Epidermal Growth Factor Receptor Inhibitor Erlotinib Research Articles

Design and Synthesis of Dual Galectin-3 and EGFR Inhibitors Against Liver Fibrosis.

Liver fibrosis, mainly arising from chronic viral or metabolic liver diseases, is a significant global health concern. There is currently only one FDA-approved drug (Resmetirom) in the market to combat liver fibrosis. Both galectin-3 and epidermal growth factor receptor (EGFR) play important roles in liver fibrosis, while galectin-3 may interact with EGFR. Galectin-3 inhibitors, typically lactose or galactose derivatives may inhibit liver fibrosis. We hypothesized that targeting both galectin-3 and EGFR may have better effect against liver fibrosis. Here, EGFR inhibitor erlotinib was used in a series of designed galectin-3 inhibitors after hybridization with the pharmacophore structure in reported galectin-3 inhibitors to impede hepatic stellate cells (HSCs) activation by a typical method of click chemistry. Bioactivity test results showed that compound 29 suppressed TGF-β-induced upregulation of fibrotic markers (α-SMA, fibronectin-1, and collagen I). The preferred compound 29 displayed better binding to galectin-3 (KD = 52.29 μM) and EGFR protein (KD = 3.31 μM) by SPR assay. Further docking studies were performed to clarify the possible binding mode of compound 29 with galectin-3 and EGFR. Taken together, these results suggested that compound 29 could be a potential dual galectin-3 and EGFR inhibitor as leading compound for anti-liver fibrosis new drug development.

Discovery of new sulfonamide-tethered 2-aryl-4-anilinoquinazolines as the first-in-class dual carbonic anhydrase and EGFR inhibitors

In today's medical field, there is a growing trend of exploiting a single small molecule to target two different molecular targets concurrently. This approach is proving to be highly effective in fighting against cancer. The 4-anilinoquinazoline scaffold, known for its potential in cancer therapy and its effectiveness as a leading class of tyrosine kinase inhibitors, was employed to develop a novel series of anilinoquinazoline-sulfonamides (AQSs) (8a-d, 9a-f, and 10a-d) as dual inhibitors of the tumor-associated carbonic anhydrases (CA) IX/XII and EGFR. 2-(3-Methoxyphenyl)quinazoline bearing p-sulfanilamide 10b elicited superior hCA IX and XII inhibition in the low nanomolar range (KIs = 38.4 and 8.9 nM, respectively). Also, 10b shined as a potent and selective EGFR inhibitor, boasting an impressive IC50 value of 51.2 ± 0.97 nM, surpassing the reference EGFR inhibitor Erlotinib (IC50 = 80 ± 2.0 nM). Compound 10b exhibited broadest-spectrum antiproliferative activity against the NCI-tumor panel with a mean GI% value of 68 %. Of special interest, 10b demonstrated potent growth inhibition (GI% ≥ 80–97 %) toward cell lines reported to express high levels of EGFR belonging to renal, colon, breast, and lung cancers. Compound 10b's molecular docking in the CA IX/XII and EGFR active sites revealed binding modes that justify its potent enzyme inhibitory effects. Additionally, molecular dynamic simulations demonstrated strong and stable interactions of 10b with the binding sites of these targets.

TGF-α/EGFR signaling promotes lipopolysaccharide-induced abnormal elastin deposition and alveolar simplification

Bronchopulmonary dysplasia (BPD) is characterized by shortened secondary septa and fewer, larger alveoli. Elastin deposition to the distal tips of the secondary septa is critical for elongation of the secondary septa. Alveolar myofibroblasts, which are thought to migrate to the septal tips during alveolarization, are mainly responsible for elastin production and deposition. Antenatal exposure to inflammation induces abnormal elastin deposition, thereby increasing the risk of developing BPD. Here, we found that lipopolysaccharide (LPS) significantly increased the expression of transforming growth factor-α (TGF-α) in an LPS-induced rat model of BPD and in LPS-treated human pulmonary epithelial cells (BEAS-2B). In addition, in vitro experiments suggested that LPS upregulated TGF-α expression via toll-like receptor 4 (TLR4)/tumor necrosis factor α-converting enzyme (TACE) signaling. Increased TGF-α levels via its receptor epidermal growth factor receptor (EGFR)-induced lysyl oxidase (LOX) overactivation and cell division cycle 42 (Cdc42) activity inhibition of myofibroblasts. Similarly, in vivo LOX overactivation and inhibition of Cdc42 activity were observed in the lungs of LPS-exposed pups. LOX overactivation led to abnormal elastin deposition, and inhibition of Cdc42 activity disturbed the directional migration of myofibroblasts and disrupted elastin localization. Most importantly, the EGFR inhibitor erlotinib partially rescued LOX overactivation and Cdc42 activity inhibition, and improved elastin deposition and alveolar development in antenatal LPS-treated rats. Taken together, our data suggest that TGF-α/EGFR signaling is critically involved in the regulation of elastin deposition and represents a novel therapeutic target.

Erlotinib combination with a mitochondria-targeted ubiquinone effectively suppresses pancreatic cancer cell survival.

Pancreatic cancer is a leading cause of cancer-related deaths. Increased activity of the epidermal growth factor receptor (EGFR) is often observed in pancreatic cancer, and the small molecule EGFR inhibitor erlotinib has been approved for pancreatic cancer therapy by the food and drug administration. Nevertheless, erlotinib alone is ineffective and should be combined with other drugs to improve therapeutic outcomes. We previously showed that certain receptor tyrosine kinase inhibitors can increase mitochondrial membrane potential (Δψm), facilitate tumor cell uptake of Δψm-sensitive agents, disrupt mitochondrial homeostasis, and subsequently trigger tumor cell death. Erlotinib has not been tested for this effect. To determine whether erlotinib can elevate Δψm and increase tumor cell uptake of Δψm-sensitive agents, subsequently triggering tumor cell death. Δψm-sensitive fluorescent dye was used to determine how erlotinib affects Δψm in pancreatic adenocarcinoma (PDAC) cell lines. The viability of conventional and patient-derived primary PDAC cell lines in 2D- and 3D cultures was measured after treating cells sequentially with erlotinib and mitochondria-targeted ubiquinone (MitoQ), a Δψm-sensitive MitoQ. The synergy between erlotinib and MitoQ was then analyzed using SynergyFinder 2.0. The preclinical efficacy of the two-drug combination was determined using immune-compromised nude mice bearing PDAC cell line xenografts. Erlotinib elevated Δψm in PDAC cells, facilitating tumor cell uptake and mitochondrial enrichment of Δψm-sensitive agents. MitoQ triggered caspase-dependent apoptosis in PDAC cells in culture if used at high doses, while erlotinib pretreatment potentiated low doses of MitoQ. SynergyFinder suggested that these drugs synergistically induced tumor cell lethality. Consistent with in vitro data, erlotinib and MitoQ combination suppressed human PDAC cell line xenografts in mice more effectively than single treatments of each agent. Our findings suggest that a combination of erlotinib and MitoQ has the potential to suppress pancreatic tumor cell viability effectively.

Secondary metabolites of Trichoderma spp. as EGFR tyrosine kinase inhibitors: Evaluation of anticancer efficacy through computational approach.

The present study explores the epidermal growth factor receptor (EGFR) tyrosine kinase inhibition efficacy of secondary metabolites in Trichoderma spp. through molecular docking, molecular dynamics (MD) simulation and MM-PBSA approach. The result of molecular docking confirmed that out of 200 metabolites screened, three metabolites such as Harzianelactone A, Pretrichodermamide G and Aspochalasin M, potentially bound with the active binding site of EGFR tyrosine kinase domain(PDB ID: 1M17) with a threshold docking score of ≤- 9.0 kcal/mol when compared with the standard EGFR inhibitor (Erlotinib). The MD simulation was run to investigate the potential for stable complex formation in EGFR tyrosine kinase domain-unbound/lead metabolite (Aspochalasin M)-bound/standard inhibitor (Erlotinib)-bound complex. The MD simulation analysis at 100 ns revealed that Aspochalasin M formed the stable complex with EGFR. Besides, the in silico predication of pharmacokinetic properties further confirmed that Aspochalasin M qualified the drug-likeness rules with no harmful side effects (viz., hERG toxicity, hepatotoxicity and skin sensitization), non-mutagenicity and favourable logBB value. Moreover, the BOILED-Egg model predicted that Aspochalasin M showed a higher gastrointestinal absorption with improved bioavailability when administered orally and removed from the central nervous system (CNS). The results of the computational studies concluded that Aspochalasin M possessed significant efficacy in binding EGFR's active sites compared to the known standard inhibitor (Erlotinib). Therefore, Aspochalasin M can be used as a possible anticancer drug candidate and further in vitro and in vivo experimental validation of Aspochalasin M of Trichoderma spp. are required to determine its anticancer potential.

