Osimertinib Resistance Mechanisms Research Articles

Abstract 4759: Targeting slow-cycling persisters in EGFR mutant non-small cell lung cancer

Abstract Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) patients are associated with clinical benefit in the metastatic and adjuvant settings when treated with EGFR inhibitors, such as osimertinib. Despite its dramatic efficacy, most patients are partial responders and refractive disease leave limited treatment options. The objective was to dissect the mechanism of osimertinib resistance in EGFR mutant NSCLC models to identify a means of re-sensitization. Imaging mass cytometric analysis of 76 NSCLC patients demonstrated EGFR expression was inversely correlated to CD105 membrane expression. CD105 is a transforming growth factor beta (TGF-β) family co-receptor that promotes bone morphogenic protein (BMP) signaling and inhibits TGF-β signaling. Significant elevation in cell surface CD105 by osimertinib was furthered by EGFR knockdown in two NSCLC lines with EGFR activating mutations. These lines were subjected to a program of increasing osimertinib concentrations to generate isogenic resistant lines. Heterogeneous drug-tolerant persister cells had significantly higher mutational load, increased CD105 cell surface expression as well as greater expression of bypass signaling factors such as ERK, PI3K, and BMP ligands compared to their parental counterparts. Single cell RNA-seq analysis of parental NSCLC revealed a small population of cells that shared elevated endothelial and pyrimidine metabolism, with a slow-cycling signature that was enriched in the osimertinib-resistant cells. Combining osimertinib with a CD105 neutralizing antibody, carotuximab, reduced the slow-cycling population and restored osimertinib sensitivity in resistant cell lines. The knockdown of CD105 had a more pronounced decrease in cell viability when combined with osimertinib. The osimertinib-resistant lines demonstrated more condensed chromatin and glycolytic state compared to the respective parental lines by ATAC-seq and Single Cell ENergetIc metabolism profiling, respectively. The addition of carotuximab reversed the chromatin and metabolic reprograming of osimertinib resistance. Carotuximab was discovered to initiate CD105 interaction with EGFR and its dominant active variant, EGFRv3. Ultimately, combination therapy of carotuximab and osimertinib resulted in significantly reduced tumor expansion compared to mice treated with either drug alone. Paradoxically, the small tumors from combination therapy had greater mitosis compared to the larger tumors of the osimertinib single agent treated mice. Inhibition of CD105 with carotuximab can overcome resistance to osimertinib and inhibit NSCLC tumor progression. This is a first-in-class pre-clinical foundation for a novel synthetic lethality treatment strategy resulting from the observation of global changes in EGFR-antagonist resistance as opposed to individual mutations identified in tumor subpopulations. Citation Format: Manish Thiruvalluvan, Sandrine Billet, Zhenqiu Liu, Joseph Lownik, Gabrielle Gonzales, Hyoyoung Kim, Anton L. Villamejor, Larry Milshteyn, Kamya Sankar, Edwin M. Posadas, Jean Lopategui, Sungyong You, Karen Reckamp, Neil A. Bhowmick. Targeting slow-cycling persisters in EGFR mutant non-small cell lung cancer [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 4759.

Abstract 7195: Repurposing non-oncology drugs with MET inhibitory effect to overcome osimertinib resistance in lung cancer

