An important mechanism by which receptor tyrosine kinases (RTKs) mediate cellular responses involves the formation of signaling complexes through direct interactions with membrane-associated docking proteins, followed by phosphorylation of multiple tyrosine residues. These docking proteins recruit and activate downstream signaling molecules and enzymes following ligand stimulation. The docking protein FRS2α has been established as a major signaling hub activated by fibroblast growth factors (FGFs), neurotrophic factors, and other extracellular cues. Here, we show that palmitoylation of FRS2α at two sites is essential for stabilizing its myristoylation-dependent association with the plasma membrane. FGF1-induced MAPK activation and other cellular responses are partially restored in cells expressing FRS2α mutants deficient in either one of the two palmitoylation sites. However full restoration of signal strength including MAPK response and other FGF1-induced cellular activities requires palmitoylation at both FRS2α sites. In addition to enhancing signaling robustness, anchoring of FRS2α to the plasma membrane creates a structural platform for assembling multi-protein complexes essential for cytoskeletal reorganization associated with membrane ruffling, macropinocytosis, and other FGF1-induced processes. Finally, we demonstrate that while PC12 cells lacking FRS2α or deficient in FRS2α palmitoylation can proliferate, FGF1-induced neuronal differentiation strictly depends on the palmitoylation of the docking protein.

Endometrial stromal sarcoma (ESS) is a rare uterine malignancy with limited treatment options. We performed integrated whole-genome, whole-exome, and transcriptome sequencing on 80 ESS tumors, comprising 32 low-grade (LG) and 48 high-grade (HG) tumors, to characterize their genetic landscape. The overall mutation burden was modest, with no significant difference between grades; however, we identified six hypermutated cases (7.5%) harboring POLE or mismatch repair mutations, genomic features predictive of immunotherapy response. We identified focal RAD54B amplifications in 15 tumors (18.8%), leading to elevated RAD54B expression and significantly shorter survival. This establishes RAD54B as an oncogenic driver in ESS. Known tumor suppressors (PTEN, TP53) were frequently mutated in HG-ESS but rare in LG-ESS, highlighting distinct grade-specific drivers of malignancy. HG-ESS exhibited widespread chromosomal gains, frequent loss of cell-cycle regulators (RB1, CDKN2A), and numerous private gene fusions arising from complex DNA rearrangements. In contrast, LG-ESS were defined by canonical fusions (e.g., JAZF1-SUZ12) and co-occurring deletions in metabolic regulator genes (TSC2, STK11). Finally, in an activating NRAS-mutant (p.Q61R) HG-ESS xenograft, the combination of MEK and FAK inhibition dramatically suppressed tumor growth and prolonged survival, highlighting a promising targeted treatment strategy. Overall, our comprehensive analysis defines the molecular basis of ESS and provides a strong preclinical rationale for precision therapies in this aggressive cancer.

Focal adhesion kinase (FAK) and the related tyrosine kinase PYK2 are signaling and scaffolding proteins co-expressed in endothelial cells (ECs) that regulate blood vessel function and tumor growth. As FAK-PYK2 share overlapping cellular roles, we generated PYK2-/-FAKfl/fl mice with tamoxifen-inducible EC-specific Cre expression. EC FAK inactivation in PYK2-/-but not PYK2+/+mice led to increased heart and lung mass, vascular leakage, and created a tumor microenvironment that was repressive to syngeneic melanoma, breast, and lung carcinoma implanted tumor growth. Tumor suppression was associated with defective vessel sprouting, enhanced p53 tumor suppressor and p21CIP1 protein expression in ECs, elevated markers of DNA damage, and altered blood cytokine levels in tumor-bearing mice. However, EC-specific hemizygous kinase-defective (KD) FAK expression in EC FAK-/KD PYK2-/-mice was not associated with elevated p53 levels. Instead, EC FAK-/KD PYK2-/-mice supported primary tumor growth but prevented metastasis, implicating EC FAK activity in tumor spread. In vitro, combined genetic or small molecule FAK-PYK2 knockdown in ECs or tumor cells elevated p21CIP1 and prevented cell proliferation in a p53-dependent manner, highlighting a linkage between EC FAK-PYK2 loss and p53 activation in tumor regulation.

