Epidermal Growth Factor Receptor Kinase Research Articles

ABCG2 Mediates Resistance to the Dual EGFR and PI3K Inhibitor MTX-211 in Cancer Cells

MTX-211 is a first-in-class dual inhibitor of epidermal growth factor receptor (EGFR) and phosphoinositide-3 kinase (PI3K) signaling pathways with a compelling pharmaceutical profile and could enhance the effectiveness of mitogen-activated protein kinase kinase (MEK) inhibitor therapy in colorectal tumors with KRAS mutations. However, the specific mechanisms contributing to the acquired resistance to MTX-211 in human cancers remain elusive. Here, we discovered that the overexpression of the ATP-binding cassette (ABC) drug transporter ABCG2, a prevalent mechanism associated with multidrug resistance (MDR), could diminish the effectiveness of MTX-211 in human cancer cells. We showed that the drug efflux activity of ABCG2 substantially decreased the intracellular accumulation of MTX-211 in cancer cells. As a result, the cytotoxicity and effectiveness of MTX-211 in suppressing the activation of the EGFR and PI3K pathways were significantly attenuated in cancer cells overexpressing ABCG2. Moreover, the enhancement of the MTX-211-stimulated ATPase activity of ABCG2 and the computational molecular docking analysis illustrating the binding of MTX-211 to the substrate-binding sites of ABCG2 offered a further indication for the interaction between MTX-211 and ABCG2. In summary, our findings indicate that MTX-211 acts as a substrate for ABCG2, underscoring the involvement of ABCG2 in the emergence of resistance to MTX-211. This finding carries clinical implications and merits further exploration.

Structural Analysis of the Macrocyclic Inhibitor BI-4020 Binding to EGFR Kinase.

A novel macrocyclic inhibitor of mutant EGFR (BI-4020) has shown promise in pre-clinical studies of T790M and C797S drug-resistant non-small cell lung cancer. To better understand the molecular basis for BI-4020 selectivity and potency, we have carried out biochemical activity assays and structural analysis with X-ray crystallography. Biochemical potencies agree with previous studies indicating that BI-4020 is uniquely potent against drug-resistant L858R/T790M and L858R/T790M/C797S variants. X-ray structures with wild-type (2.4 Å) and T790M/V948R (3.1 Å) EGFR kinase domains show that BI-4020 is likely rendered selective due to interactions with the kinase domain hinge region as well as T790M, akin to Osimertinib. Additionally, BI-4020 is also rendered more potent due to its constrained macrocycle geometry as well as additional H-bonds to conserved K745 and T845 residues in both active and inactive conformations. These findings taken together show how this novel macrocyclic inhibitor is both highly potent and selective for mutant EGFR in a reversible mechanism and motivate structure-inspired approaches to developing targeted therapies in medicinal oncology.

17-β-estradiol and phytoestrogens elicit NO production and vasodilatation through PI3K, PKA and EGF receptors pathways, evidencing functional selectivity

Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-β-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2’,7’-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.

Marine-derived EGFR inhibitors: novel compounds targeting breast cancer growth and drug resistance.

Breast cancer (BC) continues to be a major health challenge globally, ranking as the fifth leading cause of cancer mortality among women, despite advancements in cancer detection and treatment. In this study, we identified four novel compounds from marine organisms that effectively target and inhibit the Epidermal Growth Factor Receptor (EGFR), crucial for BC cell growth and proliferation. These compounds not only induced early apoptosis through Caspase-3 activation but also showed significant inhibitory effects on EGFR mutations associated with drug resistance (T790M, L858R, and L858R/T790M), demonstrating high EGFR kinase selectivity. Cell Thermal Shift Assay (CETSA) experiments indicated that Tandyukisin stabilizes EGFR in a concentration-dependent manner. Furthermore, binding competition assays using surface plasmon resonance technology revealed that Tandyukisin and Trichoharzin bound to distinct sites on EGFR and that their combined use enhanced apoptosis in BC cells. This discovery may pave the way for developing new marine-derived EGFR inhibitors, offering a promising avenue for innovative cancer treatment strategies and addressing EGFR-mediated drug resistance.

