EGFR-mutant Cancer Research Articles

Pericytes Modulate Third-Generation Tyrosine Kinase Inhibitor Sensitivity in EGFR-Mutated Lung Cancer Cells Through IL32-β5-Integrin Paracrine Signaling.

EGFR-mutated lung cancer patients sometimes display restricted responses to third-generation tyrosine kinase inhibitors (TKIs), potentially attributable to undervalued input from stromal cells, notably pericytes (PCs). The study shows that PCs isolated from EGFR-mutated patients have a unique secretome profile, notably secreting IL32 and affecting signaling pathways and biological processes linked to TKI sensitivity. Clinical evidence, supported by single-cell RNA sequencing and multiplex immunostaining of tumor tissues, confirms the presence of IL32-expressing pericytes closely interacting with β5-integrin-expressing cancer cells in EGFR-mutated patients, impacting therapeutic response and prognosis. Co-culture and conditioned medium experiments demonstrate that PCs reduce TKI effectiveness in EGFR-mutated cancer cells, a reversible phenomenon through silencing IL32 expression in PCs or depleting the IL32 receptor β5-integrin on cancer cells, thereby restoring cancer cell sensitivity. Mechanistically, it is shown that YY1 signaling upregulates IL32 secretion in PCs, subsequently activating the β5-integrin-Src-Akt pathway in EGFR-mutated cancer cells, contributing to their TKI sensitivity. In animal studies, co-injection of cancer cells with PCs compromises TKI effectiveness, independently of blood vessel functions, while inhibition of β5-integrin restores tumor cell sensitivity. Overall, the findings highlight direct crosstalk between cancer cells and pericytes, impacting TKI sensitivity via IL32-β5-integrin paracrine signaling, proposing an enhanced therapeutic approach for EGFR-mutated patients.

Loss of copy numbers of retrotransposons (HERVK) on chromosome 7p11.2 impacts EGFR (Epidermal Growth Factor Receptor)-induced phenotypes for platinum sensitivity and long-term survival in ovarian cancer-A study from the OVCAD consortium.

We analyzed variations in the epidermal growth factor receptor (EGFR) gene and 5'-upstream region to identify potential molecular predictors of treatment response in primary epithelial ovarian cancer. Tumor tissues collected during debulking surgery from the prospective multicenter OVCAD study were investigated. Copy number variations in the human endogenous retrovirus sequence human endogenous retrovirus K9 (HERVK9) and EGFR Exons 7 and 9, as well as repeat length and loss of heterozygosity of polymorphic CA-SSR I and relative EGFR mRNA expression were determined quantitatively. At least one EGFR variation was observed in 94% of the patients. Among the 30 combinations of variations discovered, enhanced platinum sensitivity (n = 151) was found dominantly with HERVK9 haploidy and Exon 7 tetraploidy, overrepresented among patients with survival ≥120 months (24/29, p = .0212). EGFR overexpression (≥80 percentile) was significantly less likely in the responders (17% vs. 32%, p = .044). Multivariate Cox regression analysis, including age, FIGO stage, and grade, indicated that the patients' subgroup was prognostically significant for CA-SSR I repeat length <18 CA for both alleles (HR 0.276, 95% confidence interval 0.109-0.655, p = .001). Although EGFR variations occur in ovarian cancer, the mRNA levels remain low compared to other EGFR-mutated cancers. Notably, the inherited length of the CA-SSR I repeat, HERVK9 haploidy, and Exon 7 tetraploidy conferred three times higher odds ratio to survive for more than 10 years under therapy. This may add value in guiding therapies if determined during follow-up in circulating tumor cells or circulating tumor DNA and offers HERVK9 as a potential therapeutic target.

Remote organ cancer adversely alters renal function and induces kidney injury, inflammation, and fibrosis.

Approximately 30% of cancer patients experience kidney complications, which hinder optimal cancer management, imposing a burden on patients' quality of life and the healthcare system. The etiology of kidney complications in cancer patients is often attributed to nephrotoxic oncological therapies. However, the direct impact of cancer on kidney health is underestimated, as most nephrotoxic oncological therapies have been studied in animal models that do not have cancer. Our previous study demonstrated that advanced lung cancer adversely alters kidney physiology and function, and exacerbates chemotherapy-induced nephrotoxicity, indicating lung cancer-kidney crosstalk. This study examines whether this phenomenon is specific to the employed cancer model. Female and male mice of various strains were injected with different cell lines representing human and mouse lung cancer, breast cancer, and melanoma, and their kidney tissues were analyzed for toxicity and fibrosis. The impact of cancer on the kidney varied by cancer type. Breast cancer and specific subtypes of lung cancer, including KRAS- and EGFR-mutant cancer, pathologically altered kidney physiology and function in a manner dependent on the metastatic potential of the cell line. This was independent of mouse strain, sex, and cancer cell line origin. Moreover, tumor DNA was not detected in the renal tissue, excluding metastases to the kidney as a causative factor for the observed pathological alterations. Lewis lung carcinoma and B16 melanoma did not cause nephrotoxicity, regardless of the tumor size. Our results confirm cancer-kidney crosstalk in specific cancer types and highlight gaps in understanding the risk of renal complications in cancer patients. In the era of precision medicine, further research is essential to identify at-risk oncology populations, enabling early detection and management of renal complications.

