Osimertinib-resistant Non-small Cell Lung Cancer Research Articles

The MEK/ERK/miR-21 Signaling Is Critical in Osimertinib Resistance in EGFR-Mutant Non-Small Cell Lung Cancer Cells.

Simple SummaryOur study provided data that the inhibition of MEK/ERK signaling could overcome Osimertinib resistance both in vitro and in vivo. Mechanistically, MEK inhibitor Trametinib suppressed the tumorigenic properties of NSCLC cells by reducing the generation of CAFs. The trametinib-mediated anti-cancer function was also associated with the significantly suppressed level of miR-21, of which primary targets included PDCD4, as shown in this study and MEK inhibitor Trametinib significantly suppressed Osimertinib-resistant NSCLC tumor growth by abolishing both processes.Background: The third-generation epidermal growth factor receptor (EGFR) inhibitor, Osimertinib, is used to treat non-small cell lung cancer (NSCLC) patients with tyrosine kinase inhibitor (TKI) resistance caused by acquired EGFR T790M mutation. However, patients eventually develop resistance against Osimertinib with mechanisms not yet fully clarified. Activated alternative survival pathways within the tumor cells and cancer-associated fibroblasts (CAFs) have been proposed to contribute to Osimertinib resistance. MET and MEK inhibitors may overcome EGFR-independent resistance. Another acquired resistance mechanism of EGFR-TKI is the up-regulation of the RAS/RAF/MEK/ERK signaling pathway, which is the key to cell survival and proliferation; this may occur downstream of various other signaling pathways. In this report, we reveal the possible regulatory mechanism and inhibitory effect of the MEK inhibitor trametinib applied to MEK/ERK/miR-21 axis and PDCD4 in Osimertinib resistance. We found a possible regulatory role of PDCD4 in ERK signaling. PDCD4 is a new type of tumor suppressor that has multiple functions of inhibiting cell growth, tumor invasion, metastasis, and inducing apoptosis. Previous bioinformatics analysis has confirmed that PDCD4 contains the binding site of miR-21 and acts as a tumor suppressor in the regulation of various processes associated with the development of cancer, including cell proliferation, invasion, metastasis, and neoplastic transformation. Based on the above analysis, we hypothesized that the tumor suppressor PDCD4 is one of the effective inhibitory targets of miR-21-5p. Methods: The expression between EGFR and ERK2 in lung adenocarcinoma was evaluated from the TCGA database. Osimertinib-sensitive and resistant NSCLC cells obtained from patients were used to co-culture with human lung fibroblasts (HLFs) to generate CAF cells (termed CAF_R1 and CAF_S1), and the functional roles of these CAF cells plus the regulatory mechanisms were further explored. Then, MEK inhibitor Trametinib with or without Osimertinib was applied in xenograft model derived from patients to validate the effects on growth inhibition of Osimertinib-resistant NSCLC tumors. Result: ERK2 expression correlated with EGFR expression and higher ERK2 level was associated with worse prognosis of patients and Osimertinib resistance. CAFs derived from Osimertinib-resistant cells secreted more IL-6, IL-8, and hepatocyte growth factor (HGF), expressed stronger CAF markers including α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP) plus platelet-derived growth factor receptor (PDGFR), and enhanced stemness and Osimertinib resistance in NSCLC cells. Meanwhile, increased MEK/ERK/miR-21 expressions were found in both CAFs and NSCLC cells. MEK inhibitor Trametinib significantly abrogated the abovementioned effects by modulating β-catenin, STAT3, and ERK. The xenograft model showed combining Osimertinib and Trametinib resulted in the most prominent growth inhibition of Osimertinib-resistant NSCLC tumors. Conclusions: Our results suggested that MEK/ERK/miR-21 signaling is critical in Osimertinib resistance and CAF transformation of NSCLC cells, and MEK inhibitor Trametinib significantly suppressed Osimertinib-resistant NSCLC tumor growth by abolishing both processes.

