Absorption, distribution, metabolism, and excretion and safety pharmacology studies were conducted with RXC004 in vitro, and pharmacokinetic exposure assessed in vivo. RXC004 effects on proliferation and tumor metabolism were explored in genetically defined colorectal and pancreatic cancer models in vitro and in vivo. RXC004 effects on immune evasion were assessed in B16F10 immune “cold” and CT26 immune “hot” murine syngeneic models, and in human cell cocultures. RXC004 showed a promising pharmacokinetic profile, inhibited Wnt ligand palmitoylation, secretion, and pathway activation, and demonstrated potent antiproliferative effects in Wnt ligand–dependent (RNF43-mutant or RSPO3-fusion) colorectal and pancreatic cell lines. Reduced tumor growth and increased cancer cell differentiation were observed in SNU-1411 (RSPO3-fusion), AsPC1 and HPAF-II (both RNF43-mutant) xenograft models, with a therapeutic window versus Wnt homeostatic functions. Additional effects of RXC004 on tumor cell metabolism were confirmed in vitro and in vivo by glucose uptake and 18fluorodeoxyglucose-PET, respectively. RXC004 stimulated host tumor immunity; reducing resident myeloid-derived suppressor cells within B16F10 tumors and synergizing with anti-programmed cell death protein-1 (PD-1) to increase CD8+/regulatory T cell ratios within CT26 tumors. Moreover, RXC004 reversed the immunosuppressive effects of HPAF-II cells cocultured with human peripheral blood mononuclear cells, confirming the multiple anticancer mechanisms of this compound, which has progressed into phase II clinical trials.Wnt signaling is implicated in the etiology of gastrointestinal tract cancers. Targeting Wnt signaling is challenging due to on-target toxicity concerns and lack of druggable pathway components. We describe the discovery and characterization of RXC004, a potent and selective inhibitor of the membrane-bound Wnt pathway dysregulation drives many gastrointestinal cancers; however, there are no approved therapies that target the pathway. RXC004 has demonstrated the potential to block both tumor growth and tumor immune evasion in a genetically defined, clinically actionable subpopulation of Wnt ligand–dependent gastrointestinal cancers. The clinical utility of RXC004, and other Porcupine inhibitors, in such Wnt ligand–dependent cancers is currently being assessed in patient trials. Wnt pathway aberrations are common in gastrointestinal cancers . The canTargeting downstream Wnt signaling therapeutically has proven challenging. Biologicals that inhibit upstream components of the pathway (such as blocking Wnt binding to Frizzled receptors), have showed limited efficacy and had dose-limiting toxicities in the clinic . Howevero-acyl transferase Porcupine, which is essential for palmitoylation and secretion of active canonical or noncanonical Wnt ligands (CTNNB1), are unlikely to be susceptible to Porcupine inhibition, these inhibitors could be effective where upstream Wnt pathway aberrations drive the pathway. The Wnt negative regulator RNF43, a transmembrane E3 ligase, targets Frizzled receptors for lysosomal destruction  and approximately 7% of pancreatic cancers  appear mutually exclusive of each other and of downstream aberrations in APC or CTNNB1  were treated with RXC004 (300 nmol/L) or DMSO for 48 hours. Wnt5a secreted into media was detected by Western blotting using anti-Wnt5a antibody .50 values determined. Recombinant Wnt3a (Bio-Techne #1324-WN-010) was used at 1 μg/mL. β-catenin expression: L-Wnt3a cells were treated with RXC004 (100 nmol/L) or DMSO (0.1%) for 24 hours. Cellular β-catenin protein levels were quantified by staining with anti-β-catenin  and assessment using an Agilent Novocyte-3000 flow cytometer.Reporter Assay: Media from L-Wnt3a cells  treated with a concentration-series of RXC004, was incubated with the LEADING LIGHT Wnt Reporter Cell Line . Luciferase was detected using the ONE-Glo Luciferase Assay System (Promega E6120), data were normalized to DMSO-treated controls and ICCells were treated with RXC004 (100 nmol/L) or DMSO (0.02%) for 7 days. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assay System (Promega #G8091) and data were plotted as a ratio relative to cell number as quantified using ATPlite 1-Step Reagent (Perkin Elmer #6016731).Aqueous solubility: A total of 1 mL of buffer was added to 1 mg of compound and incubated for 24 hours . Following filtration under positive pressure, compound concentration in solution was assessed by high-performance liquid chromatography coupled to an ultraviolet detector (LC-UV).7.4): A total of 30 μL of a 20 mmol/L stock solution of RXC004 was added to a vial containing 1.5 mL of pH7.4 phosphate buffer saturated octanol and 1.5 mL of octanol-saturated pH7.4 phosphate buffer, shaken and centrifuged to ensure phase separation. logD7.4 was calculated as the logarithm of the ratio of the concentration of RXC004 (measured by LC-UV in octanol vs. buffer phase).Lipophilicity (logDas were determined using the fast UV (spectrometric) technique. Samples were titrated under methanol-water mixture between pH2.0 and pH12.0 at 30–15 μmol/L at 25°C. The ionic strength was 0.192 mol/L (KCl). Aqueous pKas were determined per Yasuda-Shedlovsky (pKedlovsky .−1) or hepatocytes (0.5 × 106 cells · mL−1). Aliquots were removed at 0–45 minutes and RXC004 concentrations determined by LC/MS-MS analysis. Half-life (T1/2) and intrinsic clearance (CLint) were calculated as described in ref. Metabolic stability: liver microsomes and hepatocytes from human, mouse (CD1), rat (Sprague Dawley) and dog (Beagle) were obtained from Invitrogen. RXC004 (1 μmol/L) was incubated at 37°C with microsomes  assays were performed using human liver microsomes incubated with P450 specific substrates as recommended by the FDA  at a conPapp) was calculated according to standard practice (Permeability of RXC004 was assessed in MDR1-MDCKII (Netherlands Cancer Institute) and Caco-2 cells (Merck Life Sciences). RXC004 (10 μmol/L) was added to either apical or basolateral chamber for 2 hours at 37°C, then an aliquot from the opposite side of the monolayer was removed and quantified by LC/MS-MS. Apparent permeability  by intravenous and oral routes. Bioanalysis of blood and plasma samples was performed by LC/MS-MS using a Waters TQS or TQMS mass spectrometer. Pharmacokinetic information was derived using WinNonlin software (Certara).Groups of 9–10 SCID-Beige mice were dosed at Translational Drug Discovery with vehicle, 1.5 mg/kg or 5 mg/kg RXC004 orally twice daily, or 5 mg/kg RXC004 orally once daily, for 28 days. Formulation: 5% DMSO, 95% hydroxyl propyl beta cyclodextrin (25% w/v). Formalin-fixed, paraffin-embedded (FFPE) samples from the small intestine were assessed by hematoxylin and eosin (H&E) and/or Ki67 staining at Histologix.50 values determined. Colorectal cell lines used were: WiDR (APC-E853*/T1556fs*3), HCT116 , JVE-109 (RNF43-V211fs*47/G659fs*41), SNU-1411 (PRPTK-RSPO-3 fusion). Pancreatic cell lines were: CAPAN-2 (RNF43-R330fs*89), AsPC1 (RNF43-S720*), and HPAF-II (RNF43-E174*). Cell lines were from ATCC, except for SNU-1411 (Korean Cell Line Bank) and cultured for no more than 20 passages. Cells were routinely tested for Mycoplasma by the MycoAlert test (Lonza) and authenticated using short tandem repeat fingerprinting.Cells were treated with a concentration-series of RXC004 for 5 days and proliferation measured using ATPLite 1-Step Reagent (Perkin Elmer #6016731). Data were normalized to DMSO-treated controls and ICThe expression of nine potential Wnt-responsive genes was screened across cell lines by treatment with RXC004 (10 nmol/L) or DMSO (0.1%) for 3 days. RNA was extracted  and gene expression assessed by Taqman qPCR relative to appropriate housekeeping genes on the Bio-Rad CFX real-time system.50 values determined. For cell-cycle analysis, the cancer cell line panel was treated with RXC004 (100 nmol/L) or DMSO (0.1%) for 3 days. Cell-cycle profiles were determined by staining with propidium iodide (Thermo Fisher Scientific #P3566) and phospho-Ser10-Histone-H3 , and quantified using an Agilent Novocyte-3000 flow cytometer. For in vitro growth curve analysis, HPAF-II and SNU-1411 cells were seeded in 96-well plates at 2,000 cells/well and imaged on the IncuCyte (Sartorius) at 10× magnification with 4 fields of view per well. Total cell object count over time was calculated using the Adherent Cell-by-Cell analysis module.For concentration–response effects on gene expression, Single-Shot Cell Lysis kit (Bio-Rad #172-5080) was used for RNA extraction, data were normalized to DMSO-treated controls, and ICin vivo pilot studies were performed at Axis Bioservices. HPAF-II , AsPC1 , and SNU-1411  were implanted bilaterally, subcutaneously, whereas HCT116  were implanted in a single flank. Animals were randomized into the indicated groups and treatment initiated once tumor volumes reached approximately 100–150 mm3. Dosing was either 1.5 mg/kg twice daily RXC004 for 7–13 days then once daily for the remainder of study (up to 29 days), or 28 days 1.5 mg/kg twice daily RXC004 for HCT116. In these and all other animal studies, unless otherwise specified, RXC004 was dosed orally in 0.5% carboxymethylcellulose + 0.1% Tween-80. For AsPC1, an ascites model, end of study tumor weight (mg) was measured. A total of 12 hours after the final dose, RNA was extracted [GenElute RNA isolation kit ] from snap frozen tumor for qRT-PCR expression analysis of Axin2, c-Myc, RNF43, MUC4, and MUC5AC relative to appropriate housekeepers.The in vivo, SNU-1411 (1 × 107 cells) were implanted into NOD-SCID mice subcutaneously at Axis Bioservices. Animals were randomized into the indicated groups and oral dosing of RXC004 was initiated when tumors reached approximately 200 mm3. Tumor volume measurements were taken at day 19, n ≥9 per group. Pharmacokinetic/pharmacodynamic analysis in SNU1411/NOD-SCID mice was performed in a separate study at Axis Biosciences, with tissue and plasma extracted for assessments after 7 days of RXC004 dosing. Tumor was divided into two sections; one section snap frozen and the other processed by FFPE. RXC004 levels were determined in snap frozen tumor and blood samples by LC/MS-MS. Expression analysis of the indicated genes was performed on snap frozen tumor as described above. Serial FFPE tumor sections were stained with H&E, anti-Ki67 (Abcam #Ab15580) or the dual mucin stain Alcian-blue/periodic acid shift (AB-PAS) at Histologix. IHC images were quantified using HALO software and modules (Indica Labs). The AB-PAS images were analyzed with