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

PPIA dictates NRF2 stability to promote lung cancer progression

Nuclear factor erythroid 2-related factor 2 (NRF2) hyperactivation has been established as an oncogenic driver in a variety of human cancers, including non-small cell lung cancer (NSCLC). However, despite massive efforts, no specific therapy is currently available to target NRF2 hyperactivation. Here, we identify peptidylprolyl isomerase A (PPIA) is required for NRF2 protein stability. Ablation of PPIA promotes NRF2 protein degradation and blocks NRF2-driven growth in NSCLC cells. Mechanistically, PPIA physically binds to NRF2 and blocks the access of ubiquitin/Kelch Like ECH Associated Protein 1 (KEAP1) to NRF2, thus preventing ubiquitin-mediated degradation. Our X-ray co-crystal structure reveals that PPIA directly interacts with a NRF2 interdomain linker via a trans-proline 174-harboring hydrophobic sequence. We further demonstrate that an FDA-approved drug, cyclosporin A (CsA), impairs the interaction of NRF2 with PPIA, inducing NRF2 ubiquitination and degradation. Interestingly, CsA interrupts glutamine metabolism mediated by the NRF2/KLF5/SLC1A5 pathway, consequently suppressing the growth of NRF2-hyperactivated NSCLC cells. CsA and a glutaminase inhibitor combination therapy significantly retard tumor progression in NSCLC patient-derived xenograft (PDX) models with NRF2 hyperactivation. Our study demonstrates that targeting NRF2 protein stability is an actionable therapeutic approach to treat NRF2-hyperactivated NSCLC.

Representation of genomic intratumor heterogeneity in multi-region non-small cell lung cancer patient-derived xenograft models

Patient-derived xenograft (PDX) models are widely used in cancer research. To investigate the genomic fidelity of non-small cell lung cancer PDX models, we established 48 PDX models from 22 patients enrolled in the TRACERx study. Multi-region tumor sampling increased successful PDX engraftment and most models were histologically similar to their parent tumor. Whole-exome sequencing enabled comparison of tumors and PDX models and we provide an adapted mouse reference genome for improved removal of NOD scid gamma (NSG) mouse-derived reads from sequencing data. PDX model establishment caused a genomic bottleneck, with models often representing a single tumor subclone. While distinct tumor subclones were represented in independent models from the same tumor, individual PDX models did not fully recapitulate intratumor heterogeneity. On-going genomic evolution in mice contributed modestly to the genomic distance between tumors and PDX models. Our study highlights the importance of considering primary tumor heterogeneity when using PDX models and emphasizes the benefit of comprehensive tumor sampling.

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.

Personalizing non-small cell lung cancer treatment through patient-derived xenograft models: preclinical and clinical factors for consideration.

In the pursuit of creating personalized and more effective treatment strategies for lung cancer patients, Patient-Derived Xenografts (PDXs) have been introduced as preclinical platforms that can recapitulate the specific patient's tumor in an in vivo model. We investigated how well PDX models can preserve the tumor's clinical and molecular characteristics across different generations. A Non-Small Cell Lung Cancer (NSCLC) PDX model was established in NSG-SGM3 mice and clinical and preclinical factors were assessed throughout subsequent passages. Our cohort consisted of 40 NSCLC patients, which were used to create 20 patient-specific PDX models in NSG-SGM3 mice. Histopathological staining and Whole Exome Sequencing (WES) analysis were preformed to understand tumor heterogeneity throughout serial passages. The main factors that contributed to the growth of the engrafted PDX in mice were a higher grade or stage of disease, in contrast to the long duration of chemotherapy treatment, which was negatively correlated with PDX propagation. Successful PDX growth was also linked to poorer prognosis and overall survival, while growth pattern variability was affected by the tumor aggressiveness, primarily affecting the first passage. Pathology analysis showed preservation of the histological type and grade; however, WES analysis revealed genomic instability in advanced passages, leading to the inconsistencies in clinically relevant alterations between the PDXs and biopsies. Our study highlights the impact of multiple clinical and preclinical factors on the engraftment success, growth kinetics, and tumor stability of patient-specific NSCLC PDXs, and underscores the importance of considering these factors when guiding and evaluating prolonged personalized treatment studies for NSCLC patients in these models, as well as signaling the imperative for additional investigations to determine the full clinical potential of this technique.

