Epidermal Growth Factor Receptor Phosphorylation Research Articles

The effects and mechanisms of Gefitinib on airway inflammation and airway remodeling in C57BL/6 mice with asthma

Objective: To investigate the effects of the epidermal growth factor receptor(EGFR) inhibitor Gefitinib on airway inflammation and airway remodelling in asthmatic C57BL/6 mice, and to analyze its possible mechanisms. Methods: Male C57BL/6 mice, aged 6-8 weeks, were randomly assigned into five groups: Group A (control group), Group B (asthma group), Group C (asthma+20 mg/kg gefitinib group), Group D (asthma+40 mg/kg gefitinib group), and Group E (40 mg/kg gefitinib group), with seven mice per group. Mice were sensitized by intraperitoneal injection of a mixture of 0.2 ml solution containing OVA and Al(OH)3 [20 μg OVA+2 mg Al(OH)3 dissolved in 0.2 ml of physiological saline] at Day 0 and 14. Starting from Day 25 to 31, Group B, C, and D were challenged with nebulization of 1% OVA solution (8 ml) to induce asthma, once a day for approximately 40 minutes, with continuous aerosolization for 7 days. Group C and D were given 0.2 ml of Gefitinib dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) by gavage half an hour before challenging, and Group E was simultaneously given with 0.2 ml of Gefitinib dissolved in 0.5% CMCNa only. Group A and B were given an equivalent volume of 0.5% CMCNa by gavage. After 24 h of final challenge, the bronchoalveolar lavage fluid (BALF) was prepared for the determination of total cell count and eosinophil count. The levels of total immune globulin E (IgE) in serum and interleukin (IL)-4, IL-5 and IL-13 in BALF and lung tissue homogenates were measured by ELISA. The mRNA expression levels of IL-4, IL-5, IL-13 in lung were measured. Immunohistochemistry and Western blot experiments were used to detect the expression levels of EGFR in lung tissues. Results: In Group B, the level of total IgE in serum, total cell count, eosinophil count, the levels of IL-4, IL-5, IL-13 in BALF and the phosphorylation of EGFR and its downstream activation in lung were higher than those in Group A (all P<0.05). The levels of total IgE in serum [(261.32±44.38) ng/ml, (194.09±52.39) ng/ml vs (1 023.70±105.51) ng/ml], total cell count [(23.70±4.08)×105/ml, (14.92±4.06)×105/ml vs (35.36±6.30)×105/ml], eosinophil count [(108.00±13.69)×104/ml, (67.00±17.28)×104/ml vs (147.86±20.06)×104/ml], IL-4 [(36.42±4.48) pg/ml, (30.45±8.12) pg/ml vs (58.72±7.17) pg/ml], IL-5 [(16.20±4.62) pg/ml, (13.38±5.14) pg/ml vs (23.46±5.38) pg/ml], IL-13 [(18.45±7.28) pg/ml, (14.33±7.70) pg/ml vs (104.12±24.66) pg/ml] in BALF of Group C and D were lower than those in Group B (all P<0.05). The levels of IL-4, IL-5, and IL-13 as well as their mRNA levels in the lung tissue of Group C and D were lower than those in Group B (all P<0.05). In Group C and D, the positive expression rate of phosphorylated epidermal growth factor receptor (p-EGFR) in lung tissue [(40.53±6.80)%, (23.60±4.42)% vs (70.78±5.36)%], p-EGFR/EGFR (61.68±7.48, 51.13±5.19 vs 105.90±11.66), phosphorylated extracellular regulated protein kinase (p-Erk)/extracellular regulated protein kinase (Erk) (75.28±7.11, 47.54±4.83 vs 98.76±4.71), and phosphorylated protein kinase B (p-Akt)/protein kinase B (Akt) (96.24±5.40, 68.52±2.73 vs 103.30±4.52) was lower than those of Group B (all P<0.05). There was no statistically significant difference in the relevant indicators between Group A and E (all P>0.05). Conclusion: Gefitinib may alleviate airway inflammation and airway remodeling in asthmatic mice by inhibiting EGFR phosphorylation and affecting the activation of downstream Erk and Akt.

#1603 A potent protein inhibitor of HB-EGF protects kidneys from glomerular lesions in a pig model of crescentic glomerulonephritis

Abstract Background and Aims Crescentic glomerulonephritis (CGN) is a severe disease that rapidly destroys the glomerular architecture. CGN is due to immuno-inflammatory aggressions triggered by anti-neutrophil cytoplasmic autoantibody-associated vasculitides, anti-glomerular basement membrane (GBM) autoantibodies, or immune complexes deposition as occurs in systemic lupus erythematosus, IgA nephropathy or infections. Treatments are based on aggressive immunosuppression. Although required, immunosuppression doesn't directly target the deleterious glomerular response to inflammation and leads to significant morbidity. Moreover, most patients still develop chronic or end-stage kidney failure, indicating that complementary strategies are needed. We showed previously that local over-expression of the growth factor HB-EGF (Heparin-binding EGF-like growth factor) and subsequent activation of EGFR lead to dedifferentiation and proliferation of parietal epithelial cells and podocytes, leading to crescentic lesions that irreversibly obliterate the glomeruli. We aimed to evaluate the therapeutic potential of a protein inhibitor of HB-EGF in a pig model of CGN. Method We engineered a protein inhibitor of HB-EGF, DTR8, from the receptor-binding domain of diphtheria toxin, a highly selective natural ligand of HB-EGF. Twelve mutations improved solubility, led to pM affinity, and reduced immunogenicity and antigenicity. Pigs received decomplemented nephrotoxic serum (NTS) from sheep immunized against pig GBM to mimic anti-GBM antibodies disease responsible for one form of CGN. Ten pigs were treated with 0.3 mg/kg of DTR8 twice a day by intramuscular route for 20 days, and 12 pigs received vehicle only starting 6 hours after NTS. Animals were sacrificed on day 21, and blood, urine and kidneys were analyzed. Five animals that had received pre-immune sheep serum served as controls. Results Pigs developed a nephritic syndrome with high proteinuria, hypoalbuminemia, kidney failure with increased BUN and blood creatinine levels, anasarca and crescentic lesions typical of CGN along with de novo HB-EGF glomerular overexpression. The vehicle-treated group had only 24.8% of histologically normal glomeruli, while the DTR8-treated group had 57.3% of healthy glomeruli. DTR8 treatment significantly reduced the percentage of crescentic lesions (P = .0365) and preserved podocyte differentiation as assessed by the expression of nephrin that was markedly reduced in vehicle-treated animals but maintained close to normal in DTR8-treated animals (P = .0028). DTR8 administration reduced EGFR phosphorylation in glomerular cells (P = .0161), suggesting local inhibition of the HB-EGF/EGFR cascade. Lymphocyte infiltration was reduced (P = .2543). Notably, the treatment group also displayed a global reduction in interstitial fibrosis (P = .0313). Conclusion The therapeutic protein candidate DTR8 protects kidney glomeruli in a CGN model in pigs.

Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

ObjectiveOver the past decade, heat shock protein 90 (HSP90) inhibitors have emerged as promising anticancer drugs in solid and hematological malignancies. Flavokawain C (FKC) is a naturally occurring chalcone that has been found to exert considerable anti-tumor efficacy by targeting multiple molecular pathways. However, the efficacy of FKC has not been studied in nasopharyngeal carcinoma (NPC). Metabolic abnormalities and uncontrolled angiogenesis are two important features of malignant tumors, and the occurrence of these two events may involve the regulation of HSP90B1. Therefore, this study aimed to explore the effects of FKC on NPC proliferation, glycolysis, and angiogenesis by regulating HSP90B1 and the underlying molecular regulatory mechanisms.MethodsHSP90B1 expression was analyzed in NPC tissues and its relationship with patient’s prognosis was further identified. Afterward, the effects of HSP90B1 on proliferation, apoptosis, glycolysis, and angiogenesis in NPC were studied by loss-of-function assays. Next, the interaction of FKC, HSP90B1, and epidermal growth factor receptor (EGFR) was evaluated. Then, in vitro experiments were designed to analyze the effect of FKC treatment on NPC cells. Finally, in vivo experiments were allowed to investigate whether FKC treatment regulates proliferation, glycolysis, and angiogenesis of NPC cells by HSP90B1/EGFR pathway.ResultsHSP90B1 was highly expressed in NPC tissues and was identified as a poor prognostic factor in NPC. At the same time, knockdown of HSP90B1 can inhibit the proliferation of NPC cells, trigger apoptosis, and reduce glycolysis and angiogenesis. Mechanistically, FKC affects downstream EGFR phosphorylation by regulating HSP90B1, thereby regulating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. FKC treatment inhibited the proliferation, glycolysis, and angiogenesis of NPC cells, which was reversed by introducing overexpression of HSP90B1. In addition, FKC can affect NPC tumor growth and metastasis in vivo by regulating the HSP90B1/EGFR pathway.ConclusionCollectively, FKC inhibits glucose metabolism and tumor angiogenesis in NPC by targeting the HSP90B1/EGFR/PI3K/Akt/mTOR signaling axis.

Targeting pyruvate dehydrogenase kinase 1 overcomes EGFR C797S mutation-driven osimertinib resistance in non-small cell lung cancer.

Osimertinib, a selective third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), effectively targets the EGFR T790M mutant in non-small cell lung cancer (NSCLC). However, the newly identified EGFR C797S mutation confers resistance to osimertinib. In this study, we explored the role of pyruvate dehydrogenase kinase 1 (PDK1) in osimertinib resistance. Patients exhibiting osimertinib resistance initially displayed elevated PDK1 expression. Osimertinib-resistant cell lines with the EGFR C797S mutation were established using A549, NCI-H292, PC-9, and NCI-H1975 NSCLC cells for both in vitro and in vivo investigations. These EGFR C797S mutant cells exhibited heightened phosphorylation of EGFR, leading to the activation of downstream oncogenic pathways. The EGFR C797S mutation appeared to increase PDK1-driven glycolysis through the EGFR/AKT/HIF-1α axis. Combining osimertinib with the PDK1 inhibitor leelamine helped successfully overcome osimertinib resistance in allograft models. CRISPR-mediated PDK1 knockout effectively inhibited tumor formation in xenograft models. Our study established a clear link between the EGFR C797S mutation and elevated PDK1 expression, opening new avenues for the discovery of targeted therapies and improving our understanding of the roles of EGFR mutations in cancer progression.

A natural compound melatonin enhances the effects of Nimotuzumab via inhibiting EGFR in glioblastoma

Sleep disorders are prevalent and debilitating symptoms in primary brain tumor patients, notably those receiving radiation therapy. Nevertheless, the relationship between sleep disorders, melatonin - a circadian rhythm regulatory hormone, and gliomas is underexplored. Melatonin exhibits various biological functions, one of them being anti-tumor activity. In the context of gliomas, often overexpressing EGFR, the humanized monoclonal antibody Nimotuzumab targets this marker. Our research discovered that variations in circadian rhythm significantly influence tumor growth in mice through impacting melatonin secretion. Harnessing proteogenomic, we identified that melatonin could inhibit the phosphorylation of EGFR and its downstream effectors, key elements in angiogenesis and tumor progression. Building on structural simulations, we propose that melatonin may amplify Nimotuzumab's anti-glioma efficacy by inhibiting EGFR TK dimerization. This proposition was validated in our in vitro and in vivo studies where melatonin synergistically augmented cytotoxicity and apoptosis in Nimotuzumab-treated glioma cells. Thus, melatonin shows promise as a beneficial addition to Nimotuzumab treatment in glioma patients.

