Microfluidic-assisted preparation of ARV 825 and Osimertinib loaded liposomal formulation as a potential system for colorectal cancer therapy.

Long non-coding RNA LRTOR drives osimertinib resistance in non-small cell lung cancer by boosting YAP positive feedback loop.

BackgroundAt present, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment, as the first-line treatment of lung adenocarcinoma (LUAD) with EGFR mutation, has achieved good clinical efficacy, but most patients will eventually develop acquired resistance. The objective of this study is to accurately identify drug-resistant LUAD patients who can benefit from other treatments by constructing a predictive model for EGFR-TKIs resistance.MethodsBased on the differentially expressed genes (DEGs) between osimertinib (OSI)-resistant cells (PC9OR, H1975OR) and OSI-sensitive LUAD cells (PC9, H1975), the EGFR mutant LUAD prognosis-related genes from The Cancer Genome Atlas (TCGA), and the immune genes from ImmPort and InnateDB websites, we constructed an immune prognosis model of OSI resistance to predict the outcome of EGFR mutant LUAD patients. Then, according to the risk score, EGFR mutant LUAD patients were divided into high- and low-risk groups, and the molecular, immune characteristics, and responsiveness to chemotherapy and targeted drugs were analyzed. Next, PSMD11 was knocked down using small interfering RNA (siRNA) to evaluate the effects of PSMD11 on PC9OR and H1975OR cells. Finally, the correlation between PSMD11 and OSI resistance was determined in vitro via Cell Counting Kit-8 (CCK-8), colony formation assays, and flow cytometry, and in vivo via western blot and immunohistochemistry.ResultsWe constructed an immune prognostic prediction model consisting of four OSI-resistant genes (C3, PSMD11, G3BP1, TRIB2) and clarified its accuracy in predicting the prognosis of EGFR mutant LUAD patients. According to the risk score classification, high-risk EGFR mutant patients were more sensitive to traditional chemotherapy and targeted drugs, with higher expression of immune checkpoint molecules PD-1, LAG3, IDO1, and more infiltration of CD8+ T cells, M0/M1 macrophages, and natural killer (NK) cells. In addition, knockdown of PSMD11 could inhibit cell proliferation, promote cell apoptosis, and increase the sensitivity of drug-resistant cells to OSI. And compared with individual treatment, the combination treatment of PSMD11-siRNA and OSI in PC9OR and H1975OR cells could significantly inhibit cell proliferation and tumor growth. In addition, PSMD11 could promote the progression of OSI-resistant LUAD by activating the NF-κB/IL-6/STAT3 signaling pathway.ConclusionsThis signature has a high predictive effect on the prognosis of OSI-resistant LUAD patients, and can be used as a powerful predictive tool for further selection of chemotherapy and immunotherapy in OSI-resistant LUAD patients.

Objective:Osimertinib (OSI) therapy, a cornerstone in treating non-small cell lung cancer (NSCLC), has been severely limited by rapidly developing acquired resistance. Inhibition of bypass activation using a combination strategy holds promise in overcoming this resistance. Biguanides, with excellent anti-tumor effects, have recently attracted much attention for this potential. The current study investigated whether novel biguanide compounds developed by our team could overcome OSI resistance and the underlying mechanisms were explored.Methods:A comprehensive screening assay using OSI-resistant cells identified the optimal combination of biguanide compounds with OSI. Proteomics, co-immunoprecipitation mass spectrometry, RNA sequencing, and homologous recombination assays were used to elucidate the molecular mechanisms underlying combination therapy. NSCLC tumor tissues, especially OSI-resistant tissues, obtained from our clinic were used to assess the correlations between key proteins and OSI resistance.Results:SMK-010, a highly potent biguanide compound, effectively overcame OSI resistance in vitro and in vivo. Mechanistical studies showed that BMI1/FGFR1 pathway activation is responsible for OSI resistance. Specifically, silencing BMI1 promoted NEDD4-mediated FGFR1 ubiquitination and proteasomal degradation, whereas SMK-010 treatment induced FGFR1 lysosomal degradation. This reduction in FGFR1 levels impaired homologous recombination, increased DNA damage, and surmounted OSI resistance. Analysis of clinical samples revealed overexpression of BMI1 and FGFR1 in NSCLC tissues and represented potential biomarkers for OSI resistance.Conclusions:These findings highlight the crucial role of the BMI1/FGFR1 axis in OSI resistance and provide a rational basis for the future clinical application of the biguanide, SMK-010, in combination with OSI.

