Non-small Cell Lung Cancer Patient-derived Xenograft Model Research Articles

Amivantamab efficacy in wild-type EGFR NSCLC tumors correlates with levels of ligand expression

Amivantamab is an FDA-approved bispecific antibody targeting EGF and Met receptors, with clinical activity against EGFR mutant non-small cell lung cancer (NSCLC). Amivantamab efficacy has been demonstrated to be linked to three mechanisms of action (MOA): immune cell-mediated killing, receptor internalization and degradation, and inhibition of ligand binding to both EGFR and Met receptors. Among the EGFR ligands, we demonstrated that amphiregulin (AREG) is highly expressed in wild-type (WT) EGFR (EGFRWT) NSCLC primary tumors, with significantly higher circulating protein levels in NSCLC patients than in healthy volunteers. Treatment of AREG-stimulated EGFRWT cells/tumors with amivantamab or with an AREG-targeting antibody inhibited ligand-induced signaling and cell/tumor proliferation/growth. Across 11 EGFRWT NSCLC patient-derived xenograft models, amivantamab efficacy correlated with AREG RNA levels. Interestingly, in these models, amivantamab anti-tumor activity was independent of Fc engagement with immune cells, suggesting that, in this context, the ligand-blocking function is sufficient for amivantamab maximal efficacy. Finally, we demonstrated that in lung adenocarcinoma patients, high expression of AREG and EGFR mutations were mutually exclusive. In conclusion, these data 1) highlight EGFR ligand AREG as a driver of tumor growth in some EGFRWT NSCLC models, 2) illustrate the preclinical efficacy of amivantamab in ligand-driven EGFRWT NSCLC, and 3) identify AREG as a potential predictive biomarker for amivantamab activity in EGFRWT NSCLC.

PARP inhibitor synthetic lethality in ATM biallelic mutant cancer cell lines is associated with BRCA1/2 and RAD51 downregulation.

Ataxia telangiectasia-mutated (ATM) kinase is a central regulator of the DNA damage response (DDR) signaling pathway, and its function is critical for the maintenance of genomic stability in cells that coordinate a network of cellular processes, including DNA replication, DNA repair, and cell cycle progression. ATM is frequently mutated in human cancers, and approximately 3% of lung cancers have biallelic mutations in ATM, i.e., including 3.5% of lung adenocarcinomas (LUAD) and 1.4% of lung squamous cell carcinomas (LUSC). We investigated the potential of targeting the DDR pathway in lung cancer as a potential therapeutic approach. In this context, we examined whether ATM loss is synthetically lethal with niraparib monotherapy. This exploration involved the use of hATM knockout (KO) isogenic cell lines containing hATM homozygous (-/-) and heterozygous (+/-) generated via CRISPR/Cas9 gene knockout technology in DLD-1, a human colorectal adenocarcinoma cell line. Subsequently, we extended our investigation to non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) models for further validation of poly ADP-ribose polymerase inhibitor (PARPi) synthetic lethality in ATM mutant NSCLC models. Here, we demonstared that biallelic hATM deletion (-/-) in DLD-1 impairs homologous recombination (HR) repair function and sensitizes cells to the PARPi, niraparib. Niraparib also caused significant tumor regression in one-third of the NSCLC PDX models harboring deleterious biallelic ATM mutations. Loss of hATM (-/-) was concomitantly associated with low BRCA1 and BRCA2 protein expression in both the hATM (-/-) DLD-1 cell line and PARPi-sensitive ATM mutant NSCLC PDX models, suggesting a downstream effect on the impairment of HR-mediated DNA checkpoint signaling. Further analysis revealed that loss of ATM led to inhibition of phosphorylation of MRN (Mre11-Rad50-NBS1) complex proteins, which are required for ATM-mediated downstream phosphorylation of p53, BRCA1, and CHK2. Taken together, our findings highlight that the synthetic lethality of niraparib in ATM-deficient tumors can be regulated through a subsequent effect on the modulation of BRCA1/2 expression and its effect on HR function.

Abstract C053: BACE1 cleaves EGFR to drive lung adenocarcinoma brain metastasis

Abstract Brain metastasis occurs in up to 40% of patients with non-small cell lung cancer (NSCLC). Considerable genomic heterogeneity exists between the primary lung tumour and respective brain metastasis; however, the identity of the genes capable of driving brain metastasis is incompletely understood. Here, we carried out an in vivo genome-wide CRISPR activation (CRISPRa) screen to identify molecular drivers of brain metastasis from a NSCLC patient-derived xenograft model. We discovered activation of the Alzheimer’s disease associated b-site amyloid precursor protein cleaving enzyme 1 (BACE1) led to a significant increase in brain metastasis. Furthermore, BACE1 knockout or inhibition with Verubecestat treatment blocked NSCLC brain metastasis. Mechanistically, we identified BACE1 cleaves an N-terminal fragment of EGFR to activate downstream signalling through MEK and ERK and drive this metastatic phenotype. Together our data highlights the power of in vivo CRISPR screening to unveil novel molecular drivers and potential therapeutic targets of NSCLC brain metastasis. Citation Format: Shawn C Chafe, Kui Zhai, Sheila K Singh. BACE1 cleaves EGFR to drive lung adenocarcinoma brain metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C053.

