Non-small Cell Lung Cancer Models Research Articles

Inhibition of ULK1/2 and KRAS G12C controls tumor growth in preclinical models of lung cancer.

Mutational activation of KRAS occurs commonly in lung carcinogenesis and, with the recent FDA approval of covalent inhibitors of KRAS G12C such as sotorasib or adagrasib, KRAS oncoproteins are important pharmacological targets in non-small cell lung cancer (NSCLC). However, not all KRAS G12C -driven NSCLCs respond to these inhibitors, and the emergence of drug resistance in those patients that do respond can be rapid and pleiotropic. Hence, based on a backbone of covalent inhibition of KRAS G12C , efforts are underway to develop effective combination therapies. Here we report that inhibition of KRAS G12C signaling increases autophagy in KRAS G12C expressing lung cancer cells. Moreover, the combination of DCC-3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRAS G12C -driven lung cancer cell proliferation in vitro and superior tumor control in vivo . Additionally, in genetically engineered mouse models of KRAS G12C -driven NSCLC, inhibition of either KRAS G12C or ULK1/2 decreases tumor burden and increases mouse survival. Consequently, these data suggest that ULK1/2-mediated autophagy is a pharmacologically actionable cytoprotective stress response to inhibition of KRAS G12C in lung cancer.

MYTX-011: a pH-dependent anti-cMET antibody-drug conjugate designed for enhanced payload delivery to cMET expressing tumor cells.

Advances in linker payload technology and target selection have been at the forefront of recent improvements in antibody-drug conjugate (ADC) design, leading to several approvals over the last decade. In contrast, the potential of novel ADC technologies to enhance payload delivery to tumors is relatively underexplored. We demonstrate that incorporation of pH-dependent binding in the antibody component of a cMET targeting ADC (MYTX-011) can overcome the requirement for high cMET expression on tumors, an innovation that has the potential to benefit a broader population of patients with lower cMET levels. MYTX-011 drove four-fold higher net internalization than a non-pH engineered parent ADC in non-small cell lung cancer (NSCLC) cells and showed increased cytotoxicity against a panel of cell lines from various solid tumors. A single dose of MYTX-011 showed at least three-fold higher efficacy than a benchmark ADC in mouse xenograft models of NSCLC ranging from low to high cMET expression. Moreover, MYTX-011 showed improved pharmacokinetics over parent and benchmark ADCs. In a repeat dose toxicology study, MYTX-011 exhibited a toxicity profile similar to other MMAE-based ADCs. These results highlight the potential of MYTX-011 for treating a broader range of NSCLC patients with cMET expression than other cMET targeting ADCs. A first in human study is ongoing to determine the safety, tolerability, and preliminary efficacy of MYTX-011 in patients with NSCLC (NCT05652868).

Sanggenol L induces ferroptosis in non-small cell lung cancer cells via regulating the miR-26a-1-3p/MDM2/p53 signaling pathway

Ferroptosis is a regulated cell death marked by iron-dependent lipid peroxidation. Tumor cells that survive by evading chemotherapy-induced apoptosis are vulnerable to ferroptosis. Therefore, it is particularly urgent to explore active ingredients that can selectively induce ferroptosis in cancer cells. Here, we revealed that sanggenol L, the active agent of Morus Bark, predisposed non-small cell lung cancer (NSCLC) cells to ferroptosis, evidenced by reactive oxygen species (ROS) accumulation, glutathione depletion, mitochondrial shrinkage, and lipid peroxidation. Furthermore, the ferroptosis-related miRNA array showed that sanggenol L treatment upregulated the level of miR-26a-1-3p, which directly targeted the E3 ubiquitin ligase MDM2. In addition, silencing MDM2 by miR-26a-1-3p resulted in a notable increase in p53 protein levels and decrease of its downstream target SLC7A11, ultimately triggered ferroptosis. The subcutaneous xenograft model and patient-derived tumor xenograft (PDX) model of NSCLC further confirmed the anti-tumor efficacy and safety of sanggenol L in vivo. Collectively, our data suggest that miR-26a-1-3p/MDM2/p53/SLC7A11 signaling axis plays a key role in sanggenol L-induced ferroptosis, which implies that sanggenol L can serves as an anticancer therapeutic arsenal for NSCLC.