Co-occurring EGFR p.E709X mutation Mediates Primary Resistance to the Third-generation EGFR-TKIs in EGFR p.G719X-mutant Patients with Advanced NSCLC.

Current NCCN guidelines recommend afatinib or osimertinib as the preferred first-line treatment strategy for patients with advanced NSCLC harboring EGFR p.G719X mutation. However, in the absence of head-to-head trials comparing afatinib with osimertinib in EGFR p.G719X mutant patients, it is unclear which regimen is the preferred treatment option. A large cohort of 4228 treatment-naïve patients with lung cancer who underwent targeted NGS testing was screened for EGFR p.G719X mutation. A multicenter cohort involving 68 EGFR p.G719X-mutant patients with advanced NSCLC and NGS profiling was retrospectively enrolled to evaluate clinical responses to afatinib(n=37) and the third-generation EGFR-TKIs(n=31). Ba/F3 cells stably expressing the EGFR p.G719A mutation were created to investigate the response to EGFR-TKIs in vitro. Concurrent EGFR p.E709X mutations, being the most frequent co-occurring EGFR mutation in EGFR p.G719X-mutant NSCLC(~30%), exerted a detrimental effect on outcomes in patients treated with third-generation EGFR-TKI(G719X/E709X vs. G719X; ORR:0.00% vs. 47.62%, P<0.001; mPFS:7.18 vs. 14.2 months, P=0.04; respectively). Conversely, no significant difference was found in the treatment efficacy of afatinib between EGFR p.G719X/E709X and EGFR p.G719X patients(G719X/E709X vs. G719X; ORR:71.43% vs. 56.67%, P=0.99; mPFS:14.7 vs. 15.8 months, P=0.69; respectively). In vitro experiments elucidated a resistant drug sensitivity and poor inhibition of EGFR phosphorylation in Ba/F3 cells expressing EGFR p.G719A/E709K mutation upon the third-generation EGFR-TKIs treatment. Co-occurring EGFR p.E709X mutation mediates primary resistance to the third-generation EGFR-TKIs in EGFR p.G719X-mutant patients but remained sensitive to afatinib. A personalized treatment strategy should be undertaken based on the co-existing EGFR p.E709X mutation status.

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.

Abstract LB049: Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway

Abstract Lung cancer is the second most common cancer in both men and women in the United States. Even though treatments including chemotherapy, targeted therapy, and immunotherapy are available, lung cancer has been the leading cause of cancer deaths. The discovery and evaluation of new alternative medications targeting lung cancer are tremendously important for reducing lung cancer mortality. Phytochemicals are very desirable chemotherapeutic agents because of their greater safety profile. Isorhapontigenin (ISO), one of the oral bioavailable dietary stilbenes, can be found in various natural resources. Our result showed that ISO treatment suppressed the invasion and migration of human lung cancer cell lines A549, H23, and H1299 cells. Long non-coding RNA Maternally Expressed Gene 3 (MEG3), a tumor suppressor, was downregulated or lost in various primary human tumor tissues and cancer cell lines. The low expression of MEG3 is correlated with poor prognosis in non-small cell lung cancer (NSCLC). Our results showed that ISO treatment elevated MEG3 levels in these lung cancer cell lines. One of the major genetic causes of lung cancer is mutations in the epidermal growth factor receptor (EGFR) receptor tyrosine kinase, resulting in activation of EGFR which activates several signaling pathways involved in cell proliferation, drug sensitivity, and angiogenesis. Our results showed that ISO decreased the phosphorylation and protein levels of EGFR in A549 cells. Hypoxia-inducible factor-1α (HIF-1α), a transcription factor, regulates the activation of several genes involved in cell proliferation, angiogenesis, invasion, and metastasis. Our results showed that ISO decreased HIF-1α protein levels. Overexpression of MEG3 reduced p-EGFR and HIF-1α, indicating MEG3 negatively regulated EGFR and HIF-1α. Our data also showed that inhibition of EGFR by its tyrosine inhibitor decreased HIF-1α, suggesting that EGFR positively regulates HIF-1α. In cancer cells, Epithelial-Mesenchymal Transition(EMT) endows cells with increased invasiveness and tumor-initiating capacity, contributing to metastasis and resistance to therapeutics. Slug, one of the EMT transcription factors maintains cell plasticity during carcinogenesis. Our results showed that ISO treatment decreased Slugprotein levels in A549 and H23 cells. Transcription factor SOX2 regulates cancer cell proliferation, EMT, migration, invasion, metastasis, tumor initiation, cancer stem cell formation as well as resistance to apoptosis and therapy. Our results showed that ISO decreased SOX2 expression. Knockdown of MEG3 increased the expressions of Slug and SOX2, indicating that MEG3 negatively regulates Slug and SOX2. In conclusion, ISO elevated MEG3, inhibiting EGFR, HIF-1α, Slug, and SOX2, suppressing the migration and invasion of lung cancer cells. Citation Format: Sophia Shi, Zhuo Zhang, Jingxia Li, Huailu Tu, Max Costa. Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB049.

Mesenchymal glioma stem cells trigger vasectasia—distinct neovascularization process stimulated by extracellular vesicles carrying EGFR

Targeting neovascularization in glioblastoma (GBM) is hampered by poor understanding of the underlying mechanisms and unclear linkages to tumour molecular landscapes. Here we report that different molecular subtypes of human glioma stem cells (GSC) trigger distinct endothelial responses involving either angiogenic or circumferential vascular growth (vasectasia). The latter process is selectively triggered by mesenchymal (but not proneural) GSCs and is mediated by a subset of extracellular vesicles (EVs) able to transfer EGFR/EGFRvIII transcript to endothelial cells. Inhibition of the expression and phosphorylation of EGFR in endothelial cells, either pharmacologically (Dacomitinib) or genetically (gene editing), abolishes their EV responses in vitro and disrupts vasectasia in vivo. Therapeutic inhibition of EGFR markedly extends anticancer effects of VEGF blockade in mice, coupled with abrogation of vasectasia and prolonged survival. Thus, vasectasia driven by intercellular transfer of oncogenic EGFR may represent a new therapeutic target in a subset of GBMs.

