Schuerger, C. | Search bioRxiv

bioRxiv2026

Urothelial-lineage master transcription factor hub proteomics shows mechanisms impeding urothelial cancer cell differentiation

Urothelial cancer (UC) cells of the luminal subtype exhibit partial, incomplete differentiation towards umbrella cells that line bladder lumen, seen by morphology and gene expression. Differentiation is stalled even though the cells express master transcription factors (MTFs) that drive luminal urothelial differentiation, e.g., FOXA1 and CEBPB, at levels seen in normal differentiated urothelium. We therefore analyzed the FOXA1/CEBPB MTF hub by mass spectrometry. SWI/SNF coactivator complex (CoA) components, e.g., SMARCA4, ARID1A, that read the epigenetic activation mark histone 3 lysine 27 acetylation (H3K27ac) and use ATP-hydrolysis to open chromatin, were the most abundant proteins pulled-down with FOXA1/CEBPB. However, genes for these and other CoA, e.g., CREBBP, EP300 that write H3K27ac, were mutated/deleted in >95% of UCs in clinical series. Also contained in the hub were corepressors (CoR) that erase H3K27ac and close chromatin, e.g., HDAC1, CHD4 - genes for these CoR were recurrently gained in UCs. Chromatin analyses showed H3K27ac-centered remodeling was needed to activate umbrella but not constitutively accessible cell growth/division/housekeeping genes. Restoring ARID1A into ARID1A-mutated UC cells using lentiviral transduction, or inhibiting CoR with siRNA or small molecules, activated umbrella genes and terminated replications. In summary, UC-genesis selects for loss- and gain-of-function of CoA and CoR respectively in the urothelial-lineage MTF hub; small molecule CoR-inhibitors are candidate remedies to renew maturation towards terminal differentiated-fates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/744501v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@bda7e5org.highwire.dtl.DTLVardef@159ea57org.highwire.dtl.DTLVardef@28132borg.highwire.dtl.DTLVardef@1028bee_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Resistance to decitabine and 5-azacytidine emerges from adaptive responses of the pyrimidine metabolism network

Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a nucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). We report here that DNMT1 protein, although substantially depleted (~50%) in patients bone marrows at response, rebounded at relapse, and explaining this, we found pyrimidine metabolism gene expression shifts averse to the processing of each pro-drug. The same metabolic shifts observed clinically were rapidly recapitulated in leukemia cells exposed to the pro-drugs in vitro. Pyrimidine metabolism is a network that senses and preserves nucleotide balances: Decitabine, a deoxycytidine analog, and 5-azacytidine, a cytidine analog, caused acute and distinct nucleotide imbalances, by off-target inhibition of thymidylate synthase and ribonucleotide reductase respectively. Resulting expression changes in key pyrimidine metabolism enzymes peaked 72-96 hours later. Continuous pro-drug exposure stabilized metabolic shifts generated acutely, preventing DNMT1-depletion and permitting exponential leukemia out-growth as soon as day 40. Although dampening to activity of the pro-drug initially applied, adaptive metabolic responses primed for activity of the other. Hence, in xenotransplant models of chemorefractory AML, alternating decitabine with 5-azacytidine, timed to exploit compensating metabolic shifts, and addition of an inhibitor of a catabolic enzyme induced by decitabine/5-azacytidine, extended DNMT1-depletion and time-to-distress by several months versus either pro-drug alone. In sum, resistance to decitabine and 5-azacytidine emerges from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/958405v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e3f77aorg.highwire.dtl.DTLVardef@8caaa4org.highwire.dtl.DTLVardef@117236dorg.highwire.dtl.DTLVardef@16ee98_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