Microneedle (MN) technology is a rising star in the point-of-care (POC) field, which has gained increasing attention from scientists and clinics. MN-based POC devices show great potential for detecting various analytes of clinical interests and transdermal drug delivery in a minimally invasive manner owing to MNs’ micro-size sharp tips and ease of use. This review aims to go through the recent achievements in MN-based devices by investigating the selection of materials, fabrication techniques, classification, and application, respectively. We further highlight critical aspects of MN platforms for transdermal biofluids extraction, diagnosis, and drug delivery assisted disease therapy. Moreover, multifunctional MNs for stimulus-responsive drug delivery systems were discussed, which show incredible potential for accurate and efficient disease treatment in dynamic environments for a long period of time. In addition, we also discuss the remaining challenges and emerging trend of MN-based POC devices from the bench to the bedside. Wearable healthcare technologies cause significant consideration in publics and scientists, owing to the vast demand from medical laboratories and clinics ,2. AccorSkin, as one of the most significant organs in human body, plays a crucial role in protection, perception, and secretion. Biofluids below the skin provide vital indications of human health . HoweverFortunately, microneedles (MNs) with micro-scale sizes  have been emerging as minimally invasive devices for transdermal sensing, sampling, and molecule delivery ,14,15,16The preliminary study on MNs started in 1976, but it was not extensively exploited until the late 1990s because of advancements in microfabrication technology, which provided suitable tools for MNs manufacture. Up to now, MNs have been developed with various materials  ,23,24. BMany reviews on microneedle technology have been published in recent years. However, many of them typically focus on a specific aspect, such as materials science and manufacture , glucoseMaterials decide the essential properties of MNs, such as toughness, flexibility, and permeability. Therefore, it is significant to choose appropriate materials for a special application. Various materials are used to fabricate MN devices. These materials can be classified into six categories: silicon, glass, ceramics, metals, polymers, and carbohydrate, as shown in Silicon is one of commonly used materials for MN fabrication owing to its excellent properties. Firstly, as the main material used in micro-electromechanical systems (MEMSs), silicon has excellent mechanical strength and biocompatibility ,53. ThesGlass has many remarkable properties including being transparent, cheap, and chemically inert. A relatively high Young’s modulus enables glass-based MNs to pierce the derma without deformation . The cheCeramics, especially bio-ceramics, have been used in drug delivery for a long time. Porous ceramic-based MNs allow the drug to be incorporated in the interconnected pores and to diffuse directly after interposition ,58,59. IMetals are also used for the fabrication of MNs, because some metals  have outstanding mechanical strength and biocompatibility , while n2, –OH, –SO3H) possess swellability . . 164]. TAs cost-effective, reliable, minimally invasive devices, MNs conquered much more space in the therapy and diagnostics of diseases by facilitating the dynamic dosing of therapeutics, and monitoring of biomarkers of clinical interests both in blood and ISF. The MN devices can be easily obtained by changing the fabrication technology, materials, and production parameters, which can be classified into solid, hollow, coated, dissolving, and swellable MNs. Different types of MNs possess various physical and chemical characteristics, such as conductivity, morphology, wettability, swellability, and biodegradability. MNs have shown promising preclinical outcomes and are expected to contribute to personalization of treatment and management of chronic diseases. MN-based POC devices for vaccination against influenza are already commercial . Hollow Future clinical analysis devices are expected to minimize the labor cost and eliminate the restriction of operational site, thereby enabling common citizens at any cities and any levels to obtain medical care with negligible costs . Despite


Dysregulated Wnt/β-catenin activation plays a critical role in cancer progression, metastasis, and drug resistance. Genotoxic agents such as radiation and chemotherapeutics have been shown to activate the Wnt/β-catenin signaling although the underlying mechanism remains incompletely understood. Here, we show that genotoxic agent-activated Wnt/β-catenin signaling is independent of the FZD/LRP heterodimeric receptors and Wnt ligands. OTULIN, a linear linkage-specific deubiquitinase, is essential for the DNA damage-induced β-catenin activation. OTULIN inhibits linear ubiquitination of β-catenin, which attenuates its Lys48-linked ubiquitination and proteasomal degradation upon DNA damage. The association with β-catenin is enhanced by OTULIN Tyr56 phosphorylation, which depends on genotoxic stress-activated ABL1/c-Abl. Inhibiting OTULIN or Wnt/β-catenin sensitizes triple-negative breast cancer xenograft tumors to chemotherapeutics and reduces metastasis. Increased OTULIN levels are associated with aggressive molecular subtypes and poor survival in breast cancer patients. Thus, OTULIN-mediated Wnt/β-catenin activation upon genotoxic treatments promotes drug resistance and metastasis in breast cancers. Genotoxic agents have been shown to activate the Wnt/β-catenin signaling but the underlying mechanism remains unclear. Here, the authors show that upon DNA damage, the deubiquitinase OTULIN activates Wnt/β-catenin signaling by inhibiting linear ubiquitination, K48-linked polyubiquitination, and proteasomal degradation of β-catenin. Dysregulated Wnt signaling and mutation of genes involved in the Wnt signaling cascades are frequently observed in a wide range of human diseases, such as cancer and degenerative diseases2. Phosphorylated β-catenin can be recognized by F-box/WD-repeat protein β-TrCP within the SCF ubiquitin ligase complex, which facilitates assembly of Lys48 (K48)-linked polyubiquitin chains on β-catenin, leading to its proteasome-dependent degradation4. Wnt ligand binding with Fzd/LRP receptor complexes together with the recruitment of Dishevelled (Dvl) induces stoichiometric sequestration of AXIN/CK1/GSK3 onto the receptor complex and phosphorylation of LRP5/6. Receptor engagement of the destruction complex to the cell membrane inhibits GSK-3 and blocks the ubiquitination of phosphorylated β-catenin, leading to the accumulation of newly synthesized β-catenin. Increased cytoplasmic β-catenin then translocates into the nucleus where it binds to members of the T cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor family to drive transcription of Wnt/β-catenin target genes such as MYC, CCND1, and AXIN2. Wnt ligands also activate non-canonical Wnt pathways, which are independent of β-catenin5. In the planar cell polarity (PCP) pathways, non-canonical Wnt ligands  interact with FZDs and co-receptors ROR/RYK that activate the small GTPases Rho and Rac, leading to cytoskeleton rearrangement and cell polarization. Additionally, the Wnt/Ca2+ pathway depends on calcium release from the endoplasmic reticulum (ER) upon Wnt binding to FZDs and activation of phospholipase C, which activates protein kinase C and promotes CamKII -mediated activation of transcription factor nuclear factor of activated T cell (NFAT).Two major Wnt signaling cascades, β-catenin-dependent canonical Wnt pathway, and β-catenin-independent non-canonical Wnt signaling, have been well characterized. Canonical Wnt signaling is initiated by binding of Wnt ligands  to heterodimeric receptor complex formed by Frizzled (Fzd) and co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) or its close relative LRP5. The signaling output of the canonical Wnt pathway is determined by the level of cytosolic β-catenin, which is under the strict control of the destruction complex. The destruction complex is composed of AXIN, APC (Adenomatous Polyposis Coli Protein), WTX (Wilms tumor suppressor) and two constitutively active kinases (CK1α/δ and GSK3α/β), which associate with β-catenin and promote its polyubiquitination by phosphorylating the degron motif of β-catenin6. A canonical Wnt signaling-specific target gene Axin2 was upregulated in mouse intestinal crypt cells after IR8. In addition, a canonical Wnt/β-catenin gene signature was enriched in Adriamycin-treated mouse and human tumor cells9. These findings indicate that DNA damage by genotoxic treatments induces Wnt/β-catenin activation, however, the underlying mechanisms orchestrating β-catenin stabilization and transcriptional activation remain poorly understood.A previous study showed that TCF/LEF H2B-GFP reporter mice for Wnt signaling displayed robust β-catenin activation in hematopoietic stem cells (HSCs) following ionizing radiation (IR)11. Recent studies identified several hypomorphic mutations of OTULIN in humans, which lead to autoimmune responses and hyper-inflammation14. OTULIN was found to interact with the linear ubiquitin assembly complex (LUBAC), which is composed of HOIP (HOIL-1-interacting protein/RNF31), HOIL-1(heme-oxidized IRP2 ubiquitin ligase 1/RBCK1) and Sharpin. As an E3 ligase complex, LUBAC specifically attaches M1-linked polyubiquitin chains on its substrate16. The OTULIN association with LUBAC is mediated by the interaction between the PUB (peptide: N-glycanase/UBA- or UBX-containing proteins) domain of HOIP and the PIM (PUB-interaction motif) domain of OTULIN18. Phosphorylation of the highly conserved Tyr56 within the PIM was shown to disrupt the interaction between OTULIN and HOIP, which abrogates OTULIN-dependent suppression of LUBAC and enables LUBAC-regulated signaling. However, the kinase(s) phosphorylating Tyr56 and whether the Tyr56 phosphorylation is a regulated mechanism in cells were unclear19.OTULIN  is a deubiquitinase exclusively cleaving polyubiquitin chains linked with linear linkage (Met1/M1-linkage)20, how linear chain