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Human genetic evidence demonstrates that WNT1 mutations cause osteogenesis imperfecta (OI) and early‐onset osteoporosis, implicating WNT1 as a major regulator of bone metabolism. However, its main cellular source and mechanisms of action in bone remain elusive. We generated global and limb bud mesenchymal cell–targeted deletion of Wnt1 in mice. Heterozygous deletion of Wnt1 resulted in mild trabecular osteopenia due to decreased osteoblast function. Targeted deletion of Wnt1 in mesenchymal progenitors led to spontaneous fractures due to impaired osteoblast function and increased bone resorption, mimicking the severe OI phenotype in humans with homozygous WNT1 mutations. Importantly, we showed for the first time that Wnt1 signals strictly in a juxtacrine manner to induce osteoblast differentiation and to suppress osteoclastogenesis, in part via canonical Wnt signaling. In conclusion, mesenchymal cell‐derived Wnt1, acting in short range, is an essential regulator of bone homeostasis and an intriguing target for therapeutic interventions for bone diseases. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Simple Summary DNA-damaging chemotherapeutics, such as platinum drugs, are cornerstones in cancer treatment. The efficacy of such treatment is intimately linked to the DNA repair capacity of the cancer cells, as DNA damage above a tolerable threshold culminates in cell death. Cancer cells often have deregulated DNA repair mechanisms, making them initially more sensitive to DNA-damaging chemotherapies. Unfortunately, over time, cancer cells often develop resistance to such treatments by rewiring their DNA damage response pathways. Here, we identify that targeting the recognized anti-cancer target 6-phosphofructo-2-kinase/fructose-2,6,-bisphophatase 3 (PFKFB3), commonly overexpressed in cancer, with the small molecule inhibitor KAN0438757, selectively sensitizes cancer cells to platinum drugs, including treatment-resistant cancer cells, while sparing normal cells. Mechanistically, PFKFB3 promotes tolerance to and the repair of platinum-induced DNA interstrand crosslinks (ICLs) through modulation of the Fanconi anemia (FA) DNA repair pathway. Thus targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments. Abstract Replicative repair of interstrand crosslinks (ICL) generated by platinum chemotherapeutics is orchestrated by the Fanconi anemia (FA) repair pathway to ensure resolution of stalled replication forks and the maintenance of genomic integrity. Here, we identify novel regulation of FA repair by the cancer-associated glycolytic enzyme PFKFB3 that has functional consequences for replication-associated ICL repair and cancer cell survival. Inhibition of PFKFB3 displays a cancer-specific synergy with platinum compounds in blocking cell viability and restores sensitivity in treatment-resistant models. Notably, the synergies are associated with DNA-damage-induced chromatin association of PFKFB3 upon cancer transformation, which further increases upon platinum resistance. FA pathway activation triggers the PFKFB3 assembly into nuclear foci in an ATR- and FANCM-dependent manner. Blocking PFKFB3 activity disrupts the assembly of key FA repair factors and consequently prevents fork restart. This results in an incapacity to replicate cells to progress through S-phase, an accumulation of DNA damage in replicating cells, and fork collapse. We further validate PFKFB3-dependent regulation of FA repair in ex vivo cultures from cancer patients. Collectively, targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.

Simple Summary Chemotherapeutics exerting their antiproliferative actions by introducing DNA crosslinks, such as platinum drugs, are used to treat numerous cancers. Unfortunately, their therapeutic potential is limited due to adverse side effects and acquired resistance, the latter often associated with enhanced DNA repair capacity. Thus, targeting DNA repair is a promising strategy to lower effective doses and associated side effects, and to restore sensitivity to treatment. The C-X3-C motif chemokine receptor 1 (CX3CR1) is an emerging anticancer target which expression correlates with worse overall survival in cancer patients undergoing DNA damaging treatments. Here we show for the first time that the clinical-phase small molecule inhibitor KAND567 targeting CX3CR1 augments the efficacy of DNA crosslinking chemotherapeutics in cancer cell lines, including platinum resistant models, by interference of the Fanconi anemia DNA repair pathway. Hence, the interplay between CX3CR1 and FA repair provides novel potential therapeutic opportunities in cancers treated with DNA crosslinking agents. Abstract The C-X3-C motif chemokine receptor 1 (CX3CR1, fractalkine receptor) is associated with neoplastic transformation, inflammation, neurodegenerative diseases and aging, and the small molecule inhibitor KAND567 targeting CX3CR1 (CX3CR1i) is evaluated in clinical trials for acute systemic inflammation upon SARS-CoV-2 infections. Here we identify a hitherto unknown role of CX3CR1 in Fanconi anemia (FA) pathway mediated repair of DNA interstrand crosslinks (ICLs) in replicating cells. FA pathway activation triggers CX3CR1 nuclear localization which facilitates assembly of the key FA protein FANCD2 into foci. Interfering with CX3CR1 function upon ICL-induction results in inability of replicating cells to progress from S phase, replication fork stalling and impaired chromatin recruitment of key FA pathway factors. Consistent with defective FA repair, CX3CR1i results in increased levels of residual cisplatin-DNA adducts and decreased cell survival. Importantly, CX3CR1i synergizes with platinum agents in a nonreversible manner in proliferation assays including platinum resistant models. Taken together, our results reveal an unanticipated interplay between CX3CR1 and the FA pathway and show for the first time that a clinical-phase small molecule inhibitor targeting CX3CR1 might show benefit in improving responses to DNA crosslinking chemotherapeutics.