Prognostic predictive modeling of non-small cell lung cancer associated with cadmium-related pathogenic genes

Persistent exposure to low-dose of cadmium is strongly linked to both the development and prognosis of non-small cell lung cancer (NSCLC), yet the precise molecular mechanism behind this relationship remains uncertain. In this study, cadmium-related pathogenic genes (CRPGs) in NSCLC were identified via differential expression analysis. NSCLC patient clusters related to CRPGs were constructed through univariate Cox and K-means clustering algorithms. Multivariate Cox and least absolute shrinkage and selection operator (LASSO) regression analyses were employed to determine the prognosis. Sixteen CRPGs showed a significant association with NSCLC. We found biological and prognostic differences between patients in clusters A and B. A predictive prognostic risk model for NSCLC revealed that FAM83H, MSMO1, and SNAI1 are central. Hence, the 3 hub genes were named. To further elucidate the role of CRPGs in NSCLC, A549 cells were exposed to CdCl2. The mRNA and protein expression levels of the 3 hub genes and cell invasion were detected. Moreover, 10 μM CdCl2 may increase the protein expression of 3 hub genes and enhance the invasive ability of A549 cells. This risk model may have established a theoretical foundation for investigating the mechanisms, treatment, and prognosis of 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.

Construction of An Orthotopic Xenograft Model of Non-small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities.

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model. Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease's progression and facilitate the development of potential anticancer drugs.

Revealing prognostic insights of programmed cell death (PCD)-associated genes in advanced non-small cell lung cancer.

The management of patients with advanced non-small cell lung cancer (NSCLC) presents significant challenges due to cancer cells' intricate and heterogeneous nature. Programmed cell death (PCD) pathways are crucial in diverse biological processes. Nevertheless, the prognostic significance of cell death in NSCLC remains incompletely understood. Our study aims to investigate the prognostic importance of PCD genes and their ability to precisely stratify and evaluate the survival outcomes of patients with advanced NSCLC. We employed Weighted Gene Co-expression Network Analysis (WGCNA), Least Absolute Shrinkage and Selection Operator (LASSO), univariate and multivariate Cox regression analyses for prognostic gene screening. Ultimately, we identified seven PCD-related genes to establish the PCD-related risk score for the advanced NSCLC model (PRAN), effectively stratifying overall survival (OS) in patients with advanced NSCLC. Multivariate Cox regression analysis revealed that the PRAN was the independent prognostic factor than clinical baseline factors. It was positively related to specific metabolic pathways, including hexosamine biosynthesis pathways, which play crucial roles in reprogramming cancer cell metabolism. Furthermore, drug prediction for different PRAN risk groups identified several sensitive drugs explicitly targeting the cell death pathway. Molecular docking analysis suggested the potential therapeutic efficacy of navitoclax in NSCLC, as it demonstrated strong binding with the amino acid residues of C-C motif chemokine ligand 14 (CCL14), carboxypeptidase A3 (CPA3), and C-X3-C motif chemokine receptor 1 (CX3CR1) proteins. The PRAN provides a robust personalized treatment and survival assessment tool in advanced NSCLC patients. Furthermore, identifying sensitive drugs for distinct PRAN risk groups holds promise for advancing targeted therapies in NSCLC.

LILRB2 inhibition enhances radiation sensitivity in non-small cell lung cancer by attenuating radiation-induced senescence

Radiotherapy (RT) in non-small cell lung cancer (NSCLC) triggers cellular senescence, complicating tumor microenvironments and affecting treatment outcomes. This study examines the role of lymphocyte immunoglobulin-like receptor B2 (LILRB2) in modulating RT-induced senescence and radiosensitivity in NSCLC. Through methodologies including irradiation, lentivirus transfection, and various molecular assays, we assessed LILRB2’s expression and its impact on cellular senescence levels and tumor cell behaviors. Our findings reveal that RT upregulates LILRB2, facilitating senescence and a senescence-associated secretory phenotype (SASP), which in turn enhances tumor proliferation and resistance to radiation. Importantly, LILRB2 silencing attenuates these effects by inhibiting the JAK2/STAT3 pathway, significantly increasing radiosensitivity in NSCLC models. Clinical data correlate high LILRB2 expression with reduced RT response and poorer prognosis, suggesting LILRB2’s pivotal role in RT-induced senescence and its potential as a therapeutic target to improve NSCLC radiosensitivity.

Combining multi-mode thermal therapy with IL-6 and IL-17A neutralization amplifies antitumor immunity to facilitate long-term survival in LLC1-bearing mice

Non-small cell lung cancer (NSCLC) is known for rapid development and chronic inflammation-induced immunosuppression. IL-6 and IL-17A are the essential cytokines that facilitate NSCLC progression and myeloid-derived suppressive cell (MDSC)-mediated evasion. IL-6 or IL-17A targeting, especially IL-6, shown outstanding efficacy in patient NSCLC controlling, but failed to completely eradicate tumor. The local tumor multi-mode thermal therapy developed in our prior research was demonstrated to stimulate systemic and durable tumor-specific immune response thereby promoting long-term tumor-free survival of mice and prolong the progression-free survival of patients, although the therapeutic efficacy was still affected by high-level preoperative MDSCs. To further improve the efficacy, in this study, IL-6 and IL-17A neutralization were combined with multi-mode thermal therapy in mouse LLC1 NSCLC model. Study revealed that combined with single cytokine neutralization only prolonged the survival time while triple combination therapy efficiently improved the survival rate. Additionally, triple combination therapy reduced the accumulation of MDSCs but promoted their maturation with strengthened activation and function of myeloid cells, thereby triggering a Th1-dominant-CD4+ T cell-response and enhancing the malignant cell-killing capacity of immune cells. Our study highlights the extraordinary efficacy of combining multi-mode thermal therapy with IL-6 and IL-17A neutralization, revealing a new strategy for refractory NSCLC patients.Graphical

Preclinical Evaluation of Off-The-Shelf PD-L1+ Human Natural Killer Cells Secreting IL15 to Treat Non-Small Cell Lung Cancer.