the area quantification module on tumor area. A classifier was trained to detect differentiated and nondifferentiated tumor areas in RXC004-treated tumors using the AB-PAS–stained slides. Image registration was used to assign annotations from AB-PAS images to Ki67 images. The Ki67 images were analyzed with the multiplex IHC module and number of Ki67-positive cells were determined per mm2 of total or differentiated tumor area.To explore dose–response effects 3. After 9 days treatment, tumors were resected and equivalent viable tumor fragments from each tumor were reimplanted into separate naïve NOD-SCID mice, tumor volume was assessed at various subsequent timepoints.The reimplantation study was performed at Sygnature Discovery. SNU-1411–implanted NOD-SCID mice were dosed orally with RXC004  once tumors reached an average size of 350 mm5) were subcutaneously implanted in flanks of the immunocompetent male C57BL/6 mice. Treatment was initiated at day 3, once tumors were palpable with RXC004 being dosed orally as indicated. Anti-programmed cell death protein-1  or isotype control was dosed at 100 μg per dose intraperitoneal, twice weekly. Anti-CTLA-4 antibody (BioXCell BP0131) or isotype control was dosed intraperitoneally every 3 days at 100 μg per dose. Separate experiments were performed for tumor volume and flow cytometry analysis. Tumor volume was assessed in each animal and/or group until prespecified termination conditions and/or study endpoint were reached. For flow cytometry, tumors were resected at day 19, 2 hours after final dose, weighed and digested into a single-cell suspension using collagenase  and DNAase and passed through a strainer prior to flow cytometry analysis on FACS Canto II apparatus using Flow JO software. Single and fluorescence minus one stains were used for compensation setup and gating controls respectively. A myeloid analysis panel included Zombie viability dye (BioLegend #423106), CD11b (BioLegend #101228), CD45, Ly6C, and Ly6G . Myeloid myeloid-derived suppressor cells (MDSC) were gated as CD45+CD11b+ Ly6G-Ly6Chigh and polymorphonuclear MDSCs were gated as CD45+CD11b+ Ly6G+Ly6Cmed (B16F10/C57BL/6 syngeneic model was performed at Axis Bioservices. Mouse B16F10 cells (2 × 10+Ly6Cmed . For gen5) were subcutaneously implanted in flanks of the immunocompromised male SCID-Beige mice. Treatment with RXC004 (5 mg/kg once daily) was initiated at day 3, once the tumors were palpable, and tumor volume assessed over time.B16F10/SCID-Beige model was performed at Axis Bioservices. Mouse B16F10 cells (2 × 105) were subcutaneously implanted in the flanks of the immunocompetent female BALB/c mice. Treatment was initiated once tumors reached approximately 50 mm3. RXC004 was dosed orally at 1.5 or 5 mg/kg (once daily). Anti-PD-1 (BioXCell) or isotype control was dosed every 3 days at 10 mg/kg i.p. Tumor volume was measured over time. For immunophenotyping the tumor infiltrate, tumors from a separate study were resected at day 14, weighed, digested, and stained for flow cytometry analysis by Beckton Dickenson LSR Fortessa. T-cell analysis panel included viability dye, CD45, CD3, CD4, CD8, CD25, and FOXP3.CT26/BALB/c syngeneic model was performed at ProQuinase GmbH. Mouse CT26 cells (5 × 10in vitro for 48 hours with RXC004 (100 nmol/L), Latrunculin A , or DMSO prior to analysis using the Glucose Uptake Glo Assay Kit (Promega #J1341). The ATPlite assay was used to control for cell viability, confirming RXC004 has no effect on proliferation at this 48 hours timepoint.SNU-1411, HPAF-II, and HCT116 cells were treated 7 cells; NOD-SCID mice) or HPAF-II  were implanted subcutaneously and treatment with RXC004 (5 mg/kg once daily) or vehicle commenced when tumors reached approximately 200 mm3. Mice were imaged prior to dosing, day 3 and day 7 after dosing. A 10 MBq injection of 18FDG (PETNET), with a 45-minute washout was followed by a 20-minute PET scan using a Siemens Inveon PET scanner. Tissues removed on day 7 for biodistribution analysis were assessed using a PerkinElmer Wallac Wizard Gamma counter.Experiment was performed at Medicines Discovery Catapult. SNU-1411 (1 × 10HPAF-II cells were incubated for 3 days with RXC004 (100 nmol/L) or DMSO control, washed and detached using Accutase and equal number of viable cells (as assessed by Trypan Blue exclusion) per treatment were reseeded in 96-well plates. Frozen human peripheral blood mononuclear cells (PBMC), originally collected by density gradient separation using Histopaque from four healthy donors, were thawed and cultured in complete media. After approximately 40 hours, nonadherent PBMCs were added to coculture with the HPAF-II cells, at a PBMC:HPAF-II ratio of 5:1. T-cell proliferation and cytokine production was stimulated using Immunocult CD3/CD28-tetramer (Stemcell Technologies #10971). For monoculture nonadherent PBMC experiments, T cells were stimulated using Immunocult in the presence or absence of recombinant Wnt3a (Biotechne #5036-WN-010) and/or recombinant Wnt5a (Biotechne #645-WN-010), both at 100 ng/mL. After 5 days, supernatants from monoculture or coculture experiments were removed for analysis, whereas suspension cells were fixed, stained with anti-Ki67 (BD #563462), anti-CD4 (BioLegend #317420), and anti-CD8 (BD #564116) antibodies, before flow cytometry was performed on a Novocyte flow cytometer. Cytokines and chemokines in the supernatant were quantified using a 30-plex panel on Magpix (Thermo Fisher Scientific #LHC6003M) or ELISA for IFNγ (R&D Systems # DY285B-05).50 values were determined using 4-parameter curve fitting within Prism software.Unless stated otherwise, drug metabolism and pharmacokinetic (DMPK) data analysis was performed using Excel (Microsoft). All other statistical analyses were performed using Prism software (GraphPad Prism). For each analysis, residual plots were examined and where necessary a log transformation was applied prior to analysis. Where indicated, ICThe data generated in this study are available upon request from the corresponding author.in vitro screening allowed for the elucidation of structure activity relationships which ultimately resulted in the discovery of RXC004: 2-[5-methyl-4-[2-(trifluoromethyl)-4-pyridyl]imidazol-1-yl]-N-(5-pyrazin-2-yl-2-pyridyl)acetamide; structure shown in A rational design approach was used to identify potent novel Porcupine inhibitors. Routine o-acyl transferase Porcupine  and 11.2 (most acidic), meaning that the predominant species at pH 7.4 is the neutral, nonionized form of the molecule. The average percentage free of RXC004 in plasma across species ranged from 2.5% to 7.5%. Microsomal CLint values ranged from 3.9 to 31.6 μL · minute−1 · mg−1, with mouse having the lowest and dog the highest predicted clearances. Hepatocyte CLint values ranged from < 3 to 12.5 μL · minute−1 · 106 cells, with dog displaying moderate clearance while rodents and humans display low clearance. RXC004 has good intrinsic permeability, showing some evidence of efflux in MDR1-MDCKII cells but not in Caco-2 cells.The physicochemical profile of RXC004 is shown in in vitro absorption, distribution, metabolism, and excretion properties which are predictive of the low metabolic clearance and good oral bioavailability, as successfully observed in vivo in mouse, rat, and dog  or gene fusions in RSPO3 (SNU-1411), and those with downstream Wnt pathway aberrations such as LoF mutations in APC (WiDR) or gain-of-function mutations in CTNNB1  mutations in  fusions . In addi fusions . Minimal fusions .RNF43 mutations and RSPO3 fusions in a concentration-dependent manner but had no effect on cell lines with mutations in downstream Wnt signaling . A reduction in tumor growth, and inhibition of Wnt-responsive gene expression including c-Myc, was observed in the Wnt ligand–dependent SNU-1411, AsPC1, and HPAF-II models . Disruption of villi architecture  was observed only in some animals dosed at 5 mg/kg twice daily. No effects were seen at either the 1.5 mg/kg twice daily or the 5 mg/kg once daily doses previously demonstrated to inhibit tumor growth and Wnt-responsive gene expression . MoreoveIn vitro, RXC004 inhibited glucose uptake by over 50% in the Wnt ligand–dependent cell models SNU-1411 and HPAF-II cells, but had no effect in the Wnt ligand–independent HCT116 cell line (ell line . At thisell line . The posIn vivo18fluorodeoxyglucose-PET (18FDG-PET) imaging analysis in SNU-1411 xenograft models demonstrated a 31% decrease in maximum standardized uptake value in RXC004-treated tumors compared with vehicle controls at day 7. Moreover, a significant decrease in tumor:blood ratios was observed in both SNU-1411 and HPAF-II xenograft models following biodistribution analysis of the 18FDG tracer at end of study  when RXC004 was combined with anti-PD-1 treatment over control or either monotherapy. While a change in infiltrating CD8+ T cells was not observed, a significant increase in the CD8+/regulatory T cell ratio was seen for the RXC004 plus anti-PD-1 combination versus control or either monotherapy , CGX1321, ETC-159, and XNW7201. Encouragingly, there has been a lack of widespread intestinal adverse events published to date. The most consistent side effects include loss of bone density and associated fractures, and dysgeusia , the latRSPO2/3 fusions and LoF RNF43 mutations. Because of these genetically selected patient tumors likely being dependent on Wnt ligand for survival and growth, it follows that high Wnt ligand levels in the tumor microenvironment could also be responsible for increased immunosuppression. This may increase susceptibility of such patient tumors to the positive immune effects of Porcupine inhibitors such as RXC004. The synergistic effect between RXC004 and anti-PD-1 on immune infiltrate in the “immune hot” CT26 model and the reversal of immune suppression in the “immune-cold” B16F10 model supports investigating the clinical activity of Porcupine inhibitors in combination with anti-PD-1 therapy, a strategy currently being explored  analysis of RXC004 in SNU-1411/NOD-SCID mice. Supplementary Figure 4: Dose-dependent effects of RXC004 on tumor markers, intestinal Ki67 and bodyweight. Supplementary Figure 5: Effect of RXC004 with or without anti-PD-1 on body weight and survival in in vivo models. Supplementary Figure 6: Dose-dependent effects of RXC004 alone or in combination with anti-CTLA-4 in B16F10/C57BL/6 syngeneic model. Supplementary Figure 7: In vitro PBMC co-culture additional cytokines and monocultures. Supplementary Table 1: In vivo exposure data for RXC004.Click here for additional data file.


