Blockade of Discoidin Domain Receptor Signaling with Sitravatinib Reveals DDR2 as a Mediator of Neuroblastoma Pathogenesis and Metastasis.

Oncogene-driven expression and activation of receptor tyrosine kinases (RTK) promotes tumorigenesis and contributes to drug resistance. Increased expression of the kinases DDR2 (Discoid Domain Receptor 2), RET, PDGFRA, KIT, MET, and ALK (Anaplastic Lymphoma Kinase) independently correlate with decreased overall survival (OS) and event free survival (EFS) of pediatric neuroblastoma. The multikinase inhibitor sitravatinib targets DDR2, RET, PDGFRA, KIT and MET with low nanomolar activity and we therefore tested its efficacy against orthotopic and syngeneic tumor models. Sitravatinib markedly reduced cell proliferation and migration in vitro independently of MYCN (N-Myc proto-oncogene), ALK, or MYC (c-Myc proto-oncogene) status, and inhibited proliferation and metastasis of human orthotopic xenografts. Oral administration of sitravatinib to homozygous Th-MYCN transgenic mice (Th-MYCN+/+) after tumor initiation completely arrested further tumor development with no mice dying of disease while maintained on sitravatinib treatment (control cohort 57 days median time to sacrifice). Among these top kinases, DDR2 expression has the strongest correlation with poor survival and high stage at diagnosis, and the highest sensitivity to the drug. We confirmed on-target inhibition of collagen-mediated activation of DDR2. Genetic knockdown of DDR2 partially phenocopies Sitravatinib treatment, limiting tumor development and metastasis across tumor models. Analysis of single cell sequencing data demonstrated that DDR2 is restricted to mesenchymal-type tumor subpopulations and is enriched in Schwann Cell Precursor (SCP) subpopulations found in high-risk disease. These data define an unsuspected role for sitravatinib as a therapeutic agent in neuroblastoma and reveal a novel function for DDR2 as a driver of tumor growth and metastasis.

DDR2 signaling and mechanosensing orchestrate neuroblastoma cell fate through different transcriptome mechanisms.

The extracellular matrix (ECM) regulates carcinogenesis by interacting with cancer cells via cell surface receptors. Discoidin Domain Receptor 2 (DDR2) is a collagen-activated receptor implicated in cell survival, growth, and differentiation. Dysregulated DDR2 expression has been identified in various cancer types, making it as a promising therapeutic target. Additionally, cancer cells exhibit mechanosensing abilities, detecting changes in ECM stiffness, which is particularly important for carcinogenesis given the observed ECM stiffening in numerous cancer types. Despite these, whether collagen-activated DDR2 signaling and ECM stiffness-induced mechanosensing exert similar effects on cancer cell behavior and whether they operate through analogous mechanisms remain elusive. To address these questions, we performed bulk RNA sequencing (RNA-seq) on human SH-SY5Y neuroblastoma cells cultured on collagen-coated substrates. Our results show that DDR2 downregulation induces significant changes in the cell transcriptome, with changes in expression of 15% of the genome, specifically affecting the genes associated with cell division and differentiation. We validated the RNA-seq results by showing that DDR2 knockdown redirects the cell fate from proliferation to senescence. Like DDR2 knockdown, increasing substrate stiffness diminishes cell proliferation. Surprisingly, RNA-seq indicates that substrate stiffness has no detectable effect on the transcriptome. Furthermore, DDR2 knockdown influences cellular responses to substrate stiffness changes, highlighting a crosstalk between these two ECM-induced signaling pathways. Based on our results, we propose that the ECM could activate DDR2 signaling and mechanosensing in cancer cells to orchestrate their cell fate through distinct mechanisms, with or without involving gene expression, thus providing novel mechanistic insights into cancer progression.

A Novel Druggable Dual-Specificity tYrosine-Regulated Kinase3/Calmodulin Kinase-like Vesicle-Associated Signaling Module with Therapeutic Implications in Neuroblastoma.