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.

Lack of Elevated Expression of TGFβ3 Contributes to the Delay of Epithelial Wound Healing in Diabetic Corneas.

To investigate the mechanisms underlying the differential roles of TGFβ1 and TGFβ3 in accelerating corneal epithelial wound healing (CEWH) in diabetic (DM) corneas, with normoglycemia (NL) corneas as the control. Two types of diabetic mice, human corneal organ cultures, mouse corneal epithelial progenitor cell lines, and bone marrow-derived macrophages (BMDMs) were employed to assess the effects of TGFβ1 and TGFβ3 on CEWH, utilizing quantitative PCR, western blotting, ELISA, and whole-mount confocal microscopy. Epithelial debridement led to an increased expression of TGFβ1 and TGFβ3 in cultured human NL corneas, but only TGFβ1 in DM corneas. TGFβ1 and TGFβ3 inhibition was significantly impeded, but exogenous TGFβ1 and, more potently, TGFβ3 promoted CEWH in cultured TKE2 cells and in NL and DM C57BL6 mouse corneas. Wounding induced similar levels of p-SMAD2/SMAD3 in NL and DM corneas but weaker ERK1/2, Akt, and EGFR phosphorylation in DM corneas compared to NL corneas. Whereas TGFβ1 augmented SMAD2/SMAD3 phosphorylation, TGFβ3 preferentially activated ERK, PI3K, and EGFR in healing DM corneas. Furthermore, TGFβ1 and TGFβ3 differentially regulated the expression of S100a9, PAI-1, uPA/tPA, and CCL3 in healing NL and DM corneas. Finally, TGFβ1 induced the expression of M1 macrophage markers iNOS, CD86, and CTGF, whereas TGFβ3 promoted the expression of M2 markers CD206 and NGF in BMDMs from db/db or db/+ mice. Hyperglycemia disrupts the balanced expression of TGFβ3/TGFβ1, resulting in delayed CEWH, including impaired sensory nerve regeneration in the cornea. Supplementing TGFβ3 in DM wounds may hold therapeutic potential for accelerating delayed wound healing in diabetic patients.

The new N2, N4-diphenylpyridine-2,4-diamine deuterated derivatives as EGFR inhibitors to overcome C797S-mediated resistance

A series of new deuterated and non-deuterated N2, N4-diphenylpyridine − 2,4-diamine derivatives were synthesized and evaluated as EGFR C797S-mediated resistance inhibitors. Most of these compounds exhibited potent antiproliferative activity against Baf3-EGFR L858R/T790M/C797S and Baf3-EGFR Del19/T790M/C797S cancel cell lines, with IC50 values in the nanomolar concentration range. Among them, compound 14l represented the most active compound with IC50 values of 8–11 nM. Interestingly, metabolic stability assay with rat liver microsomes indicated that the half-life of the deuterated derivative 14o was significantly increased compared to that of 14l. In xenograft mice models, 14o inhibited tumor growth with excellent inhibitory rate of 75.1 % at the dosage of 40 mg/kg, comparing 73.2 % of the TGI with its non-deuterated compound 14l, at a dosage of 80 mg/kg. Mechanism studies revealed that 14o was a potent EGFR L858R/T790M/C797S and EGFR Del19/T790M/C797S kinase inhibitor, which could downregulate the protein phosphorylation of EGFR and m-TOR signaling pathways, arrest cell cycle at G2/M phase by affecting the expression of CDC25C, and promote cell apoptosis by regulating the expression of cleaved caspase-3. In summary, 14o could serve as a promising deuterated compound for the development of highly efficient anticancer agents.

Abstract 6264: Role of Nuclear ErbB3 localization in regulating ligand-dependent ErbB2-ErbB3 heterodimer activation and downstream targets in prostate cancer

Abstract Objective: Prostate cancer (PCa)-specific nuclear expression of ErbB3, a receptor tyrosine kinase of the epidermal growth factor receptor (EGFR) family, has long been recognized. While an 80kDa nuclear variant of ErbB3 has been identified, full-length 185 kDa ErbB3 also translocates to the nucleus in PCa in a bone microenvironment and androgen status-dependent manner. Here, we studied potential mechanisms by which nuclear ErbB3 may regulate PCa progression. Methods: ErbB3 localization was investigated in vitro (human prostate cancer cell lines, LNCaP, C4-2, 22Rv1) using amiloride or heregulin-1β (ErbB3-specific ligand). ErbB3 expression and subcellular localization were analyzed using immunofluorescent microscopy and subcellular fractionation followed by immunoblot. Physiological readouts included viability, migration and reporter gene assays. Results: Here we show that ErbB3 can occupy a nuclear/perinuclear location in prostate cancer cells. The hormone sensitive PCa line LNCaP expressed higher levels of nuclear ErbB3 compared to castration resistant lines C4-2 and 22Rv1. The diuretic amiloride was previously shown to inhibit ErbB3 internalization by macropinocytosis thus we investigated whether it could also modulate ErbB3 nuclear localization in PCa cells. In LNCaP cells, amiloride dose dependently prevented ErbB3 nuclear transport, whereas in C4-2 and 22Rv1, no such effect was observed. Further analysis revealed that amiloride promoted ErbB3 expression in the membrane, where ErbB3 underwent ligand-dependent phosphorylation, formed ErbB2/ErbB3 dimers, and in turn phosphorylated the downstream targets AKT and ERK1/2 (p42/p44MAPK). On the other hand, EGFR phosphorylation and dimerization were unaffected by amiloride. Our results suggest that ErbB3 is stored in the nuclear region in an inactive form, whereas upon stress induction, including androgen deprivation, ErbB3 is translocated to the plasma membrane, where it is activated by ligand binding, and causes downstream signaling. Conclusion: We previously showed that the EGFR/ErbB2 dual inhibitor lapatinib failed to affect PCa - we now show that this may be due to modulation of nuclear and cytoplasmic ErbB3 expression. Amiloride prevented translocation (and thus expression of) ErbB3 to the nucleus and retained this RTK in the membrane, resulting in the formation of active ErbB2/ErbB3 dimers. Lapatinib was then able to inhibit and inactivate its specific target, ErbB2, thereby inducing significant apoptosis. The above indicates a novel role for nuclear ErbB3 in PCa that can be regulated by the combination of amiloride and lapatinib. Citation Format: Maitreyee K. Jathal, Maria M. Mudryj, Paramita M. Ghosh. Role of Nuclear ErbB3 localization in regulating ligand-dependent ErbB2-ErbB3 heterodimer activation and downstream targets in prostate 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 6264.