We described previously a human natural killer (NK) cell population that upregulates PD-L1 expression upon recognizing and reacting to tumor cells or exposure to a combination of IL12, IL18, and IL15. Here, to investigate the safety and efficacy of tumor-reactive and cytokine-activated (TRACK) NK cells, human NK cells from umbilical cord blood were expanded, transduced with a retroviral vector encoding soluble (s) IL15, and further cytokine activated to induce PD-L1 expression. Our results show cryopreserved and thawed sIL15_TRACK NK cells had significantly improved cytotoxicity against non-small cell lung cancer (NSCLC) in vitro when compared with non-transduced (NT) NK cells, PD-L1+ NK cells lacking sIL15 expression (NT_TRACK NK), or NK cells expressing sIL15 without further cytokine activation (sIL15 NK cells). Intravenous injection of sIL15_TRACK NK cells into immunodeficient mice with NSCLC significantly slowed tumor growth and improved survival when compared with NT NK and sIL15 NK cells. The addition of the anti-PD-L1 atezolizumab further improved control of NSCLC growth by sIL15_TRACK NK cells in vivo. Moreover, a dose-dependent efficacy was assessed for sIL15_TRACK NK cells without observed toxicity. These experiments indicate that the administration of frozen, off-the-shelf allogeneic sIL15_TRACK NK cells is safe in preclinical models of human NSCLC and has potent antitumor activity without and with the administration of atezolizumab. A phase I clinical trial modeled after this preclinical study using sIL15_TRACK NK cells alone or with atezolizumab for relapsed or refractory NSCLC is currently underway (NCT05334329).

Cordyceps sinensis relieves non-small cell lung cancer by inhibiting the MAPK pathway

ObjectiveTo determine the pharmacodynamic mechanism underlying Cordyceps sinensis relief in a murine model of non-small cell lung cancer (NSCLC).MethodsWe created a murine model of NSCLC and studied the potential molecular mechanism by which C. sinensis relieved NSCLC using a combination of transcriptomics, proteomics, and experimental validation.ResultsC. sinensis markedly suppressed the fluorescence values in mice with NSCLC, improved the pathologic morphology of lung tissue, ameliorated inflammatory cytokines (tumor necrosis factor-alpha, interleukin-6, interleukin-10, and the oxidative stress indicators superoxide dismutase, malondialdehyde, and glutathione peroxidase). Transcriptomics results showed that the therapeutic effect of C. sinensis was primarily involved in the differentiation and activation of T cells. Based on the proteomic results, C. sinensis likely exerted a protective effect by recruiting immune cells and suppressing tumor cell proliferation via the MAPK pathway. Finally, the experimental validation results indicated that C. sinensis significantly decreased the VEGF and Ki67 expression, downregulated RhoA, Raf-1, and c-fos expression, which are related to cell migration and invasion, increased the serum concentration of hematopoietic factors (EPO and GM-CSF), and improved the percentage of immune cells (natural killer cells, dendritic cells, and CD4+ and CD8+ lymphocytes), which enhanced immune function.ConclusionsBased on our preclinical study, C. sinensis was shown to exert a protective effect on NSCLC, primarily by inhibiting the MAPK pathway.

Abstract 4007: AIC100 CAR-T cells targeting ICAM-1 are efficacious against solid tumors in xenograft mouse models of non-small cell lung cancer (NSCLC) and cervical cancer (CC)

