Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N-and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and ␤-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated ␣2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of ␣2-6 linked sialic acid on N-glycans. The lower ␣2-6 sialylation was caused by a decrease in activity of ␤-galactoside ␣2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting

BackgroundMutations in the gene encoding stimulator of interferon genes (STING) underlie a type I interferon (IFN) associated disease, STING-associated vasculopathy with onset in infancy (SAVI). Patients suffer cutaneous vasculopathy and interstitial lung disease, but are not known to suffer life-threatening infection.CaseWe describe a child who presented with Pneumocystis jirovecii pneumonia in early life, from which he recovered. He went on to suffer failure to thrive, developmental delay, livedo reticularis, and vesicular rash, but without cutaneous vasculitis, and with normal C-reactive protein and erythrocyte sedimentation rates. At 3 years of age, he developed life-threatening pulmonary hypertension.MethodsWhole genome sequencing (WGS) was performed using the Illumina HiSeqX10 platform and the Seave platform was used for bioinformatic analysis. mRNA expression of IFN-stimulated genes and inflammatory cytokines from peripheral blood mononuclear cells was determined by quantitative polymerase chain reaction. Luciferase assay was used to model IFNβ and NF-κB activity in vitro.ResultsWGS revealed a de novo mutation p.Arg284Ser in STING at an amino acid previously associated with SAVI. Although this mutation did not fall in the dimerization domain (DD), mRNA analysis revealed constitutive IFN-gene activation consistent with an interferonopathy, which correlated to STING activation in vitro. The patient was treated with corticosteroids and the JAK inhibitor Ruxolitinib, resulting in a rapid improvement of pulmonary hypertension, general well-being, and resolution of the IFN gene signature. However, he did go on to evolve a nasal septal erosion suggesting incomplete control of disease.ConclusionThis case provides molecular evidence to support the p.Arg284Ser variant in STING exerting pathogenicity through a gain-of-function mechanism. The lack of cutaneous vasculitis or elevated systemic inflammatory markers, and the occurrence of an opportunistic infection are notable, and raise the possibility that variants outside the STING DD may potentially manifest with an atypical SAVI phenotype. Nevertheless, there was an objective clinical improvement in response to JAK inhibition.

Urokinase plasminogen activator (uPA) and its high affinity receptor (uPAR) play crucial proteolytic and non-proteolytic roles in cancer metastasis. In addition to promoting plasmin-mediated degradation of extracellular matrix barriers, cell surface engagement of uPA through uPAR binding results in the activation of a suite of diverse cellular signal transduction pathways. Because uPAR is bound to the plasma membrane through a glycosyl-phosphatidylinositol anchor, these signalling sequelae are thought to occur through the formation of multi-protein cell surface complexes involving uPAR. To further characterize uPAR-driven protein complexes, we co-immunoprecipitated uPAR from the human ovarian cancer cell line, OVCA 429, and employed sensitive proteomic methods to identify the uPAR-associated proteins. Using this strategy, we identified several known, as well as numerous novel, uPAR associating proteins, including the epithelial restricted integrin, alphavbeta6. Reverse immunoprecipitation using anti-beta6 integrin subunit monoclonal antibodies confirmed the co-purification of this protein with uPAR. Inhibition of uPAR and/or beta6 integrin subunit using neutralizing antibodies resulted in the inhibition of uPA-mediated ERK 1/2 phosphorylation and subsequent cell proliferation. These data suggest that the association of beta6 integrin (and possibly other lynchpin cancer regulatory proteins) with uPAR may be crucial in co-transmitting uPA signals that induce cell proliferation. Our findings support the notion that uPAR behaves as a lynchpin in promoting tumorigenesis by forming functionally active multiprotein complexes.