Receptor Tyrosine Kinase Activity Research Articles

PATH-68. THE CLINICAL AND PROTEOMIC CLASSIFICATION FOR THE PRECISION TREATMENT STRATEGY OF CHORDOMA

Abstract Chordoma is a rare and heterogeneous mesenchymal malignancy, with distinct clinical and biological behaviors. Till now, its comprehensive clinical-molecular characteristics and accurate molecular classification remain obscure, leading to numerous diagnostic and therapeutic challenges. In this research, we enroll a large cohort of chordoma (102 patients) and describe their clinical, imageological and histopathological features. Through tandem mass tag-based proteomic analysis and nonnegative matrix factorization clustering, we classify chordoma into the following three molecular subtypes: bone microenvironment dominant, mesenchymal derived, and mesenchymal to epithelial transition mediated pattern. The three subtypes exhibit discrete clinical prognosis and distinct biological attributes of osteoclastogenesis and immunogenicity, oxidative phosphorylation, and receptor tyrosine kinase activation, suggesting targeted therapeutic strategies of Denosumab, S-Gboxin and Anlotinib, respectively. Notably, these approaches demonstrate positive treatment outcomes for each subtype in vivo and in vitro, as well as in patient-derived xenograft models. Altogether, this work sheds light on the clinical-proteomic characteristics of chordoma that deepens our understanding of this rare disease and provides candidate precision treatment strategy for chordoma according to molecular classification, underscoring their potential for clinical application.

CSIG-31. ANNEXIN A7 ALTERNATIVE SPLICING IN GLIOMA STEM CELLS FACILITATES THE DEGRADATION OF EGFR BY PHOSPHORYLATING TYROSINE RESIDUES EXCLUSIVE TO PROTEIN ISOFORM 1

Abstract The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling and resulting in hyperactive RTK signaling. Exon six of Annexin A7 (ANXA7) can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates in PDX glioma stem cells (GSCs). We demonstrated that ANXA7 exon skipping supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. JX14 GSCs were made to overexpress ANXA7-I1 with lentivirus to conduct immunofluorescence experiments measuring EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway for the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P>0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (R2=0.9267, P<0.0001). Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P<0.0001). On the other hand, ANXA7 siRNA KD in I1 cells impaired EGFR sorting via a 40% increase (P<0.0010) in total EGFR protein and a 40% reduction (P<0.0001) in colocalization with EEA1 to prefer recycling again while EV cells were unaffected. I1 overexpression significantly reduced EGFR signaling through degradation, which is driven by the phosphorylation of I1-exclusive tyrosine residues in phosphomimetic (p<0.01) and non-phosphorylatable protein mutants. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for GSCs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition.

CSIG-30. EXPLORING THE ROLE OF CAPICUA (CIC) IN RESISTANCE MECHANISMS OF MEK INHIBITORS IN GLIOBLASTOMA

Abstract Glioblastoma multiforme (GBM) is the most common and fatal primary brain tumor, resistant to conventional therapy. Despite its heterogeneous nature, a key feature of GBM is aberrant kinase signaling. Specifically, hyperactivation of receptor tyrosine kinases (RTK) and their downstream tumorigenic effectors Ras/MEK/Extracellular Signal Regulated Kinase (ERK) pathway are attributed to the aggressive nature of GBM. Capicua (CIC) is a High Mobility Group (HMG) box transcriptional repressor that directly binds to DNA and counteracts the transcription of genes typically expressed only in response to RTK activation such as oncogenic transcription factors ETV1/4/5. Hyperactive MEK/ERK signaling in GBM causes CIC degradation. The mechanism of CIC’s repressor function is not well understood, however, transcriptional repressors (like CIC) typically form co-repressor complexes to repress target genes. We discovered a novel interaction between CIC and the transcriptional regulator yin yang 1 (YY1), a context-dependent transcription factor that can repress or activate gene expression. We uncovered that 90-kDa ribosome S6 protein kinase (p90RSK), a downstream effector of ERK and a known CIC regulator, mediates the CIC/YY1 interaction. Interestingly, we found re-activation of p90RSK following MEK/ERK inhibition implicating p90RSK in the resistance mechanism of RTK/MEK/ERK inhibition in GBM. We hypothesize that p90RSK inhibition will stabilize the CIC/YY1 co-repressor complex and re-sensitize towards MEK/ERK inhibition. Patient-derived glioma stem-like cells (GSCs) were treated with increasing doses of BI-D1870 or LJI308 (p90RSK inhibitors) with and without selumetinib (MEK inhibitor) and various functional assays were performed to examine the effect of p90RSK inhibition on the CIC/YY1 complex and sensitization to MEK/ERK inhibition. Treatment with BI-D1870 and LJI308 markedly reduced viability and mRNA expression of ETV1/4/5 indicating that p90RSK inhibition may mediate CIC /YY1 DNA binding ability. Importantly, inhibiting p90RSK sensitized GSCs to MEK/ERK inhibition. These results highlight the importance of downstream p90RSK inhibition in stabilizing CIC/YY1 repressor complex function and sensitizing cells to RTK/MEK/ERK inhibition.