Abstract 1962: PH009-1, a highly potent, selective and brain-penetrable fourth-generation EGFR-TKI that overcome classic and resistant EGFR mutations in NSCLC

Abstract Background: Epidermal growth factor receptor (EGFR) inhibitors, especially 3rd Gen inhibitor such as osimertinib (Osi), have become the first line therapy for non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, treatments with 3rd Gen EGFR inhibitors lead to occurrence of acquired C797S mutation in 10-24% of patients. Though studies showed that double mutations 19Del/C797S (DC) and L858R/C797S (LC) could be inhibited by 1st Gen EGFR TKIs, appearance of triple mutations 19Del/T790M/C797S (DTC) and L858R/T790M/C797S (LTC) always leads to treatment failure. In recent years, 4th Gen EGFR inhibitors have been developed such as BLU-945 and PH009-1. PH009-1 had been reported on AACR annual meeting 2023. Methods: Cell activity and selectivity were determined in engineered Ba/F3 cell lines carrying EGFR wild type (WT, stimulated with EGF), 19Del (D), L858R (L), 19Del/T790M (DT), L858R/T790M (LT), DC, LC, DTC and LTC. The in vivo anti-tumor efficacy and PK-PD studies were performed in cell or patient derived xenograft (CDX or PDX) cancer models (HCC827, D; Ba/F3, LC; PDX, DTC). Brain penetration capability was tested in mice orally treated with PH009-1 for 7 days. Systemic safety of PH009-1 was evaluated in 28-day repeated dosing toxicity studies in rats and dogs following GLP guidelines. Results: PH009-1 displayed potent anti-proliferation activity against Ba/F3 cells harboring EGFR mutations (L, D, LC, DC, LT, DT, LTC, DTC) with IC50 of 2.4, 1.3, 5.2, 1.2, 1.1, 1.5, 2.8, 1.9 nM, respectively, but had very weak effect on EGFR WT Ba/F3 cells (IC50, 1135 nM), suggesting excellent potency and selectivity. In contrast, though BLU-945 potently inhibited DT, LT, DTC, LTC with IC50 less than 5 nM, but its activities on L, D, LC, DC were not strong with IC50 of 31.6, 81.2, 46.2, and 52.0 nM. In in vivo efficacy studies, PH009-1 at a dose of 20 mg/kg BID led to potent inhibition on tumor growth and EGFR phosphorylation, and tumor regression was observed at higher doses in all tested models. In HCC827 model with 19Del (first line therapy setting), mice were treated for more than 200 days, and one tumor relapsed in BLU-945 group but not in PH009-1 or Osi group. Also, deaths were observed in Osi group, but not with PH009-1. After treated mice with PH009-1 at efficacious dose for 7 days, concentrations in cerebrospinal fluids (CSF) at 0-8 h post final dose were more than enough (≥1.5 fold) to cover IC80 on all tested mutations. In GLP toxicity studies, no unexpected toxicity was observed. In dogs. the highest tested dose was determined as highest non-severely toxic dose (HNSTD) and based on high plasma exposure, wide safety margin was confirmed with PH009-1. Conclusion: Our data suggested that PH009-1 overcome T790M/C797S resistant mutations, and also have potential to become a potent and safe approach for first line therapy of NSCLC with EGFR mutations. Citation Format: Feng Gao, Jing Wang, Bin Liu, Yongyong Wu, Liandong Jing, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH009-1, a highly potent, selective and brain-penetrable fourth-generation EGFR-TKI that overcome classic and resistant EGFR mutations in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1962.

Abstract 6956: Role of soluble CD109 in the progression of head and neck squamous cell carcinoma