Abstract B092: Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy

Abstract Background: Focal high copy number oncogene amplification frequently occurs on extrachromosomal DNA (ecDNA), acentric circular DNA derived from chromosomes during neoplastic transformation and tumor progression. The unique properties of ecDNA facilitate a high degree of genomic plasticity and tumor heterogeneity, which enables rapid acquired resistance to targeted therapies and results in poor patient outcomes. Reintegrated ecDNA, also referred to as homogeneously staining regions (HSR), can exist in dynamic equilibrium with their circular form, although their potential adaptive and resistance properties have not been well characterized. Elucidating the genomic plasticity and mechanisms afforded to cancer cells harboring HSR may provide insights into new therapeutic approaches necessary to address this ecDNA-derived focal amplification. Methods: A novel model was developed that recapitulates the genomic plasticity of focal oncogene amplifications involving FGFR2 and EGFR in response to the FGFR inhibitor, infigratinib. Results: KATOIII gastric cancer cells harbor focal amplification of the FGFR2 oncogene (absolute copy number (CN) = 136 based on next-generation sequencing (NGS) that resides on an HSR as determined by FISH. Short- and long-read NGS revealed prior DNA breakpoints consistent with circular structures containing FGFR2, suggestive of reintegrated ecDNA as HSR. Despite initial sensitivity to FGFR2 inhibition (EC50 = 20 nM), cells acquired resistance following infigratinib dose escalation within 12 weeks. Infigratinib-resistant KATOIII cells retained the FGFR2 HSR while further amplifying FGFR2 on ecDNA, which correlated with increased FGFR2 CN, mRNA, and protein expression. Structural breakpoint analysis of the FGFR2 amplicon revealed conservation between baseline and ecDNA-amplified infigratinib resistant cells, suggesting that the HSR gave rise to ecDNA. Removal of infigratinib resulted in near-complete disappearance of FGFR2 ecDNA and return to near-baseline FGFR2 CN, protein expression, and signaling within 3 weeks. Strikingly, at high infigratinib concentrations that effectively inhibit FGFR2 signaling, cells rapidly switched to EGFR amplified on ecDNA, with concomitant reduction of FGFR2 ecDNA (CN = 553 to 238) and signaling and increased EGFR (CN = 3 to 30), mRNA, and EGFR signaling. Following ecDNA switching from FGFR2 to EGFR, KATOIII cells remained insensitive to infigratinib while acquiring sensitivity to the EGFR inhibitor erlotinib. Mutational analysis revealed no new oncogenic mutations including FGFR2 and EGFR, indicating the EGFR overexpression afforded by ecDNA was sufficient to drive acquired resistance to high infigratinib concentrations. Conclusions: These findings indicate that tumor cells with ecDNA-derived chromosomally reintegrated HSR retain the capacity to amplify ecDNA, thus enabling rapid adaptation to therapeutic pressure. Consequently, new treatment strategies that disable adaptive genomic plasticity are needed to address oncogene amplified cancers that leverage ecDNA biology. Citation Format: Kristen Turner, Homa Hemmati, Cory DuPai, John Bibay, Claire Klebs, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy [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 B092.

The activity of a PI3K δ-sparing inhibitor, MEN1611, in non-small cell lung cancer cells with constitutive activation of the PI3K/AKT/mTOR pathway.

Lung cancer remains the leading cause of cancer-related death worldwide. Targeted therapies with tyrosine kinase inhibitors (TKIs) result in improvement in survival for non-small cell lung cancer (NSCLC) with activating mutations of the epidermal growth factor receptor (EGFR). Unfortunately, most patients who initially respond to EGFR-TKI ultimately develop resistance to therapy, resulting in cancer progression and relapse. Combination therapy is today a common strategy for the treatment of tumors to increase the success rate, improve the outcome and survival of patients, and avoid the selection of resistant cancer cells through the activation of compensatory pathways. In NSCLC, the phosphoinositide-3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway has been heavily implicated in both tumorigenesis and the progression of disease. In this study, we investigated the efficacy of a PI3K δ-sparing inhibitor, MEN1611, in models of NSCLC sensitive and resistant to EGFR inhibitors (erlotinib and gefitinib) with a wild-type PIK3CA gene. We performed functional, biochemical, and immunohistochemistry studies. We demonstrated good efficacy of MEN1611 in NSCLC devoid of PIK3CA gene mutations but with constitutive activation of the PI3K/AKT pathway and its synergistic effect with gefitinib both in vitro and in vivo. Overall, this preclinical study indicates that the inhibitor could be a candidate for the treatment of NSCLC with an erlotinib/gefitinib-resistant phenotype and constitutive activation of the PI3K/AKT pathway, a phenotype mimicked by our model system.

Gintonin-Induced Wound-Healing-Related Responses Involve Epidermal-Growth-Factor-like Effects in Keratinocytes.