Abstract B006: High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC

Abstract KRASG12C inhibitors such as sotorasib and adagrasib have shown efficacy in patients with non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not respond. Therefore, there is a clear need for predictive biomarkers to guide treatment. The proximity ligation assay (PLA) is an immunofluorescent-based approach utilized to analyze endogenous protein-protein interactions. The method allows for the assessment of signaling-associated protein complexes, which can serve as potential targets for specific inhibitors. In particular, KRASG12C inhibitors can disrupt these complexes by interfering with the interaction between RAS and its effector RAF. By providing insights into the presence of these protein complexes, PLA offers a predictive readout for inhibitor sensitivity. We thus conducted a preclinical study to evaluate if a RAS-CRAF PLA could inform on the presence of RAS-RAF protein complexes and predict the response to KRASG12C inhibitors. To assess the specificity of the panRAS-CRAF PLA, we employed RNA interference in a KRASG12C-mutant H358 NSCLC cell line, wherein knockdown of either panRAS or CRAF resulted in a reduction in the panRAS-CRAF PLA signal. Pharmacological inhibition of RAS-RAF interaction with sotorasib also reduced the panRAS-CRAF PLA signal. We next performed panRAS-CRAF PLA in a panel of 11 KRASG12C-mutant NSCLC cell lines, revealing a higher number of panRAS-CRAF PLA spots in H358, LU65, and HCC1171. To further investigate the RAS-RAF interaction, we assessed the abundance of CRAF-bound RAS-GTP by quantifying the binding of RAS-GTP to the RAS-binding domain of CRAF. H358, LU65, and HCC1171 exhibited the highest levels of RAS-GTP among the 11 cell lines. We observed a strong correlation between the number of panRAS-CRAF PLA foci and the levels of CRAF-bound RAS-GTP (r = 0.87, P = 0.0002). These results indicated that the enhanced level of RAS-GTP for CRAF proteins facilitates the formation of RAS-CRAF complexes, thereby augmenting the panRAS-CRAF PLA signals. We then evaluated the in vitro antitumor activity of sotorasib across the G12C-mutated cell lines and found that the 50% inhibitory concentration values of sotorasib for H358, LU65, and HCC1171 were below 30 nmol/L. The panRAS-CRAF PLA was strongly associated with the sensitivity to sotorasib (r = 0.81, P = 0.002). We subsequently assessed the association of panRAS-CRAF PLA with efficacy of adagrasib in a patient-derived xenograft (PDX) cohort. PanRAS-CRAF PLA was assessed on sections of formalin-fixed paraffin-embedded tumors derived from vehicle-treated control groups at the end of efficacy studies in 18 NSCLC PDX models. Our findings revealed a significant association between high panRAS-CRAF PLA signal and increased sensitivity to adagrasib (r = 0.61, P = 0.008). Our results suggest that panRAS-CRAF PLA may serve as a predictive marker to identify patients who could derive optimal benefit from KRASG12C inhibitors, and may direct combination therapy approaches to those patients less likely to benefit from single-agent therapy. Citation Format: Ryoji Kato, Hitendra S. Solanki, Denis Imbody, Anurima Majumder, Yaakov Stern, Liznair Bridenstine, Harika Gundlapalli, Ida Aronchik, Joseph Johnson, Eric B. Haura. High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B006.

OSGIN1 is a novel TUBB3 regulator that promotes tumor progression and gefitinib resistance in non-small cell lung cancer.

Oxidative stress induced growth inhibitor 1 (OSGIN1) regulates cell death. The role and underlying molecular mechanism of OSGIN1 in non-small cell lung cancer (NSCLC) are uncharacterized. OSGIN1 expression in NSCLC samples was detected using immunohistochemistry and Western blotting. Growth of NSCLC cells and gefitinib-resistant cells expressing OSGIN1 or TUBB3 knockdown was determined by MTT, soft agar, and foci formation assays. The effect of OSGIN1 knockdown on in vivo tumor growth was assessed using NSCLC patient-derived xenograft models and gefitinib-resistant patient-derived xenograft models. Potentially interacting protein partners of OSGIN1 were identified using IP-MS/MS, immunoprecipitation, PLA, and Western blottingassays. Microtubule dynamics were explored by tubulin polymerization assay and immunofluorescence. Differential expression of signaling molecules in OSGIN1 knockdown cells was investigated using phospho-proteomics, KEGG analysis, and Western blotting. We found thatOSGIN1 is highly expressed in NSCLC tissues and is positively correlated with low survival rates and tumor size in lung cancer patients. OSGIN1 knockdown inhibited NSCLC cell growth and patient-derived NSCLC tumor growth in vivo. Knockdown of OSGIN1 strongly increased tubulin polymerization and re-established gefitinib sensitivity in vitro and in vivo. Additionally, knockdown of TUBB3 strongly inhibited NSCLC cell proliferation. Mechanistically, we found that OSGIN1 enhances DYRK1A-mediated TUBB3 phosphorylation, which is critical for inducing tubulin depolymerization. The results of phospho-proteomics and ontology analysis indicated that knockdown of OSGIN1 led to reduced propagation of the MKK3/6-p38 signaling axis. We propose that OSGIN1 modulates microtubule dynamics by enhancing DYRK1A-mediated phosphorylation of TUBB3 at serine 172. Moreover, elevated OSGIN1 expression promotes NSCLC tumor growth and gefitinib resistance through the MKK3/6-p38 signaling pathway. Our findings unveil a new mechanism of OSGIN1 and provide a promising therapeutic target for NSCLC treatment in the clinic.