Abstract 1989: Intratumor heterogeneity in EGFR-mutant lung tumors mediates targeted therapy resistance and formation of drug tolerant microenvironment

Abstract Introduction: Non-small cell lung cancers (NSCLC) harboring EGFR mutations are commonly treated with EGFR tyrosine kinase inhibitors (TKIs), but the emergence of drug resistance often leads to treatment failure. While various acquired resistance mechanisms have been identified, they are not always actionable. Furthermore, since drug resistance is typically studied after cancer relapse, little is known about the cellular or microenvironmental taking place during the formation of resistance at the stage of minimal residual disease (MRD). Therefore, it is necessary to understand the mechanisms leading to resistance during MRD and develop new ways to prevent drug resistance in EGFR-mutant cancers. Methods: In this study, we discovered pre-existing intratumor heterogeneity in EGFR expression within EGFR-mutant NSCLCs and found that patient tumors contain clones with low mutant EGFR expression. To study the effect of this intratumor heterogeneity on drug response, we isolated low and high expressing cells and conducted longitudinal drug challenge assays in ex vivo and in vivo models. To dissect how the observed heterogeneity affects drug resistance and the tumor microenvironment during MRD, we employed patient-derived tumor cells and used novel tumor-on-a-chip co-culture approaches. Results: Our findings reveal that EGFR-low cells are more tolerant to drug treatment and can survive the initial TKI insult, making them more likely to contribute to drug resistance in EGFR-mutant NSCLCs. Additionally, the surviving EGFR-low cells were able to modify the tumor microenvironment leading to fibroblast accumulation and immune suppression, contributing to the drug-tolerant tumor microenvironment. Importantly, we found that pharmacological induction of EGFR using epigenetic inhibitors was able to sensitize the drug tolerant cells to EGFR inhibition, suggesting a potential strategy to overcome TKI resistance in NSCLC patients. Conclusions: In conclusion, our study sheds light on the critical role of intratumoral heterogeneity in treatment resistance and offers valuable insights into potential strategies to overcome this challenge. Our findings suggest that combination therapies that can alter the cellular phenotype during MRD have the potential to prevent intrinsic drug resistance. This underscores the importance of gaining a better understanding of the mechanisms underlying drug-tolerant microenvironment and developing more effective treatment strategies for patients with EGFR-mutant NSCLCs. Citation Format: Heidi M. Haikala, Bassel Alsaed, Jieun Son, Linh Lin, Prafulla Gokhale, Pasi Jänne. Intratumor heterogeneity in EGFR-mutant lung tumors mediates targeted therapy resistance and formation of drug tolerant microenvironment [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 1989.

Dual inhibition of KIF11 and BCL2L1 induces apoptosis in lung adenocarcinoma cells

EGFR-mutant lung adenocarcinoma (LUAD) mostly depends on EGFR for survival and consequently responds well to EGFR inhibitors. However, resistance to the drugs develops almost universally during treatment. We previously demonstrated that EGFR-mutant LUAD cell lines, HCC827 and H1975, have subpopulations of cells, which we termed HCC827 GR2 and H1975 WR7 cells, that can thrive independently of EGFR signaling. These EGFR-independent EGFR-mutant cancer cells are difficult to treat because they lack sensitivity to EGFR inhibitors. Therefore, the development of novel strategies to target EGFR–independent EGFR-mutant LUAD is particularly important. We found that high expression of kinesin family member 11 (KIF11) correlated with poor survival in patients with LUAD. We also observed that KIF11 silencing caused cell cycle arrest at G2/M in HCC827 GR2 and H1975 WR7 cells. Furthermore, dual silencing of KIF11 plus BCL2L1, an anti-apoptotic BCL2 family member, in these two EGFR-independent sublines resulted in marked apoptosis levels. Dual inhibition of KIF11 plus BCL2L1 also induced apoptosis in HCC827 and H1975 parental cells and a KRAS-mutant LUAD cell line, H441. These findings collectively suggest that dual inhibition of KIF11 plus BCL2L1 may be a new approach for the treatment of LUAD.