Epidermal growth factor (EGF) receptor activation and related downstream signaling pathways are known to be one of the major mechanisms of the proliferation and migration of keratinocytes. The heparin-binding EGF-like growth factor (HB-EGF) binds to EGF receptors and stimulates keratinocyte proliferation and migration. Gintonin, a novel ginseng compound, is a lysophosphatidic acid (LPA) receptor ligand. Gintonin has skin-wound-healing effects. However, the underlying mechanisms for these gintonin actions remain unclear. In this study, we aimed to elucidate the involvement of EGFRs in gintonin-induced wound repair in HaCaT keratinocytes. In this study, a water-soluble tetrazolium salt-based assay, a modified Boyden chamber migration assay, and immunoblotting were performed. Gintonin increased EGF receptor activation in HaCaT cells. However, the gintonin-induced phosphorylation of the EGF receptor was markedly reduced via treatment with the LPA inhibitor Ki16425 or the EGF receptor inhibitor erlotinib. Gintonin-enhanced proliferation and migration were blocked by the EGF receptor inhibitors (erlotinib and AG1478). Additionally, gintonin stimulated the expression and release of HB-EGF in HaCaT cells. EGF receptor inhibitors blocked gintonin-enhanced HB-EGF expression. These results indicate that the wound-healing effects of gintonin are closely related to the collaboration between EGF receptor activation and HB-EGF release-mediated downstream signaling pathways.

EGFR inhibitor erlotinib plus monoclonal antibody versus erlotinib alone for first-line treatment of advanced non-small cell lung carcinoma: A systematic review and meta-analysis.

Immuno-combination therapy is emerging as an effective treatment for advanced non-small cell lung carcinoma (NSCLC). However, compared to monotherapy, such as monoclonal antibodies or kinase inhibitors, whether combination therapy can enhance antitumor efficacy or alleviate side effects remains unclear. A systematic literature search was undertaken using the PubMed, Embase, Web of Science and Cochrane Central Register of Controlled Trials databases to identify eligible studies concentrating on treatment with erlotinib or erlotinib plus monoclonal antibodies in NSCLC patients published between January 2017 and June 2022. The primary outcomes included progression-free survival (PFS), overall survival (OS), response rate (RR) and treatment-related adverse events (AEs). Seven independent randomized, controlled clinical trials including 1513 patients were obtained for the final analysis. Erlotinib plus monoclonal antibodies was significantly associated with the improvement of PFS (hazards ratio [HR], 0.60; 95% CI 0.53-0.69; z=7.59, P<0.01) and with moderate performance regarding OS (HR, 0.81; 95% CI 0.58-1.13; z=1.23, P=0.22) and RR (odds ratio [OR], 1.25; 95% CI 0.98-1.59; z=1.80, P=0.07), irrespective of EGFR mutation status. In the safety evaluation, erlotinib plus monoclonal antibodies had a markedly higher occurrence of adverse events (AEs) of Clavien grade 3 or higher (OR, 3.32; 95% CI 2.66-4.15; z=10.64, P<0.01). Compared with erlotinib alone, combination therapy (erlotinib plus monoclonal antibodies) was associated with significantly improved PFS in NSCLC therapy, accompanied by increased treatment-related AEs. Our systematic review protocol was registered in the PROSPERO international register of systematic reviews (CRD42022347667).

Extraordinary clinical response to ibrutinib in low-grade ovarian cancer guided by organoid drug testing

Low-grade serous ovarian cancer (LGSOC) typically responds poorly to standard platinum-based chemotherapy and new therapeutic approaches are needed. We describe a remarkable response to targeted therapy in a patient with platinum-resistant, advanced LGSOC who had failed standard-of-care chemotherapy and two surgeries. The patient was in rapid decline and entering hospice care on home intravenous (i.v.) opioid analgesics and a malignant bowel obstruction requiring a G-tube. Genomic analysis of the patient’s tumor did not indicate obvious therapeutic options. In contrast, a CLIA-certified drug sensitivity assay of an organoid culture derived from the patient’s tumor identified several therapeutic choices, including Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, as well as the EGFR inhibitors afatinib and erlotinib. Following off-label administration of daily ibrutinib as monotherapy, the patient had an exceptional clinical turnaround over the following 65 weeks with normalization of CA-125 levels, resolution of the malignant bowel obstruction, halting of pain medications, and improvement of performance status from ECOG 3 to ECOG 1. After 65 weeks of stable disease, the patient’s CA-125 levels began to rise, at which point the patient discontinued ibrutinib and began taking afatinib as monotherapy. The patient’s CA-125 levels remained stable for an additional 38 weeks but due to anemia and rising CA-125 levels, the patient switched to erlotinib and is currently being monitored. This case highlights the clinical utility of ex vivo drug testing of patient-derived tumor organoids as a new functional precision medicine approach to identify effective personalized therapies for patients who have failed standard-of-care treatments.

