Receptor Tyrosine Kinase Signaling Research Articles

MEK inhibitors lead to PDGFR pathway upregulation and sensitize tumors to RAF dimer inhibitors in NF1-deficient malignant peripheral nerve sheath tumor (MPNST).

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive subtype of soft tissue sarcoma with a high propensity to metastasize and extremely limited treatment options. Loss of the RAS-GAP NF1 leads to sustained RAF/MEK/ERK signaling in MPNST. However, single-agent MEK inhibitors (MEKi) have failed to elicit a sustained inhibition of the MAPK signaling pathway in MPNST. We employed pharmacological, biochemical, and genetic perturbations of the receptor tyrosine kinase (RTK) and MAPK signaling pathway regulators to investigate the mechanisms of MEKi resistance and evaluated combination therapeutic strategies in various preclinical MPNST models in vitro and in vivo. Here, we report that MEKi treatment resistance in MPNST involves two adaptive pathways: direct transcriptional upregulation of the receptor tyrosine kinase (RTK) PDGFRβ, and MEKi-induced increase in RAF dimer formation and activation of downstream signaling. While the pharmacological combination of MEKi with a PDGFRβ specific inhibitor was more effective than treatment with MEKi alone, the combination of MEKi and RAF-dimer inhibitors led to a robust inhibition of the MAPK pathway signaling. This combination treatment was effective in vitro and in vivo, as demonstrated by the significant increase in drug synergism and its high effectiveness in decreasing MPNST viability. Our findings suggest that the combination of MEKi and PDGFR and/or RAF dimer inhibitors can overcome MEKi resistance and may serve as a novel targeted therapeutic strategy for NF1-deficient MPNST patients, which in turn could impact future clinical investigations for this patient population.

The LCLAT1/LYCAT acyltransferase is required for EGF-mediated phosphatidylinositol-3,4,5-trisphosphate generation and Akt signaling.

Receptor tyrosine kinases such as EGF receptor (EGFR) stimulate phosphoinositide 3 kinases to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P2] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. PtdIns(3,4,5)P3 then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P3 partly achieves these functions by triggering activation of the kinase Akt, which phosphorylates targets like Tsc2 and GSK3β. Consequently, unchecked upregulation of PtdIns(3,4,5)P3-Akt signaling promotes tumor progression. Interestingly, 50-70% of PtdIns and PtdInsPs have stearate and arachidonate at sn-1 and sn-2 positions of glycerol, respectively, forming a species known as 38:4-PtdIns/PtdInsPs. LCLAT1 and MBOAT7 acyltransferases partly enrich PtdIns in this acyl format. We previously showed that disruption of LCLAT1 lowered PtdIns(4,5)P2 levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P3 signaling was not explored. Here, we show that LCLAT1 silencing in MDA-MB-231 and ARPE-19 cells abated the levels of PtdIns(3,4,5)P3 in response to EGF signaling. Importantly, LCLAT1-silenced cells were also impaired for EGF-driven and insulin-driven Akt activation and downstream signaling. Thus, our work provides first evidence that the LCLAT1 acyltransferase is required for receptor tyrosine kinase signaling.

A novel m6A reader RBFOX2 expression is increased in oral squamous cell carcinoma and promotes tumorigenesis