Abstract Background and Aim: Osimertinib is the only 3rd generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor approved for the 1st line therapy of advanced NSCLC patients with EGFR mutations. However, drug resistance is severely limiting its clinical efficacy. Aberrant MET activation is an important mechanism causing osimertinib resistance. There is no effective therapeutic option after osimertinib failure. This study investigated the repurposing of non-oncology MET inhibiting drug to overcome osimertinib resistance. Method: A few clinically-approved drugs with putative MET inhibitory effects were identified by a computational drug repurposing tool called “DRAR-CPI” via analysis of the chemical-protein interactome of known MET inhibitors (crizotinib, cabozantinib, foretinib, and tivantinib). Fexofenadine (FEX), originally indicated for allergic rhinitis and chronic urticaria, was chosen for detailed investigation in osimertinib-resistant NSCLC cell lines that exhibit aberrant MET activation with/without other EGFR-dependent osimertinib resistance mechanisms. The selectivity of kinase inhibition by FEX was evaluated by a KINOME profiling assay against a panel of human protein kinases. Western blot analysis was conducted to examine the activation status of Met and EGFR downstream signaling. RNA sequencing was performed to identify differentially expressed genes (DEGs) triggered by the most promising repurposed drug, presumably contributing to osimertinib resistance circumvention. Sulforhodamine B assay was used to evaluate cell proliferation. The circumvention of osimertinib resistance by FEX was further verified in a patient-derived tumor xenograft (PDX) from an advanced-stage osimertinib-refractory NSCLC patient. Result: FEX was found to inhibit recombinant MET kinase in cell-free assays in a dose- and time-dependent manner. It also inhibited MET phosphorylation and other downstream signaling molecules in osimertinib-resistant NSCLC cell lines harboring EGFR T790M+MET amplification or EGFR T790M loss+MET amplification. KINOME profiling revealed a similar kinase inhibition profile between FEX and cabozantinib using Spearman rank order correlation analysis. Among the tested osimertinib-resistant NSCLC cell lines, FEX showed the highest effectiveness in potentiating the anticancer effect of osimertinib in H820 cells (having MET amplification & EGFR T790M mutation). Transcriptome profiling analysis in H820 cells before and after FEX treatment revealed that the majority of DEGs were enriched in EMT-related biological processes and pathways. FEX was further shown to significantly potentiate the antitumor effect of osimertinib in a drug refractory PDX model, without inducing notable weight loss in the animals. Conclusion: These findings advocate the clinical evaluation of repurposing FEX to overcome osimertinib resistance. Citation Format: Kenneth K.W. To, Kwong Sak Leung, William C.S. Cho. Repurposing non-oncology drugs with MET inhibitory effect to overcome osimertinib resistance in lung cancer [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 7195.

Phosphoproteomic Analysis Identified Mutual Phosphorylation of FAK and Src as a Mechanism of Osimertinib Resistance in EGFR-Mutant Lung Cancer

IntroductionOsimertinib is a standard treatment for patients with EGFR-mutant NSCLC. Although some osimertinib resistance mechanisms have been identified, nearly 50% of the mechanisms remain to be elucidated. This study was aimed at identifying non-genetic mechanisms underlying osimertinib resistance. MethodsWe established two osimertinib-resistant cell lines from EGFR mutation-positive PC-9 and HCC827 NSCLC cell lines (PC-9OR and HCC827OR, respectively) using a stepwise method. We compared the phosphoproteomic profiles of the osimertinib-resistant and parental cells using mass spectrometry. Upstream kinases were identified using the application Kinase Enrichment Analysis version 3. ResultsPhosphoproteomic analysis revealed 80 phosphorylation sites that were mutually up-regulated in PC-9OR and HCC827OR cells. The Kinase Enrichment Analysis version 3 analysis identified focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src) as upstream kinases of these up-regulated phosphoproteins. The small-interfering RNA–mediated knockdown of FAK reduced Src phosphorylation and that of Src reduced FAK phosphorylation in both cell lines. Furthermore, FAK- or Src-specific small-interfering RNA treatments restored EGFR phosphorylation in PC-9OR and HCC827OR cells. The combination of FAK and Src inhibitors inhibited PC-9OR and HCC827OR cell proliferation in vitro and suppressed tumor growth in a xenograft mouse model. Immunohistochemistry of tumors from patients with EGFR-mutant NSCLC suggested that phosphorylated FAK and Src are involved in initial and acquired resistance to osimertinib. ConclusionsPhosphoproteomic analysis may help elucidate the mechanisms of resistance to molecular-targeted therapies in lung cancer. Mutual phosphorylation of FAK and Src is involved in osimertinib resistance. Thus, FAK and Src inhibition may be novel treatment strategies for osimertinib-resistant NSCLC.