Background: Combining pemetrexed (PEM) with Osimertinib (OSI) improves outcomes in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC), but optimal scheduling remains undefined. Sequential PEM → OSI strategies may outperform concurrent administration; however, the critical dosing interval determining synergy has not been explored. Methods: PEM pharmacodynamics were divided into an OSI-antagonized early phase (S-phase arrest and DNA damage accumulation) and OSI-synergized late phase (DNA damage peak, apoptosis initiation, and feedback EGFR activation). Time-course profiling of cell cycle, DNA damage, apoptosis, and EGFR pathways was evaluated under monotherapy or sequential combination regimens to elucidate the mechanisms underlying synergistic/antagonistic effects. Results: OSI antagonizes PEM's early phase via G1 arrest but potently enhances late-phase apoptosis through Rad51/thymidylate synthase suppression, Bim upregulation, and inhibition of EGFR signaling. The 48 h interval PEM → OSI uniquely enabled complete early-phase execution and aligned OSI exposure with late-phase initiation, yielding robust synergy across OSI-sensitive cell lines. In contrast, the 24 h interval PEM → OSI sequence demonstrated synergy only in PEM-sensitive PC9 cells. Both concurrent PEM + OSI and OSI → PEM sequence induced attenuated DNA damage and apoptotic signaling. Conclusions: The 48 h interval PEM → OSI sequence maximizes efficacy by temporally segregating antagonistic and synergistic interactions. This pharmacodynamically optimized regimen represents a promising strategy for clinical translation.

Acquired resistance to osimertinib (OSI) poses a significant challenge in the treatment of epidermal growth factor receptor mutant non-small cell lung cancer (NSCLC). Although OSI is effective as a first-line salvage therapy in T790M-positive patients following progression on first- or second-generation EGFR-TKIs (erlotinib, gefitinib, and afatinib), the inevitable development of acquired resistance limits its therapeutic efficacy. This study reveals that OSI-resistant (OSIR) NSCLC cells underwent metabolic reprogramming characterized by enhanced glycolysis and upregulation of hexokinase 2 (HK2). We demonstrated that HK2 inhibitor, benserazide, exhibited significant anticancer effects in OSIR cell models and mediated by reactive oxygen species. Our results suggested that HK2 inhibition effectively modulated the enhanced glycolysis, activated the AMPK-mTOR-autophagy axis, and unexpectedly interfered with NF-κB signaling through direct HK2-IKKβ interaction. Excitingly, the protein expression level and activity of pyruvate dehydrogenase kinase 1 (PDK1) in OSIR cells were upregulated upon HK2 inhibition, indicating a pro-survival role. Combined inhibition of HK2 and PDK1 synergistically inhibited the proliferation of OSIR cells and significantly suppressed tumor growth in an OSIR cell xenograft model, outperforming the single use of HK2 inhibitor. This combination successfully rectified aberrant glucose metabolism and enhanced oxidative phosphorylation. Our findings identified HK2 as a crucial mediator in overcoming OSI resistance and suggested that combined inhibition of HK2 and PDK1 could be a promising approach in OSIR NSCLC.

