Targeting Receptor Tyrosine Kinases Research Articles

CADD Methods for Developing Novel Compounds Synthesized to Inhibit Tyrosine Kinase Receptors.

Growth factors and their receptor tyrosine kinases play a central role in regulating vital cellular processes such as proliferation, differentiation, division, and cell survival, and they are closely associated with the development of various types of cancer, particularly in the context of angiogenesis. Although several small chemical compounds targeting tyrosine kinase receptors have been approved by the FDA for cancer treatment by inhibiting angiogenesis, there is still a need for more effective medications. in silico studies are now crucial tools for the design of new drugs, offering considerable advantages such as cost and time reduction. In this review, we examined recent in silico research carried out between 2022 and 2024, focusing on new drug candidates synthesized to fight cancer, in particular by targeting tyrosine kinase receptors involved in the process of angiogenesis.

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.

Structural Models for a Series of Allosteric Inhibitors of IGF1R Kinase.

The allosteric inhibition of insulin-like growth factor receptor 1 kinase (IGF1RK) is a potential strategy to overcome selectivity barriers for targeting receptor tyrosine kinases. We constructed structural models of a series of 12 indole-butyl-amine derivatives that have been reported as allosteric inhibitors of IGF1RK. We further studied the dynamics and interactions of each inhibitor in the allosteric pocket via all-atom explicit-solvent molecular dynamics (MD) simulations. We discovered that a bulky carbonyl substitution at the R1 indole ring is structurally unfavorable for inhibitor binding in the IGF1RK allosteric pocket. Moreover, we found that the most potent derivative (termed C11) acquires a distinct conformation: forming an allosteric pocket channel with better shape complementarity and interactions with the receptor. In addition to a hydrogen-bonding interaction with V1063, the cyano derivative C11 forms a stable hydrogen bond with M1156, which is responsible for its unique binding conformation in the allosteric pocket. Our findings show that the positioning of chemical substituents with different pharmacophore features at the R1 indole ring influences molecular interactions and binding conformations of indole-butyl-amine derivatives and, hence, dramatically affects their potencies. Our results provide a structural framework for the design of allosteric inhibitors with improved affinities and specificities against IGF1RK.

YANK2 activated by Fyn promotes glioma tumorigenesis via the mTOR-independent p70S6K activation pathway

Glioma, particularly glioblastomas (GBM), is incurable brain tumor. The most targeted receptor tyrosine kinase (RTKs) drugs did not bring benefit to GBM patients. The mechanism of glioma growth continues to be explored to find more effective treatment. Here, we reported that Ser/Thr protein kinase YANK2 (yet another kinase 2) is upregulated in glioma tissues and promotes the growth and proliferation of glioma in vitro and in vivo. Further, we confirmed that oncogene Fyn directly activated YANK2 through phosphorylation its Y110, and Fyn-mediated YANK2 phosphorylation at Y110 site promotes glioma growth by increasing its stability. Finally, YANK2 was proved to be a novel upstream kinase of p70S6K and promotes glioma growth by directly phosphorylating p70S6K at T389. Taken together, we found a new mTOR-independent p70S6K activation pathway, Fyn-YANK2-p70S6K, which promotes glioma growth, and YANK2 is a potential oncogene and serves as a novel therapeutic target for glioma.

Abstract 1637: EPHB4 is a key regulator of G1/S transition in hepatocellular carcinoma

