Pirtobrutinib induces dedifferentiation of giant cells. (A) Venn diagram showing giant cell–specific signature (Giant_UP) from bulk RNA-seq data of Mino-VEN-R and JeKo-1–IBN-R cells. (B) Graphical GSEA of Giant_UP using ShinyGo tool.²⁹ (C) Flow cytometry results show decreased levels of IgM and CD19 in giant cells compared with parental cells. ∗P < .05; ∗∗P <. 01. (D) Heat map showing B-cell lineage markers differentially regulated during the transitions of Mino-VEN-R cells with PBN (10 μM) treatment on and off. (E) UMAP overview of Mino-VEN-R cells treated with PBN for 5 days. Each dot of the UMAP plot represents a single cell. Cells are colored by PBN dose or clusters (C0-C5) with split view. (F) The RNA density and cell size plots according to PBN dose. UMAP overview with cells colored by nCount_RNA; UMAP overview with cells colored by Giant_UP signature score. (G) Split view of UMAP with cells colored by rRNA_PROCESSING score. (H) Immunofluorescence showing the expression of fibrillarin (FBL) in Mino-VEN-R parental and giant cells. Blue, DAPI; Alexa Fluor 488, FBL. Scale bar, 100 μm. (I) Immunofluorescence showing the expression of FBL selectively in enlarged cells in MCL PDOs from PT3 and PT6. Arrows indicate the presence of giant cells. Blue, DAPI; Alexa Fluor 594, FBL. Scale bar, 100 μm. (J) Split view of UMAP with cells colored by CytoTRACE score. (K) Cell composition plot of predicted cell types with bone marrow data sets as referenced³⁰ according to cell mass (nCount_RNA). DAPI, 4′,6-diamidino-2-phenylindole; FDR, false discovery rate; HSC, hematopoietic stem cell; IgM, immunoglobulin M; PBN, pirtobrutinib; pDC, plasmacytoid dendritic cells; Prog, progenitor; Prog_RBC, progenitor red blood cells; UMAP, uniform manifold approximation and projection.