EGFR-driven Non-small Cell Lung Cancer Research Articles

Breathing in danger: how particulate matter pollution is putting the public at risk of lung cancer†.

We are constantly exposed to chemicals and other agents in our environment that can influence our risk of tumorigenesis, but exactly how these factors contribute to cancer development is largely unknown. Fine particulate matter measuring ≤2.5 μm (PM2.5 ) from air pollution can accumulate in alveoli, contributing to inflammation and tissue damage. Despite prior correlative studies highlighting the mortality risk, there has been a historical reluctance to lower national standards for safe PM2.5 exposure. A recent publication further highlights the attributable risk of PM2.5 exposure with lung cancer - particularly in 'never-smokers' with EGFR-driven non-small cell lung cancer. Importantly, it also elucidates a mechanistic link between PM2.5 exposure and tumorigenesis using in vivo models of EGFR non-small cell lung cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Race-Associated Genomic Correlates of Therapeutic Response in African American Patients With Non-Small-Cell Lung Cancer.

African American individuals are disproportionately affected by lung cancer in terms of incidence and mortality. In oncogene-driven non-small-cell lung cancer (NSCLC), emerging evidence indicates that underlying molecular heterogeneity, which can be affected by ancestry, contributes to variable drug sensitivity and therapeutic responses. The purpose of this study was to evaluate race-associated differences in reported treatment decisions, therapeutic outcomes, and molecular features in KRAS- and EGFR-mutant NSCLC. This is a retrospective study using real-world clinical-genomic data from health systems in the United States to evaluate race-associated outcomes in advanced-stage KRAS- or EGFR-driven NSCLC. Our overall objectives were to evaluate race-associated therapeutic outcomes and to describe molecular features in non-Hispanic Black (NHB) and non-Hispanic White (NHW) patients with NSCLC. A total of 723 NSCLC patients with KRAS and 315 patients with EGFR oncogenic mutations were evaluated. In KRAS-mutant patients, variable outcomes were observed in NHB and NHW patients on the basis of receiving chemotherapy alone or in combination with immune checkpoint inhibitors. NHB patients received treatment at significantly lower rates compared with NHW patients. In the EGFR-mutant cohort, NHB and NHW patients received EGFR-targeted agents at similar rates, and overall survival was not significantly different. Race-associated differences in molecular features included a higher frequency of TP53 comutation in KRAS-mutant NHB patients and higher prevalence of EGFR G719S subtype in NHB patients. In a real-world cohort of patients with NSCLC, we identified race-associated differences in therapeutic outcomes and described molecular characteristics in NHB and NHW patients with NSCLC. To proactively identify patients most likely to respond to systemic therapies, a more comprehensive approach is needed to help guide therapy selection in individualized patient populations.

Abstract 4491: TY-0584: A potent, orally available small molecule YAP/TEAD inhibitor, exhibits anti-tumor effects in vitro and in vivo

Abstract Hippo/YAP pathway plays an essential role in cell proliferation, tissue regeneration, and tumorigenesis. The emerging evidence shows that hyperactivation of the Hippo/YAP pathway induces metastasis, chemoresistance, and the attribute of cancer stem cells. Dysregulated Hippo/YAP pathway can be a dominant driver of mesothelioma, meningioma, and schwannoma. It has been reported that Hippo/YAP oncogenic activation in mesothelioma is driven by NF2 loss function. In addition, it contributes to 10% of all cancers, including lung, gastric, colon, cervical, ovarian, breast, melanoma, hepatocellular, and squamous cell carcinoma. Despite the urgent need to develop a therapeutic strategy to curb the dysregulated pathway, YAP/TAZ is difficult to be directly targeted with small molecule inhibitors because of the lack of a catalytic niche. TEADs require auto-palmitoylation to become functional. Therefore, small molecules that target palmitoylation of TEAD have been explored and VT3989 (NCT04665206) and IK-930 (NCT05228015) have success to enter the clinical trials. To target cancers with dysregulated Hippo/Yap pathway, we have discovered and are developing TY-0584, which is a potent and orally available YAP/TEAD inhibitor in the IND enabling stage. The results of PK and toxicity studies of TY-0584 showed a favorable safety profile. TY-0584 had excellent efficacy in the malignant mesothelioma H226 CDX mouse model, which is driven by NF2 deletion mutation. TY-0584 treatment also demonstrated good efficacy in the head and neck cancer PDX tumor mouse model. In previous studies, Hippo/Yap signaling promotes drug resistance to EGFR-targeted therapies in non-small cell lung cancer (NSCLC). To relax the YAP resistance mechanism in EGFR treatment, we asked if combined YAP inhibition grants an extension of responses to EGFR therapy. To this end, we treated EGFR-driven NSCLC cell models with TY-0584 and TY-9591. TY-9591 is a third-generation EGFR inhibitor developed by TYK Medicines and is currently under a pivotal Phase III clinical investigation in China (NCT05382728). The results show that the combination treatment not only offers synergistic effects, but also enhances apoptosis, compared to single drug treatment. Our in vivo data further underscores this exciting finding. Our studies showed that TY-9591 had excellent efficacy in the PC9 CDX mouse model, but tumors gradually recurred as many targeted cancer therapies did. In line with the results of the in-vitro experiments, another YAP/TEAD inhibitor TY-0536 in combination with TY-9591 significantly delay the tumor regrowth in the PC9 CDX mouse model. In summary, we identified a potent and orally available YAP/TEAD inhibitor TY-0584 which is a promising candidate for further clinical validation. [Shengli Dong and Apeng Liang contributed equally to this work. Jun Li, Shengli Dong, and Apeng Liang are the corresponding authors.] Citation Format: Apeng Liang, Shengli Dong, Guangbin Liu, Zhengfei Guo, Meihua Li, Shuaibo Han, Yundi Cao, Yian Tu, Chao Zhou, Yu Yu, Linglin Xiao, Wei Huang, Xinlong Yang, Lian Fang, Haoyun Li, Chengshan Niu, Mingyu Jiang, Feng Xing, Shaoqing Chen, Jun Li, Yusheng Wu. TY-0584: A potent, orally available small molecule YAP/TEAD inhibitor, exhibits anti-tumor effects in vitro and in vivo. [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 4491.