disassembly by deubiquitinases like OTULIN influences cellular functions was less understood19. OTULIN was found to either enhance or suppress NF-κB activation in a context-dependent manner21. In mice harboring inactive Otulin/Gumby mutations (W96R or D336E), Wnt/β-catenin activation was significantly reduced, leading to angiogenic defect and embryonic lethality, whereas the underlying mechanism was largely unexplored11. Here, we find that genotoxic stress induces Wnt/β-catenin activation in an OTULIN-dependent manner. DNA damage-induced c-Abl/ABL1 activation, which depends on DNA-PK, is required for Tyr56 phosphorylation of OTULIN. OTULIN phosphorylation at Tyr56 enhances its interaction with β-catenin while diminishing the OTULIN/LUBAC association. Moreover, LUBAC promotes linear ubiquitination of β-catenin in unstimulated cells, which facilitates the proteasomal degradation of β-catenin. Upon genotoxic treatment, increased OTULIN/β-catenin association inhibits β-catenin linear ubiquitination thereby promoting its stabilization and Wnt/β-catenin signaling activation. We further show that increased OTULIN levels are associated with aggressive breast cancer subtypes and correlate with poor survival in breast cancer patients. Increased Wnt/β-catenin activation in TNBC cells upon chemotherapy, which is dependent on OTULIN, facilitates cancer cell adaptation to a drug-resistant state and enhances metastasis. Therefore, targeting genotoxic Wnt/β-catenin activation and OTULIN may mitigate the development of drug resistance and metastasis in TNBC patients receiving chemotherapy.Although much has been learned about how LUBAC-assembled linear ubiquitin chains regulate immune signaling such as NF-κB8. Enriched canonical Wnt gene signature was also observed in tumor cells treated by adriamycin9. We found significant nuclear enrichment of β-catenin in doxorubicin (Dox)-treated MDA-MB-231 cells, supporting the activation of Wnt/β-catenin signaling by genotoxic treatment lls Fig. , indicatBAC Fig. . MoreoveBAC Fig. . In contBAC Fig. , which wBAC Fig. . ImportaBAC Fig. . We also5. Interestingly, we found β-catenin was modified by M1 ubiquitin chains in cells transfected with LUBAC, which was attenuated by co-expression of OTULIN . We found that K133R mutation substantially decreased the linear ubiquitination of β-catenin compared with other mutants  and Lys 49 (K49) of β-catenin were required for its ubiquitinationnts Fig. , suggestnts Fig. . To our nts Fig. . Moreovents Fig. , suggest16. SCFβ-TrCP E3 ubiquitin ligase-mediated K48 ubiquitination is essential for proteasomal degradation of β-catenin1. LUBAC overexpression enhanced both linear and K48 ubiquitination of β-catenin in HEK293T cells, whereas co-expression of a dominant-negative β-TrCP F-box-deletion mutant selectively suppressed K48 ubiquitination of β-catenin promoted by LUBAC . This antibody detected OTULIN phosphorylation in cells exposed to genotoxic drugs, which was abrogated by phosphatase treatment or Tyr56 mutation. Tyr56 phosphorylation was not observed in cells treated with Wnt3a , ATR (VE-821) or IKK (TPCA)  significantly increased β-catenin level as well as Axin2 transcription, compared with the control PyMT cells  or CPT-11 (~6.2-fold increase in IC50)  and promoting EMT, which both contribute significantly to cancer metastasis and drug resistance50) Fig. . In contTo further validate the role of OTULIN-mediated Wnt activation in promoting breast cancer drug resistance in vivo, we established orthotopic TNBC xenograft models with WT or OTULIN-KO MDA-MB-231 LM2 cells. OTULIN deletion showed little effect in xenograft tumor growth, but it significantly sensitized MDA-MB-231 xenografts to Dox treatment and remarkably suppressed tumor growth Fig. . Moreove39. ICG-001 treatment significantly reduced basal and OTULIN-enhanced MDA-MB-231 cell migration , Immunomodulatory (IM), and Mesenchymal-like (M) subtypes compared with the others , we found genes signatures such as CTNNB1_TARGETS and MYCN_TARGETS_WITH_E-BOX, along with Basal-like breast cancer subtype genes signatures, were enriched  in breast cancer patients  had significantly shorter overall survival interval than those with lower OTULIN expression (n = 2658)  compared to those with lower OTULIN expression (n = 1324) , we found patients with high OTULIN levels (58) Fig. . Consist24) Fig. . This ob24) Fig. , and poo24) Fig. 42. These1. Here we demonstrated that β-catenin is also attached with M1-linked polyubiquitin catalyzed by the LUBAC ligase complex, which enhances β-catenin degradation. This observation is consistent with a previous report showing that overexpressing LUBAC suppressed Wnt/β-catenin activation11. Moreover, our data indicated that linear ubiquitination promotes β-catenin association with β-TrCP in resting cells, thereby promoting its proteasomal degradation. Upon genotoxic treatment, DNA damage apical kinase DNA-PK could activate c-Abl which subsequently phosphorylates OTULIN at Tyr56. The Tyr56 phosphorylation promotes OTULIN dissociation from HOIP while enhancing its interaction with β-catenin. Increased OTULIN/β-catenin interaction leads to decreased linear ubiquitination of β-catenin and reduced β-catenin association with β-TrCP-SCF ligase, resulting in decreased K48 ubiquitination and stabilization of β-catenin. Surprisingly, we found Wnt receptor proteins LRP5/6 and adapters DVL1/2/3 are dispensable for DNA damage-induced β-catenin stabilization, suggesting that genotoxic Wnt/β-catenin activation may be regulated in a Wnt ligand/receptor-independent manner. In line with this notion, inhibiting Porcupine, an essential palmitoyl transferase for Wnt protein lipidation and receptor binding, did not reduce Wnt/β-catenin activation by genotoxic agents. Taken together, our data support a critical role of OTULIN in promoting DNA damage-induced Wnt/β-catenin activation through disassembling M1 ubiquitin chains attached to β-catenin 10.5, which is earlier than the time of embryonic death (E12.5–E14) observed in Gumby mice (W96R or D336E)21. Although all these OTULIN-inactive mutations caused vasculature abnormality in embryos, the C129A mice die of aberrant cell death rather than defective Wnt signaling. It is plausible that these inactivating mutations suppressed Otulin/Gumby activity to a different extent which dysregulated multiple signaling cascades at different stages of embryo development. The hierarchy of these signaling processes during development and their reliance on Otulin activity remain to be delineated.Our data suggest that DNA-PK/ABL1-mediated OTULIN Tyr56 phosphorylation is not required for Wnt3a-induced Wnt/β-catenin activation. However, the decrease of β-catenin M1-ubiquitination was dramatically delayed in Wnt3a-treated cells compared to that by genotoxic treatments  and UBE3A  were shown to promote mutant Huntingtin protein degradation by facilitating its K48 ubiquitination49. It remains to be determined whether linear ubiquitination of activated PKC or misfolded Huntingtin also facilitates the K48-ubiquitination of the respective protein through these K48-specific E3 ligases.LUBAC-mediated linear ubiquitination plays critical roles in mediating immune response and cell death by modulating molecular signaling events such as NF-κB activation, ERK activation, inflammasome formation, RIG-I signaling, type I interferon response and necroptosisins Fig. . Accordiins Fig. , and bloins Fig. . Our res33. In the absence of DNA damage, c-Abl shuttles between the cytoplasm and nucleus32. Upon genotoxic treatment, cytoplasm-sequestered c-Abl is released from the binding of the 14-3-3 protein and translocates to the nucleus where it can be phosphorylated by DNA damage apical kinase ATM and DNA-PK50. Nuclear activated c-Abl is required for phosphorylation of histone acetyltransferase Tip60/KAT5, which could subsequently acetylate ATM and c-Abl, resulting in propagation of the DNA damage response signaling32. We found c-Abl is responsible for the Tyr56 phosphorylation of OTULIN upon DNA damage. The c-Abl-dependent OTULIN phosphorylation in response to genotoxic treatments relies on DNA-PK but not ATM or ATR. In TNBC cells treated with chemotherapeutic drugs, we detected nuclear localization of c-Abl whereas OTULIN nuclear translocation was not observed. Therefore, it is plausible that genotoxic stress-induced nuclear accumulation enables DNA-PK-dependent c-Abl activation, which may shuttle back to the cytoplasm where it phosphorylates OTULIN and promotes subsequent Wnt/β-catenin activation. Although ATM is required for DNA-PK-mediated c-Abl phosphorylation and activation by IR51, inhibiting DNA-PK, but not ATM, abolished OTULIN Tyr56 phosphorylation in response to genotoxic drug treatment. Thus, DNA-PK-dependent c-Abl phosphorylation is essential for its activation and OTULIN phosphorylation at Tyr56 in response to genotoxic drugs, which plays a critical role in promoting Wnt/β-catenin activation and development of drug resistance in TNBC upon chemotherapy.DNA damage has been shown to induce nuclear accumulation and activation of c-Abl52. Although TNBC patients overall are sensitive to cytotoxic chemotherapy initially, a significant number of TNBC patients rapidly develop drug resistance. TNBC was recently shown to adapt to neoadjuvant chemotherapy by adopting a reversible drug-tolerant state, in which a number of common gene signatures, such as glycolysis, MYC signaling, and EMT, were enriched53. We found that OTULIN is overexpressed in breast tumors, especially in the basal-like subtype  as control, or Dox (1.5 mg/kg/Week), ICG-001 (100 mg/kg/2 days), or combination of Dox and ICG-001. Primary tumors were measured by caliper every other day until 4 weeks. Lung metastasis was monitored by bioluminescent imaging in the fourth week. At endpoints, tumors were dissected and weighed, and preserved for subsequent Western blot and qPCR examination.Six-week-old immunocompromised NOD Scid Gamma (NSG) female mice were used and maintained under AAALAC-accredited specific pathogen-free housing vivarium and care, and veterinary supervision following standard guidelines for temperature and humidity with 12/12 light cycle. All