Establishment of two oxaliplatin-resistant gallbladder cancer cell lines and comprehensive analysis of dysregulated genes.

Acquired resistance to chemotherapeutic drugs in gallbladder cancer (GBC) results in therapy failure. This study is aimed to establish oxaliplatin (OXA)-resistant GBC cell lines and uncover their gene expression profiles. First, two OXA-resistant GBC cell lines (GBC-SD/OXA and SGC996/OXA) were established by gradually increasing the drug concentration, and the resistance index was 4-5. The two resistant cell lines showed slower proliferation and higher stemness, colony formation, and migration abilities. Epithelial mesenchymal transformation and increased levels of P-glycoprotein were also detected. Next RNA-sequence analysis identified 4,675 dysregulated genes (DGs) in resistant cells, and most of the 12 randomly selected DGs were verified to be consistent with the sequence results. Kyoto Encyclopedia of Genes and Genomes analysis indicated that several DGs were involved in resistance- and phenotype-related pathways, of which the activations of PD-L1 and ERK1/2 were both verified in resistant cell lines. In conclusion, this study is the first to report the gene expression profile of OXA-resistant GBC cells and provides a useful database for target development.

ALK inhibitors downregulate the expression of death receptor 4 in ALK-mutant lung cancer cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression.

The successful treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring chromosomal rearrangements of anaplastic lymphoma kinase (ALK) with ALK tyrosine kinase inhibitors (ALK-TKIs) represents a promising targeted therapy. As a result, various ALK-TKIs have been rapidly developed, some of which are approved while some are being tested in clinical trials. Death receptor 4 (DR4; also called TNFRSF10A or TRAIL-R1) is a cell surface protein, which functions as a pro-apoptotic protein that transduces TRAIL death signaling to trigger apoptosis. DR4 expression is positively regulated by MEK/ERK signaling and thus can be downregulated by MEK/ERK inhibition. This study thus focused on determining the effects of AKL-TKIs on DR4 expression and the underlying mechanisms. Three tested ALK-TKIs including APG-2449, brigatinib and alectinib effectively and preferentially inhibited Akt/mTOR as well as MEK/ERK signaling and decreased cell survival in ALK-mutant (ALKm) NSCLC cells with induction of apoptosis. This was also true for DR4 downregulation, which occurred even at 2 h post treatment. These ALK-TKIs did not affect DR4 protein stability, rather decreased DR4 mRNA expression. In parallel, they promoted degradation and reduced the levels of Fra-1 and c-Jun, two critical components of AP-1, and suppressed AP-1 (Fra-1/c-Jun)-dependent transcription/expression of DR4. Hence, it appears that ALK-TKIs downregulate DR4 expression in ALKm NSCLC cells via facilitating Fra-1 and c-Jun degradation and subsequent AP-1 suppression. Our findings thus warrant further investigation of the biological significance of DR4 downregulation in ALK-targeted cancer therapy.

CPSF6-mediated XBP1 3'UTR shortening attenuates cisplatin-induced ER stress and elevates chemo-resistance in lung adenocarcinoma.

Alternative polyadenylation (APA) is a widespread mechanism generating RNA molecules with alternative 3' ends. Herein, we discovered that TargetScan includes a novel XBP1 transcript with a longer 3' untranslated region (UTR) (XBP1-UL) than that included in NCBI. XBP1-UL exhibited a lowered level in blood samples from lung adenocarcinoma (LUAD) patients and in those after DDP treatment. Consistently, XBP1-UL was reduced in A549 cells compared to normal BEAS-2B cells, as well as in DDP-treated/resistant A549 cells relative to controls. Moreover, due to decreased usage of the distal polyadenylation site (PAS) in 3'UTR, XBP1-UL level was lowered in A549 cells and decreased further in DDP-resistant A549 (A549/DDP) cells. Importantly, use of the distal PAS (dPAS) and XBP1-UL level were gradually reduced in A549 cells under increasing concentrations of DDP, which was attributed to DDP-induced endoplasmic reticulum (ER) stress. Furthermore, XBP1 transcripts with shorter 3'UTR (XBP1-US) were more stable and presented stronger potentiation on DDP resistance. The choice of proximal PAS (pPAS) was attributed to CPSF6 elevation, which was caused by BRCA1-distrupted R-loop accumulation in CPSF6 5'end. DDP-induced nuclear LINC00221 also facilitated CPSF6-induced pPAS choice in the pre-XBP1 3'end. Finally, we found that unlike the unspliced XBP1 protein (XBP1-u), the spliced form XBP1-s retarded p53 degradation to facilitate DNA damage repair of LUAD cells. The current study provides new insights into tumor progression and DDP resistance in LUAD, which may contribute to improved LUAD treatment.

