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-γ (SNCG), SALL2, and PP1γ, 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.

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.

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.

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.

Molecular Mechanisms Associated with ROR1-Mediated Drug Resistance: Crosstalk with Hippo-YAP/TAZ and BMI-1 Pathways.

Signaling via the Wnt-related receptor tyrosine kinase-like orphan receptor 1 (ROR1) triggers tumorigenic features associated with cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT), while aberrant expression of ROR1 is strongly linked to advanced disease progression and chemoresistance. Several recent studies have shown that Wnt5a binding to ROR1 promotes oncogenic signaling by activating multiple pathways such as RhoA/Rac1 GTPases and PI3K/AKT, which in turn could induce transcriptional coactivator YAP/TAZ or polycomb complex protein BMI-1 signaling, respectively, to sustain stemness, metastasis and ultimately drug-resistance. These data point towards a new feedback loop during cancer development, linking Wnt5a-ROR1 signaling activation to YAP/TAZ or BMI-1 upregulation that could play an important role in disease progression and treatment resistance. This review focuses on the crosstalk between Wnt5a-ROR1 and YAP/TAZ or the BMI-1 signaling network, together with the current advancements in targeted strategies for ROR1-positive cancers.

Wnt5a and ROR1 activate non-canonical Wnt signaling via RhoA in TCF3-PBX1 acute lymphoblastic leukemia and highlight new treatment strategies via Bcl-2 co-targeting.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with TCF3-PBX1 fusion gene expression has constitutively elevated levels of Wnt16b and ROR1 (receptor tyrosine kinase-like orphan receptor), a ligand and a receptor from the Wnt signaling pathway, respectively. Although survival rate is usually high after the initial chemotherapy, many TCF3-PBX1 BCP-ALL patients relapse and subsequently develop treatment resistance, resulting in poor prognosis. Here, we aimed to investigate the molecular signaling associated with Wnt16b and ROR1 overexpression in TCF3-PBX1 cell lines and primary samples, and to identify effective treatment options via ROR1 targeting. We detected higher ROR1 expression on TCF3-PBX1 leukemic cells even at a later stage of patient relapse, providing a strong rationale for the use of ROR1-targeted therapy. We found that Wnt5a-ROR1 signaling enhances proliferation of TCF3-PBX1 cells via RhoA/Rac1 GTPases activation and STAT3 upregulation. Wnt16b also activated the RhoA/Rac1 signaling cascade suggesting the activation of a non-canonical Wnt pathway in TCF3-PBX1 cells. Wnt16 could interact with ROR1 but not in TCF3-PBX1 cells, suggesting that Wnt5a is the ligand signaling via ROR1 in TCF3-PBX1 cells. By high throughput drug-sensitivity testing of TCF3-PBX1 cells before and after ROR1 knockdown we found that targeting ROR1 significantly improves the therapeutic efficacy of Bcl-2 family inhibitors venetoclax and navitoclax, and this synergism was confirmed ex vivo using a drug-resistant primary sample from a relapsed TCF3-PBX1 patient. Our work underlines a new type of targeted combination therapy that could be clinically advantageous for patients with TCF3-PBX1 BCP-ALL.

Targeting Wnt signaling pseudokinases in hematological cancers.

Recent studies showed that several pseudokinases from the receptor tyrosine kinase family are important players in regulating cancer cell invasion, metastasis, and drug resistance, suggesting that targeting these proteins can play a therapeutic role in cancer treatment. Receptor Tyr kinase-like orphan receptors (RORs), protein Tyr kinase 7 (PTK7) (also called colon carcinoma kinase 4 (CCK4)), and receptor-like Tyr kinase (RYK) are Wnt ligand binding receptors within the non-canonical Wnt signaling, with important roles in development, tissue homeostasis, and organogenesis. At the cellular level, these receptors transduce signals important for cell survival, migration, polarization, and chemotaxis. Considerable progress has been made in the last decade in the field of pseudokinase signaling, improving our understanding of their structure-function mechanisms, and intracellular network of transduction components. Consequently, their role in various diseases, including cancer, is now scrutinized for therapeutic interventions to improve treatment outcome. In this article, we review findings regarding molecular mechanisms and targeted therapies for ROR1, PTK7, and RYK in hematological malignancies.

Interaction between ROR1 and MuSK activation complex in myogenic cells.

The ROR family of receptor tyrosine kinases, ROR1 and ROR2, is known to play an important role during skeletal muscle regeneration. ROR1 has a critical role in regulating satellite cell (SC) proliferation during muscle regeneration, and proinflammatory cytokines such as TNF-α and IL-1ß can induce expression of ROR1 in myogenic cells via NF-κB activation. While searching for ROR1-interacting proteins in myogenic cells, we identified MuSK as a ROR1-binding protein. MuSK interacts with and phosphorylates ROR1 at the cytoplasmic proline-rich domain. ROR1 also interacts with the MuSK activator Dok-7 independently of MuSK interaction. Collectively, our results identified ROR1 as a new interacting partner for MuSK and Dok-7, which may have an important role in myogenic cell signaling.

Targeting ROR1 identifies new treatment strategies in hematological cancers.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the ROR receptor family consisting of two closely related type I transmembrane proteins ROR1 and ROR2. Owing to mutations in their canonical motifs required for proper kinase activity, RORs are classified as pseudokinases lacking detectable catalytic activity. ROR1 stands out for its selective and high expression in numerous blood and solid malignancies compared with a minimal expression in healthy adult tissues, suggesting high potential for this molecule as a drug target for cancer therapy. Current understanding attributes a survival role for ROR1 in cancer cells; however, its oncogenic function is cancer-type-specific and involves various signaling pathways. High interest in ROR1-targeted therapies resulted in the development of ROR1 monoclonal antibodies such as cirmtuzumab, currently in a phase I clinical trial for chronic lymphocytic leukemia. Despite these advances in translational studies, the molecular mechanism employed by ROR1 in different cancers is not yet fully understood; therefore, more insights into the oncogenic role of ROR1 signaling are crucial in order to optimize the use of targeted drugs. Recent studies provided evidence that targeting ROR1 simultaneously with inhibition of B-cell receptor (BCR) signaling is more effective in killing ROR1-positive leukemia cells, suggesting a synergistic correlation between co-targeting ROR1 and BCR pathways. Although this synergy has been previously reported for B-cell acute lymphoblastic leukemia, the molecular mechanism appears rather different. These results provide more insights into ROR1-BCR combinatorial treatment strategies in hematological malignancies, which could benefit in tailoring more effective targeted therapies in other ROR1-positive cancers.

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

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-κB p65 levels and that activation of the NF-κB 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-κB pathway activation in B-cell malignancies, this study highlights key factors that can modulate ROR1-targeted treatments in hematological cancers.

Myeloid cell expressed proprotein convertase FURIN attenuates inflammation.

The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1ß levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-ß1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo.