Wild-type Epidermal Growth Factor Receptor Research Articles

Abstract 5737: Evaluation of the relationship between target expression and in vivo anti-tumor efficacy of AZD9592, an EGFR/c-MET targeted bispecific antibody drug conjugate

Abstract AZD9592 is a bispecific antibody drug conjugate (ADC) designed to deliver a topoisomerase 1 inhibitor (TOP1i) cytotoxic payload (AZ14170133) to tumor cells. AZD9592 selectively binds to epidermal growth factor receptor (EGFR) and c-MET, two cell surface receptors highly expressed in solid tumors including non-small-cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). Here we evaluate the pharmacodynamic activity of the TOP1i payload delivery by AZD9592 in an NSCLC-derived xenograft model using immunohistochemistry (IHC) approaches. Treatment-induced DNA double-strand breaks (DSB) and apoptotic cell death were measured using γH2AX, phospho-RAD-50 (pRAD50), and cleaved-caspase-3 (CC3) across increasing exposure to AZD9592. Furthermore, we report in vivo antitumor efficacy of AZD9592 in a panel of NSCLC and HNSCC patient-derived xenograft (PDX) models that were characterized for somatic tumor alterations, including oncogenic driver mutations in EGFR, tumor cell expression of EGFR and c-MET by IHC and deep-learning based image analysis, and targeted proteomics by mass spectrometry. Results demonstrate dose-dependent increases in pRAD50 and γH2AX upon treatment with AZD9592, signifying induction of DNA damage. Increased CC3 and reduced tumor volume (TV) in all treatment groups compared with control groups supports that AZD9592 induces tumor cell death due to formation of DNA DSB. In PDX experiments, tumor growth inhibition (TGI), defined as ≥30% reduction in TV from baseline after a single dose of AZD9592 8 mg/kg, was observed in 73% (16/22) of EGFR mutant NSCLC models. The models evaluated included tumors with or without prior exposure to EGFR tyrosine kinase inhibitors, and harboring diverse mutational profiles and heterogeneous expression levels of EGFR and c-MET. In EGFR wildtype NSCLC and HNSCC PDX, TGI was observed in 60% (12/20) and 44% (4/9) of models, respectively. IHC demonstrated an association of target expression and response to treatment, suggesting a potential predictive feature of response in tumors with elevated antigen expression. Targeted proteomics demonstrated an association between the expression of SLFN11, a known TOP1i sensitivity marker, and treatment response. Collectively, these results support the hypothesized mechanism of action of AZD9592: TOP1i induced tumor cell death due to formation of DNA DSB, and suggest opportunities in the treatment of tumors with a range of molecular features. AZD9592 is currently in a Phase 1 clinical trial in advanced solid malignancies. Citation Format: Lara McGrath, Ying Zheng, Simon Christ, Christian C. Sachs, Sihem Khelifa, Claudia Windmüller, Steve Sweet, Yeoun Jin Kim, Daniel Sutton, Michal Sulikowski, Arthur Lewis, Ivan Inigo, Nicolas Floch, Edward Rosfjord, Fernanda Arnaldez, Frank Comer. Evaluation of the relationship between target expression and in vivo anti-tumor efficacy of AZD9592, an EGFR/c-MET targeted bispecific antibody drug conjugate. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5737.

Abstract 3396: Discovery of BDTX-1535, a novel 4th generation, irreversible, potent, wild type sparing EGFR MasterKey inhibitor that targets oncogenic kinase domain mutations as well as extracellular domain alterations for the treatment of NSCLC and GBM