Resistance mechanisms to osimertinib and emerging therapeutic strategies in nonsmall cell lung cancer.

PURPOSE OF REVIEW: This review aims to introduce the resistance mechanisms to osimertinib, discuss the therapeutic strategies, and make clinical updates in overcoming resistance to osimertinib. RECENT FINDINGS: Osimertinib has shown favorable efficacy on second-line and first-line treatments in EGFR-mutant advanced nonsmall cell lung cancer (NSCLC). However, the presence of primary and acquired resistance to osimertinib restricts its clinical benefits. The primary resistance mainly consists of BIM deletion polymorphism and EGFR exon 20 insertions. Meanwhile, the heterogeneous mechanisms of acquired resistance include EGFR-dependent (on-target) and EGFR-independent (off-target) mechanisms. EGFR C797S mutation, MET amplification, HER2 amplification, and small cell lung cancer transformation were identified as frequent resistance mechanisms. Recently, more novel mechanisms, including rare EGFR point mutations and oncogenic fusions, were reported. With the results of completed and on-going clinical trials, the emerging therapeutic strategies of postosimertinib progression are summarized. SUMMARY: The resistance mechanisms to osimertinib are heterogeneous and gradually perfected. The combination of osimertinib with bypass targeted therapy and other therapeutic approaches emerge as promising strategies.

Genomic profiles and their associations with TMB, PD-L1 expression, and immune cell infiltration landscapes in synchronous multiple primary lung cancers.

BACKGROUND: Diagnosing and treating patients with multiple primary lung cancers (MPLCs) bring challenges to the clinic, and the preliminary evidence has revealed unsatisfying outcomes after targeted therapy and immunotherapy. Therefore, we surveyed genomic profiles of MPLCs and their possible associations with tumor mutation burden (TMB), programmed death-ligand 1 (PD-L1), and the immune cell infiltration landscape. MATERIALS AND METHODS: A total of 112 patients with MPLCs with surgically resected 294 tumors were eligible, and 255 tumors were sequenced using a 1021-gene panel. Immunohistochemistry staining was performed to evaluate the levels of PD-L1 and the density of CD3+/CD8+ tumor-infiltrating lymphocytes (TILs), and CD68+/CD163+ tumor-associated macrophages (TAMs) at the central tumor and invasive margin, and immunotypes were generated based on those variables. RESULTS: MPLCs often occur simultaneously in non-smoker women younger than 60 years and manifest as ground-glass opacities, adenocarcinoma, and stage I lung lesions. The most frequently mutated genes in the 255 tumors were EGFR (56%), ERBB2 (12%), TP53 (12%), BRAF (11%), RBM10 (11%), and KRAS (9%). We found 87 (77.7%) patients with diverse genomic profiles, and 61 (54.5%) who shared at least one putative driver gene between different tumors presented more aggressive tumors. The median TMB was 1.92 mutations/Mb, and high-TMB (≥3) lesions often harbored EGFRL858R/KRASG12C/RBM10/TP53/LRP1B mutations or wild-type ERBB2. Only 8.1% of patients and 3.9% of lesions were positive for PD-L1 on tumor cells, and this positivity was more frequent in LRP1B/TP53-mutant tumors. EGFRL858R/RBM10/TP53 mutations were positively associated with specific immune cells and an inflamed immunotype, but ERBB2 mutations were negatively correlated. TMB, CD3+TILs, and CD68+/CD163+ TAMs presented with significant heterogeneity among paired tumors (all kappa <0.2), but PD-L1 and CD8 +TILs were more uniformly present in tumor pairs. CONCLUSION: MPLCs are driven by different molecular events and often exhibit low TMB, low PD-L1, and a heterogeneous immune infiltration landscape. Specific genomic profiles are associated with TMB and the tumor immune microenvironmental landscape in MPLCs. Our findings can help to guide MPLCs diagnoses and to identify patient populations that may benefit from immunotherapy and targeted therapy.