Cyy-287, a novel pyrimidine-2,4-diamine derivative, inhibits tumor growth of EGFR-driven non-small cell lung cancer via the ERK pathway.

In recent decades, EGFR-targeted tyrosine kinase inhibitors (TKIs) have been proven to be an effective therapy for EGFR-mutant non-small cell lung cancer (NSCLC). However, resistance to EGFR-TKIs limits their clinical application. In the present study, we investigate the antitumor effect and underlying mechanism of a novel pyrimidine-2,4-diamine derivative, cyy-287, in NSCLC. We find that cyy-287 has a high affinity for lung tissue and inhibits the proliferation of NSCLC cells. Interestingly, the significant suppression of migration and induction of apoptosis by cyy-287 are only observed in EGFR-driven but not in EGFR-wild-type (wt) cells. According to the RNA sequencing and KEGG enrichment analysis results, cyy-287 markedly inhibits the MAPK pathway in EGFR-driven PC9 cells, and western blot analysis results further indicate that cyy-287 selectively blocks the ERK pathway in EGFR-driven cells. Meanwhile, apoptosis induced by cyy-287 could be partially reversed by ERK pathway inhibition. Further experiment indicates that cyy-287 inhibits the EGFR pathway in both EGFR-driven and EGFR-overexpressing cells. Interestingly, it only induces apoptosis in EGFR-driven cells, not in EGFR-overexpressing cells. The growth of EGFR-driven cells is suppressed by cyy-287 in vivo, with fewer side effects. Our results suggest that cyy-287 may be a potential therapeutic drug with promising antitumor effects against NSCLC.

EGFR Inhibition Strongly Modulates the Tumour Immune Microenvironment in EGFR-Driven Non-Small-Cell Lung Cancer.

Simple SummaryLung cancer that is driven by mutations in the epidermal growth factor receptor (EGFR) is currently treated with tyrosine kinase inhibitors (TKIs). Although patients initially respond well to TKI treatment, drug resistance against EGFR-targeted therapy emerges. Attempts to combine immunotherapy with EGFR-targeted treatment to prolong response rates or prevent the development of resistances have been limited due to insufficient knowledge about the effects of targeted therapy on the tumour microenvironment (TME) in EGFR-driven tumours and tumour-infiltrating immune cells. The aims of this study were to improve our understanding on the impact of EGFR inhibition on the immune response in EGFR-driven lung cancer and, furthermore, to gain insights into the impact of combining targeted therapy with immunotherapy on the TME.EGFR-driven non-small-cell lung cancer (NSCLC) patients are currently treated with TKIs targeting EGFR, such as erlotinib or osimertinib. Despite a promising initial response to TKI treatment, most patients gain resistance to oncogene-targeted therapy, and tumours progress. With the development of inhibitors against immune checkpoints, such as PD-1, that mediate an immunosuppressive microenvironment, immunotherapy approaches attempt to restore a proinflammatory immune response in tumours. However, this strategy has shown only limited benefits in EGFR-driven NSCLC. Approaches combining EGFR inhibition with immunotherapy to stimulate the immune response and overcome resistance to therapy have been limited due to insufficient understanding about the effect of EGFR-targeting treatment on the immune cells in the TME. Here, we investigate the impact of EGFR inhibition by erlotinib on the TME and its effect on the antitumour response of the immune cell infiltrate. For this purpose, we used a transgenic conditional mouse model to study the immunological profile in EGFR-driven NSCLC tumours. We found that EGFR inhibition mediated a higher infiltration of immune cells and increased local proliferation of T-cells in the tumours. Moreover, inhibiting EGFR signalling led to increased activation of immune cells in the TME. Most strikingly, combined simultaneous blockade of EGFR and anti-PD-1 (aPD-1) enhanced tumour treatment response in a transgenic mouse model of EGFR-driven NSCLC. Thus, our findings show that EGFR inhibition promotes an active and proinflammatory immune cell infiltrate in the TME while improving response to immune checkpoint inhibitors in EGFR-driven NSCLC.

Discovery of BLU-945, a Reversible, Potent, and Wild-Type-Sparing Next-Generation EGFR Mutant Inhibitor for Treatment-Resistant Non-Small-Cell Lung Cancer.

While 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), most patients will eventually develop resistance to TKIs. In the case of first- and second-generation TKIs, up to 60% of patients will develop an EGFR T790M mutation, while third-generation irreversible TKIs, like osimertinib, lead to C797S as the primary on-target resistance mutation. The development of reversible inhibitors of these resistance mutants is often hampered by poor selectivity against wild-type EGFR, resulting in potentially dose-limiting toxicities and a sub-optimal profile for use in combinations. BLU-945 (compound 30) is a potent, reversible, wild-type-sparing inhibitor of EGFR+/T790M and EGFR+/T790M/C797S resistance mutants that maintains activity against the sensitizing mutations, especially L858R. Pre-clinical efficacy and safety studies supported progression of BLU-945 into clinical studies, and it is currently in phase 1/2 clinical trials for treatment-resistant EGFR-driven NSCLC.