BSBM-09 IN VIVO CRISPR ACTIVATION SCREEN IDENTIFIES ß-SITE AMYLOID PRECURSOR PROTEIN CLEAVING ENZYME 1 (BACE1) AS A DRIVER OF NON-SMALL CELL LUNG CANCER BRAIN METASTASIS

Abstract Brain metastasis occurs in up to 40% of patients with non-small cell lung cancer (NSCLC). Considerable genomic heterogeneity exists between the primary lung tumour and respective brain metastasis; however, the identity of the genes capable of driving brain metastasis is incompletely understood. Here, we carried out an in vivo genome wide CRISPR activation (CRISPRa) screen to identify molecular drivers of brain metastasis from a NSCLC patient-derived xenograft model. We identified activation of the Alzheimer’s disease associated ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) led to a significant increase in brain metastasis. BACE1 is highly expressed in NSCLC and patients with BACE1 expressed in their metastatic brain tumour survived for shorter periods. Genetic loss with CRISPR-Cas9 or pharmacological inhibition of BACE1 with MK-8931 decreased cell proliferation and sphere forming capacity in vitro. Furthermore, BACE1 knockout and MK-8931 treatment blocked brain metastasis in vivo. Mechanistically, we identified BACE1 activation lead to downstream signalling through MEK and ERK. Together our data highlights the power of in vivo CRISPR screening to identify novel molecular drivers and potential therapeutic targets of NSCLC brain metastasis.

Pharmacokinetic/Pharmacodynamic Analysis of Savolitinib plus Osimertinib in an EGFR Mutation-Positive, MET-Amplified Non-Small Cell Lung Cancer Model.

Osimertinib is a third-generation, irreversible, oral EGFR tyrosine kinase inhibitor (TKI) recommended as first-line treatment for patients with locally advanced/metastatic EGFR mutation-positive (EGFRm) non-small cell lung cancer (NSCLC). However, MET amplification/overexpression is a common acquired osimertinib resistance mechanism. Savolitinib is an oral, potent, and highly selective MET-TKI; preliminary data suggest that combining osimertinib with savolitinib may overcome MET-driven resistance. A patient-derived xenograft (PDX) mouse model with EGFRm, MET-amplified NSCLC was tested with a fixed osimertinib dose [10 mg/kg for exposures equivalent to (≈)80 mg], combined with doses of savolitinib (0-15 mg/kg, ≈0-600 mg once daily), both with 1-aminobenzotriazole (to better match clinical half-life). After 20 days of oral dosing, samples were taken at various time points to follow the time course of drug exposure in addition to phosphorylated MET and EGFR (pMET and pEGFR) change. Population pharmacokinetics, savolitinib concentration versus percentage inhibition from baseline in pMET, and the relationship between pMET and tumor growth inhibition (TGI) were also modeled. As single agents, savolitinib (15 mg/kg) showed significant antitumor activity, reaching ∼84% TGI, and osimertinib (10 mg/kg) showed no significant antitumor activity (34% TGI, P > 0.05 vs. vehicle). Upon combination, at a fixed dose of osimertinib, significant savolitinib dose-related antitumor activity was shown, ranging from 81% TGI (0.3 mg/kg) to 84% tumor regression (15 mg/kg). Pharmacokinetic-pharmacodynamic modeling showed that the maximum inhibition of both pEGFR and pMET increased with increasing savolitinib doses. Savolitinib demonstrated exposure-related combination antitumor activity when combined with osimertinib in the EGFRm MET-amplified NSCLC PDX model.

Abstract 5284: M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression

Abstract Mucin 1 (MUC1) and epidermal growth factor receptor (EGFR) are co-expressed at high prevalence in multiple cancer indications such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), head and neck squamous cell carcinoma, triple negative breast cancer and ovarian cancer. Tumor-associated MUC1 is hypoglycosylated and exposes peptide epitopes within its extracellular domain, which are not accessible to antibodies in normal epithelial tissue. MUC1 co-localizes with EGFR as a result of loss of cell polarity in tumor cells. M1231 is a bispecific antibody-drug conjugate (ADC) based on a SEED (strand-exchange engineered domain)-antibody scaffold targeting tumor-associated MUC1 and EGFR. Sutro Biopharma’s cell free expression system Xpress CF+™ enables incorporation of non-natural amino acids at precise positions in the SEED-antibody for site-specific linker-payload conjugation. The cytotoxic payload of M1231 (SC209) is a hemiasterlin-related microtubule inhibitor. The cleavable linker (ValCit-PABA) maintained ADC stability in human plasma in vitro, ensuring low systemic payload release. Pre-clinical activity of M1231 was assessed in vitro in cancer cell lines and in vivo in NSCLC and ESCC patient-derived xenograft (PDX) models. M1231 inhibited tumor cell viability in vitro at sub-nanomolar IC50 values. The bispecific MUC1xEGFR antibody has demonstrated superior internalization and lysosomal trafficking compared to monospecific bivalent antibodies. Efficient ADC uptake resulted in superior antitumor activity of M1231 in PDX models compared to monospecific bivalent ADCs. M1231 demonstrated durable and dose-dependent antitumor activity in PDX models. Strong antitumor activity with complete regression was observed in NSCLC and ESCC PDX models following a single treatment of M1231. M1231 antitumor activity was associated with high target expression in NSCLC PDX models and was observed across a range of target expression levels in ESCC models. In summary, efficient payload delivery to tumor cells and strong antitumor activity of M1231 indicate that M1231 may have the potential to deliver an efficacy benefit to patients with tumors co-expressing MUC1 and EGFR. Citation Format: Christine Knuehl, Lars Toleikis, Julia Dotterweich, Jianguo Ma, Seema Kumar, Edith Ross, Claudia Wilm, Martina Schmitt, Hans Juergen Grote, Christiane Amendt. M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression [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 5284.

