Ligand-independent Activation Research Articles

Unpaired cysteine insertions favor transmembrane dimerization and induce ligand-independent constitutive cytokine receptor signaling.

Naturally occurring gain-of-function (GOF) mutants have been identified in patients for a variety of cytokine receptors. Although this constitutive activation ofcytokine receptors is strongly associated with malignant disorders, ligand-independent receptor activation is also a useful tool in synthetic biology e.g. to improve adoptive cellular therapies with genetically modified T-cells. Balanced Interleukin (IL-)7 signaling via a heterodimer of IL-7 receptor (IL-7Rα) and the common γ-chain (γc) controls T- and B-cell development and expansion, whereas uncontrolled IL-7 signaling can drive acute lymphoid leukemia (ALL) development. The ALL-driver mutation PPCL in the transmembrane domain of IL-7Rα is a mutational insertion of the four amino acids proline-proline-cysteine-leucine and leads to ligand-independent receptor dimerization and constitutive activation. We showed here in the cytokine-dependent pre-B-cell line Ba/F3 that the PPCL-insertion in a synthetic version of the IL-7Rα induced γc-independent STAT5 and ERKphosphorylation and also proliferation of the cells and that booster-stimulation by arteficial ligands additionally generated non-canonical STAT3 phosphorylation via the synthetic IL-7Rα-PPCL-receptors. Transfer of theIL-7Rα transmembrane domain with the PPCL insertion intonatural and synthetic cytokine receptor chains of the IL-6, IL-12 and Interferon families also resulted in constitutive receptor signaling. In conclusion, our data suggested that the insertion of the mutated PPCL IL-7Rα transmembrane domain is an universal approach to generate ligand-independent, constitutively active cytokine receptors.

Abstract PO1-13-06: Androgen Receptor Isoform V7 (AR-V7): A Promising Biomarker for Prognosticating Breast Cancer Aggressiveness

Abstract Androgen Receptor (AR) is an emerging endocrine therapy target in Breast Cancer (BrCa) with up to 80% expression in clinical cases. AR-V7 is a constitutively activated splice variant of AR having a truncated ligand-binding domain (LBD). This confers ligand-independent transcriptional activity to AR-V7 and makes it susceptible to nonsteroidal antiandrogens such as Bicalutamide or Enzalutamide which target the LBD of AR. Higher levels of AR-V7 in metastatic prostate cancer leads to therapeutic resistance and enhanced metastasis. We assessed AR-full length (AR-FL) and AR-V7 expression in BrCa cell lines of different molecular subtypes. This was extended for a clinical correlation of AR-FL and AR-V7 in validated treatment naïve BrCa patients undergoing surgical intervention at a tertiary care hospital in India. Transcriptomic and proteomic analysis was performed using qRT-PCR and Western Blotting. Immunocytochemistry and immunohistochemistry were used to examine protein expression and localisation in cells and tissues. AR-FL and AR-V7 were variably expressed in cell lines which got elevated with Dihydrotestosterone (DHT) treatment. Cell lines stimulated with Bicalutamide or Enzalutamide showed significant downregulation in AR-FL expression. From a cohort of 82 clinical cases, more than 50% originated from the urban setting, post-menopausal stage, and in the age groups of 61 to 70 years. ER, PR, HER2, and AR were positive in 65%, 55%, 24%, and 67% respectively. We were able to correlate aggressive clinical traits and higher pathological grades with increased expression of AR-V7 in the AR-positive BrCa fraction. This was consistent with the cell line-based preclinical research. This study affiliates AR-V7 as an important prognostic biomarker for predicting breast cancer aggressiveness. It can serve as a screening marker of poor clinical outcomes and aid in appropriate therapeutic intervention. These outcomes, however, seek extensive prospective validation. Citation Format: Tryambak Pratap Srivastava, Joyeeta Talukdar, Rohit Kumar Verma, Subhradip Karmakar. Androgen Receptor Isoform V7 (AR-V7): A Promising Biomarker for Prognosticating Breast Cancer Aggressiveness [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-06.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Recording and classifying MET receptor mutations in cancers.

