EGFR-mutant Lung Cancer Cells Research Articles

Abstract C029: PM2.5-induced drug resistance in lung cancer

Abstract Long-term exposure to PM2.5 air pollution is a significant contributor to lung cancer incidence. Our previous studies have shown that such exposure promotes lung cancer progression by activating the EGFR (epidermal growth factor receptor) and AhR (aryl hydrocarbon Receptor) pathways. We observed that in lung cancer cells with EGFR mutations, prolonged PM2.5 exposure induces EGFR activation, resulting in accelerated tumor cell growth both in vitro and in vivo. This suggests that PM2.5 may play a role in the resistance to EGFR-TKI (tyrosine kinase inhibitor) therapies, raising concerns about treatment efficacy. To explore this, we exposed the EGFR-mutant lung cancer cell line to PM2.5 for 90 days. Our results revealed that long-term exposure not only reduced the therapeutic effect of EGFR-TKIs but also increased the expression of cMyc, a gene linked to poor prognosis and drug resistance. We further confirmed that PM2.5-induced EGFR-TKI resistance is driven by the AhR-cMyc pathway. AhR, activated by environmental toxins in PM2.5, increases cMyc expression, leading to drug resistance. When we administered cMyc inhibitors, the PM2.5-exposed cells regained sensitivity to EGFR-TKI therapy. These findings indicate that targeting the AhR-cMyc pathway could be a promising approach to overcome PM2.5-induced resistance in lung cancer treatment. Further research is needed to explore the broader implications of environmental pollution on cancer therapy and to develop strategies to counteract the effects of pollutants like PM2.5. Citation Format: Chi-Yuan Chen, Chieh-Wen Chan, Tong-Hong Wang. PM2.5-induced drug resistance in lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C029.

Pericytes Modulate Third-Generation Tyrosine Kinase Inhibitor Sensitivity in EGFR-Mutated Lung Cancer Cells Through IL32-β5-Integrin Paracrine Signaling.

EGFR-mutated lung cancer patients sometimes display restricted responses to third-generation tyrosine kinase inhibitors (TKIs), potentially attributable to undervalued input from stromal cells, notably pericytes (PCs). The study shows that PCs isolated from EGFR-mutated patients have a unique secretome profile, notably secreting IL32 and affecting signaling pathways and biological processes linked to TKI sensitivity. Clinical evidence, supported by single-cell RNA sequencing and multiplex immunostaining of tumor tissues, confirms the presence of IL32-expressing pericytes closely interacting with β5-integrin-expressing cancer cells in EGFR-mutated patients, impacting therapeutic response and prognosis. Co-culture and conditioned medium experiments demonstrate that PCs reduce TKI effectiveness in EGFR-mutated cancer cells, a reversible phenomenon through silencing IL32 expression in PCs or depleting the IL32 receptor β5-integrin on cancer cells, thereby restoring cancer cell sensitivity. Mechanistically, it is shown that YY1 signaling upregulates IL32 secretion in PCs, subsequently activating the β5-integrin-Src-Akt pathway in EGFR-mutated cancer cells, contributing to their TKI sensitivity. In animal studies, co-injection of cancer cells with PCs compromises TKI effectiveness, independently of blood vessel functions, while inhibition of β5-integrin restores tumor cell sensitivity. Overall, the findings highlight direct crosstalk between cancer cells and pericytes, impacting TKI sensitivity via IL32-β5-integrin paracrine signaling, proposing an enhanced therapeutic approach for EGFR-mutated patients.

Initial AXL and MCL-1 inhibition contributes to abolishing lazertinib tolerance in EGFR-mutant lung cancer cells.

Lazertinib, a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), demonstrates marked efficacy in EGFR-mutant lung cancer. However, resistance commonly develops, prompting consideration of therapeutic strategies to overcome initial drug resistance mechanisms. This study aimed to elucidate the adaptive resistance to lazertinib and advocate novel combination treatments that demonstrate efficacy in preventing resistance as a first-line treatment for EGFR mutation-positive NSCLC. We found that AXL knockdown significantly inhibited lung cancer cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors led to residual cell proliferation and increased the MCL-1 expression level, which was mediated by the nuclear translocation of the transcription factor YAP. Triple therapy with an MCL-1 or YAP inhibitor in combination with lazertinib and an AXL inhibitor significantly reduced cell viability and increased the apoptosis rate. These results demonstrate that AXL and YAP/MCL-1 signals contribute to adaptive lazertinib resistance in EGFR-mutant lung cancer cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be a highly effective strategy in eliminating lazertinib-resistant cells.