Abstract 3335: ORIC-114, an orally bioavailable, irreversible kinase inhibitor, has superior brain penetration and antitumor activity in subcutaneous and intracranial NSCLC models

Abstract Exon 20 genomic insertions of both epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are oncogenic drivers and are most commonly found in non-small cell lung cancer (NSCLC). NSCLC patients with exon 20 insertions have a worse prognosis compared to those with other activating EGFR mutations. Moreover, approximately one-third of patients with exon 20 insertion mutations develop central nervous system (CNS) metastases over the course of their disease. Unfortunately, current therapeutics lack sufficient brain exposure for treating this patient population. ORIC-114 is a brain penetrant, orally bioavailable, irreversible small molecule inhibitor designed to target exon 20 insertions in EGFR and HER2. Notably, ORIC-114 is highly selective for the EGFR family of receptors, with excellent kinome selectivity compared to other reported exon 20 inhibitors, reducing the risk of off-target kinase liabilities. The superior brain penetration and free unbound exposure of ORIC-114 in preclinical studies also differentiates it from comparator EGFR and HER2 exon 20 targeted agents. To further characterize ORIC-114, in vivo studies were undertaken to assess activity in both subcutaneous and intracranial NSCLC tumor patient-derived xenograft (PDX) models. Consistent with in vitro potency and selectivity, once daily oral administration of 3 mg/kg ORIC-114 induced robust tumor regressions with greater than 100% tumor growth inhibition in the absence of significant body weight loss in an EGFR exon 20 insertion H773_V774insNPH NSCLC PDX model. In this subcutaneous model, ORIC-114 was superior to CLN-081 in efficacy and tolerability, and superior to BDTX-189 in efficacy. To investigate whether the brain-penetrant attributes of ORIC-114 translated into antitumor activity in the CNS, we utilized an intracranial PC-9 luciferase-labeled EGFR del 19 mutant cell line model. Once daily oral administration of ORIC-114 significantly regressed established intracranial NSCLC tumors and demonstrated greater efficacy than TAK-788, commensurate with the superior brain exposure of ORIC-114. We further explored dosing regimens in this intracranial model and found that ORIC-114 demonstrated equivalent regressions at 1.5 mg/kg twice daily and 3 mg/kg once daily, and strong efficacy with 1.5 mg/kg once daily dosing. Taken together, these data confirm ORIC-114 as a potent, selective, irreversible, brain penetrant exon 20 inhibitor, and a promising therapeutic candidate, including for patients with CNS metastases. Based upon these data, ORIC-114 is entering a Phase 1/1b clinical trial in genetically defined cancers. Citation Format: Jason E. Long, Soochan Kim, Ha Yeong Kim, Dong Guk Shin, Dong Hyun Park, Robert Warne, Akash Das, Ganapati Hegde, Padmini Narayanan, Lidia Sambucetti, Brenda Chan, Xi Chen, Jae H. Chang, Paul Gibbons, Jessica Sun, Matthew Panuwat, Lori S. Friedman, Melissa R. Junttila. ORIC-114, an orally bioavailable, irreversible kinase inhibitor, has superior brain penetration and antitumor activity in subcutaneous and intracranial NSCLC models [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 3335.

Abstract CT184: Emerging evidence of activity of BLU-945 in patients with advanced EGFR-mutant NSCLC utilizing circulating tumor DNA (ctDNA) in the phase 1/2 SYMPHONY study

Abstract Background: Despite recent advances in the treatment of EGFR-mutant (EGFRm) non-small cell lung cancer (NSCLC), most patients treated with third-generation (3G) EGFR tyrosine kinase inhibitors (TKIs), such as osimertinib, experience disease progression. The most common on-target EGFR resistance mutation is at the EGFR C797 residue (i.e., C797S), which prevents covalent binding to 3G EGFR TKIs. While 3G TKIs are effective in patients with EGFR activating and T790M mutations (commonly seen post-progression after first-/second-generation TKIs), there are no targeted therapies approved after their failure. BLU-945 is an investigational, reversible, and selective next-generation EGFR TKI intended for use as a single agent or in combination with other agents to suppress activating and on-target resistance EGFR mutants, including T790M and C797S, while sparing wildtype (wt) EGFR. BLU-945 has shown in vivo antitumor activity in 3G EGFR TKI-naïve as well as resistant NSCLC patient-derived xenograft models with EGFR T790M and C797S mutations. Based on this, we started SYMPHONY (NCT04862780), a phase 1/2 study in patients with metastatic EGFRm NSCLC, to determine the maximum tolerated dose and/or the recommended phase 2 dose (RP2D); and to assess the safety, pharmacokinetics (PK), and antitumor activity of BLU-945. Methods: In the ongoing phase 1 dose-escalation part of the study, patients aged ≥18 years with metastatic EGFRm NSCLC previously treated with ≥1 EGFR TKI (Eastern Cooperative Oncology Group performance status of 0-2) received BLU-945 once daily (QD) on a 28-day cycle following a Bayesian Optimal Interval escalation design. Adverse events (AEs), dose-limiting toxicities (DLTs), PK, ctDNA (in real-time using FoundationOne Liquid panel), and radiographic antitumor activity (by RECIST 1.1 criteria) were assessed in the first 4 treatment cohorts. Results: As of the January 7, 2022, data cut, 18 patients have been treated with BLU-945 at 25-200 mg QD. Of these, 14 (77.8%) patients had received ≥2 lines of prior systemic anticancer therapy in the metastatic setting. There were no DLTs, and most AEs were low grade. Treatment-related AEs (all Grade 1) occurred in 8 (44.4%) patients with the most common being nausea (n=3). EGFR mutation allele fraction assessment by ctDNA at C1D1 and C1D15 showed that tumors were heterogeneous at baseline. At doses of 50 mg QD and higher, reductions in EGFR resistance mutation allele fraction were seen in all patients (n=3) with EGFR C797S (n=2) and/or T790M (n=3) resistance mutations, with a median 48% reduction of these mutations. 50% of patients remain on treatment and dose escalation continues to determine the RP2D. Conclusion: As of the data cut, BLU-945 was generally well tolerated in heavily pre-treated patients with EGFRm NSCLC. There was also early evidence of a reduction in EGFR mutation allele fractions by ctDNA. Citation Format: Elaine Shum, Yasir Elamin, Karen L. Reckamp, Zofia Piotrowska, Julia Rotow, Daniel S. Tan, Koichi Goto, Jagan Parepally, Faris Albayya, Melinda Louie-Gao, Renata Sawtell, Alena Zalutskaya, David Spigel. Emerging evidence of activity of BLU-945 in patients with advanced EGFR-mutant NSCLC utilizing circulating tumor DNA (ctDNA) in the phase 1/2 SYMPHONY study [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 CT184.