Abstract 3328: Antitumor activity of BLU-945 and BLU-701 as single agents and in combination in EGFR L858R-driven models of NSCLC

Abstract BACKGROUND: Lung cancer is the leading cause of cancer death globally. EGFR mutations are the most common genomic drivers of non-small cell lung cancer (NSCLC), occurring in ~17% and up to 50% of Caucasian and Asian patients, respectively, with exon 19 deletions (ex19del) and L858R substitutions being the most prevalent activating EGFR mutations (EGFRm). Outcomes have improved in patients with EGFR-driven NSCLC since the introduction of EGFR tyrosine kinase inhibitors (TKIs); however, treatment resistance almost inevitably occurs. It has been reported that patients harboring the EGFR L858R mutation, who represent 3.4% of lung adenocarcinoma cases in North America and 23% in Asia, have poorer outcomes than those with EGFR ex19del, suggesting a significant unmet need in this population. BLU-945 and BLU-701 are investigational, reversible, selective, and orally available TKIs optimized for use as single-agent or combination therapy to effectively suppress activating and on-target resistance EGFR mutants while sparing WT EGFR, and have the potential to treat or prevent central nervous system metastases. In vivo studies previously demonstrated the antitumor activity of BLU-945 against EGFR L858R/T790M and EGFRm/T790M/C797S mutants, and BLU-701 against EGFR ex19del and EGFRm/C797S mutants. Because EGFR L858R-driven NSCLC constitutes a large population of patients who may not be receiving the same benefit from third-generation (3G) TKIs as those with EGFR ex19del, studies were conducted to evaluate the antitumor activity of BLU-945 and BLU-701, as single agents and in combination, in preclinical NSCLC tumor models driven by L858R in the absence of the T790M mutation. METHODS: The in vivo antitumor activities of BLU-945 and BLU-701, as single-agents or in combination, were evaluated in 2 EGFR L858R-driven patient-derived xenograft (PDX) subcutaneous tumor models and in an engineered EGFR L858R/C797S-driven Ba/F3 cell line-derived xenograft (CDX) subcutaneous tumor model. RESULTS: Oral daily administration of single-agent BLU-945 and single-agent BLU-701 resulted in sustained tumor regression in 2 PDX models of EGFR L858R NSCLC. Compared with single agents in an EGFR L858R/C797S Ba/F3 CDX tumor model, combination of BLU-945 with BLU-701 resulted in marked antitumor activity and prolonged tumor regression. CONCLUSION: The in vivo antitumor activities of BLU-945 and BLU-701 in preclinical tumor models suggest that both BLU-945 and BLU-701 have the potential to be used in patients with EGFR L858R-driven NSCLC, including those who are treatment naïve or previously treated with 3G TKI. Combining BLU-945 and BLU-701 may enable coverage of frequent on-target resistance mechanisms, including the EGFR C797S mutation, in addition to the common L858R activating mutation. Citation Format: Luz Tavera, Stefanie Schalm, John Campbell, Jian Guo, Clare Medendorp, Maxine Chen, Faris Albayya, Tom Dineen, Zhuo Zhang, Maria Iliou, Ebby Job, Nisha Perez, Yoav Timsit, Scott Wardwell, Katie McGinn, Richard Woessner, Chiara Conti. Antitumor activity of BLU-945 and BLU-701 as single agents and in combination in EGFR L858R-driven models of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3328.

Changes in the concentration of EGFR-mutated plasma DNA in the first hours of targeted therapy allow the prediction of tumor response in patients with EGFR-driven lung cancer.

PurposeThis study aimed to analyze changes in the plasma concentration of EGFR-mutated circulating tumor DNA (ctDNA) occurring immediately after the start of therapy with EGFR tyrosine kinase inhibitors (TKIs).MethodsSerial plasma samples were collected from 30 patients with EGFR-driven non-small cell lung cancer before intake of the first tablet and at 0.5, 1, 2, 3, 6, 12, 24, 36 and 48 h after the start of the therapy. The content of EGFR alleles (exon 19 deletions or L858R) in ctDNA was measured by ddPCR.ResultsctDNA was detected at base-line in 25/30 (83%) subjects. Twelve (50%) out of 24 informative patients showed > 25% reduction of the ctDNA content at 48 h time point; all these patients demonstrated disease control after 4 and 8–12 weeks of therapy. The remaining 12 individuals showed either stable content of EGFR-mutated ctDNA (n = 5) or the elevation of ctDNA concentration (n = 7). 10 of 12 patients with elevated or stable ctDNA level achieved an objective response at 4 weeks, but only 5 of 10 evaluable patients still demonstrated disease control at 8–12 weeks (p = 0.032, when compared to the group with ctDNA decrease). The decline of the amount of circulating EGFR mutant copies at 48 h also correlated with longer progression-free survival (14.7 months vs. 8.5 months, p = 0.013).ConclusionComparison of concentration of EGFR-mutated ctDNA at base-line and at 48 h after the start of therapy is predictive for the duration of TKI efficacy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10147-022-02128-6.

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.