Integrin αV Inhibition by GMI, a Ganoderma Microsporum Immunomodulatory Protein, Abolish Stemness and Migration in EGFR-Mutated Lung Cancer Cells Resistant to Osimertinib.

Integrins, the receptors of the extracellular matrix, are critical in the proliferation and metastasis of cancer cells. GMI, a Ganoderma microsporum immunomodulatory protein, possesses anticancer and antivirus abilities. The object of this study is to investigate the role of GMI in the integrins signaling pathway in lung cancer cells that harbor the EGFR L858R/T790M double mutation and osimertinib-resistance. Liquid chromatography-mass spectrometry and western blot assay were used to investigate the effect of GMI on inhibiting the protein expressions of integrins in H1975 cells. The migration ability and xenograft tumor growth of H1975 were suppressed by GMI. To elucidate the role of the integrin family in lung cancer resistant to osimertinib (AZD-9291, Tagrisso), H1975 cells were used to establish the osimertinib-resistant cells, named H1975/TR cells. The expressions of Integrin αV and stemness markers were much higher in H1975/TR cells than in H1975 cells. GMI suppressed cell viability, tumor spheroid growth, and the expressions of integrin αV and β1 in H1975/TR cells. Furthermore, GMI suppressed the expressions of stemness markers and formation of tumor spheres via blocking integrin αV signaling cascade. This is the first study to reveal the novel function of GMI in constraining cancer stem cells and migration by abolishing the integrin αV-related signaling pathway in EGFR-mutated and osimertinib-resistant lung cancer cells.

Integrative single-cell RNA-seq and spatial transcriptomics analyses reveal diverse apoptosis-related gene expression profiles in EGFR-mutated lung cancer

In EGFR-mutated lung cancer, the duration of response to tyrosine kinase inhibitors (TKIs) is limited by the development of acquired drug resistance. Despite the crucial role played by apoptosis-related genes in tumor cell survival, how their expression changes as resistance to EGFR-TKIs emerges remains unclear. Here, we conduct a comprehensive analysis of apoptosis-related genes, including BCL-2 and IAP family members, using single-cell RNA sequence (scRNA-seq) and spatial transcriptomics (ST). scRNA-seq of EGFR-mutated lung cancer cell lines captures changes in apoptosis-related gene expression following EGFR-TKI treatment, most notably BCL2L1 upregulation. scRNA-seq of EGFR-mutated lung cancer patient samples also reveals high BCL2L1 expression, specifically in tumor cells, while MCL1 expression is lower in tumors compared to non-tumor cells. ST analysis of specimens from transgenic mice with EGFR-driven lung cancer indicates spatial heterogeneity of tumors and corroborates scRNA-seq findings. Genetic ablation and pharmacological inhibition of BCL2L1/BCL-XL overcome or delay EGFR-TKI resistance. Overall, our findings indicate that BCL2L1/BCL-XL expression is important for tumor cell survival as EGFR-TKI resistance emerges.

Triple combination therapy comprising osimertinib, an AXL inhibitor, and an FGFR inhibitor improves the efficacy of EGFR-mutated non-small cell lung cancer

We previously reported that combined therapy with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) osimertinib and AXL inhibitor ONO-7475 is effective in preventing the survival of drug-tolerant cells in high-AXL-expressing EGFR-mutated non-small cell lung cancer (NSCLC) cells. Nevertheless, certain residual cells are anticipated to eventually develop acquired resistance to this combination therapy. In this study, we attempted to establish a multidrug combination therapy from the first-line setting to overcome resistance to this combination therapy in high-AXL-expressing EGFR-mutated NSCLC. siRNA screening assay showed that fibroblast growth factor receptor 1 (FGFR1) knockdown induced pronounced inhibition of cell viability in the presence of the osimertinib–ONO-7475 combination, which activates FGFR1 by upregulating FGF2 via the c-Myc pathway. Cell-based assays showed that triple therapy with osimertinib, ONO-7475, and the FGFR inhibitor BGJ398 significantly increased apoptosis by increasing expression of proapoptotic factor Bim and reduced cell viability compared with that observed for the osimertinib–ONO-7475 therapy. Xenograft models showed that triple therapy considerably suppressed tumor regrowth. A novel therapeutic strategy of additional initial FGFR1 inhibition may be highly effective in suppressing the emergence of osimertinib- and ONO-7475-resistant cells.