Abstract Acquired resistance to 3rd generation EGFR inhibitors used in the treatment of NSCLC is common. While the EGFR C797S substitution is a frequently reported post-osimertinib resistance mutation, real world evidence indicates the emergence of additional EGFR alterations that can drive drug resistance: kinase domain mutations (e.g., S768I), extracellular domain alterations (e.g., EGFRvIII, A289X), and EGFR amplification. Moreover, while osimertinib targets the classical EGFR mutations, primary NSCLC tumors are also driven by other oncogenic kinase domain mutations including G719X, S768I, and L861Q, which confer de novo (intrinsic) resistance to 3rd generation EGFR TKIs. A family of extracellular domain alterations occurs in nearly 50% of GBM patients, and these alterations are clinically resistant to all current generation inhibitors. Real world data in GBM demonstrate these EGFR alterations often co-occur and persist throughout treatment with current standard of care therapy. Of critical importance is the observation that the oncogenic isoform of EGFR in GBM is a covalent homo-dimer which can be formed and paradoxically activated by the binding of reversible 1st generation EGFR inhibitors. There is a significant clinical need to develop a potent CNS penetrant EGFR inhibitor that would target both acquired and intrinsic resistance mutations expressed in NSCLC and co-occurring EGFR alterations in GBM. An effective inhibitor should meet four design principles; 1) potent and selective against a broad family of intracellular, extracellular EGFR oncogenic alterations and amplification, 2) wild type EGFR sparing, 3) irreversible to avoid paradoxical activation, and 4) CNS penetrant. BDTX-1535 is a 4th generation irreversible CNS penetrant EGFR MasterKey inhibitor targeting a family of oncogenic EGFR extracellular domain alterations and amplification in GBM patients and EGFR resistance mutations in NSCLC. BDTX-1535 meets all four criteria for a highly effective EGFR inhibitor and was designed using the MAP Discovery Engine to target the activated conformations used by oncogenic EGFR to drive tumorigenesis. BDTX-1535 achieves potent anti-tumor activity against EGFR alterations and amplification across models including NSCLC and GBM PDX and intracranial tumors. BDTX-1535 is currently in a phase I clinical study in patients with NSCLC and GBM harboring sensitive EGFR alterations (NCT05256290). Citation Format: Matthew O'Connor, Matthew Lucas, Sherri Smith, Anthony Trombino, Sudharshan Eathiraj, Elizabeth Buck. Discovery of BDTX-1535, a novel 4th generation, irreversible, potent, wild type sparing EGFR MasterKey inhibitor that targets oncogenic kinase domain mutations as well as extracellular domain alterations for the treatment of NSCLC and GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3396.

Abstract 2232: The identification of novel autophagy flux-related molecules closely linked to the acquired resistance to EGFR TKIs in NSCLC patients harboring EGFR mutations

Abstract Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (mEGFR) well respond to EGFR TKIs, but the majority of patients eventually acquire drug resistance, leading to treatment failure. Recently, intracellular potential autophagy in addition to primary genetic alterations implicates the acquired drug resistance to targeted drugs in various carcinomas such as melanoma, pancreas, and breast cancer. We aimed to identify autophagy flux-related signal molecules which may be closely linked to acquired resistance in mEGFR NSCLC. Methods: We constructed the TMA composed of 579 surgically resected tumor tissues from lung adenocarcinoma patients and evaluated protein expression of autophagy flux-related signal molecules including CAGE, p-Beclin1, LC3B, p-AMPK, ULK1, p62, and ATG5 using immunohistochemistry (IHC). We established H1975OSI (166-folds) and PC9ERL (3,650-folds) resistant sub cell lines through the prolonged exposure of Osimertinib and Erlotinib to H1975 (L858R/T790M) and PC9 (Del19) cells, respectively. We also explored genes that induce autophagy flux activity using mRNA seq and qRT-PCR. Results: H-score of CAGE ( 95.6±7.9, 70±5.5), p-AMPK (73.4±7.8, 32±5.6) and p-Beclin1 (44.4±16.1, 19.7±5.5), ULK1( 57.4±4.9, 41.9±4.9) were significantly higher in mEGFR, compared to those of wild type EGFR NSCLC (P <0.001). According to EGFR mutation subtype, the expressions of CAGE (98±11.2, 70±5.5) and ULK1(60.7±5.0, 41.9±4.9) were significantly higher in Del19 (P<0.001), while the expressions of CAGE(93.2±5.6, 70±5.5,), p-AMPK(82.9±5.7, 32±5.6), and p-Beclin1(45.6±16.7, 19.7±5.5) were significantly higher in L858R (P<0.001). In advanced/metastatic mEGFR NSCLC patients treated with EGFR TKIs, p-Beclin1(13.0±10.9, 59±7.2) expression was significantly increased, when comparing before treatment and at treatment failure (P<0.018). As results of mRNA seq and qRT-PCR in H1975 parental cells and H1975OSI cells, we identified autophagy flux-related signal molecules including BECLIN1, GABAR1, NBR1, PINK1, ULK1 which were significantly increased more than 10 times. Conclusion: Taken together, our results strongly support the evidence that autophagy flux-related signal molecules as well as known genetic alterations are closely connected to the acquired resistance during period of EGFR TKI treatment in mEGFR NSCLC patients. Now, we are undergoing additional RNA profiling and function study of identified novel autophagy-related genes to examine a possibility as potential target to overcome EGFR TKI resistance. Citation Format: Jeong-Oh Kim, Jung-Young Shin, Min-Young Kim, Mi-Ran Lee, Seoree Kim, Hankyu Lee, Doyong Jeon, Seung Ryel Baek, Kim Joo Il, Kyoungjune Lee, Youn Soo Lee, Jin-Hyoung Kang. The identification of novel autophagy flux-related molecules closely linked to the acquired resistance to EGFR TKIs in NSCLC patients harboring EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2232.