Abstract Effective therapeutic regimens against advanced hepatocellular carcinoma (HCCs) have recently improved but it remains very limited. Targeting receptor tyrosine kinases (RTKs) has proven to one of the main ways of achieving better survival outcome in HCC patients, yet the precise mechanism for this success remains to be elusive. We have systematically investigated the expression profiles of RTKs transcriptomic datasets i.e. The Cancer Genome Atlas - Liver hepatocellular carcinoma (TCGA-LIHC) (n=371) and our in-house cohort of paired i.e. HKU-QMH (n=41), and identified EPHB4 as one of the most abundantly expressed and frequently mutated RTKs in human HCCs. EPHB4 expression was found to be significantly correlated with Alpha Fetoprotein (AFP) expression, a factor commonly used in the diagnosis of HCCs. Furthermore, EphB4 has been demonstrated to play a regulatory role in VEGF/VEGFR-mediated angiogenesis, critical targets for current successful drugs. We confirmed abundant membrane EphB4 expression in HCC clinical specimens and human HCC cell line models using immunohistochemistry. Clinicopathological correlation analysis of EPHB4 mRNA expression revealed that its overexpression is associated with aggressive tumor features including presence of tumor microsatellite, liver invasion, venous invasion, and absence of tumor encapsulation. Functionally, stably knocking down EPHB4 (shEPHB4) resulted in a strong reduction of proliferation and self-renewal rates of multiple HCC cell lines in vitro. It significantly reduced tumor growth rates in subcutaneous and liver orthotopic models in vivo. Furthermore, limiting dilution assay showed EPHB4 plays a role in tumor initiation property of HCC cells. Mechanistically, we performed Gene Set Enrichment Analysis (GSEA) of transcriptome profiles for shEPHB4 with respective controls and found that knocking down EPHB4 causes HCC cells undergo drastic changes in several cell cycle checkpoints including G1/S transition. Using thymidine blockade, we further confirmed that knockdown of significantly hinders the transition of HCC cells from G1 to S phase of cell cycle. Our data suggests a strong link between EphB4 and cell cycle. Conclusion: We found EphB4 plays a functional role in regulating G1/S transition in human HCCs and understanding regulatory role of EphB4 on cell cycle progression may provide mechanistic insights into the how to effectively target HCC in the clinic. Citation Format: Abdullah Husain, Elley Y. Chiu, Hoi-Tang Ma, Daniel W. Ho, Karen M. Sze, Lo-Kong Chan, Irene O. Ng. EPHB4 is a key regulator of G1/S transition in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1637.

Antibody dependent cellular cytotoxicity-inducing anti-EGFR antibodies as effective therapeutic option for cutaneous melanoma resistant to BRAF inhibitors.

About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots. By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors' and patients' peripheral blood cells. Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

Prevalence and management of proteinuria associated with vascular endothelial growth factor receptor-targeted tyrosine kinase inhibitor treatment in advanced renal cell carcinoma, hepatocellular carcinoma, and thyroid cancer.

Vascular endothelial growth factor receptor-targeted tyrosine kinase inhibitors (VEGFR-TKIs) are often used for treatment of several types of cancer; however, they are associated with an increased risk of proteinuria, sometimes leading to treatment discontinuation. We searched PubMed and Scopus to identify clinical studies examining the incidence and risk factors for proteinuria caused by VEGFR-TKIs in patients with renal cell carcinoma, thyroid cancer, and hepatocellular carcinoma. The global incidence of proteinuria ranged from 6% to 34% for all grades of proteinuria, and from 1% to 10% for grade ≥3 proteinuria. The incidence of proteinuria did not differ significantly by cancer type, but in all three cancer types, there was a trend toward a higher incidence of proteinuria with lenvatinib than with other VEGFR-TKIs. In terms of risk factors, the incidence of proteinuria was significantly higher among Asians (including Japanese) compared with non-Asian populations. Other risk factors included diabetes mellitus, hypertension, and previous nephrectomy. When grade 3/4 proteinuria occurs, patients should be treated according to the criteria for dose reduction or withdrawal specified for each drug. For grade 2 proteinuria, treatment should be continued when the benefits outweigh the risks. Referral to a nephrologist should be considered for symptoms related to decreased renal function or when proteinuria has not improved after medication withdrawal. These management practices should be implemented universally, regardless of the cancer type.

Inhibition of CSF1R and KIT With Pexidartinib Reduces Inflammatory Signaling and Cell Viability in Endometriosis.