Abstract 7203: Anti-HDGF antibody attenuates the rise of resistance to EGFR tyrosine kinase inhibitor in NSCLC patient-derived xenograft tumors

Abstract Mutant epidermal growth factor receptor (EGFR), a major oncogenic driver in non-small cell lung cancer (NSCLC), is found in the tumors of 15% (western countries) to 50% (Asian countries) of patients. EGFR-targeted therapy using tyrosine kinase inhibitors (TKIs) is a standard of care for those patients who developed metastatic or recurrent diseases. However, despite having an 80% initial response rate, most patients had partial tumor regression and their disease would progress when acquired resistance emerges. Thus, developing more effective treatment is an unmet need. The clinical response of these patients can be recapitulated in mouse models of patient-derived xenograft (PDX) tumor bearing sensitizing EGFR mutations. In this study, treating established PDX tumors with osimertinib, a third generation EGFR TKI, induced partial tumor regression (50% to 70% size reduction from baseline) followed by progression with a median progression-free survival (PFS) of 26 to 28 days. Interestingly, co-administering an antibody against hepatoma-derived growth factor (HDGF) with osimertinib resulted in enhanced tumor regression (98% to 100% size reduction from baseline) which translated into significantly extended animal survival with a median PFS of 103 or 135 days. Immunohistochemical and western blot examination of tumors collected at different treatment timepoints indicated that AKT/mTOR and MAPK pathways were reactivated after initial suppression in TKI monotherapy arm despite persistent EGFR inhibition. Since AKT/mTOR and MAPK pathways are key survival and proliferative signaling pathways, their reactivation can promote TKI tolerance of tumor cells and the emergence of TKI resistance. However, blocking HDGF attenuated the reactivation of these pathways which could partially explain the enhanced efficacy of osimertinib plus anti-HDGF antibody combination treatment seen in this study. Our results suggest tumor-derived HDGF could play a crucial role in the rise of acquired resistance to osimertinib. Identifying the exact cells and molecular pathways utilized by HDGF to promote tumor cell tolerance to TKI will help us understand how TKI sensitive tumor cells survive EGFR blockade and emerge as resistant cells. Citation Format: Cindy Q. Zhou, Ariel Li, Kaoru Ri, Ahmed Sultan, Hening Ren. Anti-HDGF antibody attenuates the rise of resistance to EGFR tyrosine kinase inhibitor in NSCLC patient-derived xenograft tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7203.