β-hydroxybutyrate ameliorates osteoarthritis through activation of the ERBB3 signaling pathway in mice.

The ketogenic diet (KD) has demonstrated efficacy in ameliorating inflammation in rats with osteoarthritis (OA). However, the long-term safety of the KD and the underlying mechanism by which it delays OA remain unclear. We found that while long-term KD could ameliorate OA, it induced severe hepatic steatosis in mice. Consequently, we developed two versions of ketogenic-based diets: KD supplemented with vitamin D and intermittent KD. Both KD supplemented with vitamin D and intermittent KD effectively alleviated OA by significantly reducing the levels of inflammatory cytokines, cartilage loss, sensory nerve sprouting, and knee hyperalgesia without inducing hepatic steatosis. Furthermore, β-hydroxybutyrate (β-HB), a convenient energy carrier produced by adipocytes, could ameliorate OA without causing liver lesions. Mechanistically, β-HB enhanced chondrocyte autophagy and reduced apoptosis through the activation of Erb-B2 receptor tyrosine kinase 3 (ERBB3) signaling pathway; a pathway which was down-regulated in the articular chondrocytes from both OA patients and mice. Collectively, our findings highlighted the potential therapeutic value of β-HB and KD supplemented with vitamin D and intermittent KD approaches for managing OA.

A multiplex single-cell RNA-Seq pharmacotranscriptomics pipeline for drug discovery.

The gene-regulatory dynamics governing drug responses in cancer are yet to be fully understood. Here, we report a pipeline capable of producing high-throughput pharmacotranscriptomic profiling through live-cell barcoding using antibody-oligonucleotide conjugates. This pipeline combines drug screening with 96-plex single-cell RNA sequencing. We show the potential of this approach by exploring the heterogeneous transcriptional landscape of primary high-grade serous ovarian cancer (HGSOC) cells after treatment with 45 drugs, with 13 distinct classes of mechanisms of action. A subset of phosphatidylinositol 3-OH kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) inhibitors induced the activation of receptor tyrosine kinases, such as the epithelial growth factor receptor (EGFR), and this was mediated by the upregulation of caveolin 1 (CAV1). This drug resistance feedback loop could be mitigated by the synergistic action of agents targeting PI3K-AKT-mTOR and EGFR for HGSOC with CAV1 and EGFR expression. Using this workflow could enable the personalized testing of patient-derived tumor samples at single-cell resolution.

Label-Free SERS Sensors for Real-Time Monitoring of Tyrosine Phosphorylation.

Dysregulation of receptor tyrosine kinases (RTKs) has been shown to correlate with cancer cell proliferation and drug resistance. Thus, monitoring the activity of RTKs at a chemical level could provide new biomedical insights and methods to assess the drug efficacy. However, direct monitoring of kinase activity is challenging and most commonly relies on in vitro techniques such as Western blotting and ELISAs. Herein, we report the development of a gold nanoparticle-based surface-enhanced Raman scattering (SERS) sensor, which allows the real-time monitoring of tyrosine phosphorylation of a reporter peptide (Axltide) by the Axl enzyme. We demonstrate that our sensor shows strong signal localization, and we are able to detect tyrosine phosphorylation of the reporter peptide through chemical phosphorylation and enzymatically with similar peak changes to those observed in the spontaneous Raman spectra. Through monitoring the SERS spectrum, we can observe changes in phosphorylation in real time.