Endometriosis is a common and debilitating disease, affecting ∼170 million women worldwide. Affected patients have limited therapeutic options such as hormonal suppression or surgical excision of the lesions, though therapies are often not completely curative. Targeting receptor tyrosine kinases (RTKs) could provide a nonhormonal treatment option for endometriosis. We determined that 2 RTKs, macrophage-colony stimulating factor 1 receptor (CSF1R) and mast/stem cell growth factor receptor KIT (KIT), are overexpressed in endometriotic lesions and could be novel nonhormonal therapeutic targets for endometriosis. The kinase activity of CSF1R and KIT is suppressed by pexidartinib, a small molecule inhibitor that was recently approved by the US Food and Drug Administration. Using immunohistochemistry, we detected CSF1R and KIT in endometriotic tissues obtained from peritoneal lesions, colorectal lesions, and endometriomas. Specifically, we show that KIT is localized to the epithelium of the lesions, while CSF1R is expressed in the stroma and macrophages of the endometriotic lesions. Given the high epithelial expression of CSF1R and KIT, 12Z endometriotic epithelial cells were used to evaluate the efficacy of dual CSF1R and KIT inhibition with pexidartinib. We found that pexidartinib suppressed activation in 12Z cells of JNK, STAT3, and AKT signaling pathways, which control key proinflammatory and survival networks within the cell. Using quantitative real-time polymerase chain reaction, we determined that pexidartinib suppressed interleukin 8 (IL8) and cyclin D1 (CCND1) expression. Lastly, we demonstrated that pexidartinib decreased cell growth and viability. Overall, these results indicate that pexidartinib-mediated CSF1R and KIT inhibition reduces proinflammatory signaling and cell viability in endometriosis.

Targeting Receptor Tyrosine Kinases as a Novel Strategy for the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) comprises a group of aggressive and heterogeneous breast carcinoma. Chemotherapy is the mainstay for the treatment of triple-negative tumors. Nevertheless, the success of chemotherapeutic treatments is limited by their toxicity and development of acquired resistance leading to therapeutic failure and tumor relapse. Hence, there is an urgent need to explore novel targeted therapies for TNBC. Receptor tyrosine kinases (RTKs) are a family of transmembrane receptors that are key regulators of intracellular signaling pathways controlling cell proliferation, differentiation, survival, and motility. Aberrant activity and/or expression of several types of RTKs have been strongly connected to tumorigenesis. RTKs are frequently overexpressed and/or deregulated in triple-negative breast tumors and are further associated with tumor progression and reduced survival in patients. Therefore, targeting RTKs could be an appealing therapeutic strategy for the treatment of TNBC. This review summarizes the current evidence regarding the antitumor activity of RTK inhibitors in preclinical models of TNBC. The review also provides insights into the clinical trials evaluating the use of RTK inhibitors for the treatment of patients with TNBC.

A review on potential heterocycles for the treatment of glioblastoma targeting receptor tyrosine kinases.

Glioblastoma, the most aggressive form of brain tumor, poses significant challenges in terms of treatment success and patient survival. Current treatment modalities for glioblastoma include radiation therapy, surgical intervention, and chemotherapy. Unfortunately, the median survival rate remains dishearteningly low at 12-15 months. One of the major obstacles in treating glioblastoma is the recurrence of tumors, making chemotherapy the primary approach for secondary glioma patients. However, the efficacy of drugs is hampered by the presence of the blood-brain barrier and multidrug resistance mechanisms. Consequently, considerable research efforts have been directed toward understanding the underlying signaling pathways involved in glioma and developing targeted drugs. To tackle glioma, numerous studies have examined kinase-downstream signaling pathways such as RAS-RAF-MEK-ERK-MPAK. By targeting specific signaling pathways, heterocyclic compounds have demonstrated efficacy in glioma therapeutics. Additionally, key kinases including phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase, cytoplasmic tyrosine kinase (CTK), receptor tyrosine kinase (RTK) and lipid kinase (LK) have been considered for investigation. These pathways play crucial roles in drug effectiveness in glioma treatment. Heterocyclic compounds, encompassing pyrimidine, thiazole, quinazoline, imidazole, indole, acridone, triazine, and other derivatives, have shown promising results in targeting these pathways. As part of this review, we propose exploring novel structures with low toxicity and high potency for glioma treatment. The development of these compounds should strive to overcome multidrug resistance mechanisms and efficiently penetrate the blood-brain barrier. By optimizing the chemical properties and designing compounds with enhanced drug-like characteristics, we can maximize their therapeutic value and minimize adverse effects. Considering the complex nature of glioblastoma, these novel structures should be rigorously tested and evaluated for their efficacy and safety profiles.