Tyrosine kinase inhibitors (TKI) directed against MET have been recently approved to treat advanced non-small cell lung cancer (NSCLC) harbouring activating MET mutations. This success is the consequence of a long characterization of MET mutations in cancers, which we propose to outline in this review. MET, a receptor tyrosine kinase (RTK), displays in a broad panel of cancers many deregulations liable to promote tumour progression. The first MET mutation was discovered in 1997, in hereditary papillary renal cancer (HPRC), providing the first direct link between MET mutations and cancer development. As in other RTKs, these mutations are located in the kinase domain, leading in most cases to ligand-independent MET activation. In 2014, novel MET mutations were identified in several advanced cancers, including lung cancers. These mutations alter splice sites of exon 14, causing in-frame exon 14 skipping and deletion of a regulatory domain. Because these mutations are not located in the kinase domain, they are original and their mode of action has yet to be fully elucidated. Less than five years after the discovery of such mutations, the efficacy of a MET TKI was evidenced in NSCLC patients displaying MET exon 14 skipping. Yet its use led to a resistance mechanism involving acquisition of novel and already characterized MET mutations. Furthermore, novel somatic MET mutations are constantly being discovered. The challenge is no longer to identify them but to characterize them in order to predict their transforming activity and their sensitivity or resistance to MET TKIs, in order to adapt treatment.

Abstract 2761: Activating RET mutations promotes osteoblastic bone metastasis by inhibiting osteoclastogenesis and stimulating osteoblast activity via regulation of osteoprotegerin

Abstract Bone metastases (BM) are a source of increased mortality and significant morbidity in patients with medullary thyroid cancer (MTC), with ~50% survival at five years post-diagnosis. MTC causes osteoblastic, osteolytic, or mixed lesions, yet the underlying mechanisms are unknown. The RET proto-oncogene encodes a transmembrane tyrosine kinase receptor and is the driver oncogene in ~60% of MTC cases where a germline or somatic activating mutation results in ligand-independent constitutive activation of the receptor. However, the role of RET mutations in the genesis of bone metastasis lesions is unknown. We show that activating RET mutations promotes an osteoblastic phenotype in mice implanted with patient-derived MTC cells by increasing bone formation and decreasing bone resorption. We found that the radiographic appearance of osteoblastic lesions differed between the types of RET mutations (C634W and M918T). MTC cells with RETC634W mutation showed a four-fold increase in bone mineral density, trabecular bone volume, number, and thickness with a decreased number of osteoblasts compared to the non-tumor-bearing femur. TRAP staining revealed that osteoclast numbers per long bone were reduced in the injected femur compared to the control. In contrast, the MTC-RETM918T mutation showed an increased cortical thickness and porosity with reduced medullary area and increased bone formation rate compared to the non-tumor bearing femur. MTC cells formed tumors with extensive mineralized tissue, composed of bone matrix surrounded by osteoblast-like cells and osteoid. The knockdown of RET in the MTC-RETM918T model significantly reduced cortical thickness and increased the number of osteoclasts per bone surface. We observed increased osteoprotegerin (OPG) expression in MTC-bearing femurs compared to controls, suggesting decreased bone resorption. The factors secreted by MTC cells, including OPG, inhibited osteoclast formation from primary bone marrow cells and Raw264 cells. In addition, RET knockdown by shRNA in MTC cells decreased OPG expression. Moreover, the MTC-conditioned media (CM) increased osteoblast differentiation and mineralization of the primary culture of pre-osteoblasts isolated from calvarial bone, long bones, and MC3T3 cells. The conditioned media of RET-depleted cells inhibits osteoblast differentiation and mineralization. The expression of alkaline phosphatase, osteocalcin, OPG, and osterix increased in pre-osteoblast incubated with MTC-CM. Compared to primary non-metastatic tumors, the OPG expression increases in tissues of sporadic MTC bone metastases harboring RETM918T mutation. These results suggest that MTC with RET-activating mutations induces osteoblastic bone metastasis due to decreased bone resorption and increased bone formation. Citation Format: Rozita Bagheri-Yarmand, Joseph L. Kidd, Gabriel M. Pagnotti, Trupti Trivedi, Leah Guerra, Xinhai Wan, Jian Song, Sue-Hwa Lin, Theresa A Guise. Activating RET mutations promotes osteoblastic bone metastasis by inhibiting osteoclastogenesis and stimulating osteoblast activity via regulation of osteoprotegerin [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 2761.