Effect of P. gingivalis on osimertinib resistance in EGFR-mutant lung cancer.

e20511 Background: While most patients with EGFR-mutant lung cancers respond to osimertinib, resistance inevitably develops. Mechanisms of osimertinib resistance remain inadequately understood. Recent studies suggest a potential link between intra-tumoral bacteria and therapy resistance in cancer. Our previous studies found that the pre-conditioned medium (PCM) of certain bacteria in the Bacteroidetes phylum conferred resistance of all EGFR-TKIs tested. In this study, we specifically investigate the effect of Porphyromonas gingivalis, a common component of the human oral microbiome and the main pathogen responsible for periodontal disease, on osimertinib sensitivity in EGFR mutant lung cancer. Methods: P. gingivalis PCM (P-CM) was applied to EGFR-mutant lung cancer cell lines for 3 days and cell viability was measured. Western blot and receptor tyrosine kinase (RTKs) assay were used to analyze the changes of cell signaling pathways, and the contribution of identified signaling pathway to P. gingivalis-mediated resistance to osimertinib were further validated by rescue experiments. To investigate the existence of P. gingivalis in human lung cancer tissues, we used Fluorescence In Situ Hybridization (FISH) probes specific to P. gingivalis and hybridized probes to a tissue microarray (TMA) containing 3 mm punches of lung adenocarcinoma tissue from 30 patients in duplicate. The underlying mechanisms of osimertinib resistance conferred by P. gingivalis were explored by a combination of bacterial genomic and cellular proteomic approaches. Results: P-PCM led to osimertinib resistance in all EGFR-mutant lung cancer cell lines tested. Both Western blot and RTKs assay showed that phosphorylated insulin-like growth factor 1 receptor (pIGF1R) level significantly increased when P-PCM was applied to PC9 EGFR mutant lung cancer cells. We found that both IGF1R inhibitor (linsitinib) and IGF1R knockdown with siRNA eliminated the effect of P-PCM-mediated osimertinib resistance. We found that two cysteine proteases secreted by P. gingivalis, lysine-specific gingipain (Kgp) and arginine-specific gingipain A (RgpA) led to osimertinib, whereas arginine-specific gingipain B (RgpB), which lacks haemagglutinin/adhesin (HA) region of RgpA had no effect on osimertinib response. We also found that gingipains-mediated osimertinib resistance is independent of enzymatic activity. We detected intra-tumoral P. gingivalis in tumor tissue of 9 (30%) patients by using FISH probes specific to P. gingivalis. Conclusions: Our studies demonstrate that P. gingivalis can induce osimertinib resistance in EGFR-mutant lung cancer cells through secreted gingipains that activate the IGF1R pathway through an enzymatic- independent mechanism. P. gingivalis is frequently detected in human lung cancer tissues , suggesting that therapies targeting P. gingivalis may improve EGFR-TKI response in certain patients with EGFR-mutant lung cancer.

RBM10 mutation as a potential negative prognostic/predictive biomarker to therapy in non-small-cell lung cancer.

e20544 Background: According to WHO, lung cancer is the leading cause of cancer-related death worldwide, but treatment has advanced in the last decade. The widespread use of Next Generation Sequencing has led to the discovery of several pathogenic mutations including RNA binding motif 10 [RBM10], a part of the spliceosome complex that regulates splicing of pre-mRNA [Pan Q, Nat Genet. 2008]. The knock down of RBM10 increases the growth of mouse tumor xenografts [Hernández J, RNA Biol. 2016]. Mutated RBM10increased spliceosome inhibition in EGFR mutated lung cancer cells thereby contributing to EGFR TKI resistance [Bao Y, Cancer Res. 2023]. Methods: Electronic medical records were utilized to create a database of patients [50 patients] seen from 2018-2023 with NSCLC and RBM10 mutations, with appropriate IRB approval. For sub-group analysis, we separated into groups by rapid progression vs stable disease defined as progression free survival earlier than respective clinical trials. Fisher’s exact tests were used to assess the association between mutation and clinical outcomes. Results: From analysis of treatment response, 71% [20 patients out of 28] of the patients with complete treatment data had rapid progression of disease. In our patient dataset, 20 out of the 50 RBM10 mutated patients had EGFR mutations while 18 had KRAS mutations. RBM10 mutations did not seem to correlate with alterations in MSI stability as all patients had MSI stable disease and there was no relationship between the tumor mutation burden and the clinical response in the RBM10 mutated patients (odds ratio (OR) = 1.33, 95% CI 0.24-7.35, p = 0.741). In comparing the mutated RBM10 population to wild type RBM10 population, controlled for driver mutations, median PFS was 6.7 (95% CI [4.5, 17.]) compared to 13.9 (95% CI [10.4, 24.2]). RBM10 mutations in EGFR mutated patients often developed later in the course of disease, commonly at progression leading to late EGFR TKI resistance. The most common non driver concurrent mutation with RBM10 was TP53, while the most common VUS were SPTA1 and ZFHX3. TP53 mutation had a higher representation in the RBM10 mutated rapid progression group, OR (odds ratio) 8.081, 95% CI [0.773, 431/717], P-value = 0. 00838). The ZFHX3 mutation had a higher representation in the RBM10 mutated stable disease group OR 0.0430, 95% CI [0.0007, 0.5506]. Conclusions: From this single institution investigation, RBM10 mutations were associated with aggressive disease with treatment progression faster than median durations of response. RBM10 mutations with concurrent ZFHX3 and EGFR mutations were associated with more stable disease, while concurrent KRAS and TP53 predicted even more aggressive disease. However, once the RBM10 mutation developed in the EGFR population, rapid progression was observed, indicating a mechanism of late resistance. More investigation is warranted into RBM10 to further certify its use as a biomarker.