procedures involving mice and experimental protocols were approved by the Institutional Animal Care and Use Committees (IACUC) of University of Tennessee Health Science Center. For xenograft models, 6-week immunocompromised NOD Scid Gamma (NSG) females were used. 10L-thyronine, 5 μM Isoproterenol hydrochloride, 50 nM Ethanolamine and 50 nM O-phosphorylethanolamine. Gene knockout cells LM2-luc_OTULIN-KO, MDA-MB-231_ABL1-KO, MDA-MB-231_OTULIN-KO, HEK293T_OTULIN-KO and 293T_HOIP-KO were generated with CRISPR-Cas9. FVB-MMTV-PyMT-Flag-OTULIN stable cell line was generated with lentiviral transduction.Human breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF7, and LM2, Human embryonic kidney 293 T and 293 A, Mouse Embryo Fibroblasts were grown in DMEM supplemented with 10% FBS and pen/strep. Human Breast cancer HCC1937 cells and HBRT1071 PDX cells were grown in RPMI-1640 supplemented with 10% FBS and pen/strep. Mouse FVB-MMTV-PyMT cells were grown in DMEM/F12 supplemented with 2% FBS and pen/strep. HCI PDX cells were grown in a special culture media composed of DMEM/F12 supplemented with 2% FBS, 10 mM HEPES, 1x Insulin-transferrin-selenium, 1x Antibiotic-Antimycotic, 5 ng/ml EGF, 0.3 μg/ml Hydrocortisone, 0.5 ng/ml Cholera toxin, 5 nM 3,3’,5-triido-Lentiviral plasmids, Vsv-g, pMDL-g, and RSV-REV were transfected together into HEK293T cells to produce viruses. After 72 h, culture media was harvested and centrifuged at 1800 rpm, 5 min at room temperature to remove cell debris, and subsequently go through 0.45 μm filter. Then, the filtered viral media plus 6 μg/ml polybrene was used to infect target cells that were seeded one day before. The target cells were selected with puromycin (2 μg/ml) after 24 h transfection. Single colonies were picked after selection.Total RNA was isolated with TRIzol (Invitrogen) and retrotranscribed with a first-strand cDNA synthesis kit (Thermo Scientific). Real-time PCR analyses were performed using the PowerUp SYBR master mix (Applied Biosystems). The housekeeping gene GAPDH was used as an internal control.p-nitrophenyl phosphate, and 10 mM sodium fluoride). 5% supernatant lysate was taken as input and the rest was incubated with 2 μg of primary antibody and protein G-Sepharose at 4 °C overnight. Protein G-Sepharose-enriched complexes were resolved on SDS-PAGE gels and transferred onto PVDF membranes. The protein signals were visualized by ECL exposure.For IP and co-IP experiments, cells were collected in 1.5 ml EP tube from 10 cm dish after treated and lysed on ice for 30 min in IP lysis buffer (20 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 2 mM DTT, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 10 mM To detect protein ubiquitination, cells were lysed with 1% SDS in IP lysis buffer at 95 °C for 30 min. The cell lysates were then diluted with IP lysis buffer to reduce SDS to 0.1% and mixed with primary antibodies and protein G-Sepharose for incubation at 4 °C overnight. The IP samples were washed five times with lysis buffer, separated by SDS-PAGE, transferred to nitrocellulose membrane, and boiled with distilled water for 30 min. The ubiquitin modification of precipitated proteins was examined by immunoblotting.Cells were seeded in 96-well plates in triplicate at an optimal density. After indicated treatment, cell viability was evaluated by adding CCK8 reagents (Dojindo) with incubation for 2 h and plates were measured at 490 nm absorbance with a 96-well plate reader.A 10 cm dish MDA-MB-231 cells were treated as indicated. Pellets were lysed in IP lysis buffer. Supernatants were added with 2 µg of anti-c-Abl antibody for each tube. Samples were rotated for 1 h at 4 °C. Protein G-Sepharose beads were then added to each tube and the samples were rotated for 2 h at 4 °C. Subsequently, beads were collected and washed four times in lysis buffer and two times in c-Abl kinase buffer (25 mM Tris-HCL (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol). The c-Abl kinase activity was assayed in kinase buffer with 1 µg of substrate  and 1 µCi of [γ-32P] ATP for 1 h at 30 °C. The reaction was terminated by adding 2x SDS loading buffer and boiled for 15 min. The proteins were separated by SDS-PAGE and then transferred to PVDF membranes which exposed to autoradiography or Phosphoimage cassette, and the radioactive signal was analyzed by Cyclone PhosphoImager. The membranes were subjected to Western blotting with the indicated antibodies.Cells for Wnt signaling reporter assay were cotransfected with 10 ng of the pRLtkLuc plasmid and 0.2 µg of the Super8X TopFlash plasmid per well in triplicate in 24-well plate. The dual-luciferase assay was performed by using a Dual-Luciferase reporter assay system  according to the manufacturer’s instructions. Luminescence was measured using a Promega GLOMAX 20/20 luminometer. Data were analyzed with Microsoft Excel.MDA-MB-231 cells were plated (5000 cells each well) in 96-well plate and reverse-transfected with Tyrosine kinase siRNAs  distributed in each well. After 48 h, cells were treated with DMSO or Dox (2 µg/ml) for 90 min. Then, the medium was removed and cells were washed with PBS twice. 2x SDS was added to each well and lysate was transferred to a tube and heated at 95 °C for 10 min. Samples were resolved on SDS-PAGE gels and transferred onto PVDF membranes. P-OTULIN (Tyr56), OTULIN, and Tubulin signals were visualized by ECL exposure. The screen was repeated independently three times. Each blot was quantified with ImageJ and the relative p-OTULIN level was calculated in mean ± SD with Excel. The result was presented in the volcano diagram.MDA-MB-231 cells were treated with MG132 (10 µM) for 16 h and lysed in the same way as described in Immunoprecipitation. Supernatant from centrifuged lysate was incubated with 2 μg of anti-β-Catenin primary antibody and protein G-Sepharose at 4 °C overnight. The next day, G-Sepharose was washed five times with IP buffer and incubated with 1 U of indicated DUB in UbiCREST Kit (Boston Biochem)-provided reaction buffer at 37 °C. After 1 h, 2× SDS was added to terminate the reaction and samples were resolved by SDS-PAGE. The ubiquitin modification of precipitated proteins was examined by immunoblotting.2, 2 mM DTT and 2 mM ATP. After the reaction, GST-β-Catenin fragment was pulldown by glutathione sepharose and subjected to SDS-PAGE and subsequent immunoblotting.Recombinant WT and K133R GST-β-Catenin (amino acid 1-153) were expressed in BL21 E. coli and purified using glutathione sepharose. Myc-HOIP was expressed in MDA-MB-231 cells and immunoprecipitated by using Myc-tag antibody and A/G agarose beads to generate active LUBAC complex. Briefly, reaction mixtures contained ubiquitin (250 μg/ml), E1 (5 µg/ml), E2 (10 µg/ml), precipitated LUBAC complex and the substrate GST-β-Catenin (100 μg/ml) and were incubated at 37 °C for 2 h in reaction buffer containing 20 mM Tris–HCl (pH 7.5), 5 mM MgClMDA-MB-231 cells were seeded on glass coverslips at the optimal density. After Dox treatment, cells were fixed in Methanol at 4°C for 20 min and then permeabilized for 5 min with 0.2% Triton X-100. Cells were blocked by 3% bovine serum albumin/PBS for 30 min before incubated with β-Catenin primary antibody (1: 200 in 1% BSA) overnight at 4 °C. The next day, cells were stained with DyLight 488 fluorescence-conjugated secondary antibody for 1 h. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and the cells were imaged with an EVOS immunofluorescence microscope (Life Technologies).To observe the subcellular location of c-Abl and OTULIN after Dox treatment, cells were transfected with GFP-OTULIN for 24 h and exposed to Dox for the indicated time. Then, cells were fixed and blocked as above before incubated with c-Abl primary antibody (1:100 in 1% BSA) overnight at 4°C. The second day, cells were stained with DyLight 594 and DAPI before imaging.MDA-MB-231 WT, OTULIN-KO, or OTULIN-KO cells reconstituted with OTULIN (WT or C129S mutant) or β-Catenin SA mutant were treated with 2 µg/ml Dox for 24 h. Then, cells were fixed with Methanol at −20 °C for 20 min and stained with 0.5% crystal violet in Methanol for 5 min. After washing off residue dye, the cell plate was imaged. Experiments were carried out at 3 times and Image J was used to quantify the stained area for each well. Cell survival percentage was calculated by the stained area in each treatment divided by that in control WT MDA-MB-231.57, which was reviewed and approved by an independent ethical committee/institutional review board (IRB) at FUSCC, and all patients gave their written informed consent before inclusion in this study. Patient specimens to generate the PDXs 1071 and 1150 were collected under an IRB protocol approved by the UTHSC IRB. TNBC specimen 1071 was obtained from 57 years old women of African American descent and the TNBC specimen 1150 was collected from 74 years old women of European ancestry. Tumor specimens (1 mm3) were implanted in female NSG mice and the growth was monitored. Once the tumors grew to 1000 mm3, the tumors were either further passaged for experiments or were frozen in liquid nitrogen for further use. Once the PDX was generated, 1071 tumors were collected, and a cell line was created.The qPCR analysis of OTULIN was performed in breast cancer patient tumor samples and normal breast tissues at Fudan University Shanghai Cancer Center (FUSCC) and with tumor cDNA library from OriGene . Biopsy samples were collected under a published protocolR ≥ 0.4 or ≤ −0.4, p < 0.001) with OTULIN levels in breast cancer patients within the TCGA-PanCAN study were retrieved through cBioPortal, enriched gene signatures were identified with GSEA. Overall survival and progression-free survival in combined breast cancer patient dataset (TCGA-TARGET-GTEx) were retrieved through UCSC-Xena, and compared between patients with high  and low  OTULIN expression. Disease-free survival in basal breast cancer patients (GSE21653) was analyzed between patients stratified by medium OTULIN level. Distant metastasis-free survival in breast cancer patients who received chemotherapies was analyzed through KM-plot42.OTULIN transcription levels in samples from TCGA-BRCA genomic dataset were