Genetic mutation profiling reveals biomarkers for targeted therapy efficacy and prognosis in non-small cell lung cancer

IntroductionThe genetic heterogeneity of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations may affect clinical responses and outcomes to EGFR tyrosine kinase inhibitors (EGFR-TKIs). This study aims to investigate the genomic factors that influence the efficacy and clinical outcomes of first-line, second-line and third-line treatments in NSCLC and explore the heterogeneity of resistance mechanisms. Materials and methodsThis real-world study comprised 65 patients with EGFR mutant NSCLC. Molecular alterations were detected using a customized DNA panel before and after administering targeted therapy. The efficacy and prognosis of each treatment line were evaluated. ResultsIn first-generation EGFR-TKIs treatment, gefitinib showed favorable efficacy compared to icotinib and erlotinib, particularly in patients with EGFR L858R mutations. The resistance mechanisms to first-generation EGFR-TKIs varied among different EGFR mutation cohorts and different first-generation EGFR-TKIs. In second-line EGFR-TKIs treatment, EPH receptor A3 (EPHA3), IKAROS family zinc finger 1 (IKZF1), p21 (RAC1) activated kinase 5 (PAK5), DNA polymerase epsilon, catalytic subunit (POLE), RAD21 cohesin complex component (RAD21) and RNA binding motif protein 10 (RBM10) mutations were markedly associated with poorer progression-free survival (PFS). Notably, EPHA3, IKZF1 and RBM10 were identified as independent predictors of PFS. The mechanisms of osimertinib resistance exhibited heterogeneity, with a higher proportion of non-EGFR-dependent resistant mutations. In third-line treatments, the combination of osimertinib and anlotinib demonstrated superior efficacy compared to other regimens. Glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A) mutation was an independent risk indicator of shorter OS following third-line treatments. ConclusionsComprehending the tumor evolution in NSCLC is advantageous for assessing the efficacy and prognosis at each stage of treatment, providing valuable insights to guide personalized treatment decisions for patients.

Sialyltransferase ST3GAL4 confers osimertinib resistance and offers strategies to overcome resistance in non-small cell lung cancer

The third-generation EGFR-TKI osimertinib is widely used in EGFR-mutated positive non-small cell lung cancer (NSCLC) patients, but drug resistance is inevitable. The currently known mechanisms only explain resistance in a small proportion of patients. For most patients, the mechanism of osimertinib resistance is still unclear, especially for EGFR-independent resistance. Herein, we thoroughly investigated the novel mechanism of osimertinib resistance and treatment strategies. We identified that ST3GAL4, a sialyltransferase, catalyzes terminal glycan sialylation of receptor protein tyrosine kinases, which induces acquired resistance to osimertinib in vitro and in vivo. In addition, ST3GAL4 is generally overexpressed in osimertinib-resistant patients with unknown resistance mechanisms. ST3GAL4 modifies MET glycosylation on N785 with sialylation, which antagonizes K48-related ubiquitin-dependent MET degradation and subsequently activates MET and its downstream proliferation signaling pathways. Meanwhile, ST3GAL4 knockdown or inhibition by brigatinib resensitizes resistant non-small cell lung cancer cells to osimertinib in vitro and in vivo This study suggests that ST3GAL4 can induce acquired resistance to osimertinib, which may be an important EGFR-independent resistance mechanism Furthermore, targeting ST3GAL4 with brigatinib provides new strategies to overcome osimertinib resistance.

Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer.

Using a novel cohort of in vivo PDX models of MET pathway activation with acquired resistance to osimertinib in EGFR-mutant lung cancer, we demonstrate that phospho-MET may be a clinically relevant assay to guide treatment selection with osimertinib and savolitinib combination. In addition, our work shows that patients with MET polysomy tumors may have subclonal MET amplification and therefore require close follow up for the use of osimertinib and savolitinib combination.

561P Mechanisms of osimertinib resistance using circulating tumor DNA analyses for EGFR-mutated non-small cell lung cancer, results from ELUCIDATOR: A prospective observational multicenter study

561P Mechanisms of osimertinib resistance using circulating tumor DNA analyses for EGFR-mutated non-small cell lung cancer, results from ELUCIDATOR: A prospective observational multicenter study

A Phase II Study of Osimertinib in Patients with Advanced-Stage Non-Small Cell Lung Cancer following Prior Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (EGFR TKI) Therapy with EGFR and T790M Mutations Detected in Plasma Circulating Tumour DNA (PLASMA Study).

