Fig 2: A case of relapsed/refractory AML (WCM254) with a novel PML-SYK fusion gene and transcript with epichaperome enrichment.a Hematoxylin and eosin (H&E) staining of the bone marrow (BM) biopsy showing hypercellular BM with unclassified myeloproliferative disorder at disease onset. Hematopathology evaluation from 45 months prior this study. b BM aspirate shows increased myeloblasts as patient progressed to acute myeloid leukemia (AML). Hematopathology evaluation 17 months prior to this study. c FISH analysis for SYK showing break apart (rearranged) green and red signals, and yellow (unrearranged) allele (for PML FISH data see Supplementary Fig. 1). d Representative gating of leukocyte subpopulations evaluated with flow cytometry. Blasts, lymphocytes and granulocytes were sorted by gating on SSC-A vs. CD45 from bone marrow (BM) sample at baseline. e Agarose gel electrophoresis of PCR amplicon showing positive PML-SYK fusion gene transcripts in bulk population and sorted populations indicated in d. f Schematic representation of cells with high (top row) or no epichaperome (bottom row) with corresponding expected flow histograms in PU-FITC biding assay. PU-FITC binds to intracellular epichaperome with high affinity and gives signal indicating abundance of epichaperome. g Preclinical evaluation of epichaperome abundance in lymphocytes (green), granulocytes (blue) and blasts (red) by performing PU-FITC-binding assay. The intensity of FITC fluorescence indicates the level of epichaperome and unstained control is shown in gray. h and i Viability assay with cells from WCM254 (red), MV4;11 (blue, control AML cell line with high epichaperome and with high sensitivity to PU-H71) or normal peripheral blood (nPB) mononuclear cells (black) treated with escalating doses of PU-H71 for 72 h in vitro. Representative flow plots (h) and graphic representation at increasing doses of PU-H71 (i). Experiments performed in triplicate, showing the mean and error bar represents the SD.

Fig 3: Identification of a CD19–IL-7R+ Progenitor(A) Fluorescence-activated cell sorting (FACS) analysis of CS17 FL showing CD19-IL-7R+ progenitor co-expressing CD34 and CD45RA. Adult BM (right) is shown for comparison. Cells were gated CD45+LIN-CD19–. Further gating as indicated. Mean percentage of total CD45+CD34+ cells.(B) Kinetics of IL-7R progenitor emergence during development as percentage of total CD45+CD34+ cells. Each dot represents one biological sample. Mean with range. Statistical comparison versus CB control.(C) B cell potential of IL-7R+KIT+ FL progenitors was tested by co-culture on MS-5 for 2 weeks. Percentage of CD19+ B cells of total CD45+ cells is shown (left) with representative FACS analysis (right). CD34+ cells were used as control. Mean ± SD, n = 2–3.(D) Agarose gel of IGH DJ rearrangements in IL-7R+ FL progenitor. Top: DH7JH recombination assay. Bottom: DH1-6JH recombination assay. Label for lanes 1–6 is shown to the right. Marker lanes (M). Predicted DH7JH recombination between 100 and 130 bp. Predicted DH1-6JH rearrangements between 110 and 290 bp, n = 1 (van Dongen et al., 2003).(E) GM and erythroid potential of IL-7R+KIT+ FL progenitor in semi-solid media. Data show distribution of colony types as percentage of plated cells versus CD34+ cells as controls. Mean ± SD, n = 2 per developmental stage.(F) Myeloid potential of IL-7R+KIT+ FL progenitor in liquid culture (20–40 cells/well). Graph shows percentage of wells expanded to >50 cells (left). Cytospin of macrophages generated from FL IL-7R+KIT+ progenitor (right). Mean ± SD, n = 3.See also Figure S2.

