Receptor Tyrosine Kinase Signaling Research Articles

GBA Regulates EMT/MET and Chemoresistance in Squamous Cell Carcinoma Cells by Modulating the Cellular Glycosphingolipid Profile.

Glycosphingolipids (GSL) are plasma membrane components that influence molecular processes involved in cancer initiation, progression, and therapeutic responses. They also modulate receptor tyrosine kinases involved in EMT. Therefore, understanding the mechanisms that regulate GSLs in cancer has important therapeutic potential. One critical regulator of GSLs is the lysosomal glucosylceramidase β1 (GBA) that catalyzes the last step in GSL degradation. We show that, in cancer, GBA copy number amplifications and increased expression are widespread. We show that depleting GBA in squamous cell carcinoma cell lines results in a mesenchymal-to-epithelial shift, decreased invasion and migration, increased chemotherapeutic sensitivity, and decreased activation of receptor tyrosine kinases that are involved in regulating EMT. Untargeted lipidomics shows that GBA depletion had significant effects on sphingolipids and GSLs, suggesting that increased GBA activity in cancer sustains EMT and chemoresistance by modulating receptor tyrosine kinase activity and signaling via effects on the cellular lipid profile.

Immune-related interaction perturbation networks unravel biological peculiars and clinical significance of glioblastoma.

The immune system is an interacting network of plentiful molecules that could better characterize the relationship between immunity and cancer. This study aims to investigate the behavioral patterns of immune-related interaction perturbation networks in glioblastoma. An immune-related interaction-perturbation framework was introduced to characterize four heterogeneous subtypes using RNA-seq data of TCGA/CGGA glioblastoma tissues and GTEx normal brain tissues. The stability and robustness of the four subtypes were validated in public datasets and our in-house cohort. In the four subtypes, C1 was an inflammatory subtype with high immune infiltration, low tumor purity, and potential response to immunotherapy; C2, an invasive subtype, was featured with dismal prognosis, telomerase reverse transcriptase promoter mutations, moderate levels of immunity, and stromal constituents, as well as sensitivity to receptor tyrosine kinase signaling inhibitors; C3 was a proliferative subtype with high tumor purity, immune-desert microenvironment, sensitivity to phosphatidylinositol 3'-kinase signaling inhibitor and DNA replication inhibitors, and potential resistance to immunotherapy; C4, a synaptogenesis subtype with the best prognosis, exhibited high synaptogenesis-related gene expression, prevalent isocitrate dehydrogenase mutations, and potential sensitivity to radiotherapy and chemotherapy. Overall, this study provided an attractive platform from the perspective of immune-related interaction perturbation networks, which might advance the tailored management of glioblastoma.

Multivalent tyrosine kinase inhibition promotes T cell recruitment to immune-desert gastric cancers by restricting epithelial-mesenchymal transition via tumour-intrinsic IFN-γ signalling

ObjectiveGastric cancer (GC) ranks fifth in incidence and fourth for mortality worldwide. The response to immune checkpoint blockade (ICB) therapy in GC is heterogeneous due to tumour-intrinsic and acquired immunotherapy...

Mass Spectrometry and Pharmacological Approaches to Measuring Cooption and Reciprocal Activation of Receptor Tyrosine Kinases.

Receptor tyrosine kinases (RTKs) can show extensive crosstalk, directly and indirectly. Elucidating RTK crosstalk remains an important goal in the clinical combination of anti-cancer therapies. Here, we present mass spectrometry and pharmacological approaches showing the hepatocyte growth factor receptor (MET)-promoting tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and other membrane receptors in MET-amplified H1993 NSCLC cells. Conversely, in H292 wt-EGFR NSCLC cells, EGFR promotes the tyrosine phosphorylation of MET. Reciprocal regulation of the EGFR and insulin receptor (IR) was observed in the GEO CRC cells, where inhibition of the EGFR drives tyrosine phosphorylation of the insulin receptor. Similarly, in platelet-derived growth factor receptor (PDGFR)-amplified H1703 NSCLC cells, inhibition of the EGFR promotes the tyrosine phosphorylation of the PDGFR. These RTK interactions are used to illustrate basic principles applicable to other RTK signaling networks. More specifically, we focus on two types of RTK interaction: (1) co-option of one RTK by another and (2) reciprocal activation of one receptor following the inhibition of a distinct receptor.