Genome-wide CRISPR screens identify the YAP/TEAD axis as a driver of persister cells in EGFR mutant lung cancer

Most lung cancer patients with metastatic cancer eventually relapse with drug-resistant disease following treatment and EGFR mutant lung cancer is no exception. Genome-wide CRISPR screens, to either knock out or overexpress all protein-coding genes in cancer cell lines, revealed the landscape of pathways that cause resistance to the EGFR inhibitors osimertinib or gefitinib in EGFR mutant lung cancer. Among the most recurrent resistance genes were those that regulate the Hippo pathway. Following osimertinib treatment a subpopulation of cancer cells are able to survive and over time develop stable resistance. These ‘persister’ cells can exploit non-genetic (transcriptional) programs that enable cancer cells to survive drug treatment. Using genetic and pharmacologic tools we identified Hippo signalling as an important non-genetic mechanism of cell survival following osimertinib treatment. Further, we show that combinatorial targeting of the Hippo pathway and EGFR is highly effective in EGFR mutant lung cancer cells and patient-derived organoids, suggesting a new therapeutic strategy for EGFR mutant lung cancer patients.

A novel dihydroacridine derivative targets epidermal growth factor receptor-expressing cancer cells in vitro and in vivo.

Small molecules are considered a source of novel medicines targeting carcinogenic intracellular pathways including epidermal growth factor receptor (EGFR) signaling. The main goal of the study is to assess whether LHT-17-19 could be considered an effective target molecule against EGFR-expressing tumor cells in silico, in vitro, and in vivo. This was an in vivo, ex vivo, and in vivo experimental study. LHT-17-19 affinity to EGFR's kinase domain was assessed by the ligand's molecular docking. EGFR-expressing Hs746T human gastric cancer cell culture and patient-derived organoid (PDO) model of EGFR-positive breast cancer (BC) were used for in vitro assessment of the molecule anticancer property. IC50 and GI50 indexes were estimated using MTT- and MTS-based tests, respectively. Anticancer activity of LHT-17-19 against EGFR-expressing mutant lung carcinoma was studied on patient-derived xenograft (PDX) model established in 10 humanized BALB/c male mice. Continuous variables were presented as a mean ± standard deviation. Intergroup differences were assessed by two-way t-test. Kaplan-Meier's curves were used for survival analysis. High affinity of LHT-17-19 for the EGFR kinase domain with dG score -7.9 kcal/mol, EDoc-5.45 kcal/mol, and Ki 101.24 uM was due to intermolecular π-σ bonds formation and the ligand intramolecular transformation. LHT-17-19 induced anti-EGFR-expressing gastric cancer cells cytotoxicity with IC50 0.32 µM (95% confidence interval [CI] 0.11-0.54 µM). The derivative inhibited growth of EGFR-expressing BC PDO with GI50 16.25 µM (95% CI 4.44-28.04 µM). 2 mg/kg LHT-17-19 intravenously daily during 7 days inhibited PDX tumor growth and metastatic activity, prolonged animals' survival, and eliminated EGFR-mutant lung cancer cells from residual tumor's node. LHT-17-19 may be considered a molecular platform for further search of promising molecules, EGFR-expressing cancer cell inhibitors.