downloaded from Firehose Broad GDAC. Genes whose expression significantly correlated (Spearman’s t-test was used to compare differences between two groups and the two-way ANOVA test was used to compare differences among multiple groups. p < 0.05 is considered significant, indicated as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Statistical parameters, including average, standard deviation, and statistical significance are presented in the figures. Statistical analysis has been performed using Prism GraphPad 7 software. Two-sided Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases  bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of  Moreov Broach, , Phox2b  Broach, , Bhlhb5  Broach, , Wnt1 , and pa Broach, , . In par Broach, , mCherry Broach, , tdTomat Broach,  fluoresc Broach, ), as welConsidering the crucial role of fibroblasts in the microenvironment, we created in the present study an original mouse model expressing the Flpo recombinase in the fibroblastic compartment ([Fsp1‐Flpo] mouse strain). We chose the Flp/Frt approach with the final goal in the future of combining this system with the Cre/lox system in dual recombinase systems (DRS), which consist in combining two recombinase systems within the same mouse .2Fsp1 promoter consists in a ~3 kbp genomic DNA fragment that drives gene expression in fibroblasts  site‐specific recombinase (SSR), which recombines DNA Frt‐sites  corresponding to the region previously described to drive the expression of Fsp1 in fibroblasts  along with pFsp1‐Flpo vector or a control vector pSICO‐Flpo constitutively expressing the Flpo recombinase . These embryos were further transferred into 14 OF1 foster females for gestation and we obtained 9 pregnant females giving birth to 53 progenies (F0 generation), 6 of which were positive for the transgene. To verify germinal transmission of the transgene, we bred these 6 F0 individuals with C57BL6/J mice and obtained 105 progenies  (F1 generation), of which 9 heterozygous Fsp1 were identified from 3 different F0  . Following another backcross in a C57BL6/J background, [Fsp1‐Flpo]‐F0#4 and its offspring were discarded as the transgene appeared to be inserted on a sexual chromosome.The s Figure . The pFsFlpo transcripts, we performed RT‐qPCR on total RNA prepared from [WT] and [Fsp1‐Flpo]‐F1#3 and [Fsp1‐Flpo]‐F1#11 cultured ear skin primary fibroblasts, and, as expected, we detected the expression of Flpo mRNA exclusively in fibroblasts bearing the Flpo transgene . We observed a slightly higher expression in [Fsp1‐Flpo]‐F1#3  a pan‐fibroblast marker , a >4‐fold enrichment being observed in PDGFRα‐positive cells compared to cells before cell sorting  transgene under the control of a Frt‐Stop‐Frt (FSF) cassette  were performed on total RNA prepared from skin and spleen samples from [WT], [Fsp1‐Flpo], [FSFhPLAP], or  mice. Flpo mRNA was detected only in organs of [Fsp1‐Flpo] and  mice  experiments on skin, spleen, and pancreas tissue sections. We observed that the Fsp1 protein was expressed in dermis fibroblasts Figure . Of noteCollectively, these data demonstrate that [Fsp1‐Flpo] mice express a functionally active Flpo recombinase specifically in Fsp1‐positive cells.Fsp1‐Flpo allele encoding the Flpo recombinase under the control of the Fsp1 promoter. We functionally validated this allele by showing that Flpo expression was restricted to Fsp1‐positive cells. We were able to observe in situ the expression of the Fsp1 protein in fibroblasts directly with an anti‐Fsp1 antibody, and Flpo recombinase activity with an in vivo reporter system . The Fsp1 promoter has been successfully used by other groups to generate [Fsp1‐Cre x TβRIILoxP/LoxP] mice carrying homozygous inactivation of type II TGFβ receptor in the stromal compartment  were cultured in complete medium  and propagated at 37°C under 5% (v/v) CO2 atmosphere. Primary ear fibroblasts were isolated and cultured as follows: mouse ears were rinsed with 70% ethanol and samples of a few square‐millimeters were harvested. Primary ear fibroblasts were isolated using mechanical dilaceration, followed by enzymatic dissociation /Dispase II  in RPMI medium) at 37°C for 1 hr. The reaction was stopped by adding 5 ml of complete medium  and cells were then incubated at 37°C overnight. The following day, after filtration through a 100 μm pore cell strainer, cells were pelleted and reseeded in complete medium . Medium was changed after 48 hr. All cells were propagated at 37°C under 5% (v/v) CO2 atmosphere.Sorted skin cells were prepared as follows: mice were sacrificed and shaved, and a piece of skin from the back of the animals was harvested and fat was removed. Five milliliter of digestion medium  was added to the skin in a petri dish. The skin was dilacerated using scissors and a cutter. Another 5 ml of digestion medium was added to help harvest the mixture. The mixture was then transferred to 50 ml tubes under agitation with a magnetic bar. One milliliter of collagenase type IA  and 200 μl of DNase1  were added. The mix was agitated 30–90 min at 37°C. The insoluble debris were eliminated by filtration onto a nylon grid. The digestion product was then centrifuged 5 min at 1,300 rpm and suspended in cell sorting buffer . Cells were labeled in cell sorting buffer with PE anti‐mouse CD140a/PDGFRα antibody (BioLegend Cat. No. 135905 1:50) and PE Rat IgG2a, κ Isotype Control Antibody (BioLegend Cat. No. 400507 1:50). Cells were sorted using the BD FACS Aria III SORP (BECTON DICKINSON [BD™]).3.1.2The [Fsp1‐Flpo] mouse strain will be available to the research community upon acceptance of the manuscript.FSFhPLAP] strain was previously described 26Sortm1(ALPP)Dym), Stock No: 009086). Mice were housed and bred in the “AniCan” specific pathogen‐free animal facility of the CRCL (Centre de Recherche en Cancérologie de Lyon), France. The experiments were performed in compliance with the animal welfare guidelines of the European Union and with the French legislation (CECCAPP protocol #CLB‐2012‐012 #CLB‐2017‐007 #CLB‐2018‐025).The ), GAPDH, mouse monoclonal primary antibody, 1:1,000 (Abcam ab8245) and the secondary antibodies were Rabbit IgG (H + L), HRP‐conjugated, 1:5,000 (Ozyme GtxRb‐003‐DHRPX), and anti‐Mouse IgG, HRP‐conjugated, rabbit polyclonal 1:20,000 (Dako P0260).3.4For immunofluorescent (IF) staining, OCT‐frozen tissues were sliced into 10 μm‐thick sections. Slides were dried 30 min at room temperature, postfixed in 4% paraformaldehyde in PBS 1X for 10 min at 4°C, then washed in cold PBS 1X, permeabilized with 0.5% Triton X‐100 for 5 min, and blocked with PBS‐4% BSA for 1 h. Labeling was performed by incubating slides overnight with rabbit polyclonal primary antibodies against FSP1/S100A4 (1:200 Abcam ab41532). After three PBS 1X washes, slides were incubated with the specific secondary antibodies Alexa647‐conjugated goat anti‐rabbit antibody  and DAPI . All antibodies were diluted in DakoReal Antibody Diluent. Slides were washed three times with PBS 1X, Samples were mounted on microscope slides with DAKO Fluorescence Mounting Medium (Agilent S3023) or Fluoromount Aqueous mounting medium (Sigma F4680). Images were acquired with a Zeiss Imager M2 AX10 and processed using Zeiss Zen.Alkaline phosphatase (AP) staining was performed as follows: 10 μm‐thick sections of frozen tissues were cut using the Cryostat HM550 (MICROM). Cryosections were dried 30 min at room temperature, postfixed in 4% paraformaldehyde in PBS 1X for 10 min at 4°C, then washed in cold PBS 1X. Sections were incubated at 70°C in preheated PBS for 90 min, then incubated in AP detection buffer  for 60 min. Sections were subsequently placed in AP staining solution (AP detection buffer with 0.8 mg/ml nitroblue tetrazolium (NBT), 0.1 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl phosphate dimethylformamide (BCIP)) at room temperature for 2–6 hr. Once color development was complete, sections were washed in PBS 1X.3.5All graphs display one representative experiment performed three times. Prism 7.0 (Graphpad) was used for statistics and to create graphs. The error bars represent the standard deviation (SD) from technical duplicates.The authors declare no conflict of interest.Victoire Cardot‐Ruffino, Stéphanie Sentis, and Laurent Bartholin designed the experiments. Victoire Cardot‐Ruffino, Véronique Chauvet, Cassandre Caligaris, Sylvie Martel, and Sophie Aires performed the experiments. All authors analyzed the experimental results. Laurent Bartholin, Victoire Cardot‐Ruffino, and Stéphanie Sentis wrote the article. All authors were involved in critical reading of the article prior to submission.Appendix S1: Supporting InformationClick here for additional data file.