SPROUTY2, a Negative Feedback Regulator of Receptor Tyrosine Kinase Signaling, Associated with Neurodevelopmental Disorders: Current Knowledge and Future Perspectives

Growth-factor-induced cell signaling plays a crucial role in development; however, negative regulation of this signaling pathway is important for sustaining homeostasis and preventing diseases. SPROUTY2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase (RTK) signaling that binds to GRB2 during RTK activation and inhibits the GRB2-SOS complex, which inhibits RAS activation and attenuates the downstream RAS/ERK signaling cascade. SPRY was formerly discovered in Drosophila but was later discovered in higher eukaryotes and was found to be connected to many developmental abnormalities. In several experimental scenarios, increased SPRY2 protein levels have been observed to be involved in both peripheral and central nervous system neuronal regeneration and degeneration. SPRY2 is a desirable pharmaceutical target for improving intracellular signaling activity, particularly in the RAS/ERK pathway, in targeted cells because of its increased expression under pathological conditions. However, the role of SPRY2 in brain-derived neurotrophic factor (BDNF) signaling, a major signaling pathway involved in nervous system development, has not been well studied yet. Recent research using a variety of small-animal models suggests that SPRY2 has substantial therapeutic promise for treating a range of neurological conditions. This is explained by its function as an intracellular ERK signaling pathway inhibitor, which is connected to a variety of neuronal activities. By modifying this route, SPRY2 may open the door to novel therapeutic approaches for these difficult-to-treat illnesses. This review integrates an in-depth analysis of the structure of SPRY2, the role of its major interactive partners in RTK signaling cascades, and their possible mechanisms of action. Furthermore, this review highlights the possible role of SPRY2 in neurodevelopmental disorders, as well as its future therapeutic implications.

Oxidative protein damage negatively affects protein-protein interaction: The case of KRAS-cRAF

Protein-protein interactions (PPIs) play crucial roles in cellular signaling, transmitting signals from the cell surface to its interior. One of the most important signaling cascades is the RAS-RAF-MEK-ERK pathway. This pathway is initiated by various upstream signaling reactions, including receptor tyrosine kinase (RTK) activation, and it controls many biological functions like cell proliferation, differentiation, and survival. Once RAS is activated, it binds RAF and relays the signal to downstream proteins. The RAS-binding domain (RBD) in RAF protein plays a crucial role in this process, facilitating the RAS-ERK pathway signaling.In this study, we explored the effect of oxidative stress induced by UV radiation on the KRAS-RBD interaction. Using the Split Intein-Mediated Protein Ligation (SIMPL) method, we assessed the impact of different UV doses on KRAS-RBD interactions and observed a disruption of this interaction at higher doses. UV-treated samples exhibited high levels of protein carbonylation, as detected by Oxime Blot and mass spectrometry (MS) analysis, indicating oxidative damage. The MS results provided detailed insights into specific carbonylation modifications on the KRAS protein.Our study demonstrates that protein oxidation and carbonylation can disrupt protein-protein interactions, specifically the KRAS/c-RAF interaction. These findings highlight the impact of oxidative stress on signaling pathways, such as those triggered by UV irradiation. A deeper understanding of these molecular changes may aid in developing therapies targeting diseases linked to oxidative stress, including cancer.