Abstract 953: Efficacy of amivantamab, a bispecific EGFR/MET antibody, correlates with EGFR expression and signaling in NSCLC models with wild-type EGFR

Abstract Small molecule EGFR inhibitors have proven effective in treatment of Non-Small Cell Lung Cancer (NSCLC) with activating EGFR mutations, however there is minimal to no efficacy in wild-type EGFR NSCLC. Amivantamab, an EGFR/MET bispecific antibody, has demonstrated clinical activity across a diverse range of EGFR activating mutations in an ongoing Phase I trial and differentiates from anti-EGFR antibodies tested previously in NSCLC clinical trials due to dual targeting and enhanced interaction with Fc receptors on immune cells. As candidate biomarkers to predict response in wt EGFR disease, EGFR and MET expression (immunohistochemistry (IHC)) and signaling (proximity ligation assays (PLA)), as well as tumor associated macrophage (TAM) content, were assessed in 39 NSCLC wt EGFR patient-derived xenograft (PDX) models. A strong correlation (EGFR r=0.63; p<0.0001; MET r=0.83; p<0.0001) between the IHC and PLA scores for both EGFR and MET confirmed the expected link between signaling level and receptor expression. Based on these data, 14 models with different levels of EGFR and MET expression and TAM infiltration were selected for amivantamab efficacy assessment. While no correlation was found between MET IHC H-score and efficacy, positive correlations were identified between EGFR H-score and PLA-score to amivantamab efficacy. Exclusion of models with known oncogenic driver mutations downstream of EGFR and MET, such as KRAS and PI3K, led to strong correlations to both EGFR expression and signaling (IHC - r=0.90; p=0.002; PLA - r=0.92; p=0.001). Indeed, models with EGFR H-score ≥170 and no alternative driver mutations achieved prolonged tumor stasis or complete tumor regressions with amivantamab treatment. No correlation between TAM content and amivantamab efficacy was observed. Previous studies have demonstrated superior amivantamab efficacy in models driven by either mutant EGFR or amplification of MET, compared to a version lacking Fc interaction; however, both antibodies demonstrated similar efficacy in 13 out of 14 NSCLC wt EGFR PDX models. This suggests that while amivantamab efficacy requires Fc-interaction in models driven by EGFR mutation or MET amplification (ligand-independent), the Fc-interaction is not required for efficacy in wt EGFR driven NSCLC models. We hypothesize that NSCLC PDX models with higher levels of wt EGFR are more growth dependent on EGFR ligands, and thus inhibited by the ligand blocking activity of amivantamab. The lack of species cross-reactivity with the MET ligand HGF, likely underestimates MET signaling dependencies in these mouse models, although this may play a role in patient tumors. These findings suggest NSCLC patients with elevated wt EGFR expression and lacking driver mutations may achieve therapeutic benefit from amivantamab treatment. Citation Format: Benjamin Henley, Kristen Chevalier, Matthew Smith, Smruthi Vijayaraghaven, Barbara Bushey, Gerald Chu, Joshua Curtin, Nataša Obermajer, Kathryn Packman, Sylvie Laquerre, Matthew Lorenzi, Eric Haura, Sheri Moores. Efficacy of amivantamab, a bispecific EGFR/MET antibody, correlates with EGFR expression and signaling in NSCLC models with wild-type EGFR [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 953.

Abstract 3004: Establishment of KRAS G12C mutant patient-derived xenograft (PDX) models for pre-clinical evaluation of KRAS G12C targeted anticancer therapy

Abstract KRAS is one of the most prevalent oncogenes in human cancers. The G12C mutation results in an amino acid substitution at position 12 in KRAS, from a glycine (G) to a cysteine (C), lead to accretion of GTP-bound activated state KRAS and activation of downstream signaling pathways. In non-small cell lung cancer (NSCLC), KRAS p.G12C is the most common mutation, comprising nearly half of all KRAS mutations. Recent breakthrough work led to the development of selective inhibitors targeting the KRAS G12C mutation, which have shown promise in early clinical trials. However, the therapeutic benefit of targeted therapies can be impaired by intrinsic or acquired resistance mechanisms, suggesting an urgent need of combinatorial strategies to overcome the resistance to drugs targeting KRAS G12C.Here we report on the establishment of a panel of KRAS G12C mutant PDX models, encompass non-small cell lung cancer, colorectal cancer and gastric cancer. The activity of AMG510, a selective KRAS G12C inhibitors in clinical trial, was characterized in these PDX models. Data showed that the validated KRAS G12C mutant models display a range of sensitivity to single treatment of AMG510. Given that in clinic only 50% of NSCLC patients with KRAS G12C mutation would benefit from KRAS G12C targeted inhibitors, in order to provide an insights for clinical therapeutic strategy, we investigated the combinatorial strategies in NSCLC PDX models: (1) AMG510 combined with Opdivo in KRAS G12C/CD274 amplification models in hPBMC humanized mice; (2) AMG510 combined with EGFR inhibitor in KRAS G12C/EGFR overexpression models. The possible mechanisms of combination effect were discussed. Altogether, the panel of KRAS G12C mutant PDX models are valuable for the pre-clinical evaluation of KRAS G12C targeted anti-cancer therapies. Citation Format: Wenting Shi, Xuzhen Tang, Jingying Zhang, Xinhong Kuang, Yan Zhang, Jingjing Wang, Qingyang Gu, Qunsheng Ji. Establishment of KRAS G12C mutant patient-derived xenograft (PDX) models for pre-clinical evaluation of KRAS G12C targeted anticancer therapy [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 3004.

