Non-small Cell Lung Cancer Tumor Samples Research Articles

Identification of genes related to PD-L1 expression in non-small cell lung cancer (NSCLC) tumor samples by GFPrint, a machine learning framework (MLF).

e13600 Background: The advent of drugs targeting the PD-1/PD-L1 axis has revolutionized NSCLC treatment. Although promising, individual responses to these therapies can vary among individuals, and research is ongoing to better understand the mechanisms involved in PD1/PD-L1 expression to optimize treatment protocols, and identify which patients are most likely to benefit from these therapies. In this study, Genetic Fingerprint (GFPrint), a MLF, was employed to identify mutations in genes linked to PD-L1 expression in NSCLC tumor samples. Methods: Whole exome sequencing (WES) was run on samples from biopsies of 46 NSCLC patients (histologic subtypes: squamous-cell carcinoma and adenocarcinoma). WES data were encoded using GFPrint, a MLF that generates virtual representations of the tumor exomes in a latent space (LS) defined by the genetic features of the samples. Clusters of patients were identified in such LS based on the genetic similarities of their tumors. Statistical univariate analysis was run to analyze differences in the distribution among both clusters of clinical (age, smoking, and metastatic status) and pathological (histotype and PD-L1 expression) features. The mutational burden (MB) of each cluster was also examined by pathway enrichment analysis (PEA). Results: Two main clusters were identified, C0 (n=16) and C1 (n=17), with 13 unassigned patients. Main differences between clusters were related to histology and PD-L1 expression. C0 included significantly less patients with adenocarcinoma (10/16; 62.5%) compared to CL1 (17/17; 100%) (p-value=.01). In addition, C0 presented more patients expressing PD-L1 (9/16; 56.2%) compared to C1 (3/17; 17.6%) (p-value=.04). Of interest, PD-L1 positivity was significantly higher (p value=.03) in adenocarcinomas of C0 patients (6/10; 60.0%) than those of C1 (3/17; 17.6%). Moreover, C0 adenocarcinomas presented higher levels of PD-L1 expression (6.3+/-9.7 %) than those of C1 (2.3+/-7.5 %) (p value=.06). MB analysis revealed that 1,357 genes were mutated exclusively in C0 patients, and PEA allowed the identification of 3 enriched cancer-related KEGG pathways: HIF-1 signaling, Ca2+ signaling and actin cytoskeleton. Selection of non-silent mutations in these pathways enabled the identification of 10 genes, mostly included in the HIF-1 signaling pathway, with previous data suggesting their involvement in PD-L1 regulation and expression: PIK3R1, HK1, NOS2, NRAS, BRAF, ITGA1, ITGA9, EGLN1, EGLN2 and AKT1, being the first 6 the only ones found in adenocarcinomas. Conclusions: The identified genes deserve further studies to validate them as possible targets of pharmacological intervention concomitant to PD-1/PD-L1 blockade, and also as potential biomarkers for patients’ selection to improve the efficiency of immunotherapy in NSCLC treatment.

Targeting lncRNA16 by GalNAc-siRNA conjugates facilitates chemotherapeutic sensibilization via the HBB/NDUFAF5/ROS pathway.

Chemoresistance is a significant barrier to effective cancer treatment. Potential mechanisms for chemoresistance include reactive oxygen species (ROS) accumulation and expression of chemoresistance-promoting genes. Here, we report a novel function of lncRNA16 in the inhibition of ROS generation and the progression of chemoresistance. By analyzing the serum levels of lncRNA16 in a cohort of 35 patients with non-small cell lung cancer (NSCLC) and paired serum samples pre- and post-treatment from 10 NSCLC patients receiving neoadjuvant platinum-based chemotherapy, performing immunohistochemistry (IHC) assays on 188 NSCLC tumor samples, using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) assays, as well as RNA immunoprecipitation (RIP) and RNA pull-down analyses, we discovered that patients with increased serum levels of lncRNA16 exhibited a poor response to platinum-based chemotherapy. The expression of hemoglobin subunit beta (HBB) and NDUFAF5 significantly increases with the development of chemoresistance. LncRNA16 binds to HBB and promotes HBB accumulation by inhibiting autophagy. LncRNA16 can also inhibit ROS generation via the HBB/NDUFAF5 axis and function as a scaffold to facilitate the colocalization of HBB and NDUFAF5 in the mitochondria. Importantly, preclinical studies in mouse models of chemo-resistant NSCLC have suggested that lncRNA16 targeting by trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA restores chemosensitivity and results in tumor growth inhibition with no detectable toxicity in vivo. Overall, lncRNA16 is a promising therapeutic target for overcoming chemoresistance, and the combination of first-line platinum-based chemotherapy with lncRNA16 intervention can substantially enhance anti-tumor efficacy.

Abstract C013: Clinical biomarkers based on PK/PD modeling to guide the development for a first-in-class, highly selective SMARCA2 (BRM) degrader, PRT3789