Tumor-infiltrating nerves play a critical role in cancer progression and treatment resistance. Our recent work identified ALKAL2, a ligand for the Anaplastic Lymphoma Kinase (ALK) receptor, as a key mediator of inflammatory pain, with its expression significantly elevated in TRPV1+ sensory neurons during inflammation. Here, we explored the regulation of neuronal ALKAL2 in a colitis-associated colorectal cancer (CAC) model. We found that neuronal ALKAL2 is upregulated at early stages of CAC, which in turn activates ALK signaling in the colonic mucosa. Notably, treating mouse colonic organoids with exogenous ALKAL2 triggered ALK activation. In vivo, mice treated with the ALK inhibitor lorlatinib at the onset of colitis exhibited a remarkable 90% reduction in tumor burden without significantly affecting overall inflammation. Moreover, activating TRPV1+ neurons using DREADD technology exacerbated tumor growth, whereas silencing these neurons significantly reduced it. These findings reveal that TRPV1+ nociceptors drive CAC progression via the ALKAL2/ALK pathway.

Focal adhesion kinase (FAK) functions as a signaling and scaffolding protein within endothelial cells (ECs) impacting blood vessel function and tumor growth. Interpretations of EC FAK-null phenotypes are complicated by related PYK2 (protein tyrosine kinase 2) expression, and to test this, we created PYK2 -/- FAK fl/fl mice with tamoxifen-inducible EC-specific Cre recombinase expression. At 11 weeks of age, EC FAK inactivation resulted in increased heart and lung mass and vascular leakage only on a PYK2 -/- background. Surprisingly, ∼90% of PYK2 -/- EC FAK -/- mice survived to 75 weeks of age. Syngeneic melanoma, breast, or lung carcinoma tumors did not grow in PYK2 -/- EC FAK -/- mice, but tumors grew normally in PYK2 -/- EC FAK fl/fl mice lacking Cre. This tumor inhibitory phenotype was associated with abortive EC vessel sprouting, enhanced EC p53 tumor suppressor and p21CIP1 (cyclin-dependent inhibitor 1) expression, and alterations in serum cytokine levels. To discern the role of FAK kinase versus scaffolding activity in ECs, we generated kinase defective (FAK K454R, KD) PYK2 -/- EC FAK fl/KD and PYK2 -/- EC FAK fl/WT (WT, wildtype) mice. Hemizygous EC FAK -/KD expression supported primary tumor growth but not metastasis, implicating EC FAK activity in tumor dissemination. In vitro , hemizygous expression of either WT or KD FAK suppressed EC p21CIP1 levels and cell death observed in primary PYK2 -/- EC FAK -/- ECs. Combined FAK and PYK2 knockdown in tumor cells also increased p21CIP1 and PARP1 (poly ADP-ribose polymerase 1) levels in a p53-associated manner impacting anchorage-independent growth. Together, these results underscore the linkage between PYK2 and FAK loss with p53 activation impacting tumor growth. Impact Statement:PYK2-null combined with endothelial cell-specific FAK transgenic mouse models show that loss of FAK activity limits tumor spread and that genetic or chemical degradation preventing combined FAK-PYK2 expression may be an approach to induce a p53-associated anti-tumor response.

