New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

ROR1, ROR2, and PTK7 are Wnt ligand-binding members of the receptor tyrosine kinase family. Despite their lack of catalytic activity, these receptors regulate skeletal, cardiorespiratory, and neurological development during embryonic and fetal stages. However, their overexpression in adult tissue is strongly connected to tumor development and metastasis, suggesting a strong pharmacological potential for these molecules. Wnt5a ligand can activate these receptors, but lead to divergent signaling and functional outcomes through mechanisms that remain largely unknown. Here, we developed a cellular model by stably expressing ROR1, ROR2, and PTK7 in BaF3 cells that allowed us to readily investigate side-by-side their signaling capability and functional outcome. We applied proteomic profiling to BaF3 clones and identified distinctive roles for ROR1, ROR2, and PTK7 pseudokinases in modulating the expression of proteins involved in cytoskeleton dynamics, apoptotic, and metabolic signaling. Functionally, we show that ROR1 expression enhances cell survival and Wnt-mediated cell proliferation, while ROR2 and PTK7 expression is linked to cell migration. We also demonstrate that the distal C-terminal regions of ROR1 and ROR2 are required for receptors stability and downstream signaling. To probe the pharmacological modulation of ROR1 oncogenic signaling, we used affinity purification coupled to mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) to map its interactome before and after binding of GZD824, a small molecule inhibitor previously shown to bind to the ROR1 pseudokinase domain. Our findings bring new insight into the molecular mechanisms of ROR1, ROR2, and PTK7, and highlight the therapeutic potential of targeting ROR1 with small molecule inhibitors binding to its vestigial ATP-binding site.Peer reviewe

Glucocorticoids induce differentiation and chemoresistance in ovarian cancer by promoting ROR1-mediated stemness

Glucocorticoids are routinely used in the clinic as anti-inflammatory and immunosuppressive agents as well as adjuvants during cancer treatment to mitigate the undesirable side effects of chemotherapy. However, recent studies have indicated that glucocorticoids may negatively impact the efficacy of chemotherapy by promoting tumor cell survival, heterogeneity, and metastasis. Here, we show that dexamethasone induces upregulation of ROR1 expression in ovarian cancer (OC), including platinum-resistant OC. Increased ROR1 expression resulted in elevated RhoA, YAP/TAZ, and BMI-1 levels in a panel of OC cell lines as well as primary ovarian cancer patient-derived cells, underlining the translational relevance of our studies. Importantly, dexamethasone induced differentiation of OC patient-derived cells ex vivo according to their molecular subtype and the phenotypic expression of cell differentiation markers. High-throughput drug testing with 528 emerging and clinical oncology compounds of OC cell lines and patient-derived cells revealed that dexamethasone treatment increased the sensitivity to several AKT/PI3K targeted kinase inhibitors, while significantly decreasing the efficacy of chemotherapeutics such as taxanes, as well as anti-apoptotic compounds such as SMAC mimetics. On the other hand, targeting ROR1 expression increased the efficacy of taxane drugs and SMAC mimetics, suggesting new combinatorial targeted treatments for patients with OC.Peer reviewe

Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.Peer reviewe

Multiomics characterization implicates PTK7 in ovarian cancer EMT and cell plasticity and offers strategies for therapeutic intervention

Most patients with ovarian cancer (OC) are diagnosed at a late stage when there are very few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or an oncogenic addiction that can be targeted pharmacologically, unlike other types of cancer. Here, we identified protein tyrosine kinase 7 (PTK7) as a potential new therapeutic target in OC following a multiomics approach using genetic and pharmacological interventions. We performed proteomics analyses upon PTK7 knockdown in OC cells and identified novel downstream effectors such as synuclein-gamma (SNCG), SALL2, and PP1 gamma, and these findings were corroborated in ex vivo primary samples using PTK7 monoclonal antibody cofetuzumab. Our phosphoproteomics analyses demonstrated that PTK7 modulates cell adhesion and Rho-GTPase signaling to sustain epithelial-mesenchymal transition (EMT) and cell plasticity, which was confirmed by high-content image analysis of 3D models. Furthermore, using high-throughput drug sensitivity testing (525 drugs) we show that targeting PTK7 exhibited synergistic activity with chemotherapeutic agent paclitaxel, CHK1/2 inhibitor prexasertib, and PLK1 inhibitor GSK461364, among others, in OC cells and ex vivo primary samples. Taken together, our study provides unique insight into the function of PTK7, which helps to define its role in mediating aberrant Wnt signaling in ovarian cancer.Peer reviewe