Background and ObjectiveOral squamous cell carcinoma (OSCC) is a significant global health concern due to its aggressive nature and poor prognosis. Recent research has highlighted the important role of RNA modifications in cancer biology, especially N6-methyladenosine (m6A) modifications, which are controlled by a complex interplay of m6A regulators. This study specifically investigates RBFOX2, a new m6A reader, and its involvement in OSCC. MethodsOur study primarily utilized OSCC tissue, adjacent normal tissue samples, OSCC cell lines, and normal healthy oral keratinocytes to validate RBFOX2 mRNA expression. Additionally, we used the TCGA-HNSCC dataset for large cohort analysis and clinicopathological characterization. Furthermore, we visualized the RBFOX2 network to identify the primary functions of the protein. ResultsOur research shows a noticeable increase in RBFOX2 expression in OSCC tissues compared to adjacent non-tumorous tissues, as determined by quantitative PCR and immunohistochemistry analyses. Functional pathway enrichment analysis revealed that RBFOX2 is involved in the receptor tyrosine kinase signaling pathway and the Hippo signaling pathway, which plays a critical role in oral cancer development and progression. Clinically, elevated RBFOX2 expression correlated with advanced tumor stages and poorer patient outcomes, demonstrating its prognostic value. These findings indicate that RBFOX2 acts as an oncogenic driver in OSCC as an m6A reader, facilitating the expression of m6A-modified oncogenes. ConclusionOur study identifies RBFOX2 as a critical player in OSCC pathogenesis and opens avenues for novel therapeutic strategies targeting the m6A regulatory machinery in this malignancy.

Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases

Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases.

Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

Receptor Tyrosine Kinase Signaling Involves Echinococcus-Host Intercommunication: A Potential Therapeutic Target in Hepatic Echinococcosis.

Echinococcosis, one of the most serious and life-threatening parasitic forms of zoonosis worldwide, is caused by the larvae of Echinococcus granulosus (E. granulosus) and Echinococcus multilocularis (E. multilocularis). Various drugs are being applied clinically to treat zoonosis; however, their therapeutic efficacy remains a great challenge, especially with albendazole as the preferred drug of choice. Receptor tyrosine kinase (RTK) signaling controls normal cellular proliferation, differentiation, and metabolism in humans and mammals, which are intermediate hosts of E. granulosus and E. multilocularis. Disruption of RTK signaling can cause various forms of carcinogenesis and exacerbate the progression of certain forms of parasitic disease. As a result, a significant number of studies on tyrosine kinase inhibitors (TKIs) have been conducted for the treatment of cancer and parasitic infection, with some TKIs already approved for clinical use for cancer. Notably, RTK signaling has been identified in the parasites E. granulosus and E. multilocularis; however, the mechanisms of RTK signaling response in Echinococcus-host intercommunication are not fully understood. Thus, understanding the RTK signaling response in Echinococcus-host intercommunication and the potential effect of RTK signaling is crucial for identifying new drug targets for echinococcosis. The present review illustrates that RTK signaling in the host is over-activated following infection by E. granulosus or E. multilocularis and can further facilitate the development of metacestodes in vitro. In addition, some TKIs exert strong parasitostatic effects on E. granulosus or E. multilocularis, both in vitro and/or in vivo, through downregulation of RTK signaling molecules. The summarized findings suggest that RTK signaling may be a promising drug target and that TKIs could be potential anti-Echinococcus drugs warranting further research.

Effects of heterologous kinase domains on growth factor receptor specificity

The kinase domains of receptor tyrosine kinases (RTKs) are highly conserved, yet they are able to discriminate among potential substrates to selectively activate downstream signaling pathways. In this study, we tested the importance of catalytic domain specificity by creating two series of chimeric RTKs. In one set, the kinase domain of insulin-like growth factor I receptor (IGF1R) was replaced by the kinase domains from insulin receptor (IR), macrophage stimulating protein 1 receptor/Ron (Ron) or Src. In the other set of chimeras, the kinase domain of epidermal growth factor receptor (EGFR) was similarly replaced by the kinase domains of IR, Ron, or Src. We expressed the wild-type and chimeric forms of the receptors in mammalian cells. For some signaling events, such as recognition of IRS1, the identity of the tyrosine kinase catalytic domain did not appear to be crucial. In contrast, recognition of some sites, such as the C-terminal autophosphorylation sites on EGFR, did depend on the identity of the kinase domain. Our data also showed that ligand dependence was lost when the native kinase domains were replaced by Src, suggesting that the identity of the kinase domains could be important for proper receptor regulation. Overall, the results are consistent with the idea that the fidelity of RTK signaling depends on co-localization and targeting with substrates, as well as on the intrinsic specificity of the kinase domain.