Epidermal growth factor receptor (EGFR) T790M mutations drive resistance in 50% of patients with advanced non-small cell lung cancer (NSCLC) who progress on first/second generation (1G/2G) EGFR tyrosine kinase inhibitors (TKIs) and are sensitive to Osimertinib. Tissue sampling is the gold-standard modality of T790M testing, but it is invasive. We evaluated the efficacy of Osimertinib in patients with EGFR mutant NSCLC and T790M in circulating tumour DNA (ctDNA). PLASMA is a prospective, open-label, multicentre single-arm Phase II study. Patients with advanced NSCLC harbouring sensitizing EGFR and T790M mutations in plasma at progression from ≥one 1G/2G TKI were treated with 80 mg of Osimertinib daily until progression. The primary endpoint was the objective response rate (ORR); the secondary endpoints included progression-free survival (PFS), overall survival (OS), disease control rate (DCR) and toxicities. Plasma next-generation sequencing was performed to determine Osimertinib resistance mechanisms and assess serial ctDNA. A total of 110 patients from eight centres in five countries were enrolled from 2017 to 2019. The median follow-up duration was 2.64 (IQR 2.44-3.12) years. The ORR was 50.9% (95% CI 41.2-60.6) and the DCR was 84.5% (95% CI 76.4-90.7). Median PFS was 7.4 (95% CI 6.0-9.3) months; median OS was 1.63 (95% CI 1.35-2.16) years. Of all of the patients, 76% had treatment-related adverse events (TRAEs), most commonly paronychia (22.7%); 11% experienced ≥ Grade 3 TRAEs. The ctDNA baseline load and dynamics were prognostic. Osimertinib is active in NSCLC harbouring sensitizing EGFR and T790M mutations in ctDNA testing post 1G/2G TKIs.

Real-World Genomic Profile of EGFR Second-Site Mutations and Other Osimertinib Resistance Mechanisms and Clinical Landscape of NSCLC Post-Osimertinib

IntroductionThe emergence of osimertinib as standard of care for EGFR-mutant NSCLC has renewed the need to understand and overcome drug resistance. We sought to understand the genomics and real-world treatment landscape of NSCLC with EGFR C797S and other on- and off-target resistance mechanisms. MethodsComprehensive genomic profiling (CGP) results from tissue or blood samples from 93,065 patients with NSCLC were queried for osimertinib EGFR second-site resistance mutations (ssEGFRms; C797, L718, G724, G796, L792). A real-world electronic health record–derived deidentified clinicogenomic database of patients with NSCLC undergoing CGP from approximately 280 U.S. cancer clinics was queried to assess post-osimertinib resistance and clinical treatment outcomes. ResultsA ssEGFRm was identified in 239 of 8845 (2.7%) EGFR-driven (L858R or exon 19 deletion) NSCLCs, most frequently C797 (71%), L718 (15%), and G724 (9.5%). ssEGFRms were not equally distributed across drivers; C797 and G724 changes strongly favored exon 19 deletion and L718, G796 and L792 favored L858R. Post-osimertinib CGP detected ssEGFRm in 19% of the cases (39 of 205); in paired pre-/post-osimertinib samples, on- and off-target resistance was largely mutually exclusive and observed in 24% and 27% of the cases, respectively. Of 391 patients with post-osimertinib treatment data, 62% received a chemotherapy-based regimen, whereas 25% received a targeted therapy or clinical study drug. Median real-world overall survival was 11.4 months from osimertinib progression. ConclusionsThe osimertinib resistance landscape is diverse with on-target ssEGFRm and off-target resistance detected in tissue and liquid biopsy. Post-osimertinib, patients are receiving primarily chemotherapy-based regimens with poor outcomes, and CGP at resistance may offer an opportunity to inform therapeutic development and improve treatment selection.

Osimertinib Induces the Opposite Effect of Proliferation and Migration in the Drug Resistance of EGFR-T790M Non-small Cell Lung Cancer Cells.