BackgroundCircular RNA (circRNA) is involved in the occurrence and development of many cancers. It has been reported that circRNA Polo like kinase 1 (PLK1) is up-regulated in non-small cell lung cancer (NSCLC), but the mechanism of circPLK1 in NSCLC remains to be further explored.MethodsTHP-1 cells were induced to M2 macrophage polarization to obtain the exosomes from M2 macrophage (M2-Exo). The uptake of M2-Exo and its role in osimertinib (OSI) sensitivity, apoptosis, and activation of Epidermal growth factor receptor (EGFR) were detected. Next, circPLK1 expression and its role on the above mentioned indicators were also detected. The targets of circPLK1 were predicted by CircInteractome. Finally, the regulatory effects of circPLK1 on miRNA and pathways were verified.ResultsM2 macrophages induced OSI resistance and reduced apoptosis of NSCLC cells. circPLK1 was up-regulated in H1975 cells after co-culture with M2 and M2-Exo. CircPLK1 overexpression decreased OSI sensitivity, inhibited apoptosis, and increased phosphorylation of EGFR in NSCLC cells. miR-186 was a target of circPLK1, and its expression was decreased in M2-Exo and circPLK1 overexpressed NSCLC cells. Further, co-culture with M2-Exo increased the phosphorylation of EGFR, AKT, and extracellular signal-regulated kinase (ERK) in NSCLC cells. miR-186 mimic decreased the levels of phosphorylation of these proteins. In OSI-treated NSCLC cells, circPLK1 overexpression increased the phosphorylation of EGFR, AKT, and ERK in NSCLC cells. miR-186 mimic eliminated the effect of circPLK1 on these proteins in NSCLC cells.ConclusioncircPLK1 in exosomes derived from M2-polarized macrophages promotes OSI resistance in NSCLC.Graphical

Background:EGFR L858R mutation is associated with poorer efficacy of EGFR-tyrosine kinase inhibitors (TKIs) than EGFR Ex19del in patients with non-small cell lung cancer (NSCLC). However, ramucirumab (RAM) + erlotinib (ERL) therapy exhibited comparable efficacy between patients with L858R mutation and Ex19del mutation (median progression-free survival (PFS): 19.6 vs 19.4 months) in the RELAY study, with favorable PFS for both gene mutations at 19.4 months in the Japanese subset. Meanwhile, the FLAURA study revealed shorter PFS with osimertinib (OSI) for L858R mutation than Ex19del mutation, with poorer PFS in the Japanese subset than in the overall population. The treatment discontinuation rates of RAM + ERL and OSI in Japanese patients were 18% and 29%, respectively. Consequently, RAM + ERL may exhibit superior efficacy and safety in Japanese patients with the L858R mutation.Objectives:To evaluate the therapeutic efficacy and safety of RAM + ERL as a first-line treatment for advanced or recurrent NSCLC harboring the L858R mutation in Japanese patients.Design:A multicenter, noninterventional, retrospective cohort study.Methods and analysis:This study will involve patients with advanced or recurrent NSCLC (ECOG PS score 0–2) with L858R mutation who received RAM + ERL between November 1, 2020 and August 31, 2023, with the planned sample size of 200 patients. The primary endpoint is time to treatment failure, and the secondary endpoints are overall survival, PFS, PFS2, time to discontinuation of any EGFR-TKI, time to failure of strategy, objective response rate, disease control rate, and safety. Exploratory endpoints are effects of ERL and RAM on PD-L1 expression and neutrophil-to-lymphocyte ratio.Discussion:To the best of our knowledge, this is the first retrospective study focusing on L858R mutations associated with the RELAY regimen, providing the corresponding real-world data.Trial registration:UMIN Clinical Trials Registry identifier: UMIN000052047.