Abstract Introduction: About five percent of global cancer deaths are attributed to Head and Neck Squamous Cell Carcinoma (HNSCC), and its annual incidence is predicted to skyrocket by thirty percent by 2030 (according to GLOBOCAN estimates). HNSCC originates from squamous cells in the oral cavity, lips, tongue, larynx, pharynx, and salivary glands. Despite the comparatively easy tumor diagnosis, the number of patients diagnosed at an advanced stage with metastatic spread is substantial. Multiple cell-signaling pathways have been proven to be dysregulated in HNSCC, including the epidermal growth factor receptor (EGFR). Our group has identified CD109, a GPI-anchored membrane-associated protein, as a TGF-β co-receptor that inhibits TGF-β signaling. CD109 expression is upregulated in SCC, and its elevated expression in premalignant lesions is associated with an increased risk of metastatic progression. Recent studies by our team have revealed that CD109 promotes tumor growth in vivo, likely by enhancing EGFR signaling. Interestingly, we have also discovered that membrane CD109 (mCD109) is endogenously released from the cell surface; however, the mechanism of action of the released/soluble form of CD109 (sCD109) remains unknown. In the current study, we examined whether sCD109 opposes the mCD109/EGFR-mediated pro-tumorigenic signaling and HNSCC progression. Research Questions: 1.Does sCD109 regulate EGF-induced EGFR signaling and downstream stem cell marker expression?2.Does sCD109 regulate EGFR expression levels on the HNSCC cell surface?3.Does sCD109 affect the nuclear localization of EGFR?4.Does sCD109 promote EGFR internalization and degradation? Methodology: HNSCC cells and patient-derived primary cells were treated with/without sCD109 recombinant protein (1nM, 5nM, and 10nM) without and with the EGF induction (10ng/ml for 15 mins) and were analyzed by western blotting, immunofluorescence, and immunocytochemistry. The role of sCD109 in regulating membrane EGFR levels was assessed by flow cytometry and surface protein biotinylation assay. Results: Our findings demonstrated that sCD109 inhibited EGF-induced phosphorylation of EGFR (Y1068), STAT3, AKT, and ERK. The sCD109 suppressed the stem cell marker expression (Nanog, OCT4, SOX2). Additionally, sCD109 hindered the nuclear localization of activated EGFR. The sCD109 decreased the membrane EGFR expression levels and activated the phosphorylation of EGFR (Y1045), suggesting a role for sCD109 in promoting EGFR degradation. Conclusion: Our team has previously reported that mCD109 is pro-tumorigenic, and our current findings suggest that sCD109 opposes the pro-tumorigenic effects of mCD109. This study aimed to determine how sCD109 regulates cellular signaling pathways and will provide key insights into the molecular mechanisms underlying the progression of HNSCC, a highly prevalent malignancy. Citation Format: Varsha Reddy Durgempudi, Tenzin Kungyal, Anie Philip. Role of soluble CD109 in the progression of head and neck squamous cell carcinoma [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 6956.

Abstract 1892: KK2260: A fully human tetravalent bispecific antibody targeting TfR1 and EGFR for the treatment of EGFR-expressing tumors

Abstract Background: TfR1 is a transmembrane glycoprotein involved in iron uptake and regulation of cell growth. High TfR1 expression has been reported in different types of cancers, therefore recognized as a target molecule for cancer therapy. Despite the promise of potent efficacy of TfR1-targeted agents, there is a risk of side effects (i.e., severe anemia), since TfR1 is also highly expressed in certain normal cells (e.g., reticulocytes). KK2260 is a fully human tetravalent bispecific antibody (bsAb) based on REGULGENTTM technology targeting TfR1 and EGFR. This bsAb was designed for EGFR-expressing cell selective anti-cancer drug which could not be achieved by conventional monospecific Ab. In this study, we evaluated the mechanism of action (MOA), anti-cancer effect, and toxicity of KK2260. Methods: Binding activity of KK2260 to TfR1 and EGFR was evaluated by surface plasmon resonance method. Ligand blocking activity was evaluated by binding competition on esophageal squamous cell carcinoma (ESCC) OE21 cells. Growth inhibitory activity of KK2260 on cancer cells or hematopoietic progenitor cells was evaluated by proliferation inhibition assay. In some studies, excessive iron or anti-EGFR parent Ab was added. Effect on the cell surface target expression levels was evaluated by flow cytometry. The intracellular iron level was evaluated by cell imaging. Effect on EGF induced EGFR phosphorylation was evaluated by Western blotting. The in vivo efficacy of KK2260 was evaluated in cell line-derived and patient-derived xenograft (PDX) models of ESCC selected for high EGFR expression level. The toxicity of KK2260 was evaluated in cynomolgus monkeys. Results: KK2260 bound to TfR1 and EGFR with no ligand binding inhibition. KK2260 inhibited cell growth of moderate to high EGFR-expressing cancer cells, but not against EGFR-negative cancer cells or hematopoietic progenitor cells. KK2260 downregulated TfR1 expression on OE21 cells and the intracellular iron level decreased, but no effect was observed on EGFR phosphorylation. The growth inhibitory activity of KK2260 was abolished by adding excessive amounts of iron or reduced by inhibiting the binding to EGFR. Potent anti-tumor activity was observed in the ESCC PDX models with tumor regression. In the intermittent-dose toxicity study of cynomolgus monkeys, KK2260 was well-tolerated. Conclusions: KK2260 demonstrated potent anti-cancer effect both in vitro and in vivo. Intriguingly, KK2260 did not inhibit EGFR phosphorylation while EGFR-binding dependent TfR1 downregulation resulted in intracellular iron deficiency, which was considered as the MOA of KK2260. KK2260 was well tolerated in non-human primates. These preclinical data suggest the unique property of KK2260 is expected to provide significant benefit to patients with advanced ESCC and other EGFR-expressing tumors and Phase 1 study is currently on going in Japan (jRCT2031230372). Citation Format: Keisuke Mitamura, Hironori Otsuki, Keiko Nakahashi, Ayaka Kitagawa, Masayo Kamigaki, Yohei Kurio, Miho Inoue, Yasunori Uemura. KK2260: A fully human tetravalent bispecific antibody targeting TfR1 and EGFR for the treatment of EGFR-expressing tumors [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 1892.