The extent and persistence of T cell responses to vaccination remains unclear. We explored SCV-2 specific Ab, B cell and T cell responses to 3rd dose vaccine (BNT162b2) we measured anti-SCV-2 receptor binding domain (RBD) Ab by electrochemiluminescence assays. Subjects reported incident COVID-19 that occurred post vaccination. In a subanalysis, we assessed SCV-2-specific plasma cell, memory B cell and T cell responses in peripheral blood mononuclear cells after stimulation with wild type (WT) RBD, WT full Spike antigen, Omicron RBD and Omicron S1 antigens by ELISpot . We compared between-group differences in immunological outcomes by incident infection by Wilcoxon signed rank or Mann–Whitney U tests. Data are median (IQR) unless specified.In plasma from adults enrolled in a multicentre prospective cohort, sampled before, 14 days and 10 months post 3rd vaccine. 76 subjects contributed to the cellular immunity subanalysis [23 of whom provided additional samples 10 months post vaccine (Table 1)]. RBD titres and B cell responses increased significantly 2 weeks post 3rd vaccine , with RBD titres and WT-specific memory B cell responses remaining significantly higher than pre-booster vaccine levels at 10 months . In contrast, there was no significant difference in T cell responses at two weeks or 10 months post 3rd dose vaccine . There was no difference in 2-week post vaccine RBD or T cell responses in those with and without incident SCV-2 infection. However, those with incident infection had significantly lower WT RBD-specific plasma and memory B cell levels .Of 132 subjects , 47 (36%) reported incident SCV-2 infection at 18 (16-21) weeks post 3rd dose vaccination induced robust antibody and B cell responses which persist at 10 months, but not T cell responses. Higher memory B cell responses post vaccination, rather than circulating antibody titres or T cells, are associated with protection from subsequent infection.3Oliver A. Cornely, MD PhD, DZIF: Advisor/Consultant|DZIF: Board Member|DZIF: Grant/Research Support|DZIF: Honoraria|DZIF: Stocks/Bonds


MiR-126-3p (miR-126) is highly expressed in normal hematopoietic stem and progenitor cells (HSPC) and maintains self-renewal capacity . Aberran−/−) mouse [−/− mice do not develop leukemia as part of their phenotype, but constitutively express higher levels of miR-126, thereby, representing a functional model of miR-126 OE [−/− versus BCR::ABL1/Spred1+/+ mice  counts and circulating pro-B blasts (B220+CD19+CD43+IgM-) and shorter survival  than the BCR::ABL1/Spred1+/+ controls  of p190-BCR::ABL1 ALL with either global or compartmentalized  miR-126 overexpression (OE) or knockout (KO). The p190-BCR::ABL1 transgenic mice develop ALL, a disease transplantable in congenic recipients , 14, and−) mouse  Fig. 1a−/− mouseR-126 OE , 9. ConsR-126 OE , in BCR:ols Fig. .Fig. 1Boflox(f)/f mice [f/fVav-icre+ (miR-126 OE in hematopoietic cells) and Spred1f/fTie2-Cre+ (miR-126 OE in ECs) mice. We then crossed these mice with p190-BCR::ABL1 mice and, respectively, obtained BCR::ABL1/Spred1f/fVav-icre+  and BCR::ABL1/Spred1f/fTie2-Cre+  mice    recipients  than the Spred1EC+/+ recipient controls /f mice  with Vavice Fig. . The BCRECs Fig. , left, hECs Fig.  and a mo01) Fig. , left th07) Fig. , right. f/f mouse with the Vav-icre+ mouse and in turn the miR-126f/fVav-icre+ (miR-126 KO in hematopoietic cells) mouse with the p190-BCR::ABL1 mouse  mouse with lower miR-126 and higher Cdkn2aip levels in the ALL blasts, lower WBC counts and pro-B blasts, and longer survival  than the BCR::ABL1/miR-126f/f/Vav-icre- (BCR::ABL1/miR-126ALL+/+) control  mouse with the p190-BCR::ABL1 mouse  mouse had significantly lower EC-miR-126 levels and lived longer  than the miR-126EC+/+ recipient controls  in PB, BM and spleen at the end of treatment and lived longer  in PB, BM or spleen, suggesting that they were potentially cured.To assess the relevance of these results to human disease, we transplanted primary human Ph+ ALL cells into NSG mice. At day 30 after transplantation, the mice were randomly divided into 4 groups and treated with SCR , miRisten , SCR + Dasatinib , or miRisten + Dasatinib for 3 weeks, followed by assessment of human cell engraftment in PB, BM and spleen and survival Fig. . MiRisteIn summary, our results support a leukemogenic role of miR-126 in BCR::ABL1 ALL cells. Of note, while we used Spred1 KO to induce endogenous miR-126 upregulation and obtain functional miR-126 OE models of BCR::ABL1 ALL, loss of Spred1, a negative regulator of the pro-leukemogenic RAS-MAPK signaling , 15, migSupplementary Information


We have previously shown that VEGF165b, an alternatively spliced VEGF-A isoform, inhibits the VEGFR-STAT3 pathway in ischemic endothelial cells (ECs) to decrease their angiogenic capacity. In ischemic macrophages (Møs), VEGF165b inhibits VEGFR1 to induce S100A8/A9 expression, which drives M1-like polarization. Our current study aims to determine whether VEGF165b inhibition promotes perfusion recovery by regulating the miR-17-92 cluster in preclinical PAD.VEGFin vitro PAD model. VEGF165b was inhibited/neutralized by an isoform-specific VEGF165b antibody.Hind limb ischemia (HLI) induced by femoral artery ligation and resection was used as a preclinical PAD model. Hypoxia serum starvation (HSS) was used as an 165b-inhibition induces miRNA-17-20a (within miR-17-92 cluster) in HSS-ECs and HSS-bone marrow derived macrophages (BMDMs) vs. respective normal and/or isotype matched IgG controls to enhance perfusion-recovery. Consistent with the bioinformatics analysis that revealed RCAN3 as a common target of miR-17 and miR-20a, Argonaute-2 pull-down assays showed decreased miR-17-20a expression and higher RCAN3 expression in the RISC complex of HSS-ECs and HSS-BMDMs vs. the respective controls. Inhibiting miR-17-20a induced RCAN3 levels to decrease ischemic angiogenesis and promoted M1-like polarization to impair perfusion recovery. Finally, using STAT3 inhibitors, S100A8/A9 silencers and VEGFR1-deficient ECs and Møs, we show that VEGF165b inhibition activates the miR-17-20a-RCAN3 pathway independent of VEGFR1-STAT3 or VEGFR1-S100A8/A9 in ischemic ECs and ischemic Møs, respectively.Systematic analysis of miR-17-92 cluster members (miR-17-18a-19a-19b-20a-92) in experimental-PAD models showed that VEGF165b inhibition in PAD.Our data revealed a hereunto unrecognized therapeutic ‘miR-17-20a-RCAN3’ pathway in the ischemic vasculature that is VEGFR1-STAT3/S100A8/A9 independent and is activated only upon VEGF In its most severe form, decreased skeletal muscle perfusion results in chronic limb-threatening ischemia (CLTI), which often results in impaired wound healing and necrosis leading to limb amputation2. Approximately 200,000 amputations occur in the US, with PAD being the major cause3. Approximately 20% of patients with CLTI are also at a high risk for cardiovascular death within the first year of diagnosis5. Currently, no approved medical therapies can revascularize the ischemic muscle and promote perfusion relief in PAD patients, indicating a greater need to identify therapies that can promote limb perfusion in PAD patients.Peripheral artery disease (PAD) occurs due to atherosclerotic occlusions in the inflow blood vessels, resulting in decreased blood flow to the lower extremities 6. Clinical trials aimed at achieving perfusion recovery in patients with PAD by activating VEGF-A-VEGFR2 signaling in PAD were not successful2. An inadequate understanding of VEGF-A signaling in ischemic-muscle vasculature could account for those failures. The signaling networks regulated by the VEGF-A family became more complex with the recognition of alternative splicing in the 8th exon of VEGF-A that results in the formation of pro-angiogenic VEGFxxxa (xxx for the no. of amino acids. VEGF165a) and anti-angiogenic VEGFxxxb (VEGF165b) isoform families8. The only difference between these 2 isoforms is a 6-aminoacid switch from CDKPRR (in humans and mice) in the VEGF165a isoform to SLTRKD (PLTGKD in mice) in the VEGF165b isoform.Vascular endothelial growth factor-A (VEGF-A) is a well-known inducer of angiogenesis165b isoforms is ~3-fold higher than the pro-angiogenic VEGF165a isoforms in human PAD muscle biopsies compared to age- and sex-matched controls. In preclinical PAD models, ischemia induced ~6-fold higher numbers of VEGF165b+ ECs9 and ~15-fold higher VEGF165b+ macrophages10 (Møs) compared to nonischemic muscle. Using a highly isoform-specific monoclonal antibody raised against the 6-amino acids that neutralizes the VEGF165b isoform (without affecting other proangiogenic VEGF165a isoforms12), we have shown that VEGF165b inhibition induces STAT3 activation in ischemic ECs to increase their angiogenic capacity but inhibits S1008/S1009 expression in ischemic Møs (independent of STAT3 activation) to induce a reparative M2-like phenotype10. Furthermore, we have shown that VEGF165b inhibition in ischemic muscle produces therapeutic perfusion recovery in multiple preclinical PAD models including eNOS-KO, Myoglobin Transgenic, and Type-2 Diabetic PAD mice12. However, significant gaps remain in our understanding of the genetic regulators downstream of VEGF165b inhibition that regulate the extent of perfusion recovery in ischemic muscle, including whether all of the effects of the antibody approach are STAT3- or S100A8/A9 dependent.In our recent studies, we have shown that the fraction of anti-angiogenic VEGF13. While lncRNAs are >200 base pairs in length and poorly conserved across species14, miRNAs are single-stranded ~16–23 nucleotide lengths that are relatively well conserved across species15. Despite recent advances in noncoding RNA biology, our understanding of the regulation or function of lncRNAs or miRNAs in human pathologies is still limited. In general, pairing of the miRNA seed sequence to the 3’ UTR of the target gene within a ribonucleotide binding region promotes target gene degradation or translational inhibition17. miRNAs can occur as individuals or as a part of a miRNA cluster. For example, the miRNA-17-92 cluster  members), also known as ‘OncomiR-1’, is one of the best-studied miRNA clusters due to its role in cancer biology19. While extensive literature is available on the miR-17-92 cluster in promoting tumor progression20, miR-92a and miR-19 within this cluster have been shown to inhibit perfusion recovery in PAD22. Hence, we wanted to determine the role of the VEGF165b-miR-17-92 cluster in regulating perfusion recovery in experimental PAD.Noncoding RNAs are becoming attractive targets to treat human disease due to their ability to modulate the expression of multiple genes and biological pathways165b inhibition induces miR-17-20a expression in ischemic vasculature that targets Calcipressin-3  independent of our previously published VEGFR1-STAT3 or VEGFR1-S100A8/A9 pathways to enhance perfusion recovery in experimental PAD.Here, we will show that VEGF165b inhibition modulates miR-17-92 cluster expression in ischemic ECs and/or Møs. qPCR analysis of skeletal muscle microvascular ECs  showed that hypoxia serum starvation  significantly decreased miR-20a expression but did not affect the expression of other miR-17-92 cluster members compared to normal controls. However, VEGF165b inhibition (10 μg/ml) induced the expression of miR-17 (P=0.058), miR-19b (P=0.075) and miR-20a (P=0.057) in HSS-SkMVECs compared to IgG , indicating a complete miR-17-92 cluster in human ECs. VEGF165b inhibition restored the expression of miR-17, miR-18, miR-19a, and miR-20a but not miR-92 to normal HUVEC levels in HSS-HUVECs vs. IgG (Supplement-1). We next examined the role of VEGF165b inhibition in regulating miR-17-92 cluster expression in bone marrow-derived macrophages (BMDMs). qPCR analysis showed that HSS numerically decreased the expression of miR-17 (P=0.068) and significantly decreased miR-20a expression but not the expression of other cluster members compared to normal BMDMs. Inhibiting VEGF165b in HSS-BMDMS restored the expression of both miR-17 and miR-20a to normal-BMDM levels  in vivo, we performed qPCR analysis of Balb/cJ mice (an inbred mouse strain with poor perfusion recovery post-HLI) ischemic muscle treated with IgG or VEGF165b-Ab  at day 3 post HLI. qPCR analysis showed differential expression within the members of this cluster in ischemic muscle vs. nonischemic muscle. A significant increase in miR-17, miR-18, and miR-20a and a numerical increase in miR-19b (P=0.076) expression were observed in ischemic vs. nonischemic muscle, and no significant differences in miR-19a or miR-92 expression were observed in ischemic vs. nonischemic