Abstract 5826: Comprehensive analysis with single-cell RNA sequence and spatial transcriptomics unravels distinctive expression profile of apoptotic-related genes in EGFR-mutated lung cancer

Abstract Despite an initial marked response to targeted therapy with tyrosine kinase inhibitors (TKIs) in EGFR-mutated lung cancer, the duration of response is limited due to the inevitable development of acquired resistance. Anti- and pro-apoptotic genes play an important role in the survival of tumor cells. Currently, variations in these gene expression levels are not well characterized due to the complex interplay between the inducement of expression changes by EGFR-TKIs and the presence of inter- and intra-tumor heterogeneity. Here, a comprehensive analysis of apoptosis-related genes, including BCL-2 family members and the IAP family members, was conducted via single-cell RNA sequence (scRNA-seq) and spatial transcriptomics. scRNA-seq captured changes in apoptosis-related gene expression after EGFR-TKI treatment using different EGFR-mutated lung cancer cell lines. Notably, BCL2L1 expression increased, whereas MCL1 expression decreased after EGFR-TKI treatment. Other anti- and pro-apoptotic genes did not display consistent trends in the cell lines used. Additionally, scRNA-seq was carried out on samples collected from patients with EGFR-mutated lung cancer after the development of resistance to EGFR-TKIs in order to validate the results obtained from cell line data and to compare gene expression levels between tumor and non-tumor cells. BCL2L1 exhibited high expression specifically in tumor cells; in contrast, MCL1 expression was lower in tumor cells compared to non-tumor cells. Moreover, BAD and XIAP were highly expressed in a tumor-specific manner. To further corroborate the scRNA-seq findings and delineate the spatial heterogeneity in tumors, spatial transcriptomics was performed using EGFR-driven lung cancer transgenic mouse specimens in three conditions: prior to treatment, during therapeutic response after short-term treatment, and following acquired resistance to EGFR-TKIs. Spatial transcriptomics data demonstrated that an increased BCL2L1 expression was observed in tumor tissues and that MCL1 expression was significantly higher in tissue adjacent to tumors than in tumors. Finally, we demonstrated that genetic ablation of BCL2L1 and pharmacological inhibition of BCL2L1 overcame or delayed resistance to EGFR-TKIs in vitro and in vivo. Overall, BCL2L1 is particularly important for tumor cell survival during the emergence of drug tolerance and the development of acquired resistance to EGFR-TKIs. Citation Format: Motohiro Izumi, Masanori Fujii, Vivian Ho, Ikei S. Kobayashi, Susumu S. Kobayashi. Comprehensive analysis with single-cell RNA sequence and spatial transcriptomics unravels distinctive expression profile of apoptotic-related genes in EGFR-mutated 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 5826.

TP53 gain-of-function mutations promote osimertinib resistance via TNF-α–NF-κB signaling in EGFR-mutated lung cancer

EGFR tyrosine kinase inhibitors (TKIs) are effective against EGFR-mutated lung cancer, but tumors eventually develop resistance to these drugs. Although TP53 gain-of-function (GOF) mutations promote carcinogenesis, their effect on EGFR-TKI efficacy has remained unclear. We here established EGFR-mutated lung cancer cell lines that express wild-type (WT) or various mutant p53 proteins with CRISPR-Cas9 technology and found that TP53-GOF mutations promote early development of resistance to the EGFR-TKI osimertinib associated with sustained activation of ERK and expression of c-Myc. Gene expression analysis revealed that osimertinib activates TNF-α–NF-κB signaling specifically in TP53-GOF mutant cells. In such cells, osimertinib promoted interaction of p53 with the NF-κB subunit p65, translocation of the resulting complex to the nucleus and its binding to the TNF promoter, and TNF-α production. Concurrent treatment of TP53-GOF mutant cells with the TNF-α inhibitor infliximab suppressed acquisition of osimertinib resistance as well as restored osimertinib sensitivity in resistant cells in association with attenuation of ERK activation and c-Myc expression. Our findings indicate that induction of TNF-α expression by osimertinib in TP53-GOF mutant cells contributes to the early development of osimertinib resistance, and that TNF-α inhibition may therefore be an effective strategy to overcome such resistance in EGFR-mutant lung cancer with TP53-GOF mutations.