Abstract Background: The SWI/SNF family of chromatin-remodeling complexes is frequently dysregulated in multiple tumor types, resulting in aberrant expression of genes. Damaging mutations resulting in loss of function of one of its core catalytic subunits SMARCA4 (BRG1) occur in multiple tumor types, including in 5-10% of non-small cell lung cancer (NSCLC). SMARCA4-deleted cells become highly dependent on the other catalytic subunit, SMARCA2 (BRM), for their survival, therefore selective degradation of SMARCA2 has therapeutic potential in these cancer types. PRT3789 is a first-in-class, potent and selective SMARCA2 degrader that is currently under evaluation in a Phase 1 study in patients with advanced solid tumors with loss of SMARCA4 (NCT05639751). We deeply characterized the degradation kinetic parameters of PRT3789 based on preclinical PK/PD studies, which enabled dose-selection. Based on this modeling, we developed a suite of biomarker assays that can be used to assess target engagement in this Phase 1 study. Methods: A sensitive and quantitative plate-based immunoassay utilizing the MSD® platform was developed for the measurement of SMARCA2 protein in human PBMCs following treatment with PRT3789. A secondary, qPCR-based assay was developed for assessment of SMARCA2 transcriptional targets in peripheral blood that may be associated with response to SMARCA2 degradation by PRT3789. Additionally, an immunohistochemistry (IHC) assay for the assessment of SMARCA2 and SMARCA4 protein expression in tumor tissue was developed on the Leica BOND III platform using commercially available antibodies. Results: Detailed in vitro enzymology characterized the degradation kinetic parameters of PRT3789 and was successfully used to predict the PRT3789 effects observed in PK/PD and xenograft efficacy studies in mice. The MSD® assay demonstrated a wide dynamic range of detection for SMARCA2 with a lower level of quantification of 23 pg/mL, as well as exhibiting acceptable spike recovery and minimal cross-reactivity to SMARCA4. Based on the qPCR assay, we developed a differential gene expression (DGE) score based on expression of an 8-gene panel that correlates with SMARCA2 protein degradation in PBMCs. The IHC assay demonstrated discrete and robust nuclear tumor staining with a dynamic intensity range and limited cytoplasmic background for both SMARCA2 and SMARCA4. A panel of NSCLC tumor samples with known SMARCA4 mutations were further evaluated in this assay. Loss of SMARCA4 protein expression in tumor cells by IHC was associated with nonsense and frameshift mutations in the SMARCA4 gene. Conclusions: We demonstrate the sensitive assessment of SMARAC2 protein degradation and effects on downstream gene expression in PBMCs, which will be used to assess target engagement following treatment with PRT3789 in a Phase 1 clinical study. Citation Format: Min Wang, Andrew Moore, Alexander Grego, Miles Cowart, Joseph Rager, Gina Paris, Koichi Ito, Naveen Babbar, Sandy Geeganage, Peegy Scherle, Neha Bhagwat. Clinical biomarkers based on PK/PD modeling to guide the development for a first-in-class, highly selective SMARCA2 (BRM) degrader, PRT3789 [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 C013.

Upregulation of Indoleamine 2,3-Dioxygenase 1 in Tumor Cells and Tertiary Lymphoid Structures is a Hallmark of Inflamed Non-Small Cell Lung Cancer.

Overexpression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) has been reported in several tumor types, including non-small cell lung cancer (NSCLC), and has been shown to promote tumor-immune evasion and inhibit T-cell activation through increased tryptophan degradation and the production of several immunosuppressive metabolites collectively known as kynurenines. However, it remains unclear whether IDO1 expression by tumor cells is detrimental specifically in the context of programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis blockade. We analyzed the transcriptome of 891 NSCLC tumor samples from patients enrolled in two large randomized clinical trials investigating the safety and activity of atezolizumab, a humanized IgG1 mAb that targets PD-L1, versus docetaxel in patients with advanced NSCLC. We complemented these transcriptomics results at the protein level by using multiplex immunofluorescence and at the functional level with in vitro experiments. The increased expression of the tryptophan-catabolizing enzyme IDO1 was significantly associated with improved objective response, progression-free survival, and overall survival in patients treated with PD-L1 inhibitors, but not in those treated with chemotherapy. Strikingly, inflamed tumors had higher levels of IDO1, and IDO1 was also expressed in tertiary lymphoid structures (TLS) by mature follicular dendritic cells. L-kynurenine impaired the differentiation of antibody-producing B cells induced by follicular helper T (Tfh)/B-cell interactions, a hallmark process within TLS. IDO1 pathway in NSCLC is driven by the immune system rather than by tumor cells. Targeting IDO1 in combination with anti-PD-1/PD-L1 might be beneficial only in patients with inflamed tumors and particularly in those bearing TLS.

Role of human epidermal growth factor receptor 3 in treatment resistance of anaplastic lymphoma kinase translocated non-small cell lung cancer

ABSTRACT Background ALK tyrosine kinase inhibitors (TKI) have revolutionized the treatment of ALK+ non-small cell lung cancer (NSCLC), and therapy resistance occurs in virtually all patients. Multiple TKI resistance mechanisms have been characterized, including ERBB receptor coactivation. In this study, we investigated the role of HER3 in ALK TKI resistance. Methods In vitro studies were carried out using ALK+ NSCLC cell lines H3122, H2228, and DFCI032. Pharmacological co-targeting of ALK and HER3 was investigated with ALK and ERBB TKIs, and HER3 knockdown was achieved using the CRISPR-Cas9 system. Co-localization of ALK and HER3 was investigated by immunoprecipitation (IP) and proximity ligation assay (PLA) in vitro and in vivo using six ALK+ NSCLC tumor samples. Results In all tested cell lines, combined targeting with ALK and pan-ERBB TKI resulted in marked inhibition of colony formation and long-term (72 h) downregulation of pAKT levels. HER3 knockdown resulted in multiple effects on ALK+ cell lines, including the downregulation of ALK expression and visible morphological changes (H2228). Co-immunoprecipitation (IP) and proximation ligation assay (PLA) experiments provided evidence that both ALK and HER3 could interact in vitro, and this finding was verified by PLA using ALK+ NSCLC tumors. Conclusions This study provides evidence that HER3 may mediate TKI resistance in ALK+ NSCLC. Interestingly, we were able to show that both translocated ALK and HER3 could interact. Joint targeting of ALK and HER3 could be further investigate in ALK+ NSCLC.

Utility of multigene panel next-generation sequencing in routine clinical practice for identifying genomic alterations in newly diagnosed metastatic nonsmall cell lung cancer.

