TKI-resistant Non-small Cell Lung Cancer Research Articles

Customised design of antisense oligonucleotides targeting EGFR driver mutants for personalised treatment of non-small cell lung cancer

Tyrosine kinase inhibitors (TKIs) are currently the standard therapy for patients with non-small cell lung cancer (NSCLC) bearing mutations in epidermal growth factor receptor (EGFR). Unfortunately, drug-acquired resistance is inevitable due to the emergence of new mutations in EGFR. Moreover, the TKI treatment is associated with severe toxicities due to the unspecific inhibition of wild-type (WT) EGFR. Thus, treatment that is customised to an individual's genetic alterations in EGFR may offer greater therapeutic benefits for patients with NSCLC. In this study, we demonstrate a new therapeutic strategy utilising customised antisense oligonucleotides (ASOs) to selectively target activating mutations in the EGFR gene in an individualised manner that can overcome drug-resistant mutations. We use extracellular vesicles (EVs) as a vehicle to deliver ASOs to NSCLC cells. Specifically guided by the mutational profile identified in NSCLC patients, we have successfully developed ASOs that selectively inhibit point mutations in the EGFR gene, including L858R and T790M, while sparing the WT EGFR. Delivery of the EGFR-targeting ASOs by EVs significantly reduced tumour growth in xenograft models of EGFR-L858R/T790M-driven NSCLC. Importantly, we have also shown that EGFR-targeting ASOs exhibit more potent anti-cancer effect than TKIs in NSCLC with EGFR mutations, effectively suppressing a patient-derived TKI-resistant NSCLC tumour. Overall, by harnessing the specificity and efficacy of ASOs, we present an effective and adaptable therapeutic platform for NSCLC treatment. This study was funded by Singapore's Ministry of Health (NMRC/OFIRG/MOH-000643-00, OFIRG21nov-0068, NMRC/OFLCG/002-2018, OFYIRG22jul-0034), National Research Foundation (NRF-NRFI08-2022, NRF-CRP22-2019-0003, NRF-CRP23-2019-0004), A∗STAR, and Ministry of Education.

Branched-chain amino acid transaminase 1 confers EGFR-TKI resistance through epigenetic glycolytic activation

Third-generation EGFR tyrosine kinase inhibitors (TKIs), exemplified by osimertinib, have demonstrated promising clinical efficacy in the treatment of non-small cell lung cancer (NSCLC). Our previous work has identified ASK120067 as a novel third-generation EGFR TKI with remarkable antitumor effects that has undergone New Drug Application (NDA) submission in China. Despite substantial progress, acquired resistance to EGFR-TKIs remains a significant challenge, impeding the long-term effectiveness of therapeutic approaches. In this study, we conducted a comprehensive investigation utilizing high-throughput proteomics analysis on established TKI-resistant tumor models, and found a notable upregulation of branched-chain amino acid transaminase 1 (BCAT1) expression in both osimertinib- and ASK120067-resistant tumors compared with the parental TKI-sensitive NSCLC tumors. Genetic depletion or pharmacological inhibition of BCAT1 impaired the growth of resistant cells and partially re-sensitized tumor cells to EGFR TKIs. Mechanistically, upregulated BCAT1 in resistant cells reprogrammed branched-chain amino acid (BCAA) metabolism and promoted alpha ketoglutarate (α-KG)-dependent demethylation of lysine 27 on histone H3 (H3K27) and subsequent transcriptional derepression of glycolysis-related genes, thereby enhancing glycolysis and promoting tumor progression. Moreover, we identified WQQ-345 as a novel BCAT1 inhibitor exhibiting antitumor activity both in vitro and in vivo against TKI-resistant lung cancer with high BCAT1 expression. In summary, our study highlighted the crucial role of BCAT1 in mediating resistance to third-generation EGFR-TKIs through epigenetic activation of glycolysis in NSCLC, thereby supporting BCAT1 as a promising therapeutic target for the treatment of TKI-resistant NSCLC.

Atractyloside inhibits gefitinib‑resistant non‑small‑cell lung cancer cell proliferation.

Atractyloside is a traditional Chinese medicine used to treat nasal congestion, and allergic rhinitis; however, its effects on cancer are unknown. Non-small cell lung cancer (NSCLC) is associated with high mortality rates worldwide, and relapse due to epidermal growth factor receptor mutations is a problem in clinical therapy. Therefore, novel biomarkers are required for the diagnosis and treatment of NSCLC. Brother of the regulator of imprinted sites (BORIS; also known as CTCFL) is a potential therapeutic target in NSCLC. BORIS promotes cisplatin resistance and it has been suggested that it may account for multidrug resistance. The present study examined BORIS expression in tyrosine kinase inhibitor (TKI)-resistant NSCLC cells. Subsequently, small interfering RNA was used to knock down BORIS expression, and the effects of this knockdown were assessed on TKI-resistant NSCLC cell viability. The present study also investigated the effect of atractyloside on the proliferation of NSCLC cells using MTT assay. The results of the present study indicated that the inhibition of BORIS or its related downstream pathways may have potential for the treatment of TKI-resistant NSCLC. In addition, atractyloside mimicked BORIS knockdown, regulated its downstream genes and inhibited the proliferation of TKI-resistant NSCLC cells. In conclusion, the findings of the present study supported the potential application of atractyloside in TKI-resistant NSCLC therapy.

