Hypoxia promotes an inflammatory phenotype of fibroblasts in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive fibroinflammatory stroma and often experiences conditions of insufficient oxygen availability or hypoxia. Cancer-associated fibroblasts (CAF) are a predominant and heterogeneous population of stromal cells within the pancreatic tumor microenvironment. Here, we uncover a previously unrecognized role for hypoxia in driving an inflammatory phenotype in PDAC CAFs. We identify hypoxia as a strong inducer of tumor IL1ɑ expression, which is required for inflammatory CAF (iCAF) formation. Notably, iCAFs preferentially reside in hypoxic regions of PDAC. Our data implicate hypoxia as a critical regulator of CAF heterogeneity in PDAC.

Acquired Resistance to Third-Generation EGFR Tyrosine Kinase Inhibitors in Patients With De Novo EGFRT790M-Mutant NSCLC.

INTRODUCTION: EGFRT790M mostly exists subclonally and is acquired as the most common mechanism of resistance to EGFR tyrosine kinase inhibitors (TKIs). Nevertheless, because de novo EGFRT790M-mutant NSCLC is rare, little is known on acquired resistance mechanisms to third-generation EGFR TKIs. METHODS: Acquired resistance mechanisms were analyzed using tumor and plasma samples before and after third-generation EGFR TKI treatment in four patients with de novo EGFRT790M-mutant NSCLC. Genetic alterations were analyzed by whole-exome sequencing, targeted sequencing, fluorescence in situ hybridization, and droplet digital PCR. MTORL1433S, confirmed for oncogenicity using the Ba/F3 system, was reproduced in H1975 cell lines using CRISPR/Cas9-RNP. RESULTS: Of seven patients with NSCLC with de novo EGFRT790M/L858R mutation, four (LC1-4) who received third-generation EGFR TKIs acquired resistance after achieving a partial response (median = 27 mo, range: 17-48 mo). Novel MTORL1433S and EGFRC797S/L798I mutations in cis, MET amplification, and EGFRC797S mutation were identified as acquired resistance mechanisms to third-generation EGFR TKIs. The MTORL1433S mutation was oncogenic in Ba/F3 models and revealed resistance to osimertinib through AKT signaling activation in NCI-H1975 cells harboring the MTORL1433S mutation edited by CRISPR/Cas9 (half-maximal inhibitory concentration, 800 ± 67 nM). Osimertinib in combination with mTOR inhibitors abrogated acquired resistance to osimertinib. CONCLUSIONS: Activation of bypass pathways and the EGFRC797S or EGFRC797S/L798I mutation were identified as acquired resistance mechanisms to third-generation EGFR TKIs in patients with NSCLC with de novo EGFRT790M mutation. In addition, MTORL1433S- and EGFRL858R/T790M-mutant NSCLC cells were sensitive to osimertinib plus mTOR inhibitors.

Preclinical Modeling of Osimertinib for NSCLC With EGFR Exon 20 Insertion Mutations.

INTRODUCTION: NSCLC with EGFR exon 20 insertion mutations is the third most common type of EGFR-mutant NSCLC and is resistant to EGFR tyrosine kinase inhibitors (TKIs). This study was conducted to evaluate the efficacies of first- to third-generation EGFR TKIs against NSCLC cells harboring EGFR exon 20 insertion mutations. METHODS: We developed seven EGFR exon 20 insertion-mutant Ba/F3 models and one patient-derived NSCLC (SNU-3173) of subtypes A763insFQEA, V769insASV, D770insSVD, D770insNPG, P772insPR, H773insH, H773insNPH, and H773insAH. Cell viability assays, immunoblotting, and N-ethyl-N-nitrosourea mutagenesis screenings were performed. EGFR exon 20 insertion-mutant structures and couplings with osimertinib, a third-generation EGFR TKI, were modeled and compared. RESULTS: EGFR exon 20 insertion-mutant NSCLC cells, excluding EGFR A763insFQEA, were resistant to first-generation EGFR TKIs (concentration that inhibits 50% [IC50], 1.1 ± 0.067 to 5.4 ± 0.115 µM). Mutants were sensitive to second-generation EGFR TKIs (IC50, 0.02 ± 0.0002 to 161.8 ± 18.7nM), except EGFR H773insH (IC50, 46.3 ± 8.0 to 352.5 ± 22.7nM). The IC50 ratios for mutant to wild-type cells were higher than those for third-generation EGFR TKIs. Third-generation EGFR TKI osimertinib was highly potent against EGFR exon 20 insertion-mutant cells (IC50, 14.7-62.7 nM), including EGFR H773insH, and spared wild-type EGFR cells. N-ethyl-N-nitrosourea mutagenesis screening of EGFR exon 20 insertion-mutant Ba/F3 cells showed various second sites for EGFR mutations, mostly at exons 20 and 21, including E762K, P794S, and G796D. In addition, osimertinib-resistant cells were established by stepwise exposure to osimertinib and harbored EGFR E762K mutation. CONCLUSIONS: Osimertinib is active against EGFR exon 20 insertion-mutant NSCLC and flexibly binds within drug-binding pockets in preclinical models.

Alterations in PD-L1 Expression Associated with Acquisition of Resistance to ALK Inhibitors in ALK-Rearranged Lung Cancer.

PURPOSE: The purpose of this study was to evaluate the relationships between the resistance of anaplastic lymphoma kinase (ALK)‒positive non-small cell lung cancer (NSCLC) to ALK inhibitors and the programmed cell death-1/programmed cell death-ligand 1 (PD-L1) pathway, we evaluated alterations in PD-L1 following acquisition of resistance to ALK inhibitors in ALK-positive lung cancer. MATERIALS AND METHODS: We established ALK inhibitor-resistant cell lines (H3122CR1, LR1, and CH1) by exposing the parental H3122 ALK-translocated NSCLC cell line to ALK inhibitors. Then, the double-resistant cell lines H3122CR1LR1 and CR1CH1 were developed by exposing the H3122CR1 to other ALK inhibitors. We compared the alterations in PD-L1 expression levels using western blotting, flow cytometry, and quantitative polymerase chain reaction. We also investigated gene expression using RNA sequencing. The expression of PD-L1 in the tumors from 26 ALK-positive metastatic NSCLC patients (11 ALK inhibitor-naïve and 15 ALK inhibitor-resistant patients) was assessed by immunohistochemistry and analyzed. RESULTS: PD-L1 was expressed at higher levels in ALK inhibitor-resistant cell lines than in the ALK inhibitor-naïve parental cell line at the total protein, surface protein, and mRNA levels. Furthermore, PD-L1 expression in the double-resistant cell lines was much higher than that in the single resistant cell lines. RNA sequencing demonstrated that expression of immune-related genes were largely involved in ALK inhibitor resistance. The mean value of the PD-L1 H-score was 6.5 pre-treatment and 35.0 post-treatment, and the fold difference was 5.42 (p=0.163). CONCLUSION: PD-L1 expression increased following acquisition of ALK inhibitor resistance in ALK-positive NSCLC cell lines and tumors.