The standard of care in newly diagnosed metastatic non-small cell lung cancer (NSCLC) is to test for aberrations in three genes for driver mutations - ALK, ROS1 and epidermal growth factor receptor (EGFR) - and also for immunohistochemistry to be performed for programmed death-ligand 1 expression level. Next-generation sequencing (NGS), with or without RNA fusion testing, is increasingly used in standard clinical practice to identify patients with potentially actionable mutations. Stratification of NGS mutation tiers is currently based on the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT) Tiers I-V and X. Our aim was to analyse NSCLC tumour samples for the prevalence of Tiers I-V mutations to establish guidance for current and novel treatments in patients with metastatic disease. NGS was performed employing the Oncomine Precision Assay (without RNA fusion testing) that interrogates DNA hotspot variants across 45 genes to screen 210 NSCLC tissue samples obtained across six Sydney hospitals between June 2021 and March 2022. In our cohort, 161 of 210 (77%) had at least one gene mutation identified, with 41 of 210 (20%) having two or more concurrent mutations. Tier I mutations included 42 of 210 (20%) EGFR mutations (EIA) and five of 210 (3%) MET exon 14 skipping mutations (EIB). Non-Tier I variants included 22 of 210 (11%) KRAS G12C hotspot mutations (EIIB), with a further 47 of 210 (22%) having non-G12C KRAS (EX) mutations. NGS testing revealed an additional 15% of cases with Tier II ESCAT mutations in NSCLC. Forty-six percent of patients also demonstrated potential Tier III and IV mutations that are currently under investigation in early-phase clinical trials. In addition to identifying patients with genomic alterations suitable for clinically proven standard-of-care therapeutic options, the 45-gene NGS panel has significant potential in identifying potentially actionable non-Tier 1 mutations that may become future standard clinical practice in NSCLC.

Revealing distinct immune landscape in EGFR mutant and EGFR wild type lung adenocarcinoma by using single-cell RNA sequencing.

e21008 Background: Non-small cell lung cancer (NSCLC) with EGFR mutations displays heterogeneity in the extent of intratumoral immunocytes infiltration compared to those with EGFR wild types, which may explain their variable responsiveness to PD-1 blockade. Here, we performed single-cell RNA sequencing (scRNA-seq) to unveil the immunescape and heterogeneity of lung adenocarcinoma (LUAD) between EGFR mutant (EGFR-mut) patients and wild type (EGFR-wt) patients. Methods: Eleven EGFR-mut and 7 EGFR-wt NSCLC tumor samples were analyzed. Immune cells (CD45+) were enriched through fluorescence-activated cell sorting (FACS) and subjected to single-cell RNA sequencing (scRNA-seq) using the 10X Genomics platform. Sequencing reads were normalized and analyzed using the R/Seurat package. Cellular components of each sample were determined based on known marker genes. Wilcox test was used to compare promotion by subtype composition and immune cell infiltrates. Results: Different immune cell compositions were observed between EGFR-mut and EGFR-wt LUAD tumor samples. We found EGFR-wt tumors are marked by an enrichment of CCL15+ tumor-associated macrophages(TAMs), which display pro-inflammatory properties and high expressions of TNF-a-signaling genes. In contrast, EGFR-mut tumors exhibit enrichment of SPARCL1+TAMs, which are involved in epithelial-mesenchymal transition (EMT) and angiogenesis. EGFR-wt tumors exhibited enrichment of CD8-Teff-MX1 and Gamma delta T cells, both of which displayed higher cytotoxic and naive features. Moreover, the high expression of Gamma delta T corresponds with a better clinical prognosis. In contrast, CD8-Teff-XCL1 and NKT-FGFBP2 were found enriched in EGFR-mut tumors, both of which contributed to higher exhaustion and decreased cytotoxic function. Additionally, NK-IL32 was found enriched in EGFR-mut tumors, showing a downregulated T cell receptor signaling and stronger exhausted features. The high expression of this subcluster correlated with poor outcomes in LUAD patients harboring EGFR mutation. Conclusions: Our study provided a deeper understanding of the immune landscape of EGFR-mut and EGFR-wt LUAD and gained better insights into the immunological mechanisms involved in the response to ICIs. The results of our study could be instructive for the development of future immunotherapy strategies for NSCLC patients harboring EGFR mutations.

Abstract 651: Revealing the role of lung cancer microbiota in the tumor progression

Abstract Lung cancer (LC) remains as the leading cause of death by cancer worldwide. In Chile, LC is the second cause of cancer-related deaths. Recently, studies in melanoma and non-small cell lung cancer (NSCLC) patients have highlighted the role of the gut microbiota as an important host factor in mediating the responses/resistance to immunotherapy, suggesting that bacteria present in the gut may modulate the immune response in these tumors. However, the role of extra-intestinal microbiota; bacteria living outside of the gut, in cancer pathogenesis and the response to anti-cancer therapies remains largely undetermined. Here we characterize the composition of intratumoral microbiota of NSCLC and seek to establish a functional relationship between it and the composition of the immune microenvironment and the clinicopathological characteristics of NSCLC patients. From the first 157 FFPE NSCLC samples, we extracted DNA from all collected samples, obtaining adequate material in quality and quantity to later be submitted for analysis to 16S sequencing. Using 16S rRNA gene sequencing, we assessed the general landscape of the NSCLC tumor microbiome, revealing the presence of large number of bacterial communities in the NSCLC tumor samples on the different histological subtypes analyzed. We have detected differences in alpha diversity in the histological subtypes studied. Even more interesting, we have detected significant differences in lung adenocarcinoma depending on the degree of histological differentiation, observing a decrease in proteobacteria and an enrichment of Bacteroidales, as cell differentiation is lost. Additionally, we identified taxonomic differences between differentiated and undifferentiated tumors. Differentiated tumors show enrichment in Akkermansia muciniphila and while undifferentiated tumors show enrichment in Actinobacter Corynebacterium. Suggesting that these taxa could contribute to maintaining a differentiated state or inducing cell dedifferentiation, respectively. Our results reveal the presence of a large number of bacterial communities in lung cancer samples in the different histological subtypes analyzed. Suggesting that these communities could play a key role in tumor differentiation and progression. Citation Format: Erick M. Riquelme, Ivania Valdes, Carlos Aravena, Ilse Valencia, Barbara Mino, Daniel Carvajal, Alberto Martin. Revealing the role of lung cancer microbiota in the tumor progression [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 651.

Circ_0006220 Contributes to NSCLC Progression through miR-342-3p/GOT2 Axis.