MA11.01 Development of Novel EGFR Mutant NSCLC Mouse Models and Murine Cell Lines: New Tools for NSCLC Research

EGFR mutant lung cancer accounts for approximately 15-30% of all non-small cell lung cancer (NSCLC) cases, with the prevalence varying in different populations. A point mutation L858R and in-frame deletions in exon 19 of the EGFR gene account for the majority of all EGFR activating mutations. EGFR tyrosine kinase inhibitors (TKIs) have led to significant advances in the treatment of EGFR mutant NSCLC; however, many patients show a partial response and all eventually develop resistance. Better preclinical models are required to design rational combination therapies. Our group has used syngeneic orthotopic mouse models of NSCLC to study interactions between cancer cells and the tumor microenvironment in the lung. These approaches are limited by the availability of murine lung cancer cell lines. To our knowledge, there are currently no EGFR mutant murine lung cancer cell lines. Therefore, we developed a novel transgenic mouse strain that conditionally expresses mutant EGFR mediated by Cre-recombinase. Administration of adeno-Cre virus into the lungs resulted in the development of EGFR-driven lung tumors. In collaboration with the Mouse Genetics Core at National Jewish Health, we developed two novel EGFR mutant mouse models (Rosa-loxP-STOP-LoxP- EGFR L860R/del19-mC3-WPRE): one with a deletion 19 and one with an L860R mutation (equivalent to the human L858R mutation) in the mouse EGFR gene. Tumors are initiated through intratracheal injections of the adeno-Cre virus. These mice were bred with p53flox mice from Jackson Laboratories to increase the diversity of tumors. Tumors were harvested and submitted to histological characterization by H&E staining or processed to create cell lines. These cell lines were characterized by in vitro growth assays, western blot analyses, and sequencing to confirm their genotype and responsiveness to EGFR TKIs. Cell lines were implanted into both the flanks and lungs of mice to determine their responsiveness to EGFR TKIs. Tumor-bearing mice were imaged by CT and treated by oral gavage with the EGFR TKIs. Intratracheal administration of adeno-Cre led to the development of multiple tumors throughout all lobes of the lung in both del19 and L860R mice. Time to tumor formation occurred within 4-12 weeks. Importantly, treatment of the mice with osimertinib resulted in tumor shrinkage and improvement in the health of mice. Moreover, we were able to successfully isolate 3 distinct cell lines (del19.1, del19.2, L860R.1) from these tumors. In vitro, these cell lines are responsive to multiple EGFR TKIs, as evidenced by a decrease in proliferation and inhibition of phospho-ERK. These cells form tumors when implanted into the flanks of nu/nu mice, del19.1-derived tumors undergo shrinkage in response to osimertinib. Orthotopically injected del19.1 cells form tumors in the left lungs of WT C57Bl/6 mice. We have developed novel mouse models as well as murine cell lines that reflect mutant EGFR-driven NSCLC which will be useful tools for studying NSCLC both in vitro and in vivo. By increasing the number of murine NSCLC cell lines, we can better recapitulate human disease to increase our understanding of NSCLC and develop novel therapeutic strategies that can be used to treat patients.

Targeting BRAF Activation as Acquired Resistance Mechanism to EGFR Tyrosine Kinase Inhibitors in EGFR-Mutant Non-Small-Cell Lung Cancer.

Osimertinib has become a standard of care in the first-line treatment of advanced-stage non-small-cell lung cancer (NSCLC) harboring exon 19 and 21 activating mutations in the EGFR gene. Nevertheless, the 18.9-month median progression-free survival emphasizes the fact that resistance to osimertinib therapy is inevitable. Acquired resistance mechanisms to osimertinib in EGFR-driven NSCLC include MET amplification, EGFR C797S mutation, neuroendocrine differentiation, small-cell lung carcinoma histologic transformation, PD-L1 and KRAS amplifications and ESR1-AKAP12 and MKRN1-BRAF translocations, as well as BRAF V600 mutation. This last one represents 3% of the acquired resistance mechanisms to osimertinib. In this review, we discuss the rationale for EGFR/BRAF/MEK co-inhibition in the light of a clinical case of EGFR-mutant NSCLC developing a BRAF V600 mutation as an acquired resistance mechanism to osimertinib and responding to the association of osimertinib plus dabrafenib and trametinib. Additionally, we discuss the acquired resistance mechanisms to osimertinib plus dabrafenib and trametinib combination in that context.

Abstract 1262: BLU-701 is a highly potent, brain-penetrant and WT-sparing next-generation EGFR TKI for the treatment of sensitizing (ex19del, L858R) and C797S resistance mutations in metastatic NSCLC