BDTX-189, a novel tyrosine kinase inhibitor, inhibits cell activity via ERK and AKT pathways in the EGFR C797S triple mutant cells

The tertiary mutation C797S in the structural domain of the EGFR kinase is a common cause of resistance to third-generation EGFR tyrosine kinase inhibitors (TKIs). In this study, we used a potent, selective and irreversible inhibitor, BDTX-189, to target EGFR C797S triple mutant cells for cell activity. The study constructed the H1975–C797S (EGFR L858R/T790 M/C797S) cell line using the CRISPR/Cas9 method and investigated its potential as a fourth-generation tyrosine kinase inhibitor via chemosensitivity approach. The results demonstrated its ability to induce cytotoxic effects, and inhibit EGFR L858R/T790 M/C797S cell growth and proliferation in a dose-dependent manner. Meanwhile, BDTX-189 reduces the protein phosphorylation levels of EGFR, ERK, and AKT, promoting apoptosis. Furthermore, BDTX-189 not only inhibits common EGFR triple mutations but also effectively inhibits EGFR L858R mutation and EGFR L858R/T790 M mutation. These findings support the cytotoxic effect of BDTX-189 and its inhibitory effect on cell division and proliferation with the EGFR C797S triple mutation.

Novel guanidine derivatives targeting leukemia as selective Src/Abl dual inhibitors: Design, synthesis and anti-proliferative activity

A new series of benzene-sulfonamide derivatives 3a-i was designed and synthesized via the reaction of N-(pyrimidin-2-yl)cyanamides 1a-i with sulfamethazine sodium salt 2 as dual Src/Abl inhibitors. Spectral data IR, 1H-, 13C- NMR and elemental analyses were used to confirm the structures of all the newly synthesized compounds 3a-i and 4a-i. Crucially, we screened all the synthesized compounds 3a-i against NCI 60 cancer cell lines. Among all, compound 3b was the most potent, with IC50 of 0.018 μM for normoxia, and 0.001 μM for hypoxia, compared to staurosporine against HL-60 leukemia cell line. To verify the selectivity of this derivative, it was assessed against a panel of tyrosine kinase EGFR, VEGFR-2, B-raf, ERK, CK1, p38-MAPK, Src and Abl enzymes. Results revealed that compound 3b can effectively and selectively inhibit Src/Abl with IC500.25 μM and Abl inhibitory activity with IC500.08 μM, respectively, and was found to be more potent on these enzymes than other kinases that showed the following results: EGFR IC500.31 μM, VEGFR-2 IC500.68 μM, B-raf IC500.33 μM, ERK IC501.41 μM, CK1 IC500.29 μM and p38-MAPK IC500.38 μM. Moreover, cell cycle analysis and apoptosis performed to compound 3b against HL-60 suggesting its antiproliferative activity through Src/Abl inhibition. Finally, molecular docking studies and physicochemical properties prediction for compounds 3b, 3c, and 3 h were carried out to investigate their biological activities and clarify their bioavailability.

Computational Analysis of Activation of Dimerized Epidermal Growth Factor Receptor Kinase Using the String Method and Markov State Model.

Epidermal growth factor receptor (EGFR) activation is accompanied by dimerization. During the activation of the intracellular kinase domain, two EGFR kinases form an asymmetric dimer, and one side of the dimer (receiver) is activated. Using the string method and Markov state model (MSM), we performed a computational analysis of the structural changes in the activation of the EGFR dimer in this study. The string method reveals the minimum free-energy pathway (MFEP) from the inactive to active structure. The MSM was constructed from numerous trajectories of molecular dynamics simulations around the MFEP, which revealed the free-energy map of structural changes. In the activation of the receiver kinase, the unfolding of the activation loop (A-loop) is followed by the rearrangement of the C-helix, as observed in other kinases. However, unlike other kinases, the free-energy map of EGFR at the asymmetric dimer showed that the active state yielded the highest stability and revealed how interactions at the dimer interface induced receiver activation. As the H-helix of the activator approaches the C-helix of the receiver during activation, the A-loop unfolds. Subsequently, L782 of the receiver enters the pocket between the G- and H-helices of the activator, leading to a rearrangement of the hydrophobic residues around L782 of the receiver, which constitutes a structural rearrangement of the C-helix of the receiver from an outward to an inner position. The MSM analysis revealed long-time scale trajectories via kinetic Monte Carlo.

Bioinformatics-driven discovery of novel EGFR kinase inhibitors as anti-cancer therapeutics: In silico screening and in vitro evaluation.