Dysregulated circular RNAs (circRNAs) have shown crucial modulatory functions in tumorigenesis, containing non-small cell lung cancer (NSCLC). The purpose of this study was to explore the biological functions and regulatory theory of circ_0006220 in NSCLC. Reverse transcription-quantitative polymerase chain reaction and Western blot assay were conducted to measure RNA and protein expression, respectively. A total of 73 cases of NSCLC tumor samples were collected for expression analysis, and A-549 and NCI-H1299 cell lines were used for functional experiments. Cell proliferation was assessed by cell counting kit-8 assay, colony formation assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry. Cell apoptosis, motility, and angiogenesis ability were analyzed by flow cytometry, transwell assays, and capillary-like network formation assay. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted to verify the target relationships. Circ_0006220 was highly expressed in NSCLC tissues and cell lines. Circ_0006220 silencing inhibited the proliferation, migration, invasion, and angiogenesis but induced the apoptosis of NSCLC cells. Circ_0006220 acted as a microRNA-342-3p (miR-342-3p) sponge, and circ_0006220 knockdown-induced changes on the phenotypes of NSCLC cells were largely overturned by the knockdown of miR-342-3p. miR-342-3p interacted with the 3' untranslated region of glutamic-oxaloacetic transaminase 2 (GOT2), and GOT2 overexpression largely diminished miR-342-3p overexpression-mediated influences in NSCLC cells. Circ_0006220 could up-regulate GOT2 expression by sponging miR-342-3p. Circ_0006220 promoted the malignant behaviors of NSCLC cells through mediating the miR-342-3p/GOT2 regulation cascade.

CircRNA has_circ_0017109 promotes lung tumor progression via activation of Wnt/β-catenin signaling due to modulating miR-671-5p/FZD4 axis

IntroductionAccumulating evidence highlights the critical roles of circular RNAs (circRNAs) in the malignant progression of cancers. In this study, we investigated the expression pattern of a newly identified circRNA (hsa_circ_0017109) in non–small cell lung cancer (NSCLC), and examined its downstream molecular targets.MethodsQuantitative real-time PCR (qRT-PCR) and Western blotting (WB) were conducted to quantify gene and protein expression. In vitro functional assays such as colony formation assay, cell counting kit-8 (CCK-8) and flow cytometry were used to study cell proliferation and apoptosis. RNA pull-down assay, luciferase reporter assay and RNA immunoprecipitation were performed to validate molecular interaction. Mouse xenograft model of NSCLC cells was used to assess the role of circ_0017109 in tumorigenesis.ResultsCirc_0017109 was upregulated in NSCLC tumor samples and cells. Silencing circ_0017109 impaired cell proliferation and promoted apoptosis in NSCLC cells, and circ_0017109 knockdown suppressed in vivo tumorigenesis of NSCLC cells in mouse xenograft model. MiR-671-5p was identified as a target of circ_0017109, and circ_0017109 negatively impacted on miR-671-5p expression. MiR-671-5p downregulated FZD4 and dampened the activity of Wnt/β-catenin signaling pathway. Circ_0017109 modulated FZD4 expression by suppressing miR-671-5p activity.ConclusionsElevated circ_0017109 expression promotes tumor progression of NSCLC by modulating miR-671-5p/FZD4/β-catenin axis.

Selected Articles from This Issue

Ceramide kinase (CERK) and its enzymatic product, ceramide-1-phosphate (C1P), have been implicated in oncogenesis, but molecular mechanisms underlying CERK/C1P-mediated oncogenesis have not been elucidated. While investigating CERK functionality in non–small cell lung cancer (NSCLC), Vu and colleagues discovered that CERK is more abundant in KRASG12 mutant NSCLC tumor samples. Further interrogation revealed that CERK inhibitor NVP-231 increases reactive oxygen species levels and ferroptotic cell death uniquely in KRASG12 mutant NSCLC cells. Accordingly, an in vivo model in which the authors used CRISPR/Cas9 to generate NSCLC cells with single CERK alleles demonstrated that limiting CERK abundance sensitizes KRASG12 mutant NSCLC cells to cisplatin. Voltage-dependent anion channel 1 (VDAC1) abrogation using siRNA reverses CERK inhibition-driven cytotoxicity and cisplatin sensitivity in vitro. Mechanistically, the authors found that CERK inhibition prevents AKT mitochondrial localization, reducing AKT-mediated VDAC1 phosphorylation and subsequent β-tubulin/VDAC1 interactions on mitochondrial outer membranes. Overall, this study demonstrates that KRASG12 mutant NSCLC survival is reliant on CERK, and that CERK dependence may be therapeutically targetable using existing chemotherapeutics.While increased Ecdysoneless (ECD) mRNA and protein abundance has been observed in breast cancer and other tumor types, molecular mechanisms by which ECD might promote breast cancer tumorigenesis have not been fully clarified. To query how ECD functions in breast cancer, Mohapatra and colleagues generated a mouse model in which an ECD transgene is overexpressed specifically in mammary epithelium (ECDTg). Mammary ECD overexpression produces mammary tumors with heterogeneous histological characteristics that occasionally metastasize to the lungs. Transcriptomic analyses of ECDTg and control tumors revealed that ECD overexpression enhances c-MYC transcriptional programming, particularly augmenting metabolic c-MYC target gene transcription. The authors demonstrated that ECD increases c-MYC abundance by facilitating c-MYC mRNA splicing and maturation. Furthermore, doxycycline disrupts ECD and c-MYC expression, unveiling a potential therapeutic vulnerability in ECD-overexpressing tumors. Taken together, this study defines novel, physiologically relevant ECD functions in mammary cancer and presents potential ECD-driven therapeutic targets.Small cell lung cancer (SCLC) is an aggressive disease for which current therapeutic options provide limited benefits, necessitating therapeutic target discovery. While it is known that MUC1-C facilitates tumorigenesis in several cancers, it has not been studied in SCLC. To assess the role of MUC1-C in SCLC, Fushimi and colleagues abrogated MUC1-C expression in classic, variant, and non-neuroendocrine SCLC cell lines using doxycycline-inducible shRNA and performed transcriptomic analyses. The authors found that MUC1-C enhances MYC transcriptional programming in each SCLC subtype. Further studies revealed that MUC1-C is required for MYC localization to the NOTCH2 promoter and enhancer, and that ablating MUC1-C or MYC expression decreases expression of NOTCH2 and master transcription factors that dictate SCLC subtypes. Genetic and pharmacological disruption of MUC1-C, as well as CRISPR-Cas9-mediated NOTCH2 elimination, inhibit SCLC cell tumorsphere formation in vitro and tumor growth in vivo, suggesting the MUC1-C/MYC/NOTCH2 axis drives stemness and tumor progression in SCLC. In sum, this study defines a novel, potentially therapeutically targetable molecular mechanism underlying SCLC tumor progression.Proteosome inhibitor bortezomib demonstrates effectiveness in many multiple myeloma patients upon initial treatment. However, patients often relapse after initial therapy and develop drug resistance. Previously, Shigeki Miyamoto's group discovered that bone marrow stromal cells (BMSC) produce cleaved forms of hyaluronan and proteoglycan link protein 1 (HAPLN1) that activate NF-κB to engender bortezomib resistance. How cleaved HAPLN1 matrikines are generated in multiple myeloma remains unknown. To test if BMSC-derived HAPLN matrikines might derive from proteolytic cleavage, Miyamoto and co-authors performed a qRT-PCR–based matrix metalloproteinase (MMP) screen in BMSC derived from healthy donors and multiple myeloma patients. The screen revealed heightened MMP2 expression in multiple myeloma patient BMSC, and gelatin zymography activity assays demonstrated increased MMP2 activity in multiple myeloma patient BMSC. Subsequent in vitro assays showed that MMP2 cleaves HAPLN1 into products similar in size to the previously discovered matrikines. Abrogating HAPLN1 and MMP2 expression via shRNA-expressing lentivirus transduction in multiple myeloma patient BMSC decreases NF-κB activation and fosters bortezomib sensitivity in multiple myeloma cells. Altogether, this study unveils a novel molecular mechanism by which BMSC support multiple myeloma cell bortezomib resistance.