Abstract BACKGROUND: Non-small cell lung cancer (NSCLC) represents 80% of lung cancers, the second leading cause of cancer deaths globally. Mutations in the kinase domain of the epidermal growth factor receptor (EGFR) are genomic drivers in a molecular sub-type of NSCLC that is sensitive to tyrosine kinase inhibitors (TKIs). Third-generation, irreversible TKIs, such as osimertinib and lazertinib, have become standard of care and are now widely used as first-line therapy in EGFR-driven metastatic NSCLC. However, resistance invariably arises during treatment. The C797S mutation is the most frequent on-target resistance mechanism in response to third-generation TKIs when used in the first-line setting. There are currently no approved therapies for patients who progress with a C797S mutation. Although first-generation TKIs inhibit EGFR with a C797S mutation, they carry liabilities of 1) insufficient selectivity over wild-type (WT) EGFR, which precludes maximal target coverage and results in dose-limiting adverse events; and 2) limited brain penetration. METHODS: BLU-701 activity was tested in biochemical assays for EGFR mutants and EGFR WT. Cellular activity was evaluated by a phosphorylation specific EGFR AlphaLisa assay in WT cell lines or those expressing EGFR mutations. The in vivo antitumor activity of BLU-701 was assessed in a PC9 ex19del cell line-derived tumor xenograft (CDX) model. RESULTS: We have developed an orally available, single-digit nanomolar, brain-penetrant, WT-sparing, reversible EGFR inhibitor that is active against the C797S resistance mutation. BLU-701 is equally potent against both the sensitizing mutations ex19del and L858R, and the resistance double mutants ex19del/C797S and L858R/C797S. BLU-701 is brain penetrant (Kpu,u >0.9) and demonstrates a selectivity over WT EGFR that compares favorably with osimertinib. Oral administration of BLU-701 to PC9 ex19del tumor-bearing mice resulted in strong and prolonged pathway suppression and tumor regression at tolerated doses. CONCLUSION: Due to its potent pharmacological activity and selectivity for mutant EGFR, BLU-701 has the potential to demonstrate activity in first-line and resistance settings as a single agent and in combination therapy, addressing potential tumor heterogeneity. The pre-clinical data described here supports the clinical development of BLU-701 in EGFR-driven NSCLC. Citation Format: Chiara Conti, John Campbell, Rich Woessner, Jian Guo, Yoav Timsit, Maria Iliou, Scott Wardwell, Alison Davis, Sharon Chicklas, John Hsieh, Meredith Eno, Omar Ahmad, Dilinie Fernando, Kevin Barvian, Joseph Kim, Steven Kazmirski, Emanuele Perola, Tom Dineen, Victoria Brown, Timothy Guzi, Ayşegül Özen, Faith Stevison, Caitlin Utt, Clare Medendorp, Robert Meissner, Marion Dorsch, Klaus Hoeflich. BLU-701 is a highly potent, brain-penetrant and WT-sparing next-generation EGFR TKI for the treatment of sensitizing (ex19del, L858R) and C797S resistance mutations in metastatic 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 1262.

Targeted ctDNA sequencing analysis reveals concurrent genomic alterations and its impact on TKI and immune checkpoint inhibitor therapy in advanced NSCLC from Asian population.

3042 Background: For advanced non-small cell lung cancer (NSCLC), liquid biopsy followed by targeted DNA sequencing is used in the clinical setting for driver-gene identification in circulating tumor DNA (ctDNA), tumor evolution and treatment monitoring. To understand the complexity and diversity of genetic alterations in advanced NSCLC, we examined the genetic landscape of late-stage NSCLC from an Asian population and its association with tyrosine kinase inhibitor (TKI) or immune checkpoint inhibitor (ICI) treatment. Methods: Plasma cell-free DNA (cfDNA) from 142 Asian patients with advanced NSCLC underwent real-world testing in a CAP-accredited and CLIA-certified central laboratory. We analyzed genetic alterations in cfDNA using an amplicon-based next-generation sequencing assay that covers 61 NSCLC related genes. 88 patients (62%) had received one or more lines of treatment. A similar cohort from Caucasian NSCLC was used for comparison (Blakely et al. Nat Genet 2017). Systematic co-mutation analysis, functional annotation and pathway enrichment analysis were performed. Results: The top 10 mutated genes in the Asian cohort were EGFR (66.2%), TP53 (48.6%), KRAS (11.3%), BRCA2 (7%), CTNNB1 (6.3%), PIK3CA (6.3%), SMAD4 (5.6%), BRCA1 (4.9%), MET (3.5%) and RB1 (3.5%). EGFR mutations occurred at hotspots in exons 18 – 21, with exon19 deletion, L858R, T790M, C797S, and G719A being the top-ranking alleles, similar to the Caucasian NSCLC cohort. Concurrent KRAS and EGFR mutations were higher in Asian cohort (8.5%) than in Caucasian cohort (0.56%). A rare PD-L1 structural variant (1.1%), reported to be related to immune evasion in other cancers (Kataoka et al. Nature 2016), was detected in the EGFR-mutated subgroup. Within the first line TKI-treated group, responders (CR+PR) harbored more alterations than non-responders (p < 0.017). Among ICI-treated NSCLC patients (anti-PD-1/PD-L1 mono- or polytherapy), responders from 1st line or 2nd line ICI treatment harbored fewer mutations than non-responders, but the distribution of mutational allele frequency (MAF) in responders was shifted to the right (more clonal than subclonal mutations). Functional annotation suggests that concurrent mutated genes and copy number alterations in advanced EGFR-mutant NSCLC were enriched in cell cycle, DNA repair, PI3K-AKT signaling pathways. Conclusions: We characterized the ctDNA-derived genetic landscape of advanced NSCLC from an Asian population and dissected mutated genes with the outcome of TKI or ICI treatment. We also report a rare PD-L1 structural variant in the EGFR mutated subgroup, which could be associated with immune evasion. Our analyses support the occurrence of clonal and subclonal driver co-alterations in EGFR-driven NSCLC, underlining the clinical utility of ctDNA detection for NSCLC diagnosis and treatment selection.

Detection of Tumor-Associated Membrane Receptors on Extracellular Vesicles from Non-Small Cell Lung Cancer Patients via Immuno-PCR.