Lung cancer has become one of the leading causes of cancer incidence and mortality worldwide. Non-small cell lung carcinoma (NSCLC) is the most common type among all lung cancer cases. NSCLC patients contained high levels of activating epidermal growth factor receptor (EGFR) mutations, such as exon 19 deletion, L858R and T790M. Osimertinib, a third-generation of EGFR tyrosine kinase inhibitor (EGFR-TKI), has therapeutic efficacy on the EGFR-T790M mutation of NSCLC patients; however, treatment of osimertinib still can induce drug resistance in lung cancer patients. Therefore, investigation of the drug resistance mechanisms of osimertinib will provide novel strategies for lung cancer therapy. The H1975OR osimertinib-resistant cell line was established by prolonged exposure with osimertinib derived from the H1975 cells. The cell proliferation ability was evaluated by the cell viability and cell growth assays. The cell migration ability was determined by the Boyden chamber assays. The differential gene expression profile was analyzed by genome-wide RNA sequencing. The protein expression and location were analyzed by western blot and confocal microscopy. In this study, we established the osimertinib-resistant H1975 (T790M/L858R) cancer cells, named the H1975OR cell line. The cell growth ability was decreased in the H1975OR cells by comparison with the H1975 parental cells. Conversely, the cell migration ability was elevated in the H1975OR cells. We found the differential gene expression profile of cell proliferation and migration pathways between the H1975OR and H1975 parental cells. Interestingly, the protein levels of phospho-EGFR, PD-L1, E-cadherin and β-catenin were decreased, but the survivin and N-cadherin proteins were increased in the H1975OR drug-resistant cells. Osimertinib induces the opposite effect of proliferation and migration in the drug resistance of EGFRT790M lung cancer cells. We suggest that differential gene and protein expressions in the cell proliferation and migration pathways may mediate the drug resistance of osimertinib in lung cancer cells. Understanding the molecular drugresistant mechanisms of proliferation and migration pathways of osimertinib may provide novel targets and strategies for the clinical treatment of EGFR-TKIs in lung cancer patients.

The potential therapeutic regimen for overcoming resistance to osimertinib due to rare mutations in NSCLC

The mechanisms of osimertinib resistance have not been well characterized. We conducted next-generation sequencing to recognize novel resistance mechanism and used cell line-derived xenograft (CDX) and patient-derived xenograft (PDX)models to evaluate the anti-proliferative effects of aspirin invivo and invitro. We observed that PIK3CG mutations led to acquired resistance to osimertinibin a patient and further confirmed that both PIK3CG and PIK3CA mutations caused osimertinib resistance. Mechanistically, the expression of PI3Kγ or PI3Kα was up-regulated after PIK3CG or PIK3CA lentivirus transfection, respectively, and which can be effectively suppressed by aspirin. Lastly, our results from invivo studies indicate that aspirin can reverse osimertinib resistance caused byPIK3CG or PIK3CA mutations in both CDX and PDX models. Herein, we first confirmed that mutations in PIK3CG can lead to resistance to osimertinib, and the combined therapy may be a strategy to reverse PIK3CG/PIK3CA mutation-induced osimertinib resistance.

Current treatment strategies for EGFR-mutated non-small cell lung cancer: from first line to beyond osimertinib resistance.

Osimertinib, a third-generation EGFR TKI, is the standard therapy for previously untreated EGFR-mutated non-small cell lung cancer patients following the landmark FLAURA study. However, resistance inevitably hinders patient prognosis, increasing the need for new therapeutic strategies beyond osimertinib. Frontline osimertinib-based combination strategies (platinum-based chemotherapy and angiogenesis inhibitors) are currently being tested primarily to prevent initial resistance. In the later-line setting after osimertinib, many next-line therapeutic candidates have been actively examined in clinical trials. Notably, several drugs with novel mechanisms of action, such as antibody-drug conjugates and EGFR -MET bispecific antibodies, have shown promising efficacy despite the resistance mechanisms and are close to clinical application. In addition, genotype-based target strategies have been investigated for a better understanding of osimertinib resistance mechanisms based on molecular profiling tests at relapse. The C797S mutation and MET gene alterations are commonly identified following osimertinib resistance, for which targeting strategies are actively tested. This review describes current pharmacotherapeutic strategies for EGFR-mutated non-small cell lung cancer based on the results of clinical trials and the latest published data, broadly grouped into two sections: 1) EGFR TKIs-based combination therapy in the front-line setting and 2) novel therapeutic strategies after osimertinib resistance.