Abstract 4019: Preclinical activity of ELVN-002: A potent, selective, and irreversible HER2 and pan-HER2 mutant small molecule inhibitor for the treatment of HER2 driven malignancies

Abstract Mutation or amplification of the human epidermal growth factor receptor 2 (HER2) gene has been identified in numerous solid tumor types, including non-small cell lung cancer (NSCLC), breast, and colorectal cancer. These genetic alterations are believed to confer elevated HER2 activity resulting in oncogenic transformation. In NSCLC, an estimated 3% of patients harbor activating mutations in the HER2 gene. Enhertu (T-DXd), a HER2-specific antibody-drug conjugate was recently approved as an important therapy for this patient population. However, for patients who are not candidates for T-DXd or have discontinued treatment due to an adverse event or disease progression, there exist few therapeutic options. Accordingly, HER2 inhibitors have the potential to provide meaningful therapeutic benefit to these patients. Due to significant structural homology between EGFR and HER2, most investigational small molecule HER2 kinase inhibitors are dual EGFR/HER2 inhibitors and are substantially dose-limited by EGFR-related toxicities in patients. These toxicities likely limit their clinical activity. Currently, tucatinib is the only HER2-selective small molecule inhibitor approved for metastatic breast cancer in combination with trastuzumab and capecitabine. However, it lacks sufficient potency against several of the key clinically relevant mutations, including HER2 YVMA, the most common exon 20 insertion mutation, prevalent in HER2 mutant NSCLC. ELVN-002 is a potent, irreversible inhibitor of HER2 with a >100-fold selectivity over EGFR. ELVN-002 potently inhibits the phosphorylation of HER2 in cell lines endogenously expressing HER2 or in those engineered to express specific clinically relevant HER2 mutants. This activity readily translates into marked anti-proliferative activity against cell lines dependent upon HER2 for their growth and survival. In addition, ELVN-002 is highly selective for HER2 and HER2 mutants versus wild-type EGFR as demonstrated via assessment of both EGFR-driven proliferation and EGFR phosphorylation in cells. Furthermore, ELVN-002 is highly active in mouse models of HER2-driven cancers, including subcutaneous models driven by wild type or mutant HER2. ELVN-002 is also active in an NCI-N87 wild-type HER2 intracranial model. In all models tested, ELVN-002 treatment is well tolerated and resulted in tumor regressions at exposures believed to be clinically achievable. Due to its selectivity and breadth of activity versus the clinically relevant HER2 mutants, ELVN-002 has the potential to be an effective treatment for HER2 mutant NSCLC as well as other HER2 mutant amplified solid tumors, including metastatic HER2 positive breast cancer. Additionally, ELVN-002 could provide a meaningful therapeutic option for patients with CNS metastases. ELVN-002 has been selected for clinical development. Citation Format: Monette Aujay, Amanda J. Broad, Stefan D. Gross, Li Ren, Joseph P. Lyssikatos, Samuel Kintz, Qi Wang, Helen Collins. Preclinical activity of ELVN-002: A potent, selective, and irreversible HER2 and pan-HER2 mutant small molecule inhibitor for the treatment of HER2 driven malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4019.

Prognostic value of CYFRA 21 − 1 and Ki67 in advanced NSCLC patients with wild-type EGFR

BackgroundThe prognostic value of cytokeratin 19 fragment (CYFRA 21 − 1) and Ki67 in advanced non-small cell lung cancer (NSCLC) patients with wild-type epidermal growth factor receptor (EGFR) remains to be explored.MethodsIn this study, 983 primary NSCLC patients from January 2016 to December 2019 were retrospectively reviewed. Finally, 117 advanced NSCLC patients with wild-type EGFR and 37 patients with EGFR mutation were included and prognostic value of CYFRA 21 − 1 and Ki67 were also identified.ResultsThe patients age, smoking history and the Eastern Corporative Oncology Group (ECOG) performance scores were significantly different between CYFRA21-1 positive and negative groups (p < 0.05), while no significant differences were found in Ki67 high and low groups. The results of over survival (OS) demonstrated that patients with CYFRA21-1 positive had markedly shorter survival time than CYFRA21-1 negative (p < 0.001, For whole cohorts; p = 0.002, For wild-type EGFR). Besides, patients with wild-type EGFR also had shorter survival times than Ki67 high group. Moreover, In CYFRA 21 − 1 positive group, patients with Ki67 high had obviously shorter survival time compared to patients with Ki67 low (median: 24vs23.5 months; p = 0.048). However, Ki67 could not be used as an adverse risk factor for patients with EGFR mutation. Multivariate cox analysis showed that age (HR, 1.031; 95%CI, 1.003 ~ 1.006; p = 0.028), Histopathology (HR, 1.760; 95%CI,1.152 ~ 2.690; p = 0.009), CYFRA 21 − 1 (HR, 2.304; 95%CI,1.224 ~ 4.335; p = 0.01) and Ki67 (HR, 2.130; 95%CI,1.242 ~ 3.652; p = 0.006) served as independent prognostic risk factor for advanced NSCLC patients.ConclusionsOur finding indicated that CYFRA 21 − 1 was an independent prognostic factor for advanced NSCLC patients and Ki67 status could be a risk stratification marker for CYFRA 21 − 1 positive NSCLC patients with wild-type EGFR.