T790M mutation sensitizes non-small cell lung cancer cells to radiation via suppressing SPOCK1

BackgroundApproximately 50% of patients harbor the T790M mutation after developing first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance. Evidence has showed the major treatment failure is local relapses and limited metastases. Several studies have demonstrated the value of radiotherapy in metastatic non-small cell lung cancer (NSCLC) with the EGFR T790M mutation after the development of TKI resistance. The aim of this study was to explore the role of radiation in T790M-mutant NSCLC and the value of early radiotherapy for NSCLC with T790M-mediated EGFR-TKI resistance. MethodsGefitinib-resistant NSCLC cell lines were established via stepwise exposure to increasing concentrations of gefitinib (PC-9-GR). Droplet digital PCR was used to determine the relative T790M subclone abundance. In vitro and in vivo models were established using different mixtures of PC-9-GR and PC-9 cells. Differentially expressed genes were identified using RNA sequencing. Two research models were constructed (salvage and prophylactic radiotherapy) to determine the effects of early radiotherapy on gefitinib-resistant cells. ResultsPC-9-GR cells exhibited higher radiosensitivity than PC-9 cells (sensitivity enhancement ratio = 1.5). Salvage radiation reduced the number of T790M-mutant subclones, and the relative T790M abundance was significantly lower than that without radiation at 90 days (10.94% vs. 21.54%). Prophylactic radiation prevented the development of T790M subclones. These results were also confirmed in vivo. qRT-PCR revealed threefold elevation of miR-1243 in PC-9-GR cells, and the increased radiosensitivity of PC-9-GR cells was inhibited when miR-1243 was knocked down. RNA sequencing revealed that SPOCK1 was downregulated in PC-9-GR cells. Interestingly, bioinformatic analysis showed that SPOCK1 was a target gene of miR-1243. SPOCK1 knockdown markedly increased the radiosensitivity of PC-9 cells. ConclusionGefitinib-resistant NSCLC with the T790M mutation had higher radiosensitivity than that without the mutation, possibly mediated by SPOCK1. Early radiotherapy can eliminate T790M subclones, providing evidence for the benefit of early local treatment in patients with TKI-resistant NSCLC.

Abstract 5843: Exploring the implications of LPAR-1 expression in TKI-resistant NSCLC

Abstract Introduction: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer in the United States with a 5-year survival rate hovering between 20-30%. Mutations in EGFR can initially cause the tumors to respond to tyrosine kinase inhibitors (TKI). Osimertinib, a third generation TKI, is the preferred first or second line of treatment and despite being effective, recent reports indicate that patients develop resistance within 10-18 months of treatment. TKI resistance limits therapeutic options for patients and therefore, alternative strategies are being explored. In this context, Lysophosphatidic acid receptors (LPARs), a family of G-protein coupled receptors consisting of LPARs 1-6 expressed differentially in prostate, breast, ovarian, pancreatic, and brain cancers are being explored as potential targets for drug resistant cancers. Specifically, LPAR-1 has been shown to play a role in tumor cell clonogenicity, migration, and response to therapy in EGFR mutant NSCLC cells. Pharmacological inhibition or CRIPSR based deletion of LPAR-1 in EGFR mutant NSCLC cell lines HCC827 and NCI-H3255, increased TKI sensitivity by reducing clonogenic potential. Further, detailed pathway analysis in TKI sensitive and resistant EGFR mutant cell lines suggests that LPAR-1 expression influences drug sensitivity. Surprisingly, the role of LPAR-1 in TKI sensitivity is not well understood. In the present study, we have performed detailed investigation on the implications of LPAR-1 expression in the development of Osimertinib resistance. Methods: Osimertinib resistant EGFR mutant cell lines (OR) were generated using dose-escalation method and drug-tolerant persisters (DTPs) were generated using a short-term high exposure method. Gene and protein expressions were examined using qPCR and western blotting. Subcutaneous tumor cell derived xenografts were generated for in vivo studies. Results: LPAR-1 is downregulated in Osimertinib resistant NSCLC cells. Further, we also observe that LPAR-1 levels are lower in DTP cells, confirming a progressive loss of LPAR-1 with Osimertinib treatment. In vivo studies in cell derived xenografts correlated with the in vitro studies. Conclusion: Our studies indicate LPAR-1 downregulation correlates to Osimertinib resistance in EGFR mutant NSCLC cell lines. LPAR-1 downregulation has a key role in development of Osimertinib resistance. Future studies will focus on delineating LPAR-1 signaling pathways related to TKI resistance and investigating the effect of LPAR-1 overexpression using CRISPR technology. Citation Format: Varsha Srinivasan, Zhaohui Li, Raghuraman Kannan, Anandhi Upendran. Exploring the implications of LPAR-1 expression in TKI-resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5843.