Targeting FGFRs Using PD173074 as a Novel Therapeutic Strategy in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an architecturally complex tumour with high heterogeneity. Discovery at later stages makes treatment challenging. However, the lack of early detection methodologies and the asymptomatic nature of CCA make early diagnosis more difficult. Recent studies revealed the fusions in Fibroblast Growth Factor Receptors (FGFRs), a sub-family of RTKs, as promising targets for targeted therapy for CCA. Particularly, FGFR2 fusions have been of particular interest, as translocations have been found in approximately 13% of CCA patients. Pursuing this, Pemigatinib, a small-molecule inhibitor of FGFR, became the first targeted therapy drug to be granted accelerated approval by the FDA for treating CCA patients harbouring FGFR2 fusions who have failed first-line chemotherapy. However, despite the availability of Pemigatinib, a very limited group of patients benefit from this treatment. Moreover, as the underlying mechanism of FGFR signalling is poorly elucidated in CCA, therapeutic inhibitors designed to inhibit this pathway are prone to primary and acquired resistance, as witnessed amongst other Tyrosine Kinase Inhibitors (TKIs). While acknowledging the limited cohort that benefits from FGFR inhibitors, and the poorly elucidated mechanism of the FGFR pathway, we sought to characterise the potential of FGFR inhibitors in CCA patients without FGFR2 fusions. Here we demonstrate aberrant FGFR expression in CCA samples using bioinformatics and further confirm phosphorylated-FGFR expression in paraffinised CCA tissues using immunohistochemistry. Our results highlight p-FGFR as a biomarker to guide FGFR-targeted therapies. Furthermore, CCA cell lines with FGFR expression were sensitive to a selective pan-FGFR inhibitor, PD173074, suggesting that this drug can be used to suppress CCA cells irrespective of the FGFR2 fusions. Finally, the correlation analysis utilising publicly available cohorts suggested the possibility of crosstalk amongst the FGFR and EGFR family of receptors as they are significantly co-expressed. Accordingly, dual inhibition of FGFRs and EGFR by PD173074 and EGFR inhibitor erlotinib was synergistic in CCA. Hence, the findings from this study provide support for further clinical investigation of PD173074, as well as other FGFR inhibitors, to benefit a larger cohort of patients. Altogether, this study shows for the first time the potential of FGFRs and the importance of dual inhibition as a novel therapeutic strategy in CCA.

Abstract 542: Synthetic lethal targeting of AURKA in head and neck squamous cell carcinoma

Abstract The purpose of this study was to identify new combination strategies for improving treatment of head and neck squamous cell carcinomas (HNSCCs). HNSCCs often express high levels of Aurora kinase A (AURKA), most studied as a kinase important for mitotic progression in normal cells. Notably the overexpression of AURKA in many cancers allows it to act throughout the cell cycle, acquiring new cancer-promoting activities. While several AURKA inhibitors are in clinical trials, limited efficacy is observed with single treatment, causing us to evaluate potential AURKA combination therapeutic combinations. Particularly relevant to HNSCC, which often overexpress and depend on EGFR, overexpression of AURKA can promote resistance to EGFR inhibitors by mechanisms variously identified as including activation of the EGFR effectors AKT, ERK, and BIM. Using viability and clonogenic assays, we have found that the AURKA inhibitor VIC-1911 synergizes with the EGFR inhibitors afatinib and erlotinib in multiple HNSCC cell lines and retains activity in HNSCC cell models selected for resistance to these EGFR inhibitors. However, AURKA inhibition increases levels of AURKA protein and its protein-stabilizing partner TPX2, counteracting drug effect. We also combined inhibition of AURKA with inhibition of WEE1, a regulator of the G2/M checkpoint, finding this dual inhibition enhanced levels of apoptosis in EGFR inhibitor resistant HNSCC cell lines. Intriguingly, one recent study reported AURKA can also modulate the glycolytic signaling pathway, increasing the cancer-promoting Warburg effect. We have found that inhibition of AURKA can significantly reduce glycolytic flux in HNSCC cell lines and alter expression and phosphorylation of key glycolytic enzymes. Dual inhibition of AURKA in combination with inhibitors of glycolytic enzymes, or enzymes regulating oxidative phosphorylation, showed marked synergy. These and other results investigating AURKA signaling mechanisms in HNSCC demonstrate that the non-canonical functions of AURKA include viable therapeutic targets that can enhance the efficacy of AURKA inhibition in HNSCC. Citation Format: Flaviane N da Silva, Theodore Nguyen, Jong Woo Lee, Barbara Burtness, Erica Golemis. Synthetic lethal targeting of AURKA in head and neck squamous cell carcinoma [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 542.