ImmunoPET Targeting Receptor Tyrosine Kinase: Clinical Applications.

Receptor tyrosine kinases, or RTKs, are one large family of cell surface receptors involved in signal transduction, which represent an integral part of the signaling pathways. They play a crucial role in most important cellular processes, starting with the cell cycle, proliferation and differentiation, as well as cell migration, metabolism and survival. The introduction of ImmunoPET evaluating the expression of RTKs by specific monoclonal antibodies (mAbs) or antibody fragments is regarded as a promising tool for imaging treatment efficacy and developing anticancer therapeutics. Our review focuses mainly on the current clinical research regarding ImmunoPET targeting RTKs, with particular interest in the epidermal growth factor family, or HER family, and vascular endothelial-derived growth factor/receptor.

Cell Membrane-Anchored DNA Nanoinhibitor for Inhibition of Receptor Tyrosine Kinase Signaling Pathways via Steric Hindrance and Lysosome-Induced Protein Degradation.

Receptor tyrosine kinase (RTK) plays a crucial role in cancer progression, and it has been identified as a key drug target for cancer targeted therapy. Although traditional RTK-targeting drugs are effective, there are some limitations that potentially hinder the further development of RTK-targeting drugs. Therefore, it is urgently needed to develop novel, simple, and general RTK-targeting inhibitors with a new mechanism of action for cancer targeted therapy. Here, a cell membrane-anchored RTK-targeting DNA nanoinhibitor is developed to inhibit RTK function. By using a DNA tetrahedron as a framework, RTK-specific aptamers as the recognition elements, and cholesterol as anchoring molecules, this DNA nanoinhibitor could rapidly anchor on the cell membrane and specifically bind to RTK. Compared with traditional RTK-targeting inhibitors, this DNA nanoinhibitor does not need to bind at a limited domain on RTK, which increases the possibilities of developing RTK inhibitors. With the cellular-mesenchymal to epithelial transition factor (c-Met) as a target RTK, the DNA nanoinhibitor can not only induce steric hindrance effects to inhibit c-Met activation but also reduce the c-Met level via lysosome-mediated protein degradation and thus inhibition of c-Met signaling pathways and related cell behaviors. Moreover, the DNA nanoinhibitor is feasible for other RTKs by just replacing aptamers. This work may provide a novel, simple, and general RTK-targeting nanoinhibitor and possess great value in RTK-targeted cancer therapy.

Abstract B093: Treating KRAS(G12D) inhibitor resistance using a KRAS- and HSP90 chaperone-targeted hetero-bispecific small molecule agent