Molecular Docking Studies and ADME Predictions on Synthesized Chalcone Compounds Targeting EGFR

In the present study, new chalcone derivatives (5a–5f) obtained from the condensation reaction of cuminaldehyde and acetophenone compounds containing different substituents were reported. Chemical characterization (1HNMR and 13CNMR analysis) and molecular docking studies of the synthesized compounds were performed against the epidermal growth factor receptor (EGFR) and reference drug (metachalcone). Erlotinib was used as the reference ligand. Compound 5 (-7.6 kcal mol-1), compound 6 (-7.38 kcal mol-1), and compound 7 (-7.44 kcal mol-1) were found to be the strongest inhibitors of EGFR when compared to Erlotinib (-7.0 kcal mol-1). In addition, an ADME estimation was made. It was determined that the synthesized compounds could be potent EGFR inhibitors compared to Erlotinib. Compounds 5-7 and the target protein showed a better binding affinity for EGFR than the reference compound (Erlotinib). The synthesized compounds can be potent inhibitors for EGFR-mutated cancers.

A first-in-human, open-label, dose escalation and expansion study of orally administered NX-019 in patients with advanced EGFR mutant cancer.

TPS9159 Background: CNS involvement, brain metastases and/or leptomeningeal disease, is common in patients with EGFR mutant NSCLC and occurs in 25-50% of patient’s journeys. Despite the activity and increased CNS penetration of later generation EGFR inhibitors, management of CNS disease remains a clinical challenge. NX-019 is a broad-spectrum EGFR inhibitor targeting both common and rare mutations including EGFR ex20ins, G719S, and L861Q. NX-019 exhibits selective activity towards mutant vs. wt. EGFR in preclinical studies. Substantial CNS exposure has been demonstrated in animal models, as well as potent anti-tumor activity toward intracranial xenografts with prolonged inhibition of EGFR signaling. Similar drug levels compared to plasma were measured in the brains of rats and mice. NX-019 was readily detectable in the CSF of cynomolgus monkeys, dogs and rats at concentrations similar to the estimated plasma free fraction. Methods: NT019-101 is a first-in-human, international, open-label study designed to evaluate single-agent NX-019 in patients with EGFR-mutant, locally advanced or metastatic NSCLC that has progressed following prior treatment. Patients with ECOG performance status 0–2 and EGFR mutant cancers including stable, asymptomatic brain metastases are eligible for Dose Escalation. Patients with known EGFR C797S mutations and secondary drivers, e.g., MET amplification and RET fusions, are excluded. Primary endpoints are to determine recommended phase 2 dose (RP2D), maximum tolerated dose (MTD), safety and tolerability (part 1), and to evaluate antitumor activity at the RP2D by RECIST v1.1 and RANO-BM (part 2). Secondary endpoints are to evaluate pharmacokinetics (PK) and antitumor activity by RECIST v1.1 (part 1) and PK, safety, tolerability, and CNS antitumor activity (part 2). Dose escalation will utilize a 3+3 design with up to 6 patients per cohort in part 1. Part 2 will enroll patients in 5 cohorts (n = 10 or 29 each), including patients with NSCLC who have (1) new or progressing CNS metastases including leptomeningeal disease after treatment with osimertinib, (2) active CNS disease and ex20ins mutations, (3) ex20ins mutations without CNS disease and (4) other rare mutations, based on a Simon 2-stage design. First patient was treated in October 2022 and enrollment is ongoing. The study is planned for approximately 35 centers in North America and Asia-Pacific region. Clinical trial information: NCT05514496 .

Association between Plasminogen Activator Inhibitor-1 and Osimertinib Tolerance in EGFR-Mutated Lung Cancer via Epithelial-Mesenchymal Transition.

Most epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) cells are killed within a few days after osimertinib treatment; however, surviving cells remain detectable and are called drug-tolerant cells. Plasminogen activator inhibitor-1 (PAI-1) was reported to be involved in chemotherapeutic or radiotherapeutic resistance. The purpose of the present study was to investigate whether PAI-1 is involved in osimertinib tolerance and whether it could be a therapeutic target for overcoming this tolerance. We showed that the PAI-1 mRNA expression levels and mesenchymal gene expression levels were significantly higher in drug-tolerant EGFR-mutated NSCLC cells than in control cells after 7 days of in vitro osimertinib treatment. Additionally, an RNA microarray analysis revealed upregulation of the integrin-induced EMT pathway in osimertinib-tolerant cells. Furthermore, we observed that PAI-1 inhibitors suppressed proliferation and the degree of epithelial-mesenchymal transition (EMT) in tolerant cells. Finally, in a subcutaneous tumor model, we showed that combining osimertinib with a PAI-1 inhibitor prevented the regrowth of tumors comprising EGFR-mutated cancer cells. The present study is the first to show PAI-1 to be involved in tolerance to osimertinib via EMT.

Localization of EGFR Mutations in Non-small-cell Lung Cancer Tissues Using Mutation-specific PNA-DNA Probes.

Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors on the localization of EGFR mutations in tumor tissues remain to be elucidated. Thus, a simple and efficient technology for the detection of mutations in tumor tissue specimens needs to be developed. Using an EGFR mutation-specific peptide nucleic acid (PNA)-DNA probe, the EGFR mutation-positive part of whole NSCLC tissues was visualized by immunofluorescence. Formalin-fixed paraffin-embedded sections obtained from A549, NCI-H1975, HCC827 and PC-9 tumors transplanted into nude mice were subjected to staining using PNA-DNA probes specific for the mRNA sequences producing the L858R, del E746-A750 and T790M mutations. The probes for the L858R mutation showed intense positive staining in H1975 cells, and the probe for the del E746-A750 mutation exhibited positive staining specifically in HCC827 and PC-9 tumors. On the other hand, A549 tumors without EGFR mutation did not show any significant staining for any PNA-DNA probe. In combination staining, the addition of cytokeratin stain increased the positive staining rate of each PNA-DNA probe. In addition, the positive staining rate of the probes for the L858R mutation was comparable to that of the antibody to EGFR L858R mutated protein. PNA-DNA probes specific for EGFR mutations might be useful tools to detect heterogeneous mutant EGFR expression in cancer tissues and efficiently evaluate the effect of EGFR signaling inhibitors on tissues of EGFR-mutant cancer.

Cost of care in EGFR mutant versus wild-type non-small cell lung cancer.

e21084 Background: Lung cancer remains the most commonly diagnosed cancer in the United States, as well as the leading cause of cancer-related mortality. Approximately 84% of all lung cancers are non-small cell lung cancer (NSCLC). Most are diagnosed at an advanced stage when the prognosis is poorest. EGFR is the most common driver mutation in NSCLC with an incidence ranging from 20% in Caucasians, and up to 50% in Asians. Oral tyrosine kinase inhibitors (TKI) are the preferred treatment for EGFR mutant cancers as they provide better outcomes and lower toxicity than chemotherapy (Ch) or immunotherapy (ICI). Therefore, it is imperative to analyze NSCLC patients for these important driver mutations. Cancer care is expensive with the treatments and attendant costs for administration and management. We sought to compare cost of care (COC) in EGFR mutant vs wild-type NSCLC as the treatments and known toxicities differ markedly. Methods: We performed a retrospective COC analysis of NSCLC using claims data from the local BCBS affiliate Highmark (HM) members. Patients were selected based on NSCLC diagnosis codes between October 2016 and September 2018. COC was calculated from claims data to compare treatment of driver mutant NSCLC with targeted therapy versus treatment of wild-type NSCLC with ICI and/or ChT. Data used to compare COC analysis included overall medical costs defined as all medical claims incurred by a patient following start of respective therapy, oncology related medical claims, therapy costs, costs related to emergency room visits and tissue biopsies. Overall medical costs, oncology related costs and therapy costs were measured in per member per month (PMPM). We extracted cost related data at 6 months, 12 months, and 18 months into treatment. Results: Patients treated with ICI and/or ChT incurred $1000-2000 more in PMPM total medical costs than patients treated with targeted therapy. Additionally, chemoimmunotherapy patients were found to have a 5% higher emergency room admission rate along with a longer median length of stay of 1/3 day, when compared to targeted therapy patients. ICI and/or ChT patients showed a 20% higher rate of tissue biopsy and a 5-10% higher rate of repeat tissue biopsy which incurred an additional $300-$400 expense for chemoimmunotherapy patients. Conclusions: The use of targeted treatment with TKIs in EGFR mutant NSCLC patients is more cost effective than ICI and/or ChT in wild-type NSCLC. Routine screening of lung cancer patients with molecular profiling is essential, as identification of such critical driver mutations can alter the treatment plan to be more efficient, providing better care at lower cost.

Dynamics of HER3 and its correlated gene expression profile in EGFR-mutated NSCLC tumor treated with EGFR-TKI toward enhancing effectiveness of patritumab deruxtecan (HER3-DXd; U3-1402).

e21175 Background: Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a standard first-line therapy for activated EGFR-mutated non-small-cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance and is hence a promising target for anticancer treatment. Patritumab deruxtecan (HER3-DXd) is an antibody drug conjugate comprised of a fully human anti-HER3 IgG1 monoclonal antibody, covalently linked to a topoisomerase 1 inhibitor payload, an exatecan derivative, via a tetrapeptide-based cleavable linker. Clinical trials of theHER3-DXd demonstrated its anticancer activity in EGFR-mutated NSCLC. However, the genomic background that regulates HER3 expression is unknown. This study was conducted with the aim to clarify the genomic background for HER3 regulation in EGFR-mutated NSCLC tumors and to explore the strategy for enhancing the anticancer activity of HER3-DXd. Methods: 48 paired samples were obtained before EGFR-TKI treatment and after the acquisition of EGFR-TKI resistance from patients with EGFR-mutated NSCLC. HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pre-treatment to post-EGFR-TKI resistance acquisition. For statistical tests, linear regression analysis was performed to investigate whether a factor, including EGFR-TKI administration, affected HER3 expression. Pearson correlation coefficients were calculated to explore the relationships between HER3 expression level and other genomic expression in the tumors. Results: We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared to paired pretreatment samples (mean H-score 100.8 vs. 155.9, paired t-test p = 0.0007). Although genomic alterations including EGFR secondary T790M mutation did not correlate with HER3 augmentation, RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation (GSEA; normalized enrichment score -1.78, p = 0.004). Multivariable regression analysis of HER3 augmentation revealed that re-biopsy under continuing EGFR-TKI treatment independently effected HER3 augmentation. Indeed, an in-vitro study also showed that EGFR-TKI increased HER3 expression via repressed AKT phosphorylation in multiple EGFR-mutated cancers, and it finally enhanced the anticancer activity of HER3-DXd. Conclusions: Our findings highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC that is currently being evaluated in a clinical trial for patients with EGFR-mutated NSCLC tumors, not only with regard to an acquired resistance to EGFR-TKI but also in EGFR-TKI-naïve tumors (NCT04676477).