Kinetics of RTK activation determine ERK reactivation and resistance to dual BRAF/MEK inhibition in melanoma

The combination of BRAF and MEK inhibitors (BRAFi/MEKi) has shown promising response rates in treating BRAF-mutant melanoma by inhibiting ERK activation. However, treatment efficacy is limited by the emergence of drug-tolerant persister cells (persisters). Here, we show that the magnitude and duration of receptor tyrosine kinase (RTK) activation determine ERK reactivation and persister development. Our single-cell analysis reveals that only a small subset of melanoma cells exhibits effective RTK and ERK activation and develops persisters, despite uniform external stimuli. The kinetics of RTK activation directly influence ERK signaling dynamics and persister development. These initially rare persisters form major resistant clones through effective RTK-mediated ERK activation. Consequently, limiting RTK signaling suppresses ERK activation and cell proliferation in drug-resistant cells. Our findings provide non-genetic mechanistic insights into the role of heterogeneity in RTK activation kinetics in ERK reactivation and BRAFi/MEKi resistance, suggesting potential strategies for overcoming drug resistance in BRAF-mutant melanoma.

Cadherins and catenins in cancer: connecting cancer pathways and tumor microenvironment.

Cadherin-catenin complexes are integral components of the adherens junctions crucial for cell-cell adhesion and tissue homeostasis. Dysregulation of these complexes is linked to cancer development via alteration of cell-autonomous oncogenic signaling pathways and extrinsic tumor microenvironment. Advances in multiomics have uncovered key signaling events in multiple cancer types, creating a need for a better understanding of the crosstalk between cadherin-catenin complexes and oncogenic pathways. In this review, we focus on the biological functions of classical cadherins and associated catenins, describe how their dysregulation influences major cancer pathways, and discuss feedback regulation mechanisms between cadherin complexes and cellular signaling. We discuss evidence of cross regulation in the following contexts: Hippo-Yap/Taz and receptor tyrosine kinase signaling, key pathways involved in cell proliferation and growth; Wnt, Notch, and hedgehog signaling, key developmental pathways involved in human cancer; as well as TGFβ and the epithelial-to-mesenchymal transition program, an important process for cancer cell plasticity. Moreover, we briefly explore the role of cadherins and catenins in mechanotransduction and the immune tumor microenvironment.

Deletion of p66Shc dysregulates ERK and STAT3 activity in embryonic stem cells, enhancing their naïve-like self-renewal in the presence of LIF.

The ShcA adapter protein is necessary for early embryonic development. The role of ShcA in development is primarily attributed to its 52kDa and 46kDa isoforms, that transduce receptor tyrosine kinase (RTK) signaling via the extracellular signal regulated kinase (ERK). During embryogenesis, ERK acts as the primary signalling effector, driving fate acquisition and germ layer specification. P66Shc, the largest of the ShcA isoforms, has been observed to antagonize ERK in several contexts, however its role during embryonic development remains poorly understood. We hypothesized that p66Shc could act as a negative regulator of ERK activity during embryonic development, antagonizing early lineage commitment. To explore the role of p66Shc in stem cell self-renewal and differentiation, we created a p66Shc knockout (KO) murine embryonic stem cell (mESC) line. Deletion of p66Shc enhanced basal ERK activity, but surprisingly, instead of inducing mESC differentiation, loss of p66Shc enhanced the expression of core and naïve pluripotency markers. Using pharmacologic inhibitors to interrogate potential signalling mechanisms, we discovered that p66Shc deletion permits the self-renewal of naïve mESCs in the absence of conventional growth factors, by increasing their responsiveness to leukemia inhibitory factor (LIF). We discovered that loss of p66Shc enhanced not only increased ERK phosphorylation but also increased phosphorylation of Signal transducer and activator of transcription (STAT3(S727)) in mESCs, which may be acting to stabilize their naïve-like identity, desensitizing them to ERK-mediated differentiation cues. These findings identify p66Shc as a regulator of both LIF-mediated ESC pluripotency and of signaling cascades that initiate post-implantation embryonic development and ESC commitment.