Abstract 3278: Analysis of the mechanism of resistance to selpercatinib in thyroid cancer cells with RET/PTC1 fusion gene

Abstract Purpose: Precision oncology, in which molecularly targeted drugs are selected based on genetic mutations, has begun in thyroid cancer, and drugs targeting BRAF, RET, and NTRK are already in clinical use. However, as with previous therapeutic agents for malignant tumors, there have been cases of resistance to these drugs either initially or during treatment, and the mechanisms of drug resistance need to be elucidated. So far, little is known about the mechanisms of resistance to molecularly targeted drugs in thyroid cancer. To overcome resistance to the RET inhibitor, selpercatinib (SEL), and to consider next therapeutic strategies, we aimed to elucidate the mechanisms of resistance to SEL. Method: We used papillary carcinoma cell lines TPC-1 and CUTC48, which harbor the RET/PTC1 fusion gene. We established an SEL-resistant subline (TPC-1/SELR) in TPC-1 by long-term exposure to SEL. The growth inhibitory effect of drugs was analyzed using the WST assay. The alteration of intracellular signal transduction was analyzed by western blotting. Result: The 50% inhibitory concentration (IC50) for SEL in the parental TPC-1 was 3 nM, while growth inhibition in TPC-1/SELR did not reach 50% even at more than 100 nM of SEL. On the other hand, growth inhibition by SEL in CUTC48 did not reach 50% even at more than 100 nM of SEL. The sensitivity to SEL in CUTC48 was comparable to that in TPC-1/SELR, and CUTC48 showed an intrinsic resistance to SEL. TPC-1/SELR showed increased phosphorylation of EGFR, Erk, Akt, and mTOR compared to TPC-1. In CUTC48, phosphorylation of EGFR and Erk was not increased compared to that of TPC-1, while phosphorylation of Akt and mTOR was increased. We investigated the growth inhibitory effect of an mTOR inhibitor everolimus and found that TPC-1 (IC50: 1.3 nM), TPC-1/SELR (5 nM), and CUTC48 (9.4 nM) were all highly sensitive to everolimus. Discussion: Our results showed that Akt and mTOR phosphorylation were elevated in the SEL-resistant TPC-1/SELR and the SEL-intrinsic resistant CUTC48 in common, suggesting the involvement of activation of the Akt-mTOR pathway in SEL resistance in thyroid cancer. The inhibition of the Akt-mTOR pathway in parallel with RET may have therapeutic potential to overcome SEL resistance in thyroid cancer. Citation Format: Masatsugu Amitani, Ken-ichi Ito. Analysis of the mechanism of resistance to selpercatinib in thyroid cancer cells with RET/PTC1 fusion gene [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 3278.

Abstract 6511: BPI-520105, a highly potent, CNS-penetrant 4th generation EGFR inhibitor overcoming major EGFR resistance mutations in NSCLC

Abstract EGFR activating mutations are prevalent in 10-50% of NSCLC patients. The most common EGFR mutations, L858R (L) and Del19 (D), initially respond to first-, second-, or third-generation (1G, 2G and 3G) EGFR-TKIs. However, on-target resistance, primarily by T790M (T) and C797X(C), inevitably emerges post-treatment and leads to disease progression. Different treatment sequence give rise to different complex mutations. In patients resistant to the 3G TKI osimertinib, double mutations L858R/C797X or Del19/C797X (LC or DC) were observed at a frequency of up to 12% after first-line osimertinib, and triple mutations L858R/T790M/C797S or Del19/T790M/C797S (LTC or DTC) were up to 20% after second-line osimertinib. There are no approved targeted therapy options for these complex resistant mutations. In this study, we developed BPI-520105, a 4th generation EGFR inhibitor capable of targeting all aforementioned major forms of EGFR activating and resistance mutations in NSCLC. It might be an optimal way to address this urgent medical need. In vitro, BPI-520105 potently inhibited the activity of a variety of EGFR kinases, as well as inhibited the proliferation and EGFR phosphorylation in cell lines bearing relative mutations, including single (D, L), double (DT, LT, and DC, LC) and triple (LTC, DTC) mutations. Meanwhile, BPI-520105 exhibited relative weakness towards wild type EGFR and IGF1R, demonstrating good selectivity in kinase and cell-based assays. Despite being a reversible EGFR inhibitor, BPI-520105 sustained the inhibition of pEGFR and pERK for up to 24 hours in the western blot assay. In vivo, oral administration of BPI-520105 displayed significant dose-dependent antitumor effect in multiple EGFR mutant xenograft models, including HCC827 (D), NCI-H1975 (LT), BaF3-EGFR-LC/DC, and BaF3-EGFR-LTC models. Remarkably, BPI-520105 significantly prolonged the lifespan of mice with brain orthotropic BaF3-EGFR-DTC-Luc2 allografts, demonstrating its activity towards brain metastasis. Additionally, BPI-520105 exhibited favorable ADME properties, with high oral exposure across various preclinical species. In summary, BPI-520105 is a highly potent, CNS-penetrant, wide type sparing, and orally bioavailable 4th generation EGFR inhibitor, holding promise for the treatment of NSCLC patients with resistance mutations post 3G progression or even at front line. Citation Format: Jing Guo, Lijia Liu, Xiangyong Liu, Bang Fu, Guangcan Bai, Changyong Qiu, Wei Ren, Xiaodong Song, Guolong Du, Xiao Sun, Yongchun Zhang, Weidong Cai, Pingping Wang, Haibo Chen, Xiaoyun Liu, Zhengyao Zou, Jiayu Zhao, Xuepeng Jv, Hong Lan, Lieming Ding, Jiabing Wang. BPI-520105, a highly potent, CNS-penetrant 4th generation EGFR inhibitor overcoming major EGFR resistance mutations in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6511.