Induction of SREBP1 degradation coupled with suppression of SREBP1-mediated lipogenesis impacts the response of EGFR mutant NSCLC cells to osimertinib.

Emergence of acquired resistance to osimertinib (AZD9291), the first-approved third-generation EGFR inhibitor that selectively and irreversibly inhibits the activating EGFR mutations and the resistant T790M mutation, is a giant and urgent clinical challenge. Fully understanding the biology underlying the response of EGFR mutant non-small cell lung cancer (NSCLC) to osimertinib is the foundation for development of mechanism-driven strategies to overcome acquired resistance to osimertinib or other third-generation EGFR inhibitors. This study focused on tackling this important issue by elucidating the critical role of sterol regulatory element-binding protein 1 (SREBP1) degradation in conferring the response of EGFR mutant NSCLC cells to osimertinib and by validating the strategy via directly targeting SREBP1 for overcoming osimertinib acquired resistance. Osimertinib facilitated degradation of the mature form of SREBP1 (mSREBP1) in a GSK3/FBXW7-dependent manner and reduced protein levels of its regulated genes in EGFR-mutant NSCLC cells/tumors accompanied with suppression of lipogenesis. Once resistant, EGFR-mutant NSCLC cell lines possessed elevated levels of mSREBP1, which were resistant to osimertinib modulation. Both genetic and pharmacological inhibition of SREBP1 sensitized osimertinib-resistant cells and tumors to osimertinib primarily through enhancing Bim-dependent induction of apoptosis, whereas enforced expression of ectopic SREBP1 in sensitive EGFR-mutant NSCLC cells compromised osimertinib's cell-killing effects. Collectively, we have demonstrated a novel connection between osimertinib and SREBP1 degradation and its impact on the response of EGFR mutant NSCLC cells to osimertinib and suggested an effective strategy for overcoming acquired resistance to osimertinib, and possibly other EGFR inhibitors, via targeting SREBP1.

Inhibition of MEK5/ERK5 signaling overcomes acquired resistance to the third generation EGFR inhibitor, osimertinib, via enhancing Bim-dependent apoptosis.

The promising therapeutic efficacy of the third generation EGFR inhibitor, osimertinib (AZD9291), for the treatment of patients with EGFR-mutant non-small cell lung cancer (NSCLC) has been demonstrated in the clinic both as first-line and second line therapy. However, inevitable acquired resistance limits its long-term benefit to patients and is thus a significant clinical challenge. The current study focuses on studying the potential role of targeting MEK5-ERK5 signaling in overcoming acquired resistance to osimertinib. Osimertinib and other third generation EGFR inhibitors exerted a rapid and sustained suppressive effect on ERK5 phosphorylation primarily in EGFR-mutant NSCLC cell lines and lost this activity in some osimertinib-resistant cell lines. Osimertinib combined with either ERK5 or MEK5 inhibitors synergistically decreased the survival of osimertinib-resistant cell lines with enhanced induction of apoptosis primarily via augmenting Bim expression. Moreover, the combination effectively inhibited the growth of osimertinib-resistant xenografts in vivo. Together, these findings suggest the potential role of MEK5-ERK5 signaling in modulating development of acquired resistance to osimertinib and value of targeting this signaling as a potential strategy in overcoming acquired resistance to osimertinib and possibly other third generation EGFR inhibitors.