Replication Study of the Association of GAS6 and PROS1 Polymorphisms with Behçet’s Disease in a Japanese Population

ABSTRACT Purpose To investigate whether polymorphisms of GAS6 and PROS1, which each encode protein ligands for a family of tyrosine kinase receptors, are associated with Behçet’s disease (BD) in a Japanese population. Methods We recruited 734 Japanese patients with BD and 1789 Japanese healthy controls. In all participants, we genotyped two single-nucleotide polymorphisms (SNPs) reportedly associated with BD: rs9577873 in GAS6 and rs4857037 in PROS1. Results We found that GAS6 rs9577873 was not significantly associated with BD. In contrast, PROS1 rs4857037, specifically the A allele, was associated with increased risk for BD. The A allele was also significantly associated with BD under additive and recessive genetic models. Expression analysis revealed that this allele was significantly associated with increased PROS1 expression. Conclusions Our findings suggest that increased PROS1 expression related to the A risk allele of rs4857037 affects tyrosine kinase receptor signaling, contributing to the development of BD.

Genome-wide profiling of patient-derived glioblastoma stem-like cells reveals recurrent genetic and transcriptomic signatures associated with brain tumors

PurposePatient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs).Methods94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively.ResultsExome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S − adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways.ConclusionThe availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

Ephrin-Eph receptor tyrosine kinases for potential therapeutics against hepatic pathologies.

Hepatic fibrosis is the common pathological change that occurs due to increased synthesis and accumulation of extracellular matrix components. Chronic insult from hepatotoxicants leads to liver cirrhosis, which if not reversed timely using appropriate therapeutics, liver transplantation remains the only effective therapy. Often the disease further progresses into hepatic carcinoma. Although there is an increased advancement in understanding the pathological phenotypes of the disease, additional knowledge of the novel molecular signaling mechanisms involved in the disease progression would enable the development of efficacious therapeutics. Ephrin-Eph molecules belong to the largest family of receptor tyrosine kinases (RTKs) which are identified to play a crucial role in cellular migratory functions, during morphological and developmental stages. Additionally, they contribute to the growth of a multicellular organism as well as in pathological conditions like cancer, and diabetes. A wide spectrum of mechanistic studies has been performed on ephrin-Eph RTKs in various hepatic tissues under both normal and diseased conditions revealing their diverse roles in hepatic pathology. This systematic review summarizes the liver-specific ephrin-Eph RTK signaling mechanisms and recognizes them as druggable targets for mitigating hepatic pathology.

Targeting neddylation as a novel approach to lung cancer treatment (Review).

As a protein that resembles ubiquitin, neural precursor cell expressed developmentally downregulated 8 (NEDD8) takes part in neddylation, which modifies substrates in a manner similar to ubiquitination and alters the activity of target proteins. Neddylation may affect the activity of multiple signaling pathways, have a regulatory role in tumor formation, progression and metastasis, and influence the prognosis of cancer treatment. The present review summarizes the regulatory roles of NEDD8 in the MDM2‑p53, NF‑κB, PI3K/AKT/mTOR, hypoxia‑inducible factor, Hippo and receptor tyrosine kinase signaling pathways, as well as in the development and progression of lung cancer.

289 Crosstalk between MET-dependent receptor tyrosine kinase signaling and oncogenic Gαq mutations in melanoma

289 Crosstalk between MET-dependent receptor tyrosine kinase signaling and oncogenic Gαq mutations in melanoma

ASAP2 interrupts c-MET-CIN85 interaction to sustain HGF/c-MET-induced malignant potentials in hepatocellular carcinoma

BackgroundSustained activation of hepatocyte growth factor (HGF)/c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression, but underlying mechanism is unclear. ArfGAP With SH3 Domain, Ankyrin Repeat And PH Domain 2 (ASAP2) can reportedly activate GTPases and promote receptor tyrosine kinase signaling. However, the exact role of ASAP2 in HCC, especially for c-MET activation, also remains elusive.MethodsASAP2 expression levels in HCC tissues and cells were quantified using qRT-PCR, western blot (WB) analysis, and immunohistochemistry staining. Cell counting kit-8 (CCK-8) and colony formation assays were performed to evaluate cell proliferation rates. Flow cytometry assays were conducted to assess apoptosis rates. Wound healing and Transwell assays were performed to determine cell migration and invasion capacities. Epithelial-mesenchymal transition (EMT)-related marker expression levels were also examined. Subcutaneous implantation and tail vein injection models were applied for in vivo growth and metastasis evaluations, respectively. Bioinformatics analyses of The Cancer Genome Atlas and STRING datasets were performed to explore ASAP2 downstream signaling. Co-immunoprecipitation and Cycloheximide chasing experiments were performed to assess protein–protein interactions and protein half-life, respectively.ResultsASAP2 had higher expression levels in HCC tissues than in normal liver, and also predicted poor prognosis. Knocking down ASAP2 significantly impaired cell proliferation, migration, and invasion capacities, but promoted apoptosis in HCC cells in vitro. However, overexpression of ASAP2 achieved the opposite effects. In vivo experiments confirmed that ASAP2 could promote HCC cell growth and facilitate lung metastasis. Interestingly, ASAP2 was essential for triggering EMT. Gene Set Enrichment Analysis demonstrated that c-MET signaling was greatly enriched in ASAP2-high HCC cases. Additionally, c-MET signaling activity was significantly decreased following ASAP knockdown, evidenced by reduced c-MET, p-AKT, and p-ERK1/2 protein levels. Importantly, ASAP2 knockdown effectively attenuated HGF/c-MET signaling-induced malignant phenotypes. c-MET and ASAP2 expression levels were positively correlated in our cohort. Mechanistically, ASAP2 can directly bind to CIN85, thereby disrupting its interaction with c-MET, and can thus antagonize CIN85-induced c-MET internalization and lysosome-mediated degradation. Notably, knocking down CIN85 can rescue the observed inhibitory effects caused by ASAP2 knockdown.ConclusionsThis study highlights the importance of ASAP2 in sustaining c-MET signaling, which can facilitate HCC progression.

Abstract CT184: Preliminary dose escalation results of ERAS-601 in combination with cetuximab in FLAGSHP-1: a phase I study of ERAS-601, a potent and selective SHP2 inhibitor, in patients with previously treated advanced or metastatic solid tumors

Abstract Background: SHP2 is an oncogenic tyrosine phosphatase that transduces receptor tyrosine kinase signaling to the RAS/MAPK pathway via its phosphatase-mediated regulation of guanine nucleotide exchange factors. ERAS-601 is a potent, selective, and orally bioavailable allosteric inhibitor of SHP2. In combination with cetuximab, an antibody that targets epidermal growth factor receptor (EGFR), ERAS-601 has demonstrated robust nonclinical activity in human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) and RAS/RAF wild-type colorectal cancer (CRC) tumors. Method: FLAGSHP-1 is the first-in-human trial of ERAS-601 administered as monotherapy and in combination with other cancer therapies in patients with advanced or metastatic solid tumors. The primary objectives are to characterize the safety profile, determine the maximum tolerated dose (MTD)/recommended dose (RD), and characterize the pharmacokinetics (PK) profile of ERAS-601 as a monotherapy and in combination with other cancer therapies. Secondary objectives include tolerability and antitumor activity in solid tumors. Presented here are results from the combination dose escalation cohorts in which patients received ERAS-601 twice a day for three weeks followed by a one-week break (BID 3/1) in combination with cetuximab (500mg/m2) administered every 2 weeks (Q2W) on a 28-day cycle. Results: As of October 31, 2022, a total of 15 patients with previously treated advanced or metastatic solid tumors received ERAS-601 BID 3/1 at the following dose levels: 20 mg BID 3/1 (n=4), 40 mg BID 3/1 (n=8), or 60 mg BID 3/1 (n=3) in combination with cetuximab. Combination therapy MTD was determined​ to be 40 mg BID 3/1. ERAS-601 Treatment Related Adverse Events (TRAEs) at or below the MTD were all Grade 1 and 2. TRAEs occurring in ≥20% of patients included diarrhea (27%), AST increase (27%), ALT increase (20%), dermatitis acneiform (20%). Grade ≥3 TRAEs included Grade 4 hypokalaemia, Grade 3 diarrhea, platelet count decreased anemia (each 7%); high grade TRAEs were only observed at 60 mg BID 3/1 (above the MTD). Dose limiting toxicities (DLTs) were only observed at the 60mg BID 3/1 dose levels and included Grade 3 platelet count decreased (n=1) and Grade 4 hypokalemia (n=1). Pharmacokinetics of ERAS-601 and cetuximab in combination were generally comparable to historical monotherapy PK values, suggesting lack of drug-drug interaction. The evaluation of clinical activity is still ongoing. Conclusions: ERAS-601 in combination with cetuximab in patients with previously treated advanced or metastatic solid tumors shows promising preliminary safety and tolerability with reversible and manageable TRAEs. Further evaluation in relevant tumor types are ongoing. Citation Format: Meredith McKean, Ezra Rosen, Minal Barve, Tarek Meniawy, Judy Wang, David S. Hong, Jennifer Yang, Zhengrong Li, Roxana Picard, Les Brail, Daniela Vecchio, Thomas John, Ezra Cohen, Gregory Obara, Aparna Parikh. Preliminary dose escalation results of ERAS-601 in combination with cetuximab in FLAGSHP-1: a phase I study of ERAS-601, a potent and selective SHP2 inhibitor, in patients with previously treated advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT184.