Efficacy of Adjuvant Chemotherapy for Stage II/III Nonsmall Cell Lung Cancer with Epidermal Growth Factor Receptor Mutations.

Adjuvant cisplatin-based chemotherapy improves the survival of patients with resected pathological stage II/III nonsmall cell lung cancer (NSCLC). However, the efficacy in patients with epidermal growth factor receptor (EGFR) mutations remains controversial. This retrospective study included 353 patients with resected pathological N1/N2 stage II/III NSCLC between 2010 and 2016. Mutant EGFR (mEGFR) was detected in 76 patients. Adjuvant chemotherapy (AC) was administered to 151 patients. We compared cancer-specific survival (CSS) and recurrence-free survival (RFS) between AC and surgery-alone (SA) groups, including patients with wild-type EGFR (wEGFR) and mEGFR. Using multivariate analysis, we evaluated the prognostic factors in patients with wEGFR and mEGFR. The median follow-up time was 4.7 years. In patients with wEGFR, the differences in CSS and RFS between the AC (n = 114) and SA (n = 163) groups were significant (CSS: 66.8% [5 years] vs. 49.4% [5 years], p = 0.001; RFS: 54.2% [5 years] vs. 39.2% [5 years], p = 0.013). The significant prognostic factors were AC (vs. SA; p < 0.0001), diffusing capacity of the lung for carbon monoxide > 60% (p = 0.028), tumor size (p < 0.001), lymphatic permeation (p = 0.041), and pN1 (vs. pN2; p < 0.001). However, the differences in CSS and RFS between the AC (n = 37) and SA (n = 39) groups were not significant (CSS: 64.0% [5 years] vs. 58.1% [5 years], p = 0.065; RFS: 45.0% [5 years] vs. 33.8% [5 years], p = 0.302). Multivariate analysis identified no significant prognostic factors in patients with mEGFR. We demonstrated the efficacy of AC in patients with mEGFR and wEGFR. The efficacy of AC may be lower in patients with mEGFR than in those with wEGFR.

Discovery of Novel EGFR Inhibitor Targeting Wild-Type and Mutant Forms of EGFR: In Silico and In Vitro Study.

Targeting L858R/T790M and L858R/T790M/C797S mutant EGFR is a critical challenge in developing EGFR tyrosine kinase inhibitors to overcome drug resistance in non-small cell lung cancer (NSCLC). The discovery of next-generation EGFR tyrosine kinase inhibitors (TKIs) is therefore necessary. To this end, a series of furopyridine derivatives were evaluated for their EGFR-based inhibition and antiproliferative activities using computational and biological approaches. We found that several compounds derived from virtual screening based on a molecular docking and solvated interaction energy (SIE) method showed the potential to suppress wild-type and mutant EGFR. The most promising PD13 displayed strong inhibitory activity against wild-type (IC50 of 11.64 ± 1.30 nM), L858R/T790M (IC50 of 10.51 ± 0.71 nM), which are more significant than known drugs. In addition, PD13 revealed a potent cytotoxic effect on A549 and H1975 cell lines with IC50 values of 18.09 ± 1.57 and 33.87 ± 0.86 µM, respectively. The 500-ns MD simulations indicated that PD13 formed a hydrogen bond with Met793 at the hinge region, thus creating excellent EGFR inhibitory activity. Moreover, the binding of PD13 in the hinge region of EGFR was the major determining factor in stabilizing the interactions via hydrogen bonds and van der Waals (vdW). Altogether, PD13 is a promising novel EGFR inhibitor that could be further clinically developed as fourth-generation EGFR-TKIs.