Abstract Purpose: Establish preclinical proof of concept efficacy with CAR T cells (AIC100) in xenograft models of solid tumors (NSCLC and CC) expressing high levels of surface ICAM-1. Experimental: AIC100 is a third generation CAR T cell with micromolar affinity to ICAM-1, tuned lower than most CARs used to date in pre-/clinical studies. Affinity tuned AIC100 drug product is expected to selectively bind and kill tumor cells while safely sparing healthy cells. AIC100 also co-expresses somatostatin receptor 2 (SSTR2), which enables in vivo monitoring of AIC100 distribution and expansion by DOTATATE PET/CT imaging. To test preclinical efficacy of AIC100 in NSCLC and CC, four xenograft mouse models were generated using tumor cell lines known to have ICAM-1 expression (verified by flow cytometry) to test efficacy of AIC100. Reporter cell lines transfected with Luciferase were H226 NSCLC adenocarcinoma, H441 NSCLC squamous cell carcinoma, HeLa CC adenocarcinoma and SW756 CC squamous cell carcinoma. AIC100 efficacy was established by in vitro cytotoxicity prior to in vivo studies. NSG mice (n=10 per cell line) were inoculated via tail vein injection (1.0x106 cells) in cell growth media. Mice were imaged to ensure solid tumor formation which occurred in all mice within 3-5 days in the lung. At D0, mice were randomized into 2 groups (n=5 per group) of vehicle (PBS) and AIC100. At D1, mice were injected with AIC100 (1.0x107 live T cells) or vehicle. Mice were followed by weight and clinical observations 2x weekly and imaging by IVIS weekly. The study continued until vehicle group was euthanized due to tumor related co-morbidities at which time the study concluded. Results: AIC100 showed clear treatment benefit in both models of NSCLC and CC. In CC HeLa model, in mice treated with AIC100, primary tumors were significantly reduced by D8 and eliminated in 5/5 mice by D22. In CC SW756 model, in mice treated with AIC100, tumor growth was reduced in size by D22. In NSCLC H441 model, in mice treated with AIC100, primary tumors were largely eliminated in 5/5 mice by D15. In NSCLC H226 model, in mice treated with AIC100, tumors were significantly reduced by D8 and largely eliminated by D57 (4/5 mice). A clear survival benefit was observed in AIC100 treated mice in NSCLC H226 xenograft model. These results show treatment benefit by AIC100 in 2 tumor models per indication and establish proof of concept for the potential development of AIC100 as a treatment for ICAM-1 expressing NSCLC and CC cancers. Conclusions: AIC100 has significant treatment benefit in NSCLC and CC tumors with ICAM-1 expression in in vivo preclinical studies. Citation Format: Alyssa Birt, Serene Josiah, Kelsey L. Joyce, Srinija Alla, Michael P. Gallagher, Janusz Puc, Hanh Nguyen, Emily Walsh Martin, Sonal Gupta. AIC100 CAR-T cells targeting ICAM-1 are efficacious against solid tumors in xenograft mouse models of non-small cell lung cancer (NSCLC) and cervical cancer (CC) [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 4007.

Abstract 5907: Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-317 targeting NaPi2b

Abstract Introduction: NaPi2b is a multi-transmembrane type II sodium-dependent phosphate transporters that regulates phosphate homeostasis in normal physiological condition [1]. It is highly expressed in ovarian cancer (OC), non-small-cell lung cancer (NSCLC), and papillary thyroid cancer, with limited expression in normal tissue, making it a promising target for antibody-drug conjugates (ADCs) [2,3]. LM-317 is a novel NaPi2b targeted humanized monoclonal antibody LM-117 conjugated to the next-generation topoisomerase I inhibitor LDX2 via a cleavable linker, with antibody-drug ratio of 8. Here, we present the preclinical in vivo and in vitro results of LM-317. Results: LM-317 is highly specific cross-reactive with rhesus monkeys, cynomolgus monkeys, mouse, and rat NaPi2b. Concentration-dependent cell binding and internalization effects of LM-317 were observed in human NaPi2b-expressing cells, and OC and NSCLC cells expressing NaPi2b, with EC-50 of 0.92 - 9.7 nM and 1.51 - 4.76 nM, respectively. The cytotoxicity effect of LM-317 in NaPi2b -positive tumor cells was superior to LM-117-DXD, with IC-50 of 0.002 - 0.52nM. The in vivo studies showed that 1 mg/kg of LM-317 almost completely eradicated tumors in the OVCAR-3 xenograft mice model of OC, and similar antitumor activity was observed in the NCI-H175-xenograft mice model of NSCLC at the dose of 3 mg/kg. Single dose of 6 mg/kg of LM-317 showed sustained tumor eradication in the NSCLC xenograft -mouse model. LM-317 also exhibited potent tumor regression in the PDX model of OC and NSCLC. The tumor growth inhibition (TGI) value was 80%-100% in OC and NSCLC PDX models with different expression degree of NaPi2b after treatment with LM-317. The antitumor activity of LM-317 in cell line- and PDX models of OC and NSLCL was comparable to those of LM-117-DXD. Conclusion: The novel ADC LM-317 targeting NaPi2b showed robust antitumor activity in vitro, and in the OC and NSCLC cell line derived models and PDX models. LM-317 could potentially provide therapeutic benefit to treat NaPi2b-positive tumors including OC and NSCLC in the future. Reference: 1.S. Banerjee et al. Cancer Treatment Reviews 112 (2023) 1024892.Finstad CL, Lloyd KO, Federici MG, et al. Clin. Cancer Res. 1997;3(8):1433-14423.Zhang et al. Tumor Biology July 2017: 1-7. Citation Format: Wentao Huang, Zhifang Liu, Yifan Li, Heng Pan, Xia Qin, Da Fei, Runsheng Li. Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-317 targeting NaPi2b [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 5907.