Abstract 1914: Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression

Abstract Amivantamab (RYBREVANT™) is an FDA-approved, low-fucose IgG1 bispecific antibody targeting EGF and Met receptor tyrosine kinases, with proven clinical activity against EGFR mutant non-small cell lung cancer (NSCLC). Preclinical studies demonstrated that three context-dependent mechanisms of action (MOA) contribute to amivantamab efficacy: immune cell-mediated killing by antibody-dependent cellular cytotoxicity and trogocytosis, receptor internalization and degradation, and inhibition of ligand binding to both EGF and Met receptors. In tumors lacking known cancer-driver genetic aberrations, ligand binding to wildtype EGFR (EGFRWT) and/or MetWT may activate downstream signaling pathways, inducing pro-tumorigenic processes. Among the EGFR ligands, we demonstrated that amphiregulin (AREG) is highly expressed in EGFRWT NSCLC primary tumors, with significantly higher circulating protein levels in NSCLC patients than in healthy volunteers, and induces EGFR phosphorylation, promoting downstream signaling and increasing cell proliferation. Importantly, treatment of AREG-stimulated EGFRWT cells/tumors with amivantamab, or an AREG-targeting antibody, inhibits ligand-induced signaling and cell/tumor proliferation/growth. Across 10 EGFRWT NSCLC patient-derived xenograft mouse models, amivantamab efficacy correlated with AREG RNA levels, suggesting AREG as a potential predictive marker for amivantamab activity in this population. Interestingly, in these xenograft models, amivantamab anti-tumor activity was independent of amivantamab's Fc engagement with immune cells, suggesting that, in this context, the amivantamab’s ligand-blocking function is sufficient for its efficacy. Juxtaposedly, we previously demonstrated that low fucose amivantamab, active Fc domain, was necessary for maximal anti-tumor activity against tumors with EGFR or Met activating mutations. Altogether, these results demonstrated amivantamab context-dependent MOA for efficacy. 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 against anti-EGFRWT therapies. Citation Format: Ricardo Rivera-Soto, Benjamin Henley, Marian Pulgar, Stacey Lehman-Tenuto, Himanshu Gupta, Megan Weindorfer, Smruthi Vijayaraghavan, Tsun-Wen Yao, Sylvie Laquerre, Sheri Moores. Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1914.

Abstract C115: Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate

Abstract STRO-002 is a novel folate receptor alpha (FolRα)-targeting antibody-drug conjugate (ADC) currently undergoing evaluation in clinical trials at various phases of development in ovarian and endometrial cancer and pediatric AML. Here, we describe the favorable properties of the STRO-002 ADC and summarize its activity in preclinical models. STRO-002 is an ADC composed of an anti-FolRα antibody conjugated to four tubulin-targeting hemiasterlin warheads. Incorporation of non-natural amino acids, using Sutro’s XpressCF® and XpressCF+® cell-free expression and conjugation systems, allows for site-specific conjugation of the hemiasterlin payload, yielding a homogenous and high-fidelity ADC. As an ADC, STRO-002 internalizes rapidly into FolRα+ tumor cells and exhibits potent target-dependent cell killing on xenograft tumor cell lines. Further characterization of STRO-002 and the hemiasterlin payload reveals significant ADC bystander activity and a lower sensitivity to drug efflux via the P-gp drug pump compared to other microtubule-targeting warheads. In addition to robust direct and bystander cell killing, STRO-002 is also capable of triggering immunogenic cell death (ICD). In in vitro cell killing assays, STRO-002 induces all three hallmarks of ICD—HMGB1 and ATP release and surface exposure of calreticulin—and in in vivo vaccination studies, injection of STRO-002-killed tumor cells was effective at establishing a protective immune response against subsequent tumor challenge, identifying STRO-002 as a bonafide ICD inducer.In vivo, STRO-002 treatment demonstrates robust efficacy in ovarian xenograft models, even when treatment is initiated in large, established tumors. In addition, combination therapy with STRO-002 and carboplatin, anti-VEGF, or anti-PD-L1 results in greater efficacy than treatment with single agents alone, demonstrating combination benefit with current standard-of-care therapies. To better understand the activity of STRO-002 in more-translatable preclinical models, we turned to patient-derived xenografts (PDX) and found that STRO-002 exhibits good anti-tumor activity in endometrial cancer and non-small cell lung cancer PDX studies, suggesting potential clinical benefit in these indications. In conclusion, STRO-002 exhibits multiple advantageous properties as an ADC and shows good anti-tumor activity, either in combination or as a single agent, in both cell-derived xenograft and PDX models. Citation Format: Robert Yuan, Andrew McGeehan, Sihong Zhou, Jennifer Smith, Dayson Moreira, Krishna Bajjuri, Cuong Tran, Gang Yin, Alice Yam, Helena Kiefel, Xiaofan Li. Preclinical characterization of STRO-002, a clinical-stage anti-FolRα antibody-drug conjugate [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 C115.

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.

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.