Expression of the non-coding RNA nc886 facilitates the development of tyrosine kinase inhibitor resistance in EGFR-mutated non-small-cell lung cancer cells

Treatment of non-small-cell lung cancer (NSCLC) patients possessing EGFR-activating mutations with tyrosine kinase inhibitors (TKIs) can confer an initial promising response. However, TKI resistance inevitably arises. Numerous TKI resistance mechanisms are identified including EGFR secondary mutations, bypass receptor tyrosine kinase (RTK) signaling, and cellular transition e.g. epithelial-mesenchymal transition (EMT). To increase the knowledge of TKI resistance we performed an epigenetic screen to identify small non-coding (nc) genes with DNA methylation alterations in HCC827 NSCLC EGFR-mutated cells with acquired TKI resistance. We analyzed Infinium Methylation EPIC 850K Array data for DNA methylation changes present in both TKI-resistant HCC827 cells with EMT and MET-amplification. Hereby, we identified that the polymorphic maternal imprinted gene nc886 (vtRNA2-1) has a decrease in promoter DNA methylation in TKI-resistant cells. This epigenetic change was associated with an increase in the expression of nc886. The induction of EMT did not affect nc886 expression. CRISPR/Cas9-mediated distortion of the nc886 sequence increased the sensitivity of HCC827 cells towards TKI. Finally, nc886 sequence distortion hindered MET RTK activation and instead was EMT the endpoint TKI resistance mechanism. In conclusion, the expression of nc886 contributes to TKI resistance in the HCC827 NSCLC cell line by supporting cell survival and selection of the endpoint TKI resistance mechanism. We propose DNA methylation and expression changes for nc886 to constitute a novel TKI resistance contributing mechanism in NSCLC.

Immunoreactivity of LMO7 and other molecular markers as potential prognostic factors in oropharyngeal squamous cell carcinoma

BackgroundDespite the better prognosis associated with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC), some patients experience relapse and succumb to the disease; thus, there is a need for biomarkers identifying these patients for intensified treatment. Leucine-rich repeats and immunoglobulin-like domain (LRIG) protein 1 is a negative regulator of receptor tyrosine kinase signaling and a positive prognostic factor in OPSCC. Studies indicate that LRIG1 interacts with the LIM domain 7 protein (LMO7), a stabilizer of adherence junctions. Its role in OPSCC has not been studied before.MethodsA total of 145 patients diagnosed with OPSCC were enrolled. Immunohistochemical LMO7 expression and staining intensity were evaluated in the tumors and correlated with known clinical and pathological prognostic factors, such as HPV status and LRIG1, CD44, Ki67, and p53 expression.ResultsOur results show that high LMO7 expression is associated with significantly longer overall survival (OS) (p = 0.044). LMO7 was a positive prognostic factor for OS in univariate analysis (HR 0.515, 95% CI: 0.267–0.994, p = 0.048) but not in multivariate analysis. The LMO7 expression correlated with LRIG1 expression (p = 0.048), consistent with previous findings. Interestingly, strong LRIG1 staining intensity was an independent negative prognostic factor in the HPV-driven group of tumors (HR 2.847, 95% Cl: 1.036–7.825, p = 0.043).ConclusionsWe show for the first time that high LMO7 expression is a positive prognostic factor in OPSCC, and we propose that LMO7 should be further explored as a biomarker. In contrast to previous reports, LRIG1 expression was shown to be an independent negative prognostic factor in HPV-driven OPSCC.

Genetic code expansion, click chemistry, and light-activated PI3K reveal details of membrane protein trafficking downstream of receptor tyrosine kinases.