muscle. VEGF165b inhibition in ischemic muscle induced miR-19a expression without affecting the expression of other cluster members vs. IgG (165b-Ab (165b inhibition in C57BL/6J mouse (an inbred mouse strain with good perfusion recovery post-HLI) ischemic muscle induced the expression of miR-17 and miR-20a with no changes in miR-19a expression vs. ischemic muscle treated with IgG (Supplement-2), suggesting that the strain of mice differentially regulates the expression of the miR-17-92 cluster to VEGF165b inhibition in ischemic muscle. No significant differences in other cluster members were observed between VEGF165b-Ab vs. IgG-treated C57BL/6J ischemic-muscle samples (Supplement-2). Based on these data, we hypothesized that VEGF165b inhibition induces the expression of the miR-17-20a cluster in ischemic ECs and Møs to promote perfusion recovery.To determine whether inhibiting VEGF vs. IgG . Interes according to our previous publications28. STAT3 inhibition did not significantly induce any changes in miR-17-20a expression in either normal- or HSS-HUVECs vs. the respective controls . However, while STAT3 inhibition induced miR-17-20a expression in normal SkMVECs (Supplement-3C), no significant difference in miR-17-20a expression was observed in HSS-SkMVECs (Supplement-3D). Since we have previously shown that inhibiting VEGF165b induces VEGFR1 activation, which activates STAT3 in HSS-ECs, we wanted to determine whether VEGFR1 regulates the miR-17-20a cluster independent of STAT3. However, no significant differences in miR-17-20a expression were observed in either normal or HSS-SkMVECs isolated from VEGFR1+/−  or HSS-BMDMs10 compared to respective VEGFR1+/+ controls). vs. VEGFR1+/+ mice , These data indicated that VEGF165b inhibition induces miR-17-20a cluster expression in HSS-ECs independent of VEGFR1-STAT3 signaling.To determine whether STAT3 activation downstream of VEGF165b inhibition induces miR-17-20a expression independent of S100A8/A9 in HSS-BMDMs. Next, we wanted to determine whether VEGFR1 regulates the miR-17-20a cluster independent of S100A8/A910 in HSS-BMDMs. qPCR analysis of miR-17-20a expression showed no significant difference in miR-17-20a expression in normal or HSS-challenged VEGFR1+/− vs. VEGFR1+/+ BMDMs . These data indicated that inhibiting VEGF165b induces miR-17-20a expression in HSS-BMDMs independent of VEGFR1-S100A8/A9 signaling.To determine whether S100A8/A9 plays a role in regulating miR-17-20a in ischemic Møs, we silenced S100A8 or S100A9  in normal or HSS-BMDMs and examined miR-17-20a expression. Silencing S100A8 or S100A9 did not affect miR-17-20a expression in normal or HSS BMDMs , indicating that VEGF165b inhibition induced miR-17-20a expression in C57BL/6J ischemic muscle (Supplement-2), we hypothesized that the ability of C57BL/6J ischemic muscle to induce miR-17-20a expression upon VEGF165b inhibition enhances perfusion recovery in experimental PAD. To test this hypothesis, we inhibited miR-17 and miR-20a in C57BL/6J skeletal muscle by i.m. delivery of miR-17+miR-20a antagomirs (100 μM miR-17 and miR-20a antagomir or equimolar concentration of nontargeting antagomir (inhibitor) in 100 μl of PBS injected at 2 nonoverlapping sites in GA and 1 site in TA) at days 0, 3, 7, 14, and 21 post-HLI29 (Supplement-8). miR-17-20a antagomirs significantly decreased perfusion recovery in C57BL/6J ischemic muscle vs. control inhibitor  in miR-17-20a inhibitor-treated C57BL/6J ischemic muscle vs. control inhibitor at day 21 post-HLI  and overexpressing miR-17 and miR-20a in HSS-HUVECs (Supplement-9B). While normal HUVECs transfected with miR-17 or miR-20a antagomirs showed a significant decrease in tube-like formation on growth factor-reduced Matrigel  and overexpressing miR-17 and miR-20a in HSS BMDMs (Supplement-10B). qPCR analysis showed no significant differences in Arg1 expression (M2 marker) but a significant increase in iNOS expression (M1 marker) in normal BMDMs transfected with miR-17 or miR-20a antagomirs , we treated ischemic muscle with control miR-17-20a inhibitors  at day 0 and examined RCAN3 expression at day 3 post HLI. qPCR analysis showed that miR-17-20a inhibition in C57BL/6J ischemic muscle significantly induced RCAN3 levels compared to the negative inhibitor at day 3 post-HLI  vs. control plasmid at day 14 post-HLI  into C57BL/6J skeletal muscle (GA and TA) and performed HLI. Laser Doppler showed a significant decrease in perfusion recovery in C57BL/6J mouse ischemic muscle treated with RCAN3-expressing plasmid vs. control plasmid (day 14: control plasmid 61.9±1.74 vs. RCAN3 plasmid 49.8±3.4), indicating that increased RCAN3 levels impair perfusion recovery in PAD . Immunohpost-HLI .29 in HSS-BMDMs, indicating an induction of the M1-like phenotype . Furthermore, silencing S100A8/A9 did not affect RCAN3 expression in normal HSS-BMDMs . No significant differences in RCAN3 expression were observed in HSS-BMDMs from VEGFR1+/− vs. VEGFR1+/+ mice (Supplement-15C). These data indicated that inhibiting VEGF165b decreases RCAN3 expression in HSS-ECs and HSS-BMDMs independent of VEGFR1-STAT3 or VEGFR1-S100A8/A9 signaling, respectively.Finally, we wanted to determine whether VEGFR1-STAT3 or VEGFR1-S100A8/A9 signaling regulates RCAN3 expression in ischemic ECs or ischemic Møs. STAT3 inhibition did not induce any significant changes in RCAN3 expression in normal or HSS-HUVECs (Supplement-14A) or in normal or HSS-SkMVECs (Supplement-14B). No significant differences in RCAN3 expression were observed in HSS-SkMVECs from VEGFR1165b isoform, our study presents a novel miR-17-20a-RCAN3 pathway that occurs specifically with the depletion of the antiangiogenic VEGF165b in PAD. The ability of skeletal muscle to recover from ischemic damage that occurs experimentally from HLI and repeatedly during daily activity in patients with PAD is directly dependent on the extent of the angiogenic response in ischemic muscle2. To the best of our knowledge, our current study is the first to demonstrate a pathway by which removing the inhibitory effect of VEGF165b in ischemic muscle allows the expression of a truncated miR-17-20a cluster in ischemic ECs and Møs that targets RCAN3 to revascularize ischemic muscle in experimental PADs. The miR-17-92 cluster is a highly studied miRNA cluster in cancer research22. While studies, in general, are focused on the whole miR-17-92 cluster, in our study, we present specific roles of miR-17 and miR-20a within the miR-17-92 cluster that are regulated by VEGF165b inhibition. Although the first evidence of an oncogenic role  in B-cell lymphoma involved a truncated miR-17-92 cluster (without miR-92), hence the name ‘OncomiR’34 was given, two paralogs of the miR-17-92 cluster are already known to occur in humans: miR106b-25  and miR-106a-363  clusters35.In our efforts to understand the mechanisms involved in regulating the angiogenic response to hind limb ischemia in general and specifically for advancing our understanding of the VEGF25 from our group showed that miR-106b, miR-93, miR-106a, and miR-17 are among the top 10 differentially regulated genes between C57BL/6J vs. Balb/cJ ischemic muscle at day 3 post-HLI. Consistent with these data, in our current study, we observed miR-17 to be one of the significantly different miRNAs in the ischemic muscle of C57BL/6J and Balb/cJ mouse strains with proangiogenic properties in the ischemic environment. Our previous studies have also shown that miR-93 within the miR-106b-25 cluster exhibits pro-angiogenic properties in an ischemic environment29, and miR-106a within the miR-106a-363 cluster induces ischemic angiogenesis (unpublished data). Taken together, these studies further indicate that miRNAs within the miR-17 seed sequence family may play critical roles in regulating the pathology of PAD.The 15 miRNAs from these 3 paralogous clusters form four seed families that include the miR-17 family ; miR-18 family ; miR-19 family  and miR-92 family . A previous study by Hazarika et al21 showed that inhibiting miR-92a improved functional recovery in PAD21. Landskroner-Eiger et al22 have shown improved limb arteriogenesis in EC-specific miR-17-92 cluster-deficient mice in experimental PAD, and this study shows that specific targeting of Frizzled Class Receptor 4 (FZD4) and LDL Receptor Related Protein 6 (LRP6) by miR-19 within the cluster plays a causal role in decreasing blood flow recovery22. In contrast, a recent report by Chamorro-Jorganes et al36. showed that EC-specific miR-17-92-deficient mice have blunted physiological retinal angiogenesis as well as diminished VEGF-induced angiogenesis36. Furthermore, deletion of the miR-17-92 cluster in renal proximal tubules or in ECs resulted in severe renal dysfunction and promoted microvascular rarefaction38. In our current study, increased expression of the miR-17-20a cluster by VEGF165b inhibition enhanced perfusion recovery. These data indicate that the phenotypic effect observed in gene knockout models is not the same as fine-tuning39 a miRNA level/function. This can be reflected in the lack of angiogenesis in miR-17-92 gene knockout models22 vs. increased ischemic-muscle revascularization when the miR-17-20a levels/function are fine-tuned  by VEGF165b inhibition. Taken together, these data indicate that VEGF165b inhibition fine-tunes the expression of the miR-17-20a cluster, which is sufficient to induce ischemic-muscle revascularization in PAD.Limited information is available on the role of the miR-17-92 cluster members in cardiovascular diseases, including PAD. Hinkel et al36 described that VEGF induces miR-17-92 expression via ELK1 -ETS-like-1), suggesting a potential role of VEGFR2 signaling in regulating the miR-17-92 cluster36. However, our recent reports have shown that VEGF165b induces VEGFR2 activation and that inhibiting VEGF165b decreases VEGFR2 activation. We anticipated that VEGF165b inhibition will induce the miR17-20a-RCAN3 pathway dependent on VEGFR1-STAT3 (in ischemic ECs) or VEGFR1-S100A8/A9 (in ischemic Møs). However, our data showed that inhibiting VEGF165b regulates the miR-17-20-RCAN3 pathway independent of VEGFR1-STAT3 or VEGFR1-S100A8/A9 signaling in ischemic ECs and ischemic Møs, respectively. While a full explanation of the involvement of alternative signaling pathways regulated by VEGF165b to control miR-17-20a expression is beyond the scope of this report, these data suggest the possibility of direct epigenetic regulation by VEGF165b isoforms in ischemic vasculature. In support of this, previous reports have shown that VEGF-A accumulates in the nucleus during wound healing and in response to hypoxia41. Since VEGF165a and VEGF165b isoforms only differ in exon 8, Further studies to determine the role of VEGF165b in nuclear vs. membrane compartments will present evidence for novel epigenetic vs. receptor-mediated signaling mechanisms downstream of VEGF165b inhibition that regulate miR-17-20a cluster expression in ways different from VEGFR2 or our previously published VEGFR1 signaling in ischemic vasculature.Interestingly, Chamorro-Jorganes et al51. Reports on the 6 miRs that comprise the miR-17-92 cluster have reported differential expression patterns. For example, miR-92 is expressed at much higher levels than the rest of the cluster members in several tumors, including glioma, colorectal cancer, and breast cancer46. Accordingly, in our current study, we consistently observed lower miR-18 expression than other cluster members in primary skeletal muscle, ECs, and BMDMs under normal or ischemic conditions. Although the miR-17-92 cluster is conserved between humans and mice, it is important to note a relatively lower expression (or lack of) of miR-18 expression in primary mouse ECs but not in human ECs in our study. This suggests a more complex evolutionary regulation of this cluster across species, and distinct context-dependent mechanisms operate in biogenesis, processing, and/or degradation within the members of this miRNA cluster53.Distinct functions and expression of the specific miRs within this cluster have been well established in several tumor studies and overexpressing RCAN3 or RCAN3-derived peptide has been shown to inhibit tumor progression23. Furthermore, RCAN3 has been shown to induce antiproliferative effects in HUVECs24. Our data showing that transcriptional and translational repression of RCAN3 by miR-17 and miR-20a enhances perfusion recovery in preclinical PAD models presents RCAN3 as a putative miR-17 and miR-20a target that regulates perfusion recovery in PAD. While further studies are needed to determine whether sex plays a role in regulating the miR-17-20a-RCAN3 pathway in experimental PAD, our study is the first report identifying RCAN3 as a downstream regulator of VEGF165b. The ability of RCAN3 to regulate ischemic EC angiogenic capacity and Mø polarization makes it an attractive PAD therapeutic.Our current study identified RCAN3 as a novel regulator of PAD. While HSS induced RCAN3 expression in SkMVECs, no significant difference in RCAN3 expression was observed in HSS-HUVECs vs. normoxic controls. Nevertheless, RCAN3 overexpression inhibited HUVEC angiogenic capacity, and silencing RCAN3 enhanced SkMVEC angiogenic capacity, indicating a functional role of the miR-17-20a-RCAN3 pathway in regulating ischemic angiogenesis. Limited information is available on the pathological roles of RCAN3. Recent studies have shown that RCAN3 decreases arthritis development in collagen-induced murine models165b is necessary to induce miR-17 and miR-20a expression that revascularizes ischemic muscle by targeting RCAN3, a novel regulator of PAD. Further studies are needed to understand the molecular mechanisms that regulate the transcriptional control of miR17-92 cluster members that results in their distinct expression pattern post VEGF165b inhibition. Advances in miRNA therapeutics and monoclonal antibody-based therapeutics point toward the potential to target miR-17-20a and RCAN3 for clinical applications.We present new evidence that removal of VEGF