Abstract 1871: Discovery and characterization of NXV01c, an EGFR × cMET bispecific nanobody drug conjugate with potent anti-tumor activity

Abstract EGFR and cMET are proven cancer targets co-expressed in diverse tumor types. EGFR × cMET bispecific antibody has been approved for the treatment of NSCLC, supporting a simultaneous targeting strategy. In addition to this dual targeting benefit, bispecific antibody drug conjugates (ADCs) targeting EGFR and cMET have also been developed to further improve anti-tumor activity and tissue selectivity. Sufficient target affinity, good cellular internalization and high tumor infiltration are critical for an ADC to mediate therapeutic activity. To this end, we developed the first EGFR × cMET bispecific nanobody conjugated with monomethyl auristatin E (MMAE) as payload (NXV01c). Lead nanobodies against EGFR and cMET were respectively selected from immune libraries by phage display, followed by highly efficient humanization and optimization by neoX’s computation platform. The bispecific nanobody with Fc (NXV01) was constructed by “knobs-into-holes” heterodimerization and then homogeneously conjugated with MMAE via a lysosomal cleavable valine-citrulline dipeptide linker. The resulting bispecific nanobody drug conjugate (NDC), NXV01c, was evaluated in multiple tumor cell lines and tumor xenograft models. NXV01, the pre-conjugate bispecific nanobody, bound EGFR and cMET with nanomolar potency (BLI) and inhibited the phosphorylation of cellular EGFR and cMET with nanomolar IC50 (ELISA). Moreover, it did not activate cellular cMET. Importantly, the NDC NXV01c is highly homogeneous: it has an average DAR of 3.87 and &amp;gt;95% of NXV01c has a DAR of 4. To examine stability of conjugation, NXV01C was incubated in human plasma (37°C) for 14 days, the maximum free drug release rate (by LC/MS/MS) is 0.68%, which is much lower than that of DS8201. In vitro, NXV01c was rapidly internalized into H1975 cells which co-expressed EGFR and cMET. It inhibited the growth of H1975 (lung) and SNU5 (gastric) cancer cells with picomolar activity but spared normal keratinocytes. NXV01c also inhibited the proliferation of additional cell lines derived from lung, gastric, esophageal, and liver cancer, and the level of inhibition positively associated with the expression density of both targets. In an H1975 cell line-derived xenograft model, NXV01C exhibited potent and dose-dependent anti-tumor activity. Treatments at 3 and 10 mg/kg once per week for 2 weeks led to shrinkage and complete regression of tumor, respectively. In patient-derived xenograft models of NSCLC and esophageal cancer, NXV01C led to tumor regression and elimination without noticeable toxic effects. EGFR × cMET bispecific nanobody drug conjugate NXV01c has favorable drug-like properties and demonstrated superior anti-tumor effect in vitro and in vivo. The results suggest NXV01C can be an effective solution for EGFR/cMET bearing tumors commonly found in diverse malignancies. Citation Format: Ran Wu, Puwei Yuan, Yang Xie, Jianxiu Guo, Fei Zhang, Fan Liu, Taylor B. Guo. Discovery and characterization of NXV01c, an EGFR × cMET bispecific nanobody drug conjugate with potent anti-tumor activity [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 1871.

Abstract 615: NXP900, a novel YES1/SRC kinase inhibitor in phase 1, demonstrates potent inhibition of proliferation in cell lines resistant to ALK and EGFR inhibitors

Abstract Background: NXP900 (eCF506) is a novel potent and selective SRC family kinase (SFK) inhibitor, (IC50 of 0.47 nM against YES1). NXP900 locks its target into its native “closed” conformation (type 1.5), thereby inhibiting both kinase activity and complex formation with protein partners (Temps et al. Cancer Res. 2021, 81, 5438). In contrast, multi-kinase inhibitors, including dasatinib and bosutinib, block SRC in the active “open” conformation (type 1) promoting the association of SFK and signaling partners via allosteric facilitation (Higuchi et al. Cell Rep. 2021, 34, 108876). Activation of SFK suggests that NXP900 may have therapeutic potential in cancers with acquired resistance to EGFR and ALK inhibitors. Methods: Cell lines: Alectinib sensitive (NCI-H2228, ATCC) and osimertinib sensitive (PC9, RIKEN). Alectinib resistant (H2228-ALR1, H2228-ALR2, H2228-ALR3, H2228-ALR4) and osimertinib resistant (PC9-OR1, PC9-OR3) cell lines were generated by treatment of NCI-H2228 or PC9 with increasing concentrations of, respectively, alectinib or osimertinib. Cell proliferation assay: Intracellular ATP (Oncolines B.V.); cells were treated with inhibitors for 120 hours. Reverse Phase Protein Array (RPPA): Protein extracts were prepared from cell lines at sequential timepoints following treatment with NXP900 or a combination of NXP900 and osimertinib. Extracts were printed as a concentration series onto nitrocellulose coated slides across multiple sub-arrays using an Aushon2470 arrayer, each sub-array was addressed with individual mono-specific antibodies to quantify the abundance of SRC, EGFR and other canonical cancer signaling pathway phosphorylation events. Results: NXP900 single agent potently inhibited cell proliferation of ALK sensitive (GI50=83nM) and all ALK resistant cell lines (GI50=5.8-16nM). GI50 for EGFR sensitive and EGFR resistant cells were 605 nM and 826-4665 nM, respectively. In combination with a fixed concentration of osimertinib (160nM), NXP900 potently reversed osimertinib resistance in both EGFR resistant cell lines (GI50= 43-121 nM). Conclusions: Despite high response rates to osimertinib and alectinib, acquired resistance almost universally arises. Here we demonstrate that NXP900 can potently inhibit cell proliferation of ALK resistant cell lines as a single agent and EGFR resistant cell lines in combination with osimertinib. Activation of SFK and YAP1 has been shown to be important in the development of resistance to ALK and EGFR treatment. We have previously demonstrated that NXP900 potently inhibits YAP1 nuclear localization and induces tumor regressions in squamous models in vivo providing additional proof of concept for targeting solid tumors with YES1/SRC and Hippo pathway alterations. A FIH, Phase1 dose escalation study for NXP900 has been initiated. Citation Format: Neil O. Carragher, Enrique Poradosu, Ben King, Alison F. Munro, John C. Dawson, Asier Unciti-Broceta. NXP900, a novel YES1/SRC kinase inhibitor in phase 1, demonstrates potent inhibition of proliferation in cell lines resistant to ALK and EGFR inhibitors [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 615.