These findings highlight how small, but significant changes at critical positions on a protein surface are exploited in evolution to alter function.Alternative splicing can expand the diversity of proteomes. Homologous mutually exclusive exons (MXEs) originate from the same ancestral exon and result in polypeptides with similar structural properties but altered sequence. Why would some genes switch homologous exons and what are their biological impact? Here, we analyse the extent of sequence, structural and functional variability in MXEs and report the first large scale, structure-based analysis of the biological impact of MXE events from different genomes. MXE-specific residues tend to map to single domains, are highly enriched in surface exposed residues and cluster at or near protein functional sites. Thus, MXE events are likely to maintain the protein fold, but alter specificity and selectivity of protein function. This comprehensive resource of MXE events and their annotations is available at:  http://gene3d.biochem.ucl.ac.uk/mxemod/.A single gene can produce more than one protein through processes such as Alternative Splicing. Mutually exclusive splicing is one type of splicing by which cells produce isoforms that are often very similar in their three-dimensional shape. Different transcript isoforms are usually produced in different cell tissues and cell environments. The structural impacts of this splicing are still not well-studied. In this study, we compare proteins generated from mutually exclusive exon switching from 5 organisms. Most of these proteins do not have their three-dimensional shape solved by experimental methods. We therefore calculate the most probable three-dimensional structural models. We found small regions that are different between the proteins and occur near important residue sites that determine the functions of the proteins. We speculate mutually exclusive splicing may be involved in tuning the protein function. We have built a webserver to present information on these exon switching events:  Alternative splicing (AS) refers to the assembly and rearrangement of different exons of a gene during pre-mRNA splicing such that different mRNAs and thus proteins are produced from the same gene. Alternative splicing is common in humans and other animals producing different transcripts in different developmental stages, tissues or disease states ,2. AlterAmongst the most important exon-switching events are those that generate homologous mutually exclusive exons (MXEs), such that only one out of the two exons is retained while the other one is always spliced out. This type of exon switching is less likely to be disruptive to highly organised globular protein structure ,15 compaIt is possible to align a pair of homologous MXE events and identify conserved and variable amino acid residues, the latter of which could be considered responsible for any functional shifts of the MXE . That isGiven that MXEs are likely to affect protein function, how often and what types of functions are altered? Here, we analyse multiple genomes and use large scale protein structure modelling data to assess the functional impact of these processes and their likely biological role. We describe the mechanisms by which MXE events trigger dynamic switching of protein surface patches associated with key solvent exposed functions . We subsequently illustrate how aberrations in the same functional switching regions altered by MXE events may be mutated leading to deregulation in cancer. Unlike MXE events, many AS events such as cassette exon removal in globular regions, are likely to lead to significant disruption of the protein structure. However, for a subset of cassette exon splicing events where there is less likely to be structural disruption, and microexons in general we do not identify the same functional enrichments as for MXE events. Finally, we present a comprehensive online resource of the structural and functional annotations of MXE events to allow users to search for MXEs of interest to investigate the structural/functional consequences of these events.Materials and Methods). In this way we could compare results across genomes to examine any potential similarities and variations in the different species. We find that in our dataset, each organism has over one hundred MXE containing genes  . The results showed that whilst only a very small number of MXE events can be mapped to known structures, we can massively expand our structural coverage using our structural modelling strategy, from 14 to 691 MXE events.CATH superfamilies are groups of homologous protein domains  . Each CAferences ,26) and The MXE regions structurally mapped to a relatively small number (115 out of a possible 2700) of CATH superfamilies . Prelimi-6, Fold enrichment = 2.05). Brain and neural tissue enrichments can also be found for the other classes of splicing . In total 13 of the 672 FDA approved genes available from HPA, could be mapped to one of the 125 human MXE genes . We also analysed single exon loss events  and saw a significant enrichment  but with a substantially lower odds-ratio of 1.6, whilst microexons showed no enrichment.Finally, using a set of FDA druggable proteins from the Human Protein Atlas (HPA) , we founMaterials and Methods) provided structural information to assess this . The level of the difference was striking and indicates that MXE events are strongly biased to altering residues with surface exposure. This picture is consistent across multiple organisms and indicates this is a prominent and consistent feature of MXE splicing.The function of a protein domain is largely dictated by the shape and nature of the residues on the protein surface. Enrichment of MXE events on surface residues would indicate absence of an effect on the protein fold but an effect on function such as altered protein interactions, protein-small molecule binding, etc. Our structural mapping approach . These observations further demonstrate that the amino acid changes in MXE events consistently alter surface exposed residues on the protein, much more than we would expect by chance. Together these findings collectively suggest a role for these surface variable regions in modulating protein functional sites.Are the variable residues involved in MXE events more exposed to the solvent than the MXE region itself? A more detailed analysis, involving buried surface area calculations for all the MXEs revealed that variable (i.e. MXE-specific) residues are much more significantly exposed than non-variable MXE residues . Briefly, we mapped the MXE region onto CATH functional families (FunFams). Positions that are conserved within and unique to a particular FunFam but not conserved across the superfamily are likely to be specificity-determining positions and responsible for a specific function that is distinct from other superfamily members. Finally, we also identified putative allosteric sites using the A-SITE approach .Do the MXE variable residues cluster together on the protein structure (or are they dispersed) and do they tend to affect functionally relevant sites? A detailed analysis of the positioning of the MXE variable residues revealed that they tend to form a single cluster in space in over 70% of the MXE events. For those MXE events that formed multiple clusters, they usually formed 2–3 clusters . Next, wMaterials and Methods) shows that there is a significant tendency for clusters of MXE variable residues to lie close to protein-protein interaction, protein-small molecule, specificity determining positions and allosteric sites for all species . We calculated the minimum distance between the atoms of the residues involved. A comparison with a random model , they are usually surface exposed and therefore unlikely to affect the ability to produce stable structures but more likely to alter surface functions.Materials and Methods) and found no enrichment of cassette exon near functional sites . We found similar properties with the Hatje dataset further supporting the generality of our observations See –S24 FigsMaterials and Methods). We therefore investigated whether MutFam cancer mutation residues coincide with both MXE variable residues and functional residues. We mapped putative cancer driver genes from MutFams to the 143 human MXE events. We found carcinoma  and melanoma to be the common cancers that have mutations coinciding with MXE events . Functional analysis by the PANTHER pipeline [How often do variable regions lie at or close to cancer mutation sites? Recent studies have suggested an association between human MXE events and inherited pathogenic mutations . Furtherpipeline  on canceMaterials and Methods). We obtained the same results for both per-residue and per-cluster analyses. We checked if these MXE events were close to functional residues and found 35 out of 40 of the events were close to functional residues. Similarly, there is a significant tendency for cancer mutation and variable residues to lie close to protein-protein interaction, protein-small molecule, specificity determining positions and allosteric sites. Hence, MutFam cancer mutations target a similar set of functional sites as are altered by MXE events, suggesting some advantage to cancer in modifying the same set of specific functional residues on proteins that are dynamically regulated by MXE events. We found similar properties with the Hatje dataset further supporting the generality of our observations (See We could annotate 43 of these events with structures and we then determined if the variable residues/clusters in these MXE events were close to MutFam mutation residues. We found that 40 of the 43 MXE events had variable residues that were significantly closer (in terms of distance) to cancer mutations than expected by random .A total of 391 MXE events were associated with allosteric functions in our analysis. Analysis of PKM MXE structures  idenInterestingly, we find another tightly regulated glycolytic enzyme, that is also allosterically regulated, called Phospho Fructo Kinase-platelet (PFKP)  and agaiFurthermore, the MXE regions of PKM1/PKM2 and of PFKP were found to overlap with pan-cancer mutations in structural regions close to functional sites , reinforMaterials and Methods), some of which overlap the MXE variable regions, it suggests that MXE switching could also switch CRMP protein interactions.A total of 235 MXE events were associated with protein-small molecule interaction functions. An example of this is the Collapsin Response Mediator Protein CRMP gene (FBgn0023023), a dihydropyrimidinase enzyme (EC 3.5.2.2) that has been implicated in biological processes including Notch signalling . An MXE We identify 9 MXE events that affect catalytic sites (CSA residues) in our dataset. For example, CG42249 a poorly functionally characterised fly gene is likely to be a nucleotidase ). We identify switches in key catalytic residues between its MXE isoforms with major differences in physicochemical properties . Such chIntegrins are an important class of transmembrane proteins in animals mediating cell interactions and signalling in many processes (e.g. embryonic development). We identified an MXE in integrin-B1, where one isoform (ENSMUSP00000119699) has MXE variable residues substituting two residues (Thr788-Thr789 in ENSMUSP00000087457) from Thr to Asn, abolishing a known phosphorylation site. Hence even though the kinase may be expressed and active, only one isoform is capable of recruiting the kinase and so switching isoforms can produce changes in downstream signalling.KLRC2 is a transmembrane protein important for regulating Natural Killer (NK) cell-mediated immunity and