Clinical-proteomic classification and precision treatment strategy of chordoma

Chordoma is a rare and heterogeneous mesenchymal malignancy, with distinct clinical and biological behaviors. Till now, its comprehensive clinical-molecular characteristics and accurate molecular classification remain obscure. In this research, we enroll 102 patients with chordoma and describe their clinical, imageological, and histopathological features. Through tandem mass tag-based proteomic analysis and nonnegative matrix factorization clustering, we classify chordoma into three molecular subtypes: bone microenvironment-dominant, mesenchymal-derived, and mesenchymal-to-epithelial transition-mediated pattern. The three subtypes exhibit discrete clinical prognosis and distinct biological attributes of osteoclastogenesis and immunogenicity, oxidative phosphorylation, and receptor tyrosine kinase activation, suggesting targeted therapeutic strategies of denosumab, S-Gboxin, and anlotinib, respectively. Notably, these approaches demonstrate positive treatment outcomes for each subtype in vitro and in vivo. Altogether, this work sheds light on the clinical-proteomic characteristics of chordoma and provides a candidate precision treatment strategy for chordoma according to molecular classification, underscoring their potential for clinical application.

Nivolumab and Cabozantinib Immunotherapy Induced Thyroid Dysfunction Detected on 18 F-FDG PET/CT in Renal Cell Carcinoma.

Targeted immunotherapy became the most advanced approach for cancer treatment. Programmed death-1 (PD-1) expressed on activated T cells can reverse immune suppression and cause T-cell activation. Nivolumab, a PD-1 immune checkpoint inhibitor antibody that is a fully human immunoglobulin G4, blocks PD-1 and promotes antitumor immunity. Cabozantinib (tyrosine kinase inhibitor) inhibits the tyrosine kinase activity of vascular endothelial growth factor receptors 1, 2, and 3. As a result of enhancing immune response in normal tissues, immune-related adverse events can occur. Thyroid dysfunction is a common form of immune-related adverse event and seen on 18 F-FDG PET/CT scans post therapy.

Cutting-Edge Approach to Targeted Therapy: Repositioning of Old Drugs in Combination with Standard Clinical Chemotherapeutics Potentiates a Propitious Novel Targeted Therapy for Human Pancreatic Cancer

Metastatic pancreatic cancer leads to a fatal outcome, with a median progression-free survival of approximately six months when utilizing the most successful combination of chemotherapeutic regimens. When drug resistance develops, it facilitates an increase in primary tumor growth and new and growing metastases. Patients inevitably and quickly succumb to their disease and die. Notably, chemotherapy has an unintended impact on the development of drug resistance through the enhancement of EMT development and the enrichment of cancer stem cells (CSC). Recent report discovered that neuraminidase-1 (Neu-1) regulates EMT induction, angiogenesis, and cellular proliferation by the activation of several receptor tyrosine kinases. Here, the continual therapeutic inhibition of Neu-1 through intravenous administration of oseltamivir phosphate (OP) and aspirin (ASA) alongside GEM treatment significantly inhibits tumor progression, crucial compensatory signaling pathways, EMT program, CSC, and metastasis progression in a preclinical RAG2xCy double mutant BALB/c mouse model of human PANC-1 pancreatic cancer. The tumorigenic and metastatic potential of the xenotumors from the animals treated with the experimental protocols were significantly ablated when transferred into the mammary fat pads of NSG (NOD SCID gamma) branded mice. Keywords: pancreatic cancer; chemoresistance; drug repurposing; EMT.

Abstract IA-08: Syndecan 1 is a therapeutic target for KRAS-driven pancreatic cancer