Circadian gene ARNTL initiates circGUCY1A2 transcription to suppress non-small cell lung cancer progression via miR-200c-3p/PTEN signaling

BackgroundAs a subclass of endogenous stable noncoding RNAs, circular RNAs are beginning to be appreciated for their potential as tumor therapeutics. However, the functions and mechanisms by which circRNAs exert protective functions in non-small cell lung cancer (NSCLC) remain largely elusive.MethodsThe prognostic role of circGUCY1A2 was explored in lung adenocarcinoma specimens. The overexpressed and knockdown plasmids were used to evaluate the effect of circGUCY1A2 on NSCLC cell proliferation and apoptosis efficacy. Luciferase reporter system is used to prove that circGUCY1A2 could bind to miRNA. Chip-PCR was used to prove that circGUCY1A2 could be initiated by transcription factors ARNTL. Subcutaneous tumorigenicity grafts models were established to validate findings in vivo.ResultsThe expression of circGUCY1A2 were significantly reduced (P < 0.001) and negatively correlated with tumor size (P < 0.05) in non-small cell lung cancer (NSCLC). CircGUCY1A2 upregulation promoted apoptosis and inhibits cell proliferation and growth of subcutaneous tumorigenicity grafts in nude mice (P < 0.01). In addition, intra-tumor injection of pLCDH-circGUCY1A2 inhibited tumor growth in patient-derived NSCLC xenograft models (PDX). Mechanism studies showed that circGUCY1A2 could act as a sponge to competitively bind miR-200c-3p, promote PTEN expression, and thereby inhibit PI3K/AKT pathway. In addition, we found that the circadian gene ARNTL, which was reduced in NSCLC and prolonged the overall survival of patients, could bind to the promoter of circGUCY1A2, thereby increasing its expression.ConclusionsThis study is an original demonstration that ARNTL can inhibit the development of lung adenocarcinoma through the circGUCY1A2/miR-200c-3p/PTEN axis, and this finding provides potential targets and therapeutic approaches for the treatment of lung adenocarcinoma.

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.

Use of a systems-biology informed machine learning model to predict drug response using clinically available NGS data.

3136 Background: Tremendous progress towards understanding the molecular underpinnings of cancer has ushered in an era of precision medicine, where actionable biomarkers inform rational design of novel drugs, repurposing of existing medications or new combination therapies, agnostic of tumor type. However less than 20% of cancer patients match to therapies using existing predictive biomarkers. There is therefore an acute need for new methods to expand the population of patients who could benefit from existing drugs and improve patient selection and design of clinical trials evaluating new or repurposed drugs. Methods: We developed systems biology-informed AI methods trained on vast multi-omics and multi-modal datasets from DepMap, TCGA and other publicly available sources, to uncover latent vulnerabilities existing in tumors. These vulnerabilities map to pharmacological sensitivities and can be assigned to individual patients using clinically available molecular data. To evaluate our model, we focused on predicting sensitivities to a third-generation tyrosine kinase inhibitor (TKI), which we assessed in non-small cell lung cancer (NSCLC) cell lines, patient derived xenografts (PDXs) and a clinicogenomics cohort from AACR Project GENIE (n=2004 patients, n=273 patients treated with TKI of interest). Where applicable, whole exome data used as input for validation model predictions were filtered to ~300 genes included on commercial NGS panels to reflect data available in RWD cohorts. Results: Our model accurately predicts response to the TKI across diverse cancer cell lines with high accuracy (r2= 0.768; rho= 0.83; mean absolute error = 0.065). Using NSCLC cancer cell lines and PDX model systems heterogeneous for this TKI’s current FDA molecular label (but within the scope of the drug’s current cancer indication), we were able to further validate our model’s predictions, show they were not dependent on obvious molecular confounders, and demonstrate a potential label expansion opportunity for this drug beyond the current narrow molecular label. While all NSCLC patients from AACR Project GENIE treated with this TKI were “on label”, NSCLC patients our model predicted to be sensitive to the TKI had a 4-month increased median PFS compared to patients predicted to be less sensitive (Log-rank test p-value&lt; 0.05; HR= 1.64; 1.23-2.22, 95% CI). The PFS partition was not due to obvious clinical confounders such as stage (p= 0.91), age (p=0.86), sex (p=0.63), smoking status (p=0.57) or on-target mutations (p=0.70). Biological networks associated with each prediction provide rich context and highlight a putative rational drug combination strategy for this drug. Conclusions: We have developed an ML model that uses clinically obtainable NGS data to accurately predict increased sensitivity to a third-generation TKI validated in NSCLC cell lines, PDX models and a RWD patient cohort.