FGFR1 overexpression in non-small cell lung cancer is mediated by genetic and epigenetic mechanisms and is a determinant of FGFR1 inhibitor response

Amplification of fibroblast growth factor receptor 1 (FGFR1) in non-small cell lung cancer (NSCLC) has been considered as an actionable drug target. However, pan-FGFR tyrosine kinase inhibitors did not demonstrate convincing clinical efficacy in FGFR1-amplified NSCLC patients. This study aimed to characterise the molecular context of FGFR1 expression and to define biomarkers predictive of FGFR1 inhibitor response.In this study, 635 NSCLC samples were characterised for FGFR1 protein expression by immunohistochemistry and copy number gain (CNG) by in situ hybridisation (n = 298) or DNA microarray (n = 189). FGFR1 gene expression (n = 369) and immune cell profiles (n = 309) were also examined. Furthermore, gene expression, methylation and microRNA data from The Cancer Genome Atlas (TCGA) were compared. A panel of FGFR1-amplified NSCLC patient-derived xenograft (PDX) models were tested for response to the selective FGFR1 antagonist M6123.A minority of patients demonstrated FGFR1 CNG (10.5%) or increased FGFR1 mRNA (8.7%) and protein expression (4.4%). FGFR1 CNG correlated weakly with FGFR1 gene and protein expression. Tumours overexpressing FGFR1 protein were typically devoid of driver alterations (e.g. EGFR, KRAS) and showed reduced infiltration of T-lymphocytes and lower PD-L1 expression. Promoter methylation and microRNA were identified as regulators of FGFR1 expression in NSCLC and other cancers. Finally, NSCLC PDX models demonstrating FGFR1 amplification and FGFR1 protein overexpression were sensitive to M6123.The unique molecular and immune features of tumours with high FGFR1 expression provide a rationale to stratify patients in future clinical trials of FGFR1 pathway-targeting agents.

Patient-derived non-small cell lung cancer xenograft mirrors complex tumor heterogeneity.

Objective:Patient-derived xenograft (PDX) models have shown great promise in preclinical and translational applications, but their consistency with primary tumors in phenotypic, genetic, and pharmacodynamic heterogeneity has not been well-studied. This study aimed to establish a PDX repository for non-small cell lung cancer (NSCLC) and to further elucidate whether it could preserve the heterogeneity within and between tumors in patients.Methods:A total of 75 surgically resected NSCLC specimens were implanted into immunodeficient NOD/SCID mice. Based on the successful establishment of the NSCLC PDX model, we compared the expressions of vimentin, Ki67, EGFR, and PD-L1 proteins between cancer tissues and PDX models using hematoxylin and eosin staining and immunohistochemical staining. In addition, we detected whole gene expression profiling between primary tumors and PDX generations. We also performed whole exome sequencing (WES) analysis in 17 first generation xenografts to further assess whether PDXs retained the patient heterogeneities. Finally, paclitaxel, cisplatin, doxorubicin, atezolizumab, afatininb, and AZD4547 were used to evaluate the responses of PDX models to the standard-of-care agents.Results:A large collection of serially transplantable PDX models for NSCLC were successfully developed. The histology and pathological immunohistochemistry of PDX xenografts were consistent with the patients’ tumor samples. WES and RNA-seq further confirmed that PDX accurately replicated the molecular heterogeneities of primary tumors. Similar to clinical patients, PDX models responded differentially to the standard-of-care treatment, including chemo-, targeted- and immuno-therapeutics.Conclusions:Our established PDX models of NSCLC faithfully reproduced the molecular, histopathological, and therapeutic characteristics, as well as the corresponding tumor heterogeneities, which provides a clinically relevant platform for drug screening, biomarker discovery, and translational research.

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

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

Abstract 1059: A case study: OncoRat is a viable patient avatar for a NSCLC patient with a Y1248H Met activating mutation

Abstract Human cancer xenografts in rodents can provide predictive data on the success of candidate drugs in clinical trials and have been a pivotal tool in moving new drugs from the bench to the clinic. However, currently available immunodeficient mouse models have shown some limitation and variability in tumor take rates and growth kinetics in cancer cell lines. In addition, commercially available human cancer cell lines aren’t representative of the genomic and molecular diversity of cancers found in patients. Patient Derived Xenograft (PDX), in which tumor tissue is transplanted directly into rodents after biopsy from the patient, better represents that molecular signature, heterogeneity, and pathology of the original tumor. Therefore, in vivo efficacy studies with PDX models could be highly predictive for treatment sensitivity. Despite the many advantages of PDXs for preclinical research, PDX mouse models are hindered by low engraftment rates and slow tumor growth kinetics. The loss of patient tumor heterogeneity and stromal cells as the PDX is passaged multiple times to generate sufficient tumor tissue to inoculate a cohort of animals for efficacy studies is also a disadvantage in the immunodeficient mouse models. To address these limitations, we have introduced the OncoRat®; built on the SRGTM Platform, a Sprague-Dawley Rag2/Il2rg double knockout rat that lacks mature B cells, T cells, and circulating NK cells. We have demonstrated that the OncoRat has improved tumor take rate and growth kinetics for non-small cell lung cancer (NSCLC) PDXs. The NSLSC PDXs in the OncoRat have a much larger tumor volume, over 20,000 mm3 in the first passage (P0) in the rat, which provides an ample source of tissue for characterization and/or subsequent passage (P1) into OncoRat for drug efficacy studies. This leads to fewer animals used for study and faster timelines to drug efficacy data, resulting in a reduction in cost. In addition, we have used genomic analysis for guidance in planning in vivo efficacy studies. One of our NSCLC PDX models harbors a novel mutation in the MET pathway, suggesting this tumor would not be responsive to standard of care treatment. An efficacy study we performed in the OncoRat suggests that this particular tumor would respond well to Type II MET inhibitors, such as Cabonzantinib. This proof of concept study demonstrates that genomic and molecular analysis can provide insight into treatment outcomes and that PDX models in the OncoRat could serve as patient avatars for predicting treatment outcomes. Citation Format: Fallon K. Noto, Bisoye Towobola Adedeji, Sam Moody, Chris Brenzel, Jack Crawford, Goutham Narla, Tseten Yeshi Jamling. A case study: OncoRat is a viable patient avatar for a NSCLC patient with a Y1248H Met activating mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1059.