Abstract 946: Landscape of HRD aberration in EGFR mutated lung cancer and the role of PARP-inhibitor in EGFR mutated lung cancer with HRD aberration

Abstract Introduction: Olaparib, an oral poly polymerase (PARP) inhibitor has received approval for treating malignancies displaying homologous recombination deficiency (HRD). Current data is insufficient in establishing the role of PARP inhibitors in non-small cell lung cancer (NSCLC) in the setting of third generation EGFR tyrosine kinase inhibitor (TKI) use. Preclinical investigation suggests heightened PARP inhibitor sensitivity of TKI-resistant EGFR mutant NSCLC cells compared to TKI-sensitive cells. Methods: We utilized The Cancer Genome Atlas (TCGA) to investigate the prevalence of HRD gene mutations in patients with NSCLC harboring EGFR mutations. We also present a case demonstrating a favorable response to the dual therapy of olaparib and osimertinib in NSCLC harboring EGFR, RAD50, and ARID1A mutations that progressed on osimertinib. Results: There were 2298 patients in the NSCLC cohort (MSKCC database). Among 687 patients with EGFR mutations, 24 patients (3.5%) exhibited ARID1A mutations, 8 patients (1.2%) had BRCA1 mutations, 11 patients (1.6%) had BRCA2 mutations, and 5 (0.7%) had RAD50 mutations. A 71-year-old female presented with stage IV adenocarcinoma of the lung. She had recurrent malignant pericardial effusion requiring pericardiocentesis for previous cardiac tamponade. Computed tomography (CT) of the chest revealed a 3cm irregular left hilar mass with bilateral mediastinal and hilar lymph nodes. Tissue next generation sequencing (NGS) at baseline identified EGFR exon19 deletion along with RAD50 R726H which is a HRD pathway gene. Circulating tumor DNA (ct-DNA) NGS (Guardant360) showed EGFR E746_A750del 4.8%. The patient achieved partial response to osimertinib for 28 months. Follow-up CT showed disease progression. Olaparib 200mg twice daily was added to osimertinib based on repeat ct-DNA NGS indicating a new ARID1A alteration (splice site SNV) along with the known BRCA1 mutation. Follow up scans currently have consistently demonstrated ongoing partial response for 19 months. Combining olaparib with osimertinib can be effective in an osimertinib-resistant NSCLC patient with EGFR mutation and HRD aberration. The favorable response could be attributed not only to pre-existing RAD50 mutation but also to acquired mutations in ARID1A HRD genes. Conclusion: HRD aberrations are uncommon in EGFR mutated lung cancer patients. Further investigation on the role of PARP inhibitor in EGFR mutated lung cancer is warranted. Citation Format: Jinah Kim, Horyun Choi, Liam Il-Young Chung, Young Kwang Chae. Landscape of HRD aberration in EGFR mutated lung cancer and the role of PARP-inhibitor in EGFR mutated lung cancer with HRD aberration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 946.

YES1 as a potential target to overcome drug resistance in EGFR-deregulated non-small cell lung cancer.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) such as gefitinib and osimertinib have primarily been used as first-line treatments for patients with EGFR-activating mutations in non-small cell lung cancer (NSCLC). Novel biomarkers are required to distinguish patients with lung cancer who are resistant to EGFR-TKIs. The aim of the study is to investigate the expression and functional role of YES1, one of the Src-family kinases, in EGFR-TKI-resistant NSCLC. YES1 expression was elevated in gefitinib-resistant HCC827 (HCC827/GR) cells, harboring EGFR mutations. Moreover, HCC827/GR cells exhibited increased reactive oxygen species (ROS) levels compared to those of the parent cells, resulting in the phosphorylation/activation of YES1 due to oxidation of the cysteine residue. HCC827/GR cells showed elevated expression levels of YES1-associated protein 1 (YAP1), NF-E2-related factor 2 (Nrf2), cancer stemness-related markers, and antioxidant proteins compared to those of the parent cells. Knockdown of YES1 in HCC827/GR cells suppressed YAP1 phosphorylation, leading to the inhibition of Bcl-2, Bcl-xL, and Cyclin D1 expression. Silencing YES1 markedly attenuated the proliferation, migration, and tumorigenicity of HCC827/GR cells. Dasatinib inhibited the proliferation of HCC827/GR cells by targeting YES1-mediated signaling pathways. Furthermore, the combination of gefitinib and dasatinib demonstrated a synergistic effect in suppressing the proliferation of HCC827/GR cells. Notably, YES1- and Nrf2-regulated genes showed a positive regulatory relationship in patients with lung cancer and in TKI-resistant NSCLC cell lines. Taken together, these findings suggest that modulation of YES1 expression and activity may be an attractive therapeutic strategy for the treatment of drug-resistant NSCLC.