Simple SummaryLung cancer is often detected at late stages when metastases are present and the genomic make-ups of the tumors are heterogeneous. Analyses of genomic alterations in non-small-cell lung cancer (NSCLC) have revealed mutated tumor-associated membrane receptors and fusion proteins, which can be targeted via tyrosine kinase inhibitors (TKIs). TKIs initially often have a good effect, but a fraction of the tumor lesions may develop resistance through additional mutations in the targeted kinases or by increased expression/function of other membrane receptors. Detection of TKI-bypassing mechanisms is difficult in tissue biopsies as these analyze only a subpart of tumors or lesions. Liquid biopsies based on tumor-secreted small extracellular vesicles (sEVs) into body fluids can assess tumor heterogeneity. We present an immuno-PCR method for the detection of the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2), and the insulin-like growth factor 1 receptor (IGF-1R) on sEVs. Initial investigations of sEVs from EGFR-mutant NSCLC tumor cells or pleural effusion (PE) fluid from patients with NSCLC or benign diseases showed different protein profiles for individual sEV samples. Further development of the immuno-PCR could complement DNA/mRNA-based assays detecting kinase mutations to allow longitudinal treatment monitoring of diverse TKI-bypassing mechanisms.Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an EGFR-driven NSCLC adenocarcinoma than in sEVs from PE samples of non-EGFR driven adenocarcinoma patients or in samples from patients with benign lung disease. In summary, we have developed a diagnostic method for sEVs in liquid biopsies of cancer patients which may be used for longitudinal treatment monitoring to detect emerging bypassing resistance mechanisms in a noninvasive way.

Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth

Epidermal growth factor receptor (EGFR) activation plays a pivotal role in EGFR-driven non-small cell lung cancer (NSCLC) and is considered as a key target of molecular targeted therapy. EGFR tyrosine kinase inhibitors (TKIs) have been canonically used in NSCLC treatment. However, prevalent innate and acquired resistances and EGFR kinase-independent pro-survival properties limit the clinical efficacy of EGFR TKIs. Therefore, the discovery of novel EGFR degraders is a promising approach towards improving therapeutic efficacy and overcoming drug resistance. Here, we identified a 23-hydroxybetulinic acid derivative, namely DPBA, as a novel EGFR small-molecule ligand. It exerted potent in vitro and in vivo anticancer activity in both EGFR wild type and mutant NSCLC by degrading EGFR. Mechanistic studies disclosed that DPBA binds to the EGFR extracellular domain at sites differing from those of EGF and EGFR. DPBA did not induce EGFR dimerization, phosphorylation, and ubiquitination, but it significantly promoted EGFR degradation and repressed downstream survival pathways. Further analyses showed that DPBA induced clathrin-independent EGFR endocytosis mediated by flotillin-dependent lipid rafts and unaffected by EGFR TKIs. Activation of the early and late endosome markers rab5 and rab7 but not the recycling endosome marker rab11 was involved in DPBA-induced EGFR lysosomal degradation. The present study offers a new EGFR ligand for EGFR pharmacological degradation and proposes it as a potential treatment for EGFR-positive NSCLC, particularly NSCLC with innate or acquired EGFR TKI resistance. DPBA can also serve as a chemical probe in the studies on EGFR trafficking and degradation.

Circulating Proteoglycan Endocan Mediates EGFR-Driven Progression of Non-Small Cell Lung Cancer.

Although new generations of EGFR-tyrosine kinase inhibitors (EGFR-TKI) have been developed for the treatment of patients with non-small cell lung cancer (NSCLC) with EGFR-mutant tumors, TKI resistance often returns as a result of additional EGFR mutations. In addition to seeking for next-generation EGFR-TKI, developing novel EGFR-targeting strategies may hold the key to overcome the vicious cycle of TKI resistance. Endocan is known as a receptor tyrosine kinase ligand enhancer in tumorigenesis, but the impact of endocan on EGFR-driven NSCLC progression remains unknown. In this study, higher endocan levels were found in lung tumors compared with cancer-free tissues and correlated with poor prognosis in patients with NSCLC harboring mutant EGFR; circulating endocan levels were also significantly higher in patients with mutant EGFR. Endocan facilitated EGFR signaling via direct binding and enhancing of the EGF-EGFR interaction and supported the growth of tumors driven by mutated EGFR. Activated EGFR in turn upregulated expression of endocan via JAK/STAT3 and ERK/ELK cascades, thus forming a positive regulatory loop of endocan-EGFR signaling. On the basis of the binding region between endocan and EGFR, we designed therapeutic peptides and demonstrated promising therapeutic effects in xenografts harboring EGFR mutations including TKI-resistant T790M. Together, our findings highlight the novel interaction between endocan and EGFR and new opportunities to effectively target endocan-EGFR regulatory axis in patients with TKI-resistant NSCLC. SIGNIFICANCE: Endocan is a novel and critical regulator of EGF/EGFR signaling and serves as an alternative target of EGFR-TKI resistance in NSCLC.

Urea Cycle Sustains Cellular Energetics upon EGFR Inhibition in EGFR-Mutant NSCLC.