Nanoparticles for co-delivery of osimertinib and selumetinib to overcome osimertinib-acquired resistance in non-small cell lung cancer.

Osimertinib (OSI) is the first FDA-approved third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). It can be used for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutation and for patients who are resistant to first-generation EGFR TKIs due to T790M resistance mutation. However, patients treated with OSI ultimately develop acquired resistance, which prevents its long-term benefit for patients. Therefore, the development of effective strategies to overcome OSI resistance will address a significant clinical challenge and benefit patients by prolonging their survival time. Our previous studies indicated that combination therapy was a promising strategy for overcoming OSI resistance. In this study, we developed nanoparticle (NP) formulations for co-delivery of osimertinib (OSI) and selumetinib (SEL) to treat OSI-resistant NSCLC effectively. We conjugated SEL with PEG through a reactive oxygen species (ROS)-responsive linker to generate polyethylene glycol (PEG)-SEL conjugate prodrug (PEG-S-SEL). Due to the amphiphilic nature of PEG-S-SEL, it can self-assemble in an aqueous solution to form micelle NP and serve as a delivery carrier for OSI. The ROS-responsive linker can facilitate the release of drugs in the tumor microenvironment with elevated ROS levels. OSI and SEL combination NP can overcome OSI resistance by simultaneously inhibiting both EGFR and mitogen-activated protein kinase (MEK), thus effectively inducing apoptosis in OSI-resistant NSCLC cells and inhibiting OSI-resistant tumors in vivo. In conclusion, the OSI+SEL NP combination therapy showed promising anticancer efficacy and demonstrated potential for treating NSCLC patients with OSI acquired resistance. STATEMENT OF SIGNIFICANCE: Osimertinib (OSI) is the first FDA-approved third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. It has been successfully used for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutation. However, patients treated with OSI ultimately develop acquired resistance. This study developed OSI and selumetinib (SEL) co-delivering nanoparticles to overcome OSI-acquired resistance in NSCLC. PEG-SEL conjugate functions as reactive oxygen species (ROS)-responsive prodrug and forms micelle nanoparticles through self-assembly to deliver OSI. The combination NP can simultaneously inhibit EGFR and mitogen-activated protein kinase (MEK), thus effectively inducing apoptosis in OSI-resistant NSCLC cells. In summary, the OSI and SEL nanoparticle combination therapy showed promising anticancer efficacy and demonstrated potential for treating NSCLC patients with OSI acquired resistance.

Managing Acquired Resistance to Third-Generation EGFR Tyrosine Kinase Inhibitors Through Co-Targeting MEK/ERK Signaling.

Although epidermal growth factor receptor (EGFR)-targeted therapy has improved clinical outcomes of patients with advanced non-small-cell lung cancer (NSCLC) carrying activating EGFR mutations, the development of acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs), including the promising third-generation ones, results in disease progression and has become an unavoidable problem that limits patient long-term benefit. The third-generation EGFR-TKIs, osimertinib and almonertinib, are now approved for the treatment of advanced NSCLC patients harboring activating EGFR mutations (first-line) and/or the resistant T790M mutation (second-line). Clinically, appropriate management of acquired resistance to third-generation EGFR-TKIs will substantially improve their long-term efficacy against EGFR-mutant NSCLC. Recent preclinical and clinical studies suggest that activation of the Ras/Raf/MEK/ERK signaling pathway may be an important resistance mechanism and accordingly co-targeting this pathway effectively overcomes and abrogates acquired resistance to third-generation EGFR-TKIs. This review focuses on discussing the scientific rationale for and potential of co-targeting MEK/ERK signaling in delaying and overcoming acquired resistance to third-generation EGFR-TKIs, particularly osimertinib.