Structure-based virtual screening and molecular dynamics simulations for detecting novel candidates for allosteric inhibition of EGFRT790M

Structure-based virtual screening (SBVS) was applied to predict lead compounds for the allosteric inhibition of epidermal growth factor receptor (EGFR) by screening the library of chemical compounds prepared from the e-molecules chemical database. The library of chemical compounds consisting of 133,083 ligands was composed by evaluating the chemical and physical properties of e-molecules chemicals. The prepared library was screened by CCDC Gold software in the allosteric binding site of EGFRT790M using the library and virtual screening default parameters to filter out, respectively. The GOLD fitness scores 75 and 80 were selected as threshold values for the library and virtual screening processes, respectively. After the docking study, molecular dynamics simulations (MDS) of the top 25 compounds were built for calculating binding free energies from their MDS trajectories. MM-GBSA binding free energies for the compounds were computed from 20 ns MDS, 50 ns MDS and 200 ns MDS trajectories to filter out the candidates. Following MM-GBSA/MM-PBSA binding free energy calculations, six compounds were detected as the most promising candidates for allosteric inhibition of EGFRT790M. The dynamic behaviors of final compounds inside EGFR T790M were searched using structure stability, binding modes and energy decomposition analysis. Besides, the estimated inhibitors were exposed to docking study and MM-GBSA/MM-PBSA binding free energy calculations inside wild-type EGFR, respectively, to be determined their selectivity towards mutant form. Five of the estimated inhibitors displayed estimated selectivity towards EGFRT790M. Besides the ADMET properties of the estimated inhibitors were predicted by PreAdmet tools. Communicated by Ramaswamy H. Sarma

Proximity proteome mapping reveals PD-L1-dependent pathways disrupted by anti-PD-L1 antibody specifically in EGFR-mutant lung cancer cells

BackgroundPD-L1, a transmembrane ligand for immune checkpoint receptor PD1, has been successfully targeted to activate an anti-tumor immune response in a variety of solid tumors, including non-small cell lung cancer (NSCLC). Despite the success of targeting PD-L1, only about 20% of patients achieve a durable response. The reasons for the heterogeneity in response are not understood, although some molecular subtypes (e.g., mutant EGF receptor tumors) are generally poor responders. Although PD-L1 is best characterized as a transmembrane PD1 ligand, the emerging view is that PD-L1 has functions independent of activating PD1 signaling. It is not known whether these cell-intrinsic functions of PD-L1 are shared among non-transformed and transformed cells, if they vary among cancer molecular subtypes, or if they are impacted by anti-PD-L1 therapy.MethodsHere we use quantitative microscopy techniques and APEX2 proximity mapping to describe the behavior of PD-L1 and to identify PD-L1's proximal proteome in human lung epithelial cells.ResultsOur data reveal growth factor control of PD-L1 recycling as a mechanism for acute and reversible regulation of PD-L1 density on the plasma membrane. In addition, we describe novel PD-L1 biology restricted to mutant EGFR cells. Anti-PD-L1 antibody treatment of mutant EGFR cells perturbs cell intrinsic PD-L1 functions, leading to reduced cell migration, increased half-life of EGFR and increased extracellular vesicle biogenesis, whereas anti-PD-L1 antibody does not induce these changes in wild type EGFR cells.ConclusionsGrowth factor acute regulation of PD-L1 trafficking, by contributing to the control of plasma membrane density, might contribute to the regulation of PD-L1's immune checkpoint activity, whereas the specific effects of anti-PD-L1 on mutant EGFR cells might contribute to the poor anti-PD-L1 response of mutant EGFR tumors.5zzQ_mo5e1oawe5DznU6q-Video

A 30-year-old Woman with Multiple-Sites Metastasis Lung Adenocarcinoma and Coronavirus-19 Disease (COVID-19)

Background: Adenocarcinoma is the most common Non-Small Cell Lung Cancer (NSCLC) in non-smokers in Indonesia. It often metastasizes in multiple sites. Metastasis is a negative predictor for prognosis in lung adenocarcinoma. In this covid pandemic era we present a case of a 30-years-old woman with adenocarcinoma and multiple-sites metastasis, and Coronavirus-19 Disease (COVID-19).&#x0D; Case: A 30-year-old woman came to our hospital with multiple complaints of hearing loss, visual loss, and occasional shortness of breath. The patient had a history of wild-type EGFR adenocarcinoma and has been treated with 4 cycles of gemcitabine. She also presented with a lump in her neck. Further examination revealed multiple metastasis tumours in her brain, right eye, neck, and spine. Pleural fluid examination also showed a malignant pleural effusion. Nasopharyngeal swab using reverse transcriptase-polymerase chain reaction (rt-PCR) method revealed that she also suffers from COVID-19 with positive swab result. Despite adequate treatment, she rapidly deteriorates and passed away in hospital.&#x0D; Discussion: Management in this patient is complex due to multiple complications and limitation of management given in COVID-19 isolation ward. She presented with multiple equally complicated metastasis, in which treatment priority should be carefully assessed. Despite treatment by multiple specialists, her problems still persist and proved to be fatal.&#x0D; Conclusion: Although metastasis is common in adenocarcinoma, multiple metastasis is uncommon, and this case highlights the need of careful management priority in such patients in COVID-19 pandemic setting.&#x0D; Keywords: Adenocarcinoma, Multiple Metastasis, Lung Cancer, COVID-19

