Janus Kinases in Leukemia

Simple Summary Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is a crucial cell signaling pathway that drives the development, differentiation, and function of immune cells and has an important role in blood cell formation. Mutations targeting this pathway can lead to overproduction of these cell types, giving rise to various hematological diseases. This review summarizes pathogenic JAK/STAT activation mechanisms and links known mutations and translocations to different leukemia. In addition, the review discusses the current therapeutic approaches used to inhibit constitutive, cytokine-independent activation of the pathway and the prospects of developing more specific potent JAK inhibitors. Janus kinases (JAKs) transduce signals from dozens of extracellular cytokines and function as critical regulators of cell growth, differentiation, gene expression, and immune responses. Deregulation of JAK/STAT signaling is a central component in several human diseases including various types of leukemia and other malignancies and autoimmune diseases. Different types of leukemia harbor genomic aberrations in all four JAKs (JAK1, JAK2, JAK3, and TYK2), most of which are activating somatic mutations and less frequently translocations resulting in constitutively active JAK fusion proteins. JAKs have become important therapeutic targets and currently, six JAK inhibitors have been approved by the FDA for the treatment of both autoimmune diseases and hematological malignancies. However, the efficacy of the current drugs is not optimal and the full potential of JAK modulators in leukemia is yet to be harnessed. This review discusses the deregulation of JAK-STAT signaling that underlie the pathogenesis of leukemia, i.e., mutations and other mechanisms causing hyperactive cytokine signaling, as well as JAK inhibitors used in clinic and under clinical development.Peer reviewe

Evaluating Targeted Therapies in Ovarian Cancer Metabolism: Novel Role for PCSK9 and Second Generation mTOR Inhibitors

Background: Dysregulated lipid metabolism is emerging as a hallmark in several malignancies, including ovarian cancer (OC). Specifically, metastatic OC is highly dependent on lipid-rich omentum. We aimed to investigate the therapeutic value of targeting lipid metabolism in OC. For this purpose, we studied the role of PCSK9, a cholesterol-regulating enzyme, in OC cell survival and its downstream signaling. We also investigated the cytotoxic efficacy of a small library of metabolic (n = 11) and mTOR (n = 10) inhibitors using OC cell lines (n = 8) and ex vivo patient-derived cell cultures (PDCs, n = 5) to identify clinically suitable drug vulnerabilities. Targeting PCSK9 expression with siRNA or PCSK9 specific inhibitor (PF-06446846) impaired OC cell survival. In addition, overexpression of PCSK9 induced robust AKT phosphorylation along with increased expression of ERK1/2 and MEK1/2, suggesting a pro-survival role of PCSK9 in OC cells. Moreover, our drug testing revealed marked differences in cytotoxic responses to drugs targeting metabolic pathways of high-grade serous ovarian cancer (HGSOC) and low-grade serous ovarian cancer (LGSOC) PDCs. Our results show that targeting PCSK9 expression could impair OC cell survival, which warrants further investigation to address the dependency of this cancer on lipogenesis and omental metastasis. Moreover, the differences in metabolic gene expression and drug responses of OC PDCs indicate the existence of a metabolic heterogeneity within OC subtypes, which should be further explored for therapeutic improvements

Evaluating Targeted Therapies in Ovarian Cancer Metabolism: Novel Role for PCSK9 and Second Generation mTOR Inhibitors