The novel MET inhibitor, HQP8361, possesses single agent activity and enhances therapeutic efficacy of AZD9291 (osimertinib) against AZD9291-resistant NSCLC cells with activated MET.

HQP8361 (MK8033) is a novel and selective MET kinase inhibitor that has completed a phase I clinical trial. AZD9291 (osimertinib) represents the first-approved third generation EGFR-tyrosine kinase inhibitor (EGFR-TKI) for the treatment of non-small cell lung cancer (NSCLC) with activating EGFR mutations and resistant T790M mutation, but faces the giant challenge of acquired resistance developed in patients in the clinic. The current study focuses on determining the activity and mechanism of action of HQP8361 as a single agent and in combination with AZD9291 against human NSCLC cells, particularly those with acquired resistance to AZD9291. The majority of human NSCLC cell lines tested had very low levels of MET and p-MET and were insensitive to HQP8361. However, AZD9291-resistant (AR) cell lines with high levels of MET and p-MET responded to HQP8361 single agent and particularly to the combination of HQP8361 and AZD9291. The HQP8361 and AZD9291 combination synergistically decreased the survival of these HCC827/AR cell lines with enhanced induction of apoptosis that involved alteration of Bim and Mcl-1 levels via modulating their degradation. Moreover, the combination also very effectively inhibited the growth of HCC827/AR xenografts in nude mice. These preclinical findings support the potential of HQP8361 in the treatment of NSCLCs with MET amplification or highly activated MET and, when combined with AZD9291, in overcoming acquired resistance to EGFR-TKIs due to MET amplification.

Submucosal tunneling and endoscopic resection of submucosal tumors at the esophagogastric junction.

AIM: To evaluate the safety and efficacy of submucosal tunneling and endoscopic resection (STER) for treating submucosal tumors (SMTs). METHODS: Between August 2012 and October 2013, 21 patients with SMTs originating from the muscularis propria (MP) layer at the esophagogastric junction were treated by STER of their tumors. Key steps of the procedure include: (1) mucosal incision: a 2-cm longitudinal mucosal incision was made 5 cm proximal to the tumor; (2) submucosal tunneling: a submucosal tunnel was created 5 cm proximal to and 1 to 2 cm distal to the tumor; (3) tumor resection: the SMT was resected under direct endoscopic viewing; (4) hemostasis: while finishing the tumor resection, careful hemostasis of the MP defect and the tunnel was performed; and (5) mucosal closure: the mucosal incision site was closed by using hemostatic clips. During the operation, equipment used included a cap-fitted endoscope, an insulated-tip knife, a hook knife, hemostatic forceps, an injection needle, a snare, an endoclip, and a high-frequency generator. Carbon dioxide (CO2) insufflation was achieved by using a CO2 insufflator. RESULTS: The median age of the patients was 46.2 years (range, 35-59 years), and the majority were male (18 male vs 3 female). Complete resection rate was 100% (21/21). Eighteen lesions were resected en bloc. Mean tumor size was 23 mm (range, 10-40 mm), and mean procedure time was 62.9 min (range, 45-90 min). Pathological diagnosis of these tumors included leiomyoma (15 out of 21) and gastrointestinal stromal tumor (6 out of 21). Full-thickness MP resection was performed in 9 of 21 patients (42.9%), with mediastinal and subcutaneous emphysema occurring in all nine. At the completion of the procedure, all patients received closure of the incision with hemoclips. One patient required percutaneous drainage. The remaining 20 patients required no further endoscopic or surgical intervention. There were no incidents of massive or delayed bleeding. The median follow-up period after the procedure was 6 mo (range, 2-14 mo). During follow-up, no patients were found to have residual or recurrent tumor or esophageal stricture. CONCLUSION: STER is safe, effective and feasible, which provides accurate histopathologic evaluation and curative treatment for SMTs originating from the MP layer at the esophagogastric junction.