Mutations in oncogenes and tumor suppressor genes engender unique metabolic phenotypes crucial to the survival of tumor cells. EGFR signaling has been linked to the rewiring of tumor metabolism in non-small cell lung cancer (NSCLC). We have integrated the use of a functional genomics screen and metabolomics to identify metabolic vulnerabilities induced by EGFR inhibition. These studies reveal that following EGFR inhibition, EGFR-driven NSCLC cells become dependent on the urea cycle and, in particular, the urea cycle enzyme CPS1. Combining knockdown of CPS1 with EGFR inhibition further reduces cell proliferation and impedes cell-cycle progression. Profiling of the metabolome demonstrates that suppression of CPS1 potentiates the effects of EGFR inhibition on central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism, coinciding with reduced glycolysis and mitochondrial respiration. We show that EGFR inhibition and CPS1 knockdown lead to a decrease in arginine levels and pyrimidine derivatives, and the addition of exogenous pyrimidines partially rescues the impairment in cell growth. Finally, we show that high expression of CPS1 in lung adenocarcinomas correlated with worse patient prognosis in publicly available databases. These data collectively reveal that NSCLC cells have a greater dependency on the urea cycle to sustain central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism to meet cellular energetics upon inhibition of EGFR. IMPLICATIONS: Our results reveal that the urea cycle may be a novel metabolic vulnerability in the context of EGFR inhibition, providing an opportunity to develop rational combination therapies with EGFR inhibitors for the treatment of EGFR-driven NSCLC.

Upfront EGFR TKI with or without brain radiotherapy (RT) in EGFR-driven non-small cell lung cancer (NSCLC) with brain metastases.

e20614 Background: The central nervous system (CNS) remains a common site of metastatic disease for NSCLC, especially in EGFR-driven disease. Surgery and RT are upfront treatment options but have potential early and late onset complications. Osimertinib (Osi) is now approved for use in the front-line setting for EGFR-mutated NSCLC and is highly CNS penetrant. A prior retrospective study showed that the use of early generation EGFR TKIs had an inferior overall survival (OS) compared to upfront RT in newly diagnosed brain metastases, but the applicability of this data in the Osi-era is unknown. Methods: This was a single institution retrospective analysis of patients with EGFR mutated NSCLC and brain metastases who were referred to Radiation Oncology from 1/1/2012 to 12/31/2018. We separated EGFR TKIs between Osi and non-Osi. The primary objectives were to evaluate OS, intracranial progression free survival (icPFS), and intracranial response (icORR) among upfront or delayed RT, and type of EGFR TKI. Results: 67 patients with a median age of 64 years old (33-89) were divided into one of four groups: non-Osi TKI with (N = 38) or without RT (N = 12), and Osi with (N = 14) or without RT (N = 3). Fourteen patients who did not get upfront Osi, received Osi with (N = 7) or without RT (N = 7) after intracranial progressive disease (icPD). Patients were predominantly female, never-smokers, and with an ECOG PS 0-1. Brain metastases were mostly asymptomatic and < 10 mm. The OS for the entire population was 26.7 months (95% CI, 23.9-29.5); there was no difference between groups, and use or type of RT versus TKI. The icPFS was 14.3 months (95% CI, 9.1-19.5), icORR was 64.2%, and icDCR was 82.7%, without any difference between groups. Among those patients who did not receive upfront Osi, use in the post-progression setting resulted in a significantly longer OS (54.8 vs. 23.0 months, p = 0.001). Conclusions: We found no statistical difference in OS, icPFS, or icORR in patients treated with upfront RT or EGFR TKI. Results should be confirmed with a prospective study.

1497P - JNJ-61186372 (JNJ-372), an EGFR-cMET bispecific antibody, in advanced non-small cell lung cancer (NSCLC): An update on phase I results

Background: JNJ-372 has demonstrated activity in EGFR and cMET-driven tumor models. In an ongoing phase 1 study of JNJ-372 in patients (pts) with advanced NSCLC, 1050 mg was identified as a recommended phase 2 dose in dose escalation (Part 1) and is being explored in the dose expansion of pts with EGFR-mutated (mut) NSCLC (Part 2). Methods: Pts were treated with JNJ-372 IV weekly x 4W for cycle 1, then biweekly thereafter. Pharmacokinetic (PK) sampling was taken at multiple time points for all pts in Part 1 and for the first 6 pts in Part 2. Disease evaluations were performed every 8 weeks. Tumors were characterized at baseline through next-generation sequencing of ctDNA and/or tumor tissue. Results: As of April 13th, 2018, 55 pts received JNJ-372 in Part 1 (n = 30) or Part 2 (n = 25), with 21 pts (38%) continuing therapy. Median age was 63y, 40% were male, 96% were Asian, and median prior systemic therapies was 2. The most frequent (≥15%) adverse events (AEs) were infusion-related reaction (56%), rash/acneiform dermatitis (53%), dyspnea (20%), paronychia (24%), nausea (22%), constipation (18%), stomatitis (18%), decreased appetite (18%), pruritus (16%), fatigue (16%), and peripheral edema (16%). AEs were mostly grade 1-2, with 3 ≥grade 3 treatment-related AEs (myalgia, neutropenia, and peripheral edema, all grade 3). PK was linear and dose proportional at doses ≥350 mg, with faster clearance PK at the 140-mg dose. Of the 30 response-evaluable pts with EGFRmut disease, treated at doses ≥700 mg, there are 8 partial responses (PRs; 4 confirmed PRs). This includes 6 pts with L858R or Exon19del primary mutations, and acquired resistance to first and/or third-generation EGFR tyrosine kinase inhibitors (TKIs), and 2 pts with primary Exon20ins disease. Activity was also observed in 2 pts with EGFRwt disease (squamous cell carcinoma and pleomorphic adenocarcinoma), each with -20% change in sum of largest diameter. The longest duration of treatment was 13.6 months. Conclusions: JNJ-372 is a novel EGFR-cMET bispecific antibody with a manageable safety profile consistent with EGFR and cMET inhibition. Preliminary evidence suggests JNJ-372 can have activity in EGFR-driven NSCLC, including pts resistant to EGFR TKIs. Clinical trial identification: NCT02609776 First posted: November 20, 2015. Editorial acknowledgement: Writing support was provided by Tracy T. Cao, PhD of Janssen Global Services, LLC. and funded by Janssen Global Services, LLC. Legal entity responsible for the study: Janssen Research & Development. Funding: Janssen Research & Development. Disclosure: B.C. Cho: Research funding: Janssen, Novartis, AstraZeneca, Yuhan, Champions Oncology, Abbvie, Dong-A ST, Ono, MOGAM; Honoraria: AstraZeneca, Novartis, MSD, Ono, Roche; Consulting or Advisory role: Janssen, AstraZeneca, Novartis, MSD, Roche, Yuhan; Speakers’ bureau: Novartis J-Y. Han: Research funding: Roche. E. Haura: Research funding: Lilly, Novartis, Boehringer Ingelheim, Janssen, Ignyta, Forma Therapeutics; Consulting or Advisory role: Janssen; Patent: Patent pending on PLA technology related to kinase inhibitor sensitivity biomarkers; Travel/Accommodations: Bristol-Myers Squibb, Roche. J. Bauml: Research funding: Merck, Novartis, Carevive Systems, Janssen, Bayer; Consulting or Advisory Role: Clovis, Bristol-Myers Squibb, Celgene, Boehringer-Ingelheim, AstraZeneca, Genentech, Guardant Health. R.E. Sanborn: Research funding: Merck (investigator-initiated study), Bristol-Myers Squibb (institutional research support), MedImmune (institutional research support); Honoraria: AstraZeneca; Consulting or Advisory Role: AstraZeneca, Seattle Genetics, Takeda, Genentech, Abbvie, Celldex; Travel/Accommodations: Janssen. M. Curtis, E. Attiyeh, N. Haddish-Berhane, K. Bae, R. Knoblauch, L. Sherman, M.V. Lorenzi: Employment: Janssen Stock: Johnson & Johnson. All other authors have declared no conflicts of interest.