Fig 4: Lymphoid Commitment in hPSC Culture Recapitulates Human Fetal B Lymphopoiesis(A) hPSC in vitro B cell differentiation protocol. hPSCs are harvested onto a layer of overgrown OP9 and co-cultured for 10 days. After CD34 enrichment, cells were either analyzed (D10) or co-cultured for an additional 21 days on MS-5 in the presence of lymphoid cytokines as indicated (D31) (Carpenter et al., 2011).(B) Differentiated MIFF3 hPSCs were analyzed for CD19+ B cells (top) and CD19–IL-7R+ progenitor (bottom) at D10 (left) and D31 (right). Cells were gated CD45+ or CD45+IGM-; further gating as indicated. Mean percentages of CD45+ cells, n = 5–7.(C) qPCR of D31 hPSCs. CD34+CD45RA-, IL-7R+ progenitor, and CD34+CD19+ proB cells were analyzed for expression of lymphoid (IL7R, RAG2, EBF1, and PAX5), megakaryocytic/erythroid (GATA1), and fetal-specific (LIN28B) genes. Data are normalized to GAPDH, mean ± SD, n = 2–3.(D) PCA of RNA-seq data. Left: PCA based on primary cells only; LIN-CD34+CD45RA-, IL-7R+ progenitor, and proB cells from adult BM (n = 3) and FL (CS21-22, n = 2). Right: hPSC-derived cells and the early CS17 FL IL-7R+ progenitor (outlier) were placed on the PCA calculation shown to the left. hPSCs (MIFF3 and H1) are from D31 (CD34+CD45RA-, IL-7R+ progenitor, and proB cells) of differentiation. PCA1 and PCA2 are shown and each dot represents one sample. The ellipses show the 90% density function for that cell type.(E) Single-cell qPCR analysis of IL-7R+ progenitor from D10 and D31 of differentiation (MIFF3). Each column represents a single cell. Colored by CT value. Only cells expressing GAPDH are shown. Genes in red are myeloid, blue are lymphoid, and green are B cell genes. A total of 35–58 cells per time point, n = 2/population.(F) PCA calculated from single-cell qPCR data from the human primary cells (Figure 3D) and differentiated hPSCs (MIFF3 and H1). hPSCs are from D10 (IL-7R+ progenitor only) and D31 (IL-7R+ progenitor and proB cells). Each dot represents a single cell (only hPSCs shown). Highlighted ellipses represent primary human cell clusters, 36 genes were used for the PCA, n = 2–3.See also Figure S3.

Fig 5: PU-H71 productively engages the epichaperome during treatment of patient WCM254.a During treatment with PU-H71, the patient WCM254 was monitored for complete blood counts (CBCs), signaling in leukocytes subsets, and epichaperome abundance in cells from peripheral blood and bone marrow. b The clinical course of patient WCM254 during the first ten doses of PU-H71. Absolute leukocyte (blue circle) and blast counts (red triangle) are indicated on each day they were assessed. Days on which the patient received a dose of PU-H71 are indicated with green squares. Gray shaded area indicates the normal range of leukocyte counts. c Chronological changes of percentages of lymphocytes (green) and blasts (red) in mononuclear cells at baseline and on day 2, 5, 11, and 15. Lymphocytes and blasts were gated on SSC-A vs. CD45. d, Epichaperome abundance determined by flow cytometry on day 0, 5, 11, and 15 is shown as a biomarker of response to PU-H71. Graph, mean ± SD of MFI of blasts (i.e., epichaperome +ve)/MFI of CD3 + lymphocytes (i.e., epichaperome -ve). e Isoelectric focusing shows long-lived multimeric HSP90 species (a biochemical signature of epichaperomes) in sorted CD34 + baseline (BL) blasts that decrease by day 5 of treatment (D5). Negative control, ASPC1 (epichaperome + ) homogenate; positive control, MDA-MB-468 homogenate (epichaperome-)1. f Cell subsets as in c and d were evaluated by flow cytometry for levels of intracellular phospho-Syk(Y525/526), phospho-STAT5(Y694), phospho-S6 (S235/S236) and phospho-ERK(T202/Y204). Graph, mean ± SD of MFI of blasts/MFI of CD3 + lymphocytes.