Background: Dysregulated lipid metabolism is emerging as a hallmark in several malignancies, including ovarian cancer (OC). Specifically, metastatic OC is highly dependent on lipid-rich omentum. We aimed to investigate the therapeutic value of targeting lipid metabolism in OC. For this purpose, we studied the role of PCSK9, a cholesterol-regulating enzyme, in OC cell survival and its downstream signaling. We also investigated the cytotoxic efficacy of a small library of metabolic (n = 11) and mTOR (n = 10) inhibitors using OC cell lines (n = 8) and ex vivo patient-derived cell cultures (PDCs, n = 5) to identify clinically suitable drug vulnerabilities. Targeting PCSK9 expression with siRNA or PCSK9 specific inhibitor (PF-06446846) impaired OC cell survival. In addition, overexpression of PCSK9 induced robust AKT phosphorylation along with increased expression of ERK1/2 and MEK1/2, suggesting a pro-survival role of PCSK9 in OC cells. Moreover, our drug testing revealed marked differences in cytotoxic responses to drugs targeting metabolic pathways of high-grade serous ovarian cancer (HGSOC) and low-grade serous ovarian cancer (LGSOC) PDCs. Our results show that targeting PCSK9 expression could impair OC cell survival, which warrants further investigation to address the dependency of this cancer on lipogenesis and omental metastasis. Moreover, the differences in metabolic gene expression and drug responses of OC PDCs indicate the existence of a metabolic heterogeneity within OC subtypes, which should be further explored for therapeutic improvements