Increased expression of SSEA-4 on TKI-resistant non–small cell lung cancer with EGFR-T790M mutation

Non-small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme β3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

PTPN23 ubiquitination by WDR4 suppresses EGFR and c-MET degradation to define a lung cancer therapeutic target

Aberrant overexpression or activation of EGFR drives the development of non-small cell lung cancer (NSCLC) and acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) by secondary EGFR mutations or c-MET amplification/activation remains as a major hurdle for NSCLC treatment. We previously identified WDR4 as a substrate adaptor of Cullin 4 ubiquitin ligase and an association of WDR4 high expression with poor prognosis of lung cancer. Here, using an unbiased ubiquitylome analysis, we uncover PTPN23, a component of the ESCRT complex, as a substrate of WDR4-based ubiquitin ligase. WDR4-mediated PTPN23 ubiquitination leads to its proteasomal degradation, thereby suppressing lysosome trafficking and degradation of wild type EGFR, EGFR mutant, and c-MET. Through this mechanism, WDR4 sustains EGFR and c-MET signaling to promote NSCLC proliferation, migration, invasion, stemness, and metastasis. Clinically, PTPN23 is downregulated in lung cancer and its low expression correlates with WDR4 high expression and poor prognosis. Targeting WDR4-mediated PTPN23 ubiquitination by a peptide that competes with PTPN23 for binding WDR4 promotes EGFR and c-MET degradation to block the growth and progression of EGFR TKI-resistant NSCLC. These findings identify a central role of WDR4/PTPN23 axis in EGFR and c-MET trafficking and a potential therapeutic target for treating EGFR TKI-resistant NSCLC.

EGFR-T790M Mutation–Derived Interactome Rerouted EGFR Translocation Contributing to Gefitinib Resistance in Non-Small Cell Lung Cancer

Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer.

SKB264 (TROP2-ADC) for the treatment of patients with advanced NSCLC: Efficacy and safety data from a phase 2 study.

9114 Background: TROP2 (trophoblast cell surface antigen 2) is commonly overexpressed in non-small cell lung cancer (NSCLC) and associated with poor prognosis. SKB264 is a novel anti-TROP2 ADC developed using sulfonyl pyrimidine-CL2A-carbonate linker to conjugate its payload, a belotecan-derivative topoisomerase I inhibitor, to achieve an average Drug-to-antibody Ratio (DAR) of 7.4. The design was to achieve a more effective balance between stability in circulation and release of the ADC payload in tumor cells. Here we report clinical efficacy and safety results of SKB264 in the treatment of patients (pts) with NSCLC from a Phase 2 expansion cohort. TROP2 expression level by immunohistochemistry was assessed retrospectively. Correlation analyses between response and TROP2 level will be provided. Methods: This is a Phase 1/2, multicenter dose-escalation/expansion study in pts with relapsed or refractory locally advanced/metastatic NSCLC and other tumor types (NCT04152499). All NSCLC pts received SKB264 at 5 mg/kg IV Q2W. Tumor assessments based on RECIST 1.1 were performed every 8 weeks by investigators. Results: As of February 9th, 2023, 43 pts (63% male, 88% ECOG PS 1, median age 58 yrs [44-74]) were enrolled. Median follow-up was 11.5 months (mo; 95% CI, 10.4-12.2). Median treatment duration was 5.7 mo (range, 0.5-14.1). Among 39 response-evaluable pts, the ORR was 44% (17/39, 15 confirmed and 2 pending confirmation), median DoR was 9.3 mo (range, 1.3+ to 11.2+), 6-month DoR rate was 77%. For EGFR wild type subgroup (previously received median 2 lines of therapy including anti-PD-1/L1), the ORR was 26% (5/19), DCR was 89% (17/19), median PFS was 5.3 mo, and 9-month OS rate was 80.4%. For subgroup with TKI resistant EGFR mutant NSCLC (50% also failed at least one line of chemotherapy), the ORR was 60% (12/20), DCR was 100% (20/20), median PFS was 11.1 mo, and 9-month PFS rate was 66.7%. 67.4% (29/43) of pts had Grade ≥ 3 treatment-related adverse events (TRAEs). The most common Grade ≥3 TRAEs (occurred in ≥5% of pts) were neutrophil count decreased (32.6%), anemia (30.2%), white blood cell count (WBC) decreased (23.3%), stomatitis (9.3%), rash (7.0%), and lymphocyte count decreased (7.0%). Grade 4 TRAEs occurred only for neutropenia and WBC decreased. Most of the hematology toxicity occurred within the first two months of treatment and resolved after treatment with granulocyte colony stimulating factor or erythropoietin without blood transfusions. 23.3% (10/43) of the pts experienced dose reduction due to TRAEs. No neuropathy or drug-related ILD/pneumonitis was reported. No TRAEs led to treatment discontinuation or death. Conclusions: SKB264 at 5 mg/kg Q2W demonstrated encouraging anti-tumor activity and manageable safety profile in pts with relapsed or refractory locally advanced/metastatic NSCLC. TRAEs were mainly hematologic. Phase 3 studies of SKB264 in pts with advanced NSCLC have been planned. Clinical trial information: NCT04152499 .