Abstract KRAS inhibitors targeting G12C mutation have been approved by FDA for the treatment of non-small cell lung cancers and have shown promising clinical responses in pancreatic ductal adenocarcinoma (PDAC). RAS inhibitors targeting additional KRAS mutations or multiple RAS isoforms are under clinical development. Although targeting KRAS exhibits anti-tumor effect in PDAC, majority of tumors inevitably develop resistant disease, as is the case for most targeted therapies. In this new era of targeting KRAS driven cancer, understanding mechanisms driving this resistance to RAS targeted therapies and further devising combination strategies to overcome it have become a necessary, though challenging, task to achieve long term disease control. To tackle this problem, we employed various in vitro and in vivo preclinical model systems to explore the development of resistance to both genetic and pharmacological KRAS blockade. Our previous work identified the membrane proteoglycan Syndecan 1 (SDC1) as a KRAS downstream surrogate whose cell surface localization is tightly controlled by oncogenic KRAS signaling and is critical for KRAS-driven tumor development. Here, our analysis of KRAS driven genetically engineered PDAC mouse model and human pancreatic or colorectal cancer (CRC) cells with KRASG12C mutation discovered that, while cell surface SDC1 is reduced upon acute genetic or pharmacological inhibition of oncogenic KRAS, plasma membrane SDC1 is recovered in tumor cells that become resistant to KRAS inhibition (KRAS-bypass cells). Surface localization of SDC1 leads to the activation of multiple receptor tyrosine kinases (RTKs) and macropinocytosis, resulting in the resistance to KRAS blockade. We further identified that the recovery of plasma membrane SDC1 in KRAS-bypass cells is driven by YAP1 oncogene, which induces the downregulation of ARF6 GAPs, leading to the activation of ARF6, a small GTPase required for SDC1 membrane recycling. To therapeutically targeting SDC1, we have successfully developed a monoclonal antibody (22B mAb), which exhibits remarkable sensitivity and specificity for human SDC1. Our results indicate that 22B mAb exerts substantial inhibition of PDAC cell growth in vitro and desirable tumor inhibitory effects in vivo in murine PDAC tumors and human PDAC cell-derived xenograft tumors. Importantly, our 22B mAb synergizes with KRAS inhibitors, chemotherapy, or immunotherapy, resulting in significantly enhanced therapeutic effects. Taken together, our study identified SDC1 as a functional driver for the bypass of KRAS-dependence and therapeutically targeting SDC1 with a novel mAb will overcome the acquired resistance to KRAS targeted therapy. As such, our research yields important new insights in the fields of cancer biology regarding mechanisms of resistance employed by human cancers, and identifies new actionable therapeutic targets and approaches to overcome resistance to KRAS inhibition. Citation Format: Mitsunobu Takeda, Zecheng Yang, Madeline S Theardy, Alexey Sorokin, Oluwadara Coker, Shuaitong Chen, Long T Vien, Laura Bover, Haoqiang Ying, Scott Kopetz, Wantong Yao. Syndecan 1 is a therapeutic target for KRAS-driven pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-08.

A Probe-Free Occupancy Assay to Assess a Targeted Covalent Inhibitor of Receptor Tyrosine-Protein Kinase erbB-2.

Establishing target engagement is fundamental to effective target-based drug development. It paves the way for efficient medicinal chemistry design and definitive answers about target validation in the clinic. For irreversible targeted covalent inhibitor (TCI) drugs, there is a unique opportunity to establish and quantify the target engagement or occupancy. This is typically accomplished by using a covalent molecular probe, often a TCI analogue, derivatized to allow unoccupied target sites to be tracked; the difference of total sites minus unoccupied sites yields the occupied sites. When such probes are not available or the target is not readily accessible to covalent probes, another approach is needed. Receptor tyrosine-protein kinase erbB-2 (HER2) occupancy by afatinib presents such a case. Available HER2 covalent probes were unable to consistently modify HER2 after sample preparation, resulting in inadequate data. We demonstrate an alternative quantitative probe-free occupancy (PFO) method. It employs the immunoprecipitation of HER2 and direct mass spectrometer analysis of the cysteine-containing peptide that is targeted and covalently occupied by afatinib. Nontarget HER2 peptides provide normalization to the total protein. We show that HER2 occupancy by afatinib correlates directly to the inhibition of the receptor tyrosine kinase activity in NCI-N87 cells in culture and in vivo using those cells in a mouse tumor xenograft mode.

Extracellular Matrix Sulfation in the Tumor Microenvironment Stimulates Cancer Stemness and Invasiveness.

Tumor extracellular matrices (ECM) exhibit aberrant changes in composition and mechanics compared to normal tissues. Proteoglycans (PG) are vital regulators of cellular signaling in the ECM with the ability to modulate receptor tyrosine kinase (RTK) activation via their sulfated glycosaminoglycan (sGAG) side chains. However, their role on tumor cell behavior is controversial. Here, it is demonstrated that PGs are heavily expressed in lung adenocarcinoma (LUAD) patients in correlation with invasive phenotype and poor prognosis. A bioengineered human lung tumor model that recapitulates the increase of sGAGs in tumors in an organotypic matrix with independent control of stiffness, viscoelasticity, ligand density, and porosity, is developed. This model reveals that increased sulfation stimulates extensive proliferation, epithelial-mesenchymal transition (EMT), and stemness in cancer cells. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K) signaling axis is identified as a mediator of sulfation-induced molecular changes in cells upon activation of a distinct set of RTKs within tumor-mimetic hydrogels. The study shows that the transcriptomic landscape of tumor cells in response to increased sulfation resembles native PG-rich patient tumors by employing integrative omics and network modeling approaches.