FBXL2 counteracts Grp94 to destabilize EGFR and inhibit EGFR-driven NSCLC growth

Abnormal activation of epidermal growth factor receptor (EGFR) drives non-small cell lung cancer (NSCLC) development. EGFR mutations-mediated resistance to tyrosine-kinase inhibitors (TKIs) is a major hurdle for NSCLC treatment. Here, we show that F-box protein FBXL2 targets EGFR and EGFR TKI-resistant mutants for proteasome-mediated degradation, resulting in suppression of EGFR-driven NSCLC growth. Reduced FBXL2 expression is associated with poor clinical outcomes of NSCLC patients. Furthermore, we show that glucose-regulated protein 94 (Grp94) protects EGFR from degradation via blockage of FBXL2 binding to EGFR. Moreover, we have identified nebivolol, a clinically used small molecule inhibitor, that can upregulate FBXL2 expression to inhibit EGFR-driven NSCLC growth. Nebivolol in combination with osimertinib or Grp94-inhibitor-1 exhibits strong inhibitory effects on osimertinib-resistant NSCLC. Together, this study demonstrates that the FBXL2-Grp94-EGFR axis plays a critical role in NSCLC development and suggests that targeting FBXL2-Grp94 to destabilize EGFR may represent a putative therapeutic strategy for TKI-resistant NSCLC.

PAR2 blockade reverses osimertinib resistance in non-small-cell lung cancer cells via attenuating ERK-mediated EMT and PD-L1 expression

Osimertinib, as the third-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs), is a first-line molecularly targeted drug for non-small cell lung cancer (NSCLC). However, the emergence of therapeutic resistance to osimertinib markedly impairs its efficiency and efficacy, leading to the failure of clinical applications. Novel molecular targets and drugs are urgently needed for reversing osimertinib resistance in NSCLC. Protease-activated receptor 2 (PAR2) that belongs to a subfamily of G protein-coupled receptors can stimulate the transactivation of EGFR to regulate multiple cellular signalling, actively participating in tumour progression. This study firstly discovered that PAR2 expression was notably enhanced when NSCLC cells became resistant to osimertinib. A PAR2 inhibitor facilitated osimertinib to attenuate EGFR transactivation, ERK phosphorylation, EMT and PD-L1 expression which were associated to osimertinib resistance. The combination of the PAR2 inhibitor and osimertinib also notably blocked cell viability, migration, 3D sphere formation and in vivo tumour growth whereas osimertinib itself lost such inhibitory effects in osimertinib-resistant NSCLC cells. Importantly, this reversal effect of PAR2 blockade was uncovered to depend on ERK-mediated EMT and PD-L1, since inhibition of β-arrestin or ERK, which could be modulated by PAR2, sensitized osimertinib to prevent EMT, PD-L1 expression and consequently overcame osimertinib resistance. Thus, this study demonstrated that PAR2 antagonism could limit ERK-mediated EMT and immune checkpoints, consequently attenuating EGFR transactivation and reactivate osimertinib. It suggested that PAR2 may be a novel drug target for osimertinib resistance, and PAR2 inhibition may be a promising strategy candidate for reversing EGFR-TKI resistance in NSCLC.

Cytotoxicity of combinations of the pan-KRAS inhibitor BAY-293 against primary non-small lung cancer cells

KRAS is mutated in approximately 25% of Non-small Cell Lung Cancer (NSCLC) patients and first specific inhibitors showed promising responses that may be improved by concurrent interference with downstream signaling pathways. Cell lines exhibiting KRAS mutations show specific sensitivities to modulators affecting glucose utilization, signal transduction and cell survival. Novel SOS1-directed inhibitors with a broader anticancer coverage such as BAY-293 and BI-3406 inhibit KRAS through the hindrance of SOS1-KRAS interactions. The aim of this study was to check the putative synergy of BAY-293 with modulators having revealed specific vulnerabilities of KRAS-mutated cell lines. The present investigation tested the cytotoxicity of BAY-293 combinations against a series of Osimertinib-resistant primary NSCLC cell lines using MTT tests and calculation of combination indices according to the Chou-Talalay method. The results show that BAY-293 synergizes with modulators of glucose metabolism, inhibitors of cellular proliferation, several chemotherapeutics and a range of diverse modulators, thus corroborating the chemosensitivities of cells expressing mutated KRAS. In conclusion, BAY-293 exerts cytotoxicity with a wide range of drugs against Osimertinib-resistant primary NSCLC cell lines. The administration of pan-KRAS inhibitors alone may be limited in vivo by toxicity to normal tissues but made feasible by its use as part of suitable drug combinations. This study shows that BAY-293 combinations are active against NSCLC cells not further amenable to mutated EGFR-directed targeted therapy and results likewise hold relevance for pancreatic and colon cancer.

GZ17-6.02 and Pemetrexed Interact to Kill Osimertinib-Resistant NSCLC Cells That Express Mutant ERBB1 Proteins.