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

Abstract 4991: Phosphorylation of MERTK is required for nuclear localization in non-small cell lung cancer (NSCLC)

Abstract MERTK is a transmembrane receptor that belongs to the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases. The role of MERTK in cancer progression and resistance to therapies are reported in many cancer types, including non-small cell lung cancer (NSCLC). In canonical signaling, MERTK is localized at the cell membrane and mediates intracellular signal transduction pathways. Non-canonical signaling of receptor tyrosine kinases (RTKs) has been reported, including signaling which occurs when RTKs translocate to the nucleus. Our group previously reported nuclear localization of MERTK in leukemia cells and we now extend these findings to non-small cell lung cancer. We found exogenous MERTK localized to the nucleus in cells transfected with a plasmid expressing wild-type MERTK. Additionally, endogenous nuclear MERTK was phosphorylated, suggesting that MERTK may have a functional role in the nucleus. MERTK kinase inhibitor MRX-2843 modulates its nuclear translocation. Introducing point mutations to MERTK autophosphorylation sites diminished nuclear translocation in comparison to cells transfected with wild-type MERTK. Our analysis of proteins that may interact with MERTK to regulate nuclear localization and function demonstrated co-immunoprecipitation of nuclear MERTK and STAT1. Current studies are underway to further explore nuclear MERTK functions and how these roles may be modulated upon interaction with STAT1. Citation Format: K.M. Tanim, Deborah DeRyckere, Douglas K. Graham. Phosphorylation of MERTK is required for nuclear localization in non-small cell lung cancer (NSCLC). [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 4991.

Abstract 3876: Kinetics of RTK activation determine ERK signaling dynamics and resistance to BRAF and MEK inhibitors

Abstract Combination treatment of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) produces remarkable response rates and is expected to prevent ERK activation in BRAF-mutant melanoma. However, sporadic ERK reactivation occurs in melanoma models, and efficacy is severely limited by the emergence of drug resistance. The underlying molecular mechanisms that drive ERK reactivation and adaptation to BRAFi/MEKi remain elusive. Here, using single-cell imaging and optogenetic approaches, we show that the strength and duration of receptor tyrosine kinase (RTK) activation determines ERK reactivation and the development of BRAFi/MEKi-resistant cells. Single-cell data reveal that despite global RTK activation by the same external stimuli, only small subsets of melanoma cells induce effective RTK and ERK activation to develop BRAFi/MEKi resistance. Furthermore, by directly controlling RTK activity, we demonstrate that the kinetics of RTK activation determine ERK signaling dynamics and the percentage of drug-resistant cells. While these drug-resistant cells are rare during early stages of drug treatment, they eventually become the dominant population through sustained RTK-mediated ERK activation that drives cell proliferation. Thus, RTK activation in established drug-resistant cells causes effective ERK activation and cell proliferation. Importantly, limiting RTK signaling activation reverses the BRAFi/MEKi-resistant state. We show that targeting SHP2, a mediator of RTK signaling, effectively suppresses RTK-mediated ERK activation and cell proliferation in BRAFi/MEKi-resistant cells. Our results provide mechanistic insights into the contribution of heterogeneity in RTK activation kinetics to ERK reactivation and BRAFi/MEKi resistance, also highlighting SHP2 activity as a promising target to overcome drug resistance in BRAF-mutant melanoma. Citation Format: Sungsoo Kim, Richard Carvajal, Minah Kim, Hee Won Yang. Kinetics of RTK activation determine ERK signaling dynamics and resistance to BRAF and MEK 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 3876.