The MET Family of Receptor Tyrosine Kinases Promotes a Shift to Pro-Tumor Metabolism.

The development and growth of cancer is fundamentally dependent on pro-tumor changes in metabolism. Cancer cells generally shift away from oxidative phosphorylation as the primary source of energy and rely more heavily on glycolysis. Receptor tyrosine kinases (RTKs) are a type of receptor that is implicated in this shift to pro-tumor metabolism. RTKs are important drivers of cancer growth and metastasis. One such family of RTKs is the MET family, which consists of MET and RON (MST1R). The overexpression of either MET or RON has been associated with worse cancer patient prognosis in a variety of tumor types. Both MET and RON signaling promote increased glycolysis by upregulating the expression of key glycolytic enzymes via increased MYC transcription factor activity. Additionally, both MET and RON signaling promote increased cholesterol biosynthesis downstream of glycolysis by upregulating the expression of SREBP2-induced cholesterol biosynthesis enzymes via CTTNB1. These changes in metabolism, driven by RTK activity, provide potential targets in limiting tumor growth and metastasis via pharmacological inhibition or modifications in diet. This review summarizes pro-tumor changes in metabolism driven by the MET family of RTKs. In doing so, we will offer our unique perspective on metabolic pathways that drive worse patient prognosis and provide suggestions for future study.

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.

A Tetravalent Bispecific Antibody Selectively Inhibits Diverse FGFR3 Oncogenic Variants.

The receptor tyrosine kinase FGFR3 is frequently mutated in bladder cancer and is a validated therapeutic target. Although pan-FGFR tyrosine kinase inhibitors (TKI) have shown clinical efficacy, toxicity and acquired resistance limit the benefit of these agents. While antibody-based therapeutics can offer superior selectivity than TKIs, conventional ligand-blocking antibodies are usually ineffective inhibitors of constitutively active receptor tyrosine kinases. Furthermore, the existence of multiple oncogenic variants of FGFR3 presents an additional challenge for antibody-mediated blockade. Here, we developed a tetravalent FGFR3×FGFR3 bispecific antibody that inhibited FGFR3 point mutants and fusion proteins more effectively than any of the conventional FGFR3 antibodies that we produced. Each arm of the bispecific antibody contacted two distinct epitopes of FGFR3 through a cis mode of binding. The antibody blocked dimerization of the most common FGFR3 oncogenic variant (S249C extracellular domain mutation) and inhibited the function of FGFR3 variants that are resistant to pan-FGFR TKIs. The antibody was highly effective in suppressing growth of FGFR3-driven tumor models, providing efficacy comparable to that of the FDA-approved TKI erdafitinib. Thus, this bispecific antibody may provide an effective approach for broad and highly selective inhibition of oncogenic FGFR3 variants. Significance: Development of a bispecific antibody that broadly inhibits gain-of-function FGFR3 variants provides a therapeutic strategy to target tumors with oncogenic FGFR3 point mutations and fusions, a particularly difficult case for antibody blockade.

Gastro Intestinal Stromal Tumor: Cases Series Report

Gastrointestinal stromal tumors (GISTs) are rare mesenchymal tumors of the alimentary tract. Now a day GISTs represents 0.1-3% of all gastrointestinal malignancies, making it a diagnostic challenge. Lesions are frequently located in stomach and proximal small intestine but rarely elsewhere in the abdomen. They are believed to result from mutations of protooncogenes c-Kit or platelet-derived growth factor receptor alpha polypeptide, this increase tyrosine kinase receptor activity, leading to uncontrolled proliferation of stem cells that differentiate into cells of Cajal. They can occur at any age but predominantly in middle-aged people and in elderly. We reported the cases presented to our hospital with progressively distension of the abdomen, finding in diagnostic image suggested GIST without evidence of metastasis disease, total excision was done, Cytologic and immunohistochemistry analysis confirm diagnosis of GISTs.