Molecular basis of VEGFR1 autoinhibition at the plasma membrane

Ligand-independent activation of VEGFRs is a hallmark of diabetes and several cancers. Like EGFR, VEGFR2 is activated spontaneously at high receptor concentrations. VEGFR1, on the other hand, remains constitutively inactive in the unligated state, making it an exception among VEGFRs. Ligand stimulation transiently phosphorylates VEGFR1 and induces weak kinase activation in endothelial cells. Recent studies, however, suggest that VEGFR1 signaling is indispensable in regulating various physiological or pathological events. The reason why VEGFR1 is regulated differently from other VEGFRs remains unknown. Here, we elucidate a mechanism of juxtamembrane inhibition that shifts the equilibrium of VEGFR1 towards the inactive state, rendering it an inefficient kinase. The juxtamembrane inhibition of VEGFR1 suppresses its basal phosphorylation even at high receptor concentrations and transiently stabilizes tyrosine phosphorylation after ligand stimulation. We conclude that a subtle imbalance in phosphatase activation or removing juxtamembrane inhibition is sufficient to induce ligand-independent activation of VEGFR1 and sustain tyrosine phosphorylation.

Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias.

Delayed fatherhood results in a higher risk of inheriting a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early- or late-onset effects in the offspring. Here, we used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline. Our functional assessment of the receptor signaling of the variants with biophysical methods showed that 9 of these variants resulted in a higher activation of the receptor´s downstream signaling, resulting in 2 different expansion behaviors. Variants that form larger subclonal expansions in a dissected postmortem testis also showed a positive correlation of the substitution frequency with the sperm donor's age, and a high and ligand-independent FGFR3 activation. In contrast, variants that measured high FGFR3 signaling and elevated substitution frequencies independent of the donor's age did not result in measurable subclonal expansions in the testis. This suggests that promiscuous signal activation might also result in an accumulation of mutations before the sexual maturation of the male gonad with clones staying relatively constant in size throughout time. Collectively, these results provide novel insights into our understanding of the mutagenesis of driver mutations and their resulting mosaicism in the male germline with important consequences for the transmission and recurrence of associated disorders.

Calcium Activation of the Androgen Receptor in Prostate Cells.

Although prostate cancer patients initially respond to androgen deprivation therapy, most patients progress to a resistant phenotype. Castration resistance is due, in part, to intratumoral and/or adrenal synthesis of androgens, overexpression or mutation of the androgen receptor (AR), stabilization of AR by chaperones, and ligand-independent activation of AR. Increasing evidence also links disruption of calcium homeostasis to progression of prostate cancer. Our previous study shows that heavy metal cadmium activates the AR through a ligand-independent mechanism. Cadmium mimics calcium in biological systems due to their similar ionic charge and radius. This study determines whether calcium activates AR and whether first- and second-generation antiandrogens block the ability of calcium to activate the receptor. The expression of androgen-responsive genes and calcium channels was measured in prostate cells using a quantitative real-time polymerase chain reaction assay. Cell growth was measured. To ask whether calcium activates AR, prostate cells were treated with calcium in the absence and presence of the first-generation antiandrogens hydroxyflutamide and bicalutamide and the second-generation antiandrogen enzalutamide, and the expression of androgen-responsive genes and cell growth was measured. In the normal PWR-1E cells and HEK293T cells transiently expressing AR, treatment with calcium increased the expression of androgen-responsive genes by approximately 3-fold. The increase was blocked by enzalutamide but was not consistently blocked by the first-generation antiandrogens. In LNCaP cells which contain a mutant AR, treatment with calcium also increased the expression of androgen-responsive genes by approximately 3-fold, and the increase was more effectively blocked by enzalutamide than by hydroxyflutamide or bicalutamide. Treatment with calcium also increased cell growth that was blocked by enzalutamide. To ask whether dysregulation of calcium channels is associated with castration resistance, calcium channels were measured in the normal PWR-1E prostate cells, the hormone-responsive LNCaP cells, and the castration-resistant VCaP and 22RV1 cells. Compared to normal prostate cells, the hormone-responsive and hormone-resistant cells overexpressed several calcium channels. The results of this study show that calcium activates AR and increases cell growth and that calcium channels are overexpressed in hormone-responsive and hormone-resistant prostate cancer cells. Taken together, the results suggest a novel role of calcium in the castration-resistant phenotype.