1066P Tumor microenvironment (TME) of HRAS mutated non-small cell lung cancer (NSCLC)

RAS pathway alterations in NSCLC have been linked to worse prognosis and remain a challenging therapeutic target. HRAS, a RAS family members, is under-investigated and its activation depends on farnesylation, making it an appealing target for farnesyltransferase inhibitors. Although KRAS has been associated with immune modulation in NSCLC, the role of HRAS remains unclear. We aimed to investigate the relationship of HRAS gene mutations and the TME. Molecular profiling of 29,767 NSCLC tumor samples was performed at Caris Life Sciences to obtain whole exom sequencing, whole transcriptome sequencing and immunohistochemistry. MAPK signaling activity (MPAS) and immune cell fraction were assessed by mRNA analysis. Wilcoxon, Fisher’s exact were used for statistical significance. Overall survival was calculated using the Kaplan-Meier method. Comparisons were conducted between HRAS mutated (mt) tumors and the entire NSCLC general cohort (GC). HRAS mt were detected in 0.4% of NSCLC tumors (n=128), with a majority among smokers. HRAS mt displayed less B cell, macrophage M2, NK cell, CD4+ and CD8+ T cells infiltrates compared to GC (q<0.05 ). In addition, HRAS Q61 was found to be correlated with worse prognosis in pts treated with pembro (HR = 2.779, 95% CI [1.04-7.423], p =0.03) but not G12 or G13, and displayed the highest MPAS score (p = 0.05), which had previously been demonstrated to indicate immune evasion. The median difference for survival curves was -246 days between HRAS mt squamous cell carcinomas (SCC, all smokers) and GC SCC in pts treated with pembro (HR 1.72, 95% CI 0.85-3.64, p =0.126). HRAS mt SCC showed less infiltration with M1, B cells, myeloid dendritic cells (mDC) and also displayed a significantly higher MPAS score and LAG3 expression as compared to GC (0.53 vs -0.31, p = 0.03; 2.10 vs 1.03 TPM, p = 0.04). No similar trends were observed in adenocarcinoma histology. HRAS mt NSCLC displayed a relatively immune-cold pattern with less CD4+ and CD8+ T cells infiltrates compared to GC. HRAS mt SCC also showed an immune-cold TME associated with high MAPK pathway activation and high LAG3 expression. This warrants further investigation, in particular in HRAS Q61 or HRAS mt SCC, with combination therapies targeting MAPK pathway or LAG3 protein.

Abstract 48: Molecular characteristics of HRAS mutated non-small cell lung cancer (NSCLC)