Abstract 2665: Biochemical inhibition profiles of 370 wild type human kinases provide a basis for selecting alternative combinations of EGFR and VEGFR inhibitors

Abstract Drug combinations targeting the vascular endothelial growth factor receptor (VEGFR) and the epidermal growth factor receptor (EGFR) have been used to treat EGFR-expressing cancers. In addition, we observed that the combination of the EGFR inhibitor erlotinib and the VEGFR2 inhibitor cediranib had greater activity in patient-derived xenografts (PDX) from rare cancers compared to the single agents alone. To better understand the combination activity and seek potential alternatives for each drug, their inhibition of 370 wild type human kinases was measured with a biochemical enzymatic assay. The single agents and combinations were tested at the IC50 value for their primary targets, 500 nM as a common reference concentration, and Cmax as achievable clinical concentrations. At the clinical Cmax, cediranib (148 nM) inhibited 5 kinases, including VEGFR2, by &amp;gt;90% and inhibited EGFR by 75%. Erlotinib (clinical Cmax 3.15 µM) inhibited 9 kinases, including EGFR, by ≥89% and inhibited VEGFR2 by 82%. When combined at their clinical concentrations (Cmax), cediranib + erlotinib inhibited 18 kinases by ≥89%. Kinase inhibition profiles were measured for 7 agents that were candidate alternatives for cediranib or erlotinib, including: vandetanib, erdafitinib, axitinib, lenvatinib, cabozantinib, poziotinib, and mobocertinib (note: Cmax values varied 58-fold among the drugs). At clinical Cmax, cediranib showed the greatest similarity to axitinib and lenvatinib based on their kinase inhibition profiles, whereas erlotinib showed the greatest similarity to poziotinib and vandetanib. The rank order of drug similarities based on biochemical kinase inhibition profiles aligned with the NCI-60 cell-based activities of the drugs according to a COMPARE Analysis. The Bliss independence model was used to calculate the combination activity of cediranib + erlotinib from single agent data for comparison with the experimental results of the drug combination. The high correlation between the calculated and experimental datasets indicated that the model could be used to assess other drug combinations from single agent data. Comparisons of the calculated kinase inhibition profiles of the novel drug combinations to the profile of cediranib + erlotinib indicated that erlotinib would be difficult to replace; however, cediranib might be substituted by either lenvatinib or axitinib. Similarities in the activities of erlotinib in combination with cediranib, lenvatinib and axitinib were observed in complex spheroids containing patient-derived cells as well as PDX models. These results suggest that the additive effect of VEGFR inhibition on erlotinib activity may not solely be due to erlotinib’s EGFR inhibitory activity, and therefore may not be seen with other EGFR inhibitors. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Nathan P. Coussens, Thomas S. Dexheimer, Thomas Silvers, Shannon Uzelac, Kyle Georgius, John Carter, Tia Shearer, Rekha Rao, Karen Gray, Apurva K. Srivastava, Robert J. Kinders, Yvonne A. Evrard, Melinda G. Hollingshead, Joel Morris, Jeffrey A. Moscow, Ralph E. Parchment, Beverly A. Teicher, James H. Doroshow. Biochemical inhibition profiles of 370 wild type human kinases provide a basis for selecting alternative combinations of EGFR and VEGFR inhibitors [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 2665.

Single-Cell Analysis Reveals Transcriptomic Features of Drug-Tolerant Persisters and Stromal Adaptation in a Patient-Derived EGFR-Mutated Lung Adenocarcinoma Xenograft Model.

Targeted therapies require life-long treatment, as drug discontinuation invariably leads to tumor recurrence. Recurrence is mainly driven by minor subpopulations of drug-tolerant persister (DTP) cells that survive the cytotoxic drug effect. In lung cancer, DTP studies have mainly been conducted with cell line models. We conducted an invivo DTP study using a lung adenocarcinoma patient-derived xenograft tumor driven by an EGFR mutation. Daily treatment of tumor-bearing mice for 5 to 6 weeks with the EGFR inhibitor erlotinib markedly shrunk tumors and generated DTPs, which were analyzed by whole exome, bulk population transcriptome, and single-cell RNA sequencing. The DTP tumors maintained the genomic clonal architecture of untreated baseline (BL) tumors but had reduced proliferation. Single-cell RNA sequencing identified a rare (approximately 4%) subpopulation of BL cells (DTP-like) with transcriptomic similarity to DTP cells and intermediate activity of pathways that are up-regulated in DTPs. Furthermore, the predominant transforming growth factor-β activated cancer-associated fibroblast (CAF) population in BL tumors was replaced by a CAF population enriched for IL6 production. Invitro experiments indicate that these populations interconvert depending on the levels of transforming growth factor-β versus NF-κB signaling, which is modulated by tyrosine kinase inhibitor presence. The DTPs had signs of increased NF-κB and STAT3 signaling, which may promote their survival. The DTPs may arise from a specific preexisting subpopulation of cancer cells with partial activation of specific drug resistance pathways. Tyrosine kinase inhibitor treatment induces DTPs revealing greater activation of these pathways while converting the major preexisting CAF population into a new state that may further promote DTP survival.