Antineoplastic effects of pharmacological inhibitors of aurora kinases in CSF3RT618I-driven cells

Myeloproliferative neoplasms (MPN) are consolidated as a relevant group of diseases derived from the malfunction of the hematopoiesis process and have as a particular attribute the increased proliferation of myeloid lineage. Among these, chronic neutrophilic leukemia (CNL) is distinguished, caused by the T618I mutation of the CSF3R gene, a trait that generates ligand-independent receptor activation and downstream JAK2/STAT signaling. Previous studies reported that mutations in BCR::ABL1 and JAK2V617F increased the expression of the aurora kinase A (AURKA) and B (AURKB) in Ba/F3 cells and their pharmacological inhibition displays antineoplastic effects in human BCR::ABL1 and JAK2V617F positive cells. Delimiting the current scenario, aspects related to the AURKA and AURKB as a potential target in CSF3RT618I-driven models is little known. In the present study, the cellular and molecular effects of pharmacological inhibitors of aurora kinases, such as aurora A inhibitor I, AZD1152-HQPA, and reversine, were evaluated in Ba/F3 expressing the CSF3RT618I mutation. AZD1152-HQPA and reversine demonstrated antineoplastic potential, causing a decrease in cell viability, clonogenicity, and proliferative capacity. At molecular levels, all inhibitors reduced histone H3 phosphorylation, aurora A inhibitor I and reversine reduced STAT5 phosphorylation, and AZD1152-HQPA and reversine induced PARP1 cleavage and γH2AX expression. Reversine more efficiently modulated genes associated with cell cycle and apoptosis compared to other drugs. In summary, our findings shed new insights into the use of AURKB inhibitors in the context of CNL.

Effect of N-glycosylation on constitutive signal transduction by mutated cytokine receptor-like factor 2

BackgroundCytokine receptor-like factor 2 (CRLF2) is a subunit of the receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation (insEIM) in the transmembrane domains of CRLF2 has been identified in acute lymphocytic leukemia (ALL), and Glu-Ile-Met (EIM) CRLF2 induces constitutive activation of signals. However, the signaling mechanism remains unclear. MethodsHEK293 cells were transfected with expression vectors encoding wild-type (WT), insEIM CRLF2, or their mutants which N-glycosylation site was replaced with a glutamine. Cell surface expression of CRLF2 was assessed by flow cytometry. Total CRLF2 and phosphorylated signal transducer and activator of transcription 5 (STAT5) were detected by western blotting. ResultsThree major species of CRLF2 (53-, 57- and 58-kDa) were identified. Deglycosylation analysis revealed that they were modified with complex-type and oligomannose-type glycans. The expression of both WT and EIM CRLF2 decreased in N-acetylglucosaminyltransferase (GnT)-I (MGAT1) knockout (KO) cells and slightly decreased in α1,6-fucosyltransferase (Fut8) KO cells compared to that in the control cells. In GnT-I or Fut8 KO cells, WT CRLF2 did not induce ligand-independent activation. Both WT and EIM CRLF2 contained four N-glycosylation sites. N55 of CRLF2 was required for the cell surface expression and activation by EIM CRLF2. ConclusionsWe found that N-glycosylation of CRLF2 plays crucial roles for its cell surface expression and signaling. However, N-glycan processing in the Golgi apparatus does not seem to be essential for ligand-independent activation of EIM CRLF2. General significanceOur studies provide a crucial role of glycosylation in the cell surface expression of receptors.

Ligand-independent Activation of Aryl Hydrocarbon Receptor and Attenuation of Glutamine Levels by Natural Deep Eutectic Solvent.