Epidermal growth factor receptor EGFR inhibitors are widely used as first line therapy for the treatment of non-small-cell lung cancer (NSCLC) in patients harboring EGFR mutation. However, the acquisition of a second-site mutation (T790 M) limited the efficacy and developed resistance. Therefore, discovery and development of specific drug target for this mutation is of urgent needs. In our study we used the ChemDiv diversity database for receptor-based virtual screening to secure EGFR-TK inhibitors chemotherapeutics. We identified four compounds that bind to the ATP-binding region of the EGFR-TK using AutoDock 4.0 and AutoDock Vina1.1.2 and post-docking investigations. The ligand showed hydrophobic interactions to the hydrophobic region of the binding site and engaged in hydrogen bonding with Met793. The ligands also explored π-cation interactions between the π-system of the ligand-phenyl ring and the positive amino group of Lys745. Molecular mechanics Poisson-Boltzmann surface area MM/PBSA per-residue energy decomposition analyses revealed that Val726, Leu792, Met793, Gly796, Cys797, Leu798, and Thr844 contributed the most to the binding energy. Biological evaluation of the retrieved hit compounds showed suppressing activity against EGFR auto phosphorylation and selective apoptosis-induced effects toward lung cancer cells harboring the EGFR L858R/T790M double mutation. Our work anticipated into novel and specific EGFR-TKIs and identified new compounds with therapeutic potential against lung cancer.

PDZK1 suppresses TNBC development and sensitizes TNBC cells to erlotinib via the EGFR pathway

Epidermal growth factor receptor (EGFR)-targeted drugs (erlotinib, etc.) are used to treat multiple types of tumours. EGFR is highly expressed in most triple-negative breast cancer (TNBC) patients. However, only a small proportion of TNBC patients benefit from EGFR-targeted drugs in clinical trials, and the resistance mechanism is unclear. Here, we found that PDZ domain containing 1 (PDZK1) is downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation is related to erlotinib resistance in TNBC. PDZK1 binds to EGFR. Through this interaction, PDZK1 promotes EGFR degradation by enhancing the binding of EGFR to c-Cbl and inhibits EGFR phosphorylation by hindering EGFR dimerisation. We also found that PDZK1 is specifically downregulated in TNBC tissues and correlated with a poor prognosis in TNBC patients. In vitro and in vivo functional assays showed that PDZK1 suppressed TNBC development. Restoration of EGFR expression or kinase inhibitor treatment reversed the degree of cell malignancy induced by PDZK1 overexpression or knockdown, respectively. PDZK1 overexpression sensitised TNBC cells to erlotinib both in vitro and in vivo. In conclusion, PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for reversing erlotinib resistance in TNBC cells.

Design, synthesis, biological assessment and molecular modeling studies of novel imidazothiazole-thiazolidinone hybrids as potential anticancer and anti-inflammatory agents

A new series of imidazothiazole derivatives bearing thiazolidinone moiety (4a-g and 5a-d) were designed, synthesized and evaluated for potential epidermal growth factor receptor (EGFR) kinase inhibition, anticancer and anti-inflammatory activity, cardiomyopathy toxicity and hepatotoxicity. Compound 4c inhibited EGFR kinase at a concentration of 18.35 ± 1.25 µM, whereas standard drug erlotinib showed IC50 value of 06.12 ± 0.92 µM. The molecular docking, dynamics simulation and MM-GBSA binding energy calculations revealed strong interaction of compound 4c with binding site of EGFR. The synthesized compounds were evaluated for their anticancer activity by MTT assay against three human cancer cell lines A549 (Lung), MCF-7 (Breast), HCT116 (Colon), one normal human embryonic kidney cell line HEK293 and also for their EGFR kinase inhibitory activity. Few compounds of the series (4a, 4b, 4c) showed promising growth inhibition against all the tested cancer cell lines and against EGFR kinase. Among these, compound 4c was found to be most active and displayed IC50 value of 10.74 ± 0.40, 18.73 ± 0.88 against cancer cell lines A549 and MCF7 respectively whereas it showed an IC50 value of 96.38 ± 1.79 against HEK293 cell line indicating lesser cytotoxicity for healthy cell. Compounds 4a, 4b and 4c were also examined for their apoptosis inducing potential through AO/EB dual staining assay and it was observed that their antiproliferative activity against A549 cells is mediated via induction of apoptosis. Cardiomyopathy studies showed normal cardiomyocytes with no marked sign of pyknotic nucleus of compounds 4b and 4c. Hepatotoxicity studies of compounds 4b and 4c also showed normal architecture of hepatocytes. Compounds 4a-g and 5a-d were also evaluated for their in-vitro anti-inflammatory activity by protein albumin denaturation assay. Among the tested compounds 4a-d and 5a-b showed promising activity and were selected for in-vivo inflammatory activity against carrageenan rat paw edema test. Among these compounds, 4b was found to be most active in the series showing 84.94% inhibition, whereas the standard drug diclofenac sodium showed 84.57% inhibition. Compound 4b also showed low ulcerogenic potential and lipid peroxidation. Thus, compounds 4c and 4b could be a promising lead compounds for developing anticancer and anti-inflammatory agents with low toxicity and selectivity.