Nonetheless, conventional imaging tools cannot accurately assess response in immunotherapy-treated patients. Using a lung cancer syngeneic mouse model responder to immunotherapy, we aimed to demonstrate that [18F]fluoro-D-glucose ([18F]-FDG) PET scans. Additionally, tumor lymphocyte infiltration was analyzed by the use of an [89Zr]-labeled anti-PD-1 antibody and measured as 89Zr tumor uptake.A syngeneic mouse model responder to anti-PD-1 therapy was used. Tumor growth and response to PD-1 blockade were monitored by conventional 2-deoxy-2-[18F]-FDG-PET scans failed to detect the antitumor activity exerted by anti-PD-1 therapy. However, [89Zr]-anti-PD-1 uptake was substantially higher in mice that responded to PD-1 blockade. The analysis of tumor-infiltrating immune cell populations and interleukins demonstrated an increased anti-tumor effect elicited by activation of effector immune cells in PD-1-responder mice. Interestingly, a positive correlation between [89Zr]-anti-PD-1 uptake and the proportion of tumor-infiltrating lymphocytes (TILs) was found .Conventional [Our data may support the clinical implementation of immuno-PET as a promising novel imaging tool to predict and assess the response of PD-1/PD-L1 inhibitors in patients with NSCLC. Lung cancer is the leading cause of cancer deaths . Over thAlthough immunohistochemical PD-L1 expression in NSCLC is a predictive biomarker of response to PD-1/PD-L1-inhibiting mAbs, other potential predictive biomarkers such as PD-1 expression in tumor-infiltrating lymphocytes (TILs) or the ability of the PD-1 antibody to reach its target have not been evaluated . MoreoveId1) is a negative transcription regulator that belongs to the Id (Id1-Id4) gene family  tumors though Id1 downregulation (unpublished data).Inhibitor of differentiation-1  PET is useful for proper initial staging and response monitoring of patients on systemic treatment. However, this imaging tool may be suboptimal in immunotherapy-treated patients in whom a metabolic uptake increase does not necessarily mean disease progression , positron-emission tomography (PET) and magnetic resonance imaging (MRI), have been established as powerful tools for the staging of lung cancer and the accurate assessment of therapeutic response , 18. PETgression . Respons18F]-FDG is emerging as a non-invasive method to monitor in real time the immune landscape of patients receiving ICIs. This approach is currently under evaluation and may potentially enter routine clinical practice if proven effective . Lentiviral production of shRNA against Id1  was performed as previously described (Murine LUAD cell line Lewis lung carcinoma (LLC) was purchased from the American Type Culture Collection . Cells were cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum (FBS) , 1% penicillin-streptomycin  and HEPES  and routinely tested for escribed , 16. pLKinterleukin-1β (Il-1β); tumor necrosis factor alpha (Tnf-α) and interferon gamma (Ifn-γ) gene expression was determined by real-time quantitative PCR as previously described  under the protocol number CEEA 054-19E1.Id1+/+) , and Id1-deficient (-/-Id1 or IDKO) mice with C57BL/6J background. -/-Id1 mice were kindly provided by Dr. Robert Benezra .This study included 8–12-week-old female C57BL/6J mice (6) with constitutive Id1 expression (LLC- pLKO-Sc) or Id1-silenced (LLC-sh-Id1) cells were injected subcutaneously in the flank of C57BL/6J (Id1+/+ and -/-Id1) mice. Tumor-bearing mice were treated with DPBS or anti-PD-1 monoclonal antibody  7, 10 and 14 days after cell inoculation . In the active treatment group, the last injection (day 14) consisted of the anti-PD-1 antibody radiolabeled with zirconium-89 ([89Zr]-anti-PD-1). Tumors were measured periodically using a digital caliper , and tumor volume was calculated using the formula: volume = π/6 x length x width2. At the end of the experiment tumors were harvested and fixed in 4% formaldehyde (pH = 7) for immunohistochemical analyses .Murine LLC cells  system to obtain the corresponding anatomical correlate of the tumors.All PET images were acquired on a Mosaic  small animal dedicated tomograph and reconstructed applying dead time, decay, random and scattering corrections into a 128×128 matrix with a 1 mm voxel size. Additionally, on the days that [18F]-FDG images, mice were fasted overnight with ad libitum access to drinking water. On the day of the study, a dose of 9.3 ± 0.8 MBq was injected intravenously in the tail vein. After 50 minutes, the animals were anesthetized with 2% isoflurane in 100% O2 gas and placed prone on the scanner bed for a 15-minute image acquisition. Images with [89Zr]-anti-PD-1 were acquired during 30 minutes and 24, 72 and 144 hours post-injection of an intravenous single dose (3.8 ± 0.02 MBq).To obtain PET [18F]-FDG and 89Zr uptake in the tumors was analyzed by drawing volumes of interest (VOI) manually containing the entire tumor, guided by CT when available. Semi-automatic segmentation was then performed including voxels with a value greater than 50% of the maximum value of the tumor. Finally, the average of the SUV values within the semi-automatic VOI was calculated (SUV mean).PET data were exported and analyzed using the PMOD software  and transformed to standardized uptake value (SUV) units using the formula SUV = [tissue activity concentration (Bq/cm3)/injected dose (Bq)] × body weight (g). -FDG-PET scans for accurately assessing the response to immunotherapy treatments, we used a lung cancer syngeneic mouse model exposed to PD-1/PD-L1 axis blockade, as previously published  and mice with Id1 silenced at the host microenvironment (IDKO)   (We investigated the synergistic impact of anti-PD-1 treatment combined with Id1 expression (tumor cells and host microenvironment). Id1 silencing  significantly reduced tumor growth     Table 1.Id1 blockade in the tumor microenvironment (Id1-/- mice) significantly enhanced CD3+ T cell infiltration in tumor samples as compared to Id1+/+ mice. However, no significant differences were observed when additional Id1 genetic silencing in LLC cells was added to Id1 inhibition in the tumor microenvironment in terms of CD3+ T cells infiltration in tumor samples; (p > 0.0001)     and CD8+ T cells  analyzed in tumor samples by IHC  and non-responding mice (mice with constitutive Id1 expression). Id1 absence in the tumor microenvironment in Id1-deficient mice significantly increased the tumor infiltration of CD3+ T cells, and more importantly, effector CD8+ T cells, as compared to Id1-expressing mice    Table 3.Il-1β, Tnf-α and Ifn-γ using RT-PCR. Id1 absence in the host microenvironment enhanced the expression of Il-1b, Ifn-γ and Tnf-α, factors implicated in immune T cell activation  , hyperprogression, and dissociated responses following treatment. These patterns, however, are not reflected in the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1), which is the standard for response assessment in oncology. Therefore, new response evaluation tools are required.89Zr]-anti-PD-1 radiotracer as a strategy to overcome the limitations of conventional [18F]-FDG-PET scans in assessing immunotherapy antitumor responses. Our results show that an [89Zr]-anti-PD-1-PET-CT could accurately assess tumor response to ICIs and may constitute a potential biomarker which directly labels effector T cells and predicts the efficacy of PD-1 inhibition in real time, non-invasively and safely.In this paper, we propose the use of a PET-CT scan based on an [18F]-FDG-PET-CT is a powerful tool for monitoring lung cancer initial staging and antitumor response due to its ability to detect small metastatic lesions and regional lymph node tumor spread more accurately than the conventional CT and MRI imaging methods  or PERCIMT   . However89Zr]-nivolumab and [18F]-BMS-986192 were studied in 13 patients. The median [18F]-BMS-986192 and [89Zr]-nivolumab SUVpeak was higher for lesions with ≥50% tumor PD-L1 than for lesions with <50% expression ; and aggregates of PD-1 positive tumor-infiltrating immune cells   , but also with progression-free survival (PFS) (p = 0.0025) and overall survival (OS) (p = 0.026)  .. More r= 0.066) . The NCT= 0.026) . More re therapy .89Zr]-anti-PD-1 uptake in tumor lesions was significantly higher among treatment-responding Id1 knock-out mice, than among non-responding mice maintaining a constitutive Id1 expression. More importantly, the novelty of our study relies on the demonstration of a clear correlation between that [89Zr]-anti-PD-1 uptake and the proportion of TILs (CD3+ T and effector CD8+ T cells) infiltrating tumor lesions and with a proinflammatory tumor microenvironment. As we previously demonstrated, Id1 and PD-1 combine blockade synergy was generated mainly through CD8+ T cells infiltration  and PD-1 blockade showed complete regression of their syngeneic lung cancer tumors, so they could not be measured. Moreover, our experiments were only performed in immunologically competent murine models, so these results should be confirmed in humanized murine models.The main limitation of our study is the relatively small tumor sample size analyzed, since most mice with combined complete 89Zr]-anti-PD-1 immuno-PET as a novel imaging tool for the non-invasive, real-time detection of antitumor effector TILs in a lung cancer mouse model responder to anti-PD-1 therapy. In this regard, [89Zr]-anti-PD-1 (immuno-PET) may probably perform similarly well in other lung cancer subtypes for which immunotherapy has been approved in frontline treatment or as subsequent lines (KRAS or BRAF mutant lung tumors).We have demonstrated the utility of [89Zr]-anti-PD-1 immuno-PET as a safe and feasible imaging modality to monitor PD-1/PD-L1 inhibitor antitumor response in NSCLC. We show that [89Zr]-anti-PD-1 uptake is significantly higher in anti-PD-1-responding mice as compared to non-responding mice. Moreover, our data may confirm the capacity of [89Zr]-anti-PD-1 immuno-PET to label PD–expressing immune cells, such as effector CD8+ TILs. Nevertheless, further investigation is warranted.This study proposes the potential use of [The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was approved by Committee on Animal Research and Ethics . The study was conducted in accordance with the local legislation and institutional requirements.AP: Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. MR-R: Formal Analysis, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing. IL: Data curation, Validation, Visualization, Writing – review & editing. FI: Formal Analysis, Writing – review & editing. JS: Writing – review & editing. ME: Data curation, Writing – review & editing. MC: Data curation, Formal Analysis, Writing – review & editing. AV-L: Formal Analysis, Writing – review & editing. AF-C: Data curation, Formal Analysis, Writing – review & editing. JS: Data curation, Formal Analysis, Writing – review & editing. SS: Writing – review & editing, Project administration, Supervision. IP: Project administration, Supervision, Writing – review & editing. AC: Project administration, Supervision, Writing – review & editing. DA: Project administration, Supervision, Writing – review & editing. IG-B: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review & editing.


Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) clinical strains to assess cefiderocol (CFD) antimicrobial activity. Whole-genome sequencing was performed on KPC-Kp strains by Illumina iSeq100 platform. The Categorical Agreement (CA) between the BMD method and DD test was 92% (69/75) with a Major Error (ME) of 16.7% (6/36). Additionally, the CA between the BMD method and test strip was 90.7% (68/75) with a Very Major Error (VME) of 17.9% (7/39) and 82.7% (62/75) between the strip test and DD with a ME of 30.2%. KPC-Kp strains showing resistance to CFD were 27 out of 75 (36%) by three methods. Specifically, 51.9% (14/27) of KPC-Kp resistant to CFD harbored blaKPC-3, while 48.1% (13/27) harbored mutated blaKPC-3. Moreover, KPC-Kp strains carrying a mutated blaKPC-3 gene exhibited high MIC values  compared to wild-type blaKPC-3. In conclusion, the DD test resulted as a valid alternative to the BMD method to determine the in vitro susceptibility to CFD, while the strip test exhibited major limitations.The aim of this study was to compare the reference broth microdilution (BMD) method with the Disk Diffusion (DD) test and strip test against a collection of 75 well-characterized  Infections caused by carbapenem-resistant Enterobacteriales (CRE) organisms have become one of the greatest threats to public health over the past decade ,2. CRE-rIn order to overcome these limitations, cefiderocol (CFD), formerly S-649266, an intravenously infused siderophoric cephalosporin ,8, was aK. pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii [CFD exhibited structural stability against most β-lactamase resistance mechanisms of gram-negative bacteria ,10, sincaumannii  and showaumannii , oxacillaumannii ,10. HoweHence, the aim of this work is to compare the broth microdilution reference method (BMD) currently used to assess CFD antimicrobial activity against KPC-producing Enterobacteriales and disk diffusion (DD) test and strip test sensitivity methods, describing the degree of concordance  and errors  observed between the three different techniques analyzed.In this study, we selected 75 strains of KPC-producing K. pneumoniae (KPC-Kp) genotypically characterized by WGS.The phenotypic profiles of KPC-Kp strains showed that 32 out of 75 (42.6%) KPC-Kp strains included in this study were resistant to CAZ-AVI and/or MER-VAB and/or IMI-REL. In particular, 10 out of 32 (31.3%) KPC-Kp strains were resistant only to CAZ-AVI, while five out of 32 (15.6%) were resistant only to MER-VAB, and one (3.1%) KPC-Kp strain was resistant exclusively to IMI-REL. Deeper analysis of KPC-Kp strains resistant to novel βL-βLICs showed that 21.9% (7/32) exhibited cross-resistance to CAZ-AVI, MER-VAB and IMI-REL; 12.5% (4/32) showed cross-resistance to CAZ-AVI and MER-VAB; 12.5% (4/32) showed cross-resistance to MER-VAB and IMI-REL; and 3.1% (1/32) showed cross-resistance to CAZ-AVI and IMI-REL.blaKPC-3; 50% (16/32) harbored a mutated blaKPC-3 gene; and 3.1% (1/32) carried blaKPC-2. MLST analysis showed that the KPC-Kp strains included in this study that were resistant to CAZ-AVI and/or MER-VAB and/or IMI-REL belonged to different sequence types (ST) including ST101, ST258, ST307, ST512 and ST1519.Genotypic analysis revealed that 46.9% (15/32) of strains resistant to CAZ-AVI and/or MER-VAB and/or IMI-REL harbored According to the EUCAST interpretative criteria, antimicrobial susceptibility testing (AST) performed with the BMD method on the 75 selected KPC-Kp strains included in this study exhibited MICs ranging between 0.25 and >16 mg/L. KPC-Kp strains CFD-susceptible (MIC ≤ 2 mg/L) were 48% (36/75), while 52% (39/75) of KPC-Kp strains were resistant to CFD . ApplyinblaKPC-3 and exhibited a median MIC for CFD of 8 (IQR 4–8). Conversely, 13 out of 27 (48.1%) KPC-Kp resistant to CFD harbored a mutated blaKPC-3 gene and exhibited a median MIC for CFD equal to 32 (IQR 8–32).KPC-Kp strains showing resistance to CFD by the BMD, DD and strip test methods were 27 out of 75 (36%) with a median MIC for CFD equal to 8 (IQR 32–4). Interestingly, genetic analysis showed that 14 out of 27 (51.9%) KPC-Kp strains resistant to CFD harbored a wild-type n = 19) was the most represented Sequence Type followed by ST307 (n = 5), ST1519 (n = 2) and ST101 (n = 1). Comparing MIC values between mutated and wild-type blaKPC-3 showed that KPC-Kp strains carrying mutation within Ω-loop exhibited high MIC values for CFD . It is noteworthy that 12 out of 27 KPC-Kp strains resistant to CFD harbored a mutated blaKPC and showed cross-resistance to CAZ-AVI within a median MIC of 144 (IQR 112–256). Specifically, 58.3% (7/12) carried the blaKPC-31 variant. Furthermore, 15 out of 27 KPC-Kp strains displaying resistance to CFD were susceptible to CAZ-AVI within a median MIC of 4 (IQR 4–8). In particular, 14 out of 15 carried the wild-type blaKPC-3 gene, while one KPC-Kp strain carried the blaKPC-85 gene , the categorical agreement rises to 98.6%, and the ME drops to 2.8%, positioning the DD test as a notable alternative to the BMD method. However, particular attention must be placed on strains that fall inside the ATU range, which should be retested by the reference method, reported as resistant or ignored . MoreoveIn conclusion, our results demonstrated that the DD test is a valid alternative for determining in vitro susceptibility to CFD, since the BMD method has proved to be challenging for routine laboratory analysis, especially for those laboratories managing large numbers of patients. The ATU values can be used as an indication of uncertainty in the test procedure and therefore as an index of difficulty in interpreting results. In these cases, it may be useful to subsequently confirm uncertain MIC values with the reference method, in order to have reliable results.K. pneumoniae strains included in the study were isolated from patients admitted at IRCCS Policlinico di Sant’Orsola, a 1.420-bed university hospital with an average of 72,000 admissions per year. Bacterial strains were identified by MALDI-TOF MS assay . AST was performed using the MicroScan Walkaway system. MICs for novel βL-βLICs were confirmed with MIC test strips . Carbapenemase production was detected by NG-Test CARBA 5  and confirmed with molecular assay Xpert Carba-R . AST for cefiderocol was performed using broth microdilution with ID-CAMHB. DD and MIC test strips  were used on regular non-supplemented Mueller–Hinton agar . MIC results were interpreted following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints v12.0  [points/) .®100 binding resin . The broth was then filtered through a 0.22 μm pore size filter. CaCl2 (22.5 mg/L), MgCl2 (11.2 mg/L) and ZnSO4 (5.6 mg/L) were supplemented to the depleted broth. Cefiderocol powder  was dissolved in sterile saline solution (320 mg/L). Strains were inoculated at a concentration of 1.5 × 108 CFU/mL and incubated for 20 h at 35 °C [AST for CFD was performed removing iron from Muller–Hinton broth using Chelexat 35 °C ,18. MIC at 35 °C .K. pneumoniae using DNeasy Blood&Tissue Kit  by following the manufacturer’s instructions and further cleaned up with AMPure XP magnetic beads (Beckman Coulter). Whole genome analysis was performed as previously described [https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 10 January 2023). Genome assemblies were performed using with SPAdes v.3.10 with careful settings and were polished with Pilon v.1.23. Annotation was automatically carried out using the RAST server  and manually curated using Artemis v.17.0.1. Antimicrobial resistance genes and MLST analysis were assessed using the online platform . β-lactamase content was confirmed by using BLAST analysis against CARDB and Beta-Lactamase-Database .Genomic DNA was extracted from purified cultures of escribed . Briefly


Elraglusib (formerly 9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3β (GSK3β) undergoing clinical trials for the treatment of various cancers including non-Hodgkin lymphoma (NHL). The drug reduces proliferation of several NHL cell lines and has efficacy in xenograft models of the disease. To confirm the importance of its action on GSK3β, we treated 3 lymphoma cell lines with selective, structurally distinct GSK3 inhibitors: CT99021, SB216763, LY2090314, tideglusib, and elraglusib. Stabilization of β-catenin and reduced phosphorylation of CRMP2, two validated targets of GSK3, were used as functional read-outs for GSK3 inhibition. CT99021, SB216763, and LY2090314 failed to reduce proliferation or viability in any cell line at concentrations that stabilized β-catenin and reduced CRMP2 phosphorylation. There was partial reduction of CRMP2 phosphorylation but no significant effect on β-catenin at cytotoxic doses of elraglusib. There was no indication of GSK3 inhibition at doses of tideglusib that affected cell viability and apoptosis. Cell-free kinase screening confirmed several other targets of elraglusib, distinct from the GSK3 inhibitors with no anti-lymphoma actions, including PIM kinases and MST2. These data question GSK3 as the target of elraglusib in lymphoma, and hence the utility of GSK3 expression as a ‘stand-alone’, therapeutic biomarker in NHL.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01147-8. Despite improvements in outcomes, high-grade B- and T-cell non-Hodgkin lymphoma (NHL) represent a significant area of unmet therapeutic need, particularly in the context of relapsed or refractory disease , 2.Glycogen synthase kinase 3 (GSK3) is a ubiquitously expressed, serine/threonine kinase involved in diverse cellular functions . Two parA recently developed GSK3 inhibitor, elraglusib (formerly 9-ING-41), is particularly noteworthy for its anti-lymphoma effects in both B- and T-cell NHL lines and immunocompromised mouse patient-derived lymphoma xenograft models , 12.Elraglusib has now progressed to early-phase clinical trials for several malignant conditions, including lymphoma, with clinical efficacy reported in case studies of refractory adult T-cell leukemia/lymphoma , 14. TheDoxorubicin, SB216763, elraglusib (9-ING-41) and tideglusib were purchased from Cambridge Bioscience, LY2090314 from Stratech, CT99021 was synthesized as previously described . All comHS-Sultan cells were gifted from Dr Banerjee, University of Dundee. Karpas-299 cells were purchased from Public Health England and HH cells from American Type Culture Collection. Cells were grown in RPMI-1640 media containing glycine, 10% fetal bovine serum supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin .To measure changes in protein expression, cells in the exponential phase of growth were cultured in serum-free media overnight, prior to 4 h of drug treatment. For PARP (Poly [ADP-ribose] polymerase) cleavage assays, cells were cultured in serum-containing media for 24 h prior to protein extraction .Expression of β-catenin and phosphorylation of collapsin response-mediator protein 2 (CRMP2) were used as functional read-outs of GSK3 inhibition and PARP cleavage for apoptosis, all measured by immunoblotting. Rabbit anti-PARP (9542), rabbit anti-CRMP2 (9393) and rabbit anti-β-catenin (9562) were from Cell Signaling. Mouse anti-actin (A3853) was from Sigma-Aldrich. Sheep anti-phospho-CRMP2 (Thr509/514) was made in-house as described previously .p =  < 0.05 for statistical significance.Anti-rabbit (Licor Goat anti-Rabbit (926–32211)) and anti-mouse (Thermofisher Invitrogen Goat anti-Mouse (A21057)) secondary antibodies were used at 1:10,000. Rabbit anti-sheep HRP (horseradish peroxidase) secondary (ThermoFisher 31480) was used at 1:2500. All antibodies were diluted in 1% BSA in TBS-T. Cell lysates were prepared for SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) as previously described , and proThis was assessed using CellTiter 96® Aqueous One Solution Cell Proliferation Assay Kit (Promega) according to manufacturer’s instructions. In brief, 8000 cells/well were seeded into 96-well plates. Compounds were incubated with cells for 72 h prior to the addition of MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent for 2 h followed by absorbance assessment at 450 nM by Modulus Microplate reader. Analysis was performed using Graphpad Prism 9.33P cell-free screens were performed by the MRC Protein Phosphorylation Unit, University of Dundee as previously described [escribed .Elraglusib 1-5 µM) reduced cell viability and induced apoptosis in both B- and T-cell NHL cell lines in keeping with its reported anti-lymphoma properties  produced a weak reduction in cell viability and only induced apoptosis in one of three cell lines, while none of the other GSK3 inhibitors reduced cell viability or induced apoptosis at concentrations previously reported to result in GSK3 inhibition  at concentrations that generate changes in cell viability.TP53-mutant Burkitt lymphoma cell lines to anthracycline chemotherapy [Wu et al. were unable to generate GSK3β knockouts in four of five lymphoma cell lines, an intrinsic dependence upon GSK3β for lymphoma survival was suggested [These are important observations as GSK3 is increasingly recognized as a potential therapeutic target in hematological malignancy. Structurally distinct GSK3 inhibitors can induce apoptosis in patient-derived CLL cells, reduce proliferation in AML cell lines and re-sensitize otherapy –11. Sincuggested . These duggested . Howeveruggested –24. Simiuggested .In conclusion, we re-affirm the anti-lymphoma effects of elraglusib but argue that this action cannot be attributed to GSK3 inhibition. Hence, we caution against the assumption that selective GSK3 inhibitors will have clinical benefit in lymphoma and against GSK3 activity as a mechanistic biomarker.Additional file 1.


Correction: Arthritis Res Ther 25, 108 (2023)https://doi.org/10.1186/s13075-023-03085-9Following publication of the original article , the autThe author group has been updated above and the original article [1] has been corrected.