Abstract 5889: TWIST1 is a key mediator of HGF-MET-driven resistance to targeted therapies in EGFR mutant and MET-driven lung cancer

Abstract The c-Met (MET) receptor and its ligand, hepatocyte growth factor (HGF), have been shown to mediate epithelial-mesenchymal transition (EMT), proliferation, invasion, motility, and angiogenesis. The HGF/MET pathway is frequently altered in non-small cell lung cancer (NSCLC) and has emerged as a targetable oncogenic driver, as patients with MET amplification and/or mutations have demonstrated marked responses to the MET tyrosine kinase inhibitor (TKI), crizotinib. However, long-term efficacy of MET TKIs is limited as acquired resistance is inevitable and almost half of patients with MET alterations fail to respond to MET TKIs. HGF overexpression has been identified as a mechanism of resistance to both MET and EGFR TKIs in MET altered and EGFR mutant NSCLC. Furthermore, MET amplification has been implicated in EGFR TKI resistance. However, the mechanism(s) by which the HGF-MET pathway causes resistance are poorly understood. We have previously shown that the EMT-transcription factor, TWIST1, is required for MET-driven NSCLC. Here, we investigated the requirement of TWIST1 in HGF-mediated resistance to MET and EGFR TKIs and the role of TWIST1 in de novo and acquired resistance to MET and EGFR TKIs. We found that HGF treatment induced EMT in NSCLC cell lines and increased TWIST1 protein expression through a post-translational mechanism. We demonstrated that targeting TWIST1 pharmacologically with the TWIST1 inhibitor, harmine, overcame HGF-mediated resistance to both MET and EGFR TKIs in MET and EGFR-driven NSCLC. This suggests that TWIST1 is specifically required for HGF-mediated resistance to targeted therapies. We also found that TWIST1 is overexpressed in a subset of MET and EGFR altered cell lines and TWIST1 overexpression was sufficient to cause resistance to MET and EGFR TKIs. In MET-driven and EGFR mutant cell lines that express TWIST1 and are resistant to targeted therapies, we demonstrated that harmine treatment resensitized resistant cells to MET and EGFR TKIs, respectively. To investigate the role of TWIST1 overexpression in Hgf-driven lung cancer, we utilized a CCSP-Hgf (CH) mouse model that constitutively overexpresses Hgf in the lung and develops crizotinib-sensitive tumors following treatment with the tobacco carcinogen, nicotine-derived nitrosamine ketone (NNK). We demonstrated that the Twist1 overexpressing CTH (CCSP-rtTA/Twist1-tetO-luc/CCSP-Hgf) mice developed significantly larger tumors in response to NNK as compared to CH and CCSP-rtTA/Twist1-tetO-luc (CT) mice. In summary, we established that HGF-regulated TWIST1 expression and that TWIST1 expression is required for resistance to MET and EGFR TKIs in the presence and absence of HGF overexpression. These studies suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome HGF-MET-driven resistance in EGFR mutant NSCLC as well as MET TKI resistance in MET-driven NSCLC. Citation Format: Zachary A. Yochum, Suman Chatterjee, Eric H. Huang, Deena M. Maurer, Myriam A. Attar, Sanja Dacic, Laura P. Stabile, Timothy F. Burns. TWIST1 is a key mediator of HGF-MET-driven resistance to targeted therapies in EGFR mutant and MET-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5889.