Decursin inhibits EGFR-ERK1/2 signaling axis in advanced human prostate carcinoma cells.

We have shown that decursin, a coumarin compound, induces cell cycle arrest and apoptosis in human prostate cancer cells (PCa); however, its molecular mechanisms are largely unexplored. We studied the mechanisms associated with its anticancer activity in advanced human prostate carcinoma cells. We found that decursin inhibited epidermal growth factor receptor (EGFR) signaling by inhibiting its activating phosphorylation at tyrosine 1068 residue in DU145 and 22Rv1 cells. This inhibition of EGFR was associated with the downregulation of ERK1/2 phosphorylation. Both EGFR and ERK1/2 are known to be deregulated/activated in many human malignancies. Consistent with our earlier study, decursin (25-100 µM) treatment for 24-72 h inhibited DU145 cell proliferation by 49%-87% (p < 0.001) which was associated with strong G1 phase arrest and cell death. It also decreased (p < 0.001) the number of surviving colonies. Decursin moderately increased the expression of Rb-related proteins p107 and p130 but decreased the levels of E2F family transcription factors including E2F-3, E2F-4 and E2F-5. Further, decursin strongly inhibited the growth of androgen-dependent prostate carcinoma 22Rv1 cells from 61% to 79% (p < 0.001) and arrested these cells at G1 phase via induction of cyclin-dependent kinase inhibitor p27/Kip1 and downregulation of CDK2 and CDK4 protein expression. Additionally, EGFR inhibitor erlotinib- and EGF ligand-modulated EGFR activation validated EGFR signaling as a target of decursin-mediated cell growth inhibition and cytotoxicity. Decursin decreased EGF ligand-induced phosphorylation of EGFR (Y-1068) as well as activation of its downstream mediator, ERK1/2. Furthermore, inhibitory targeting of EGFR-ERK1/2 axis by combinatorial treatment of decursin and erlotinib further sensitized DU145 cells for the decursin-induced growth inhibition and cell death. Overall, these findings strongly suggest that anticancer efficacy of decursin against human PCa involves inhibitory targeting of EGFR-ERK1/2 signaling axis, a pathway constitutively active in advanced PCa.

Transcriptomic changes underlying EGFR inhibitor resistance in human and mouse models of basal-like breast cancer.

The goals of this study were to identify transcriptomic changes that arise in basal-like breast cancer cells during the development of resistance to epidermal growth factor receptor inhibitors (EGFRi) and to identify drugs that are cytotoxic once EGFRi resistance occurs. Human patient-derived xenografts (PDXs) were grown in immunodeficient mice and treated with a set of EGFRi; the EGFRi erlotinib was selected for more expansive in vivo studies. Single-cell RNA sequencing was performed on mammary tumors from the basal-like PDX WHIM2 that was treated with vehicle or erlotinib for 9weeks. The PDX was then subjected to long-term erlotinib treatment in vivo. Through serial passaging, an erlotinib-resistant subline of WHIM2 was generated. Bulk RNA-sequencing was performed on parental and erlotinib-resistant tumors. In vitro high-throughput drug screening with > 500 clinically used compounds was performed on parental and erlotinib-resistant cells. Previously published bulk gene expression microarray data from MMTV-Wnt1 tumors were contrasted with the WHIM2 PDX data. Erlotinib effectively inhibited WHIM2 tumor growth for approximately 4weeks. Compared to untreated cells, single-cell RNA sequencing revealed that a greater proportion of erlotinib-treated cells were in the G1 phase of the cell cycle. Comparison of WHIM2 and MMTV-Wnt1 gene expression data revealed a set of 38 overlapping genes that were differentially expressed in the erlotinib-resistant WHIM2 and MMTV-Wnt1 tumors. Comparison of all three data types revealed five genes that were upregulated across all erlotinib-resistant samples: IL19, KLK7, LCN2, SAA1, and SAA2. Of these five genes, LCN2 was most abundantly expressed in triple-negative breast cancers, and its knockdown restored erlotinib sensitivity in vitro. Despite transcriptomic differences, parental and erlotinib-resistant WHIM2 displayed similar responses to the majority of drugs assessed for cytotoxicity in vitro. This study identified transcriptomic changes arising in erlotinib-resistant basal-like breast cancer. These data could be used to identify a biomarker or develop a gene signature predictive of patient response to EGFRi. Future studies should explore the predictive capacity of these gene signatures as well as how LCN2 contributes to the development of EGFRi resistance.