Osimertinib (OSI) is a third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) and the standard treatment for non-small cell lung cancer with EGFR mutation. OSI contains active metabolites such as AZ5104 and AZ7550. The correlation between the blood concentrations of OSI, AZ5104, and AZ7550 with their efficacy and safety is not clear; hence, we examined it. This was a single-center, retrospective study. We measured the blood concentrations of OSI, AZ5104, and AZ7550 in 46 patients who received OSI between March 2016 and December 2022 and examined their relationships with progression-free survival (PFS), overall survival (OS), and adverse events. The high OSI group had a longer PFS than the low OSI group (26.5 vs. 17.9 months, p=0.058); however, blood levels of AZ7550 and AZ5104 were not correlated with PFS. In the multivariate analysis, blood OSI concentration was an independent prognostic factor for PFS. Overall survival was not different between patients with high or low blood levels of OSI, AZ5104, or AZ7550. In terms of safety, AZ7550 blood levels were higher in patients with higher grades of neutropenia, lymphopenia and anemia, whereas AZ5104 blood levels were higher in patients with higher grades of lymphopenia. Higher OSI blood concentrations were associated with a longer PFS, while higher blood concentrations of AZ5104 and AZ7550 were observed in patients with higher hematological toxicity grades. Further research is needed to determine the optimal blood levels of OSI that improve prognosis and minimize toxicity.

Purpose: Combinatorial therapies are essential for treating advanced non-small cell lung cancer (NSCLC), particularly overcoming resistance to third-generation epidermal growth factor receptor (EGFR) like osimertinib (OSI). The Hippo signaling pathway, a critical regulator of cell proliferation, apoptosis, and tumor progression, is often dysregulated in NSCLC and contributes to chemo-resistance. This study investigated the potential of epigallocatechin-3-gallate (EGCG), a green tea polyphenol, to overcome OSI resistance by modulating the Hippo signaling pathway, specifically through inhibition of the YAP-1 (Yes-associated protein)-TEAD (TEA domain transcription factor)-CTGF (connective tissue growth factor) axis. Methods: Using stepwise dose escalation, OSI-resistant (OR) clones were developed from EGFR T790M-mutated H460 cells. The anti-proliferative effects of EGCG were assessed, and synergistic interactions between OSI and EGCG were analysed using combination index (CI) values and the median effect concept. Mechanistic studies evaluated the co-treatment’s impact on the Hippo signaling pathway, focusing on the inhibition of the YAP/TEAD/CTGF signaling axis.Results: The OR clones exhibited significantly higher IC50 values for OSI (25.12–28.48 µM) compared to parental H460 cells (2.74±0.2µM). EGCG treatment reduced cell viability in a concentration-dependent manner, with IC50 values of 102.54±0.23μM for H460 cells and 225.79–237.36 µM for OR clones. Combination treatment of OSI and EGCG showed strong synergy at a 1:2 molar ratio, with CI values indicating synergism across a range from IC50 to IC95. Mechanistically, co-treatment suppressed the overexpression of the YAP/TEAD/CTGF axis, restoring Hippo pathway activity and reversing OSI resistance. Conclusion: This study provides evidence that EGCG effectively targets the Hippo signaling pathway to overcome OSI resistance in NSCLC. The inclusion of EGCG in combinatorial therapies holds promise as a novel approach to combat therapeutic resistance and improve outcomes for patients with EGFR-mutated NSCLC.

Abstract Osimertinib (Tagrisso, AstraZeneca Pharmaceuticals) is the inaugural third-generation irreversible EGFR-TKI that specifically targets both the EGFR T790M resistant mutation and EGFR-TKI-sensitizing mutations. On September 25, 2024, the Food and Drug Administration authorized osimertinib (OSM) for adult patients with locally advanced, unresectable (stage III) non-small cell lung cancer (NSCLC). The target of this work was to establish a fast, accurate, environmentally friendly, and highly sensitive UPLC-MS/MS methodology for detecting OSM levels in human liver microsomes (HLMs). The separation of OSM and zanubrutinib (ZNB) was accomplished utilizing a C8 column and an isocratic mobile phase. The linearity of the OSM calibration curve spanned from 1 to 3,000 ng mL−1. The AGREE score of 0.76 validates the efficacy of the current approach. The brief in vitro half-life (23.72 min) and moderate intrinsic clearance (34.18 mL min−1 kg−1) of OSM indicate that it resembles drugs with a moderate extraction ratio. The present LC-MS/MS technique is regarded as the primary analytical methodology for quantifying OSM in HLM matrices. The characterization of OSM metabolic stability and in silico ADME features are crucial for progressing the discovery of novel drugs with improved metabolic stability.