Abstract 6487: The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models

Abstract KRASG12C is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling and unchecked oncogenic growth, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. KRASG12C-specific inhibitors are approved and/or recommended in metastatic non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and while responses are robust, some patients are inherently resistant, and most responders acquire resistance. Resistance mechanisms include reactivation of MAPK signaling, and cancer cells which escape MAPKi are reported to have high levels of genomic instability. WEE1 is a kinase involved in cell cycle progression, allowing cells to repair damaged DNA before entering the next cell cycle phase. Consequently, inhibition of WEE1 leads to premature cell cycle entry and RS, thereby driving cancer cells through the cell cycle with unchecked accumulation of DNA damage, ultimately resulting in mitotic catastrophe and cell death. We hypothesized that high levels of genomic instability and RS in KRASG12C mutant and KRASG12C inhibitor-resistant tumors present a vulnerability to WEE1 inhibition and provide rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with KRASG12C inhibitors. Here, we report azenosertib in combination with KRASG12C inhibitors as an effective therapeutic strategy for KRASG12C tumors. In vitro combination of azenosertib with multiple KRASG12C inhibitors demonstrated synergistic cell growth inhibition across a panel of KRASG12C cell lines in both 2D and 3D assays. In vivo treatment with azenosertib in KRASG12C inhibitor-sensitive cell line-derived xenograft (CDX) models of NSCLC (NCI-H358, NCI-H2122, NCI-H1792), CRC (SW837, SW1463), and pancreatic cancer (MIA PaCa-2) demonstrated significant monotherapy activity as well as synergistic tumor growth inhibition (TGI) when combined with KRASG12C inhibitors, including tumor regression in some models. Importantly, the KRASG12C inhibitor-resistant SW1573 NSCLC CDX model demonstrated synergistic TGI in the combo arm (84%) when compared against monotherapy azenosertib (31%) or adagrasib (58%). Similarly, treatment of a KRASG12C inhibitor-resistant patient-derived xenograft (PDX) model of CRC resulted in synergistic TGI in the combination arm (61%) relative to monotherapy azenosertib (17%) or adagrasib (2%). Finally, analysis of biomarkers from both in vitro and in vivo tumor samples demonstrated synergistic increases in protein markers of RS, DNA damage, and apoptosis with combination therapy. Taken together, our results suggest that the combination of azenosertib with KRASG12C inhibitors enhances tumor growth inhibition over single agent therapy and may be an effective treatment option for patients with KRASG12C tumors. Citation Format: Nathan M. Jameson, Gabriel Kim, Catherine Lee, Blake Skrable, Alexandra Shea, Monah Abed, Olivier Harismendy, Jianhui Ma, Doris Kim, Mark R. Lackner. The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models [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 6487.

Abstract 5825: Non-genetic determinants driving the evolutional trajectories of drug-tolerant-persisters in KRAS Non-small cell lung cancer

Abstract Rare cell populations manage to evade the effects of cancer therapies, ultimately leading to drug resistance and subsequent cancer relapse. These resilient populations, often referred to as "persisters," exhibit distinct molecular characteristics when compared to the broader tumor population. In the current standard of clinical care for non-small cell lung cancer (NSCLC), patients typically undergo a series of standard treatments, such as surgery followed by chemotherapy/immunotherapy, and then targeted therapy, based on the disease stages. This continues until drug resistance emerges, with little attention given to the state of these persister cells in response to these treatments. In the case of drug resistance in KRAS-mutated NSCLC, ongoing clinical investigations are exploring the combined efficacy of KRAS G12Ci inhibitors with drugs that target mitogen-activated protein kinase (MAPK) feedback reactivation, immunotherapy, and chemotherapy. Despite the myriad of combination therapies, persister populations invariably emerge. The manner in which these persisters evolve to withstand therapies remains elusive. We employed a DNA barcode lineage tracing analysis coupled with mathematical modeling to decode persister evolution to therapies targeting different pathways, e.g., KRAS+ SHP2, KRAS+CDK4/6, and KRAS+MCL-1. Our model reveals that rapidly emerging persister populations are predetermined and do not exhibit clear genetic alterations. In some cells, common persisters exist in response to different therapies. Single-cell ATAC-RNA sequencing showed that these persisters are enriched in chromatin-binding of transcription factors that regulate classic non-homologous end-joining (cNHEJ) DNA repair (ZNF384), interferon response (IRF1), and stem-like characteristics (FOXP1), along with the activation of an IFN-γ transcriptional signature. These findings underscore the connection between specific DNA repair processes, chromosomal instability, and resistance to KRAS G12Ci. In other cells, however, distinct persister populations exist to different therapies, leading to the hypothesis that these diverse persister populations can be targeted orthogonally or sequentially to achieve tumor regression. In summary, our study unveils the heterogeneity in persister evolution trajectories in KRAS NSCLC. Our platform identifies targetable persister populations in NSCLC models and offers a proof-of-concept for non-standard strategies to combat drug resistance based on persister cell states. Citation Format: Chendi Li, Christopher J. Graser, Qian Qin, Anahita Nimbalkar, Usman Syed, Barbara Karakyriakou, Sarah Clark, Anne Y. Saiki, Paul E. Hughes, Christopher Ott, Luca Pinello, Franziska Michor, Aaron Hata. Non-genetic determinants driving the evolutional trajectories of drug-tolerant-persisters in KRAS Non-small cell lung cancer [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 5825.