Betacellulin promotes tumor development and EGFR mutant lung cancer growth by stimulating the EGFR pathway and suppressing apoptosis

SummaryOncogenic mutations in the EGFR gene account for 15–20% of lung adenocarcinoma (LUAD) cases. However, the mechanism for EGFR driven tumor development and growth is not fully understood. Here, using an mRNA expression profiling-based approach we identified betacellulin (BTC) as one the gene upregulated by oncogenic EGFR in an MAP kinase-dependent manner. BTC protein expression was markedly increased in LUAD patient samples compared to normal lung tissue, with higher expression in EGFR-mutant LUAD. BTC was sufficient to transform immortalized mouse cells, initiate tumor development in mice, and promote the survival of immortalized human lung epithelial cells. Conversely, knockdown of BTC inhibited the growth of EGFR-mutant human LUAD cells in culture and their tumor-forming ability in mice. Mechanistically, BTC knockdown resulted in attenuated EGFR signaling and apoptosis induction. Collectively, these results demonstrate a key role of BTC in EGFR-mutant LUAD, with potential therapeutic implications in LUAD and other EGFR-mutant cancers.

HER3 Augmentation via Blockade of EGFR/AKT Signaling Enhances Anticancer Activity of HER3-Targeting Patritumab Deruxtecan in EGFR-Mutated Non-Small Cell Lung Cancer.

EGFR-tyrosine kinase inhibitor (TKI) is a standard first-line therapy for activated EGFR-mutated non-small cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance. Clinical trials of the HER3-targeting antibody-drug conjugate patritumab deruxtecan (HER3-DXd) demonstrated its anticancer activity in EGFR-mutated NSCLC; however, the mechanisms that regulate HER3 expression are unknown. This study was conducted with the aim to clarify the mechanisms underlying HER3 regulation in EGFR-mutated NSCLC tumors and explored the strategy for enhancing the anticancer activity of HER3-DXd in EGFR-mutated NSCLC. Paired tumor samples were obtained from 48 patients with EGFR-mutated NSCLC treated with EGFR-TKI(s). HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pretreatment to post-EGFR-TKI resistance acquisition. The anticancer efficacy of HER3-DXd and osimertinib was evaluated in EGFR-mutated NSCLC cells. We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared with paired pretreatment samples. RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation, especially in tumors from patients who received continuous EGFR-TKI therapy. An in vitro study also showed that EGFR-TKI increased HER3 expression, repressed AKT phosphorylation in multiple EGFR-mutated cancers, and enhanced the anticancer activity of HER3-DXd. Our findings help clarify the mechanisms of HER3 regulation in EGFR-mutated NSCLC tumors and highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC.

Abstract P212: Systems biology-guided indication selection to inform the clinical development of a novel TEAD inhibitor

