Summary of PK data for EZH2 inhibitors used in KARPAS-422 and HT1376 xeograft mmouse models

Studies on glycans of glycoproteins are hampered by the lack of standards that reflect the wide diversity in structure typically observed. To this end we have exploited a large library of N-glycan standards comprised of a unique collection of 226 N-glycans including oligomannose, hybrid, and complex-type. We generated a method employing porous graphitized carbon (PGC) and liquid chromatography mass spectrometry (PGC-LC-MS), which can provide a high degree of resolution of underivatized N-glycan structures. Chromatogram libraries arising from these studies include retention time data, diagnostic fragments, and validated structural assignments, providing a robust platform for both targeted and discovery-based glycomics. We refer to this as an N-glycopedia, the first type of resource in which researchers can compare this collective data to N-glycans under study and overcome the limitations of only having compositional data and predicted structures. The technology is easily expandable to include additional N-glycans as new standards become available.

Tulmimetostat increases PRC2-repressed gene expression in ARID1A mutant bladder cancer cells. (

In cancer patients tulmimetostat plasma exposure levels correlate with PRC2 target gene expression changes in peripheral blood.

Table with cell line information including source and RRID

List of 365 genes (used in main Fig. 7F) that are induced in patient’s whole blood upon treatment with tulmimetostat

Tulmimetostat treatment results in greater PRC2 target gene expression induction in phenotypically sensitive ARID1A LOF cell lines

List of gene names corresponding to 132 tulmimetostat upregulated genes in responder versus non responder cell lines

Tulmimetostat mediated cancer cell phenotypic responses are increased in ARID1A LOF contexts.

Tulmimetostat improves cisplatin responsiveness in chemotherapy-resistant bladder cancer cells.

Synthetic pathway and characterization of tulmimetostat, a long residence time EZH2 inhibitor.

Tulmimetostat demonstrates superior level of tumor PRC2 target gene inducon and correlation with efficacy in a bladder cancer xenograft mouse model.

Table listing experimentally determined compound binding affinities for EZH2 and EZH1 containing PRC2 protein complexes

Additional method information to describe technical aspects of the experiments described in this manuscript

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), a leading cause of death by an infectious disease globally, has no efficacious vaccine. Antibodies are implicated in M. tuberculosis control, but the mechanisms of action remain poorly understood. We assembled a library of monoclonal antibodies (mAb) and screened for M. tuberculosis-restrictive activity in mice, identifying protective antibodies targeting diverse antigens. To dissect the mechanism of mAb-mediated M. tuberculosis restriction, we optimized a protective lipoarabinomannan-specific mAb, generating Fc variants. In vivo analysis of these Fc variants revealed a role for Fc-effector function in M. tuberculosis restriction. Restrictive Fc variants altered distribution of M. tuberculosis across innate immune cells. Single-cell transcriptomics highlighted distinctly activated pathways within innate immune cell subpopulations, identifying early activation of neutrophils as a key signature of mAb-mediated M. tuberculosis restriction. Therefore, antibody-mediated restriction of M. tuberculosis is associated with reorganization of the tissue-level immune response to infection and depends on the collaboration of antibody Fab and Fc.