Highlights of This Issue

Surgery and chemoradiation are the standard of care for glioblastoma multiforme (GBM), a devastating and incurable cancer with few clinical options. Small molecule inhibitors targeting ATM, a master regulator of the DNA damage response, are currently in clinical development. Karlin, Allen, and colleagues report an experimental, blood-brain barrier penetrating ATM kinase inhibitor that prolongs the survival of mice with orthotopic tumors in combination with radiation. The enhanced response seen with tumors having dysfunctional p53 signaling is attributed to increased mitotic catastrophe. These results define a mechanism for preferential responses in p53-defective cancers and may lead to improved, selective treatment of GBM.Development of EGFR-tyrosine kinase inhibitor (TKI) with selectivity against exon 20 insertion mutations over wild-type is a major challenge for the treatment of non-small-cell lung cancer (NSCLC) driven by EGFR harboring such mutations. Hasako and colleagues identified TAS6417 which is a novel small molecule EGFR-TKI and exhibits selective inhibition of exon 20 insertion mutations over wild-type in vitro and in vivo, achieving remarkable antitumor efficacy in preclinical model including NSCLC patient-derived xenograft model. These findings support clinical evaluation of TAS6417 as an efficacious drug candidate for patients with NSCLC harboring EGFR exon 20 insertion mutations.EYA3 is a protein tyrosine phosphatase that promotes survival of cells after DNA damage. In this study, Wang and colleagues show that host vascular endothelial cell EYA3 promotes tumor angiogenesis, and that tumor cell EYA3 promotes survival and proliferation of tumor cells. Pharmacological inhibition of the EYA3 protein tyrosine phosphatase activity attenuates tumor growth and tumor angiogenesis by targeting both host and tumor cells. Simultaneously targeting the tumor vasculature and tumor cells is an attractive therapeutic strategy since it could counter the development of the more aggressive phenotype known to emerge from conventional anti-angiogenic agents.Rising incidence and extreme mortality rate of PDAC, engender a major unmet clinical need that urgently requires novel therapeutic strategies. Small molecule inhibitors targeting DNA damage response pathways hold promise, but rational combinations with conventional therapies require investigations. Here, Wallez and colleagues report that the ATR inhibitor, AZD6738, and gemcitabine potently synergize to inhibit proliferation of PDAC cell lines, and demonstrate that this combination is well tolerated, inducing tumor regression in preclinical cancer models. Together, these data support clinical testing of this innovative combination in patients with PDAC, and more generally in solid tumors with high levels of replication stress.

Abstract 832: An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC

Abstract Enhanced AXL expression has been observed in tumor tissues obtained from non-small cell lung (NSCLC) patients, both in drug-treatment naïve patients and in patients with acquired resistance to EGFR tyrosine kinase inhibitors (EGFRi). In this study, we have evaluated the antitumor activity of the novel AXL-targeting antibody-drug conjugate (ADC) AXL-107-MMAE (HuMax-AXL-ADC) in non-small cell lung cancer (NSCLC) in vitro and in vivo. The antitumor activity of AXL-107-MMAE (4 mg/kg) was evaluated in vivo in a mouse patient-derived xenograft (PDX) clinical trial (1 mouse per group), using a collection of 57 NSCLC-derived PDX models, encompassing the typical NSCLC histological subtypes and mutational genotypes. AXL-107-MMAE induced responses, defined by a decrease in relative tumor growth compared to an untreated control tumor, in 35/57 (61%) of PDX models. Potent anti-tumor activity, i.e. tumor stasis or tumor regression, was observed in 16 out of 57 (28%) models. Potent anti-tumor activity was associated with higher AXL RNA expression compared to models showing intermediate response or non-responders (p<0.001). The therapeutic activity of AXL-107-MMAE (2 and 4 mg/kg) was confirmed in a panel of NSCLC PDX or cell line-derived xenograft models using 6-8 mice per group. AXL-107-MMAE induced dose-dependent, single agent anti-tumor activity in 8 out of 9 models. These included 2 EGFR-mutant PDX models that were resistant to the EGFR inhibitor erlotinib, including one model containing L858R/T790M EGFR mutations and one model with a L858R EGFR mutation. To specifically evaluate opportunities for AXL-107-MMAE in EGFR-mutant, treatment-resistant NSCLC, we established a panel of EGFR-mutant NSCLC cell lines with acquired resistance to the EGFRi erlotinib, gefitinib, or osimertinib. In general, cell lines showed enhanced AXL protein expression upon acquiring resistance to EGFRi. Moreover, the AXL-expressing, EGFRi-resistant cell lines were efficiently killed by AXL-107-MMAE in vitro, whereas the parental, EGFR-TKI sensitive NSCLC cell lines, which expressed little or no AXL, did not respond to treatment with AXL-107-MMAE. In summary, we show that AXL-107-MMAE has therapeutic activity as a single agent in the majority of NSCLC-derived models evaluated in vivo, representing different NSCLC histological and mutational subtypes. Moreover, AXL-107-MMAE induced efficient cytotoxicity in NSCLC cell lines that showed enhanced AXL expression upon acquired resistance to EGFRi. Finally, AXL-107-MMAE induces anti-tumor activity in EGFR-mutant, EGFRi resistant NSCLC PDX models. Citation Format: Louise A. Koopman, Maarten L. Janmaat, Kirstine Jacobsen, Mikkel Green Terp, Elke Gresnigt-van den Heuvel, Ulf Forssman, Andreas Lingnau, Paul W. Parren, Henrik Ditzel, Esther C. Breij. An AXL-specific antibody-drug conjugate shows preclinical anti-tumor activity in non-small cell lung cancer, including EGFR-inhibitor resistant NSCLC [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 832.