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

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

Molecular Regulation of Janus Kinases (JAKs) : Focus on the Pseudokinase Domain

JAK-STAT-(vapaasti suomennettuna Janus-kinaasi - signaalinvälittäjä ja transkriptioaktivaattori) reitti välittää yli 50 sytokiinin signaaleja, jotka säätelevät solun selviytymistä, jakaantumista, migraatiota, geeniekspressiota, sekä muita elintärkeitä prosesseja kuten immuunivastetta. Siksi myös virheellisesti toimiva JAK- STAT signalointi aikaansaa vakavia seurauksia. Aktivoivat JAK-mutaatiot aiheuttavat hematologisia syöpiä sekä myeloproliferatiivisia tauteja, kun taas vajaatoimintainen JAK-signalointi voi johtaa muun muassa vakavaan immuunivajaukseen sekä autoimmuunisairauksiin. Tämä tutkimus keskittyi JAKeissa (JAK1-3 ja tyrosiinikinaasi 2, TYK2) olevaan pseudokinaasiosaan (JH2) joka ei ole kinaasiaktiivinen, kuten sitä muistuttava kinaasiosa (JH1). Biokemialliset tutkimuksemme osoittavat, että JAK pseudokinaasiosat eroavat muun muassa nukleotidin (ATP:n) sitoutumisominaisuuksien osalta. Solupohjaisten kokeiden avulla näytimme, että mutatoimalla kohdennetusti tiettyjä JH2 alueita, pystymme vaikuttamaan JAK- aktiivisuuteen. Vertailimme myös näiden mutanttien vaikutusta signalointiin, riippuen siitä mihin JAK-perheen jäseneen mutaatio kohdentuu. Havaitsimme, että yksittäisen JAKin ja sen pseudokinaasiosan rooli on keskeinen toiminnallisessa signaloinnissa, mutta se voi vaihdella riippuen reseptorikompleksista, jossa JAK kulloinkin toimii. Koska hyvälaatuista täyspitkää JAK rakennetta ei ole saatavilla, D.E. Shaw research (N.Y.) toteutti laskennallisen mallin JAK2-erytropoientin -reseptori kompleksista, jonka me yhteistyössä vahvistimme rakenneperustaisen mutaatioanalyysin avulla. Mallimme kattaa sekä aktiivisen dimeerin, että inaktiivisen monomeerisen JAK2 rakenteen. Inaktiivisessa konformaatiossa JAKin sisäisen JH2-JH1 interaktio sulkee rakenteen ja estää aktiivisten osien transfosforylaation. Aktiivisessa, aukinaisessa rakenteessa JH2-JH2 interaktio kahden JAK2 proteiinin välillä vahvistaa aktiivisen reseptorikompleksin muodostumista. Myös viimeaikaset tutkimukset tukevat pseudokinaasiosan osallistumista dimerisaatioon, ja sitä kautta aktivaatioon. Mallimme antaa myös teoreettista taustaa huomiollemme, jonka mukaan ATP:n sitoutuminen pseudokinaasiosaan mahdollistaa patogeenisten mutaatioiden aktivoitumisen: ATP-sitoutumiskohta on rakenteellisesti tärkeä osa pseudokinaasia, ja vaikuttaa suoraan sen rakenteeseen, sekä dynaamiseen vaihteluun aktiivisen ja inaktiivisen konformaation välillä. Edellä kuvatut tulokset lisäävät ymmärrystä niistä ominaisuuksista, jotka määrittävät moninaisten JAK-signalointireittien spesifisyyden. Lisäksi työmme valottaa mekanismeja, joilla patogeeniset JAK-mutaatiot aiheuttavat pysyvän signaloinnin aktivoitumisen, sekä tukee uusien, entistä vaikuttavampien JAK-inhibiittoreiden kehitystä.The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway mediates the transduction of over 50 cytokines that regulate cell survival, proliferation, migration, gene expression and other vital processes such as immune response. On the other hand, defects in the JAK-STAT signalling have severe impacts. Activating JAK (JAK1-3 and tyrosine kinase 2, TYK2) mutations cause haematological cancers and myeloproliferative disorders while impaired JAK- signalling leads to severe combined immunodeficiency (SCID) and autoimmune diseases. The results presented in this thesis focus on the JAK pseudokinase domain (JH2) that is inactive but has a crucial role in regulating the JAK activity. Our biochemical and cell-based studies show that all JAK JH2s bind ATP, but that the binding properties vary among the JAK-family. Clinical and structure-based mutation studies show that modulation of JH2 can be used to effectively alter the activity. In addition, by introducing homologous mutations into all JAK members, we observed that an individual JAK can have varying functions depending of the signaling systems it is attached to. The results highlight that each JAK within the signaling complex, and specifically JH2, is important for the signaling. As no well-defined structures exist of the full-length JAKs, a molecular dynamic (MD) simulation model of the full-length JAK2 with erythropoietin receptor was constructed in collaboration with D.E. Shaw research (N.Y.). The model depicts JAK2 in different states of activation and is supported by structure-based mutation analysis. In the inactive, monomeric conformation the interaction between JH2 and the kinase domain (JH1) in part closes the JAK2 structure and hinders the transphosphorylation of the active kinase domains. The active conformation is a more open, dimeric structure formed partly via JH2-JH2 interactions between the opposing JAK2 proteins. The model also gives theoretical background to our observation that ATP-binding to JH2 is crucial for the pathogenic JAK activation. It implies that the ATP-binding site is structurally important region within the pseudokinase that directly affects to the dynamic shifting between the active and inactive JH2 conformations. The results summarized above allow us to better comprehend the characteristics that dictate the specificity among JAK-signaling pathways. Moreover, they bring insight into the mechanism of pathologic JAK activation and support the development of novel, more potent JAK inhibitors