Abstract 4005: HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against NSCLC

Abstract Approximately 3% of non-small cell lung cancer (NSCLC) cases harbor ERBB2/HER2 mutations. Notably, the majority of actionable HER2 mutations in NSCLC are in-frame insertions in exon 20. Current HER2-targeting approaches, including antibody-drug conjugates (ADCs) such as trastuzumab deruxtecan, have shown promising response rates. To explore novel strategies for targeting HER2-positive NSCLC, we investigated chimeric antigen receptor (CAR)-based therapies to enhance the anti-tumor activity of immune effector cells. We engineered a series of HER2 CAR constructs utilizing scFvs derived from trastuzumab, pertuzumab, and FRP5, recognizing distinct domains of HER2. These scFvs were integrated into second-generation BB3z (with 4-1BB and CD3z intracellular domains) and third-generation 28BB3z (with CD28, 4-1BB, and CD3z intracellular domains) CAR backbones, which were subsequently transduced into activated T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. High transduction efficiencies were achieved, with over 80% in CAR-T cells and over 50% in CAR-NK cells. We next evaluated their activity against a panel of NSCLC cell lines expressing varying levels of HER2 in vitro using firefly luciferase assays. All HER2 CAR-T cells demonstrated more than 90% killing against H2170 (HER2 high) and H322 (HER2 medium) at an effector:target (E:T) ratio of 10:1, whereas HER2 CAR-T cells yielded a killing effect of 35% against A549 (HER2 low) cells. HER2-targeting CAR-NK cells also displayed potent cytotoxicity against HER2-expressing NSCLC cells. Moreover, we observed that treatment of HER2 mutant and HER2 amplified NSCLC cells with low doses of HER2 tyrosine kinase inhibitors (TKIs) such as poziotinib enhanced the expression of HER2 on the cell surface. This upregulation resulted in enhanced activation of HER2 CAR-T and CAR-NK cells in coculture experiments as measured by CD107a expression on effector cells. Additionally, treatment with the HER2 TKI poziotinib potentiated the killing activity mediated by HER2 CAR-T and CAR-NK cells. Using a HER2 YVMA duplication patient-derived xenograft (PDX) NSCLC model, we observed that the in vivo combination of poziotinib with HER2 CAR-NK cells exhibited superior anti-tumor activity as compared to poziotinib treatment alone. In conclusion, HER2 CAR-T and CAR-NK cells effectively target HER2-mutant and HER2-positive NSCLC, and their combination with HER2 TKIs enhances vulnerability to HER2-targeting strategies. Citation Format: Yan Yang, Monique B. Nilsson, Alissa Poteete, Junqin He, Qian Huang, John V. Heymach. HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against NSCLC [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 4005.

Abstract 1935: The FAK/SRC axis mediates resistance to KRAS G12C inhibitors and its blockade can overcome KRAS inhibitor resistance