Abstract ND10: Discovery of MRT-2359, an orally bioavailable GSPT1 molecular glue degrader, for MYC-driven cancers

Abstract MYC transcription factors have been demonstrated to be drivers of many cancers, yet have remained recalcitrant to new drug development. We hypothesized that MYC-driven cancer cell lines are addicted to protein translation, and therefore are vulnerable to the loss of the translation termination factor, GSPT1. We have discovered MRT-2359, a selective GSPT1 molecular glue degrader, that demonstrates potent and preferential antiproliferative activity in MYC-driven cell lines, such as high N- and L-MYC mRNA expressing non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) lines. MRT-2359 has been optimized for differential toxicity between MYC- and non-MYC-driven cancer cell lines, kinetics and depth of degradation, oral bioavailability, and in vivo efficacy. The anti-tumor activity of MRT-2359 was assessed in more than 80 lung patient-derived xenografts (PDXs) that confirmed the preferential anti-tumor activity in N- and L-MYC high NSCLC and SCLC PDXs when dosed orally daily or intermittently. Similar levels of activity were also observed in neuroendocrine lung cancer PDXs and lymphoma models. Oral MRT-2359 is currently in a Phase 1/2 clinical trial in selected cancer patients with MYC-driven NSCLC, SCLC, high grade neuroendocrine cancers and diffuse large B-cell lymphoma (NCT05546268). Citation Format: Owen B. Wallace, Gerald Gavory, Mahmoud Ghandi, Anne-Cecile d’Alessandro, Debora Bonenfant, Maciej Cabanski, Lisa Cantagallo, Agustin Chicas, Qian Chen, Anna Diesslin, Christopher King, Vittoria Massafra, Rajiv Narayan, Arnaud Osmont, Dave Peck, Carolina Perdomo Ortiz, Martin Schillo, Ambika Singh, Ralph Tiedt, Simone Tortoioli, Silvia Buonamici, Markus Warmuth, Filip Janku, Bernhard Fasching. Discovery of MRT-2359, an orally bioavailable GSPT1 molecular glue degrader, for MYC-driven cancers [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 ND10.

Outcome comparison of pyrotinib with current standard of care in the second/third line setting in advanced non-small cell lung cancer patients with HER2 mutation.

Outcome comparison of pyrotinib with current standard of care in the second/third line setting in advanced non-small cell lung cancer patients with HER2 mutation.