Abstract Background: Alterations in the RAS pathway have been linked to tumorigenesis, apoptosis, metabolism and angiogenesis. Mutations of KRAS in NSCLC are more frequent and better characterized, however, other family members such as HRAS remain under investigated and RAS remains a challenging therapeutic target. HRAS has been indirectly targeted with tipifarnib, a farnesyltransferase inhibitor rendering HRAS inactive in head and neck tumors. Here, we characterize the incidence, genomic landscape, and clinical context of HRAS alterations in NSCLC. Methods: A total of 29,767 NSCLC tumor samples underwent comprehensive molecular profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq, or whole exome sequencing, NovaSeq), RNA (NovaSeq, whole transcriptome sequencing, WTS) and immunohistochemistry. MAPK activation was assessed using the MPAS gene expression signature. Wilcoxon, Fisher’s exact, or Dunnett’s tests were used to determined statistical significance (displayed as p value without and q value with multi-comparison correction). Overall survival was calculated from date of tissue collection to last contact from insurance claims data and used for Kaplan-Meier analysis. Comparisons were conducted between HRAS mutated tumors and the entire NSCLC general cohort (GC). Results: HRAS mutations (Hm) were detected in 128 of 29767 NSCLC samples (0.4%) and were significantly enriched in older patients (median age, 71 vs. 69 years; q&amp;lt;0.01) and squamous histology (57.8% vs 21.8%, q&amp;lt;0.0001) compared to GC. Smoking status was not associated with HRAS mutational status (p=0.19). The most prevalent loci of hotspot mutations in Hm tumors were G13 (42.2%), followed by Q61 (33.6%) and G12 (21.1%). HRAS was not prognostic for overall survival (HR = 1.06, 95% CI [0.83-1.35], p=0.64), but for HRAS G13 mutant subset of NSCLC there was a trend towards worse prognosis (HR = 1.31, 95% CI [0.91-1.88], p =0.14). Hm-positive tumors harbored significantly more PIK3CA mutations (16.9% vs 5.5%, q&amp;lt;0.05) but less KRAS (3.2% vs 27.4%, q&amp;lt;0.05) and EGFR mutations (0.8% vs 11.9%, q&amp;lt;0.05) compared to GC. In addition, Hm displayed dMMR/MSI deficiency (2.6% vs 0.7%, p &amp;lt;0.05) more frequently, but had a comparable percentage of TMB-H tumors (34.2% vs 40.2%, p = 0.16) and similar median PD-L1 expression (54.7% vs 60.3%, p = 0.21) when comparing to GC. Lastly, MAPK pathway activation score was relatively higher in Hm compared to GC (-0.26 vs -0.42) with Q61 being the highest group (0.04 vs -0.42, p = 0.37). Conclusions: HRAS mutations are detectable but uncommon events in NSCLC and significantly enriched in squamous histology. HRAS mutations often occur with PIK3CA co-mutations and trended towards higher activation of MAPK pathway and MSI-H frequency. This warrants further investigation on possible clinical applications of HRAS pathway inhibitors and utility of immune checkpoint inhibitors for this subset of NSCLC. Citation Format: Asaad Trabolsi, Estelamari Rodriguez, Samuel Alexander Kareff, Michael Korn, Joanne Xiu, Stephen Liu, Philip walker, Patrick Ma, Hirva Mamdani, Jorge Nieva, Hossein Borghaei, Chadi Nabhan, Misako Nagasaka, Sonam puri, Gilberto Lopes. Molecular characteristics of HRAS mutated non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 48.

Association of PAK4 expression with overall survival in patients with non-small cell lung cancer (NSCLC).

e21149 Background: P21-activated kinase 4 (PAK4) is a crucial effector of the Rho GTPases. It acts as a regulatory switch that controls a wide range of cellular functions and plays a pivotal role in cancer progression and metastasis. Very little is known about the expression and prognostic value of PAK4 in NSCLC. Methods: 17,689 NSCLC tumor samples were analyzed using next-generation sequencing (NextSeq, 592 Genes, or WES NovaSeq), immunohistochemistry (IHC), and whole transcriptome sequencing (NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was analyzed by IHC (Dako 22c3; PD-L1 positive: TPS &gt;1%). TMB was measured by counting all somatic mutations found per tumor (TMB-high: &gt;10 mutations/MB). Tumors with PAK4-high (H) and PAK4-low (L) expression were classified as those in top quartile and bottom 3 quartiles, respectively. Immune cell infiltrates were calculated by Quantiseq. Significance was determined using chi-square and Wilcoxon rank sum test and adjusted for multiple comparisons (q-value &lt; 0.05). Survival was extracted from insurance claims data and calculated from the time of tissue collection to the last contact using Kaplan-Meier estimates. Results: There was no difference in median age, gender, smoking status, and histologic distribution between PAK-H and PAK-L tumors. Compared to PAK4-L, the PAK4-H was associated with higher frequency of co-mutations in TP53 (76.3% vs 63.9%, p &lt; 0.0001) and RB1 (13.6% vs 8.1%, p &lt; 0.0001). PAK4-H tumors were associated with greater genomic loss of heterozygosity (24.1% vs 16.4%, p &lt; 0.0001), and expression of immune checkpoint genes ( LAG3, PDCD1, PDCD1LG2, CD274, IDO1, CTLA4, CD80, HAVCR2; p &lt; 0.05). KRAS (including KRAS G12C) , BRAF, STK11, and EGFR mutations, and ALK and ROS1 fusions were less frequent in PAK4-H tumors (p &lt; 0.001). A greater proportion of PAK4-H tumors were TMB-H (40.3% vs 33.3%, p &lt; 0.0001) and PD-L1 negative (48.2% vs 40.8%, p &lt; 0.001). PAK4-H tumors had lower infiltration of B cells, M1/M2 macrophages, CD8+ T-cells, and Tregs (p &lt; 0.001). Overall survival (OS) was inferior among patients with PAK4-H tumors (median, 14.9 vs 21.5 months, HR = 1.28, 95% CI, 1.21-1.36, p &lt; 0.001), which was driven by adenocarcinoma histology. Survival with immunotherapy was also inferior in patients with PAK4-H adenocarcinoma (median, 23.6 vs 30.3 months, HR = 1.23, 95% CI, 1.02-1.48, p = 0.03), but not squamous cell carcinoma. A similar trend in survival was noted in patients who received EGFR- and ALK inhibitors. Conclusions: PAK4-H expression in NSCLC is associated with a higher frequency of alterations predisposing to genomic instability, differentially modulated immune phenotype, and a lower frequency of actionable genomic alterations. Patients with PAK4-H adenocarcinoma have inferior OS, and survival with immunotherapy and targeted therapy. PAK4-H expression defines a subgroup of patients with un unmet need for novel treatment strategies.

Characterization of MET exon 14 skipping alterations (METex14) in non–small cell lung cancer (NSCLC) using whole transcriptome sequencing (WTS).