High-grade neuroendocrine cervical cancers (NETc) are exceedingly rare, highly aggressive tumors. We analyzed 64 NETc tumor samples by whole-exome sequencing (WES). Human papillomavirus DNA was detected in 65.6% (42/64) of the tumors. Recurrent mutations were identified in PIK3CA, KMT2D/MLL2, K-RAS, ARID1A, NOTCH2, and RPL10. The top mutated genes included RB1, ARID1A, PTEN, KMT2D / MLL2, and WDFY3, a gene not yet implicated in NETc. Somatic CNV analysis identified two copy number gains (3q27.1 and 19q13.12) and five copy number losses (1p36.21/5q31.3/6p22.2/9q21.11/11p15.5). Also, gene fusions affecting the ACLY-CRHR1 and PVT1-MYC genes were identified in one of the eight samples subjected to RNA sequencing. To resolve evolutionary history, multiregion WES in NETc admixed with adenocarcinoma cells was performed (i.e., mixed-NETc). Phylogenetic analysis of mixed-NETc demonstrated that adenocarcinoma and neuroendocrine elements derive from a common precursor with mutations typical of adenocarcinomas. Over one-third (22/64) of NETc demonstrated a mutator phenotype of C > T at CpG consistent with deficiencies in MBD4 , a member of the base excision repair (BER) pathway. Mutations in the PI3K/AMPK pathways were identified in 49/64 samples. We used two patient-derived-xenografts (PDX) (i.e., NET19 and NET21) to evaluate the activity of pan-HER (afatinib), PIK3CA (copanlisib), and ATR (elimusertib) inhibitors, alone and in combination. PDXs harboring alterations in the ERBB2/PI3K/AKT/mTOR/ATR pathway were sensitive to afatinib, copanlisib, and elimusertib ( P < 0.001 vs. controls). However, combinations of copanlisib/afatinib and copanlisib/elimusertib were significantly more effective in controlling NETc tumor growth. These findings define the genetic landscape of NETc and suggest that a large subset of these highly lethal malignancies might benefit from existing targeted therapies.

Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.

While important insights were gained about how FGF21 and other endocrine fibroblast growth factors (FGFs) bind to Klotho proteins, the exact mechanism of Klotho/FGF receptor assembly that drives receptor dimerization and activation has not been elucidated. The prevailing dogma is that Klotho proteins substitute for the loss of heparan sulfate proteoglycan (HSPG) binding to endocrine FGFs by high-affinity binding of endocrine FGF molecules to Klotho receptors. To explore a potential role of HSPG in FGF21 signaling, we have analyzed the dynamic properties of FGF21-induced FGF21-βKlotho-FGFR1c complexes on the surface of living wild-type (WT) or HSPG-deficient Chinese hamster ovary (CHO) cells by employing quantitative single-molecule fluorescence imaging analyses. Moreover, detailed analyses of FGF21 and FGF1 stimulation of cellular signaling pathways activated in WT or in HSPG-deficient CHO cells are also analyzed and compared. These experiments demonstrate that heparin is required for the formation of FGF21-βKlotho-FGFR1c complexes on the cell membrane and that binding of heparin or HSPG to FGFR1c is essential for optimal FGF21 stimulation of FGFR1c activation, mitogen-activated protein kinase responses, and intracellular Ca2+ release. It is also shown that FGF1 binding stimulates assembly of βKlotho and FGFR1c on cell membranes, resulting in endocytosis and degradation of βKlotho. We conclude that heparin or HSPG is essential for FGF21 signaling and for regulation of βKlotho cellular stability by acting as a coligand of FGFR1c.

Introduction Uterine leiomyosarcomas (uLMS) are rare, highly aggressive tumors. Up to 30% of uLMS may harbor gain of function (GOF) in the MAP2K4 gene, important for tumor cell proliferation, differentiation and metastasis. We investigated the in vivo activity of a novel MAP2K4 inhibitor, PLX8725, against uLMS harboring MAP2K4 gene-amplification. Methods Two fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or PLX8725 (50 mg/kg) were given via oral gavage daily on weekdays for up to 60 days. Tumor volume differences were calculated with two-way ANOVA. Pharmacokinetic (PK) and mechanistic studies of PLX8725 in uLMS PDX models were also performed. Results Both uLMS tumors evaluated demonstrated GOF in MAP2K4 (i.e., 3 CNV in both LEY-11 and LEY-16). Tumor growth inhibition was significantly greater in both PDX LEY-11 and PDX LEY-16 treated with PLX8725 when compared to controls (p < 0.001). Median overall survival was also significantly longer in both PDX LEY-11 (p = 0.0047) and PDX LEY-16 (p = 0.0058) treatment cohorts when compared to controls. PLX8725 oral treatment was well tolerated, and PK studies demonstrated that oral PLX8725 gives extended exposure in mice. Ex vivo tumor samples after PLX8725 exposure decreased phosphorylated-ATR, JNK and p38, and increased expression of apoptotic molecules on western blot. Conclusion PLX8725 demonstrates promising in vivo activity against PDX models of uLMS harboring GOF alterations in the MAP2K4 gene with tolerable toxicity. Phase I trials of PLX8725 in advanced, recurrent, chemotherapy-resistant uLMS patients are warranted.