An electrochemical sensor based on molecularly imprinted copolymer for the detection of Osimertinib

Comparison of efficacy of gefitinib and osimertinib for untreated EGFR mutation-positive non-small-cell lung cancer in patients with poor performance status

396 Background: For patients with advanced EGFR-mutant non-small cell lung cancer (EGFRm aNSCLC) progressing after osimertinib (OSI) and platinum-based chemotherapy (PBC) no uniformly accepted standard of care exists. The study was to provide updated results with a refreshed dataset for a previous study describing clinical outcomes of treatment regimens initiated after OSI and PBC in real-world patients with similar baseline characteristics to those in HERTHENA-Lung01 (HL-01), a multinational single-arm phase II trial evaluating the safety and efficacy of patritumab deruxtecan [1] . Methods: This study analyzed data from the US nationwide Flatiron Health electronic health record–derived aNSCLC de-identified database from approximately 280 US cancer clinics (~800 sites of care). Adult patients with an aNSCLC diagnosis (after 01 January 2011) and prior treatment with OSI and PBC, who initiated a subsequent line of therapy (index LOT) before 01 August 2022 were included in the study. Patients were chosen to closely match the HL-01 eligibility criteria (documented exon 19 deletion or L858R mutation; Eastern Cooperative Oncology Group Performance Status of 0 or 1; and absence of relevant comorbidities at index date). To further balance patient characteristics, the sample was propensity-score (PS) weighted based on nine covariates. Real-world overall survival (rwOS), progression-free survival (rwPFS) and overall response (rwORR, defined as partial or complete response with ≥2 assessments ≥28 days apart) were assessed from the start of the index LOT with corresponding 95% confidence intervals (CI). Results: 179 patients in the database fulfilled the eligibility criteria, increasing the sample size by 42% compared to the previous study 1 . Median age of the unweighted sample at index was 68 years, 65.9% of patients were female, 40.2% had a history of smoking and 26.3% received more than 3 LOTs prior to the index LOT. In the PS-weighted cohort (standard mean difference of all covariates was ≤0.127) the median rwOS was 9.1 months (95% CI: 7.3 – 11.3) and median rwPFS was 3.5 months (95% CI: 2.7 – 4.6). For a subsample of 75 patients with qualifying response assessments, rwORR was 12.7% (95% CI: 4.8 – 25.7). Conclusions: These observations in patients with EGFRm NSCLC after OSI and PBC demonstrate a limited clinical benefit with currently available therapies. The results are consistent with the previously published study and increase precision of the outcome estimates 1 . They highlight the high unmet need and provide further context on clinical outcomes of HL-01 trial results. [1] Patel et al. Clinical Outcomes of Real-World Treatment for Metastatic EGFRm NSCLC after Osimertinib and Platinum-Based Chemotherapy. 2023 Sep 9-12 Singapore. P2.31.