Abstract KRAS is the most frequently mutated oncoprotein in human cancers. Although long considered “undruggable”, recent breakthroughs in medicinal chemistry have led to FDA-approval of the KRAS(G12C) mutation-specific inhibitors sotorasib and adagrasib for the treatment of KRAS(G12C)-positive non-small cell lung cancer. However, illustrating the need to develop additional novel agents targeting mutated KRAS, the 5-year relative survival rate for pancreatic cancer, which is commonly associated with a variety of different KRAS mutations, is only 12% (all SEER stages) due to poor early detection and a lack of effective treatments. In particular, KRAS(G12D) is the most common KRAS mutation, being found in 37% of pancreatic ductal adenocarcinomas (PDACs), as well as in 12.5% of colorectal cancer and 4.9% of lung adenocarcinoma (LUAD) patients. MRTX1133 is a highly-selective, non-covalent KRAS(G12D) inhibitor that has recently entered a phase 1 clinical trial. However, here we show that exposure of KRAS(G12D)-mutated PDAC (PANC-1 and AsPC-1) and LUAD (SK-LU-1) cell lines and a patient-derived organoid (PDO) PDAC model (RPAN001) to MRTX1133 resulted in varying degrees of in vitro efficacy. Decreased downstream KRAS signaling in the form of reduced phospho-ERK1/2 levels was observed to rapidly recover within 24 hours of treatment with 500 nM MRTX1133. Moreover, this rebound coincided with increased expression of KRAS, NRAS andHRAS mRNAs. Concurrently, activation of the receptor tyrosine kinases (RTKs) EGFR and MET was observed in PANC-1 and SK-LU-1 cells that displayed innate resistance to MRTX1133, while the sensitive AsPC-1 cells showed no such RTK activation. These results suggest that targeting of both KRAS(G12D) and RTKs may be needed to treat KRAS(G12D) inhibitor-resistant cancers. To address this, we employed a novel hetero-bispecific CHAMP molecule, RNK08179, that simultaneously targets both KRAS(G12D) and HSP90, an RTK-regulating chaperone protein. RNK08179 treatment demonstrated a striking reduction in phospho-ERK1/2 levels, RTK activation and cell viability in MRTX1133-resistant PANC-1 and SK-LU-1 cells. Furthermore, similar efficacy was observed in the MRTX1133-resistant RPAN001 PDO model. In summary, RNK08179 displayed promising efficacy in cancer models harboring KRAS(G12D) by suppressing both mutated KRAS and HSP90-supported RTK signaling. Citation Format: Ines Pulido, Qiyue Luan, Chenghao Yin, Zimo Yang, Jinhua Lin, Yaya Wang, Yuetong Sun, Chuche Liu, Haoxin Zhou, Marek Massad, Ian Papautsky, Thomas L. Prince, Guoqiang Wang, Kevin P. Foley, Weiwen Ying, Takeshi Shimamura. Treating KRAS(G12D) inhibitor resistance using a KRAS- and HSP90 chaperone-targeted hetero-bispecific small molecule agent [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B093.

TAM family kinases as therapeutic targets at the interface of cancer and immunity.

Novel treatment approaches are needed to overcome innate and acquired mechanisms of resistance to current anticancer therapies in cancer cells and the tumour immune microenvironment. The TAM (TYRO3, AXL and MERTK) family receptor tyrosine kinases (RTKs) are potential therapeutic targets in a wide range of cancers. In cancer cells, TAM RTKs activate signalling pathways that promote cell survival, metastasis and resistance to a variety of chemotherapeutic agents and targeted therapies. TAM RTKs also function in innate immune cells, contributing to various mechanisms that suppress antitumour immunity and promote resistance to immune-checkpoint inhibitors. Therefore, TAM antagonists provide an unprecedented opportunity for both direct and immune-mediated therapeutic activity provided by inhibition of a single target, and are likely to be particularly effective when used in combination with other cancer therapies. To exploit this potential, a variety of agents have been designed to selectively target TAM RTKs, many of which have now entered clinical testing. This Review provides an essential guide to the TAM RTKs for clinicians, including an overview of the rationale for therapeutic targeting of TAM RTKs in cancer cells and the tumour immune microenvironment, a description of the current preclinical and clinical experience with TAM inhibitors, and a perspective on strategies for continued development of TAM-targeted agents for oncology applications.

LITESPARK-024: A randomized phase 1/2 study of belzutifan with or without palbociclib for previously treated advanced renal cell carcinoma