Natural deep eutectic solvents (NADESs) are emerging sustainable alternatives to conventional organic solvents. Beyond their role as laboratory solvents, NADESs are increasingly explored in drug delivery and as therapeutics. Their increasing applications notwithstanding, our understanding of how they interact with biomolecules at multiple levels - metabolome, proteome, and transcriptome - within human cell remain poor. Here, we deploy integrated metabolomics, proteomics, and transcriptomics to probe how NADESs perturb the molecular landscape of human cells. In a human cell line model, we found that an archetypal NADES derived from choline and geranic acid (CAGE) significantly altered the metabolome, proteome, and transcriptome. CAGE upregulated indole-3-lactic acid and 4-hydroxyphenyllactic acid levels, resulting in ligand-independent activation of aryl hydrocarbon receptor to signal the transcription of genes with implications for inflammation, immunomodulation, cell development, and chemical detoxification. Further, treating the cell line with CAGE downregulated glutamine biosynthesis, a nutrient rapidly proliferating cancer cells require. The ability of CAGE to attenuate glutamine levels is potentially relevant for cancer treatment. These findings suggest that NADESs, even when derived from natural components like choline, can indirectly modulate cell biology at multiple levels, expanding their applications beyond chemistry to biomedicine and biotechnology.

Knockout of all ErbB-family genes delineates their roles in proliferation, survival and migration.

The ErbB-family receptors play pivotal roles in the proliferation, migration and survival of epithelial cells. Because our knowledge on the ErbB-family receptors has been largely obtained by the exogenous application of their ligands, it remains unknown to what extent each of the ErbB members contributes to these outputs. We here knocked out each ErbB gene, various combinations of ErbB genes or all ErbB genes in Madin-Darby canine kidney cells to delineate the contribution of each gene. ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) activation waves during collective cell migration were mediated primarily by ErbB1 and secondarily by the ErbB2 and ErbB3 heterodimer. Either ErbB1 or the ErbB2 and ErbB3 complex was sufficient for the G1/S progression. The saturation cell density was markedly reduced in cells deficient in all ErbB proteins, but not in cells retaining only ErbB2, which cannot bind to ligands. Thus, a ligand-independent ErbB2 activity is sufficient for preventing apoptosis at high cell density. In short, systematic knockout of ErbB-family genes has delineated the roles of each ErbB receptor.

Ligand-independent activation of platelet-derived growth factor receptor β promotes vitreous-induced contraction of retinal pigment epithelial cells

BackgroundEpiretinal membranes in patients with proliferative vitreoretinopathy (PVR) consist of extracellular matrix and a number of cell types including retinal pigment epithelial (RPE) cells and fibroblasts, whose contraction causes retinal detachment. In RPE cells depletion of platelet-derived growth factor (PDGF) receptor (PDGFR)β suppresses vitreous-induced Akt activation, whereas in fibroblasts Akt activation through indirect activation of PDGFRα by growth factors outside the PDGF family (non-PDGFs) plays an essential role in experimental PVR. Whether non-PDGFs in the vitreous, however, were also able to activate PDGFRβ in RPE cells remained elusive.MethodsThe CRISPR/Cas9 technology was utilized to edit a genomic PDGFRB locus in RPE cells derived from an epiretinal membrane (RPEM) from a patient with PVR, and a retroviral vector was used to express a truncated PDGFRβ short of a PDGF-binding domain in the RPEM cells lacking PDGFRβ. Western blot was employed to analyze expression of PDGFRβ and α-smooth muscle actin, and signaling events (p-PDGFRβ and p-Akt). Cellular assays (proliferation, migration and contraction) were also applied in this study.ResultsExpression of a truncated PDGFRβ lacking a PDGF-binding domain in the RPEM cells whose PDGFRB gene has been silent using the CRISPR/Cas9 technology restores vitreous-induced Akt activation as well as cell proliferation, epithelial-mesenchymal transition, migration and contraction. In addition, we show that scavenging reactive oxygen species (ROS) with N-acetyl-cysteine and inhibiting Src family kinases (SFKs) with their specific inhibitor SU6656 blunt the vitreous-induced activation of the truncated PDGFRβ and Akt as well as the cellular events related to the PVR pathogenesis. These discoveries suggest that in RPE cells PDGFRβ can be activated indirectly by non-PDGFs in the vitreous via an intracellular pathway of ROS/SFKs to facilitate the development of PVR, thereby providing novel opportunities for PVR therapeutics.ConclusionThe data shown here will improve our understanding of the mechanism by which PDGFRβ can be activated by non-PDGFs in the vitreous via an intracellular route of ROS/SFKs and provide a conceptual foundation for preventing PVR by inhibiting PDGFRβ transactivation (ligand-independent activation).