9122 Background: Multiple DNA alterations in exon 14 splice sites have been identified in NSCLC and result in skipping of the juxtamembrane domain Cbl-E3 ubiquitin ligase binding region, leading to increased MET stability and oncogenesis. The effects of these alterations on transcriptome-level have not been fully characterized. We present the largest cohort study of METex14 using WTS and identify key cellular pathways associated with invasion and metastases in METex14. Methods: 17,666 NSCLC tumor samples underwent genomic profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq, whole exome sequencing, NovaSeq) and RNA (NovaSeq, WTS). METex14 was captured via WTS. ssGSEA analysis was used to evaluate pathway enrichment. Wilcoxon, Fisher’s exact were used for statistical significance (p without and q values with multiple comparison correction). Results: 440 patients (2.5%) with METex14 were identified. METex14 patients were of older age, female gender, and enriched in sarcomatoid histology (Table 1). The most common alterations were point mutations (51.5%) and deletions (17.3%) at donor splice sites. Splice site alterations except point mutations at splice acceptor site translated to increased mRNA expression compared to wild-type MET (WT). MET amplification translated to higher mRNA expression compared to METex14 and WT with synergistic expression when co-altered with METex14 (q&lt;0.05). The most common co-alterations were amplifications of MDM2 (18.5% vs. 1.8% WT), HMGA2 (13.7% vs 0.9% WT), and CDK4 (10.4% vs 1.4% WT) (q &lt; 0.05). METex14 were mutually exclusive to mutations in KRAS and EGFR. High PD-L1 (22c3) &gt; 50% (53% vs. 27.6% WT, q&lt;0.001) and lower TMB (4 mut/Mb vs. 7 mut/Mb WT, p&lt;0.001) were observed with METex14 and pathways associated with skipping variants included IFNγ signaling, angiogenesis, and apical junction pathways on univariate analysis (q&lt;0.05). Conclusions: We present the largest WTS analysis of METex14. Splicing alterations and MET co-amplification translated to higher and synergistic MET expression at transcriptome level, respectively. Association with upregulated angiogenic and apical junction pathways support preclinical observation of vascular and cytoskeletal remodeling as potential mechanisms of invasion and metastases in MET ex14 NSCLC.[Table: see text]

Long Non-Coding RNA NANCI/NKX2-1 Duplex Impacts Prognosis in Stage I Non-Small-Cell Lung Cancer

BackgroundNANCI, an intergenic long non-coding RNA (lncRNA) is essential for buffering NKX2-1 expression during embryonic development and in adult tissue. We analyzed NANCI and NKX2-1 in human lung embryonic samples and adult lung tissues and evaluated their potential as prognostic markers in stage I non-small cell lung cancer (NSCLC). Methods and resultsNANCI and NKX2-1 expression was assessed by TaqMan assays in 18 human embryonic samples from 8 to 13 weeks, 59 non-tumoral (NT) lung tissue samples, and 98 stage I NSCLC tumor samples. NANCI and NKX2-1 expression in embryonic and NSCLC samples were downregulated in comparison to adult NT tissue. Patients with low expression of NANCI had shorter disease-free survival (DFS) and overall survival (OS) than those with high levels (47.6 vs 69.3 months, P=0.032 and 57.7 vs 77.6 months, P=0.021, respectively). When the expression levels of NANCI and NKX2-1 were evaluated in combination, four groups were identified (high NANCI/high NKX2-1, low NANCI/high NKX2-1, high NANCI/low NKX2-1 and low NANCI/low NKX2-1) with differential impact on DFS (P=0.042) and OS (P=0.024). Interestingly, the high NANCI/high NKX2-1 duplex group had longer DFS and OS than the other three groups (71.25 vs 46.3 months, P=0.009 and 81.3 vs 56.1 months, P=0.004, respectively). In the multivariate analysis, the high NANCI/high NKX2-1 duplex was identified as an independent prognostic factor for longer DFS (HR 0.346, 95% CI, 0.169–0.709; P=0.004) and OS (HR 0.309, 95% CI, 0.121–0.786; P=0.014). ConclusionsNANCI and the NANCI-NKX2-1 duplex impacts prognosis in stage I NSCLC patients.

Abstract 4311: PPIB expression associates with tumor progression and unfavorable disease-free survival of non-small cell lung cancer

Abstract Background: PPIB (Peptidylprolyl isomerase B) is involved in cellular transcriptional regulation, immune response, chemotaxis, and proliferation. Recent studies have shown that PPIB is linked to tumor progression and chemoresistance. The role of PPIB in non-small cell lung cancer (NSCLC) has not been explored. We aimed to investigate the significance of PPIB in NSCLC and its mechanism of action. Methods: The role of PPIB was assessed in 5 human NSCLC cell lines. PPIB expression was examined using RNA in situ hybridization (RNA-ISH) on tissue microarrays consisting of 544 NSCLC cases and Ki-67 expression was evaluated by immunohistochemical assays (IHC). RNA-ISH and IHC scores were evaluated by digital image analysis and used to compare patients' prognostic outcomes. Results: PPIB overexpression promoted proliferation, colony formation and migration of NSCLC tumor cells. We also confirmed the oncogenic properties of PPIB expression in PPIB transfected human bronchogenic epithelial cells. In NSCLC tumor samples, high PPIB expression correlated with lymph node metastasis and recurrence (p = 0.018 and 0.026, respectively), while Ki-67 overexpression correlated with advanced tumor stage (p &amp;lt; 0.001). Patients with high PPIB expression assessed by RNA-ISH (PPIBhigh), exhibited significantly shorter disease-free survival (DFS) time than those who were PPIBlow [HR (95% CI) = 1.53 (1.11-2.10), p = 0.008]. Multivariate analysis revealed that combined Ki-67high/PPIBhigh expression was an independent predictor of shorter DFS [HR (95% CI) = 1.71 (1.26-2.31), p &amp;lt; 0.001]. Conclusions: These findings suggest that PPIB plays a role in NSCLC tumor growth and is associated with unfavorable disease-free survival. PPIB may have value as a prognostic marker and a potential target molecule for NSCLC treatment. Citation Format: Jeongwon Kim, Joon-Yong Chung, Stephen M. Hewitt, Ilseon Hwang, Joon Seon Song, Chang-Min Choi, Eunho Cho, Kwon-Ho Song, Tae Woo Kim, Kris Ylaya. PPIB expression associates with tumor progression and unfavorable disease-free survival of non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4311.