Ligands such as insulin, epidermal growth factor, platelet derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell- impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

N-phenyl pyrazoline derivative inhibits cell aggressiveness and enhances paclitaxel sensitivity of triple negative breast cancer cells

Protein kinase dysregulation induces cancer cell aggressiveness leading to rapid tumor progression and poor prognosis in TNBC patients. Many small-molecule kinase inhibitors have been tested in clinical trials to treat TNBC patients. In the previous study, we found that N-phenylpyrazoline small molecule acts as a protein kinase inhibitor in cervical cancer cells. However, there remains unknown about N-phenyl pyrazoline potency as a kinase inhibitor and its anti-cancer activity in TNBC cells. In this study, we investigated the activity of N-phenyl pyrazoline against TNBC cells via tyrosine kinase inhibition. Based on the MTT assay, the IC50 values for the N-phenyl pyrazoline 2, 5, A, B, C, and D against Hs578T were 12.63 µM, 3.95 µM, not available, 18.62 µM, 30.13 µM, and 26.79 µM, respectively. While only P5 exhibited the IC50 against MDA MB 231 (21.55 µM). Further, N-phenyl pyrazoline 5 treatment significantly inhibited the cell proliferation rate of Hs578T and MDA MB 231 cells. The migration assay showed that treatment with the compound N-phenyl pyrazoline 5 with 4 µM concentration significantly reduced cell migration of Hs578T cells. N-phenyl pyrazoline 5 treatment at 1 µM and 2 µM was able to reduce the tumorsphere size of Hs578t cells. A combination treatment of P5 and paclitaxel showed a synergistic effect with a combination index score > 1 in both TNBC cells. Further, the P5 predictively targeted the protein kinases that significantly correlated to breast cancer prognosis. The GSEA analysis result shows that receptor tyrosine kinase, Notch3, Notch4, and Ephrin signaling pathways were targeted by P5. The P5 treatment reduced the EGFR expression level and activation in TNBC cells.

Decoding the anti-cancer potential of Pexidartinib (PLX3397), a Fms-like tyrosine kinase 3 inhibitor, using next-generation knowledge discovery methods.

Acute Myeloid Leukemia (AML) is a complex hematologic malignancy characterized by the rapid proliferation of abnormal myeloid precursor cells. The FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase, plays a pivotal role in regulating cell survival, proliferation, and differentiation within the hematopoietic system. Mutations in FLT3, particularly internal tandem duplications (ITDs) and point mutations within the tyrosine kinase domain (TKD), are prevalent in AML and are associated with poor prognosis and increased risk of relapse. The development of targeted therapies has revolutionized the landscape of cancer treatment by focusing on the inhibition of kinase signalling. Small-molecule inhibitors designed to selectively target receptor tyrosine kinases, such as PLX3397, have shown promising results in preclinical studies and early phase clinical trials. PLX3397 exerts its inhibitory effects by targeting CSF1R and KIT, leading to the disruption of receptor tyrosine kinase signalling cascades, suppression of leukemic cell growth, and induction of apoptosis. This study emphasizes the significance of FLT3 as a receptor tyrosine kinase as a therapeutic target for PLX3397. After evaluating the usefulness of PLX3397 as an enzyme inhibitor using ADMET prediction, PLX3397 was prepared for molecular docking in the FLT3 crystal structure (PDB: 4XUF). A molecular dynamics simulation was performed on PLX3397 to evaluate its binding affinity and protein stability in a simulated physiological environment. In conclusion, targeting FLT3 as a receptor tyrosine kinase with PLX3397 represents a promising therapeutic strategy for improving outcomes in patients with FLT3-mutated AML. Further clinical investigations are warranted to validate the efficacy and safety of PLX3397 and to optimize treatment strategies for AML patients based on the FLT3 mutational status.