Abstract Activating mutations in KRAS are found in approximately 30% of non-small cell lung cancer (NSCLC), and among all KRAS mutations, KRAS G12C is the most frequent variant in NSCLC, with a prevalence of approximately 14%. KRAS G12C inhibitors, sotorasib and adagrasib are FDA approved for locally advanced or metastatic KRAS G12C-positive NSCLC. However, clinical evidence suggests that there are various factors limiting the efficacy of KRAS G12C inhibitors in patients with KRAS G12C-positive NSCLC, highlighting the urgent need for novel treatment strategies or combinations to improve efficacy. Therefore, we sought to identify effective combinations to enhance the efficacy of KRAS G12C inhibitors using in vitro and in vivo models of KRAS G12C-positive NSCLC. Employing a high throughput in vitro drug screening effort using KRAS mutant NSCLC cell lines, we observed that Focal Adhesion Kinase (FAK) and Steroid Receptor Coactivator (SRC) inhibitors produced a synergistic effect when combined with KRAS G12C inhibitors adagrasib or sotorasib. As determined by reverse phase array and Western blotting, in H358, H23, HCC44, and H2122 cells, KRAS inhibitor treatment alone resulted in an initial suppression of RAS/MAPK and AKT pathway activation which was followed by upregulation of multiple receptor tyrosine kinases and integrins which contributed to the re-activation of the RAS/MAPK and AKT pathways after 24-48 hours. We identified FAK as a key downstream mediator of these bypass pathways. FAK/SRC inhibitors enhanced the efficacy of KRAS G12C inhibitors by preventing this feedback activation. Moreover, we observed increased FAK activation in H358 NSCLC cell lines with acquired resistance to adagrasib or sotorasib as compared to parental cells, and FAK/SRC inhibitors re-sensitized resistant cells to KRAS G12C inhibitors. Using a xenograft model of human KRAS mutant NSCLC, we determined that the combination of repotrectinib, a FAK/SRC inhibitor, and a KRAS G12C inhibitor significantly suppressed tumor growth as compared to KRAS G12C inhibitors alone, and this combination was well tolerated. Overall, our findings indicate that activation of FAK is a key mechanism of adaptive feedback and acquired resistance to KRAS G12C inhibitors in KRAS G12C-positive NSCLC and highlight the therapeutic potential of FAK/SRC inhibitors in combination with KRAS G12C inhibitors. These data support the clinical testing of the combination of FAK/SRC inhibitors and KRAS G12C inhibitors in patients with KRAS G12C-positive NSCLC. Citation Format: Yuji Shibata, Hibiki Udagawa, Monique B. Nilsson, Jacqulyne Robichaux, Ana Galan-Cobo, Marcelo Negrao, David P. Molkentine, Junqin He, Alissa Poteete, Yu Qian, Qian Huang, Ferdinandos Skoulidis, John V. Heymach. The FAK/SRC axis mediates resistance to KRAS G12C inhibitors and its blockade can overcome KRAS inhibitor resistance [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 1935.

Abstract 3916: TNG260, a small molecule CoREST inhibitor, sensitizes STK11-mutant NSCLC to anti-PD1 immunotherapy

Abstract Loss of function (LOF) of the serine-threonine kinase STK11/LKB1, which has been reported in ~15% of non-small cell lung cancer (NSCLC), drives resistance to immune checkpoint therapies such as PD1. In vivo CRISPR screens in tumor-bearing immune competent mice identified the Corepressor of Repressor Element 1 Silencing Transcription (CoREST) complex as a synthetic lethal immune evasion target for STK11- mutant tumors. Here, we demonstrate that TNG260, a small molecule drug specifically targeting the CoREST complex, can sensitize STK11 mutant lung cancers to anti-PD1 immunotherapy. Combination treatment with TNG260 and anti-PD1 arrested KRAS/STK11 mutant NSCLC tumor growth with no major toxicity observed, as confirmed in both allograft and genetically engineered mouse models (GEMMs) of STK11 mutant NSCLC. Further transcriptional analysis via bulk RNA-seq revealed that treatment of KRAS/STK11 mutant lung tumors with TNG260 promotes genes related to inflammatory responses and suppresses genes related to cell cycle and mitotic checkpoint(s). This phenotype was not observed in STK11 wildtype KRAS/P53 mutant lung tumors. Furthermore, combinational treatment of TNG260 with PD1 antibody showed increased macrophages, neutrophils and dendritic cells signatures in vivo. Our data suggests that epigenetic reprogramming via TNG260 sensitizes STK11 mutant NSCLC tumors to anti- PD1 treatment via targeting of the CoREST complex. This work identifies new vulnerabilities in STK11 LOF cancers that can be exploited therapeutically. TNG260 is currently being investigated in combination with pembrolizumab for the treatment of STK11-mutant cancer (NCT05887492), including NSCLC. Citation Format: Ayushi Patel, Soumyadip Sahu, Ke Geng, Salman Punekar, Janaye Stephens, Jiehui Deng, Ting Chen, Leanne Ahronian, Minjie Zhang, Jannik Andersen, Brian Haines, Kwok-Kin Wong. TNG260, a small molecule CoREST inhibitor, sensitizes STK11-mutant NSCLC to anti-PD1 immunotherapy [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 3916.

Abstract 4238: ModelingRET-rearranged non-small cell lung cancer (NSCLC): Generation of lung cell progenitors from patient-derived induced pluripotent stem cells (iPSCs)