Abstract TEAD transcription factors are the final effectors of the Hippo pathway, a signaling cascade comprising multiple tumor suppressors (NF2, MST1/2, LATS1/2) critical in regulating proliferation, survival, and tissue homeostasis. Activated Hippo signaling suppresses TEAD-dependent transcription through the phosphorylation and degradation of TEAD co-activators YAP1 and TAZ in the cytosol. Consistently, genetic alterations in NF2, LATS1/2, YAP1 or TAZ lead to aberrant TEAD activation and are implicated in tumor initiation, progression, and therapeutic resistance in cancer. Here we present a unique systems biology approach integrating genomic, transcriptional and tissue-based analysis, to inform clinical development, indication and patient selection biomarkers for a novel inhibitor of TEAD being developed by Ikena. To identify cancers dependent on TEAD activity, tumor types were evaluated based on the cumulative incidence of genetic alterations in Hippo pathway genes including NF2, LATS1/2, YAP1, TAZ and others. This novel analysis pointed to a subset of tumors with high frequency of genetic alterations driving aberrant Hippo signaling including mesothelioma and NSCLC. This same subset was found to highly express a transcriptional signature indicative of YAP1/TEAD-dependency, confirming increased activation of TEAD transcription and pointing to the dependency of these tumors on TEAD activity. A proprietary IHC method was used to further assess YAP1/TAZ activation by assessing expression of either protein in the nucleus in multiple tumor tissue microarrays. Tissue-based analysis showed high YAP1 nuclear expression in tumors with frequent genetic alterations and high YAP1/TEAD-signature. Mesothelioma in particular ranks top among these indications, due to high frequency of NF2 deficiency and other Hippo pathway alterations. Consistently, TEAD inhibition showed single agent activity in two xenograft models of mesothelioma with NF2 deficiency and LAST1/2 alterations respectively. Hippo pathway alterations frequently co-occur with mutations in other oncogenic signaling pathways, e.g. EGFR. Importantly, YAP1/TAZ have been implicated in acquired resistance to targeted therapies in cancer including EGFR inhibitors in EGFR mutant tumors. Indeed, PDX models derived from patients who relapsed on osimertinib treatment showed high YAP1 protein expression in the nucleus. In addition, combination of TEAD and EGFR inhibitors induced apoptosis in vitro and greater antitumor activity than either drug as a single agent in vivo in EGFR mutant cancer models. In summary, the integration of multi-disciplinary bioinformatics, pharmacologic and tissue-based approaches enabled the identification of cancer types with high dependency on Hippo signaling. Moreover, these studies support the for monotherapy and combination of TEAD inhibitors with other targeted therapies including EGFR inhibitors. Altogether this unique approach has identified cancer patients who may benefit from TEAD inhibition and has informed the clinical development plan of a novel TEAD inhibitor. Citation Format: Marta Sanchez-Martin, Sakeena Syed, Hyejin Frosch, Chelsea Turcotte, Benjamin Amidon, Karen McGovern, Jeffrey Ecsedy, Michelle X. Zhang. Systems biology-guided indication selection to inform the clinical development of a novel TEAD inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P212.

High Tumor Mutational Burden in Rare and Complex EGFR Mutant Non-Small Cell Lung Cancer

<h3>Purpose/Objective(s)</h3> Classical EGFR mutant lung cancer with exon 19 deletions, exon 21 L858R and de novo exon 20 T790M mutations are associated with good response to tyrosine kinase inhibitors. In contrast, rare and complex mutations have uncertain or poor response to targeted therapy. Recent retrospective analyses have observed a potential benefit of rare and complex EGFR mutations from immunotherapy, in contrast to cancers harboring classical EGFR mutations. We hypothesized that a larger proportion of rare and complex EGFR mutant lung cancers may have molecular differences that would promote immune checkpoint blockade as a putative combination or alternative treatment. <h3>Materials/Methods</h3> Patients (pts) with nonsynonymous EGFR mutant lung cancers from two datasets (The Cancer Genome Atlas, Clinical Proteomic Tumor Analysis Consortium) were identified. Rare EGFR mutations were defined as non-sensitizing somatic mutations occurring within the tyrosine kinase (TK) domain of exons 18 through 21. Complex EGFR mutant cases (i.e., G719X + S768I) were tumors with two mutations within the TK region where at least one mutation was non-sensitizing. Tumor mutational burden (TMB) was calculated as the number of somatic mutations per million bases (human exome was estimated to be 38 Mb). High-TMB was denoted as a TMB over 10. Gene expression of PD-L1 was evaluated by RNA-sequencing as well as bioinformatic assessment of tumor purity, immune infiltration and stroma contribution within the tumor microenvironment using the ESTIMATE (Estimation of STromal and Immune cells in MAlignant Tumours using Expression data) algorithm. <h3>Results</h3> Of the 675 pts evaluated, 97 (14.3%) had tumors which harbored EGFR mutations. Patients with EGFR mutant tumors were more likely to be female (64 vs 48%, <i>P</i> = 0.006) and non-smokers (49 vs 12%, <i>P</i> < 0.001). Tumors from non-smokers were less likely to have high-TMB (<i>P</i> < 0.001). Twenty four of 97 (25%) EGFR mutant cancers had either rare (n = 12) or complex mutations (n = 12). Tumors with rare/complex EGFR mutations had a higher average TMB (5.4 compared to 2.3) and were significantly more likely to have a TMB over 10 (17% vs 1.4%, <i>P</i> = 0.003). 25% of rare EGFR mutant tumors (L861Q, E866K, K754I) had a TMB > 10 (<i>P</i> = 0.0003). There were no significant differences in PD-L1 expression (<i>P</i> = 0.46) or smoking history (<i>P</i> = 0.71) between classical and rare/complex EGFR mutant cancers. Tumors with rare EGFR mutations had significantly higher tumor purity (0.759 versus 0.459, <i>P</i> = 0.0038), and corresponding lower proportion of immune (<i>P</i> = 0.0045) and stromal cells (<i>P</i> = 0.0077). <h3>Conclusion</h3> Our data suggests that lung cancers harboring rare EGFR mutations may have mutation-specific dependencies that would make a non-significant proportion amenable to immunotherapy. While PD-L1 appears to be a poor biomarker, we observed an increase in high-TMB and distinct changes in the tumor microenvironment in rare EGFR mutant lung cancer.