Characterization of JAK1 Pseudokinase Domain in Cytokine Signaling

The Janus kinase-signal transducer and activator of transcription protein (JAK-STAT) pathway mediates essential biological functions from immune responses to haematopoiesis. Deregulated JAK-STAT signaling causes myeloproliferative neoplasms, leukaemia, and lymphomas, as well as autoimmune diseases. Thereby JAKs have gained significant relevance as therapeutic targets. However, there is still a clinical need for better JAK inhibitors and novel strategies targeting regions outside the conserved kinase domain have gained interest. In-depth knowledge about the molecular details of JAK activation is required. For example, whether the function and regulation between receptors is conserved remains an open question. We used JAK-deficient cell-lines and structure-based mutagenesis to study the function of JAK1 and its pseudokinase domain (JH2) in cytokine signaling pathways that employ JAK1 with different JAK heterodimerization partner. In interleukin-2 (IL-2)-induced STAT5 activation JAK1 was dominant over JAK3 but in interferon-gamma (IFN gamma) and interferon-alpha (IFN alpha) signaling both JAK1 and heteromeric partner JAK2 or TYK2 were both indispensable for STAT1 activation. Moreover, IL-2 signaling was strictly dependent on both JAK1 JH1 and JH2 but in IFN gamma signaling JAK1 JH2 rather than kinase activity was required for STAT1 activation. To investigate the regulatory function, we focused on two allosteric regions in JAK1 JH2, the ATP-binding pocket and the alpha C-helix. Mutating L633 at the alpha C reduced basal and cytokine induced activation of STAT in both JAK1 wild-type (WT) and constitutively activated mutant backgrounds. Moreover, biochemical characterization and comparison of JH2s let us depict differences in the JH2 ATP-binding and strengthen the hypothesis that de-stabilization of the domain disturbs the regulatory JH1-JH2 interaction. Collectively, our results bring mechanistic understanding about the function of JAK1 in different receptor complexes that likely have relevance for the design of specific JAK modulators.Peer reviewe

Characterization of JAK1 Pseudokinase Domain in Cytokine Signaling

The Janus kinase-signal transducer and activator of transcription protein (JAK-STAT) pathway mediates essential biological functions from immune responses to haematopoiesis. Deregulated JAK-STAT signaling causes myeloproliferative neoplasms, leukaemia, and lymphomas, as well as autoimmune diseases. Thereby JAKs have gained significant relevance as therapeutic targets. However, there is still a clinical need for better JAK inhibitors and novel strategies targeting regions outside the conserved kinase domain have gained interest. In-depth knowledge about the molecular details of JAK activation is required. For example, whether the function and regulation between receptors is conserved remains an open question. We used JAK-deficient cell-lines and structure-based mutagenesis to study the function of JAK1 and its pseudokinase domain (JH2) in cytokine signaling pathways that employ JAK1 with different JAK heterodimerization partner. In interleukin-2 (IL-2)-induced STAT5 activation JAK1 was dominant over JAK3 but in interferon-γ (IFNγ) and interferon-α (IFNα) signaling both JAK1 and heteromeric partner JAK2 or TYK2 were both indispensable for STAT1 activation. Moreover, IL-2 signaling was strictly dependent on both JAK1 JH1 and JH2 but in IFNγ signaling JAK1 JH2 rather than kinase activity was required for STAT1 activation. To investigate the regulatory function, we focused on two allosteric regions in JAK1 JH2, the ATP-binding pocket and the αC-helix. Mutating L633 at the αC reduced basal and cytokine induced activation of STAT in both JAK1 wild-type (WT) and constitutively activated mutant backgrounds. Moreover, biochemical characterization and comparison of JH2s let us depict differences in the JH2 ATP-binding and strengthen the hypothesis that de-stabilization of the domain disturbs the regulatory JH1-JH2 interaction. Collectively, our results bring mechanistic understanding about the function of JAK1 in different receptor complexes that likely have relevance for the design of specific JAK modulators

Selective JAKinibs : Prospects in Inflammatory and Autoimmune Diseases

Cytokines, many of which signal through the JAK–STAT (Janus kinase–Signal Transducers and Activators of Transcription) pathway, play a central role in the pathogenesis of inflammatory and autoimmune diseases. Currently three JAK inhibitors have been approved for clinical use in USA and/or Europe: tofacitinib for rheumatoid arthritis, psoriatic arthritis and ulcerative colitis, baricitinib for rheumatoid arthritis, and ruxolitinib for myeloproliferative neoplasms. The clinical JAK inhibitors target multiple JAKs at high potency and current research has focused on more selective JAK inhibitors, almost a dozen of which currently are being evaluated in clinical trials. In this narrative review, we summarize the status of the pan-JAK and selective JAK inhibitors approved or in clinical trials, and discuss the rationale for selective targeting of JAKs in inflammatory and autoimmune diseases.Peer reviewe