Targeting NG2 relieves the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitors

BRAFV600E represents a constitutively active onco-kinase and stands as the most prevalent genetic alteration in thyroid cancer. However, the clinical efficacy of small-molecule inhibitors targeting BRAFV600E is often limited by acquired resistance. Here, we find that nerve/glial antigen 2 (NG2), also known as chondroitin sulfate proteoglycan 4 (CSPG4), is up-regulated in thyroid cancers, and its expression is increased with tumor progression in a BRAFV600E-driven thyroid cancer mouse model. Functional studies show that NG2 knockout almost does not affect tumor growth, but significantly improves the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4720. Mechanistically, the blockade of ERK-dependent feedback by BRAF inhibitor can activate receptor tyrosine kinase (RTK) signaling, causing the resistance to this inhibitor. NG2 knockout attenuates the PLX4720-mediated feedback activation of several RTKs, improving the sensitivity of BRAF-mutant thyroid cancer cells to this inhibitor. Based on this finding, we propose and demonstrate an alternative strategy for targeting NG2 to effectively treat BRAF-mutant thyroid cancers by combining multiple kinase inhibitor (MKI) Sorafenib or Lenvatinib with PLX4720. Thus, this study uncovers a new mechanism in which NG2 contributes to the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitor, and provides a promising therapeutic option for BRAF-mutant thyroid cancers.

Abstract LT05: METTL8-MEDIATED EPITRANSCRIPTOMIC REGULATION OF GBM CYCLING STATE IS DEPENDENT ON RTK/PI3K/AKT SIGNALING

Abstract Glioblastoma (GBM) is a lethal brain tumor with limited treatment options. Treatment failures are often attributed to the existence of glioma stem cells (GSCs), which confer intratumoral heterogeneity and therapy resistance. Contrary to common belief that cancer cells employ aerobic glycolysis (Warburg effect) to sustain their metabolic requirements, GSCs rely on oxidative phosphorylation (OXPHOS) for energy production. As a result, GSCs are uniquely sensitive to OXPHOS inhibitors (Gboxin), as well as mitochondrial translation inhibitors which disrupt OXPHOS. METTL8 is a newly discovered mitochondrial methyltransferase which catalyzes m3C32 modifications in mitochondrial tRNAsThr/Ser(UCN) (mt-tRNAs). This post-transcriptional modification ensures the optimal structure of mt-tRNAs to facilitate efficient translation of mitochondrial proteins involved in electron transport chain (ETC). Although the epitranscriptomic functions of METTL8 have been well characterized, its roles in cancers (i.e. glioblastoma) remain largely unexplored. In this study, we showed that METTL8 is overexpressed in GBM and high METTL8 expression portends poor patient prognosis. In GSC, METTL8 is under the transcriptional regulation of HIF-1α, and the chromatin accessibility at METTL8 promoter is facilitated by the deposition of histone variant H2AZ. METTL8 depletion downregulated m3C32 modifications in mt-tRNAsThr/Ser(UCN) in GSC, leading to impaired mitochondrial translation and OXPHOS defects. Phenotypically, METTL8 knockdown (KD) compromised GSCs’ self-renewal, proliferation, invasiveness and tumorigenicity. Interestingly, METTL8-KD GSC acquired a slow cycling state which is linked to reduced receptor tyrosine kinase (RTK) signaling. Consistently, METTL8-KD GSC showed lower Akt phosphorylation and exhibited increased sensitivity to mTOR/Akt inhibitors. Finally, our mechanistic findings led to the identification of a novel HIF1-α/Akt inhibitor drug combination which faithfully recapitulates the phenotypes of METTL8-KD GSCs. In conclusion, our findings provide the first in-depth study of METTL8 roles in GBM pathogenesis, and highlights a novel drug combination which could potentially be used to treat this dreadful disease. Citation Format: Bernice Woon Li Lee, You Heng Chuah, Jeehyun Yoon, Oleg V. Grinchuk, Jia Feng, Yajing Liang, Jayshree L Hirpara, Ya Ting Shen, Loo Chien Wang, Yan Ting Lim, Tianyun Zhao, Radoslaw M Sobota, Tan Boon Toh, Shazib Pervaiz, Zhewang Lin, Derrick Sek Tong Ong. METTL8-MEDIATED EPITRANSCRIPTOMIC REGULATION OF GBM CYCLING STATE IS DEPENDENT ON RTK/PI3K/AKT SIGNALING [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT05.

Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN’s involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization.Graphical

Exploring Emerging Therapeutic Targets and Opportunities in Neuroendocrine Tumors: Updates on Receptor Tyrosine Kinases

Neuroendocrine tumors (NETs) represent a diverse group of neoplasms originating from neuroendocrine cells, presenting varied clinical behaviors and posing significant challenges in management. This review explores the emerging roles of receptor tyrosine kinases (RTKs) in the pathogenesis and progression of NETs, including vascular endothelial growth factor receptors (VEGFRs), insulin-like growth factor receptors (IGF-1R), RET, epidermal growth factor receptor (EGFR), and ALK. The dysregulation of RTK signaling pathways contributes to key cellular processes such as proliferation, survival, and invasion in NETs. We discuss the potential of targeting RTKs as therapeutic strategies in NETs, with a focus on recent developments in RET inhibitors and the therapeutic implications of RTK alterations.

Abstract 3940: Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8

Abstract Breast cancer is a highly complex multifactorial disease, which can be driven by the aberrant regulation of different signaling pathways including receptor tyrosine kinase (RTK) signaling. Notably, while RTKs have been classically described as transmembrane receptors, they can also translocate to the nucleus, even though the functional importance of this process remains largely unexplored for most RTKs. Here, we report a novel role for the nuclear form of the RTK human epidermal growth factor receptor 3 (HER3) in driving primary breast cancer growth and metastasis. Firstly, using different patient-derived organoids (PDOs) established from metastatic breast cancer patients, we demonstrated the robust translocation of the activated phosphorylated HER3 receptor (pHER3) from the plasma membrane to the nucleus in response to its ligand Neuregulin 1 (NRG1). Interestingly, the nuclear translocation was observed not only in the HER2-positive breast cancer subtype but also in luminal and triple-negative breast cancer-derived cells. In order to decipher the functional role of nuclear HER3 (nHER3), we identified and mutated its nuclear localization signal, resulting in decreased nuclear translocation of the receptor while its membrane form remained intact. This reduction in nHER3 remarkably impeded primary tumor growth and metastatic spread in different patient-derived xenograft (PDX) models. Conversely, selective overexpression of HER3 in the nucleus led to increased primary tumor growth and metastatic burden in vivo. In order to decipher the mechanism of action of nHER3, we performed co-immunoprecipitation followed by global mass spectrometric analysis to identify the protein binding partners of the receptor in the nucleus. Specifically, nHER3 was found to interact with different transcription factors such as the AP-2 family of transcription factors and with major chromatin remodeling complexes like the nucleosome remodeling and deacetylase (NuRD) complex, implicating its role in transcriptional regulation. Furthermore, gene expression analysis of PDOs expressing the wild-type or the mutated form of HER3 revealed the modulation of several key growth regulators such as early growth response 3 (EGR3) by nHER3. The nHER3-EGR3 axis further controlled downstream effectors like the immune chemoattractants CXCL1 and CXCL8. Accordingly, PDX from breast cancer cells overexpressing nHER3 showed significantly increased immune infiltration, suggesting a possible role of nHER3 in regulating the tumor microenvironment. Altogether, we report here a novel non-canonical role of the nuclear form of HER3 receptor in driving breast tumorigenesis, with key implications for our understanding and targeting of RTK signaling in breast cancer. Citation Format: Tasneem Cheytan, Roberto Würth, Nina Hahnen, Elisa Donato, Corinna Klein, Rebecca Weber, Daniele Colombo, Jeroen Krijgsveld, Martin Sprick, Andreas Trumpp. Nuclear translocation of HER3 promotes breast cancer progression and dissemination recruiting immune cells via CXCL1 and CXCL8 [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 3940.