We determined the molecular mechanisms by which the novel therapeutic GZ17-6.02 killed non-small cell lung cancer (NSCLC) cells. Erlotinib, afatinib, and osimertinib interacted with GZ17-6.02 to kill NSCLC cells expressing mutant EGFR proteins. GZ17-6.02 did not interact with any EGFR inhibitor to kill osimertinib-resistant cells. GZ17-6.02 interacted with the thymidylate synthase inhibitor pemetrexed to kill NSCLC cells expressing mutant ERBB1 proteins or mutant RAS proteins or cells that were resistant to EGFR inhibitors. The drugs interacted to activate ATM, the AMPK, and ULK1 and inactivate mTORC1, mTORC2, ERK1/2, AKT, eIF2α; and c-SRC. Knockdown of ATM or AMPKα1 prevented ULK1 activation. The drugs interacted to cause autophagosome formation followed by flux, which was significantly reduced by knockdown of ATM, AMPKα1, and eIF2α, or by expression of an activated mTOR protein. Knockdown of Beclin1, ATG5, or [BAX + BAK] partially though significantly reduced drug combination lethality as did expression of activated mTOR/AKT/MEK1 or over-expression of BCL-XL. Expression of dominant negative caspase 9 weakly reduced killing. The drug combination reduced the expression of HDAC2 and HDAC3, which correlated with lower PD-L1, IDO1, and ODC levels and increased MHCA expression. Collectively, our data support consideration of combining GZ17-6.02 and pemetrexed in osimertinib-resistant NSCLC.

Overcoming Osimertinib Resistance in Advanced Non-small Cell Lung Cancer

Osimertinib is used as a first-line treatment for metastatic non-small cell lung cancer with positive epidermal growth factor receptor mutations based on the results of the FLAURA trial. However, as with any other epidermal growth factor receptor tyrosine kinase inhibitor, resistance also develops for osimertinib. Various genetic aberrations associated with the molecular heterogeneity of cancer cells contribute to osimertinib resistance. It is also important to choose an appropriate subsequent treatment for osimertinib-resistant non-small cell lung cancer. In this overview, we discuss the underlying mechanisms of osimertinib resistance and the efficacy of possible subsequent treatment measures.

Abstract 1467: BLU-945, a fourth-generation, potent and highly selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with intracranial activity, demonstrates robust in vivo antitumor activity in models of osimertinib-resistant non-small cell lung cancer (NSCLC)

Abstract Background: EGFR-activating mutations (L858R and ex19del) are major drivers (15-47%) of lung adenocarcinoma. EGFR TKI development has changed the treatment paradigm for EGFR-mutant (EGFR+) NSCLC. However, tumors often develop acquired resistance mutations to current TKIs leading to clinical progression. EGFR+/T790M is the most prevalent acquired resistance mutation after first- and second-generation TKI treatment. Osimertinib, a third-generation TKI which targets EGFR+/T790M, can overcome this resistance, but faces further resistance within 10-18 months. EGFR+/C797S is the most significant on-target resistance mechanism to osimertinib, leading to EGFR+/T790M/C797S double resistant mutants. There are currently no approved targeted therapies for patients with EGFR+/T790M/C797S NSCLC. BLU-945 is a potent and highly selective inhibitor of the EGFR+/T790M/C797S and EGFR+/T790M mutations, with in vivo activity in subcutaneous and intracranial EGFR-mutant tumor models. Method: In vivo antitumor activity of BLU-945 was evaluated in engineered triple mutant cell line-derived xenograft (CDX) models and osimertinib-resistant patient-derived xenograft (PDX) models of NSCLC. Plasma and tumor samples were collected for future pharmacokinetic and pharmacodynamic analyses. In vivo activity was also evaluated in an intracranial implantation model using luciferase-expressing YU-1097 patient-derived-cells harboring EGFRex19del/T790M/C797S resistance mutations, with the tumor burden of intracranial lesions measured by bioluminescence imaging. Results: Oral administration of BLU-945 to tumor-bearing mice showed potent EGFR pathway inhibition and significant single-agent antitumor activity at well-tolerated doses in the engineered osimertinib-resistant EGFRL858R/T790M/C797S CDX model, and an EGFRex19del/T790M/C797S PDX model. In addition, combination of BLU-945 with osimertinib showed enhanced antitumor activity compared with single-agent treatment in the EGFRex19del/T790M/C797S PDX model. Pharmacodynamic assays revealed treatment with BLU-945 resulted in marked inhibition of EGFR, AKT and ERK phosphorylation in the EGFRL858R/T790M/C797S CDX and EGFRex19del/T790M/C797S PDX models. BLU-945 also demonstrated potent activity in a patient-derived model of EGFRex19del/T790M/C797S implanted intracranially. Conclusion: BLU-945 is a potent, selective, and orally available fourth-generation EGFR inhibitor with robust antitumor activity in osimertinib-resistant NSCLC models grown subcutaneously and intracranially. BLU-945 shows activity as a single agent and in combination with other EGFR inhibitors. Clinical development of BLU-945 is expected in 2021. Citation Format: Sun Min Lim, Chae Won Park, Zhuo Zhang, Rich Woessner, Tom Dineen, Faith Stevison, John Hsieh, Meredith Eno, Doug Wilson, John Campbell, Caitlin Utt, Faris Albayya, Nicolas Lamontagne, Marion Dorsch, Klaus Hoeflich, Byoung Chul Cho, Stefanie Schalm. BLU-945, a fourth-generation, potent and highly selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with intracranial activity, demonstrates robust in vivo antitumor activity in models of osimertinib-resistant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1467.