The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.

Progressive myoclonus epilepsy of Unverricht–Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by incapacitating stimulus-sensitive myoclonus and tonic-clonic epileptic seizures with onset at the age of 6 to 16 years. EPM1 patients also exhibit a range of skeletal changes, e.g., thickened frontal cranial bone, arachnodactyly and scoliosis. Mutations in the gene encoding cystatin B (CSTB) underlie EPM1. CSTB is an inhibitor of cysteine cathepsins, including cathepsin K, a key enzyme in bone resorption by osteoclasts. CSTB has previously been shown to protect osteoclasts from experimentally induced apoptosis and to modulate bone resorption in vitro. Nevertheless, its physiological function in bone and the cause of the bone changes in patients remain unknown. Here we used the CSTB-deficient mouse (Cstb−/−) model of EPM1 to evaluate the contribution of defective CSTB protein function on bone pathology and osteoclast differentiation and function. Micro-computed tomography of hind limbs revealed thicker trabeculae and elevated bone mineral density in the trabecular bone of Cstb−/− mice. Histology from Cstb−/− mouse bones showed lower osteoclast count and thinner growth plates in long bones. Bone marrow-derived osteoclast cultures revealed lower osteoclast number and size in the Cstb−/− group. Cstb−/− osteoclasts formed less and smaller resorption pits in an in vitro assay. This impaired resorptive capacity was likely due to a decrease in osteoclast numbers and size. These data imply that the skeletal changes in Cstb−/− mice and in EPM1 patients are a result of CSTB deficiency leading to impaired osteoclast formation and consequently compromised resorptive capacity. These results suggest that the role of CSTB in osteoclast homeostasis and modulation of bone metabolism extends beyond cathepsin K regulation., Highlights • μCT reveals changes in trabecular bone of the Cstb−/− mouse model of EPM1, compatible with findings in human patients. • Bone histology in Cstb−/− mice shows lower osteoclast number and thinner growth plates in long bones. • Cultured osteoclasts of Cstb−/− mice show decreased size and number of mature osteoclasts with impaired bone resorption. • Impaired osteoclast formation and resorption are likely to underlie the bone phenotype associated with CSTB deficiency.

Background: Receptor tyrosine kinase (RTK) (ROR1) is normally expressed during embryogenesis but absent in most normal tissues. However, ROR1 is overexpressed in several cancers (onco-fetal RTK) and of importance for various tumor cell functions such as proliferation and survival. In patients with diffuse large B-cell lymphomas (DLBCL) there is a great medical need to develop new treatment alternatives for those not responding to primary treatment as well as for patients with relapse as effective treatments are warranted. Inhibition of ROR1 by a small molecule ROR1 inhibitor (KAN0439834) abrogated downstream kinase activities as well as induced apoptosis of various tumor cells as CLL and pancreatic carcinoma (Leukemia, Oct;32(10):2291-2295, 2018) (PLoS One. 13(6): e0198038, 2018). A 2nd generation of ROR1 inhibitor (KAN0441571C) has been synthesized with the aim to bind to the ROR1-TK domain and inhibit ROR1 signaling. Aim: To examine the expression of ROR1 in DLBCL cell lines (RC-KB, SUDHL4, MS, OCL-LY3, U2932) and in patients´ samples at different stages of DLBCL as well as effects of KAN0441571C on survival of DLBCL cells and ROR1 signaling. Methods: Flow cytometry, tissue microarray and immunohistochemistry assays were used to check ROR1 expression. MTT and Annexin V/PI assays were applied to analyse cytotoxicity and apoptosis of KAN0441571C alone or in combination with ibrutinib (BTK inhibitor) and venetoclax (BCL-2 inhibitor) on DLBCL cell lines. Western blot was performed to evaluate ROR1 phosphorylation and associated signaling pathways. DLBCL cells were also cultured with HS-5 stromal cells (ROR1 neg.) to evaluate the apoptosis inhibitory effects of stromal cells. Results: ROR1 expression was significantly more frequently noted in patients with advanced disease (Richter´s, transformation, transformed