IL6 Mediates Suppression of T- and NK-cell Function in EMT-associated TKI-resistant EGFR-mutant NSCLC.

Patients with advanced non-small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype. We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens. We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell-mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells. These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.

The Ethanolic Extract of Trichosanthes Kirilowii Root Exerts anti-Cancer Effects in Human Non-Small Cell Lung Cancer Cells Resistant to EGFR TKI

The aim of this study was to investigate whether the ethanol extract of the Trichosanthes kirilowii root (ETK), traditionally used to treat lung diseases, exhibits anticancer activity in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) cells. ETK treatment suppressed the growth of EGFR TKI-resistant NSCLC cells, including H1299, H1975, PC9/ER (erlotinib-resistant PC9) and PC9/GR (gefitinib-resistant PC9) cells, in a concentration- and time-dependent manner. Dose-dependent decline in anchorage-dependent and -independent colony formation was also detected following ETK treatment. We demonstrate that the growth-inhibitory effect of ETK was related to apoptosis induction, based on flow cytometry results showing ETK-induced increase in the percentage of cells with sub-G1 DNA and the population of annexin V-positive cells. Consistently, ETK induced chromatin condensation and cleavage of poly(ADP-ribose) polymerase (PARP). As a molecular mechanism, the phosphorylation level of signal transducer and activator of transcription 3 (STAT3) and Src was decreased by ETK. ETK-induced apoptosis was partially reversed by transfection of constitutively activated STAT3, indicating that STAT3 inactivation mediated ETK-induced apoptosis in EGFR TKI-resistant NSCLC cells. Our results provide basic evidence supporting the role of ETK as a novel therapeutic in EGFR TKI-resistant NSCLC.

Extracellular Vesicles from EGFR T790M/L858R-mutant Non-small Cell Lung Cancer Promote Cancer Progression.

Tyrosine kinase inhibitors (TKIs) are the first-line therapy for non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. Unfortunately, most patients quickly develop an acquired resistance to EGFR-TKIs. However, the effects of NSCLC harboring EGFR-T790M mutation on aggressive NSCLC phenotypes is still unclear. This study aimed to investigate the extracellular vesicles (EVs) involvement in promoting the aggressiveness of NSCLC cells. EVs were isolated from the culture media of TKI-sensitive (HCC827) and TKI-resistant (H1975) NSCLC cells using ultracentrifugation. Cell viability, proliferation, migration, and invasion were examined following incubation with indicated EVs. HCC827 and H1975 cells showed time-dependent uptake of PKH67 dye labeled EVs. Incubation of EVs derived from H1975 cells (EV-H1975) did not alter the TKI sensitivity of HCC827 cells. Interestingly, EV-H1975 significantly increased HCC827 cells proliferation, invasion, and migration. By a phospho-kinase array, EV-H1975 increased phosphorylation of several proteins related to cell proliferation, invasion, and migration, including FAK, AKT, and ERK1/2, in HCC827 cells. EGFR-T790M NSCLC cells promote TKI-sensitive NSCLC cell aggressiveness, at least partially, through mechanisms associated with EVs.