Abstract 1109: A novel strategy to cope with osimertinib resistance in non-small cell lung cancer by treatment with a PIM kinase inhibitor in combination with a MERTK-selective kinase inhibitor

Abstract Osimertinib is currently the preferred treatment for EGFR-mutated non-small cell lung cancer (NSCLC) patients due to its superior therapeutic efficacy and prolonged overall survival compared to earlier generation EGFR tyrosine kinase inhibitors, but durable responses to osimertinib treatment are rare due to acquired drug resistance. Thus, there is an urgent need for novel strategies to treat osimertinib-resistant NSCLC. Recently, we found that treatment with MRX-2843, a novel MERTK-selective kinase inhibitor currently in Phase I clinical trials, resulted in dose-dependent inhibition of cell expansion and colony formation in an osimertinib-resistant (osiR) H4006 derivative cell line. An unbiased screen of 378 kinase inhibitors was carried out to identify compounds that synergized with MRX-2843 to inhibit expansion of an osiR derivative of the EGFR-mutated H4011 cell line. Treatment with 1µM PIM kinase inhibitor SGI-1776 or 100nM MRX-2843 alone reduced cell density by 5±3% and 44±7%, respectively, while treatment with MRX-2843 and SGI-1776 combined mediated an 82±0.4% decrease. Synergy was also observed in H4006 osiR and H1650 osiR derivative cell lines. Furthermore, treatment with PIM447, a structurally distinct PIM kinase inhibitor, and MRX-2843 decreased cell expansion more effectively than either agent alone. Mechanistically, treatment with a PIM kinase inhibitor in combination with MRX-2843 decreased downstream PI3K-AKT and MAPK-ERK signaling more effectively than single agents. Additionally, combined treatment with MRX-2843 and SGI-1776 prevented colony formation, while single agents had limited effect. In all, these data indicate that combining MRX-2843 and a PIM TKI may control osimertinib resistant tumor growth, providing a potential treatment strategy for osimertinib resistant EGFR-mutated NSCLC patients for whom the choices are still limited. Citation Format: Dan Yan, Zikang Tan, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, III, Deborah DeRyckere, Douglas K. Graham. A novel strategy to cope with osimertinib resistance in non-small cell lung cancer by treatment with a PIM kinase inhibitor in combination with a MERTK-selective kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1109.

Dihydroartemisinin overcomes the resistance to osimertinib in EGFR-mutant non-small-cell lung cancer

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), is commonly used to treat EGFR-mutant non-small-cell lung cancer (NSCLC). However, acquired resistance to mutant EGFR (T790M) can evolve following osimertinib treatment. High reactive oxygen species (ROS) levels in lung cancer cells can influence heme levels and have an impact on osimertinib resistance. Here, we found that heme levels were increased in osimertinib resistant EGFR-mutant NSCLC cell lines and plasma heme levels were also elevated in osimertinib-treated EGFR-mutant NSCLC patients. The antimalarial drug dihydroartemisinin (DHA), which has anticancer effects and requires heme, was tested to determine its potential to revert osimertinib resistance. DHA downregulated the expression of heme oxygenase 1 and inhibited cell proliferation in osimertinib-resistant EGFR-mutant NSCLC cells (PC9-GR4-AZD1), which was further enhanced by addition of 5-aminolevulinic acid, protoporphyrin IX and hemin. DHA was synergistic with osimertinib in inhibiting cell proliferation and colony formation of all osimertinib-resistant cell lines tested. Combination treatment with osimertinib and DHA also increased the levels of ROS, downregulated the phosphorylation or protein levels of several RTKs that often are overexpressed in osimertinib-resistant EGFR-mutant NSCLC cells, and inhibited tumor growth without toxicity in a PC9-GR4-AZD1 xenograft mouse model. The results suggest that DHA is able to reverse the resistance to osimertinib in EGFR-mutant NSCLC by elevating ROS level and impair heme metabolism.