Abstract 3875: Glioblastoma mutational profiles drive cancer cell signaling and immune evasion

Abstract Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with a median survival below two years. Characterization of GBM through gene expression profiling and DNA sequencing has identified disease subgroups and commonly altered genes and pathways, including alterations in the genes encoding neurofibromin-1 (NF1) and the epidermal growth factor receptor (EGFR). NF1 and EGFR are involved in receptor tyrosine kinase (RTK), RAS/MEK/ERK, and PI3K signaling, and together, are genetically altered in over 60% of adult GBMs. Interestingly, NF1 and EGFR alterations in GBM are almost always mutually exclusive. Here, we designed a multimodal approach integrating spatial proteomics, transcriptomics, and genomics to measure cell composition, immune cell function, cancer cell signaling, cellular spatial organization, gene expression, transcriptional subtypes, and genome alterations in 80 NF1 and EGFR altered human GBMs with extensively curated treatment and survival data. We quantified the spatial expression of 42 proteins in over 31 million single cells measuring RTK/RAS/PI3K and Rb signaling and features of the TME, including 23 cell types covering tumor cells, immune cells, and brain resident cells. In addition to tumor genotype labels, we classified each GBM tumor by its transcriptional subtype (i.e., mesenchymal, classical, or proneural). We identified genotype-phenotype associations in GBM including hyperactive RAS signaling cellular niches, a macrophage-rich TME, and an epithelial-mesenchymal signature in NF1 altered tumors and hyperactive RTK signaling and an immune desert TME in EGFR amplified tumors. Furthermore, we observed spatial heterogeneity in cancer cell signaling patterns suggestive of the presence of different tumor cell clones within different regions of each GBM tumor. Together, we identify several genotype-associated features of GBMs, reinforcing the value of integrating spatial multi-omic data for generation of genotype-driven therapeutic hypotheses, with implications for GBM genotype-based therapeutic stratification. Citation Format: Maryam Pourmaleki, Brian D. Greenstein, Caitlin J. Jones, Subhiksha Nandakumar, Daniel A. Navarrete, Smrutiben Mehta, Carl Campos, Travis J. Hollmann, Nicholas D. Socci, Tejus Bale, Sohrab P. Shah, Ingo K. Mellinghoff. Glioblastoma mutational profiles drive cancer cell signaling and immune evasion [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 3875.

Abstract 5947: Discovery of ACE-47228211, a highly potent and selective SOS1 inhibitor

Abstract Dysregulation of receptor tyrosine kinase (RTKs) and KRAS signaling is the most frequent causes of cancer development and progression. SOS1 is one of the major guanine nucleotide exchange factors (GEFs) that facilitates the conversion of the inactive GDP-bound KRAS “off” state to the activated GTP-bound KRAS “on” state. SOS1 is therefore required for dysregulated RTKs, and wildtype and mutant KRAS proteins to drive cancer cells. In addition, SOS1 plays a crucial role in modulating the reactivation of the RAS/MAPK signaling upon the treatment of RAS/MEK/ERK inhibitors. Thus, blocking SOS1 and KRAS protein-protein interactions could be an effective strategy for targeting KRAS-MAPK driven cancers. Here, we report the discovery of a highly potent and orally bioavailable small molecule SOS1 inhibitor, ACE-47228211. This molecule has a unique novel scaffold. It exhibits an IC50 of 8.5 nM in the SOS1-KrasG12D (GDP) exchange assay and 5.0 nM in cellular ERK phosphorylation assay in H1957 cells. ACE-47228211 at 3 mg/kg (BID) demonstrated significant tumor growth inhibition in MiaPaca-2 KRAS G12C tumor model than MRTX0920 at 50 mg/kg (BID). When combined with Sotorasib (5 mg/kg QD), a Kras G12C inhibitor, ACE-47228211 at 3 mg/kg exhibited synergistic effect similar to MRTX0920 at 50 mg/kg. Further, ACE-47228211 demonstrated synergistic effects with EGFR and FGFR2 inhibitors in RTK mutant tumor models. ACE-47228211 has excellent ADME and physical properties with low risks of hERG, CYP inhibition and drug-drug interactions. The evaluation of ACE-47228211 in a safety panel of 87 targets and a kinase panel of >500 targets shows that this molecule has favorable selectivity and safety profiles. The molecule is currently in IND enabling stage of development. Citation Format: Junmei Wang, Wenqian Li, Sanjeev Kumar, Weitao Pan, Zhiming Wen, Tinggui Yin, Genshi Zhao, Kuo-Long Yu. Discovery of ACE-47228211, a highly potent and selective SOS1 inhibitor [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 5947.