Abstract 3260: Enhanced lineage plasticity in RTK-independent TKI-resistant EGFR-mutant NSCLC

Abstract Background: Resistance to targeted tyrosine kinase inhibitors (TKI) inevitably develops in metastatic EGFR-mutant non-small cell lung cancer (NSCLC). Resistance mechanisms are diverse, and mechanisms beyond receptor tyrosine kinase (RTK) pathway mutations are poorly understood. We hypothesized that the use of osimertinib as first-line therapy is increasing the prevalence of RTK-independent resistance mechanisms, and that RTK-independent resistant tumors undergo enhanced tumor cell lineage plasticity as an escape mechanism to EGFR TKI therapy. Methods: We identified patients who developed osimertinib resistance (OR) after first line (1L, n=54) and second line (2L, n=42) treatments and determined the resistance mechanisms based on clinical sequencing and histopathology. We also performed single-cell RNA-seq of 24 samples from 13 patients with EGFRm NSCLC at TKI treatment-naïve (TN, n=2), residual disease (RD, n=4), and progression disease (PD, n=7) stages. Results: Compared to 2L OR tumors, 1L OR tumors had increased RTK-independent mechanisms of resistance (76% vs. 46%, p=0.002), including 8% with small cell transformation (n=4), 2% with squamous transformation (n=1) and 66% with unknown mechanisms (n=34). To understand inter- and intra-tumor heterogeneity, we analyzed transcriptomic profiles of 76,266 single cells. Lung developmental lineages were assigned to 10,250 EpCAM+ cells, including 4,735 cells classified as malignant cells by inferCNV and RTK signaling analysis. In the two EGFRm TN tumors, the malignant cells demonstrated bronchoalveolar lineage and moderate EGFR expression. In the TKI resistant cases (PD, n=7), both RTK-dependent and RTK-independent resistance were observed. The RTK-dependent tumors (EGFR T790M n=1; ERBB2 amplification n=1) demonstrated preserved bronchoalveolar lineage identity. In the RTK-independent resistant tumors (n=5), one had complete lineage switch from epithelial to small cell neuroendocrine and very low expression level of EGFR. The remaining 4 PD tumors displayed varying expression of epithelial-to-mesenchymal transformation (EMT) features. One tumor had sarcomatoid histology and a high proportion of cells having positive VIM expression (84%) and 92% of cells having complete loss of NAPSA expression; 3 tumors had partial EMT demonstrated by heterogeneous proportion of cells having VIM expression (18-56%) and loss of NAPSA (26-67%). Interestingly, some of the cells with EMT and partial-EMT had moderate levels of EGFR expression, similar to the levels in the TN tumors. Conclusion: With osimertinib use at 1L, the incidence of RTK-independent resistance has increased to become the dominant mechanism, whereas RTK-dependent resistance has decreased. Increased lineage plasticity (small cell neuroendocrine, squamous and EMT) potentially serves as an RTK-independent TKI-resistance mechanism in EGFRm NSCLC. Citation Format: Xiuning Le, Ruiping Wang, Natalie Vokes, Yasir Elamin, Neda Kalhor, Daniel McGrail, Yuanxin Xi, Santiago Treviño III, Lingzhi Hong, Robyn Du, George Blumenschein, Carl Gay, Marcelo Negrao, Mehmet Altan, Hai Tran, Limei Hu, Jing Wang, Simon Heeke, Monique Nilsson, Jacqulyne Robichaux, Minghao Dang, Guangchun Han, Lauren Byers, Anne Tsao, Boris Sepesi, Chantale Bernatchez, Jianjun Zhang, Linghua Wang, John Heymach. Enhanced lineage plasticity in RTK-independent TKI-resistant EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3260.

Abstract 6138: High Globo-H expression associated with poor survival of gastric cancer patients and enriched PD-L1 expression