Furiinipuutteisen hiiren suolistotulehduksen karakterisointi : Geeniekspressioanalyysi hiiren paksusuolen kudosnäytteistä

Tulehdukselliset suolistosairaudet ovat kroonisia ja uusiutuvia tulehdustiloja suolistossa, eikä niiden syytä vielä tarkasti tunneta. Kaksi yleisintä tulehduksellista suolistosairautta ovat Crohnin tauti ja haavainen paksusuolitulehdus. Sairauksien oletetaan syntyvän normaalin mikrobiflooran aiheuttamasta tavallista voimakkaammasta immuunivasteesta suoliston limakalvoilla, mistä seuraa kudosten vahingoittuminen. Lisätekijöitä ovat geneettinen alttius sekä ympäristön ja ravinnon vaikutukset. Tiettyjen T-soluvasteiden tiedetään vaikuttavan suolistotulehduksen syntyyn. Lisäksi T-soluaktivaation ja proinflammatoristen sytokiinien erityksen on osoitettu kroonistuvan suolistosairauksissa. Hiirille on todettu syntyvän suolistotulehdus, kun niiden T-soluista on poistettu proproteiinikonvertaasientsyymi furiini. Furiinin on osoitettu säätelevän immuunijärjestelmää, mutta sen toimintaa suolistotulehduksessa ei vielä kuitenkaan täysin tunneta. Opinnäytetyön tavoitteena oli tutkia ennalta valittujen sytokiinien ja transkriptiotekijöiden ekspressoitumista suolistotulehduksessa. Työn avulla oli mahdollista saada osaltaan selville tulehduksen mekanismeja sekä nähdä millaisia eroja sytokiiniekspressiossa muodostuu, kun furiini on poistettu T-soluista ja myeloisista soluista. Tarkoituksena oli karakterisoida suolistotulehdus hiirilinjasta, jossa furiini on poistettu T-soluista ja tarkastella mahdollisia tulehduksen merkkejä linjassa, jossa furiini on poistettu hiiren myeloisista soluista. Karakterisointi tehtiin kvantitatiivisen sytokiini- ja transkriptiotekijäekspressioanalyysin avulla. Sytokiiniekspressio oli suurinta niillä hiirillä, joilla furiini oli poistettu T-soluista. Villityypin hiirillä ekspressiotasot olivat odotetusti pienemmät. Myeloisen furiinipuutteisen hiirilinjan sytokiiniekspressiot olivat niin ikään furiini-T-solupuutteisia hiiriä pienemmät, eikä niillä todettu erityisiä tulehduksen merkkejä. Saadut tulokset tukevat aiempia oletuksia siitä, että proinflammatoristen sytokiinien tuotanto tulehduksellisissa suolistosairauksissa on lisääntynyt. Myös furiinilla on tulehduksen kehittymisessä merkitystä, mutta sen toiminta on rajoittunut T-imusoluihin, sillä poistettaessa furiini T-soluista syntyy tulehdus. Sen sijaan furiinin puuttuminen myeloisista soluista ei merkitsevästi kiihdytä tulehdusta. Tulevissa tutkimuksissa voitaisiin selvittää suoliston mikrobikantaa kudosspesifisesti poistogeenisistä furiinilinjoista, jolloin furiinin mahdollisesta yhteydestä mikrobistoon saataisiin lisää tietoa.Inflammatory bowel diseases (IBDs) are chronic and recurrent disorders whose origin is yet unclear. Two most common versions of IBD are Crohn’s disease and ulcerative colitis. The main cause behind the diseases is thought to be a person’s own luminal flora triggering overactive mucosal immune response which leads to intestinal tissue damage. Genetic susceptibility, environment and nutriment also play a role in this. Certain T cell responses are known to have an effect on IBDs. The activation of T cells and the secretion of proinflammatory cytokines have been shown to become chronic in these diseases. Furin T cell deficient mice have got an inflammatory bowel disease. Furin is a notable factor in the specimens’ evolution and it has several target proteins. Its function in IBD is not fully understood although it has been shown that it regulates the immune system. The aim of the thesis was to research the expression of cytokines and transcription factors in IBD. The cytokines and transcription factors were chosen beforehand. By the results of the work it was possible to get some information about the mechanisms of the IBD and see if there were any differences in cytokine expression between two different mouse models. The purpose of the thesis was to characterise the inflammatory bowel disease from mouse models which had the furin knocked out from T cells. A further goal was to observe potential inflammation marks from mice which had furin removed from myeloid cells. The characterisation was made with the help of quantitative cytokine and transcription factor expression analysis. Cytokine expressions were highest with those mice that had furin removed from T cells. The wild type mice had lower expression levels as expected. The expression levels for the myeloid furin deficient mice were lower and there were no particular inflammation marks. The results support the theory that the production of proinflammatory cytokines in inflammatory bowel disease has increased. Moreover furin has some significance with the progress of inflammation because when it was removed from T cells the mice had inflammation, whereas the deletion of furin from myeloid cells did not cause notable inflammation. In future the microbiota of the intestines could be researched with furin knock out mice in which case we could get more information about the possible connection with furin and microbiota