Clean glycopeptides were obtained by performing acetyl removalfrom the l-arabinose groups prior to deprotection of theneopentyl-protected sulfated tyrosine.Post-translationallymodified peptides are important regulatingmolecules for living organisms. Here, we report the stereoselectivetotal synthesis of β-1,2-linked  To achieve that, we applya previously reported glycosylation strategy that involves the useof mild activator iodonium dicollidine perchlorate (IDCP) in orderto stereoselectively couple the arabinose donor to Fmoc-Hyp-OBn.15It was envisioned that synthesis of the polyamidebackbone of PSY1would be accessible with Fmoc/l-arabinose 5 into its corresponding furanoside by Fischer glycosylation, whichthen underwent a thioglycosylation to form ethyl thioarabinofuranoside 6. Simultaneous protection of the 3- and 5-OH of furanoside 6 with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDS)was achieved, after which the 2-OH was protected using freshly prepared p-methoxybenzyl (PMB) bromide, to form intermediate 7. We found reported procedures that used commercially availablePMB bromide were ineffective.16 However,using freshly prepared PMB bromide (see ESI) for installation of the PMB moiety at C2–O was successful(52% yield). Lastly, the TIPDSprotecting group was removed and replaced with benzyl groups by treatmentof 7 with tetrabutylammonium fluoride followed by benzylationusing benzyl bromide and NaH to obtain arabinose donor 8. In this step, both l-α (8) and l-β (S4) arabinofuranoside were obtained. Specifically, l-α-arabinofuranoside 8 was identified bya doublet of the anomeric proton at 5.31 ppm with a J-coupling constant of 2.38 Hz, whereas the anomeric proton of l-β arabinofuranoside S4 was found at 5.35ppm with a J-coupling constant of 4.94 Hz.17 Formation of both anomers most probably originatedfrom a yet not understood furanose-ring mutarotation process in whichthe thioethyl group inverts.18 Importantly,both enantiomers led to the same 1,2-cis-linked productwhen applied in the next step (vide infra).Synthesis of β-1,2-linked triarabinosylatedPSY1 startedfrom the generation of the arabinose donor 1a and th10 wasobtained from commercially availableFmoc-Hyp(tBu)–OH 9 that was firstbenzylated on the carboxylic acid moiety and subsequently subjectedto tert-butyl removal using TFA, providing the secondaryalcohol.Fmoc-Hyp-OBn 8 and hydroxyprolineacceptor 10 in hand, stereoselective 1,2-cis glycosylationwas performed to monoarabinosylate hydroxyproline  led to the transientmixed acetal intermediate. Subsequently, freshly prepared IDCP wasused to activate the IAD mechanism to afford β-linked hydroxyprolinemonoarabinofuranoside 11. The resulting free 2-OH of 11 was reacted with arabinose donor 8 using DDQto form the transient mixed acetal, which was converted into the β-1,2-linkedhydroxyproline diarabinofuranoside 12 by IDCP-initiatedIAD. Similarly, treatment of 12 with arabinose donor 8 and the aforementioned reagents resulted in β-1,2-linkedhydroxyproline triarabinofuranoside 13.As such, treatmentof donor 11–13 were debenzylated in order to liberate carboxylicacid for peptide coupling, and to exchange the glycosyl O-benzyl groups with O-acetyl groups.15 Consequently, Fmoc-Hyp[Ara(OBn)]1-OBn 11, Fmoc-Hyp[Ara(OBn)]2-OBn 12, and Fmoc-Hyp[Ara(OBn)]3-OBn 13 were subjectedto hydrogenolysis using Pd(OH2)/C and H2 gas.15 Interestingly, striking differences in debenzylationrates were observed for the different monomers. Whereas debenzylationof Ara1Hyp 11 required multiple Pd(OH)2/C refreshing and extended reaction times (48 h), the di-and triarabinosylated building blocks did not require refreshing ofPd(OH)2/C and were completed overnight. Also, whereas debenzylationof 11 and 13 was accompanied by significantFmoc-removal, this was much less the case for 12.Prior totheir application in SPPS, the various arabinosylatedhydroxyproline building blocks that were collected along the previouslydescribed synthesis path were converted into Fmoc-protected peracetylatedamino acid constructs. For this, the three AraHyp moieties 1–OH 14, Fmoc-Hyp[Ara(OAc)]2–OH 15, and Fmoc-Hyp[Ara(OAc)]3–OH 16, were obtained in yields of 66%, 59%,and 58%, respectively. Flash column chromatography purification ofthe arabinosylated building blocks resulted in higher yields of purecompound, when compared to previous reported procedure involving reverse-phaseHPLC purification.15Fortunately, removal of the fluorenylmethyloxycarbonyl (Fmoc) protectinggroup from the secondary amine could be remedied by treatment of theproduct with Fmoc-OSu in the presence of sodium bicarbonate in orderto protect the amino functionality again. After protection of theglycosyl OH groups with acetyl groups using acetic anhydride and pyridine,the desired mono-, di-, and triarabinosylated hydroxyproline buildingblocks Fmoc-Hyp[Ara(OAc)]Brassica PSY1 peptidewas commenced (tBu)–OH.For coupling of the arabinosylated hydroxyproline  monomers we used the couplingreagents 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate(HBTU) and 1-hydroxy-benzotriazole (HOBt). Introduction of arabinosylatedFmoc-Hyp 14–16 to the dipeptide wasmonitored by ninhydrin/chloranil tests and high-resolution mass spectrometry(HRMS) after cleavage of small aliquots of resins. As we used a slightexcess of resin-bound amine groups with respect to the arabinosylatedbuilding blocks, remaining amine-groups were permanently acetylatedusing Ac2O and pyridine. After this, Fmoc-Val-OH couplingwas conducted manually in order to monitor coupling of this beta-substitutedamino acid to the different sterically hindered glycosylated tripeptides.Once formation of tetrapeptides 17–19 was confirmed, subsequent chain elongation was conducted using anautomated peptide synthesizer. After acidic cleavage from the resin,the crude (glyco)peptides were purified using reversed-phase preparative-HPLC,yielding analytically pure Np- and Ac-protected (glyco)peptides 20–23 (see ESI).With these Fmoc/Ac-protectedarabinosylated hydroxyproline buildingblocks in hand, SPPS of the ommenced 3. Starti3Np) and acetyl groups at the 16-Hyp[Ara(OAc)]0–3 (21–23). For thenonarabinosylated control peptide PSY1 16-Hyp 20 onlyNp removal in 2 M ammonium acetate solution fulfills the completesynthesis of this peptide resulting in Brassica PSY116-Hyp 1. However, for glycopeptides 21–23 the order of deprotection was found important. Specifically,starting with Np removal from acetylated glycopeptide 21 using a 2 M ammonium acetate solution resulted in multiple peaksin liquid chromatography trace, only one of which corresponded tothe target compound (24–26) and subsequent removal of the tyrosine sulfateNp protecting group with 2 M ammonium acetate afforded the desiredPSY1 peptides 16-Ara1Hyp 2 .Once the octadeca-(glyco)peptideswere prepared, we focused onremoval of the two acid-stable protecting groups, namely, neopentyl(Np) at the 2-Tyr using a PMB ether on the C2hydroxyl group. This resulted in three Fmoc-protected building blocksthat are compatible with Fmoc-based solid-phase peptide synthesis(SPPS) using HBTU/HOBt as coupling reagents for the introduction ofFmoc-Hyp[(l-Ara)]1–3–OH 14–16 and Fmoc-Hyp(tBu)–OH 9. Elongation to the PSY1 (glyco)peptide to form derivatives 20–23 was done using an automated peptidesynthesizer. Target peptides were obtained by initial removal of acetylgroups and subsequent Np removal. With this, we disclose a robustsynthesis approach for the preparation of complex post-translationallymodified peptides, especially those containing a sulfated tyrosineand arabinosylated hydroxyproline units, that will be useful for thepreparation of peptides for phenotypical biological evaluation, suchas their role in intercellular signaling.To conclude,


Introduction: UNC-6/Netrin is a conserved bi-functional guidance cue which regulates dorsal-ventral axon guidance in C. elegans. In the Polarity/Protrusion model of UNC-6/Netrin mediated dorsal growth away from UNC-6/Netrin, The UNC-5 receptor first polarizes the VD growth cone such that filopodial protrusions are biased dorsally. Based on this polarity, the UNC-40/DCC receptor stimulates growth cone lamellipodial and filopodial protrusion dorsally. The UNC-5 receptor maintains dorsal polarity of protrusion, and inhibits growth cone protrusion ventrally, resulting in net dorsal growth cone advance.Methods: Growth cone imaging in mutants, combined with Cas9 genome editing and genetic analysis, were used to analyze the role of a novel short isoform on unc-5 in growth cone polarity and protrusion.Results: Work presented here demonstrates a novel role of a previously undescribed, conserved short isoform of UNC-5 (UNC-5B). UNC-5B lacks the cytoplasmic domains of UNC-5 long, including the DEATH domain, the UPA/DB domain, and most of the ZU5 domain. Mutations that specifically affect only the unc-5 long isoforms were hypomorphic, suggesting a role of unc-5B short. A mutation specifically affecting unc-5B caused loss of dorsal polarity of protrusion and reduced growth cone filopodial protrusion, the opposite of unc-5 long mutations. Transgenic expression of unc-5B partially rescued unc-5 axon guidance defects, and resulted in large growth cones. Tyrosine 482 (Y482) in the cytoplasmic juxtamembrane region has been shown to be important for UNC-5 function, and is present in both UNC-5 long and UNC-5B short. Results reported here show that Y482 is required for the function of UNC-5 long and for some functions of UNC-5B short. Finally, genetic interactions with unc-40 and unc-6 suggest that UNC-5B short acts in parallel to UNC-6/Netrin to ensure robust growth cone lamellipodial protrusion.Discussion: These results demonstrate a previously-undescribed role for the UNC-5B short isoform, which is required for dorsal polarity of growth cone filopodial protrusion and to stimulate growth cone protrusion, in contrast to the previously-described role of UNC-5 long in inhibiting growth cone protrusion. Caenorhabditis elegans, UNC-6/Netrin is a conserved, extracellular laminin-like guidance cue, and is expressed in ventral cells and axonal processes   . In any The 19 GABAergic DD and VD motor neuron cell bodies reside along the ventral nerve cord  [from G]. The VDC. elegans VD growth cones during their outgrowth in UNC-6/Netrin signaling mutants led to the Polarity/Protrusion model of growth cone migration away from UNC-6  from being delivered to the growth cone plasma membrane.Previous studies implicate three distinct pathways utilized by UNC-5 to inhibit growth cone protrusion. The FMO flavin monooxygenases act downstream of UNC-5 to inhibit protrusion , possiblunc-5 locus is predicted to encode for multiple isoforms (The isoforms .unc-5B) differs at the 3’ end of unc-5 gene, and is predicted to encode a molecule lacking most of the cytoplasmic domains, including the DEATH domain, the UPA/DB domain, and most of the ZU5 domain. ZU5 domains interact with several molecules including ankyrin. The ZU5 domain in Unc5A binds to the melanoma-associated antigen D1 (MAGE-D1/NRAGE) and participates in apoptosis (C. elegans (unc-5 isoforms (unc-5 strong loss-of-function mutants)  and Thrombospondin (TSP) repeats. One isoform  .unc-5B short isoform in axon guidance and growth cone morphology. unc-5 mutant alleles that affect only the long isoforms and not the short isoform were found to be hypomorphic for unc-5 function compared to unc-5 strong loss-of-function mutants: they displayed less severely uncoordinated locomotion and weaker VD/DD axon guidance defects. However, growth cone polarity and protrusion largely resembled strong unc-5 mutants, with loss of dorsal polarization of filopodial protrusion and a larger growth cone area and longer filopodia compared to wild-type. Using CRISPR/Cas9 genome editing, an unc-5 allele was generated that was predicted to affect only the unc-5B short isoform. This unc-5B mutant displayed weakly uncoordinated locomotion and weak VD/DD axon defects. unc-5B mutant growth cones lacked dorsal polarity of filopodial protrusion, indicating that UNC-5B is required for VD growth cone polarity. unc-5B mutant growth cones were also smaller and less protrusive than wild-type, indicating a possible pro-protrusive role for UNC-5B. unc-5B failed to complement a strong loss-of-function unc-5 mutant for these phenotypes, but complemented unc-5 hypomorphic mutants, consistent with the idea that unc-5B short encodes a distinct genetic function unc-5 long isoforms. Transgenic expression of unc-5B rescued uncoordinated locomotion and axon defects of unc-5 mutants, and perturbed VD growth cone polarity and protrusion consistent with a pro-protrusive role.Work reported here is an analysis of the role of the unc-5B short isoform in VD/DD axon guidance that is genetically separable from the function of the unc-5 long isoforms. Both are required for VD growth cone dorsal polarity of protrusion, UNC-5 long isoforms are required to inhibit growth cone protrusion, and UNC-5B short isoform might have a pro-protrusive