The combined action of the intracellular regions regulates FGFR2 kinase activity

Receptor tyrosine kinases (RTKs) are typically activated through a precise sequence of intracellular phosphorylation events starting with a tyrosine residue on the activation loop (A-loop) of the kinase domain (KD). From this point the mono-phosphorylated enzyme is active, but subject to stringent regulatory mechanisms which can vary dramatically across the different RTKs. In the absence of extracellular stimulation, fibroblast growth factor receptor 2 (FGFR2) exists in the mono-phosphorylated state in which catalytic activity is regulated to allow rapid response upon ligand binding, whilst restricting ligand-independent activation. Failure of this regulation is responsible for pathologic outcomes including cancer. Here we reveal the molecular mechanistic detail of KD control based on combinatorial interactions of the juxtamembrane (JM) and the C-terminal tail (CT) regions of the receptor. JM stabilizes the asymmetric dimeric KD required for substrate phosphorylation, whilst CT binding opposes dimerization, and down-regulates activity. Direct binding between JM and CT delays the recruitment of downstream effector proteins adding a further control step as the receptor proceeds to full activation. Our findings underscore the diversity in mechanisms of RTK oligomerisation and activation.

Real-World Use of Highly Sensitive Liquid Biopsy Monitoring in Metastatic Breast Cancer Patients Treated with Endocrine Agents after Exposure to Aromatase Inhibitors.

Endocrine-resistant, hormone receptor-positive, and HER2-negative (HR+/HER2-) metastatic breast cancer (mBC) is largely governed by acquired mutations in the estrogen receptor, which promote ligand-independent activation, and by truncal alterations in the PI3K signaling pathway, with a broader range of gene alterations occurring with less prevalence. Circulating tumor DNA (ctDNA)-based technologies are progressively permeating the clinical setting. However, their utility for serial monitoring has been hindered by their significant costs, inter-technique variability, and real-world patient heterogeneity. We interrogated a longitudinal collection of 180 plasma samples from 75 HR+/HER2- mBC patients who progressed or relapsed after exposure to aromatase inhibitors and were subsequently treated with endocrine therapy (ET) by means of highly sensitive and affordable digital PCR and SafeSEQ sequencing. Baseline PIK3CA and TP53 mutations were prognostic of a shorter progression-free survival in our population. Mutant PIK3CA was prognostic in the subset of patients receiving fulvestrant monotherapy after progression to a CDK4/6 inhibitor (CDK4/6i)-containing regimen, and its suppression was predictive in a case of long-term benefit with alpelisib. Mutant ESR1 was prognostic in patients who did not receive concurrent CDK4/6i, an impact influenced by the variant allele frequency, and its early suppression was strongly predictive of efficacy and associated with long-term benefit in the whole cohort. Mutations in ESR1, TP53, and KRAS emerged as putative drivers of acquired resistance. These findings collectively contribute to the characterization of longitudinal ctDNA in real-world cases of HR+/HER2- mBC previously exposed to aromatase inhibitors and support ongoing studies either targeting actionable alterations or leveraging the ultra-sensitive tracking of ctDNA.

The 2023 Updated Classification and Diagnostic Criteria of Mastocytosis

Mastocytosis is an uncommon hematologic malignancy characterized by accumulation of abnormal mast cells in various organs or tissues. A somatic point mutation in the KIT gene at codon 816 is detected in more than 90% of patients with systemic Masto cytosis (SM). The 2022 WHO classification continues to recognize three disease types: systemic mastocytosis, cutaneous mastocytosis and mast cell sarcoma. Bone marrow mastocytosis is a new separate subtype of SM. The classification also recognizes well-differentiated systemic mastocytosis, a morphologic pattern that can occur in any SM subtype. Diagnostic criteria for SM have been modified. The expression of CD30 and the presence of any KIT mutation causing ligand-independent activation have been accepted as minor diagnostic criteria. Classical B-findings and C-findings have undergone minor refinements. Most notably, variant allele frequency of D816V mutation ≥ 10% in bone marrow cells or peripheral blood leukocytes is qualified as a B-finding.

Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of 6 amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together these data allow us to develop a general model for activation of TpoR and to explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

A phase 1b/2 dose escalation and expansion study of OP-1250 in combination with ribociclib or alpelisib in patients with advanced and/or metastatic estrogen receptor–positive (ER+)/HER2-negative (HER2-) breast cancer.