Crosstalk between ROR1 and BCR pathways defines novel treatment strategies in mantle cell lymphoma

International audienceKey Points • Targeting ROR1 downregulates NF-kB p65 expression and sensitizes MCL cells to BCR-or Bcl-2–targeted drugs. • Inhibition of BCR signaling by BTK-specific inhibitors such as ibrutinib impairs ROR1 levels and consecutively ROR1-targeted therapies. Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin B-cell lymphoma with poor prognosis due to drug resistance. Introduction of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib has markedly improved MCL therapy outcome, but drug resistance remains a challenge. The selective cell-surface expression of oncogenic receptor tyrosine kinase–like orphan receptor 1 (ROR1) pseudokinase in hematological malignancies has made this receptor a promising candidate for targeted therapy. We sought to identify the molecular mechanism underlying divergent ROR1-mediated apoptotic responses in MCL cell lines and primary samples. We show that targeting ROR1 expression resulted in downregulation of NF-kB p65 levels and that activation of the NF-kB pathway can antagonize ROR1-mediated apoptotic responses. High-throughput drug-sensitivity testing of MCL cells before and after ROR1 targeting revealed synergistic effects between cotargeting of ROR1 and the B-cell antigen receptor (BCR) or Bcl-2 family, underlining the high potential for ROR1-targeted therapies in overcoming MCL drug resistance. However, inhibition of the BCR pathway by targeted drugs such as ibrutinib can impair ROR1 expression and consequently ROR1-targeted treatments, underscoring the importance of inhibiting both pathways to augment cancer cell killing. Considering the central role of NF-kB pathway activation in B-cell malignancies, this study highlights key factors that can modulat

New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome

Abstract ROR1, ROR2, and PTK7 are Wnt ligand-binding members of the receptor tyrosine kinase family. Despite their lack of catalytic activity, these receptors regulate skeletal, cardiorespiratory, and neurological development during embryonic and fetal stages. However, their overexpression in adult tissue is strongly connected to tumor development and metastasis, suggesting a strong pharmacological potential for these molecules. Wnt5a ligand can activate these receptors, but lead to divergent signaling and functional outcomes through mechanisms that remain largely unknown. Here, we developed a cellular model by stably expressing ROR1, ROR2, and PTK7 in BaF3 cells that allowed us to readily investigate side-by-side their signaling capability and functional outcome. We applied proteomic profiling to BaF3 clones and identified distinctive roles for ROR1, ROR2, and PTK7 pseudokinases in modulating the expression of proteins involved in cytoskeleton dynamics, apoptotic, and metabolic signaling. Functionally, we show that ROR1 expression enhances cell survival and Wnt-mediated cell proliferation, while ROR2 and PTK7 expression is linked to cell migration. We also demonstrate that the distal C-terminal regions of ROR1 and ROR2 are required for receptors stability and downstream signaling. To probe the pharmacological modulation of ROR1 oncogenic signaling, we used affinity purification coupled to mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) to map its interactome before and after binding of GZD824, a small molecule inhibitor previously shown to bind to the ROR1 pseudokinase domain. Our findings bring new insight into the molecular mechanisms of ROR1, ROR2, and PTK7, and highlight the therapeutic potential of targeting ROR1 with small molecule inhibitors binding to its vestigial ATP-binding site