Abstract Background Immunotherapy is a standard-of-care first-line treatment for advanced clear cell renal cell carcinoma (ccRCC). However, many patients will develop resistance to first-line therapy, and effective second- and later-line treatment options are therefore needed. The von Hippel-Lindau (VHL) gene is inactivated in approximately 90% of patients with RCC and results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α), a key oncogenic driver in RCC. Belzutifan, a first-in-class HIF-2α inhibitor, has demonstrated antitumor activity with manageable safety in previously treated patients with advanced ccRCC. The cyclin-dependent kinase (CDK) pathway is also associated with poor clinical outcomes in ccRCC. In RCC cell lines, the CDK 4/6 inhibitor palbociclib inhibited cell growth. CDK 4/6 inhibition has shown synergistic antiproliferative effects with HIF-2α inhibition in HIF-2α–dependent VHL -/- RCC cell lines. Palbociclib could therefore potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods LITESPARK-024 is a 2-part, open-label, multicenter, phase 1/2 randomized study (NCT05468697). Part 1 is intended to establish the recommended phase 2 dose (RP2D) of belzutifan plus palbociclib using a modified toxicity probability interval design. After the RP2D is established, part 2 will directly compare the safety and efficacy of belzutifan monotherapy with belzutifan + palbociclib in patients with advanced ccRCC. In both parts, patients with measurable disease per RECIST v1.1, a Karnofsky Performance Status score of ≥70%, and histologically confirmed unresectable stage IV RCC with a clear cell component and disease progressing on or after receiving at least 2 systemic treatments (both an anti–PD-1/L1 monoclonal antibody and a VEGF receptor–targeted tyrosine kinase inhibitor, in sequence or in combination) will be enrolled. Up to 30 patients will be enrolled into 3 dose groups in part 1 and will receive belzutifan 120 mg once daily plus palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 patients will be randomly assigned 2:1 to receive belzutifan 120 mg once daily plus palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Patients will be stratified by International metastatic RCC Database Consortium risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events and to determine the RP2D of belzutifan plus palbociclib. The primary end point for part 2 is objective response rate per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are clinical benefit rate, duration of response, and progression-free survival per RECIST v1.1 by investigator assessment; overall survival, and safety and tolerability. Enrollment began in July 2022. ©2023 American Society of Clinical Oncology, Inc. Reused with permission. This abstract was accepted and previously presented at the 2023 ASCO Genitourinary Cancers Symposium. All rights reserved. CDMRP DOD Funding: no

Hijacking of GPCRs and RTKs by pathogens

Pathogens exploit multiple cellular and molecular pathways in the host organisms for their entry, survival and dissemination. The cell surface receptors such as G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) constitute the targets of many pathogens. This is due to the ubiquitous expression of these two receptor families in the organism and their pivotal role in various cellular and physiological processes. At the molecular level, receptor hijacking implies either direct or indirect interactions between pathogens' effectors or toxins with GPCRs and RTKs at the cell surface thereby interfering with their activation and their downstream signaling pathways inside the host cells. As a result, the pathogens manipulate and redirect GPCR/RTK-mediated signaling pathways and different aspects of cell function for their benefit. The review presents a compilation of the major examples of pathogen infections where GPCRs and RTKs and their related intracellular signaling pathways are targeted. This provides a molecular basis for pathogens hijacking cell signaling and their virulence. Our understanding of such complex host-pathogen interactions at the molecular level will open new opportunities to develop new prophylactic and therapeutic approaches against infections. In this context, the pharmacological targeting of GPCRs and RTKs may be a promising approach.

LITESPARK-024: Randomized phase 1/2 study of belzutifan with or without palbociclib for treatment of advanced renal cell carcinoma (RCC).