Abstract 1758: Resistance to cancer therapy via upregulation of the NRP2/VEGF-C axis can be neutralized by ATYR2810

Abstract Introduction: Intrinsic or acquired resistance to cancer therapy is a major problem in the treatment of many cancers. Therapy-resistant cancer cells are able to evade the effect of cancer drugs, resulting in highly aggressive clones that can cause poor long-term prognosis. Neuropilin-2 (NRP2) is a cell surface receptor that acts as a co-receptor for vascular endothelial growth factor (VEGF) and has been shown to be associated with therapy resistance. The VEGF-C signaling pathway has also been linked to chemoresistance in several cancer types including breast, bladder, and gastric cancer. Previously we have shown that ATYR2810—a highly specific humanized monoclonal antibody that blocks NRP2/VEGF-C signaling—sensitizes triple negative breast cancer (TNBC), a highly aggressive and chemoresistant subtype of breast cancer, to chemotherapy and targeted anti-VEGF therapy both in vitro and in vivo. In this study we examined the role of enhanced VEGF-C signaling in tumor growth and therapeutic resistance, and the ability of ATYR2810 to neutralize these effects using in vivo models for lung and kidney cancers. Results: We generated a stable VEGF-C overexpressing non-small cell lung cancer (NSCLC) cell line and found that tumor growth was enhanced by increased VEGF-C signaling. Importantly, ATYR2810 monotherapy counteracted this VEGF-C-driven enhanced tumor growth as tumor growth inhibition was increased in the VEGF-C overexpressing tumors compared to control tumors. Further, VEGF-C overexpression was also found to enhance resistance to platinum-based chemotherapy in this model. However, sensitivity to chemotherapy was restored when ATYR2810 was used in combination with cisplatin. In clear cell renal cell carcinoma (ccRCC), the use of sunitinib—a small molecule inhibitor that inhibits the signaling of several receptor tyrosine kinases (RTKs), including vascular endothelial growth factor receptors (VEGFRs)—has reportedly been associated with the upregulation of VEGF-C and NRP2 expression as well as increased metastasis. In line with this, we found that the use of sunitinib leads to increased levels of circulating VEGF-C in the serum of ccRCC xenograft mice. The use of ATYR2810 in combination with sunitinib was found to act synergistically to inhibit tumor growth in ccRCC xenograft tumors leading to complete tumor regression in some animals. Conclusions: Here we demonstrate that high levels of VEGF-C expression increase tumor dependency on the VEGF-C/NRP2 signaling axis thereby promoting tumor growth and chemoresistance. ATYR2810 counteracts the aggressive traits of tumors expressing high levels of VEGF-C resulting in enhanced tumor growth inhibition and sensitivity to both chemotherapy and targeted therapy. Citation Format: Alison Barber, Zhiwen Xu, Lisa Eide, Clara Polizzi, Max Pastenes, Lauren Guy, Jasmine Stamps, Kristina Hamel, Zachary Fogassy, Sofia Klopp-Savino, Esther Chong, Yang Qing, Lara Glendening, Christoph Burkart, Leslie Nangle. Resistance to cancer therapy via upregulation of the NRP2/VEGF-C axis can be neutralized by ATYR2810 [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 1758.

Abstract 2547: YAP1 associates with the EPHA3 receptor tyrosine kinase to promote prostate cancer progression