Genomic profiling of cerebrospinal fluid ctDNA to characterize molecular features and monitor intracranial responses of patients with osimertinib-resistant non-small cell lung cancer with leptomeningeal metastases.

e21095 Background: Leptomeningeal metastasis (LM) indicates an unfavorable prognosis in non-small cell lung cancer (NSCLC). Due to the blood-brain barrier, plasma is not an ideal medium to evaluate osimertinib resistance mechanisms in patients with LM. This study aimed to use cerebrospinal fluid (CSF) as a source of ctDNA for characterizing molecular features and monitoring intracranial responses from patients with leptomeningeal metastases who progressed on osimertinib. Methods: From Jan 2020 to Nov 2022, 65 patients with advanced NSCLC, including 32 osimertinib-treated patients, 6 other TKI-treated patients, and 27 treatment-naïve patients, in Beijing Tiantan Hospital were enrolled in this study. CSF obtained via lumbar puncture was used for targeted sequencing and cytologic analysis. In parallel, paired plasma from 35 patients was also subjected to targeted sequencing. Results: Among 65 patients, the overall positive rate of CSF ctDNA was 77% (50/65), 94% (34/36) in patients with LM, and 55% (16/29) in patients with brain parenchymal metastases (p = 0.0002). CSF ctDNA analysis had a higher consistency with the cytologic diagnosis of LM than imaging. Among the patients cytologic diagnosed with LM, 94% (32/34) of the patients had positive CSF ctDNA analysis at the same time, while the positive diagnostic rate of imaging was only 39% (13/33). EGFR alteration was found in 97% (29/30) CSF of patients with LM who progressed on Osimertinib, including L858R (67%), exon19 deletion (17%), T790M (10%), L861Q (7%), G719X (7%) and L747X (7%). Other recurrent genes discovered in the CSF were TP53 (73%), PIK3CA (43%), ERBB2 (33%), POLE (33%), ALK (27%), BRCA2 (27%), BRAF (23%), BRCA1 (23%), RB1 (20%) and PALB (20%). In the 35 paired CSF-plasma pairs, more alterations were uniquely found in CSF than in plasma (71% vs 17%), and only 12% of alterations were shared. Serial surveillance of CSF ctDNA in 7 cases of LM patients treated with osimertinib found that the VAF of CSF ctDNA showed a highly consistent trend with the tumor cell fraction of cytology. Conclusions: For NSCLC patients with LM, liquid biopsy of CSF is more sensitive than plasma to characterize osimertinib-resistant mechanisms and guide follow-up treatment. Serial surveillance of CSF ctDNA dynamics may reflect the intracranial tumor responses, which still needs to be validated in a large sample size.

First report of ELUCIDATOR study: A prospective observational multicenter study on resistance mechanisms in patients with early disease progression during osimertinib.