Abstract Rearranged during Transfection (RET) oncogenic rearrangements can occur in 1-2% of lung adenocarcinomas. While RET-driven NSCLC models have been developed using various approaches, no model based on patient-derived induced pluripotent stem cells (iPSCs) has been described yet. Patient-derived iPSCs hold great promise for disease modeling. However, generating iPSCs with specific oncogenic drivers, like RET rearrangements, presents challenges due to reprogramming efficiency and genotypic variability within tumors. To address this issue, we aimed to generate lung progenitor cells (LPCs) from patient-derived iPSCs carrying the somatic mutation RETC634Y which is commonly associated with hereditary medullary thyroid carcinoma. Additionally, we have established a RETC634Y knock-in iPSC model to validate the effect of this oncogenic mutation during LPC differentiation. We successfully generated LPCs from RETC634Y iPSCs using a 16-days protocol and showed an overexpression of cancer-associated markers as compared to control iPSCs. Transcriptomic analysis revealed a distinct signature of NSCLC tumor repression, suggesting a lung multilineage lung dedifferentiation, along with an upregulated signature associated with RETC634Ymutation potentially linked to poor NSCLC prognosis. These findings were validated using the RETC634Y knock-in iPSC model, highlighting key cancerous targets such as PROM2 and C1QTNF6, known to be associated with poor prognostic outcomes. Furthermore, the LPCs derived from RETC634Y iPSCs exhibited a positive response to the RET inhibitor Pralsetinib, evidenced by the downregulation of the cancer markers. This study provides a novel off-the-shelf iPSC model of RET-driven NSCLC, paving the way for exploring the molecular mechanisms involved in RET-driven NSCLC to study disease progression and to uncover potential therapeutic targets. Citation Format: Paul Marcoux, Jinwook Hwang, Christophe Desterke, Jusuf Imeri, Annelise Bennaceur Griscelli, Ali Turhan. ModelingRET-rearranged non-small cell lung cancer (NSCLC): Generation of lung cell progenitors from patient-derived induced pluripotent stem cells (iPSCs) [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 4238.

Abstract 101: A novel humanized mouse model recapitulates the unique TME found in patients with HIV-associated NSCLC

Abstract Among people with HIV (PWH), non-small cell lung cancer (NSCLC) is increasing in incidence, presents with more advanced disease, and portends a worse prognosis compared to the general NSCLC population. Despite effective control of viral replication with antiretroviral therapy (ART), PWH have evidence of immune dysfunction, and it is unknown whether these immune perturbations impact the tumor microenvironment (TME) to influence disease disparities.Here we preclinically model HIV-associated NSCLC using MISTRG6-A2, a humanized mouse system that is highly optimized for development of functional innate and adaptive immune cells. We compare tumor growth and immune features of HIV-infected, ART-suppressed MISTRG6-A2 hosts to uninfected hosts, with parameters informed by parallel analyses of human tissue samples obtained from PWH with NSCLC, and non-HIV controls.MISTRG6-A2 mice were engrafted with human CD34+ HSPCs from HLA-A*02-expressing donors on post-natal day 2 and intravenously infected with HIV-1 at 6 weeks of age. When HIV viral titers were >106 copies of viral RNA/ml plasma and hCD4 T cells were depleted, ART was initiated (RAL/FTC/TDF). When plasma viral RNA was undetectable and CD4 T cells recovered, NSCLC PDX tissue from HLA-A*02-expressing tumor was implanted into these HIV-infected, ART-suppressed MISTRG6-A2 mice as well as non-infected littermate controls which had received HSPCs from the same donor. HIV-infected mice displayed enhanced growth of PDX tissue (mean tumor size 206.7 mm3 vs 110.2 mm3; p < 0.05).Quantitative immunofluorescence of the TME from these hosts revealed significantly increased infiltration of CD4 and CD8 T cells in tumors of HIV-NSCLC mice (p<0.01 and 0.05, respectively); of note, enhanced T cell infiltration was restricted to tumors, with similar frequency of CD4 and CD8 T cells detected in spleen, lung and liver tissues of HIV-infected vs uninfected hosts. Staining of tumor epitopes revealed increased expression of B2M and EGFR in tumors from HIV-NSCLC mice (mean qIF scores in NSCLC vs HIV-NSCLC 3.8 × 106 vs 1.2 × 107 for B2M, p<0.001; 440 vs 1,971 for EGFR, p<0.01), consistent with our findings in samples from HIV-NSCLC tumor tissues vs NSCLC tumor tissues. Of note, elevated EGFR staining was only present in tumor tissue, not found in stromal cells or other tissues. Single cell transcriptomic analyses of the TME revealed prominent interferon and antigen presentation signatures in HIV-NSCLC tumors, as well as differential expression of immunoregulatory molecules.These results demonstrate the fidelity of the HIV-NSCLC MISTRG6-A2 system as a model for HIV-associated NSCLC and suggest EGFR-directed therapies as potentially relevant in this neglected disease. Citation Format: Melani Juric, Gabriel Kaufmann, Li Zhu, Kishu Ranjan, Kriti Agrawal, Barani Kumar Rajendran, Jyothi K. Rajashekar, Hongyu Zhao, Yuval Kluger, Brinda Emu, Kurt A. Schalper, Priti Kumar, Richard A. Flavell, Michael Chiorazzi. A novel humanized mouse model recapitulates the unique TME found in patients with HIV-associated NSCLC [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 101.