Abstract P1-11-03: VRN101099, Brain Permeable HER2 Kinase Inhibitor, Shows the Anti-tumor Activity in Preclinical Models of HER2-positive Cancers

Abstract The brain is a common target organ for breast cancer metastasis, and the risk of brain metastasis is usually high for patients with HER2-positive breast cancer. While T-DXd showed positive results in HER2-positive breast cancer brain metastasis (BCBM), its activity and blood-brain barrier (BBB) permeability in active BCBM need to be further validated (TUXEDO-1 included 15 patients with active brain metastasis). Tucatinib plus Trastuzumab and Capecitabine triplet showed efficacy in patients with brain metastasis, but Tucatinib alone has poor BBB penetration. There is still an unmet medical need for active HER2-positive BCBM. VRN101099 is a highly selective kinase inhibitor of HER2 (Eurofins scanMAX Kinase Profiling, S-score (35) of 0.01). VRN101099 has single- to double-digit nanomolar (IC50) cellular potency in HER2-dependent cancer cells, and Ba/F3 cells expressing HER2 wild type or mutations with selectivity over wildtype EGFR. VRN101099 inhibited proliferation of BT474 with 3.6 nM IC50 but HaCaT with 829.2 nM IC50. VRN101099 binds HER2 kinase by forming a covalent bond to the Cys805 and its irreversible inhibition resulted in a longer target resident time than Tucatinib, confirmed by in vitro washout experiments. Robust in vivo activity of VRN101099 was observed in HER2-positive BT474 xenograft models. Moreover, once daily oral administration of VRN101099 significantly regressed intracranial BT474 tumor, demonstrating greater efficacy than twice daily oral administration of Tucatinib. These results were well explained by the superior brain to plasma exposure of VRN101099 to Tucatinib. Also, VRN101099 showed high exposure in the target organ, the fat pad, which potentiated better clinical translation. These anti-tumor efficacies are correlated with pharmacodynamic responses, as confirmed by decreased HER2, AKT, and ERK phosphorylation. In summary, VRN101099 is a brain penetrant, orally bioavailable, irreversible, and highly selective inhibitor of HER2 with therapeutic potential in HER2-positive BCBM. These data support the clinical development of VRN101099 in HER2-driven cancers. Citation Format: Yikyung Ko, Jihye yoo, Hong-ryul Jung, Hyerim Lim, YeongDeok Lee, Se Hyuk Kim, Dong Guk Shin, Serin Cho, Myung hoe Heo, Haelee Kim, Ha Yeon Cho, Ah Reum Han, Eunhwa Ko, Hwan Geun Choi, Deakwon Kim, Sunghwan Kim. VRN101099, Brain Permeable HER2 Kinase Inhibitor, Shows the Anti-tumor Activity in Preclinical Models of HER2-positive Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-03.

Increased EGFRvIII Epitope Accessibility after Tyrosine Kinase Inhibitor Treatment of Glioblastoma Cells Creates More Opportunities for Immunotherapy.

The number of glioblastoma (GB) cases is increasing every year, and the currently available therapies remain ineffective. A prospective antigen for GB therapy is EGFRvIII, an EGFR deletion mutant containing a unique epitope that is recognized by the L8A4 antibody used in CAR-T (chimeric antigen receptor T cell) therapy. In this study, we observed that the concomitant use of L8A4 with particular tyrosine kinase inhibitors (TKIs) does not impede the interaction between L8A4 and EGFRvIII; moreover, in this case, the stabilization of formed dimers results in increased epitope display. Unlike in wild-type EGFR, a free cysteine at position 16 (C16) is exposed in the extracellular structure of EGFRvIII monomers, leading to covalent dimer formation in the region of L8A4-EGFRvIII mutual interaction. Following in silico analysis of cysteines possibly involved in covalent homodimerization, we prepared constructs containing cysteine-serine substitutions of EGFRvIII in adjacent regions. We found that the extracellular part of EGFRvIII possesses plasticity in the formation of disulfide bridges within EGFRvIII monomers and dimers due to the engagement of cysteines other than C16. Our results suggest that the EGFRvIII-specific L8A4 antibody recognizes both EGFRvIII monomers and covalent dimers, regardless of the cysteine bridging structure. To summarize, immunotherapy based on the L8A4 antibody, including CAR-T combined with TKIs, can potentially increase the chances of success in anti-GB therapy.

Dihydromyricetin suppresses tumor growth via downregulation of the EGFR/Akt/survivin signaling pathway.