Novel 2-[thio]acetamide linked quinazoline/1,2,4-triazole/chalcone hybrids: Design, synthesis, and anticancer activity as EGFR inhibitors and apoptotic inducers.

Novel triazoloquinazolines carrying the 2-[thio]acetamide entity (4 and 5a-d) and triazoloquinazoline/chalcone hybrids incorporating the 2-[thio]acetamide linker (8a-b and 9a-f) were developed as anticancer candidates. NCI screening of the synthesized compounds at 10 μM concentration displayed growth inhibition not only up to 99.74% as observed for 9a but also a lethal effect could be achieved as stated for compounds 9c (RPMI-8226 and HCT-116) and 8b, 9a, and 9e on the HCT-116 cell line. The antiproliferative activity was determined for the chalcone series on three cell lines: RPMI-8226, HCT-116, and MCF-7. Compounds 8b, 9a, 9b, and 9f were the most active ones. To understand the mechanistic study, the inhibitory effect on the epidermal growth factor receptor (EGFR) kinase was evaluated. The results stated that the activity of compound 8b (IC50 = 0.07 μM) was near that of the reference drug erlotinib (IC50 = 0.052 μM) whereas compound 9b (IC50 = 0.045 μM) was found to be more potent than erlotinib. Both compounds 8b and 9b were selected for cell cycle analysis and apoptotic assays. Moreover, molecular docking results of the selected chalcone hybrids showed high binding scores and good binding affinities especially for 8b and 9b, which were consistent with the biological activity (EGFR).

Structural determinants for membrane binding of the EGFR juxtamembrane domain.

Overactivation of the epidermal growth factor receptor (EGFR) is critical for the development of multiple cancers. Previous studies have shown that the cell membrane is a key regulator of EGFR kinase activity through its interaction with the EGFR juxtamembrane domain (JM). However, the lipid recognition specificity of EGFR-JM and its interaction details remain unclear. Using lipid strip and liposome pulldown assays, we showed that EGFR-JM could specifically interact with PI(4,5)P2-or phosphatidylserine-containing membranes. We further characterized the JM-membrane interaction using NMR-titration-based chemical shift perturbation and paramagnetic relaxation enhancement analyses, and found that residues I649 - L659 comprised the membrane-binding site. Furthermore, the membrane-binding region contains the predicted dimerization motif of JM, 655LRRLL659, suggesting that membrane binding may affect JM dimerization and, therefore, regulate kinase activation.

DRD1 suppresses cell proliferation and reduces EGFR activation and PD-L1 expression in NSCLC.

Dopamine (DA) acts in various key neurological and physiological processes as both a neurotransmitter and circulating hormone. Over the past several decades, the DA signaling network has been shown to regulate the progression of several types of solid tumors, and considerable evidence has shown it is a druggable pathway in the cancer cell context. However, the specific activity and effect of these pathway components appears to be tissue-type and cell-context-dependent. In the present study, expression and methylation of dopamine receptor D1 (DRD1) were measured using RNA sequencing (RNAseq) and reverse transcription polymerase chain reaction (RT-PCR) in non-small cell lung cancer (NSCLC) samples, and validated using publicly available datasets, including The Cancer Genome Atlas (TCGA). In vitro and in vivo functional experiments were performed for cell proliferation and tumor growth, respectively. Mechanistic analyses of the transcriptome and kinome in DRD1-modulated cells informed further experiments, which characterized the effects on the epidermal growth factor receptor (EGFR) pathway and programmed cell death 1 ligand 1 (PD-L1) proteins. Through these experiments, we identified the DRD1 gene as a negative regulator of disease progression in NSCLC. We show that DRD1, as well as other DA pathway components, are expressed in normal human lung tissue, and that loss of DRD1 expression through promoter hypermethylation is a common feature in NSCLC patients and is associated with worse survival. At the cellular level, DRD1 affects proliferation by inhibiting the activation of EGFR and mitogen-activated protein kinase 1/2 (ERK1/2). Interestingly, we also found that DRD1 regulates the expression of PD-L1 in lung cancer cells. Taken together, these results suggest that DRD1 methylation may constitute a biomarker of poor prognosis in NSCLC patients while other components of this pathway could be targeted to improve response to EGFR- and PD-L1-targeted therapies.