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

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

Abstract 6276: SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid tumors

Abstract Background: Protein arginine methyltransferase 5 (PRMT5) is overexpressed in various human malignancies and induces epigenetic and post-translational changes, which have a significant impact on cell growth, proliferation, apoptosis, and DNA damage repair. SKL27969 is a potent and selective brain-penetrating PRMT5 inhibitor demonstrating antitumor activity in the preclinical models of glioblastoma (GBM) and non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC) brain metastasis models. In this study, we evaluated the antitumor activity of SKL27969 alone or in combination with DNA-damaging agents in various cancer models. Methods: Cell cycle analysis, apoptosis, proliferation, and invasion assays were conducted to determine in vitro profiles of SKL27969. In vivo efficacy of SKL27969 was evaluated in human cancer cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) models. Immunohistochemistry and RNA-sequencing analysis in tumor samples from animal models were conducted to confirm the in vivo mechanism of action (MoA). In vitro synergistic effects of SKL27969 with combination partner drugs were analyzed using Combenefit software. Results: SKL27969 exhibits strong anti-proliferative effect by inducing cell cycle arrest and apoptosis and also inhibits invasion of cancer cells. An evaluation of SKL27969 in a panel of over 100 cancer cell lines revealed a broad spectrum of anti-proliferative activity in various cancer types, and an especially more sensitive response in cell lines harboring genetic alterations in the mitotic DNA damage checkpoint, DNA repair, or RNA processing pathways. Administration of SKL27969 demonstrated significant tumor growth inhibition, with a decrease in tumor symmetric dimethylarginine (SDMA) in NSCLC and TNBC subcutaneous xenograft models. Transcriptome and immunohistochemical analysis of SKL27969-treated tumors revealed that the cell cycle checkpoint pathways and DNA damage repair genes were downregulated, supporting the rationale for combination treatment with DNA-damaging agents such as chemotherapeutic agents. Robust antitumor effects were observed after treatment with SKL27969 in combination with paclitaxel or gemcitabine in NSCLC PDX or pancreatic cancer CDX models. Further ex vivo studies to confirm the in vivo MoA of this synergism, and in vivo combination studies of SKL27969 with standard chemotherapeutic agents in other solid cancer CDX models, are ongoing. Conclusion: This is the first preclinical study that suggests the therapeutic potential of SKL27969 in combination with currently approved DNA-damaging agents, and supports further clinical investigation of SKL27969’s role in improving the therapeutic benefit of standard therapies. Citation Format: Mijin Moon, Yongje Shin, Soyoung Ki, Jungtae Na, Ho Yeon Lee, Ilkyoo Koh, Beomjin Hong, Jiyeon Yun, Janice Laramy, Vijaykumar Vashi, Sook-Kyung Park. SKL27969, an oral selective PRMT5 inhibitor, sensitizes the effect of DNA-damaging agents in preclinical models of multiple solid tumors. [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 6276.

Abstract 4678: Characterization of a PDX panel covering molecular diversity of non-small cell lung cancer to accelerate the development of precision therapy

Abstract Lung cancer remains the first cause of cancer-related deaths worldwide, of which Non-Small Cell Lung Cancer (NSCLC) represents more than 80% of patients with advanced disease at the time of diagnosis. NSCLC is a highly heterogenous disease, and the identification of its main actionable oncogenic drivers (i.e. EGFR, ALK, PI3K/AKT/mTOR, RET, MET, BRAF and NTRK/ROS1) and the development of specific inhibitors against these targets has transformed therapeutic care. In addition, immune-checkpoint therapy has emerged as an indispensable treatment modality, especially for patients lacking actionable oncogenic drivers, although biomarkers for predicting response to immune-checkpoint inhibition have remained elusive. Despite these new therapeutic options, NSCLC remains a lethal disease in the majority of patients due to tumor plasticity and selection leading to frequent resistance development and disease progression. Efforts are therefore needed to identify drugs and drug combinations that can prevent or overcome these resistance pathways. Patient-Derived Xenografts (PDX) models developed in immune-compromised mice recapitulate the disease more faithfully than any other in vivo model in terms of histopathologic and genomic features. They have proven their relevance in the study of pathways leading to the development and progression of cancer, to the mechanisms linked to tumor resistance and to the identification of novel effective therapies, facilitating the translation of preclinical results in the clinical setting. We describe a platform of over 35 NSCLC PDX models covering most of the molecular diversity of the disease, that have been fully characterized at the molecular level and for their response to a panel of cytotoxic chemotherapies and targeted therapies. These NSCLC PDX models have been established in immune-deficient mice from tumor biopsies collected in treatment-naïve patients or in patients having acquired resistance following an initial objective response to a variety of targeted inhibitors (EGFRi, ALKi, ROSi, BRAFi,⋯) in the MATCH-R clinical trial. In addition, 4 PDX models were established from circulating tumor cells (CTC) isolated from the blood of advanced NSCLC patients. Finally, some of these NSCLC PDX models were established in highly immunodeficient mice humanized with human PBMCs or CD34+ cells allowing testing of efficacy of bispecific T-Cell engager antibody or immune-checkpoint inhibitors. This panel of NSCLC PDX models provides a powerful preclinical platform to improve our knowledge on the mechanisms underlying resistance to treatment and to rapidly evaluate response to new treatments and translate this knowledge to the clinic. Citation Format: Delphine Nicolle, Laura Brulle-Soumare, Katell Mevel, Ludovic Bigot, Tala Tayoun, Benjamin Besse, Françoise Farace, Luc Friboulet, Didier Decaudin, Erwan Corcuff, Anaïs Joachim, Bernard Malissen, Ana Zarubica, Hervé Luche, Jean-Gabriel Judde, Olivier Deas. Characterization of a PDX panel covering molecular diversity of non-small cell lung cancer to accelerate the development of precision therapy. [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 4678.