Deregulation of epidermal growth factor receptor (EGFR) signaling is frequently observed in non-small cell lung cancer (NSCLC). The present study aimed to determine the impact of dihydromyricetin (DHM) on NSCLC, a natural compound extracted from Ampelopsis grossedentata with various pharmacological activities. Results of the present study demonstrated that DHM may act as a promising antitumor agent for NSCLC therapy, inhibiting the growth of cancer cells in vitro and in vivo. Mechanistically, results of the present study demonstrated that exposure to DHM downregulated the activity of wild-type (WT) and mutant EGFRs (mutations, exon 19 deletion, and L858R/T790M mutation). Moreover, western blot analysis indicated that DHM induced cell apoptosis via suppression of the antiapoptotic protein, survivin. Results of the present study further demonstrated that depletion or activation of EGFR/Akt signaling may regulate survivin expression though modulating ubiquitination. Collectively, these results suggested that DHM may act as a potential EGFR inhibitor, and may provide a novel choice of treatment strategy for patients with NSCLC.

Dual kinase inhibitor for EGFR mutants and ErbB2 limit breast cancer

Mutations in the epidermal growth factor receptor (EGFR) have been found in more than 10% of non-small cell lung cancer (NSCLC) patients in North America. The vast majority of these differences are L858R point mutations in Exon 21. Currently, monoclonal antibodies directed against the extracellular domain of EGFR or small molecule/tyrosine kinase inhibitors (TKI) are the stalwarts of NSCLC therapy. Resistance, however, gradually develops because of the T790 mutation towards first and second generation TKIs. The third generation TKI AZD9291 (Osimertinib) has a high affinity for both activating and the acquired resistant mutation (T790 M) in EGFR, with a low affinity towards wild-type EGFR. Recent research, however, suggests that the EGFR (C797S) mutation in the tyrosine kinase domain is a likely cause of resistance to AZD9291. Another significant transformation mechanism associated with this resistance is erbB2 amplification. Our laboratory has developed a small kinase inhibitor, ER121 (MW: ∼500), that inhibits the erbB2/HER2 tyrosine kinases in addition to the EGFR C797S mutations. We have identified a TKI, ER121 targeting the mutant EGFR(T790 M). Using in vitro and in vivo models, examined the efficacy of ER121 on mutant EGFR cell lines. This has enabled us to establish that ER121 is well tolerated when administered orally and produces significant inhibitory activity against human cancers generated by mutant EGFR and amplified ErbB2.

Immunotoxin-αCD40 therapy activates innate and adaptive immunity and generates a durable antitumor response in glioblastoma models.

D2C7-immunotoxin (IT), a dual-specific IT targeting wild-type epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII) proteins, demonstrates encouraging survival outcomes in a subset of patients with glioblastoma. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. To improve the response rate and reverse immunosuppression, we combined D2C7-IT tumor cell killing with αCD40 costimulation of antigen-presenting cells. In murine glioma models, a single intratumoral injection of D2C7-IT+αCD40 treatment activated a proinflammatory phenotype in microglia and macrophages, promoted long-term tumor-specific CD8+ T cell immunity, and generated cures. D2C7-IT+αCD40 treatment increased intratumoral Slamf6+CD8+ T cells with a progenitor phenotype and decreased terminally exhausted CD8+ T cells. D2C7-IT+αCD40 treatment stimulated intratumoral CD8+ T cell proliferation and generated cures in glioma-bearing mice despite FTY720-induced peripheral T cell sequestration. Tumor transcriptome profiling established CD40 up-regulation, pattern recognition receptor, cell senescence, and immune response pathway activation as the drivers of D2C7-IT+αCD40 antitumor responses. To determine potential translation, immunohistochemistry staining confirmed CD40 expression in human GBM tissue sections. These promising preclinical data allowed us to initiate a phase 1 study with D2C7-IT+αhCD40 in patients with malignant glioma (NCT04547777) to further evaluate this treatment in humans.

Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation

Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.

Abstract A023: Analysis of survival and prognostic factors for patients with non-small cell lung cancer and brain metastasis: 10-year experience in a single institution