Abstract 3220: Osimertinib-induced epigenetic and transcriptional reprogramming drives a BRD4-POUF-PDGFRB-YAP-EMT axis of resistance in NSCLC

Abstract Acquired resistance to Osimertinib continues to pose a major limitation to achieving durable therapeutic responses in EGFR mutant non-small cell lung cancer (NSCLC). While multiple actionable genetic mutations have been identified as the underlying molecular basis of resistance, a genomic mechanism cannot be identified in >50% of Osimertinib resistant tumors and thus an effective combinatorial treatment strategy cannot be deployed for these patients. Recently, we identified YAP/TEAD activation and ERK1/2 reactivation as two major non-mutational counter-regulatory pathways that diminish the efficacy of Osimertinib, promoting establishment of the drug tolerant persister (DTP) and resistant states. Moreover, it is well known that cancer cells escape tyrosine kinase inhibitor (TKI) therapy by developing dependence on alternate, non-mutated receptor tyrosine kinases (RTKs) through a treatment-induced “RTK switch” phenomenon. The link however between alternate RTK signaling and YAP/TEAD/ERK1/2 has never been explored in the context of Osimertinib resistance. In this study, we systemically uncover transcriptional de-repression of, and critical dependence on PDGFRβ as part of a novel “RTK switch” mechanism deployed by cells to evade Osimertinib-induced death. Genetic or pharmacologic co-inhibition of PDGFRβ renders NSCLC cells more sensitive to Osimertinib during drug naïve, DTP and resistant phases of treatment through suppression of ERK1/2 reactivation, YAP activity/nuclear translocation and YAP-mediated induction of EMT factors, SLUG and Zeb2. Importantly, compared to Osimertinib alone, co-targeting EGFR and PDGFRβ robustly suppresses tumor regrowth following treatment cessation both in vitro and in vivo which is consistent with a reduction in the DTP cell population. These data auspiciously demonstrate a two-fold utility of concomitant PDGFRβ inhibition: to enhance the initial efficacy of Osimertinib and to eliminate the DTP and resistant cells following chronic Osimertinib treatment. Further mechanistic studies from integration of ATAC-seq, CUT&RUN and RNA-seq reveal that Osimertinib induces the dynamic cooperativity of chromatin modulator BRD4 with pluripotent POU-domain transcription factors Oct-4, Pit-1 and Brn-3 at putative proximal enhancer elements, facilitating upregulation of PDGFRβ. Collectively, we demonstrate a novel treatment-induced BRD4/POUF epigenetic/transcriptional mediated “RTK switch” that involves co-opting PDGFRβ signaling to activate YAP/TEAD and ERK1/2 as a means of driving adaptive resistance to Osimertinib. These data provide compelling rationale for combined anti-EGFR and PDGFRβ targeted therapy to improve patient outcomes. Citation Format: Antja-Voy Hartley, Caitlyn Weston, Simon Baldacci, William Feng, Mustafa Al-Dulaimi, Claudia Gentile, Matthew Booker, Michael Tolstorokuv, Cigall Kadoch, Pasi Janne. Osimertinib-induced epigenetic and transcriptional reprogramming drives a BRD4-POUF-PDGFRB-YAP-EMT axis of resistance 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 3220.