LP2, a cyclic angiotensin-(1−7) analog extended with an N-terminal D-lysine, impairs growth of patient-derived xenografts of colorectal carcinoma in mice

LP2 is a 4, 7 D, L lanthionine-stabilized analog of angiotensin-(1−7), with an N-terminal D-lysine, resistant to breakdown by peptidases. It is a specific agonist of the angiotensin II type 2 receptor. Consistent with its high specificity and stability, LP2 has shown excellent safety and pharmacokinetics in a first-in-human clinical phase Ia trial. Here, based on strong rationales, we studied the capacity of LP2 to inhibit the growth of patient-derived xenografts of colorectal cancer in mice. Prior to efficacy studies, immunohistochemistry on an untreated tissue array demonstrated that the AT2R expression is reduced in human colorectal cancer and in stroma when compared to tumor adjacent tissue. Subsequent studies demonstrated that LP2 at a subcutaneously injected dose as low as 0.2 µg/kg/day inhibited patient-derived xenografts of colorectal carcinoma in mice. Kinome analyses and validation of elected kinase inhibition indicated that LP2-mediated AT2R stimulation inhibited PI3K/AKT/mTOR which resulted in apoptosis via CDKs. LP2 acted synergistically with 5-FU and the EGFR inhibitor erlotinib. Taken together, the extremely low dose of LP2 at which antitumor activity is exerted, the synergism with selected drugs and, together with its excellent specificity, safety and stability, warrant further evaluation of LP2′s inhibitory potential of colorectal cancer.

Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation invitro and invivo.

Hypoxia activated HGF expression in pancreatic stellate cells confers resistance of pancreatic cancer cells to EGFR inhibition.

Epidermal growth factor receptor (EGFR) is an essential target for cancer treatment. However, EGFR inhibitor erlotinib showed limited clinical benefit in pancreatic cancer therapy. Here, we showed the underlying mechanism of tumor microenvironment suppressing the sensitivity of EGFR inhibitor through the pancreatic stellate cell (PSC). The expression of alpha-smooth muscle actin (α-SMA) and hypoxia marker in human pancreatic cancer tissues were detected by immunohistochemistry, and their correlation with overall survival was evaluated. Human immortalized PSC was constructed and used to investigate the potential effect on pancreatic cancer cell lines in hypoxia and normoxia. Luciferase reporter assay and Chromatin immunoprecipitation were performed to explore the potential mechanisms invitro. The combined inhibition of EGFR and Met was evaluated in an orthotopic xenograft mouse model of pancreatic cancer. We found that high expression levels of α-SMA and hypoxia markers are associated with poor prognosis of pancreatic cancer patients. Mechanistically, we demonstrated that hypoxia induced the expression and secretion of HGF in PSC via transcription factor HIF-1α. PSC-derived HGF activates Met, the HGF receptor, suppressing the sensitivity of pancreatic cancer cells to EGFR inhibitor in a KRAS-independent manner by activating the PI3K-AKT pathway. Furthermore, we found that the combination of EGFR inhibitor and Met inhibitor significantly suppressed tumor growth in an orthotopic xenograft mouse model. Our study revealed a previously uncharacterized HIF1α-HGF-Met-PI3K-AKT signaling axis between PSC and cancer cells and indicated that EGFR inhibition plus Met inhibition might be a promising strategy for pancreatic cancer treatment. This study was supported by The National Natural Science Foundation of China.

TNBC Therapeutics Based on Combination of Fusarochromanone with EGFR Inhibitors.

Fusarochromanone is an experimental drug with unique and potent anti-cancer activity. Current cancer therapies often incorporate a combination of drugs to increase efficacy and decrease the development of drug resistance. In this study, we used drug combinations and cellular phenotypic screens to address important questions about FC101's mode of action and its potential therapeutic synergies in triple negative breast cancer (TNBC). We hypothesized that FC101's activity against TNBC is similar to the mTOR inhibitor, everolimus, because FC101 downregulates the phosphorylation of two mTOR substrates, S6K and S6. Since everolimus synergistically enhances the anti-cancer activities of two known EGFR inhibitors (erlotinib or lapatinib) in TNBC, we performed analogous studies with FC101. Phenotypic cellular assays helped assess whether FC101 acts similarly to everolimus, in both single and combination treatments with the two inhibitors. FC101 outperformed all other single treatments in both cell proliferation and viability assays. However, unlike everolimus, FC101 produced a sustained decrease in cell viability in drug washout studies. None of the other drugs were able to maintain comparable effects upon removal. Although we observed slightly additive effects when the TNBC cells were treated with FC101 and the two EGFR inhibitors, those effects were not truly synergistic in the manner displayed with everolimus.