TPS1127 Background: Therapies targeting the estrogen receptor (ER) are standard of care in the management of ER+/HER2- metastatic breast cancer (MBC) and when administered concurrently with cyclin-dependent kinase (CDK) inhibitors are the most effective first-line therapy. CDK 4/6 inhibitors plus endocrine therapy improve progression-free survival and, for some agents, overall survival for patients with MBC. However, most patients will acquire resistance, highlighting an unmet need for more effective endocrine options in ER+ disease. OP-1250 is a small molecule oral complete estrogen receptor antagonist (CERAN) and selective estrogen receptor degrader (SERD) that binds the ligand binding domain of ER and completely blocks ER-driven transcriptional activity. OP-1250 potently inactivates both wild-type and mutant forms of ER, the latter of which confers ligand independent activity as a mechanism of resistance to standard of care endocrine therapies. In preclinical studies, OP-1250 in combination with CDK4/6 inhibitors demonstrated synergistic activity in models of both wild-type ER and those with ESR1 (the gene that encodes ER) activating mutations, and in brain metastasis. OP-1250 is orally bioavailable with a favorable pharmacokinetic (PK) profile supportive of once-daily dosing, and a low probability of drug:drug interactions, making it an attractive agent for patients with ER+/HER2- MBC. Methods: The study (NCT05508906) is an open-label, multicenter, 2-part study of OP-1250 in combination with either the CDK4/6 inhibitor ribociclib or the PI3K inhibitor alpelisib for patients with MBC. Eligibility includes adults with evaluable (measurable or non-measurable) ER+/HER2- recurrent, locally advanced or MBC confirmed by histology/cytology, ≤2 prior hormonal regimens (prior CDK4/6 inhibitors allowed) and ≤ 1 prior line of chemotherapy. Patients in the alpelisib arm must have a PIK3CA mutation. A total of 30 patients will be enrolled in each arm. The dose escalation cohorts will evaluate the safety and PK of varied OP-1250 doses (30mg, 60mg, or 120mg) administered orally (PO) every day (QD) in combination with approved dosages of either ribociclib 600 mg PO QD or alpelisib 300 mg PO QD to identify the recommended phase 2 doses (RP2D). The dose expansion cohorts will assess additional safety and PK parameters and further explore the anti-tumor activity of OP-1250 in combination with ribociclib or alpelisib. Tolerability, safety and PK of OP-1250 in combination with ribociclib or alpelisib constitute the primary endpoints. Overall response rate, clinical benefit rate and duration of response are key secondary endpoints. Enrollment at approximately 20 sites in the United States and Australia is ongoing. Clinical trial information: NCT05508906 .

Phase separation propensity of the intrinsically disordered AB region of human RXRβ

RXRβ is one of three subtypes of human retinoid X receptor (RXR), a transcription factor that belongs to the nuclear receptor superfamily. Its expression can be detected in almost all tissues. In contrast to other subtypes – RXRα and RXRγ – RXRβ has the longest and unique N-terminal sequence called the AB region, which harbors a ligand-independent activation function. In contrast to the functional properties of this sequence, the molecular properties of the AB region of human RXRβ (AB_hRXRB) have not yet been characterized. Here, we present a systematic biochemical and biophysical analysis of recombinant AB_hRXRB, along with in silico examinations, which demonstrate that AB_hRXRB exhibits properties of a coil-like intrinsically disordered region. AB_hRXRB possesses a flexible structure that is able to adopt a more ordered conformation under the influence of different environmental factors. Interestingly, AB_hRXRB promotes the formation of liquid–liquid phase separation (LLPS), a phenomenon previously observed for the AB region of another human subtype of RXR – RXRγ (AB_hRXRG). Although both AB regions seem to be similar in terms of their ability to induce phase separation, they clearly differ in the sensitivity to factors driving and regulating LLPS. This distinct LLPS response to environmental factors driven by the unique amino acid compositions of AB_hRXRB and AB_hRXRG can be significant for the specific modulation of the transcriptional activation of target genes by different subtypes of RXR.89Jx1wwVvjJQ45uAcpVb5SVideo