ID1 mediates resistance to osimertinib in EGFR T790M-positive non-small cell lung cancer through epithelial\u2013mesenchymal transition

BackgroundID1 is associated with resistance to the first generation of EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). However, the effect of ID1 expression on osimertinib resistance in EGFR T790M-positive NSCLC is not clear.MethodsWe established a drug-resistant cell line, H1975/OR, from the osimertinib-sensitive cell line H1975. Alterations in ID1 protein expression and Epithelial–mesenchymal transition (EMT)-related proteins were detected with western blot analysis. RT-PCR was used to evaluate the differences of gene mRNA levels. ID1 silencing and overexpression were used to investigate the effects of related gene on osimertinib resistance. Cell Counting Kit-8 (CCK8) was used to assess the proliferation rate in cells with altered of ID1 expression. Transwell assay was used to evaluate the invasion ability of different cells. The effects on the cell cycle and apoptosis were also compared using flow cytometry.ResultsIn our study, we found that in osimertinib-resistant NSCLC cells, the expression level of the EMT-related protein E-cadherin was lower than that of sensitive cells, while the expression level of ID1 and vimentin were higher than those of sensitive cells. ID1 expression levels was closely related to E-cadherin and vimentin in both osimertinib-sensitive and resistant cells. Alteration of ID1 expression in H1975/OR cells could change the expression of E-cadherin. Downregulating ID1 expression in H1975/OR cells could inhibit cell proliferation, reduce cell invasion, promote cell apoptosis and arrested the cell cycle in the G1/G0 stage phase. Our study suggests that ID1 may induce EMT in EGFR T790M-positive NSCLC, which mediates drug resistance of osimertinib.ConclusionsOur study revealed the mechanism of ID1 mediated resistance to osimertinib in EGFR T790M-positive NSCLC through EMT, which may provide new ideas and methods for the treatment of EGFR mutated NSCLC after osimertinib resistance.

Exosomal microRNAs as potential biomarkers for osimertinib resistance of non-small cell lung cancer patients.

Osimertinib is an epidermal growth factor receptor-tyrosine kinase inhibitor that specifically targets the T790M mutation in cancer.Unfortunately, most non-small cell lung cancer (NSCLC) patients develop osimertinib resistance. Currently, the molecular biomarkers for monitoring osimertinib resistance are not available. This study aimed to examine the profile of exosomal miRNA in the plasma of osimertinib-resistant NSCLC patients. Plasma exosomal miRNA profiles of 8 NSCLC patients were analyzed by next-generation sequencing at osimertinib-sensitive and osimertinib-resistance stage.The expression of dysregulated exosomal miRNAs was validated and confirmed in another cohort of 19 NSCLC patients by qPCR. The relationship between exosomal miRNA upregulation and clinical prognosis, survival analysis was evaluated by Kaplan-Meier curves. In osimertinib-resistant NSCLC patients, 10 exosomal miRNAs were significantly dysregulated compared to baseline. Upregulation of all 10 candidate exosomal miRNAs tended to correlate with increased latency to treatment failure and improved overall survival. Among them, 4 exosomal miRNAs, miR-323-3p, miR-1468-3p, miR-5189-5p and miR-6513-5p were essentially upregulated and show the potential to be markers for the discrimination of osimertinib-resistance from osimertinib-sensitive NSCLC patients with high accuracy (p< 0.0001). Our results highlight the potential role of these exosomal miRNAs as molecular biomarkers for the detection of osimertinib resistance.

MET inhibitor, capmatinib overcomes osimertinib resistance via suppression of MET/Akt/snail signaling in non-small cell lung cancer and decreased generation of cancer-associated fibroblasts.