TPS4603 Background: Immunotherapy is a standard-of-care first-line treatment for advanced clear cell RCC (ccRCC). However, many patients (pts) will develop resistance to first-line therapy, and effective second- and later-line treatment options are therefore needed. The von Hippel-Lindau ( VHL) gene is inactivated in approximately 90% of ccRCC cases and results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α), a key oncogenic driver in RCC. Belzutifan, a first-in-class HIF-2α inhibitor, has demonstrated antitumor activity with manageable safety in previously treated pts with advanced ccRCC. The cyclin-dependent kinase (CDK) pathway is also associated with poor clinical outcomes in ccRCC. In RCC cell lines, the CDK 4/6 inhibitor palbociclib inhibited cell growth. CDK 4/6 inhibition has shown synergistic antiproliferative effects with HIF-2α inhibition in HIF-2α–dependent VHL -/- RCC cell lines. Palbociclib could therefore potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods: LITESPARK-024 is a 2-part, open-label, multicenter, phase 1/2 randomized study (NCT05468697). Part 1 is intended to establish the recommended phase 2 dose (RP2D) of belzutifan + palbociclib using a modified toxicity probability interval design. After the RP2D is established, part 2 will directly compare the safety and efficacy of belzutifan monotherapy with belzutifan + palbociclib in pts with advanced ccRCC. In both parts, pts with measurable disease per RECIST v1.1, a KPS score of ≥70%, and histologically confirmed unresectable stage IV RCC with a clear cell component with disease progressing on or after receiving at least 2 systemic treatments (both an anti–PD-1/L1 monoclonal antibody and a VEGF receptor–targeted tyrosine kinase inhibitor, in sequence or in combination) will be enrolled. Up to 30 pts will be enrolled into 3 dose groups in part 1 and will receive belzutifan 120 mg once daily + palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 pts will be randomly assigned 2:1 to receive belzutifan 120 mg once daily + palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Pts will be stratified by IMDC risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events and to determine the RP2D of belzutifan + palbociclib. The primary end point for part 2 is ORR per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are CBR, DOR, and PFS per RECIST v1.1 by investigator assessment, OS, and safety and tolerability. Enrollment began in July 2022. Clinical trial information: NCT05468697 .

Abstract LB226: FGFR2 modulates ALK inhibition response in high-risk neuroblastoma

Abstract High-risk neuroblastoma (NB) has a poor prognosis despite multimodal treatment. To improve survival and minimise treatment side-effects, research has focused on developing more effective therapeutic strategies. Anaplastic lymphoma kinase (ALK) is a promising druggable target as its expression rapidly decreases in healthy tissues postnatally, and it is expressed as a hyperactivated mutant form in ~14% of high-risk NB. The ALK tyrosine kinase inhibitor lorlatinib is a promising treatment, but resistance has been reported, hindering long-term benefits. With a trimodal approach, consisting of genome-wide CRISPR-Cas9-overexpression (CRISPRa) screens, RNA sequencing and high-throughput drug screens, we have identified genes whose expression is associated with decreased sensitivity to lorlatinib creating novel therapeutic vulnerabilities. In particular, we validate the CRISPRa screen hit FGFR2 as a bypass signalling mechanism desensitising mutant ALK-expressing NB cells to lorlatinib; overexpression of FGFR2 increased, while silencing decreased resistance of NB cells to lorlatinib. Furthermore, RNA sequencing of lorlatinib resistant (LR) NB cells developed in our lab, compared to parental cell lines, showed FGFR2 to be expressed to a higher level in the LR cells. High-throughput drug screens exposing LR and parental NB cell lines to an FDA-approved drug library of 1430 compounds showed that drugs targeting receptor tyrosine kinases, including FGFR2, were amongst the compounds most significantly effective in reducing the viability of LR NB cells compared to parental cells. The multi-kinase inhibitor ponatinib and the selective FGFR inhibitor erdafitinib acted synergistically with lorlatinib in treating both parental and LR NB cells although both inhibitors were more effective in inhibiting the growth of LR compared to parental cells when given alone, suggesting that FGFR2 may represent a novel vulnerability to treat lorlatinib resistant NB. In vivo studies using patient-derived xenograft (PDX) models of high-risk NB (MYCN-amplified and ALKF1174L mutant) showed that combinations of either ponatinib or erdafitinib with lorlatinib decreased tumour growth and increased survival compared to PDXs treated with vehicle or either agent alone. In conclusion, these findings suggest that FGFR2 alters NB sensitivity to lorlatinib and modulation of this pathway in combination with ALK inhibition is a promising approach to improve NB treatment response and ultimately patient survival. Citation Format: Perla Pucci, Charlotte Barrett, Ricky Trigg, Leila Jahangiri, Jamie D. Matthews, Luca Mologni, G. A. Amos Burke, Suzanne D Turner. FGFR2 modulates ALK inhibition response in high-risk neuroblastoma [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 LB226.