EGFR Inhibition by Erlotinib Rescues Desmosome Ultrastructure and Keratin Anchorage and Protects against Pemphigus Vulgaris IgG–Induced Acantholysis in Human Epidermis

Pemphigus is a severe blistering disease caused by autoantibodies primarily against the desmosomal cadherins desmoglein (DSG)1 and DSG3 which impair desmosome integrity. Especially for the acute phase, additional treatment options allowing to reduce corticosteroids would fulfill an unmet medical need. Here, we provide evidence that epidermal growth factor receptor (EGFR) inhibition by erlotinib ameliorates pemphigus vulgaris immunoglobulin G (PV-IgG) -induced acantholysis in intact human epidermis. PV-IgG caused phosphorylation of EGFR (Y845) and SRC in human epidermis. In line with that, a phosphotyrosine kinome analysis revealed a robust response associated with EGFR and SRC family kinase signaling in response to PV-IgG but not pemphigus foliaceus autoantibodies. Erlotinib inhibited PV-IgG-induced epidermal blistering and EGFR phosphorylation, loss of desmosomes as well as ultrastructural alterations of desmosome size, plaque symmetry, keratin filament insertion and restored the desmosome midline considered as hallmark of mature desmosomes. Erlotinib enhanced both single molecule DSG3 binding frequency and strength and delayed DSG3 fluorescence recovery supporting that EGFR inhibition increases DSG3 availability and cytoskeletal anchorage. Our data indicate that EGFR is a promising target for pemphigus therapy due to its link to several signaling pathways known to be involved in pemphigus pathogenesis.

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.

KRASG12C Inhibitor as a Treatment Option for Non-Small-Cell Lung Cancer with Comorbid Interstitial Pneumonia.

Non-small-cell lung cancer (NSCLC) with comorbid interstitial pneumonia (IP) is a population with limited treatment options and a poor prognosis. Patients with comorbid IP are at high risk of developing fatal drug-induced pneumonitis, and data on the safety and efficacy of molecularly targeted therapies are lacking. KRAS mutations have been frequently detected in patients with NSCLC with comorbid IP. However, the low detection rate of common driver gene mutations, such as epidermal growth factor receptor and anaplastic lymphoma kinase, in patients with comorbid IP frequently results in inadequate screening for driver mutations, and KRAS mutations may be overlooked. Recently, sotorasib and adagrasib were approved as treatment options for advanced NSCLC with KRASG12C mutations. Although patients with comorbid IP were not excluded from clinical trials of these KRASG12C inhibitors, the incidence of drug-induced pneumonitis was low. Therefore, KRASG12C inhibitors may be a safe and effective treatment option for NSCLC with comorbid IP. This review article discusses the promise and prospects of molecular-targeted therapies, especially KRASG12C inhibitors, for NSCLC with comorbid IP, along with our own clinical experience.

Abstract 4474: Novel analogs of 4-substituted pyrrolo[2,3-d]pyrimidines as dual-targeted mEGFR/AURKA inhibitors for lung cancer