YAP mediates resistance to EGF-induced apoptosis in EGFR-mutated non-small cell lung cancer cells

Mechanisms underlying the growth and survival of non-small cell lung cancer (NSCLC) cells positive for activating mutations of the epidermal growth factor receptor gene (EGFR) have remained unclear. We here examined the functional relation between such mutant forms of EGFR and Yes-associated protein (YAP), a transcriptional coactivator of the Hippo signaling pathway that regulates cell proliferation and survival. Under the condition of serum deprivation, epidermal growth factor (EGF) induced activation of YAP in NSCLC cell lines positive for mutated EGFR but not in those wild type (WT) for EGFR. Similar EGF-induced activation of YAP was apparent in A549 lung cancer cells forcibly expressing mutant EGFR but not in those overexpressing the WT receptor. Furthermore, EGF induced apoptotic cell death in serum-deprived A549 cells overexpressing the WT form of EGFR but not in those expressing mutant EGFR, and knockdown of YAP rendered the latter cells sensitive to this effect of EGF. Our results thus suggest that activation of YAP mediates resistance of EGFR-mutated NSCLC cells to EGF-induced apoptosis and thereby contributes specifically to the survival of such cells.

SLC12A8 mediates TKI resistance in EGFR-mutant lung cancer via PDK1/AKT axis.

Epidermal growth factor receptor (EGFR) mutation is a prominent driver of lung cancer. Tyrosine kinase inhibitors (TKIs) have shown efficacy in treating EGFR-mutant lung cancer, but the emergence of drug resistance poses a significant challenge. Recent research has highlighted solute carrier family 12 member 8 (SLC12A8) as one of the highly upregulated genes in various cancer types. However, its oncogenic function remains largely unexplored. 343 consecutive lung cancer patients were prospectively recruited and were followed for over 10years. SLC12A8 expression in lung cancer tissues was measured by qPCR and was associated with patient survival. The association of SLC12A8 with TKI resistance was studied in in vitro EGFR-mutant lung cancer cell line as well as in in vivo xenograft tumor model. High-throughput kinome screening was employed to investigate SLC12A8-mediated oncogenic signaling pathway in lung cancer. SLC12A8 is a predictive biomarker of poor prognosis in lung cancer, particularly in patients with EGFR mutations. SLC12A8 overexpression diminishes the effectiveness of TKIs in EGFR-mutant lung cancer, resulting in treatment failure and disease progression. More importantly, SLC12A8-induced TKI resistance is mediated by the PDK1/AKT signaling axis, while silencing SLC12A8 expression inhibits oncogenic PDK1/AKT signaling, restoring TKI sensitivity in lung cancer cells. SLC12A8 mediates TKI resistance in EGFR-mutant lung cancer via PDK1/AKT axis. These findings not only advance our understanding of the molecular mechanisms driving TKI resistance, but also offer novel alternative strategies for the treatment of lung cancer.

The combination of osimertinib with Raf inhibitor overcomes osimertinib resistance induced by KRAS amplification in EGFR-mutated lung cancer cells

Osimertinib is a third-generation epidermal growth factor receptor (EGFR)1 tyrosine kinase inhibitor (TKI) approved for the treatment of EGFR-positive patients exhibiting a T790 M resistance mutation after treatment with an earlier generation of EGFR TKIs. However, resistance to osimertinib inevitably develops despite its efficacy, and the resistance mechanisms are complex and not fully understood. We established cell lines with acquired resistance to osimertinib from gefitinib- or erlotinib-resistant NSCLC cells using a dose-escalation method, and found that they had upregulated levels of phosphorylated ERK1/2. Targeted next-generation sequencing of 143 genes was performed, and interestingly, amplification of KRAS was observed in osimertinib-resistant cells. Transfection of siRNA against the KRAS gene notably reduced the activation of ERK1/2 and AKT and significantly enhanced the induction of apoptosis by osimertinib treatment in osimertinib-resistant cells. LY3009120, a RAF inhibitor, showed a significant synergistic effect with osimertinib on apoptotic cell death in osimertinib-resistant cells. Combined treatment with osimertinib and LY3009120 also demonstrated remarkable synergistic anti-tumor activity in mouse xenografts of these cells. This could be a potential new treatment option for KRAS amplification-induced osimertinib failure.

Inhibition of PLK1 Destabilizes EGFR and Sensitizes EGFR-Mutated Lung Cancer Cells to Small Molecule Inhibitor Osimertinib.

Tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) have significantly prolonged survival in EGFR-mutant non-small cell lung cancer patients. However, the development of resistance mechanisms prohibits the curative potential of EGFR TKIs. Combination therapies emerge as a valuable approach to preventing or delaying disease progression. Here, we investigated the combined inhibition of polo-like kinase 1 (PLK1) and EGFR in TKI-sensitive EGFR-mutant NSCLC cells. The pharmacological inhibition of PLK1 destabilized EGFR levels and sensitized NSCLC cells to Osimertinib through induction of apoptosis. In addition, we found that c-Cbl, a ubiquitin ligase of EGFR, is a direct phosphorylation target of PLK1 and PLK1 impacts the stability of c-Cbl in a kinase-dependent manner. In conclusion, we describe a novel interaction between mutant EGFR and PLK1 that may be exploited in the clinic. Co-targeting PLK1 and EGFR may improve and prolong the clinical response to EGFR TKI in patients with an EGFR-mutated NSCLC.

Abstract 3856: Evaluation of replication protein A inhibitor, NERx329 in combination with EGFR mutant targeted therapy

Abstract Lung cancer is the leading cause of cancer related deaths worldwide with the majority (~80%) of patients diagnosed with non-small lung cancer (NSCLC). Targeted therapies directed against tumor initiating growth factor receptors are effective treatment options for driver mutation positive NSCLC. Osimertinib is a third-generation EGFR-Tyrosine Kinase inhibitor (TKI) that has activity against EGFR exon 19, exon 21, and T790M mutations. Unfortunately, Osimertinib treated patients acquire resistance and median progression free survival is 18 months. Moreover, Osimertinib does not cure stage IV EGFR mutant NSCLC. The acquisition of secondary mutations leading to activation of bypass pathways, MET amplification, EMT, compensatory pathway activation and histological transformation to small cell phenotype are major causes of acquiring resistance to EGFR TKIs. Proliferating cancer cells regularly experience a low level of replication stress. Additionally, receptor tyrosine kinases such as EGFR are known to interact with DNA repair proteins and impact DNA damage repair following chemotherapy, radiation therapy, and EGFR TKI treatment. However, the role of DNA repair pathways in EGFR-TKI resistance is unknown and the interaction between EGFR pathway, DNA damage, and repair pathways has not been fully elucidated. At low-level replicative stress promotes genomic instability but at a high level through mitotic catastrophe it causes cell death. Replication protein A is a critical sensor of the DNA damage response detecting replication stress. The small molecule inhibitor of RPA, NERx 329, sequesters active RPA and induces replication catastrophe and cell death. Here we demonstrate that NERx329 in combination with Osimertinib enhanced Osimertinib mediated cell death. CCk-8 assays performed after 48h of NERx329 and Osimertinib combination treatment result in a robust decrease in IC50 compared to single agent therapy. Dissection of signaling pathways leading to this death-promoting effect of NERx329 showed a combination of bypass pathway marker (AXL) and compensatory pathway marker (STAT) inhibition in AXL-high expressing EGFR mutant lung cancer cells. Whereas inhibition of STAT pathway was responsible for the death-promoting effect of NERx329 in AXL-low expressing EGFR mutant lung cancer cells. AXL and STAT inhibition are associated with induced DNA damage and impaired DNA repair. From these data, we infer that DNA damage repair pathways could be involved in TKI resistance and NERx329 could be a promising drug candidate for combination targeted therapy. Citation Format: Reshma Bhowmick, John J. Turchi, Shadia I. Jalal. Evaluation of replication protein A inhibitor, NERx329 in combination with EGFR mutant targeted therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3856.

Abstract 4726: KMT5C-dependent regulation of mesenchymal-related genes and identification of KMT5C interactome using BioID

Abstract Epigenetic dysregulation has recently been recognized as a new hallmark of cancer. Dysregulation of chromatin remodelers is estimated to affect 10-20% of cancer. Thus, understanding how epigenetic-based mechanisms contribute to cancer initiation and progression is critical. KMT5C is a histone methyltransferase that catalyzes histone H4 lysine 20 trimethylation (H4K20me3), which has historically been associated with formation and maintenance of constitutive heterochromatin regions. Albeit recent evidence from our lab also suggests regulation of gene-rich euchromatin regions. In this previous body of work we discovered that loss of KMT5C promotes development of resistance to tyrosine kinase inhibitors (TKI) in EGFR-mutant non-small cell lung cancer (NSCLC) cells (PC9 and HCC827). Patient data further supports that the KMT5C transcript level is downregulated following resistance to osimertinib, a third-generation TKI. Our data suggests that KMT5C is involved in non-canonical regulation of genes outside of heterochromatic regions through its methyltransferase activity. Analysis of data deposited into the Cell Miner database and publicly available RNA-sequencing and ChIP-sequencing data revealed that the KMT5C-H4K20me3 axis has significant negative correlations with several genes involved in epithelial-to-mesenchymal transition (EMT). Our validation studies support this observation. However the mechanisms involved in KMT5C facilitating this change at EMT-related genes is unknown. KMT5C is well understood to be recruited to heterochromatin regions through its interaction with HP1, yet the mechanism for recruitment of KMT5C to gene-rich euchromatin regions remains to be elucidated. We hypothesize that KMT5C is recruited to its target genes in euchromatin in an HP1-independent mechanism. In this study, we aim to identify the interactome of KMT5C using an unbiased proximity labeling BioID approach. This study is expected to uncover a novel epigenetic mechanism that contributes to development of resistance to TKIs and may inform of strategies to target KMT5C-downregulated TKI-resistant cancers. Citation Format: Jihye Son, Alejandra Agredo, Ally Glaws, Andrea L. Kasinski. KMT5C-dependent regulation of mesenchymal-related genes and identification of KMT5C interactome using BioID. [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 4726.