DIPG-68. FLAG-003, A NOVEL, DUAL-ACTION THERAPEUTIC, DEMONSTRATES IN-VITRO ACTIVITY AGAINST DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Abstract BACKGROUND FLAG-003 is a novel, rationally-designed, CNS-penetrant, dual-acting agent that inhibits receptor tyrosine kinase activity and microtubule polymerization. Preclinical studies in glioblastoma (GBM) demonstrated decreased cell viability, reduced tumor volumes, decreased microvessel density, and increased survival. Remarkably, using an orthotopic DIPG model, all mice treated with FLAG-003 at 275 mg/day/orally displayed tumor regression, tumor reduction and increased survival. Given the significant preclinical activity against DIPG, as well as the known heterogeneity of DIPG, we evaluated the efficacy of FLAG-003 in vitro against a panel of DIPG cell lines. METHODS Eight DIPG cell lines hosted at the Center for Patient Derived Models (CPDM) were selected based on genomic and growth characteristics. Cells were plated on 384-well plates; 24 hours after plating, cells were dosed with 1nM to 50uM (12 step log curve) FLAG-003. Three technical replicates were performed per step. Controls consisted of positive kill control Staurosporine, negative growth control of 0.5% DMSO, or no DMSO for baseline growth. ONC201, a small molecule antagonist of DRD2/3 currently in clinical trials for DIPG, was used as a comparator. We also evaluated two GBM patient-derived models. RESULTS FLAG-003 demonstrated efficacy in all DIPG and GBM cell lines tested with IC50 less than 100nM. FLAG-003 was more potent compared to ONC201 in all models evaluated. CONCLUSIONS Single agent FLAG-003 demonstrated activity against a panel of DIPG cell lines. As next steps, we are exploring the efficacy of this drug in orthotopic DIPG PDX models. Continued development of FLAG-003 as a potential therapeutic candidate for DIPG patients is warranted and underway.

Targeting Tyro3, Axl, and MerTK Receptor Tyrosine Kinases Significantly Sensitizes Triple-Negative Breast Cancer to CDK4/6 Inhibition.

Triple-negative breast cancer (TNBC) is the most aggressive subtype with high metastasis and mortality rates. Given the lack of actionable targets such as ER and HER2, TNBC still remains an unmet therapeutic challenge. Despite harboring high CDK4/6 expression levels, the efficacy of CDK4/6 inhibition in TNBC has been limited due to the emergence of resistance. The resistance to CDK4/6 inhibition is mainly mediated by RB1 inactivation. Since our aim is to overcome resistance to CDK4/6 inhibition, in this study, we primarily used the cell lines that do not express RB1. Following a screening for activated receptor tyrosine kinases (RTKs) upon CDK4/6 inhibition, we identified the TAM (Tyro3, Axl, and MerTK) RTKs as a crucial therapeutic vulnerability in TNBC. We show that targeting the TAM receptors with a novel inhibitor, sitravatinib, significantly sensitizes TNBC to CDK4/6 inhibitors. Upon prolonged HER2 inhibitor treatment, HER2+ breast cancers suppress HER2 expression, physiologically transforming into TNBC-like cells. We further show that the combined treatment is highly effective against drug-resistant HER2+ breast cancer as well. Following quantitative proteomics and RNA-seq data analysis, we extended our study into the immunophenotyping of TNBC. Given the roles of the TAM receptors in promoting the creation of an immunosuppressive tumor microenvironment (TME), we further demonstrate that the combination of CDK4/6 inhibitor abemaciclib and sitravatinib modifies the immune landscape of TNBC to favor immune checkpoint blockade. Overall, our study offers a novel and highly effective combination therapy against TNBC and potentially treatment-resistant HER2+ breast cancer that can be rapidly moved to the clinic.