3032 Background: Osimertinib (Osi) is the key standard therapy for patients (pts) with chemo-naive advanced non-small cell lung cancer (NSCLC) harboring sensitizing epidermal growth factor receptor ( EGFR) mutations. However, many of cases treated with Osi experience the disease progression (PD). Therefore, understanding first-line osimertinib resistance mechanisms is essential for future therapeutic strategies. Here, we addressed the underlying mechanisms of resistance to Osi using paired plasma DNA samples and particularly focused on early PD (PD within 6 months) cases. Methods: This trial is the multicenter prospective research, and we prospectively collected plasma DNA samples prior Osi treatment and at PD. Next-generation sequencing (NGS) implemented with the molecular barcoding method was performed to analyze gene mutations in 197 cancer-associated genes. Then using these ct-DNA, we analyzed the Osi resistance by ultra-sensitive NGS. In this interim analysis, we specially analyze the early PD (PD within 6 months) cases. Results: One hundred eighty-eight pts were enrolled between May 2019 and January 2021. Among these pts, 125 (66%) pts were female, 96 (51%) were EGFR exon 19 deletion mutation, and 109 (58%) were never smoker. Seventy-eight pts experienced PD and 36 pts experienced early PD at interim analysis. Baseline shedders, patients with plasma positivity for EGFR-activating mutation were more frequently observed in early PD pts than non-PD (86.1% vs 63.4%, p = 0.009). Early PD patients had significantly more co-occurring gene mutations along with EGFR mutations than non-early PD patients (2.89±1.49 vs 1.97±1.37, p = 0.002). Most notably, 3 among 36 early PD pts and 8 among 78 PD pts had significant reduction of plasma EGFR mutant allele frequency at PD than that at baseline. Among those, 1 had germline BRCA1 mutation and 1 had BRINP3 mutation in 3 early PD pts, and 1 had germline BRCA2 mutation, and 1 had germline TP53 mutation in 5 PD pts excluding early PD pts. Co-occurring TP53 mutations were not relevant to early PD in this study cohort. Conclusions: Germline mutations in BRCA1/2, TP53 and BRINP3 may confer early and unprecedented resistance to Osi monotherapy. Plasma-based serial comprehensive gene profiling may help predict and identify patients unlikely to benefit from Osi treatment, though further study is warranted. Clinical trial information: jRCTs031180051 .

Tackling Osimertinib Resistance in EGFR-Mutant Non-Small Cell Lung Cancer.

The current landscape of targeted therapies directed against oncogenic driver alterations in non-small cell lung cancer (NSCLC) is expanding. Patients with EGFR-mutant NSCLC can derive significant benefit from EGFR tyrosine kinase inhibitor (TKI) therapy, including the third-generation EGFR TKI osimertinib. However, invariably, all patients will experience disease progression with this therapy mainly due to the adaptation of cancer cells through primary or secondary molecular mechanisms of resistance. The comprehension and access to tissue and cell-free DNA next-generation sequencing have fueled the development of innovative therapeutic strategies to prevent and overcome resistance to osimertinib in the clinical setting. Herein, we review the biological and clinical implications of molecular mechanisms of osimertinib resistance and the ongoing development of therapeutic strategies to overcome or prevent resistance.

Novel CDK12/13 Inhibitors AU-15506 and AU-16770 Are Potent Anti-Cancer Agents in EGFR Mutant Lung Adenocarcinoma with and without Osimertinib Resistance.

Osimertinib is a third-generation epidermal growth factor receptor and tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of lung adenocarcinoma patients harboring EGFR mutations. However, acquired resistance to this targeted therapy is inevitable, leading to disease relapse within a few years. Therefore, understanding the molecular mechanisms of osimertinib resistance and identifying novel targets to overcome such resistance are unmet needs of cancer patients. Here, we investigated the efficacy of two novel CDK12/13 inhibitors, AU-15506 and AU-16770, in osimertinib-resistant EGFR mutant lung adenocarcinoma cells in culture and xenograft models in vivo. We demonstrate that these drugs, either alone or in combination with osimertinib, are potent inhibitors of osimertinib-resistant as well as -sensitive lung adenocarcinoma cells in culture. Interestingly, only the CDK12/13 inhibitor in combination with osimertinib, although not as monotherapy, suppresses the growth of resistant tumors in xenograft models in vivo. Taken together, the results of this study suggest that inhibition of CDK12/13 in combination with osimertinib has the potential to overcome osimertinib resistance in EGFR mutant lung adenocarcinoma patients.

Therapeutic strategies for EGFR-mutated non-small cell lung cancer patients with osimertinib resistance

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the preferential options for advanced non-small cell lung cancer (NSCLC) patients harboring EGFR mutations. Osimertinib is a potent irreversible third-generation EGFR-TKI targeting EGFR mutations but has little effect on wild-type EGFR. In view of its remarkable efficacy and manageable safety, osimertinib was recommended as the standard first-line treatment for advanced or metastatic NSCLC patients with EGFR mutations. However, as the other EGFR-TKIs, osimertinib will inevitably develop acquired resistance, which limits its efficacy on the treatment of EGFR-mutated NSCLC patients. The etiology of triggering osimertinib resistance is complex including EGFR-dependent and EGFR-independent pathways, and different therapeutic strategies for the NSCLC patients with osimertinib resistance have been developed. Herein, we comprehensively summarized the resistance mechanisms of osimertinib and discuss in detail the potential therapeutic strategies for EGFR-mutated NSCLC patients suffering osimertinib resistance for the sake of the improvement of survival and further achievement of precise medicine.