has an MXE event that switches exon 3, changing 2 amino acids in the extracellular region of the protein. The substitution of Serine 102 to Phenylalanine by this MXE event would remove the glycosylation of amino acid N100 , via a previously published metric called the Transcript Switch score  which caHomologous mutually exclusive exons are enriched in proteomics data, suggesting a functional role. In this study, we used a large-scale structural modelling analysis to investigate how the homologous exons could be altering function. Previous analyses by other groups have demonstrated that MXE events are evolutionary conserved among vertebrates ,18 and tThe fact that so many of the 3D modelled domains for MXE events produced reliable structural models  suggests that the MXE sequence changes generally do not affect the stable folding of the proteins, but rather tune the functions of the proteins. Although this lack of disruption to the fold has been shown for individual structures previously , this isWe examined cancer mutations in an MXE structural context and focused on specific so-called cancer-driver domain families , which can be statistically associated with cancer mutations. We find a significant proximity (in structure) between cancer mutations in these families and the MXE variable residues, showing that cancer may dysregulate the same functional regions altered by MXE events.Examination of the recently published MXE-splicing dataset (the Hatje dataset) from human See  showed sAnalysis of cassette exons and microexons showed none of the functional enrichments seen for homologous MXE datasets See  Texts hiWhy MXE events have evolved in some proteins, altering their regulation or functional specificity, to give genes with separate functionalities, remains an open question, but for whatever reason it appears to be particularly important for functional innovations in membrane proteins. Similar mechanisms to those we describe for MXE events can occur in the functional divergence of paralogs  i.e. witA detailed characterisation of MXE events can help us to better understand cell regulation, various diseases and possibly develop highly specific therapeutics. For example, one reported MXE event that could be targeted therapeutically is the MXE switch between 8a and 8 in the CaV1.2 Calcium Channel , which iA further advantage of MXE splice variants as drug targets is that, as we have demonstrated here, we can obtain reliable structural models for a considerable number of the MXE events, providing a good starting point for rational drug design. Furthermore, since the physicochemical properties of the variable residues show strong shifts between MXE events this should also help in designing isoform specific drugs. Finally, much of the above discussion for therapeutic drug development could equally be applied to developing more targeted insecticides, especially if some of the putative lineage-specific MXEs were validated (e.g. Multidrug-Resistance like Protein 1 in drosophila is one such MXE we identify).Drosophila melanogaster from the Kassiopeia resource [We downloaded the annotations of predicted MXEs for resource  which coresource  and storresource , settingAt the time of data preparation, the Kassiopeia resource did not provide MXE download annotations for the other four species . In order to identify MXEs for these species, we did the following. Based on Ensembl version 87 , we obtaOrthologs were obtained using Ensembl Compara  includinWe used the PANTHER functional annotation tool, last updated in February 2020, to identify functional enrichments of the MXE genes. We used 5 different background sets  for testing the enrichment.We mapped the splice isoforms to CATH functional families (FunFams). FunFams are a sub-classification of CATH protein domain superfamilies . Each FuThe domains within a given FunFam have been demonstrated to be structurally coherent ,61. The All the MXE events were scanned against the library of CATH v4.1 FunFam HMMs ,64 usingThe two splice isoforms for an MXE event were aligned to other relatives in the FunFam using the multiple sequence alignment tool MAFFT  and the For those MXE events which are not included in FunFams, we scanned the isoforms against the libraries of HMMs built from non-redundant structures in various resources, CATH v4.1 HMMs (i.e. including non-FunFam CATH domains), SCOPe 2.06 HMMs  and PDB7For those MXE events which we failed to annotate with structure information, we predicted if they were intrinsically disordered using IUPRED . We usedTo compare the MXE events with other types of alternative splicing we examined the impacts of single cassette exon events and microexon events, both of which like MXE events would be expected to have a relatively small effect on the protein structure compared to other AS events.Loss of exons is likely to disrupt protein structure, especially if the loss is in the globular folded region . By restricting to single exon events, we hoped to identify cassette exons that were least likely to alter protein structure and should therefore be easier to analyse from a structural point of view. Conversely, we can imagine that in general more extreme splicing events leading to greater losses would be more disruptive to structure and more difficult to analyse from a homology modelling point of view. To minimise this, for each protein coding gene we took the longest protein isoform and found the next longest isoform where the only difference was the loss of a single coding, non-terminal exon. In this way, we were more likely to identify a minimally disruptive cassette exon event. We used this set of minimally disruptive cassette exons for the domain/ protein function enrichments and the cassette exon structural models. We only analysed cassette exon events that could be mapped to a globular domain in one of our FunFams.Microexons have been defined previously as having ≤27 nucelotides (nt). The most comprehensive set of microexons can be found in the VASTDB database . We obtaWe compared the physicochemical properties of variable residues using the McLachlan physicochemical similarity matrix . A pair NACCESS  is a staWe also investigated random models to examine the solvent exposure for the splice region itself relative to background. For every MXE pair, we randomly selected from the same structure regions that have the same size as the splice region, 10,000 times, and determined the percentage of exposed residues in this region. This percentage is then compared with the splice region solvent exposure. We used the Wilcoxon signed-rank test to compute the statistical significance.We investigated whether exposed variable residues in MXE events lie on or in the vicinity of functional residues. To reduce the noise and identify those variable residues likely to be having similar impacts, we clustered variable residues into structural clusters. For each pair of MXE events, we calculated the all-atom-versus-all-atom distances of the variable residue atoms of the structural representative and determined the minimum distance between residues. We used an in-house multi-linkage clustering program which clusters residues that are within 8Å distance of each other (i.e. between the centres of the residues). We made sure there are at least three residues present in a cluster.Subsequently, we calculated the distance from the structural clusters to known functional residues of the structural representative. We calculated the centre-of-mass of the clusters. The minimum distance from the centre-of-mass of the cluster to the closest functional residue was calculated. If the functional residue was in the cluster the distance was simply set to zero. We used a distance cut-off of 6Å to define if the structural cluster lies close to a functional site.We also investigated random models to examine the proximity of randomly selected residue regions to functional sites, to evaluate statistical significance. For every MXE pair, we randomly selected structural clusters that have the same size as the largest MXE structural cluster, 10,000 times, and determined the percentage of clusters that lie close to functional sites. This was done by randomly selecting a residue and then identifying the residues that are close  to the selected residue, and each other, until they made up the same volume as the largest MXE cluster. Then, the overall percentage for all random events was computed. This percentage is then compared with the actual MXE percentage. We used the Z-score test to compute the statistical significance.The functional sites considered were known experimentally characterised sites: catalytic residues taken from CSA , proteinhttps://github.com/amoyag/protein-structure-network.We also used the A-SITE allosteric site predictor , based on the centrality of nodes (in a protein i.e. residue nodes) in terms of their capability to transfer signals through the protein, to predict allosteric sites in the protein, in order to determine whether variable residues lay on or close to allosteric sites. The program identifies residues with high betweenness centrality. The depletion of residues having high betweenness centrality values would be expected to interrupt the allosteric communication among regions of the protein that lie far apart. For each structure, we defined its allosteric residues as the top 5 percentile residues . The source code for A-SITE program is available at COSMIC is a database which collects somatic mutations from human cancer patients . AccumulRecently, mutationally enriched domain functional families (MutFams) have been identified by in-house studies . MutFamsTherefore, we determined if MutFam cancer mutation residues lie close to MXE variable residues. We mapped all the MutFam mutation residues onto the human MXE events. For those where we have structural information, we determined if the MXE variable residue or residue clusters are close to MutFam mutation residues using a distance cut-off of 4Å (between any atoms in the residue pairs) or 6Å (to the centre of the MXE cluster).The set of FDA druggable proteins downloaded from the Human Protein Atlas (HPA) and we checked for enrichment against the gene lists from the MXE datasets. The gene background for the Fisher Exact test was set to the set of protein coding genes. The TopAnat enrichments were calculated through the website using the default parameters ) but using only protein coding genes (from GRCh38) as the genomic background and we report the FDR scores from the website, as described in the methods publication ,29.We used Adaptive Poisson-Boltzmann Solver (APBS)  to deterS1 FigFor every CATH superfamily identified in each species, we obtained the number of MXE genes mapped to the superfamily, the number of MXE genes that do not map to the superfamily, the number of non-MXE genes mapped to the superfamily and the number of non-MXE genes that do not map to this superfamily. This information was then used to compute a 2x2 contingency table. We then performed the Fisher exact test using the contingency table. We adjusted the p-value using Benjamini-Hochberg correction to account for multiple hypothesis testing. We check the odds ratio is > 1 and p-value < 0.05 for significance. MXE domain families are enriched in important Metazoan functions such as membrane proteins involved in cell-cell adhesion and signal transduction.