Background:Immunoglobulin G (IgG) plays a critical role in immune defense yet our understanding of its role in cardiovascular disease (CVD) is evolving. Observational studies have correlated statin use with changes in IgG N-glycan structures. However, statin effects on IgG N-glycan changes have not been tested in randomized controlled trials, and their direct association with CVD remains unclear. Methods:IgG N-glycans were measured at baseline and after one year of randomized high-intensity statin interventions in 2 sub-studies of randomized trials: JUPITER (Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin; NCT00239681; primary prevention; discovery, n = 239 participants); and TNT (Treating to New Targets; NCT00327691; secondary prevention; validation, n = 711). Using linear regression adjusted for baseline levels of IgG N-glycans and clinical risk factors (e.g., age, sex) as well as the occurrence of CVD during the year of follow-up, we investigated the one-year randomized effects of high-intensity rosuvastatin v. placebo on IgG N-glycans in JUPITER. Significant statin-IgG N-glycan associations were then validated in TNT with one-year randomized effects of high- v. low-intensity atorvastatin intervention. We examined the architecture of IgG N-glycan connectivity at baseline using a data-driven Bayesian network and compared it with the architecture after one year of randomized statin intervention. We then investigated whether the changes in IgG N-glycans triggered by statins were associated with incident CVD events. Results:We identified 5 IgG N-glycans (corresponding to core fucosylated, monosialylated, and disialylated IgG N-glycans) in JUPITER whose levels decreased significantly with statin versus placebo (false discovery rate < 0.05), with an approximate 11.3-25.9% reduction in the individual IgG N-glycan levels. Four out of the five IgG N-glycans altered by statin were validated in TNT. Furthermore, monosialylation and core fucosylation (glycan peaks, GP 16 and 18) were inversely associated with CVD in JUPITER (OR = 0.87 and 0.73 per standard deviation increase, 95% CI: (0.57, 0.98) and (0.55, 0.96) respectively), and validated in TNT. Despite the effect of statin therapy on certain IgG N-glycans, the overall architecture of the IgG N-glycan network remained unchanged after one year of statin intervention. Conclusion:High-intensity statin interventions decreased several specific IgG N-glycan levels without changing the overall architecture of IgG N-glycan connectivity. Two IgG N-glycans that were decreased by statins were inversely associated with CVD outcomes, suggesting that statins have effects on monosialylated and core fucosylated IgG N-glycans, which may affect their cardioprotective properties. These findings highlight a potential immunomodulatory role of statins through IgG N-glycan alterations that should be further investigated in relation to CVD.

Synthesis of glycopeptide libraries is often limited by the cost and availability of the glycoamino acids. To circumvent this, we have devised a method of using SPOT synthesis for production of such libraries using cellulose filter paper as the solid support. We termed this method Glyco-SPOT synthesis, and it can be used to produce libraries of glycopeptides in microgram quantities in a parallel manner using microgram amounts of glycoamino acids. This chapter provides details on the method to perform this synthesis process.

A major challenge in the glycosciences is the scarcity of sensitive and specific glycan-binding reagents, such as monoclonal antibodies, for detecting and isolating glycans. Here we report the development and characterization of new monoclonal antibodies (mAbs) that bind carbohydrate-based red blood cell (RBC) antigens including the ABO(H) antigens. This approach exploits the immune system of the sea lamprey (Petromyzon marinus), which strongly responds to human glycans to enable the generation of high affinity antibodies. To develop these mAbs, we immunized the lamprey with RBCs and designed a targeted antibody enrichment and screening process using intact RBCs and a custom microarray displaying blood group antigens. Through multiple rounds of enrichment and testing we identified two mAbs; A_25 and A_39. Glycan binding analysis of the mAbs using glycan microarrays, the Luminex platform and western blot analysis revealed their binding to H antigens and terminal N-acetyllactosamine Galβ1-4GlcNAc (LacNAc, a type 2 sequence). Mechanistic insights into antigen specificity were gained through glycan inhibition assays, sequence homology analysis, and nanomolar-range affinity measurements. mAb binding to RBCs was determined using flow cytometry. Both mAbs bound RBCs of all ABO blood groups, whereas strongest binding was observed for blood group O RBCs. Our findings highlight the efficacy of the lamprey system to develop glycan-specific mAbs. These reagents allow investigation of expression of the H antigen and LacNAc-containing glycans in human tissues. In the future, they could also be modified using molecular engineering techniques to generate mAbs specific to other understudied blood group antigens.

The H antigen (O blood group), the precursor to A and B blood groups, is expressed on human erythrocytes and other cells (e.g., endothelial cells). Remarkably, the specific expression of the H antigens is uncertain due to the lack of specific reagents for its detection. Here, we describe two monoclonal antibodies (mAbs), Tn4-31L and OmcFL3-02, generated by immunizing sea lampreys with human cells. Both mAbs are highly specific in comparison with the lectin Ulex europaeus agglutinin-I (UEA-I). We identified expression of H antigens in mammalian glycoproteins, human cells, and normal and malignant human tissues, discovering that different types of human carcinomas exhibit unexpected H antigen expression. Interestingly, H antigen detection in many cases was distinctly elevated by enzymatic desialylation, as well as being elevated in cells engineered to lack sialic acid. These highly specific mAbs will be valuable reagents in blood testing and for exploring the expression and function of H antigens.