follicular lymphoma and refractory DLBCL) compared to less advanced disease (recurrent or de novo DLBCL) (p=0.0001). In primary refractory and relapsing DLBCL 5-years survival was 45% in ROR1- patients (n=17) while in ROR1+ patients (n=16) the corresponding figure was KAN0441571C induced a dose-dependent cytotoxic effect in all ROR1+ DLBCL cell lines (EC50=50-100 nM) while no effect could be noted in the ROR1- U2932 cell line (EC50>10000 nM). EC50 for venetoclax in the ROR1+ DLBCL cell lines varied between 100 and 500 and 5000 - 10000 nM for ibrutinib. In comparison to venetoclax, KAN0441571C induced a similar or significantly higher cytotoxic effect. KAN0441571C and venetoclax seemed to be the most promising drug combination approaching 100% killing at the EC50 dose for each drug. Apoptosis was confirmed by Annexin V/PI staining as well as by downregulation of BCL-2 and MCL-1 as well as cleavage of PARP and caspase 3. KAN0441571C dephosphorylated ROR1 as well as the co-receptor LRP6 and the SRC protein which binds to phosphorylated ROR1. The downstream molecules PI3Kδ/AKT/mTOR was also dephosphorylated and the transcription factor CREB. CK1δ and GSK3B were also dephosphorylated and β-catenin downregulated indicating involvement of both the non-canonical and canonical Wnt pathways. When DLBCL and HS-5 cells (ROR1 neg.) were co-cultured, HS-5 cells could partially prevent induction of apoptosis of DLBCL cells at low concentrations of KAN0441571C, while at higher concentrations the presence of stromal cells was less effective. Zebrafish embryos transplanted with the OCI-Ly3 cell line were treated for 3 days with KAN0441571C (25-1000 nM). No toxic effects of the drug could be noted. A significant dose and time-dependent decrease in the tumor area were noted. Conclusion: KAN0441571C is the 2nd generation of a novel class of ROR1-inhibiting small molecule drugs. The molecule was more effective in inducing apoptosis of DCBCL cells than venetoclax or ibrutinib. New anti-cancer drugs with other mechanisms of action than those clinically available for DLBCL are warranted to improve the prognosis. ROR1 inhibitors in combination with other targeted drugs as venetoclax and ibrutinib might improve the therapeutic effects. KAN0441571C may be a novel drug candidate which needs further exploration in DLBCL. Disclosures Lehto: Kancera AB: Employment. Vågberg:Kancera AB: Employment. Olsson:Kancera AB: Employment. Löfberg:Kancera AB: Employment. Norström:Kancera AB: Employment. Schultz:Kancera AB: Employment, Equity Ownership. Norin:Kancera AB: Employment. Olin:Kancera AB: Employment, Equity Ownership. Österborg:Kancera AB: Research Funding; Janssen: Research Funding; Abbvie: Research Funding; Gilead: Research Funding; BeiGene: Research Funding. Mellstedt:Kancera AB: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Fractures still present a significant burden to patients due to pain and periods of unproductivity. Numerous growth factors have been identified to regulate bone remodeling. However, to date, only the bone morphogenetic proteins (BMPs) are used to enhance fracture healing in clinical settings. Activins are pleiotropic growth factors belonging to the TGF-beta superfamily. We and others have recently shown that treatment with recombinant fusion proteins of activin receptors greatly increases bone mass in different animal models by trapping activins and other ligands thus inhibiting their signaling pathways. However, their effects on fracture healing are less known. Twelve-week old male C57Bl mice were subjected to a standardized, closed tibial fracture model. Animals were divided into control and treatment groups and were administered either PBS control or a soluble activin type IIB receptor (ActRIIB-Fc) intraperitoneally once a week for a duration of two or four weeks. There were no significant differences between the groups at two weeks but we observed a significant increase in callus mineralization in ActRIIB-Fc-treated animals by microcomputed tomography imaging at four weeks. Bone volume per tissue volume was 60%, trabecular number 55% and bone mineral density 60% higher in the 4-week calluses of the ActRIIB-Fc-treated mice (p