(TIF)Click here for additional data file.S2 FigWe used organism whole genome as background. We were unable to include Fugu fish because PANTHER does not include Fugu fish in their analysis datasets.(TIF)Click here for additional data file.S3 FigFor our background dataset, we only used genes that have no paralogs. Removing the paralogs was to counter potential effects from MXE events being retained after a gene duplication, which could lead to overestimates of functional coherence of the gene set. We were unable to include Fugu fish because PANTHER does not include Fugu fish in their analysis datasets.(TIF)Click here for additional data file.S4 FigWe used only multi-exon genes as background. Removing single exon genes was to counter potential effects of any functional bias, since by definition our MXE genes require more than one exon. We were unable to include Fugu fish because PANTHER does not include Fugu fish in their analysis datasets.(TIF)Click here for additional data file.S5 FigWe used only multi-protein isoform genes as background. Removing single protein isoform genes was to counter potential effects of any functional bias. We were unable to include Fugu fish because PANTHER does not include Fugu fish in their analysis datasets.(TIF)Click here for additional data file.S6 FigWe used only MXE or CE genes as background to counter potential effects of any functional bias. We were unable to include Fugu fish because PANTHER does not include Fugu fish in their analysis datasets.(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 FigFor each FunFam, with high DOPS, and a known structure we took all known functional sites , PPI (protein-protein interactions), PSI ) and identified other residues within 4Å from the functional site. We then determined the residue usage and calculated the chemical changes using McLachlan score. We used Mann-Whitney U test to compute the statistical significance.(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 FigFor every pair of MXE events, we determined if the variable residues within the splice regions lie close to any functional sites. We calculated the minimum distance between the atoms of the residues involved. We used a distance cut-off of 4Å to determine if residues are close. For every pair of MXE events, we created 10000 random models , and determined the percentage of highlighted residues that lie close to functional sites. We used the z-score test to compute the statistical significance. This reports a p-value for the level of significance. There is a statistically significant tendency for MXE events with variable residues to lie close to protein-protein interaction, protein-small molecule and FunSites for both fly and human datasets. There is also a tendency for variable residues to be in the vicinity of allosteric sites for the fly dataset. We had less than 10 events annotated with catalytic sites but these did not usually lie close to a MXE region using a 4Å distance cut-off.(TIF)Click here for additional data file.S12 FigWe used organism whole genome as background.(TIF)Click here for additional data file.S13 Fig(A) Exon structures of PKM isoforms. (B) Sequences from domain 4 for the 2 isoforms. The splice regions are coloured purple or yellow. (C)The sequence alignment of the splice region. Variable residues are coloured as red.(TIF)Click here for additional data file.S14 FigThe FBP molecule is coloured in cyan.(TIF)Click here for additional data file.S15 Fig(TIF)Click here for additional data file.S16 Fig(TIF)Click here for additional data file.S17 FigThe analysis was done using PFKP structure (PDB id 4XZ2).(TIF)Click here for additional data file.S18 FigPositively charged residues are coloured as blue, negatively charged residues are coloured as red.(TIF)Click here for additional data file.S19 Fig(A) Distributions of MXE sizes. (B) Sequence identity between MXE pairs. (C) Number of variable residues between MXE pairs.(TIF)Click here for additional data file.S20 FigWe used organism whole genome as background.(TIF)Click here for additional data file.S21 FigFor our background dataset, we only used genes that have no paralogs. Removing the paralogs was to counter potential effects from MXE events being retained after a gene duplication, which could lead to overestimates of functional coherence of the gene set.(TIF)Click here for additional data file.S22 FigWe used only multi-exon genes as background. Removing single exon genes was to counter potential effects of any functional bias, since by definition our MXE genes require more than one exon.(TIF)Click here for additional data file.S23 FigWe used only multi-protein-isoform genes as background. Removing single protein-isoform exon genes was to counter potential effects of any functional bias, since by definition our MXE genes require more than one exon.(TIF)Click here for additional data file.S24 FigWe used only MXE or CE genes as background to counter potential effects of any functional bias.(TIF)Click here for additional data file.S25 Fig(A) Proportion of splice isoforms with structural information. (B) Distribution of McLachlan scores for the variable MXE residues. (C) The surface exposure of the MXE events compared to random expectation. (D)The surface exposure of the MXE variable residues compared to the MXE event as a whole. (E) Proximity of MXE variable residue clusters to amino acids assigned to different functional classes (indicated by Icons) CSA = catalytic residues from the Catalytic Site Atlas, PPI = protein interaction sites and PSI = protein-small molecule interactions).(TIF)Click here for additional data file.S26 Fig(A) Number of cassette exons identified (B) Distribution of cassette exon sizes. (C) Distribution of cassette exon coverage (whole sequence).(TIF)Click here for additional data file.S27 FigWe used organism whole genome as background.(TIF)Click here for additional data file.S28 FigFor our background dataset, we only used genes that have no paralogs. Removing the paralogs was to counter potential effects from MXE events being retained after a gene duplication, which could lead to overestimates of functional coherence of the gene set.(TIF)Click here for additional data file.S29 FigWe used only multi-exon genes as background. Removing single exon genes was to counter potential effects of any functional bias, since by definition our MXE genes require more than one exon.(TIF)Click here for additional data file.S30 Fig(TIF)Click here for additional data file.S31 FigWe only show cases where an acceptable quality model can be built for the longer isoform.(TIF)Click here for additional data file.S32 FigThe difference in nDOPE score is given by a ΔnDOPE value .(TIF)Click here for additional data file.S33 Fig(TIF)Click here for additional data file.S34 Fig(A) Number of MIC events identified (B) Distributions of MIC size.(TIF)Click here for additional data file.S35 FigWe used organism whole genome as background.(TIF)Click here for additional data file.S36 FigFor our background dataset, we only used genes that have no paralogs. Removing the paralogs was to counter potential effects from MXE events being retained after a gene duplication, which could lead to overestimates of functional coherence of the gene set.(TIF)Click here for additional data file.S37 FigWe used only multi-exon genes as background. Removing single exon genes was to counter potential effects of any functional bias, since by definition our MXE genes require more than one exon.(TIF)Click here for additional data file.S38 Fig(A) Percentage of splice isoforms with structural information. (B) The surface exposure of the MIC residues compared to non-MIC residues.