Background: Patients with non-small cell lung cancer (NSCLC) initially responding to tyrosine kinase inhibitors (TKIs) eventually develop resistance due to accumulating mutations in the EGFR and additional lesser investigated mechanisms such as the participation of the tumor microenvironment (TME).Methods: Here, we examined the potential for MET inhibitor capmatinib for the treatment of osimertinib-resistant NSCLCs and normalizing the TME.Results: We first established that HCC827 and H1975 cells showed increased resistance against osimertinib when co-cultured with CAFs isolated from osimertinib-resistant patients. Additionally, we showed that CAFs promoted epithelial-mesenchymal transition (EMT) and self-renewal ability in both HCC827 and H1975 cells. We subsequently found that both CAF-cultured HCC827 and H1975 showed a significantly higher expression of MET, Akt, Snail and IL-1β, which were associated with survival and inflammatory responses. These cells in turn, promoted the generation of CAFs from normal lung fibroblasts. Subsequently, we observed that the treatment of capmatinib resulted in the re-sensitization of CAF-co-cultured H1975 and HCC827 to osimertinib, in association with reduced EMT and self-renewal ability. MET-silencing experiment using siRNA supported the observations made with capmatinib while with a greater magnitude. MET-silenced cell exhibited a severely hindered expression of inflammatory markers, IL-1β and NF-κB; EMT markers, Snail and Vimentin, while increased E-cadherin. Finally, we demonstrated that the combination of capmatinib and osimertinib led to an increased tumor inhibition and significantly lower number of CAFs within the patient derived xenograft (PDX) model.Conclusion: Taken together, our findings suggested that an increased MET/Akt/Snail signaling was induced between the NSCLC cells and their TME (CAFs), resulting in osimertinib resistance. Suppression of this pathway by capmatinib may bypass the EGFR activating mutation and overcomes osimertinib resistance by targeting both tumor cells and CAFs.

Efficacy of the CDK4/6 Dual Inhibitor Abemaciclib in EGFR-Mutated NSCLC Cell Lines with Different Resistance Mechanisms to Osimertinib.

Simple SummaryOsimertinib, a third-generation irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has shown marked clinical benefit for non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, resistance to osimertinib inevitably develops and heterogeneous mechanisms of acquired resistance have been documented. Therefore, new strategies to bypass resistance are urgently needed. In this study, we investigated the potential activity of abemaciclib as second-line therapeutic approach after osimertinib progression and the effect of combining abemaciclib with osimertinib on the appearance of resistance in osimertinib-sensitive models. Abemaciclib is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6 that inhibits the transition from the G1 to the S phase of the cell cycle by blocking downstream CDK4/6-mediated phosphorylation of Rb. The effects of abemaciclib alone or combined with the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib were examined in a panel of PC9 and HCC827 osimertinib-resistant non-small cell lung cancer (NSCLC) cell lines carrying EGFR-dependent or -independent mechanisms of intrinsic or acquired resistance. Differently from sensitive cells, all the resistant cell lines analyzed maintained p-Rb, which may be considered as a biomarker of osimertinib resistance and a potential target for therapeutic intervention. In these models, abemaciclib inhibited cell growth, spheroid formation, colony formation, and induced senescence, and its efficacy was not enhanced in the presence of osimertinib. Interestingly, in osimertinib sensitive PC9, PC9T790M, and H1975 cells the combination of abemaciclib with osimertinib significantly inhibited the onset of resistance in long-term experiments. Our findings provide a preclinical support for using abemaciclib to treat resistance in EGFR mutated NSCLC patients progressed to osimertinib either as single treatment or combined with osimertinib, and suggest the combination of osimertinib with abemaciclib as a potential approach to prevent or delay osimertinib resistance in first-line treatment.

384P BLU-945, a highly potent and selective 4th generation EGFR TKI for the treatment of EGFR T790M/C797S resistant NSCLC