High-risk neuroblastoma is a very aggressive pediatric cancer, accounting for ~15% of childhood cancer mortality. Therefore, novel therapeutic strategies for the treatment of neuroblastoma are urgently sought. Here, we focused on the potential implications of the Dual-specificity tYrosine-Regulated Kinase (DYRK) family and downstream signaling pathways. We used bioinformatic analysis of public datasets from neuroblastoma cohorts and cell lines to search correlations between patient survival and expression of DYRK kinases. Additionally, we performed biochemical, molecular, and cellular approaches to validate and characterize our observations, as well as an in vivo orthotopic murine model of neuroblastoma. We identified the DYRK3 kinase as a critical mediator of neuroblastoma cell proliferation and in vivo tumor growth. DYRK3 has recently emerged as a key regulator of several biomolecular condensates and has been linked to the hypoxic response of neuroblastoma cells. Our data suggest a role for DYRK3 as a regulator of the neuroblastoma-specific protein CAMKV, which is also required for neuroblastoma cell proliferation. CAMKV is a very understudied member of the Ca2+/calmodulin-dependent protein kinase family, originally described as a pseudokinase. We show that CAMKV is phosphorylated by DYRK3, and that inhibition of DYRK3 kinase activity induces CAMKV aggregation, probably mediated by its highly disordered C-terminal half. Importantly, we provide evidence that the DYRK3/CAMKV signaling module could play an important role for the function of the mitotic spindle during cell division. Our data strongly support the idea that inhibition of DYRK3 and/or CAMKV in neuroblastoma cells could constitute an innovative and highly specific intervention to fight against this dreadful cancer.

RUN(X) out of blood: emerging RUNX1 functions beyond hematopoiesis and links to Down syndrome.

BACKGROUND: RUNX1 is a transcription factor and a master regulator for the specification of the hematopoietic lineage during embryogenesis and postnatal megakaryopoiesis. Mutations and rearrangements on RUNX1 are key drivers of hematological malignancies. In humans, this gene is localized to the 'Down syndrome critical region' of chromosome 21, triplication of which is necessary and sufficient for most phenotypes that characterize Trisomy 21. MAIN BODY: Individuals with Down syndrome show a higher predisposition to leukemias. Hence, RUNX1 overexpression was initially proposed as a critical player on Down syndrome-associated leukemogenesis. Less is known about the functions of RUNX1 in other tissues and organs, although growing reports show important implications in development or homeostasis of neural tissues, muscle, heart, bone, ovary, or the endothelium, among others. Even less is understood about the consequences on these tissues of RUNX1 gene dosage alterations in the context of Down syndrome. In this review, we summarize the current knowledge on RUNX1 activities outside blood/leukemia, while suggesting for the first time their potential relation to specific Trisomy 21 co-occurring conditions. CONCLUSION: Our concise review on the emerging RUNX1 roles in different tissues outside the hematopoietic context provides a number of well-funded hypotheses that will open new research avenues toward a better understanding of RUNX1-mediated transcription in health and disease, contributing to novel potential diagnostic and therapeutic strategies for Down syndrome-associated conditions.

PRMT5 activates AKT via methylation to promote tumor metastasis.

Protein arginine methyltransferase 5 (PRMT5) is the primary methyltransferase generating symmetric-dimethyl-arginine marks on histone and non-histone proteins. PRMT5 dysregulation is implicated in multiple oncogenic processes. Here, we report that PRMT5-mediated methylation of protein kinase B (AKT) is required for its subsequent phosphorylation at Thr308 and Ser473. Moreover, pharmacologic or genetic inhibition of PRMT5 abolishes AKT1 arginine 15 methylation, thereby preventing AKT1 translocation to the plasma membrane and subsequent recruitment of its upstream activating kinases PDK1 and mTOR2. We show that PRMT5/AKT signaling controls the expression of the epithelial-mesenchymal-transition transcription factors ZEB1, SNAIL, and TWIST1. PRMT5 inhibition significantly attenuates primary tumor growth and broadly blocks metastasis in multiple organs in xenograft tumor models of high-risk neuroblastoma. Collectively, our results suggest that PRMT5 inhibition augments anti-AKT or other downstream targeted therapeutics in high-risk metastatic cancers.