The purpose of this study was to develop and validate a physiologically based pharmacokinetic (PBPK) model combined with an EGFR occupancy (EO) model for osimertinib (OSI) to predict plasma trough concentration (Ctrough) and the intracranial time-course of EGFR (T790M and L858R mutants) engagement in patient populations. The PBPK model was also used to investigate the key factors affecting OSI pharmacokinetics (PK) and intracranial EGFR engagement, analyze resistance to the target mutation C797S, and determine optimal dosing regimens when used alone and in drug-drug interactions (DDIs). A population PBPK-EO model of OSI was developed using physicochemical, biochemical, binding kinetic, and physiological properties, and then validated using nine clinical PK studies, observed EO study, and two clinical DDI studies. The PBPK-EO model demonstrated good consistency with observed data, with most prediction-to-observation ratios falling within the range of 0.7 to 1.3 for plasma AUC, Cmax, Ctrough and intracranial free concentration. The simulated time-course of C797S occupancy by the PBPK model was much lower than T790M and L858R occupancy, providing an explanation for OSI on-target resistance to the C797S mutation. The PBPK model identified ABCB1 CLint,u, albumin level, and EGFR expression as key factors affecting plasma Ctrough and intracranial EO for OSI. Additionally, PBPK-EO simulations indicated that the optimal dosing regimen for OSI in patients with brain metastases is either 80 mg once daily (OD) or 160 mg OD, or 40 mg or 80 mg twice daily (BID). When used concomitantly with CYP enzyme perpetrators, the PBPK-EO model suggested appropriate dosing regimens of 80 mg OD with fluvoxamine (FLUV) itraconazole (ITR) or fluvoxamine (FLUC) for co-administration and an increase to 160 mg OD with rifampicin (RIF) or efavirenz (EFA). In conclusion, the PBPK-EO model has been shown to be capable of simulating the pharmacokinetic concentration–time profiles and the time-course of EGFR engagement for OSI, as well as determining the optimum dosing in various clinical situations.

Abstract Lung cancer will account for 21% of US cancer deaths in 2023 with 238,340 cases and 127,070 deaths. Treatment with EGFR-TKI (epidermal growth factor receptor tyrosine kinase) inhibitors initially decrease tumor growth, but often leads to TKI resistance. According to studies, non-small cell lung cancer (NSCLC) tumors express more SphK2 (Sphingosine kinase 2) than normal lung tissue and patients with overexpression have worse survival chances. However, SphK2's role in TKI resistance is unclear. We hypothesize that inhibiting/downregulating SphK2 could reduce tumorigenicity and overcome ER (Erlotinib) and OR (Osimertinib) resistance. We studied the expression of SphK2 in both drug-resistant (OR/ER) and drug sensitive (parental) H358, H2170, H3255, and PC9 NSCLC cell lines using Western blotting, qRT-PCR, and immunofluorescence. H3255OR cells were also bioprinted with GelMA hydrogel using an R-Gen 200 bioprinter to mimic the physiological tumor environment and treated with SphK2 siRNA or ABC294640 (SphK2i). Immunoblotting was done to analyze changes in the expression of SphK2 in H358 (WT-EGFR) and H3255 (mutant EGFR- L858R) parental and OR-resistant cell lines. Results indicated a 1.5-1.8-fold upregulation of SphK2. Increased SphK2 gene expression was observed using qPCR in TKI-resistant H3255OR (4.3-fold), PC9ER/OR with mutant EGFR (E746-A750del) (1.8-3.3-fold), and H2170ER/OR with WT EGFR (1.3-1.5-fold) compared to parental cells. Immunofluorescence studies showed increased fluorescence of SphK2 in H3255OR (2.6-fold), H358 ER (2.4-fold) and OR cells (2.3-fold) compared to parental cells. Spheroids formed from single cancer cells can "re-create" the growth of a tumor. The spheroid count of H3255OR was reduced by 73.98% and 56%, respectively, when SphK2i was added to the 3D-cell culture with Matrigel, compared to the diluent and OR alone (p≤0.01). We used 3D bioprinting to precisely stack cells on biological scaffolds to recreate lung tumors from patients and boost the therapeutic efficacy of these findings. When bioprinted spheroids of H3255OR were treated with SphK2i in combination with OR, the number of spheroids per microscopic field were reduced by 87.3% compared to OR alone. Bioprinted H3255OR spheroids treated with SphK2 siRNA and OR revealed a 75.8% and 84% reduction of spheroid count, respectively, compared to treatments with siRNA alone or OR alone (p≤0.05). This data supports the hypothesis that SphK2 suppression or downregulation can assist in mitigating oncogenesis. We also used H3255OR transwell migration assays to examine how SphK2 inhibition affects cell migration. Combining OR with SphK2i resulted in a 94.7% and 91.4% decrease in cell migration compared to diluent and OR alone (p≤0.01). In H358OR-resistant cells, combining OR with SphK2i resulted in a 94.97% inhibition compared to diluent (p≤0.01). These results suggest that targeting SphK2 could be a valuable approach for treating lung cancer. Citation Format: Ayesha Khan, Bamby Dieng, Namrata Dube, Mohammad Fazle Alam, Neelu Puri. Reduction in NSCLC tumorigenesis by targeting sphingosine kinase 2 [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 4675.