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment landscape for EGFR-mutant (L858R and ex19del) driven non-small-cell lung cancer (NSCLC). T790M is the most prevalent acquired EGFR resistance mutation to treatment with 1st and 2nd generation TKIs. 3rd generation TKIs, such as osimertinib, were developed to treat patients with tumors driven by this mutation. The C797S mutation is the most significant on-target resistance mechanism to osimertinib, leading to EGFR T790M/C797S double-resistant mutants. There are currently no approved targeted therapies for NSCLC patients with T790M/C797S mutations. BLU-945 is designed to target and selectively inhibit the EGFR T790M/C797S and T790M resistant mutations. BLU-945 activity on EGFR mutants and EGFR wild-type (WT) was tested in biochemical assays and cellular phosphorylation specific EGFR AlphaLisa assays. The in vivo anti-tumor activity of BLU-945 was evaluated in an NCI-H1975 cell line-derived tumor xenograft (CDX) model, as well as in osimertinib-resistant CDX- and patient-derived xenograft (PDX) models of NSCLC. BLU-945 inhibits EGFRex19del/T790M/C797S, EGFRL858R/T790M/C797S, EGFRex19del/T790M, and EGFRL858R/T790M mutants with sub-nanomolar IC50 values in an enzyme assay with a >1000-fold window over EGFRWT enzyme activity. BLU-945 achieves potent EGFR pathway inhibition in NCI-H1975 EGFRL858R/T790M, Ba/F3 EGFRL858R/T790M/C797S, and Ba/F3 EGFRex19del/T790M/C797S cell lines and a large window relative to EGFRWT inhibition. Oral administration of BLU-945 to tumor-bearing mice demonstrated potent EGFR pathway inhibition and anti-tumor activity at well-tolerated doses in the subcutaneous NCI-H1975 CDX model, and osimertinib-resistant CDX and PDX models, as well as in the intracranial luc-H1975 model of NSCLC. BLU-945 is a potent, selective, and orally available EGFR inhibitor that shows robust anti-tumor activity in osimertinib-resistant EGFR xenograft models. Because of its pharmacological activity and selectivity for mutant EGFR, BLU-945 has the potential to demonstrate activity in osimertinib-resistant EGFR-mutant NSCLC.

Allosteric SHP2 Inhibitor, IACS-13909, Overcomes EGFR-Dependent and EGFR-Independent Resistance Mechanisms toward Osimertinib.

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non-small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. SIGNIFICANCE: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

A Novel Serum Exosomes-Based Biomarker hsa_circ_0002130 Facilitates Osimertinib-Resistance in Non-Small Cell Lung Cancer by Sponging miR-498.

PurposeExosomes are the effective delivery system for biological compounds, including circular RNAs. In this research, we aimed to explore the role of circular RNA hsa_circRNA_0002130 in osimertinib-resistant non-small cell lung cancer (NSCLC).Materials and MethodsIn our study, the relative protein expression of glucose transporter 1 (GLUT1), hexokinase-2 (HK2) and lactate dehydrogenase A (LDHA) was detected by Western blot, while the expression of hsa_circ_0002130 and microRNA-498 (miR-498) was detected by quantitative real-time PCR (qRT-PCR). The biological functions of hsa_circ_0002130 in osimertinib-resistant NSCLC were analyzed by cell viability assay, flow cytometry analysis, luciferase reporter assay, RNA pull-down assay, and tumor xenograft model in vivo. Moreover, glucose uptake, lactate production and extracellular acidification (ECAR) levels were measured by glucose uptake colorimetric assay kit, lactate assay kit II, and Seahorse Extracellular Flux Analyzer XF96 assay, respectively. hsa_circ_0002130 identification and localization were confirmed by RNase R digestion and subcellular localization assay, respectively. Exosomes were isolated from the sera collected from NSCLC patients and identified using a transmission electron microscopy and nanoparticle tracking analysis.ResultsOsimertinib-resistance was closely related to glycolysis. hsa_circ_0002130 was highly expressed in osimertinib-resistant NSCLC cells and hsa_circ_0002130 deletion inhibited osimertinib-resistance both in vitro and in vivo. Moreover, hsa_circ_0002130 targeted miR-498 to regulate GLUT1, HK2 and LDHA. The inhibitory effects of hsa_circ_0002130 deletion on osimertinib-resistant were reversed by downregulating miR-498. Importantly, hsa_circ_0002130 was upregulated in serum exosomes from osimertinib-resistant NSCLC patients.ConclusionOur findings confirmed that hsa_circ_0002130 served as a promotion role in osimertinib-resistant NSCLC.

Ginsenoside Rg3 attenuates the osimertinib resistance by reducing the stemness of non-small cell lung cancer cells.