Abstract 2412: MET inhibition enhances the effect of radiation in MET dysregulated non-small cell lung cancer models

Abstract Objective: Alterations in the MET receptor are seen in up to 10% of patients with non-small cell lung cancer (NSCLC). A mutation in exon 14, which results in deletion of the intracellular juxtamembrane domain of the receptor, leading to enhanced signaling, is the most common MET mutation in NSCLC. MET is involved in multiple pathways associated with apoptosis and radiation response. We investigated the effects of inhibiting MET with capmatinib, a potent and clinically relevant ATP-competitive kinase inhibitor, in combination with radiation in MET-dependentNSCLC models. Methods: MET exon 14 mutated and MET amplified NSCLC cell lines treated with radiation or capmatinib were assessed with proliferation, clonogenic survival, apoptosis, and DNA damage signaling and repair assays. MET signaling proteins were evaluated by Western blotting. In vivo growth responses to the MET inhibitor capmatinib with and without radiation were evaluated in patient derived xenograft (PDX) models established from both MET amplified and MET exon 14 skipping mutation containing patient tumors. Radiation was delivered in 10 daily fractions of 2 Gy and capmatinib was administered by oral gavage daily 1 hour prior to radiation at a dose 20 mg/kg for capmatinib. Immunohistochemistry (IHC) was performed to evaluate MET signaling and proliferation. Results: Radiation increased the expression and capmatinib inhibited phospho-AKT, phospho-s6,phospho-ERK, and phospho-STAT3 in our MET-driven NSCLC cell lines. Combination of capmatinib and radiation in clonogenic assays increased sensitivity to radiation in MET dysregulated cell lines. No radiation-enhancing effect was observed in MET wild-type and human bronchial epithelial cell lines. Campatinib alone significantly inhibited tumor growth in both ourMET exon 14 mutated and MET amplified xenograft models (p&amp;lt;0.01). Combination of capmatinib and radiation significantly delayed growth compared to vehicle control, capmatinib alone or radiation alone (p&amp;lt;0.01). IHC demonstrated inhibition of phospho-MET and phospho-S6 and a decrease in Ki67 with inhibition of MET. A significant decrease Ki67 proliferation was observed in the combination arm compared to radiation along or drug alone. Conclusion:Inhibition of MET enhanced the effect of radiation in multiple MET-altered NSCLC PDX models.Additional studies are currently underway evaluating the mechanisms of radiation sensitization with a focus on MET-induced apoptosis. Citation Format: Shrey K. Ramesh, Saahil N. Javeri, Kwangok P. Nickel, Ahmet Cifci, Rachel L. Minne, Randall J. Kimple, Andrew M. Baschnagel. MET inhibition enhances the effect of radiation in MET dysregulated non-small cell lung cancer models [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 2412.

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.