Feiyanning formula modulates the molecular mechanism of osimertinib resistance in lung cancer by regulating the Wnt/β-catenin pathway.

Feiyanning Formula (FYN), a Chinese herbal formula derived from summarized clinical experience, is proven to have anti-tumor effects in lung cancer patients. Osimertinib, a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), can improve progression-free survival and overall survival of patients but drug resistance is inevitable. The current study evaluated the effects of FYN in osimertinib-resistant HCC827OR and PC9OR cells. FYN preferentially inhibited the proliferation and migration of HCC827OR and PC9OR cells. Moreover, FYN and osimertinib exhibited synergistic inhibitory effects on proliferation and migration. Real-time qPCR (RT-qPCR) and western blotting results indicated that FYN downregulated gene and protein levels of GSK3β and SRFS1, which are enriched in the Wnt/β-catenin pathway. Besides, FYN inhibited tumor growth and exhibited synergistic effects with osimertinib in vivo. Collectively, the results suggested that FYN exerted an anti-osimertinib resistance effect via the Wnt/β-catenin pathway.

EP08.02-022 Investigating Partners in Crime: Osimertinib Resistance Mechanisms in Non-small Cell Lung Cancer Using Focused CRISPR Screen

Non-small cell lung cancer (NSCLC) is the most prevalent and detrimental cancer type, accounting for 85% of all lung cancer cases. EGFR mutations, which can be targeted by tyrosine kinase inhibitors (TKIs), have been addressed as one of the cancer driving mechanisms in NSCLC adenocarcinomas. Osimertinib, a third generation EGFR-TKI, has been recently approved for clinical use in the first-line treatment of advanced NSCLC. Unfortunately, the long-standing obstacle in EGFR-TKI treatments, including Osimertinib, is the inevitable resistance.

L718Q/V mutation in exon 18 of EGFR mediates resistance to osimertinib: clinical features and treatment

Osimertinib, a mutant-specific third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is emerging as the preferred first-line of treatment for EGFR-mutant lung cancer. However, osimertinib resistance inevitably develops among patients treated with the drug. The modal resistance mechanisms of osimertinib include the occurrence of epithelial transition factor (c-MET) amplification and C797S mutation, whereas rare mutations are presented as case reports. Recently, the L718Q/V mutation in exon 18 of EGFR has been reported to contribute to one of the possible mechanisms of resistance. The clinical features and subsequent treatment strategies for this mutation require further research. This study retrospectively enrolled NSCLC patients with the L718Q/V mutation from 2017 to 2021 at the Cancer Hospital of the University of the Chinese Academy of Sciences (Zhejiang Cancer Hospital), as well as additional patients with the same mutation from PubMed literature, to summarize the clinical features of the mutation. The association between the detection of L718Q/V and resistance to osimertinib, as well as impacts on the therapeutic process and outcome, was analyzed. We included a total of two patients diagnosed at Zhejiang Cancer Hospital and twelve patients from the literature. Of the fourteen total patients, 64.3% were male and 35.7% were female. The average age of the group was 60.2 years (range 45–72). A history of tobacco use was common among the group. In all of the cases we considered, the L718Q/V mutation was secondary to the L858R mutation. The second-generation TKI afatinib was found to provide a high disease control rate (DCR) (85.7%, 6/7) and relatively low objective response rate (ORR) (42/9%, 3/7). The median progression free survival (mPFS) for this treatment reached 2 months (1–6 months). The patients failed to benefit from chemotherapy combined with immunotherapy or other TKI medications. Due to the limited number of cases considered in this study, future studies should explore drugs that more precisely target the L718Q/V mutation of EGFR exon 18.