Systematic review of the receptor tyrosine kinase superfamily in neuroblastoma pathophysiology.

BACKGROUND: Neuroblastoma is a devastating disease accounting for 15% of all childhood cancer deaths. Yet, our understanding of key molecular drivers such as receptor tyrosine kinases (RTKs) in this pathology remains poorly clarified. Here, we provide a systematic analysis of the RTK superfamily in the context of neuroblastoma pathogenesis. METHODS: Statistical correlations for all RTK family members' expression to neuroblastoma patient survival across 10 independent patient cohorts were annotated, synthesized, and ranked using the R2: Genomics Analysis and Visualization Platform. Gene expression of selected members across different cancer cell lines was further analyzed in the Cancer Cell Line Encyclopedia, part of the Cancer Dependency Map portal (depmap portal ( http://depmap.org )). Finally, we provide a detailed literature review for highly ranked candidates. RESULTS: Our analysis defined two subsets of RTKs showing robust associations with either better or worse survival, constituting potential novel players in neuroblastoma pathophysiology, diagnosis, and therapy. We review the available literature regarding the oncogenic functions of these RTKs, their roles in neuroblastoma pathophysiology, and potential utility as therapeutic targets. CONCLUSIONS: Our systematic analysis and review of the RTK superfamily in neuroblastoma pathogenesis provides a new resource to guide the research community towards focused efforts investigating signaling pathways that contribute to neuroblastoma tumor establishment, growth, and/or aggressiveness and targeting these druggable molecules in novel therapeutic strategies.

DYRK1A Kinase Positively Regulates Angiogenic Responses in Endothelial Cells.

Angiogenesis is a highly regulated process essential for organ development and maintenance, and its deregulation contributes to inflammation, cardiac disorders, and cancer. The Ca2+/nuclear factor of activated T cells (NFAT) signaling pathway is central to endothelial cell angiogenic responses, and it is activated by stimuli like vascular endothelial growth factor (VEGF) A. NFAT phosphorylation by dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) is thought to be an inactivating event. Contrary to expectations, we show that the DYRK family member DYRK1A positively regulates VEGF-dependent NFAT transcriptional responses in primary endothelial cells. DYRK1A silencing reduces intracellular Ca2+ influx in response to VEGF, which dampens NFAT activation. The effect is exerted at the level of VEGFR2 accumulation leading to impairment in PLCγ1 activation. Notably, Dyrk1a heterozygous mice show defects in developmental retinal vascularization. Our data establish a regulatory circuit, DYRK1A/ Ca2+/NFAT, to fine-tune endothelial cell proliferation and angiogenesis.

Loss of Sprouty1 rescues renal agenesis caused by Ret mutation.

Renal morphogenesis requires a balance between positive and negative signals, which are provided in part by the receptor tyrosine kinase Ret and the putative tumor suppressor Sprouty1, respectively. Tyrosine 1062 of Ret is a binding site for several adaptor and effector proteins, such as Grb2/Sos/Ras, which activate the ERK pathway. Mice lacking Ret tyrosine 1062 nearly mimic the phenotype of Ret-knockout mice, which includes renal agenesis. Sprouty1 regulates Ret activity by modulating the ERK pathway, but the mechanism by which this occurs is uncertain. Here, we show that loss of Sprouty1 rescues the renal agenesis and early postnatal lethality caused by lack of Ret tyrosine 1062. The kidneys and lower urinary tracts of double-mutant mice developed normally. This effect was specific to the urinary system, because loss of Sprouty1 did not rescue the defects in the enteric nervous system characteristic of animals lacking Ret tyrosine 1062. These results suggest that Sprouty1 can modulate ERK signaling downstream of Ret, independent of Grb2/Sos/Ras, during renal morphogenesis.