Intercellular transfer of exosomal wild type EGFR triggers osimertinib resistance in non-small cell lung cancer

BackgroundEpidermal growth factor receptor (EGFR)-mutated lung cancer constitutes a major subgroup of non-small cell lung cancer (NSCLC) and osimertinib is administrated as first-line treatment. However, most patients with osimertinib treatment eventually relapse within one year. The underlying mechanisms of osimertinib resistance remain largely unexplored.MethodsExosomes isolation was performed by differential centrifugation. Co-culture assays were conducted to explore the alteration of drug sensitivity by cell viability and apoptosis assays. Immunofluorescence and flow cytometry were performed to visualize the formation or absorption of exosomes. Exosomes secretion was measured by Nanoparticle Tracking Analysis or ELISA. The xenograft tumor model in mice was established to evaluate the effect of exosomes on osimertinib sensitivity in vivo.ResultsIntercellular transfer of exosomal wild type EGFR protein confers osimertinib resistance to EGFR-mutated sensitive cancer cells in vitro and in vivo. Co-culture of EGFR-mutated sensitive cells and EGFR-nonmutated resistant cells promoted osimertinib resistance phenotype in EGFR-mutated cancer cells, while depletion of exosomes from conditioned medium or blockade of exosomal EGFR by neutralizing antibody alleviated this phenotype. Mechanistically, osimertinib promoted the release of exosomes by upregulated a Rab GTPase (RAB17). Knockdown of RAB17 resulted in the decrease of exosomes secretion. Moreover, exosomes could be internalized by EGFR-mutated cancer cells via Clathrin-dependent endocytosis and then the encapsulated exosomal wild type EGFR protein activated downstream PI3K/AKT and MAPK signaling pathways and triggered osimertinib resistance.ConclusionsIntercellular transfer of exosomal wild type EGFR promotes osimertinib resistance in NSCLC, which may represent a novel resistant mechanism of osimertinib and provide a proof of concept for targeting exosomes to prevent and reverse the osimertinib resistance.

Marked synergy by vertical inhibition of EGFR signaling in NSCLC spheroids shows SOS1 is a therapeutic target in EGFR-mutated cancer.

Drug treatment of 3D cancer spheroids more accurately reflects in vivo therapeutic responses compared to adherent culture studies. In EGFR-mutated lung adenocarcinoma, EGFR-TKIs show enhanced efficacy in spheroid cultures. Simultaneous inhibition of multiple parallel RTKs further enhances EGFR-TKI effectiveness. We show that the common RTK signaling intermediate SOS1 was required for 3D spheroid growth of EGFR-mutated NSCLC cells. Using two distinct measures of pharmacologic synergy, we demonstrated that SOS1 inhibition strongly synergized with EGFR-TKI treatment only in 3D spheroid cultures. Combined EGFR- and SOS1-inhibition markedly inhibited Raf/MEK/ERK and PI3K/AKT signaling. Finally, broad assessment of the pharmacologic landscape of drug-drug interactions downstream of mutated EGFR revealed synergy when combining an EGFR-TKI with inhibitors of proximal signaling intermediates SOS1 and SHP2, but not inhibitors of downstream RAS effector pathways. These data indicate that vertical inhibition of proximal EGFR signaling should be pursued as a potential therapy to treat EGFR-mutated tumors.

MTOR Promotes Tissue Factor Expression and Activity in EGFR-Mutant Cancer.

Mechanistic target of rapamycin (mTOR) signaling pathway mediates the function of oncogenic receptor tyrosine kinases (RTKs). We aimed to elucidate new role of mTOR in EGFR-mutant (EGFR-mut) non-small cell lung cancer (NSCLC) and glioblastoma (GBM) with a focus on tumor microenvironments. Here, we report a novel regulatory link between mTOR complexes (mTORCs) and tissue factor (TF), an initiator of tumor-derived thrombosis. TF is elevated in EGFR-mut NSCLC/GBM cell lines and tumors from patients with poor prognosis. Application of mTORC1/2 inhibitors (AZD8055, WYE-125132, MTI-31, and rapamycin) or genetic mTORC-depletion all reduced TF expression, which appeared to be differentially mediated depending on cellular context. In U87MG and HCC827 cells, mTORC1 exerted a dominant role via promoting TF mRNA transcription. In EGFR-TKI-resistant H1975 and PC9 cells, it was mTORC2 that played a major role in specific repression of lysosomal-targeted TF protein degradation. Successful inhibition of TF expression was demonstrated in AZD8055- or MTI-31-treated H1975 and U87MG tumors in mice, while a TF-targeted antibody antagonized TF activity without reducing TF protein. Both the mTOR- and TF-targeted therapy induced a multifaceted remodeling of tumor microenvironment reflecting not only a diminished hypercoagulopathy state (fibrin level) but also a reduced stromal fibrosis (collagen distribution), compromised vessel density and/or maturity (CD31 and/or α-SMA) as well as a substantially decreased infiltration of immune-suppressive M2-type tumor-associated macrophages (CD206/F4/80 ratio). Thus, our results have identified TF as a functional biomarker of mTOR. Downregulation of mTOR-TF axis activity likely contributes to the therapeutic mechanism of mTORC1/2- and TF-targeted agents in EGFR-mut advanced NSCLC and GBM.

