycogen synthase kinase (GSK)‐3/AKT axis in osimertinib‐sensitive and osimertinib‐resistant cells. EMT, epithelial‐mesenchymal transition.
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Patient‐derived xenograft (PDX) models are a useful tool in cancer biology research. However, the number of lung cancer PDX is limited. In the present study, we successfully established 10 PDX, ...including three adenocarcinoma (AD), six squamous cell carcinoma (SQ) and one large cell carcinoma (LA), from 30 patients with non‐small cell lung cancer (NSCLC) (18 AD, 10 SQ, and 2 LA), mainly in SCID hairless outbred (SHO) mice (Crlj:SHO‐PrkdcscidHrhr). Histology of SQ, advanced clinical stage (III‐IV), status of lymph node metastasis (N2‐3), and maximum standardized uptake value ≥10 when evaluated using a delayed 18F‐fluoro‐2‐deoxy‐d‐glucose positron emission tomography (FDG‐PET) scan was associated with successful PDX establishment. Histological analyses showed that PDX had histology similar to that of patients’ surgically resected tumors (SRT), whereas components of the microenvironment were replaced with murine cells after several passages. Next‐generation sequencing analyses showed that after two to six passages, PDX preserved the majority of the somatic mutations and mRNA expressions of the corresponding SRT. Two out of three PDX with AD histology had epidermal growth factor receptor (EGFR) mutations (L858R or exon 19 deletion) and were sensitive to EGFR tyrosine kinase inhibitors (EGFR‐TKI), such as gefitinib and osimertinib. Furthermore, in one of the two PDX with an EGFR mutation, osimertinib resistance was induced that was associated with epithelial‐to‐mesenchymal transition. This study presented 10 serially transplantable PDX of NSCLC in SHO mice and showed the use of PDX with an EGFR mutation for analyses of EGFR‐TKI resistance.
We successfully established 10 PDX. Next‐generation sequencing analyses showed that after two to six passages, PDX preserved the majority of the somatic mutations and mRNA expressions of the corresponding SRT. Two out of three PDX with AD histology had EGFR mutations (L858R or exon 19 deletion) and were sensitive to EGFR tyrosine kinase inhibitors. Furthermore, in one of the two PDX with an EGFR mutation, osimertinib resistance was induced. This study presented 10 serially transplantable PDX of NSCLC in SHO mice and showed the use of PDX with an EGFR mutation for analyses of EGFR‐TKI resistance.
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Mutations in the
gene are detectable in approximately 40% of
-rearranged lung cancers resistant to ALK inhibitors. Although epithelial-to-mesenchymal transition (EMT) is a mechanism of resistance to ...various targeted drugs, its involvement in ALK inhibitor resistance is largely unknown. In this study, we report that both
-mutant L1196M and EMT were concomitantly detected in a single crizotinib-resistant lesion in a patient with
-rearranged lung cancer. Digital PCR analyses combined with microdissection after IHC staining for EMT markers revealed that
L1196M was predominantly detected in epithelial-type tumor cells, indicating that mesenchymal phenotype and
mutation can coexist as independent mechanisms underlying ALK inhibitor-resistant cancers. Preclinical experiments with crizotinib-resistant lung cancer cells showed that EMT associated with decreased expression of miR-200c and increased expression of ZEB1 caused cross-resistance to new-generation ALK inhibitors alectinib, ceritinib, and lorlatinib. Pretreatment with the histone deacetylase (HDAC) inhibitor quisinostat overcame this resistance by reverting EMT
and
. These findings indicate that HDAC inhibitor pretreatment followed by a new ALK inhibitor may be useful to circumvent resistance constituted by coexistence of resistance mutations and EMT in the heterogeneous tumor. SIGNIFICANCE: These findings show that dual inhibition of HDAC and ALK receptor tyrosine kinase activities provides a means to circumvent crizotinib resistance in lung cancer.
In
-rearranged non-small cell lung cancer (NSCLC), impacts of concomitant genetic alterations on targeted therapies with ALK-tyrosine kinase inhibitors (ALK-TKI) are not yet well understood. Here, we ...investigated genetic alterations related to ALK-TKI resistance using clinico-genomic data and explored effective therapies to overcome the resistance in preclinical models through the identification of underlying molecular mechanisms.
We used integrated clinical and next-generation sequencing data generated in a nationwide lung cancer genome screening project (LC-SCRUM-Japan).
-rearranged NSCLC cell lines expressing wild-type or mutant
were used to evaluate cellular apoptosis induced by ALK-TKIs.
In 90 patients with
-rearranged NSCLC who were treated with a selective ALK-TKI, alectinib,
comutated patients showed significantly worse progression-free survival (PFS) than
wild-type patients median PFS, 11.7 months (95% confidence interval, CI, 6.3-not reached, NR) vs. NR (23.6-NR);
= 0.0008; HR, 0.33 (95% CI, 0.17-0.65).