Abstract Background: Globo series antigens have been implicated in playing important roles in enhancing tumor growth through protecting tumor cells from apoptosis, suppressing T cell activity in the tumor microenvironment and promoting endothelial cell angiogenesis. Globo-H (GH), a globo-series glycosphingolipid (GSL) antigen that is synthesized by key enzymes β1,3-galactosyltransferase V (β3GalT5), fucosyltransferase (FUT) 1 and 2, is highly expressed on a variety of epithelial cancers rendering it a promising target for cancer immunotherapy. GH is reported to associate the EGFR mutant and PD-L1 expression in non-small cell lung cancer (NSCLC) patients. In addition, GH-targeting antibody-drug conjugate (ADC) OBI-999 has been demonstrated an excellent tumor growth inhibition potency in animal models across multiple cancer types including gastric cancer (GC). This study aims to further investigate the GH roles in GC. Methods: Prognostic roles of β3GalT5, FUT1 and FUT2 in GC patients were accessed through the “Kaplan-Meier plotter” database. The level of GH expression was evaluated in clinical adenocarcinoma samples from 105 patients with GC by immunohistochemistry (IHC) using H-score. Microwestern protein array analysis was performed in sorted GC NCI-N87 cells. FACS methods were also employed to measure the levels of PD-L1 and phospho-EGFRs in multiple cancer cell lines. Results: Significant correlations were observed between high mRNA expression of synthesized key enzymes and worse overall survival (OS)/post-progression survival for all 498 GC patients. Positive GH expression (H score≥20; 33.3%) was significantly associated with a poor disease specific survival in all samples (at 15 years; p=0.029), a poor OS in poorly differentiated tumors (p=0.033), and invasiveness (p=0.013), indicating GH expression was a potential prognostic factor in GC. Sorted NCI-N87 cells with high level of endogenous GH expression showed a relatively greater proliferative activity compared with cells expressing low level of GH. Microwestern array analysis on sorted cells indicated upregulations of phospho-AKT/P38/JNK, Cyclin D1 and Cyclin E1 protein in high GH expression NCI-N87 cells, which implicated a mechanistic link between high GH and tumor promoting signals in GC. In addition, GH level was shown to be correlated with cell surface expression level of PD-L1 in NCI-N87, SNU-16, HCC-1428, ACHN, and TKI-resistant H1975 NSCLC cancer cells. Higher phospho-EGFRs (Tyr1068 and Tyr1173) were detected in high GH level of H1975 cells, which is consistent with the results from previous studies showing association between GH level and EGFR mutant/PD-L1 expression in NSCLC. Conclusions: GH level was associated with the survival of GC patients and positively correlated with cell surface PD-L1 expression in vitro. Therefore, GH-targeting therapy has a potential application for the treatment of GC patients. Citation Format: Tzer-Min Kuo, Hui-Ting Hsu, Cheng-Yen Wei, Ming-Tain Lai, Chun-Te Chen. High Globo-H expression associated with poor survival of gastric cancer patients and enriched PD-L1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6138.

Abstract 548: EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC

Abstract The leading cause of cancer-related deaths in the US is non-small cell lung cancer (NSCLC), and about 10-15% of NSCLC cases harbor EGFR activating mutations. While these patients are initially responsive to EGFR tyrosine kinase inhibitors (TKIs), they eventually experience progression and develop acquired TKI resistance. Currently, there are no approved therapies for NSCLC patients with acquired resistance to the third-generation EGFR TKI osimertinib (OSI). Moreover, EGFR-TKI resistant NSCLCs are refractory to immune checkpoint inhibitors. Therefore, novel treatment strategies are urgently needed. Chimeric antigen receptors (CARs) have been used to enhance the anti-tumor activity of immune effector cells. Here, we developed CAR-based therapies targeting EGFR as a novel strategy to overcome immune suppression and target EGFR TKI-resistant NSCLC. We designed multi-generational EGFR CAR constructs and transduced them into T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. We then evaluated their activity against NSCLC cell lines in vitro using firefly luciferase assays. EGFR CAR-T and CAR-NK cells showed potent cytotoxicity against NSCLC cells expressing EGFR including EGFR wild-type and mutant NSCLCs, as well as NSCLC cells with acquired resistance to OSI. However, EGFR CAR-T cells showed decreased killing activity against OSI-resistant NSCLC cells compared to their parental cells. In contrast, EGFR CAR-NK cells showed stronger cytotoxicity against NSCLC with acquired OSI resistance as compared to parental cells. Transcriptomic analysis revealed that NSCLC cells with acquired resistance to TKI had undergone epithelial-mesenchymal transition and had downregulated expression of genes related to antigen presentation and upregulated expression of B7-H6 (NCR3LG1), which have been reported to be associated with increased responsiveness to NK cells. Moreover, treatment of TKI resistant cells with OSI resulted in upregulation of cell surface EGFR and potentiated CAR-NK cells-mediated killing. TGF-β is a critical immune-suppressive cytokine. We found that EGFR-TKI resistant NSCLC cells elaborated expression of TGF-β as compared to parental cells. Blockade of the TGF-β pathway using galunisertib significantly enhanced the activity of EGFR CAR-NK cells against OSI-resistant NSCLC cells. Next, we engineered the dominant-negative TGF-β receptor II (DNTGFBRII) into our EGFR CAR constructs. The expression of DNTGFBRII inhibited the phosphorylation of SMAD2 and the downregulation of granzyme B and perforin induced by TGF-β. DNTGFBRII-CAR-NK cells showed higher killing activity against OSI-resistant NSCLC cells and were resistant to TGF-β-mediated immunosuppression. In conclusion, EGFR CAR-NK cells show significant anti-tumor activity to EGFR TKI-resistant NSCLC cells, and the cytotoxicity is enhanced in combination with OSI treatment and blockade of the TGF-β pathway. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Jacqulyne Robichaux, Alissa Poteete, Xiaoxing Yu, Fahao Zhang, Simon Heeke, Yu Qian, Junqin He, John Heymach. EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 548.