Abstract Small molecule, mutant epidermal growth factor receptor kinase (mEGFR) inhibitors such as osimertinib and erlotinib constitute the recommended therapy for mEGFR-positive non-small cell lung cancer (NSCLC). However, mEGFR inhibitors are ineffective in NSCLC over time due to mutations within mEGFR and redundant signaling pathways such as mutant KRAS (mKRAS) that bypass mEGFR inhibition. Simultaneous inhibition of multiple kinases has been suggested to provide synergistic effects on tumor growth inhibition and tumor resistance. Studies have shown that resistance to mEGFR inhibitors was overcome when mEGFR inhibitors were combined with aurora kinase (AURK) inhibitors. Additionally, mEGFR inhibitors were found to retain their anticancer effects in mKRAS-positive NSCLC cells if given concurrently with AURK inhibitors. Kurup et al. previously reported a series of 4-substituted pyrrolo[2,3-d]pyrimidines as dual mEGFR/AURKB inhibitors. Novel analogs of 1-5 have been designed to incorporate AURKA inhibition while maintaining mEGFR inhibition. Molecular modeling was utilized to guide selectivity for AURKA over AURKB. This study led to the identification of novel dual-targeted mEGFR/AURKA inhibitors with demonstrated inhibition of mKRAS+ NSCLC and mEGFR+ NSCLC. The design, synthesis, kinase inhibitory activities, and anticancer effects for these novel analogs will be presented. Structure-activity relationships for dual mEGFR/AURKA inhibition will be defined. Citation Format: Sonali Kurup, Gabe Carpenter, Samantha Satawa, Felix Amissah, Erika Lisabeth, Matthew Giletto, Edmund Ellsworth. Novel analogs of 4-substituted pyrrolo[2,3-d]pyrimidines as dual-targeted mEGFR/AURKA inhibitors for 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 4474.

Abstract 1866: Discovery of mutation-independent EGFR degrading bispecific antibodies that suppress tumor growth in preclinical tumor models

Abstract Extracellular targeted protein degradation (eTPD) has emerged as a promising new drug modality focused on targeted elimination of extracellular and transmembrane proteins. In contrast to intracellular protein degraders, such as proteolysis targeting chimeras (PROTACs) and molecular glues which require ubiquitin-proteosome cellular degradation pathways, extracellular protein degraders can additionally harness endosomal-lysosomal protein degradation. Two recently published examples of extracellular protein degraders include AbTACs (antibody-based PROTAC) which co-engage a protein of interest (POI) and transmembrane E3 ligases, and KineTACs (cytokine receptor-targeting chimeras) which utilize endogenous cytokine receptors to degrade extracellular POIs. These bispecific antibody degrader platforms not only have advantageous pharmacological and drug-like manufacturing properties, but can also be engineered for tissue-specificity and to address multiple complementary targets, with the goal of increased efficacy and decreased toxicity. To that end, we have greatly expanded the extracellular degrader repertoire beyond AbTACs and KineTACs, with a novel bispecific antibody degrader platform called TrainTACs (tissue receptor antigen internalization targeting chimeras). To demonstrate the potential of this novel extracellular degrading platform, we developed degraders for the canonical receptor tyrosine kinase epidermal growth factor receptor (EGFR). EGFR is an oncogenic driver, that has been clinically validated in lung, colorectal and head and neck cancers, but patient benefit has been limited by treatment-related acquired resistance mutations and on-target/off-tumor dose-limiting toxicities. The potential of our TrainTACs, AbTACs and KineTACs to overcome the limitations of current therapeutic modalities was assessed. Gene expression profiles from tumor and normal tissues were evaluated to identify potential degraders co-expressed with EGFR in tumors. More than 70 unique bispecific antibody constructs spanning 20 receptors were generated and screened in tumor cell-based assays to evaluate EGFR antagonism, internalization, and degradation. TrainTACs, AbTACs and KineTACs degraded EGFR in multiple tumor cell lines covering a range of EGFR mutations. Degradation of EGFR led to deep inhibition of EGFR signaling, robust inhibitory effects on tumor spheroids, and in xenograft mouse tumor models. In conclusion, eTPD represents a promising new drug modality, and TrainTACs, AbTACs and KineTACs have expanded the toolbox of extracellular targeted protein degraders that can be utilized in a target-, tissue- and disease-specific manner. Citation Format: Jonathan Sitrin, Lisa Marshall, Hai Tran, Kenneth Ng, Kimberly Hoi, Josef Gramespacher, Zhong Huang, Andy Goodrich, Filomena Housley, May Dayao, Man-Tzu Wang, Katarina Pance, Aleysha Chen, Kevin Carlin, Lichao Zhang, James Lee, Rami Hannoush, Ken Flanagan, Maia Vinogradova, Isaac Rondon, Shyra Gardai. Discovery of mutation-independent EGFR degrading bispecific antibodies that suppress tumor growth in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1866.