Abstract The aims of the study were 1) to perform the review of non-small cell lung cancer (NSCLC) patients with brain metastasis in our institution, 2) to investigate the response and prognostic factor of brain metastasis after institution’s best treatment, and 3) to suggest the shift of treatment paradigms for brain metastasis. From Jan. 2007 to Oct. 2018, A total 214 NSCLC patients with brain metastasis were enrolled. Female to male ratio was 0.65 and median age at diagnosis of NSCLC was 64 years (range, 30-90). 54 patients had a solitary brain metastasis and 76 patients had more than 10 lesions. Supratentorial, infratentorial and both locations were 106, 17, and 91 patients, respectively. 116 patients (54%) had symptoms associated with brain metastasis at the diagnosis of brain metastasis. Radiation only, radiosurgery, craniotomy with whole brain radiotherapy (WBRT), and supportive care were given for brain metastasis in 111 (52%), 10 (5%), 13 (6%) and 61 (29%) patients, respectively. Overall survival (OS) was associated with age, treatment modality, histology, egfr mutation. Median OS of Radiation only, radiosurgery, craniotomy with WBRT, and supportive care was 13, 15, 15 and 9 months, respectively (P-value 0.044). Median OS of adenocarcinoma and squamous cell carcinoma was 15 and 4 months, respectively (P-value &amp;lt;0.05). Median OS of egfr mutation and wild type was 18 and 9 months, respectively (P-value 0.016). The number and location of brain metastasis, symptoms and sex were not associated with OS Serial brain imaging was performed in 99 patients. 61 out of them (62%) developed shrinkage of brain metastasis (response group). Median OS of response and non-response group were 23 and 17 months (p-value 0.618). Age is significantly associated with response of brain metastasis to the institution’s best treatment (p-value 0.047). Interestingly, 3 patients developed complete remission of brain metastasis following WBRT and chemotherapy. The response duration was 431, 390, 641 days, respectively. Our data show that the prognosis of brain metastasis primarily depends on the age of NSCLC patients rather than the number, location of brain metastasis, treatment modality. Survival of NSCLC patients with brain metastasis was about 15 months despite the institution’s best treatment. The authors suggest that we should update our institution’s treatment policy and provide the latest treatments for patients. Citation Format: Seung Ho Yang. Analysis of survival and prognostic factors for patients with non-small cell lung cancer and brain metastasis: 10-year experience in a single institution [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A023.

Rapid determination of the presence of EGFR mutations with DNA-based nanocalipers.

Selecting 1st-line treatment for lung cancer is currently a binary choice, either chemotherapy or targeted medicine, depending on whether EGFR mutations exist. Next-generation sequencing is fully capable of accurately identifying EGFR mutations and guiding the usage of tyrosine kinase inhibitors, but it is highly expensive. Moreover, as the sequencing is not helpful for patients with wild-type EGFR, the long wait for sequencing may delay the chemotherapy and correspondingly increase the risks of cancer progression. To address this issue, a new method for rapidly determining the presence of EGFR mutations is developed in this study. A series of DNA origami-engineered nanocalipers are designed and constructed to determine the EGFR spatial distribution of either mutated EGFR or wild-type EGFR lung cancer cells. The experimental results on cancer cell lines and 9 clinical tissue samples show that compared with wild-type EGFR cells, mutated EGFR cells have narrower EGFR spacing. Hence, the DNA nanocalipers are demonstrated to be capable of determining the presence of EGFR mutations and shrinking the detection period from weeks to hours, compared with sequencing. For determining EGFR mutation status in 9 clinical samples, DNA nanocalipers show 100% consistency with next-generation sequencing.

The Co-expression of HER Family Members and CD109 is common in Pancreatic Cancer.

In the absence of biomarkers for the early detection of pancreatic cancer and the development of more effective therapeutic interventions, pancreatic cancer is projected to become the second leading cause of death from cancer in the Western world. In the past four decades, aberrant expression and activation of human epidermal growth factor receptor (EGFR, also called HER) family members have been reported in a wide range of human cancers. Currently, of the various types of drugs targeting one or more members of the HER family, only the EGFR-specific tyrosine kinase inhibitor erlotinib and more recently Zenocutuzumab, a HER-2/HER3 bispecific antibody, were granted breakthrough therapy designation by the FDA for the treatment of patients with pancreatic cancer. However, the therapeutic benefit may be modest in some patients. Hence there is an urgent need for the identification of biomarkers as prognostic indicators, therapeutic targets, and for the response to therapy and the development of more effective therapeutic agents for this highly heterogeneous cancer. In this study, for the first time, we investigated the relative expression and prognostic significance of all members of the HER family, the type-III EGFR mutant (EGFRvIII), and CD109 in tissue microarrays (TMAs) and whole tumour specimens (WTS) from pancreatic cancer patients by immunohistochemistry. We found the positive expression of wild-type EGFR (wt-EGFR) (63%, 4.7%), HER2 (75%, 14%), HER3 (none, 14%), HER4 (45%, 21%), EGFRvIII (5%, 3%), and CD109 (67%, 55%) in TMAs (USA) and WTS (UK) from pancreatic cancer patients respectively. Our results also show that the co-expression of HER family members with CD109 occurs frequently in patients with pancreatic cancer. In addition, the co-expression of HER4/CD109 may also be associated with poorer overall survival in such patients. In this article, we shall discuss these findings and their implications and future opportunities for more effective targeting of HER positive pancreatic cancer using the HER inhibitors in combination with other drugs and therapeutic interventions.

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.