In the present study, we found that Ginsenoside Rg3 attenuated the stemness of non-small cell lung cancer (NSCLC) cells, evident by decreasing spheroid formation ability, ALDH1 activity and stemness marker expression. Furthermore, osimertinib-resistant NSCLC cells displayed a stronger stemness than the parental cells. Ginsenoside Rg3 reduced the stemness and osimertinib resistance of osimertinib-resistant cells. RNA-sequencing revealed that Hippo signaling was shown on the top of the most upregulated pathways regulated by Ginsenoside Rg3 in NSCLC cells, and YAP/TAZ expression was suppressed by Ginsenoside Rg3. Notably, the inhibitor of Hippo signaling attenuated the effects of Ginsenoside Rg3 on the stemness of NSCLC cells. Therefore, Ginsenoside Rg3 attenuates the osimertinib resistance of NSCLC cells via suppressing the stemness, which is dependent on Hippo pathway.

Enapotamab vedotin, an AXL-specific antibody-drug conjugate, shows preclinical antitumor activity in non-small cell lung cancer.

Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin-mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor-resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.

35P - Hsp90 inhibitors enhance the antitumoral effect of osimertinib and overcome osimertinib resistance in non-small cell cell lung cancer cell models

BackgroundThe epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) osimertinib improves therapy for non-small cell lung cancer (NSCLC) patients who possess EGR mutations. However, invariable acquired resistance appears due to several molecular mechanisms. Hsp90 protein clients are involved in these processes. MethodsCell viability and colony formation assays were performed in PC9, H1975 and PC9-derived osimertinib-resistant NSCLC cell lines tested with osimertinib plus hsp90 inhibitors, luminespib or ganetespib. To analyze the mechanism of action of these compounds and its combination, western blot analysis was carried out to study the protein expression and activation. ResultsIn our laboratory five osimertinib-resistant cell lines were generated from PC9 NSCLC cell line and overexpression or activation of several proteins, such as, AXL, Yap, bcl2, Akt, Stat3 and IGF-1R were detected. Hsp90 inhibitors, Ganetespib and Luminespib, inhibited cell viability and colony formation in H1975, PC9 and PC9-derived osimertinib-resistant cell lines, and the combination of these inhibitors with osimertinib achieved enhanced cell viability reduction. Luminespib downregulated the expression of several proteins involved in osimertinib-resistance and the combination of this compound plus osimertinib caused an important decrease of expression in several of these proteins, such as, Stat3, Yap, Akt, EGFR and Met. In addition, osimertinib activated the phosphorylation of several membrane receptors and downstream molecules, such as, Met, Yap, Stat3, Akt and Src, and its activation was partially inhibited by Luminespib. ConclusionsWe conclude that hsp90 inhibitors and osimertinib exhibit good efficiency in inhibiting cell viability and colony formation, while at the same time, inhibiting expression and activation of proteins involved in osimertinib-resistance. Our preclinical study shows that the combination of hsp90 inhibitors and osimertinib may represent an effective strategy for NSCLC patients with resistance to osimertinib treatment. Legal entity responsible for the studyPangaea Oncology. FundingPangaea Oncology. DisclosureAll authors have declared no conflicts of interest.

Hsp90 inhibitors enhance the antitumoral effect of osimertinib in parental and osimertinib-resistant non-small cell lung cancer cell lines

Osimertinib improve therapy for non-small cell lung cancer (NSCLC). However, invariable acquired resistance appears. MTT assay was used to analyze cell viability. Protein expression and activation was detected by Western blotting. In addition, the effects of heat shock protein 90 (Hsp90) inhibitors and osimertinib were studied in colony formation assays. Our laboratory generated osimertinib resistant cell lines from PC9 cell line and overexpression or activation of several proteins was detected. Hsp90 inhibitors, ganetespib and luminespib, inhibited cell viability and colony formation in H1975, PC9 and PC9-derived osimertinib-resistant cell lines and combination of these inhibitors with osimertinib achieved to enhance this cell viability and colony formation inhibition. Luminespib downregulated the expression of the several proteins involved in osimertinib-resistance and the combination of this compound plus osimertinib caused an important decrease of expression of several of these proteins, such as Stat3, Yap, Akt, EGFR and Met. Osimertinib activated the phosphorylation of several membrane receptors and downstream molecules that was partially inhibited by luminespib. In addition, a lung cancer patient with an EGFR eon 20 mutation had a partial radiographic response to ganetespib. Hsp90 inhibitors and osimertinib exhibits a good efficiency to inhibit cell viability, colony formation and inhibits expression and activation of proteins involved in osimertinib-resistance and may represent an effective strategy for NSCLC with intrinsic resistance to osimertinib inhibition.