Long Non-Coding RNA NANCI/NKX2-1 Duplex Impacts Prognosis in Stage I Non-Small-Cell Lung Cancer

BackgroundNANCI, an intergenic long non-coding RNA (lncRNA) is essential for buffering NKX2-1 expression during embryonic development and in adult tissue. We analyzed NANCI and NKX2-1 in human lung embryonic samples and adult lung tissues and evaluated their potential as prognostic markers in stage I non-small cell lung cancer (NSCLC). Methods and resultsNANCI and NKX2-1 expression was assessed by TaqMan assays in 18 human embryonic samples from 8 to 13 weeks, 59 non-tumoral (NT) lung tissue samples, and 98 stage I NSCLC tumor samples. NANCI and NKX2-1 expression in embryonic and NSCLC samples were downregulated in comparison to adult NT tissue. Patients with low expression of NANCI had shorter disease-free survival (DFS) and overall survival (OS) than those with high levels (47.6 vs 69.3 months, P=0.032 and 57.7 vs 77.6 months, P=0.021, respectively). When the expression levels of NANCI and NKX2-1 were evaluated in combination, four groups were identified (high NANCI/high NKX2-1, low NANCI/high NKX2-1, high NANCI/low NKX2-1 and low NANCI/low NKX2-1) with differential impact on DFS (P=0.042) and OS (P=0.024). Interestingly, the high NANCI/high NKX2-1 duplex group had longer DFS and OS than the other three groups (71.25 vs 46.3 months, P=0.009 and 81.3 vs 56.1 months, P=0.004, respectively). In the multivariate analysis, the high NANCI/high NKX2-1 duplex was identified as an independent prognostic factor for longer DFS (HR 0.346, 95% CI, 0.169–0.709; P=0.004) and OS (HR 0.309, 95% CI, 0.121–0.786; P=0.014). ConclusionsNANCI and the NANCI-NKX2-1 duplex impacts prognosis in stage I NSCLC patients.

Fluzoparib increases radiation sensitivity of non-small cell lung cancer (NSCLC) cells without BRCA1/2 mutation, a novel PARP1 inhibitor undergoing clinical trials

Poly (ADP-ribose) polymerase 1 inhibitors were originally investigated as anti-cancer therapeutics with BRCA1/2 genes mutation. Here, we investigate the effectiveness of a novel PARP1 inhibitor fluzoparib, for enhancing the radiation sensitivity of NSCLC cells lacking BRCA1/2 mutation. We used MTS assays, western blotting, colony formation assays, immunofluorescence staining, and flow cytometry to evaluate the radiosensitization of NSCLC cells to fluzoparib and explore the underlying mechanisms in vitro. Through BRCA1 and RAD50 genes knockdown, we established dysfunctional homologous recombination (HR) DNA repair pathway models in NSCLC cells. We next investigated the radiosensitization effect of fluzoparib in vivo using human NSCLC xenograft models in mice. The expression of PARP1 and BRCA1 in human NSCLC tumor samples was measured by immunohistochemistry. Furthermore, we sequenced HR-related gene mutations and analyzed their frequencies in advanced NSCLC. In vitro experiments in NSCLC cell lines along with in vivo experiments using an NSCLC xenograft mouse model demonstrated the radiosensitization effect of fluzoparib. The underlying mechanisms involved increased apoptosis, cell-cycle arrest, enhanced irradiation-induced DNA damage, and delayed DNA-damage repair. Immunohistochemical staining showed no correlation between the expression of PARP1 and BRCA1. Moreover, our sequencing results revealed high mutation frequencies for the BRCA1/2, CHEK2, ATR, and RAD50 genes. The potential therapeutic value of fluzoparib for increasing the radiation sensitivity of NSCLC is well confirmed. Moreover, our findings of high mutation frequencies among HR genes suggest that PARP1 inhibition may be an effective treatment strategy for advanced non-small cell lung cancer patients.

Inhibition of Aurora A enhances radiosensitivity in selected lung cancer cell lines

BackgroundIn mammalian cells, Aurora serine/threonine kinases (Aurora A, B, and C) are expressed in a cell cycle-dependent fashion as key mitotic regulators required for the maintenance of chromosomal stability. Aurora-A (AURKA) has been proven to be an oncogene in a variety of cancers; however, whether its expression relates to patient survival and the association with radiotherapy remains unclear in non-small cell lung cancer (NSCLC).MethodsHere, we first analyzed AURKA expression in 63 NSCLC tumor samples by immunohistochemistry (IHC) and used an MTS assay to compare cell survival by targeting AURKA with MLN8237 (Alisertib) in H460 and HCC2429 (P53-competent), and H1299 (P53-deficient) cell lines. The radiosensitivity of MLN8237 was further evaluated by clonogenic assay. Finally, we examined the effect of combining radiation and AURKA inhibition in vivo with a xenograft model and explored the potential mechanism.ResultsWe found that increased AURKA expression correlated with decreased time to progression and overall survival (p = 0.0447 and 0.0096, respectively). AURKA inhibition using 100 nM MLN8237 for 48 h decreases cell growth in a partially P53-dependent manner, and the survival rates of H460, HCC2429, and H1299 cells were 56, 50, and 77%, respectively. In addition, the survival of H1299 cells decreased 27% after ectopic restoration of P53 expression, and the radiotherapy enhancement was also influenced by P53 expression (DER H460 = 1.33; HCC2429 = 1.35; H1299 = 1.02). Furthermore, tumor growth of H460 was delayed significantly in a subcutaneous mouse model exposed to both MLN8237 and radiation.ConclusionsTaken together, our results confirmed that the expression of AURKA correlated with decreased NSCLC patient survival, and it might be a promising inhibition target when combined with radiotherapy, especially for P53-competent lung cancer cells. Modulation of P53 function could provide a new option for reversing cell resistance to the AURKA inhibitor MLN8237, which deserves further investigation.