role. Vertebrate genomes encode four Unc5 genes, A, B, C, and D, with Unc5A having a predominant role in axon guidance   (unc-5B. These results indicate that unc-5B constituted 3.6% (9/251) of unc-5 transcripts in mixed-stage animals.The e WS245) . Six of d 3′ end . The sho intron) . The ope intron) , resulti intron) . NeighboThe UNC-5A long isoform encodes a molecule with two extracellular immunoglobulin (Ig) domains, two extracellular thrombospondin type I repeats (TSPI), a transmembrane domain, and cytoplasmic zona occludens I/UNC-5 domain (ZU5), UNC- 5/PIDD/Ankyrin (UPA), and DEATH domains . A tyrosunc5 genes, A-D. The human Unc5A locus encodes a truncated isoform similar to C. elegans UNC-5B, called Unc5A X4  .Vertebrates, including humans, have four 32989.1) . Unc5A X32989.1) . unc5A Xunc-5 isoforms A, C, D, E and F are collectively referred to as unc-5 long isoforms, and unc-5B the short isoform. The unc-5 alleles e791, e53, e553, and e152 introduce premature stop codons that affect all unc-5 isoforms  phenotype  from the end of the predicted unc-5B reading frame. This is downstream of the transmembrane domain and Y482. Any truncated molecule made by translation of e152 would include both  displayed growth cones that were excessively protrusive compared to wild-type, with larger growth cone area and longer filopodial protrusions (unc-5(e53) (unc-5(e791) showed a similar loss of filopodial dorsal polarity (unc-5(e152) hypomorphic allele also displayed increased growth cone area and filopodial length, and loss of dorsal bias of filopodial protrusion (unc-5(ev480) hypomorphic mutant  . Strong polarity  and excepolarity  and incrpolarity . The uncotrusion    . PreviouMethods) . MicrotuMethods) . UNC-5 wMethods) . These sunc-5(e152) and unc-5(ev480) also displayed loss of dorsal F-actin accumulation  was produced by precise removal of intron 7, which is predicted to preclude the formation of the unc-5B short isoform by fusing together exons seven and eight  mutants displayed wild-type locomotion and weak but significant total axon defects (3%) including axon wandering (unc- 5B(lq190) did not significantly affect VD growth cone area (unc-5B(lq190)  . MicrotuB(lq190) .unc-5B(lq190) was due to the unc-5 genome edit and not to a background mutation, we analyzed trans-heterozygotes of unc-5B(lq190) and the strong loss-of-function allele unc-5(tmIs1241) found on the balancer chromosome tmC25 (. tmIs1241 is an insertion of a myo- 2::gfp transgene in the unc-5 exon seven shared by long and short isoforms (unc-5(e791) (data not shown). Thus, unc-5(tmIs1241) a strong loss-of-function unc-5 mutant. VD growth cones of lq190/tmC25 trans-heterozygotes resembled unc- 5B(lq190) alone, with significantly reduced filopodial length compared to wild-type and loss of dorsal polarity of filopodial protrusion (unc- 5(tm1241) failed to complement unc-5B(lq190) as expected.To ensure that the phenotype observed in me tmC25 . tmIs124isoforms , and hasotrusion . This inlq190 trans-heterozygote with the hypomorphic ev480 allele displayed filopodial length similar to wild-type and dorsally-polarized filopodial protrusion (unc-5(ev480) complemented unc-5B(lq190), consistent with the idea that these isoforms encode distinct functions. In sum, these results indicate that unc-5 long and unc-5B short encode genetically distinct and separable functions. unc-5B short isoform is required for dorsal polarity of filopodial protrusion, as well as robust filopodial protrusion, suggesting a pro-protrusive role.An otrusion . Thus, uunc-5::gfp minigene is composed of the unc-5 promoter driving the unc-5A cDNA fused to gfp  short isoform having a distinct role from the long isoforms, which inhibit protrusion.A MYR::UNC-5B short isoform transgene was constructed with the 3’ end of unc- 5B . Expresspolarity . This isunc-40(n324) null mutant displayed a slight but not significant reduction in growth cone area, and significantly shorter filopodial protrusions (unc-40(n324) did not significantly affect dorsal polarity of filopodial protrusions  (unc-5(lq190); unc-40(n324) double mutant growth cones resembled the additive effect of each mutant alone: slight but not significant reduction in growth cone area; significantly reduced filopodial length, and loss of dorsal polarity of filopodial protrusion (unc-5B(lq190) synergistically increased unc-40(n324) VD/DD axon guidance defects  . unc-5 strong loss-of-function resulted in unpolarized growth cones with no significant effect on growth cone and filopodial protrusion (unc-6(ev400); unc-5(lq190) growth cones showed significantly reduced growth cone area compared to either single mutant alone. Filopodial length resembled the short filopodia of unc- 5(lq190) alone. This suggests that unc-6 and unc-5B short might act in parallel to promote growth cone protrusion and is consistent with a pro-protrusive role of unc-5B short isoform.otrusion . unc-6 did not significantly reduce increased growth cone area or filopodial length in unc-5(ev480) hypomorphic mutant (unc-5(ev480) hypomorph is not solely due to overactivity of UNC-40. Possibly, the UNC-5B short isoform has pro-protrusive activity that is increased in the absence of the long isoforms. This is consistent with unc-5B(lq190) short isoform mutants having reduced filopodial length.otrusion . unc-40(c mutant . This suunc-40 mutation suppressed the VD/DD axon guidance defects of unc-5(e53) strong loss-of-function and of unc-5(e152) hypomorphic mutants (unc-5(ev480) hypomorph was not suppressed by unc-40(n324) and in fact showed synergistic enhancement for failure of lateral midline crossing and total axon guidance defects (unc-5(ev480) hypomorphic mutants driving excess protrusion, possibly involving UNC-5B.Previous results showed that  mutants . In cont defects . This isunc-5(e791) mutants  mutants  mutants  VD/DD axon guidance defects by unc- 5A(Y482F) was significantly reduced compared to unc-5A(+) (Tyrosine 482 was previously shown to be required for the ability of an  mutants . Tyrosinnc-5A(+) .unc-5A(Y482F) caused a significant increase in growth cone area and filopodial length similar to unc-5(e791) mutants (unc-5(e791) mutants  failed to rescue VD/DD axon guidance defects in unc-5 mutants, indicating that Y482 is important in UNC-5B function  caused severely uncoordinated locomotion and severe VD/DD axon guidance defects (unc-5 strong loss-of-function). Very few axons were observed emanating from the ventral nerve cord, and those that did failed to extend dorsally past the lateral midline   . Howeverunc-5B short isoform was generated by CRISPR/Cas9 genome editing. The unc-5B 3’ end results from failure to splice intron 7 from the unc-5 transcript, resulting in a stop codon after 16 additional amino acid coding codons (unc-5B reads in intron 7 (unc-5B(lq190) was a precise removal of intron 7, resulting in the fusion of exons 7 and 8, leaving unc-5 long isoform coding potential unchanged. unc-5b(lq190) mutants were only slightly uncoordinated with weak VD/DD axon guidance defects  failed to complement an unc-5 strong loss-of-function mutation for these phenotypes, but did complement an unc-5 hypomorphic long isoform-specific mutant  resulted in unpolarized F-actin, but did not affect microtubule + end abundance  double mutants with unc-6 had severely reduced VD growth cone area compared to wild-type and single mutants alone, and transgenic expression of unc-5B resulted in significantly increased growth cone area compared to wild type  does not rescue the uncoordinated locomotion or VD/DD axon defects of unc- 5 mutants as well as the wild-type transgene (unc-5 mutants (Previous studies showed that tyrosine 482 (Y482) is phosphorylated and that it is required for the function of UNC-5 in axon guidance  expression alone still resulted in increased growth cone area and loss of filopodial protrusion. Thus, in UNC-5B short, Y482 is required for some functions but not others. While it is unclear which tyrosine kinase might phosphorylate Y482, the SH2 domain of SRC-1 binds to phosphorylated Y482, SRC-1 is required for robust UNC-5 tyrosine phosphorylation, and SRC-1 functions with UNC-5 in gonadal distal tip cell migration and axon guidance ; unc-40 displayed an additive phenotype, and no significant genetic interaction. This suggests that UNC-5B short might act independently of UNC-40, or in the same pathway. UNC- 5B short is lacking the cytoplasmic UPA/DB domain that mediates physical interaction with the UNC-40 P1 cytoplasmic domain. This suggests that UNC-5B short might act independently of UNC-40.These results indicate that the UNC-5B short isoform is required for VD growth cone dorsal polarity of filopodial protrusion and for robust growth cone lamellipodial and filopodial protrusion. This is in contrast to the UNC-5A long isoform, which inhibits growth cone protrusion. Double mutants of unc-5B(lq190); unc-6 double mutants displayed growth cones that were smaller than wild-type and either single mutant alone. This suggests that UNC-5B short might act in parallel to UNC-6 to promote growth cone lamellipodial protrusion. In the Polarity/Protrusion model, UNC-6 acts through UNC-40 to stimulate growth cone protrusion. However, this effect is independent of UNC-40 as unc-5B(lq190); unc-40 double mutants did not display small growth cones similar to unc-5B(lq190); unc-6 mutants. This suggests that an unidentified ligand might interact with UNC-5B short to stimulate protrusion in parallel to UNC-6, and that an unidentified receptor for UNC-6 might act in parallel to UNC-5B short ], lqIs387 [Punc-5::unc-5B(Y482F)], lqIs326 [Punc-5::myr::unc- 5A], lqIs315 [Punc-5::myr::unc-5B].Experiments were performed at 20°C using standard chniques . The prePunc-5::unc-5B short isoform transgene was constructed by removing the coding sequence of exons eight and nine, and replacing with the first 45 bases of intron 7 .  as previously described   . Resultin 2.1.0)   . Sequencunc-5(lq190) was generated by CRISPR/Cas9 genome editing which precisely deleted the entire intron 7 of unc-5A, between exon 7 and 8  co-CRISPR reagents . Deletion of unc-5 intron 7 was confirmed by PCR and sequencing and outcrossed to N2 three times. Genome editing reagents were produced by In Vivo Biosystems : 7 and 8 . This resgRNA1: 5′ CTCTCCCCAGTCATCGTCAT 3’sgRNA2: 5′ TAACAGATCAACCTCACCTC 3’lq190 mutation): 5′GAAAATGCTTTATCTGGCTGTTTCGGACACCTTAACCGACCAGCCACATCTTAAGCCAATTGAGTCTGCCCTTTCTCCTGTTATTGTCATCGGACAATGTGATGTTTCAATGTCAGCTCATG3’ (recoded exon sequence is underlined).ssODN repair template , with an ⍺ false positive value of 0.05.Proportional data were analyzed using Fisher’s Exact test. Continuous data were analyzed with Student’s Punc-25::gfp transgene, juIs76 , which is expressed in 13 VD and 6DD GABAergic motor neurons. Axon guidance defects were scored as previously described  is much lower than 0.2. Growth cones were imaged with Qimaging Retiga EXi camera on a Leica DM5500 microscope at ×100 magnification. Projections less than 0.5 um in width were scored as filopodia. Growth cone area and filopodial length were quantified using ImageJ software as previously described (t-test with unequal variance.Growth cones of VD axons were imaged as previously described . Late L1escribed . SignifiPolarity of growth filopodial protrusions was determined as previously described . Growth t-test with unequal variance on these average normalized ratios of multiple growth cones of each genotype.Growth cone F-actin was analyzed as previously described , using tt-test with unequal variance.Growth cone EBP-2::GFP puncta  was anal


A 45-year-old male patient presented a 2-week history of progressive gait imbalance. He also presented impaired proprioception, symmetric distal quadriparesis (grade 4/5), gait ataxia with a positive Romberg sign, bilateral upper limb dysmetria, and dysdiadochokinesia. The patient did not present nystagmus and/or dysarthria. A cervical spine magnetic resonance imaging (MRI) scan revealed severe degenerative disk disease and compressive spondylotic myelopathy at C3-C4 and C5-C6 . He was


The live oral rotavirus RV1 (Rotarix) vaccine is formulated from the human G1P[8] RIX4414 virus. Based on RIX4414 sequences, T7 expression plasmids were constructed that supported recovery of recombinant RIX4414-like viruses by reverse genetics. These plasmids will advance the study of the RV1 vaccine, possibly allowing improvements to its efficacy. RV1 , the most widely used rotavirus vaccine, is formulated from the human G1P[8] virus RIX4414 . To protEcoRV site of pUC-GW-Amp plasmids (Azenta).The rotavirus genome consists of 11 segments of double-stranded RNA . The rot5 MA104 cells. Three days later, the BHK-T7/MA104 cells was overseeded with 105 Vero cells. Eight days later, viruses in the cell lysates were amplified on Vero cells and plaque isolated on MA104 cells (The pT7/RIX4414 plasmids supported recovery of rRIX4414-like virus using a modified reverse genetics procedure , 12. Bri04 cells . The RNA04 cells , making