-rearranged NSCLC cell lines that lost p53 function were resistant to alectinib-induced apoptosis, but a proteasome inhibitior, ixazomib, markedly induced apoptosis in the alectinib-treated cells by increasing the expression of a proapoptotic protein, Noxa, which bound to an antiapoptotic protein, Mcl-1. In subcutaneous tumor models, combination of ixazomib and alectinib prominently induced tumor regression and apoptosis even though the tumors were generated from
-rearranged NSCLC cells with nonfunctional p53.
These clinical and preclinical results indicate concomitant
mutations reduce the efficacy of alectinib for
-rearranged NSCLC and the combined use of a proteasome inhibitor with alectinib is a promising therapy for
-rearranged/
-mutated NSCLC.
Leptomeningeal carcinomatosis occurs frequently in
-mutant lung cancer, and develops acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). This study aimed to clarify the mechanism of ...EGFR-TKI resistance in leptomeningeal carcinomatosis and seek for a novel therapeutic strategy. We examined
mutations, including the T790M gatekeeper mutation, in 32 re-biopsy specimens from 12 leptomeningeal carcinomatosis and 20 extracranial lesions of
-mutant lung cancer patients who became refractory to EGFR-TKI treatment. All the 32 specimens had the same baseline
mutations, but the T790M mutation was less frequent in leptomeningeal carcinomatosis specimens than in extracranial specimens (8% vs. 55%,
< 0.01). To study molecular mechanisms of acquired EGFR-TKI resistance in leptomeningeal carcinomatosis, we utilized our previously developed mouse model of leptomeningeal carcinomatosis with the
-mutant lung cancer cell line PC-9/ffluc cells, in which acquired resistance to gefitinib was induced by continuous oral treatment. Compared with subcutaneously inoculated gefitinib-resistant tumors, the T790M mutation was less frequent in leptomeningeal carcinomatosis that acquired resistance to gefitinib. PC-9/LMC-GR cells were established from the gefitinib-resistant leptomeningeal carcinomatosis model, and they were found to be intermediately resistant to gefitinib and osimertinib (third-generation EGFR-TKI). Although
-T790M was negative, gefitinib resistance of PC-9/LMC-GR cells was related to
copy number gain with MET activation. Moreover, combined use of EGFR-TKI and crizotinib, a MET inhibitor, dramatically regressed leptomeningeal carcinomatosis with acquired resistance to gefitinib or osimertinib. These findings suggest that combination therapy with MET inhibitors may be promising for controlling leptomeningeal carcinomatosis that acquires resistance to EGFR-TKIs.
.
Small‐cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers, and is characterized as extremely aggressive, often displaying rapid tumor growth and multiple organ metastases. In ...addition, the clinical outcome of SCLC patients is poor due to early relapse and acquired resistance to standard chemotherapy treatments. Hence, novel therapeutic strategies for the treatment of SCLC are urgently required. Accordingly, several molecular targeted therapies were evaluated in SCLC; however, they failed to improve the clinical outcome. The receptor tyrosine kinase MET is a receptor for hepatocyte growth factor (HGF), and aberrant activation of HGF/MET signaling is known as one of the crucial mechanisms enabling cancer progression and invasion. Here, we found that the HGF/MET signaling was aberrantly activated in chemoresistant or chemorelapsed SCLC cell lines (SBC‐5, DMS273, and DMS273‐G3H) by the secretion of HGF and/or MET copy number gain. A cell‐based in vitro assay revealed that HGF/MET inhibition, induced either by MET inhibitors (crizotinib and golvatinib), or by siRNA‐mediated knockdown of HGF or MET, constrained growth of chemoresistant SCLC cells through the inhibition of ERK and AKT signals. Furthermore, treatment with either crizotinib or golvatinib suppressed the systemic metastasis of SBC‐5 cell tumors in natural killer cell‐depleted SCID mice, predominantly through cell cycle arrest. These findings reveal the therapeutic potential of targeting the HGF/MET pathway for inhibition, to constrain tumor progression of SCLC cells showing aberrant activation of HGF/MET signaling. We suggest that it would be clinically valuable to further investigate HGF/MET‐mediated signaling in SCLC cells.
In the present study, we demonstrated that HGF/MET signaling was aberrantly activated in chemo‐resistant or chemo‐relapsed SCLC cell lines by the secretion of HGF and/or MET copy number gain. Furthermore, the MET inhibitors crizotinib and golvatinib were able to inhibit cell growth, and prevent systemic metastasis of SCLC cells in vivo, predominantly via cell cycle arrest. These findings indicate that inhibition of HGF/MET signaling may constrain tumor progression of SCLC cells exhibiting aberrant activation of the HGF/MET pathway.
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