Abstract CT120: The randomized phase 1/2 LITESPARK-024 study of belzutifan with or without palbociclib in patients with advanced renal cell carcinoma (RCC)

Abstract Background: First-line treatment with immunotherapy alone or in combination with antiangiogenic agents is a standard of care for advanced RCC. Many patients (pts) develop resistance to first-line treatment and effective second-line and beyond options are needed. The von Hippel-Lindau (VHL) gene is inactivated in approximately 90% of RCC cases, which results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α), a key oncogenic driver in RCC. The first-in-class HIF-2α inhibitor belzutifan has demonstrated promising antitumor activity with manageable safety in previously treated pts with advanced RCC. The cyclin-dependent kinase (CDK) pathway has also been implicated in RCC and is associated with poor clinical outcomes. In RCC cell lines, the CDK 4/6 inhibitor palbociclib inhibited cell growth. The addition of CDK 4/6 inhibition had synergistic antiproliferative effects with HIF-2α inhibition in HIF-2α-dependent VHL -/- RCC cell lines. Palbociclib could therefore potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods: The two-part, open-label, multicenter, phase 1/2 randomized LITESPARK-024 study (NCT05468697) is intended to establish the recommended phase 2 dose (RP2D) of belzutifan + palbociclib in combination with a modified toxicity probability interval design (Part 1), followed by a direct comparison of belzutifan monotherapy to the combination with respect to safety and efficacy in pts with advanced RCC (Part 2). Pts with histologically confirmed unresectable stage IV RCC with a clear cell component, whose disease progressed on or after having received at least 2 systemic treatments (both an anti-PD-1/PD-L1 monoclonal antibody and a VEGF receptor-targeted tyrosine kinase inhibitor, in sequence or in combination), have measurable disease per RECIST v1.1 by blinded independent central review, and have KPS score of ≥70% will be enrolled. Up to 30 pts will be enrolled into 3 dose groups in Part 1 and will receive belzutifan 120 mg once daily + palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 pts will be randomly assigned 2:1 to receive belzutifan 120 mg once daily + palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Pts will be stratified by IMDC risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events to determine the RP2D of belzutifan + palbociclib. The primary end point for part 2 is ORR per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are CBR, DOR, PFS, OS, and safety and tolerability. This abstract was accepted and previously presented at the 2023 ASCO Genitourinary Cancers Symposium. All rights reserved. Citation Format: David F. McDermott, Avivit Peer, Neeraj Agarwal, Michael B. Atkins, Jerry Cornell, Rodolfo F. Perini, Kenneth F. Grossmann, Howard Gurney. The randomized phase 1/2 LITESPARK-024 study of belzutifan with or without palbociclib in patients with advanced renal cell carcinoma (RCC) [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 CT120.

Gene Fusion Detection in NSCLC Routine Clinical Practice: Targeted-NGS or FISH?

The ability to identify the broadest range of targetable gene fusions is crucial to facilitate personalized therapy selection for advanced lung adenocarcinoma (LuADs) patients harboring targetable receptor tyrosine kinase (RTK) genomic alterations. In order to evaluate the most effective testing approach for LuAD targetable gene fusion detection, we analyzed 210 NSCLC selected clinical samples, comparing in situ (Fluorescence In Situ Hybridization, FISH, and ImmunoHistoChemistry, IHC) and molecular (targeted RNA Next-Generation Sequencing, NGS, and RealTime-PCR, RT-PCR) approaches. The overall concordance among these methods was high (>90%), and targeted RNA NGS was confirmed to be the most efficient technique for gene fusion identification in clinical practice, allowing the simultaneous analysis of a large set of genomic rearrangements at the RNA level. However, we observed that FISH was useful to detect targetable fusions in those samples with inadequate tissue material for molecular testing as well as in those few cases whose fusions were not identified by the RNA NGS panel. We conclude that the targeted RNA NGS analysis of LuADs allows accurate RTK fusion detection; nevertheless, standard methods such as FISH should not be dismissed, as they can crucially contribute to the completion of the molecular characterization of LuADs and, most importantly, the identification of patients as candidates for targeted therapies.