Abstract Metastatic castration-resistant prostate cancer is lethal, and the mechanisms contributing to this deadly disease are largely unknown. The disparity in our knowledge poses a significant barrier to developing effective therapeutics against advanced prostate cancer in humans. Evidence suggests that the ephrin family of receptor tyrosine kinase signaling may play a critical role in the pathobiology of prostate cancer progression. However, the mechanism is elusive. Our initial study showed that YAP1, in concert with the TEAD1 transcription factor, regulated EPHA3 expression. However, the significance of EPHA3 signaling in prostate cancer remains elusive. Here we investigated the role of EPHA3 signaling in prostate cancer cells. We showed that the expression of EPHA3 correlated with YAP1 in prostate cancer clinical samples. We also showed that EPHA3 knockout reduced cell growth in 2D and 3D cultures. In addition, EPHA3 loss sensitized castration-resistant prostate cancer cells to enzalutamide. Moreover, EPHA3 knockout reduced the GTPase activity of RHOA compared to cells with EPHA3-WT, which coincided with reduced phospho-ERK1/2 levels. Consistently, EPHA3 knockout reduces phosphor-ERK1/2, similar to the inhibition of phospho-ERK1/2 by an inhibitor of ROCK1 (Rho-associated coiled-coil-containing protein kinase 1). Besides, EPHA3 knockout reduced the expression of EMT markers ZEB1/2 and the cancer stem cell marker CD44 compared to the wild type. Furthermore, the levels of EPAH3 transcripts were higher in high-grade PC of AA patients compared to other races. Finally, AA and Caucasian Americans (CA) PC cell lines differentially express the member of ephrin-A and ephrin-B receptors. These observations suggest that the YAP1-EPHA3 signaling is a viable therapeutic target for patients with advanced prostate cancer. Citation Format: Marwah M. Al-Mathkour, Abdulrahman M. Dwead, Kezhan Khazaw, Bekir Cinar. YAP1 associates with the EPHA3 receptor tyrosine kinase to promote prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2547.

Abstract 2659: Anti-HER3 antibody, HMBD-001, in combination with an EGFR inhibitor effectively inhibits tumor growth in biomarker-selected pre-clinical models of squamous cell carcinomas

Abstract Squamous cell carcinomas (SCC) originate from stratified squamous epithelium in diverse anatomical locations that share common histological features and genetic mutations. Non-melanoma skin cancer, head and neck cancer (HNSCC), esophageal cancer (ESCC), and non-small cell lung cancer (sqNSCLC) make up the majority of SCC cases and together represent the largest subtype of cancer. There is a significant unmet need for effective treatment options post-progression of standard-of-care therapies. Alterations in receptor tyrosine kinase signaling pathways are commonly found in SCCs, e.g. EGFR is expressed in 90% of HNSCC, 76% of ESCC and 82% of sqNSCLC. However, the combination of EGFR targeting antibody cetuximab with chemotherapy confers limited clinical benefit and even initially sensitive tumors often develop resistance. Studies in SCC have implicated HER3, a key dimerization partner of HER family members, as one likely cause of treatment failure, through HER3 heterodimer activation of PI3K/AKT and MAPK/ERK pathways. In this study, we investigated the potential of combining HER3 and EGFR inhibitors to improve efficacy and overcome resistance. Previous attempts to target HER3 have shown limited clinical efficacy due to suboptimal HER3 inhibition, especially the lack of efficacy in ligand-independent activation mechanisms. They also lacked biomarker stratification for patients where HER3 inhibition would confer benefit. HMBD-001 is a clinical-stage anti-HER3 antibody which is being evaluated in a first-in-human, open-label, multi-center, dose escalation and expansion Phase I/IIa trial in patients with HER3 expressing advanced solid tumors (NCT05057013). HMBD-001 was rationally developed to uniquely block the HER3 heterodimerization interface to potently inhibit all HER3 dimer formation, including ligand-dependent and independent HER3 dimerization. Here, we show that dual blockade of EGFR and HER3 using cetuximab and HMBD-001 robustly inhibits tumor growth and is superior to either monotherapy. In multiple cell and patient derived SCC xenograft models, including models of HNSCC, ESCC and sqNSCLC, selected based on a novel gene signature that is robustly predictive of response, HMBD-001 in combination with cetuximab demonstrated up to 100% tumor growth inhibition and the abrogation of PI3K signalling. This combination was well tolerated with no relapse observed, even after a prolonged period of over 100 days in selected models. This study lays the foundation for testing HMBD-001 in combination with EGFR inhibitors in clinical settings. Better combination strategies of SoC and targeted therapeutics alongside a potent anti-HER3 mAb, in biomarker-selected patient populations, have the potential to meaningfully improve patient outcomes and prolong clinical benefit. Citation Format: Weiyi Toy, Dipti Thakkar, Khang Luu, Bui Ngoc Linh Chi, Jason Lai, Debleena Ray, Namita Gandhi, Alejandro Mas, Konrad Paszkiewicz, Piers Ingram, Jerome Boyd-Kirkup. Anti-HER3 antibody, HMBD-001, in combination with an EGFR inhibitor effectively inhibits tumor growth in biomarker-selected pre-clinical models of squamous cell carcinomas [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 2659.