Abstract Lung cancer is the second most common cancer worldwide with 1.8 million deaths in 2020 being the leading cause of cancer related death. With 86% of cases, non-small cell lung cancer (NSCLC) is the most prominent subtype. The survival rate improved over the past 20 years by application of targeted therapies. Driver mutations in protein kinases such as the epidermal growth factor receptor (EGFR) are frequent and have evolved as therapeutic targets in anti-cancer therapy. Inhibitors of receptor tyrosine kinase (RTKIs), prominently EGFR-TKIs, are effective in anti-cancer therapy but tumor evolution inevitably generates resistance. Thus, strategies for the treatment of patients with acquired resistance to RTKIs remains an urgent need. Resistance in solid tumors is overcome by a combination of bortezomib with the BH3-mimetic ABT-199 (Muenchow et al. 2020). Also, overcoming ALK-TKI resistance in NSCLC was described by Tanimoto et al. (2021) by proteasome inhibiton that mediates induction of NOXA. Furthermore, the novel described BCL-2 independent activity of ABT-199 to transactivate NOXA expression (Weller et al. 2022) represents a generally active principle by combining drugs with independent mechanism of action. We hypothesize, that combined treatment of EGFR-TKI Osimertinib (OSI) with BH3-mimetics enhances sensitivity to apoptosis induction. To explore this hypothesis, we investigated cell death induction by combined administration of OSI and BH3-mimetics or PIs in NSCLC differing in their mutational status. In addition, we analyzed the expression of BCL-2 family proteins and their relevance and regulation during RTKI-induced cell death by RNA sequencing and Western Blot. These analyses will be performed in cellular model systems of NSCLC with specific resistance mediating mutations of EGFR or activated parallel survival pathways. We found that BCL-2 inhibitors enhance efficacy of OSI in NSCLC, resulting in synergistic cell death induction. Additionally, the sensitivity to BCL-2 inhibitors tends to increase in OSI-resistant NSCLC. Western Blot analysis and RNA sequencing data of OSI-resistant NSCLC identified BCL-2 overexpression and an EMT signature as resistance-mediating mechanisms, which can be overcome by combination of OSI with BCL-2 inhibitors. Indeed, our data suggest that combination of OSI with BCL-2 inhibitors disrupts resistance to OSI in NSCLC. We propose that BCL-2 inhibitors sensitize OSI-resistant NSCLC to effectively induce cell death. Thus, studying the molecular mechanism of synergistic efficacy of RTKI/BCL-2 inhibitor combination therapies, should prompt future efforts in the clinical evaluation of combinatorial regimens. We hypothesize that a general therapeutic concept which relies on simultaneous blocking of driver mutations and survival-promoting BCL-2 proteins represents a generally active principle of a tumor-agnostic mechanism of action. Citation Format: Sandra Weller, Tobias Beigl, Frank Essmann, Annika Harsch, Hans-Georg Kopp, Tom Ekstrom. Overcoming resistance to EGFR-TKI in NSCLC by simultaneously inhibition of anti-apoptotic proteins [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 6484.