Acetyltanshinone IIA reduces the synthesis of cell cycle-related proteins by degrading p70S6K and subsequently inhibits drug-resistant lung cancer cell growth

Targeted therapies using tyrosine kinase inhibitors (TKIs) against epidermal growth factor receptor (EGFR) have improved the outcomes of patients with non-small cell lung cancer (NSCLC). However, due to genetic mutations of EGFR or activation of other oncogenic pathways, cancer cells can develop resistance to TKIs, resulting in usually temporary and reversible therapeutic effects. Therefore, new anticancer agents are urgently needed to treat drug-resistant NSCLC. In this study, we found that acetyltanshinone IIA (ATA) displayed much stronger potency than erlotinib in inhibiting the growth of drug-resistant NSCLC cells and their-derived xenograft tumors. Our analyses revealed that ATA achieved this effect by the following mechanisms. First, ATA could bind p70S6K at its ATP-binding pocket to prevent phosphorylation, and second by increasing the ubiquitination of p70S6K to cause its degradation. Since phosphorylation of S6 ribosome protein (S6RP) by p70S6K can induce protein synthesis at the ribosome, the dramatic reduction of p70S6K after ATA treatment led to great reductions of new protein synthesis on several cell cycle-related proteins including cyclin D3, aurora kinase A, polo-like kinase, cyclin B1, survivin; and reduced the levels of EGFR and MET. In addition, ATA treatment increased the levels of p53 and p21 proteins, which blocked cell cycle progression in the G1/S phase. Taken together, as ATA can effectively block multiple signaling pathways essential for protein synthesis and cell proliferation, ATA can potentially be developed into a multi-target anti-cancer agent to treat TKI-resistant NSCLC.

The Root Extract of Peucedanum praeruptorum Dunn Exerts Anticancer Effects in Human Non-Small-Cell Lung Cancer Cells with Different EGFR Mutation Statuses by Suppressing MET Activity.

The aim of this study was to investigate the anticancer effects of the root extract of Peucedanum praeruptorum Dunn (EPP) in human non-small-cell lung cancer (NSCLC) cells and explore the mechanisms of action. We used four types of human lung cancer cell lines, including H1299 (epidermal growth factor receptor (EGFR) wild-type), PC9 (EGFR Glu746-Ala750 deletion mutation in exon 19; EGFR tyrosine kinase inhibitor (TKI)-sensitive), H1975 (EGFR L858R/T790M double-mutant; EGFR TKI-resistant), and PC9/ER (erlotinib-resistant) cells. EPP suppressed cell growth and the colony formation of NSCLC cells in a concentration-dependent manner. EPP stimulated chromatin condensation, increased the percentage of sub-G1 phase cells, and enhanced the proportion of annexin V-positive cells, demonstrating that EPP triggered apoptosis in NSCLC cells regardless of the EGFR mutation and EGFR TKI resistance status. The phosphorylation level of the signal transducer and activator of transcription 3 (STAT3) and AKT was decreased by EPP. The expression of STAT3 target genes was also downregulated by EPP. EPP reversed hepatocyte growth factor (HGF)-induced MET phosphorylation and gefitinib resistance. Taken together, our results demonstrate that EPP exerted anticancer effects not only in EGFR TKI-sensitive NSCLC cells, but also in EGFR TKI-resistant NSCLC cells, by suppressing MET activity.

LPCAT1 promotes gefitinib resistance via upregulation of the EGFR/PI3K/AKT signaling pathway in lung adenocarcinoma.

Currently, the mechanisms of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance have been a focus of clinical research. Despite that most of the mechanisms of acquired EGFR TKI resistance have been revealed, about 30% of non-small-cell lung cancer (NSCLC) cases have not been fully elucidated, especially for lung adenocarcinoma (LUAD). Recently, LPCAT1, an important enzyme of phospholipid metabolism, has been found to bridge the gap between the oncogene and metabolic reprogramming. In NSCLC, LPCAT1 has been shown to participate in progression and metastasis. However, little is known about the role of LPCAT1 in acquired EGFR TKI resistance. In this study, elevated LPCAT1 expressions were observed in an EGFR TKI-resistant cell line (PC-9R) relative to a corresponding EGFR TKI-sensitive cell line (PC-9). In vivo and in vitro gene functional studies showed that LPCAT1 contributed to the pathogenesis of gefitinib resistance in LUAD, where an LPCAT1-EGFR positive feedback loop formed and then regulated its downstream signaling molecules of the EGFR/PI3K/AKT signaling pathway. The results provided novel insights into the acquired resistance mechanism of EGFR TKI from the perspective of phospholipid metabolism. These findings suggest LPCAT1 may serve as a potential therapeutic target for patients with EGFR TKI-resistant NSCLC.