The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as acute myeloid leukemia, gastrointestinal stromal tumor, and mast cell leukemia, are driven by somatic gain-of-function KIT mutations. Here, we report cryo electron microscopy (cryo-EM) structural analyses of full-length wild-type and two oncogenic KIT mutants, which show that the overall symmetric arrangement of the extracellular domain of ligand-occupied KIT dimers contains asymmetric D5 homotypic contacts juxtaposing the plasma membrane. Mutational analysis of KIT reveals in D5 region an “Achilles heel” for therapeutic intervention. A ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stable D5 asymmetric conformation. A constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. Binding of SCF to this mutant fully restores the conformation of wild-type KIT dimers, including the formation of salt bridges responsible for D4 homotypic contacts and other hallmarks of SCF-induced KIT dimerization. These experiments reveal an unexpected structural plasticity of oncogenic KIT mutants and a therapeutic target in D5.

Groups and dosing description of the in vivo drug study on USC-ARK1 xenograft.

Ex vivo c-Myc expression assessed by IHC on the ARK2 xenograft tumor samples, excised 1 hour and 6 hours after a single dose of GS-5829 at 40, 20, 10 mg/kg, GS-626510 at 10 mg/kg and vehicle for oral gavage studies.

Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways. Highlights De novo designed cyclic oligomers with tunable geometric properties Cyclic, homo-oligomeric FGFR binding modules induce geometry- and valency-dependent activity of isoform-specific FGF signaling Modulation of FGFR isoform activity controls bifurcation of endothelial and mesenchymal fate during vascular development C-isoform activation favors arterial endothelial cell formation while B-isoform induces pericyte differentiation Graphical Abstract

Cell lines characteristics and tissue source.

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge1-5. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder-target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

Augmentor α and β (Augα and Augβ) are newly discovered ligands of the receptor tyrosine kinases Alk and Ltk. Augα functions as a dimeric ligand that binds with high affinity and specificity to Alk and Ltk. However, a monomeric Augα fragment and monomeric Augβ also bind to Alk and potently stimulate cellular responses. While previous studies demonstrated that oncogenic Alk mutants function as important drivers of a variety of human cancers, the physiological roles of Augα and Augβ are poorly understood. Here, we investigate the physiological roles of Augα and Augβ by exploring mice deficient in each or both Aug ligands. Analysis of mutant mice showed that both Augα single knockout and double knockout of Augα and Augβ exhibit a similar thinness phenotype and resistance to diet-induced obesity. In the Augα-knockout mice, the leanness phenotype is coupled to increased physical activity. By contrast, Augβ-knockout mice showed similar weight curves as the littermate controls. Experiments are presented demonstrating that Augα is robustly expressed and metabolically regulated in agouti-related peptide (AgRP) neurons, cells that control whole-body energy homeostasis in part via their projections to the paraventricular nucleus (PVN). Moreover, both Alk and melanocortin receptor-4 are expressed in discrete neuronal populations in the PVN and are regulated by projections containing Augα and AgRP, respectively, demonstrating that two distinct mechanisms that regulate pigmentation operate in the hypothalamus to control body weight. These experiments show that Alk-driven cancers were co-opted from a neuronal pathway in control of body weight, offering therapeutic opportunities for metabolic diseases and cancer.

Probe molecules that covalently modify the JAK2 pseudokinase domain (JH2) are reported. Selective targeting of JH2 domains over the kinase (JH1) domains is a necessary feature for ligands intended to evaluate JH2 domains as therapeutic targets. The JH2 domains of three Janus kinases (JAK1, JAK2, and TYK2) possess a cysteine residue in the catalytic loop that does not occur in their JH1 domains. Starting from a non-selective kinase binding molecule, computer-aided design directed attachment of substituents terminating in acrylamide warheads to modify Cys675 of JAK2 JH2. Successful covalent attachment was demonstrated first through observation of enhanced binding with increasing incubation time in fluorescence polarization experiments. Covalent binding also increased selectivity to as much as ca. 30-fold for binding the JAK2 JH2 domain over the JH1 domain after a 20-h incubation. Covalency was confirmed through HPLC electrospray quadrupole time-of-flight HRMS experiments, which revealed the expected mass shifts.