Purpose: This study aimed to develop and validate a physiologically based pharmacokinetic (PBPK) model for osimertinib (OSI) to predict plasma trough concentration (Ctrough) and pulmonary EGFRm+ (T790M and L858R mutants) inhibition in Caucasian, Japanese, and Chinese populations. The PBPK model was also utilized to investigate inter-ethnic and inter-patient differences in OSI pharmacokinetics (PK) and determine optimal dosing regimens. Methods: Population PBPK models of OSI for healthy and disease populations were developed using physicochemical and biochemical properties of OSI and physiological parameters of different groups. And then the PBPK models were validated using the multiple clinical PK and drug-drug interaction (DDI) study data. Results: The model demonstrated good consistency with the observed data, with most of prediction-to-observation ratios of 0.8-1.25 for AUC, Cmax, and Ctrough. The PBPK model revealed that plasma exposure of OSI was approximately 2-fold higher in patients compared to healthy individuals, and higher exposure observed in Caucasians compared to other ethnic groups. This was primarily attributed to a lower CL/F of OSI in patients and Caucasian. The PBPK model displayed that key factors influencing PK and EGFRm+ inhibition differences included genetic polymorphism of CYP3A4, CYP1A2 expression, plasma free concentration (fup), albumin level, and auto-inhibition/induction on CYP3A4. Inter-patient PK variability was most influenced by CYP3A4 variants, fup, and albumin level. The PBPK simulations indicated that the optimal dosing regimen for patients across the three populations of European, Japanese, and Chinese ancestry was OSI 80mg once daily (OD) to achieve the desired range of plasma Ctrough (328-677nmol/L), as well as 80mg and 160mg OD for desirable pulmonary EGFRm+ inhibition (>80%). Conclusion: In conclusion, this study's PBPK simulations highlighted potential ethnic and inter-patient variability in OSI PK and EGFRm+ inhibition between Caucasian, Japanese, and Chinese populations, while also providing insights into optimal dosing regimens of OSI.

Drug resistance is the main cause of high mortality of lung cancer. This study was conducted to investigate the effect of folic acid (FA) on the resistance of non-small cell lung cancer (NSCLC) cells to Osimertinib (OSM) by regulating the methylation of dual specificity phosphatase 1 (DUSP1). The OSM resistant NSCLC cell line PC9R was establishd by gradually escalation of OSM concentration in PC9 cells. PC9R cells were randomly grouped into Control group, OSM group (5 μmol/L OSM), FA group (600 nmol/L FA), methylation inhibitor decitabine (DAC) group (10 μmol/L DAC), FA+OSM group (600 nmol/L FA+5 μmol/L OSM), and FA+OSM+DAC group (600 nmol/L FA+5 μmol/L OSM+10 μmol/L DAC). CCK-8 method was applied to detect cell proliferation ability. Scratch test was applied to test the ability of cell migration. Transwell assay was applied to detect cell invasion ability. Flow cytometry was applied to measure and analyze the apoptosis rate of cells in each group. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) method was applied to detect the expression level of DUSP1 mRNA in cells. Methylation specific PCR (MSP) was applied to detect the methylation status of the DUSP1 promoter region in each group. Western blot was applied to analyze the expression levels of DUSP1 protein and key proteins in the DUSP1 downstream mitogen-activated protein kinase (MAPK) signaling pathway in each group. Compared with the Control group, the cell OD450 values (48 h, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of extracellular regulated protein kinases (ERK) were obviously increased (P<0.05); the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the DAC group were obviously increased (P<0.05); the apoptosis rate, the expression of p38 MAPK protein, the phosphorylation level of ERK, and the methylation level of DUSP1 were obviously reduced (P<0.05). Compared with the OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously increased (P<0.05). Compared with the FA+OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM+DAC group were obviously increased; the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously reduced (P<0.05). FA may inhibit DUSP1 expression by enhancing DUSP1 methylation, regulate downstream MAPK signal pathway, then promote apoptosis, inhibit cell invasion and metastasis, and ultimately reduce OSM resistance in NSCLC cells.

P2.09-29 Efficacy of Osimertinib (OSI) vs 1st-Generation TKI Followed by OSI by Clinical Profile in EGFR-Mutant NSCLC (SMILE STUDY)