Abstract 549: PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation

Abstract Background: Triple-negative breast cancer (TNBC) is lack of effective targeted therapies. Epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. However, TNBC is resistant to EGFR-targeted drugs in clinical trials, and the tolerance mechanism remains unclear. This study aimed to elucidate the possible resistance mechanism for TNBC to EGFR-targeted therapy and how sensitize TNBC cells to erlotinib. Methods: PDZ domain containing 1 (PDZK1) correlated with TNBC development was screened out by bioinformatics analysis and its correlation with resistance to erlotinib was identified by western blotting (WB). Its interaction with EGFR and structural basis were determined by GST-Pull down and coimmunoprecipitation. Its regulation on EGFR signaling activation and expression level were detected by WB in PDZK1 overexpression and knockdown TNBC cells, xenograft tissues and correlation analyses. Correlation was performed by gene set enrichment analysis (GSEA) and immunohistochemistry of tissue microarray followed by Pearson analysis. TNBC-suppressing effects of PDZK1 and its sensitizing effects on erlotinib were investigated using cell viability assay, colony-forming assay, wound healing assay, Matrigel invasion assay and xenograft model in combination with PDZK1 wild type/mutant transfection and rescue experiment. Results: Here we found that PDZK1 was correlated with TNBC development and its level was downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation was related to erlotinib resistance in TNBC. PDZK1 bound with EGFR. Through the interaction, PDZK1 promoted EGFR degradation by enhancing the binding of EGFR with c-Cbl and inhibited EGFR phosphorylation by hindering EGFR dimerization. PDZK1 was specifically downregulated in TNBC tissues and was correlated with poor prognosis of TNBC. Functional assays in vitro and in vivo showed that PDZK1 suppressed TNBC development. Restoring EGFR expression and kinase inhibitor treatment reversed the malignancy caused by PDZK1 overexpression and knockdown, respectively. PDZK1 wild type, but not mutant overexpression enhanced the inhibitory effect of erlotinib on EGFR signaling and TNBC malignancy in vitro and in vivo. Conclusion: PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for TNBC to reverse the tolerance of TNBC cells to erlotinib. Citation Format: Junfang Zheng, Yuanzhen Ma, Zhiyu Fang, Yijun Qi. PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation [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 549.

Abstract 6266: Neurotensin receptor 1 regulates dimerization of HER2-HER4 and proliferation of non-small cell lung cancer cells

Abstract The neurotensin receptor 1 (NTSR1) is a 418 amino acid type A G protein-coupled receptor which causes phosphatidylinositol turnover and proliferation of non-small cell lung cancer (NSCLC) cells (Moody et al., Peptides 2021; 137: 710480). Adding the agonist NTS to NSCLC cells increases transactivation of the receptor tyrosine kinases (RTK) EGFR, HER2 and HER3 (Moody et al., Biology 2023 12: 957). The RTK tyrosine phosphorylation and growth caused by NTS is impaired by SR48692, a small molecule NTSR1 antagonist. The effects of NTS were investigated on HER4 using NSCLC cells. By Western blot, cell lines NCI-H522 and H661 had high levels of NTSR1, HER 4 and neuregulin 1 (NRG1), a ligand for HER4 but not NTSR2, HER3 or NRG2. By RT-PCT, high levels of HER4 and its isoforms JM-a, CYT1 and CYT2 were present in NCI-H522 and H661 cells. Adding NTS (0.1 μM) or NRG1 (0.01 μg/ml) to NCI-H522 or H661 cells increased P-Tyr1284-HER4 and P-ERK 3-fold. Using immunoprecipitation techniques, adding NTS to NSCLC cells increased formation of HER4-HER2 heterodimers which leads to cancer proliferation (Lucas et al., Pharmacol Rev 2022; 74:18). The increase in PY1284-HER4 caused by NTS was impaired by SR48692, HER4 siRNA, NRG1 siRNA or ibrutinib (TKI). The NTSR1 regulation of HER4 transactivation is impaired by GM6001 (MMP inhibitor]), PP2 (Src inhibitor) or N-acetylcysteine (antioxidant). The clonal growth of NSCLC cells is increased by NTS or NRG1 but decreased by SR48692 or ibrutinib. The results indicate that NTS increases the formation of HER2/HER4 heterodimers leading to the proliferation of NSCLC cells. Citation Format: Terry W. Moody, Irene Ramos-Alvarez, Robert T. Jensen. Neurotensin receptor 1 regulates dimerization of HER2-HER4 and proliferation of non-small cell lung cancer cells [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 6266.