Abstract 2859: High-resolution lineage tracing for the study of cancer drug persistence at the single-cell level

Abstract Cancer drug persistence allows small fractions of otherwise drug-sensitive populations of cancer cells to survive treatment with anti-cancer drugs. Unlike drug resistance, persistence mechanisms are not driven by genetic mutations, and are thus considered reversible after a ‘drug holiday’ period. While most persistent cells remain in cell cycle arrest under drug treatment, recent work with EGFR-mutant Non-Small Cell Lung Cancer (NSCLC) cell lines has demonstrated that a small fraction of persister cells continue to undergo cell divisions under treatment with EGFR inhibitors. Thus, persister cells have a two-fold potential clinical significance: (i) they can serve as an intermediate state, allowing a non-resistant cancer cell to survive long enough and continue to divide under drug treatment to acquire a resistance mutation; and (ii) cycling persister cells might directly contribute to relapse, without a secondary resistance-driving mutation. Despite this, little is known about the underlying mechanisms by which affect the potential of a given cell to persist, or about the epigenetic traits that govern the distribution of persistence potential in a cell population. In this work, we use high-resolution lineage tracing to study lineages of drug persistent cells within a cancer cell population. Using the PC9 NSCLC cell line as a model, we demonstrate that while most lineages within the PC9 cell population can generate persisters, certain branches within the lineage tree have significantly higher persistence potentials than other branches, suggesting that inherited events determine the probability of a cell to persist under treatment. Moreover, we analyze the rate of change of the potential to persist within a single-cell-derived lineage of NSCLC cells and assess which branches within the lineage tree generate cycling persisters. Furthermore, we couple single-cell RNAseq data with lineage tracing information to identify gene expression patterns that correlate with the probability of drug-naïve cells to persist under drug treatment, allowing for insight into the cellular and molecular mechanisms underlying the continuum of the probability to persist. Finally, we demonstrate that while persistent-derived PC9 cells are indeed resensitized after a drug holiday, they give rise to a significantly higher proportion of cycling persister cells upon resumption of drug treatment, allowing for a rapid repopulation of the cell culture by cycling persisters. These results suggest that lineage tracing is a powerful tool to study drug-induced persistence and resistance and we will apply it to multiple other cell lines and tumor types. Citation Format: Benny Zhitomirsky, Jideofor Ezike, Elizabeth Hopkins, Brian P. Danysh, Laxmi Parida, Yaara Oren, Gad Getz. High-resolution lineage tracing for the study of cancer drug persistence at the single-cell level [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 2859.

Integrated CRISPR screening and drug profiling identifies combination opportunities for EGFR, ALK, and BRAF/MEK inhibitors

Anti-tumor efficacy of targeted therapies is variable across patients and cancer types. Even in patients with initial deep response, tumors are typically not eradicated and eventually relapse. To address these challenges, we present a systematic screen for targets that limit the anti-tumor efficacy of EGFR and ALK inhibitors in non-small cell lung cancer and BRAF/MEK inhibitors in colorectal cancer. Our approach includes genome-wide CRISPR screens with or without drugs targeting the oncogenic driver ("anchor therapy"), and large-scale pairwise combination screens of anchor therapies with 351 other drugs. Interestingly, targeting of a small number of genes, including MCL1, BCL2L1, and YAP1, sensitizes multiple cell lines to the respective anchor therapy. Data from drug combination screens with EGF816 and ceritinib indicate that dasatinib and agents disrupting microtubules act synergistically across many cell lines. Finally, we show that a higher-order-combination screen with 26 selected drugs in two resistant EGFR-mutant lung cancer cell lines identified active triplet combinations.