(TIF)Click here for additional data file.S39 Fig(TIF)Click here for additional data file.S40 Fig(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S4 Text(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Leukemia is an aggressive hematologic neoplastic disease. Therapy-resistant leukemic stem cells (LSCs) may contribute to the relapse of the disease. LSCs are thought to be protected in the leukemia microenvironment, mainly consisting of mesenchymal stem/stromal cells (MSC), endothelial cells, and osteoblasts. Canonical and noncanonical Wnt pathways play a critical role in the maintenance of normal hematopoietic stem cells (HSC) and LSCs. In this review, we summarize recent findings on the role of Wnt signaling in leukemia and its microenvironment and provide information on the currently available strategies for targeting Wnt signaling. Leukemia represents a heterogeneous group of blood neoplasia commonly characterized by an abnormal production of blood cells. Common leukemia subtypes include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphoblastic leukemia (CLL) ,2,3,4. IThe survival and maintenance of normal hematopoietic stem cells (HSC) relies on interactions with the bone marrow (BM) microenvironment, also known as BM niches . LeukemiStudies suggest a fundamental role for the Wnt signaling pathway in HSC , cancer Wnt ligands are secreted glycoproteins that bind to Frizzled (Fzd) receptors, which are G-protein-coupled receptors (GPCRs) with seven transmembrane domains . Upon biUpon the binding of Wnt ligands to Fzd receptors and LRP5/6, a disheveled segment polarity protein (Dvl) is recruited to the intracellular domain of Fzd. Subsequently, Dvl acts as a platform for Axin to interact with the cytoplasmic domain of LRP5/6 and forms the foundation for the signalosome. This interaction disassembles the APC–Axin–CK1α–GSK3β degradation complex, leading to the accumulation and translocation of unphosphorylated β-catenin to the nucleus. In the nucleus, another multiprotein complex called the enhancesome forms when β-catenin displaces the corepressor Groucho and interacts with the T-cell factor/lymphoid enhancer factor (T-cell factor (TCF)/LEF) family of transcription factors to regulate the transcription of several target genes  Figure B.The dynamic polymerization of multiprotein assemblies such as the degradation complex in the presence of Wnt and the signalosome in the absence of Wnt is complex . Dvl and2+ pathway [N-terminal kinase (JNK) expression, triggering the expression of downstream target genes  pathway and Wnt–Ca pathway . In the et genes C. The PC pathway , Wnt lig pathway ,31. CalcFurthermore, Wnt signaling is regulated at different levels by a wide range of effectors including agonists and antagonists which act either intracellularly to modulate components of the signal transduction or extracellularly to modulate ligand–receptor interactions. Six families of secreted and four families of transmembrane Wnt antagonists are known to date: the Dickkopf proteins (Dkks) , secreteWnt signaling plays a critical role in the maintenance of HSC homeostasis ,47. To bBCR–ABL1 oncogene, which plays a critical role in chronic myeloid leukemia (CML), can stimulate β–catenin in the blast phase of CML [FOXM1 gene is directly promoted by NF-κB and that the nuclear translocation of FOXM1/β-catenin requires NF-κB activation; meanwhile, Wnt–β-catenin activation facilitates the nuclear translocation of NFκB-p65 [It has been shown that the enhancement of self-renewal activity is associated with the activation of the Wnt–β-catenin pathway . The BCRe of CML . Moreovee of CML . Meanwhie of CML . Similare of CML . In addie of CML . It was NFκB-p65 .+CD38− fraction. Furthermore, CD34+ AML LSCs are primarily detected within lymphoid-primed multipotent progenitors (LMPP-like CD34+CD38−CD90−CD45RA+) and granulocyte-macrophage progenitor (GMP-like CD34+CD38+CD123+CD45RA+) subpopulations when xenografted into mice. Less frequently, they are detected in dominant multipotent progenitor (MPP-like CD34+CD38−CD90−CD45RA−) subpopulations. Interestingly, there are CD34− AML LSCs that are primarily detected within a precursor granulocyte-macrophage (GM-like CD34−CD117+CD244+/−) subpopulation [Acute myeloid leukemia (AML) LSCs predominantly reside in the CD34pulation .First of all, the Wnt–β-catenin signaling pathway was notably required for the self-renewal of LSCs in mouse models of AML . TranscrStem cell markers CD82 , CD70/CDCCND1 (cyclin D1) and C-MYC, via noncanonical Wnt signaling [LEF1, as a central transcriptional mediator of Wnt signaling, has been able to cause AML in mice ,70. Addiignaling .As a synergistic strategy of targeting LSC and other applicable inhibitors, Wnt–β-catenin signaling plays an important role. For example, LSC-mediated resistance to BET inhibitors while Wnt–β-catenin signaling is inhibited, in turn, restores the sensitivity to BET inhibition .AXIN1, C-MYC, BIRC5/Survivin, TCF1, and LEF1 in ALL [The Wnt–β-catenin pathway is upregulated in both B-cell-ALL (B-ALL)  and T-ce1 in ALL ,77,78,791 in ALL . Similar1 in ALL ,82. Othe1 in ALL . It has 1 in ALL .del/+Apc mouse model in MSCs, showing that Wnt signaling in the BM niche is responsible for MDS, rather than hematopoietic cells [+ cells in an in vivo mouse model via a COX2–PG-NR4A–Wnt signaling axis [There is accumulating evidence that functional alteration of stromal cells contributes to the development of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) ,85,86. Hic cells . Interesic cells  and coulic cells  and coulic cells . Fanconiing axis . Moreoveing axis .MSCs also contribute to drug resistance through cell-adhesion-mediated drug resistance (CAM-DR) ,92, in wIn vitro co-culture experiments showed that stromal cells provide acute leukemia cells with drug resistance through the upregulation of galectin-3 and stabilization of β-catenin . FurtherStudies have indicated that leukemic cells could alter the BM microenvironment and result in a bulk of growth factors to facilitate LSC propagation. First, it has been demonstrated that AML cells induced osteogenic differentiation in MSCs through activation of Smad1/5 signaling . MoreoveTaken together, Wnt signaling is one of many critical pathways by which MSCs facilitate leukemia cell survival, proliferation, and drug resistance. Meanwhile, leukemia cells may modify the MSC to support themselves.Both paracrine (EC-dependent) and autocrine (EC-independent) vascular endothelial growth factor (VEGF)–VEGF receptor (VEGFR) signaling pathways have been demonstrated to be important in a human leukemia model . A recen2+ pathway, resulting in the nuclear import of Nfatc1 and activation of Sox11, which induces osteoblast differentiation in human BM MSCs [Osteoblasts, which are derived from MSCs, are bone-forming cells that line the endosteum and act together with osteoclasts, the bone-reabsorbing cells, to maintain bone homeostasis . The mai BM MSCs .Moreover, taurine, an osteocyte metabolite, was shown to have a protective effect in osteocytes against cell death due to reactive oxygen species in the IDG-SW3 cell line. In this study, taurine also remarkably inhibited Dkk1, an inhibitor of the Wnt–β-catenin signaling pathway, although only at higher doses . SimilarAlp, Runx2, LEF1, and Axin, and is associated with the development of several cancers, such as leukemia [A recent study using the human osteoblastic cell line SaOS-2 showed that mineralization and downregulated osteoblast marker genes, including alkaline phosphatase, osteocalcin, and osterix, and that genes associated with the proosteogenic Wnt signaling pathway were significantly inhibited by TKIs . C-type leukemia ,125. It leukemia . For exaleukemia  through leukemia . throughleukemia .Taken together, these studies emphasize that nonautonomous Wnt signaling in leukemia can be dependent on cues from the BM microenvironment.Preclinical studies have indicated that Wnt signaling pathways can be targeted, and recent reviews have broadly discussed the applicable compounds in various cancers ,130,131.The available targets in this section include Wnt ligands and Fzd receptors. Porcupine (PORCN) is a membrane-bound-O-acetyltransferase enzyme that palmitoylates Wnts, which is important for its interactions with Fzd receptors . PORCN iDickkopf-1 (DKK1), a soluble inhibitor of Wnt–β-catenin signaling, regulates Wnt signaling by binding to the Wnt coreceptor LRP5/6. Since the levels of Wnt3a, Wnt5b, Wnt10a, Wnt14, Wnt16, Fzd3, Fzd6, and LRP5/6 were significantly higher in a vincristine-resistant BALL-1 ALL cell line (BALL-1/VCR), soluble DKK-1 selectively suppressed the Wnt signaling pathway and sensitized the response of BALL-1/VCR to chemotherapy .Wnt inhibitors such as CGX1321, Foxy-5, and ipafricept (OMP-54F28), and Fzd inhibitors such as OSTA101 and vantictumab OMP18RS) have been evaluated in clinical trials in cancers other than leukemia . Dvl inhRS have bRelated components in the cytoplasmic portion of Wnt signaling are applicable targets, such as Axin, Dvl, CK1, and GSK. Tankyrases, the members of the poly (ADP-ribose) polymerase (PARP) family of enzymes, mediate the ubiquitin-based proteasomal degradation of Axin . TankyraThe inhibition of CK1 using PF-670462 significantly prolonged the overall survival of a CLL mouse model and had synergistic effects with the B-cell receptor (BCR) inhibitor ibrutinib in both in vitro and in vivo CLL .CREB-binding protein (CBP)/catenin antagonists were recently reviewed as a critical strategy to target LSCs . XX-650-In addition, a synergistic increase of apoptotic effects and restored chemosensitivity from prednisolone treatment in B-/T-ALL cells were confirmed by using traditional chemotherapeutic drugs and iCRT14, a β-catenin/TCF inhibitor . A LEF1/N-(4-hydroxybenzyl)-1,3,4-triphenyl-4,5-dihydro-1H-pyrazole-5-carboxa-mide), a novel canonical Wnt–β–catenin-signaling inhibitor, was recently evaluated in the CML cell line K562, showing inhibition of cell proliferation in a dose-dependent manner and the induction of apoptosis [Furthermore, BHX (poptosis .+CD38−CD90−CD45RA+ leukemic stem cell population in AML [An increasing number of studies have demonstrated that compounds may exert their effects by modulating Wnt signaling. For example, ARV-825, a BET/BRD4 inhibitor targeting c-Myc, can downregulate Wnt–β-catenin signaling, thereby decreasing the CD34n in AML . Pyrvinin in AML . Emodin n in AML . The metn in AML .Interestingly, cotreatment with celecoxib, a specific COX-2 inhibitor, and doxorubicin significantly inhibited cell proliferation and induced apoptosis in AML ,160. Sul+ CML LSCs, while ex vivo exposure of CML CD34+ cells decreased long-term engraftment in mice. Furthermore, in vivo treatment of p-Niclosamide alone prolonged survival of CML mice compared to placebo group, and combination therapy of p-Niclosamide with imatinib further prolonged survival of CML bearing mice. Anti-leukemic potential of Niclosamide was achieved by interfering the interplay between p65 of NF-κB and FOXM1/β-catenin [Niclosamide is an antihelmenthic drug that is known for modulation of Wnt signaling, along with other signaling pathways such as mTORC1, STAT3, NF-kB, and Notch pathways, in many types of cancers ,168,169.-catenin .Lastly, clofazimine, an antileprosy drug, is known for its anti-cancer potential via inhibition of canonical Wnt signaling . ClofazaHerein, we reviewed the complex relationship between Wnt signaling and many factors in the leukemia microenvironment . As disc