Abstract 5279: HSP72 expression and suppression of resistance is associated with survival in EGFR mutated NSCLC

Abstract Background: Patients with non-small cell lung cancer (NSCLC) harboring EGFR activating mutations initially respond favorably to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Acquired resistance develops in most patients, usually due to new DNA mutations. Our previous work has shown that EGFR-TKI therapy blocked phosphorylation of Hsp72, leading to degradation and suppression of polymerase α error editing and base excision repair, causing an increase in the overall mutation rate. This study evaluated baseline Hsp72 expression in EGFR mutated NSCLC and assessed Hsp72 ability to suppress EGFR-TKI resistance and improve overall survival (OS) of patients with EGFR mutated NSCLC. Methods: A retrospective cohort of patients was identified with known EGFR mutation positive NSCLC and available tissue from the City of Hope Comprehensive Cancer Center seen between 2008 and 2016. Patients were classified into two groups based on HSP72 levels: less than the mean HSP72 level (low expression, n = 40) and greater than or equal to the mean HSP72 level (high expression, n = 22), and examined OS. A chi-square test for categorical data and multivariate logistic regression analyses was used to study the relation between HSP72 expression and other clinical and demographic data. In vitro, HSP72 overexpression was induced by BPG-15 and degradation was blocked with bortezomib. We analyzed resistance in EGFR mutant NSCLC with osimertinib alone, and in combination with osimertinib and BPG-15 or bortezomib. Results: Sixty-three patients with EGFR mutated NSCLC and available tissue were identified. Median survival of high and low HSP72 expression were 51.5 and 28.8 months respectively (p = 0.003, log-rank test), with HR of 0.47 (95% CI 0.29-0.77). When stratifying by stage (removal of stage I and II patients), the HR associated with a high HSP72 expression remained 0.47 (95% CI 0.29-0.77). In vitro studies showed that in the absence of bortezomib or BGP-15, time to resistance to osimertinib in HCC827 cells was 13 days. In the presence of bortezomib or BGP-15, time to resistance was 29 and 25 days, respectively. A similar phenomenon was observed with the EGFR-TKI, erlotinib. Conclusions: In EGFR mutated NSCLC, a high level of HSP72 expression is associated with improved survival. Furthermore, HSP72 inducing drugs delays the development of resistance in EGFR-mutated NSCLC cancer cells, and mechanisms are under investigation. Supported by R01 CA073764 (BHS), R50 CA211397 (LZ), the William and Anna Tenenblatt Foundation (BHS, KR) and the STOP Cancer Carrie Scott Seed grant (KR). Citation Format: Li Zheng, Angel R. Baroz, Erminia Massarelli, Joy Huang, Ravi Salgia, Binghui Shen, Karen L. Reckamp. HSP72 expression and suppression of resistance is associated with survival in EGFR mutated NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5279.

EGFR-independent EGFR-mutant lung adenocarcinoma cells depend on Bcl-xL and MCL1 for survival

Although most EGFR-mutant lung adenocarcinomas initially respond to EGFR inhibitors, disease progression almost inevitably occurs. We previously reported that two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, contain subpopulations of cells that display an epithelial-to-mesenchymal phenotype and can thrive independently of EGFR signaling. In this study, we explored to what extent these two sublines, HCC827 GR2 and H1975 WR7, depended on the anti-apoptotic BCL2 family members, Bcl-xL and/or MCL1, for survival. Although HCC827 GR2 cells were hardly affected by Bcl-xL or MCL1 knockdown alone, dual inhibition of Bcl-xL and MCL1 caused the cells to undergo apoptosis, resulting in decreased viability. In H1975 WR7 cells, not only dual inhibition, but also MCL1 silencing alone, induced the cells to undergo apoptosis. Interestingly, the two sublines markedly declined in number when autophagy flux was suppressed, because they depend, in part, on active autophagy for survival. However, autophagy inhibition was inferior to dual inhibition of Bcl-xL plus MCL1 for GR2 cells, or MCL1 inhibition alone, for decreasing the viability of WR7 cells. Collectively, these findings suggest that inhibiting Bcl-xL plus MCL1, or MCL1 alone, may represent a new approach to treat EGFR-independent EGFR-mutant cancer cells.