Effects of siRNA Silencing of TUG1 and LCAL6 Long Non-coding RNAs on Patient-derived Xenograft of Non-small Cell Lung Cancer.

The aim of the present study was to establish a patient-derived xenograft (PDX) mouse model of non-small cell lung cancer (NSCLC) and investigate the anti-tumor efficacy of silencing of TUG1 and LCAL6 long non-coding RNA in the PDX model. PDXs were established by subcutaneously implanting NSCLC surgical tumor fragments into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical and real-time polymerase chain reaction (RT-PCR) analyses for NSCLC subtype-specific markers and expression of LCAL6 and TUG1. Anti-tumor efficacy of siRNA silencing of TUG1 and LCAL6 was also investigated in the PDX model. The effect of TUG1 and LCAL6 silencing on protein expression of proliferation marker Ki67 and HOX-gene family HOXB7 in the tumors was assessed by immunohistochemical staining and Western blotting. Establishment of NSCLC PDX models resulted in 9 of 26 cases (34.6%). Lung squamous cell carcinomas (SCC) had a higher engraftment rate (58.3%) than lung adenocarcinomas (ADC) (18.2%) (p<0.05). Comparative analysis indicated these established PDX models of NSCLC closely resembled the original tumors with regard to NSCLC subtype-specific markers TTF-1, napsin A, p63 and expression of LCAL6 and TUG1. The tumor volume and weight were significantly reduced in the TUG1-silenced group as compared to the control group (p<0.05). However, no significant tumor growth inhibition was found in the LCAL6-silenced group (p>0.05). Expression of both TUG1and LCAL6 was reduced by siRNA treatment. Expression of Ki67 and HOXB7 was significantly suppressed in both the TUG1- and LCAL6-silenced groups compared to the control group (p<0.01). The TUG1-silenced group showed more reduced Ki67 expression than the LCAL6-silenced group (p<0.05). PDX NSCLC models were established with a high degree of similarity with the original tumor with regard to histological, immunohistochemical features and RNA expression of TUG1 and LCAL6. Silencing of TUG1 inhibited both tumor growth and expression of the proliferation marker Ki67 and HOX-gene family HOXB7 in the PDX model of NSCLC.

Abstract A018: Patient-derived xenograft (PDX) models of NSCLC reflect clinical drug responses and predict effective treatments for patients

Abstract Background: Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality and prognosis remains poor despite the availability of numerous therapies. Integration of drug screening and sequencing in PDX models may allow for improved understanding of mechanisms of resistance (de novo and acquired), identification of biomarkers, and optimization of therapeutic strategies for NSCLC patients. In this study, we evaluated the response of NSCLC PDX models to multiple therapies and correlated responses to known clinical outcomes and molecular characteristics. Methods: PDX models were developed from 86 patients with NSCLC and evaluated by next-generation sequencing for genomic alterations (mutations, amplifications/deletions, fusions, and gene expression changes). Models were screened against different therapies including first-line platinum and nonplatinum doublets and triplets, second-line single agent docetaxel and pemetrexed (second-line therapies), and EGFR-targeted inhibitors. Tumor regression (TR) values and RECIST criteria were determined and correlated with known literature-based response rates (RR) as well as individual patient outcomes. Results: Eighty-eight PDX models from 86 patients were interrogated. To date, 63 (72%) models have been sequenced and 39 screened against standard-of-care therapies. There was robust concordance in mutational and allelic frequency profiles between patient tumors and corresponding PDX models. Based on PDX tumor growth, regression (CR/PR RECIST) was observed for at least one regimen in 50% (12/24) of models screened against first-line therapies and 25% (2/8) screened against second-line docetaxel or pemetrexed. No tumor regression was observed in any NSCLC model treated with the EGFR inhibitor, erlotinib (0%, 0/15), despite EGFR amplification and the absence of KRAS mutations. Nine PDX responses could be correlated to clinical outcomes, with 89% (8/9) accurately reflecting patient responses to the same treatment. Conclusion: Our study demonstrated the strong alignment between PDX model response to standard-of-care therapies and patient clinical outcomes, which highlights the potential application of PDX models for translational modeling and utilizing cohorts of PDX models for clinical trial simulation. The responses of these models to different lines of therapy reflected corresponding patient outcomes both at an individual and population level. Comprehensive sequencing (WES and RNA) and standard-of-care drug testing of these PDX models is planned and could allow a deeper understanding of such mechanisms. In this context, application of PDX models to drug development and stratification of clinical trial patients for treatment will continue to evolve. Citation Format: Daniel Ciznadija, Igor Astaturov, Haiying Cheng, Nir Peled, Jennifer Jaskowiak, Angela Davies, David Sidransky. Patient-derived xenograft (PDX) models of NSCLC reflect clinical drug responses and predict effective treatments for patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A018.