Fig 1: Osimertinib directly binds to CD34 protein. A, Chemical structure of biotin-tagged osimertinib. B, Pulldown assays were conducted using biotin-osimertinib against recombinant CD34 (rCD34) in the presence/absence of free osimertinib as a competitor. C, Pulldown assays were conducted using biotin-osimertinib against KG-1 cell lysates in the presence/absence of free osimertinib as a competitor. D, MS/MS analysis of the cysteine 199 (C199) tryptic peptide of rCD34 protein incubated with (bottom) or without (top) osimertinib for 30 minutes. E, Predicted conformations of compound osimertinib (magenta) and ibrutinib (cyan) in the binding pocket of CD34. The protein of CD34 is shown in wheat surface and cartoon. Compounds are shown as sticks. Cys177 and Cys199 of CD34 are shown as sticks. The dark dash lines show the distance between Cys199/Cys177 to the atom that is involved in Michael addition (in dash cycles). F, KG-1, Kasumi-1, and primary AML cells were infected with CD34-targeting CRISPR-Cas9 to deplete CD34 expression. Expression of CD34 was analyzed with immunoblot assay. G, CD34 was knocked out by CRISPR-Cas9 system in CD34+ cells purified from patients with AML (n = 3) and was exposed to 1 μmol/L osimertinib for 72 hours. Apoptosis was measured by flow cytometry. H, KG-1 and Kasumi-1 cells were infected with CD34-targeting CRISPR-Cas9 to deplete endogenous CD34 and then infected with plasmids encompassing WT, C199S, or C199S/C177S mutant CD34. These cells were treated with 2 μmol/L osimertinib for 72 hours and apoptosis was determined by flow cytometry. *, P < 0.05; ***, P < 0.001, between the line-pointed group. EV, empty vector; sgCD34, specific guide CD34; sgNS, specific guide nonspecific.
Fig 2: CD34 is an adverse prognosis marker in AML and associates with specific pathogenic events. A, Differences in OS in de novo patients with AML from the BeatAML dataset (n = 200; left) and TCGA dataset (n = 173; right) by expression of CD34. P values, HRs, and 95% confidence interval (CI) are shown from univariate Cox analysis. B, Differences in OS in de novo patients with AML from the BeatAML dataset (n = 200) by combination of CD34 expression with presence of FLT3, NPM1, CEBPA, and CBF fusion. P values, HRs, and 95% CI are for the CD34 high expression from the multivariate Cox analysis. CBF fusion, RUNX1-RUNX1T1 fusion or CBFB-MYH11 fusion. C, Landscape and percentage of mutations and fusions in patients with AML with high or low CD34 expression level. D and E, RNA-seq and quantitative proteomics analysis of sorted CD34+ cells samples collected from de novo patients with AML and healthy donors (Normal). The mRNA (D) and protein (E) expression level of CD34 are shown. F, HALLMARK genesets from the GSEA analysis of RNA-seq and quantitative proteomic data of CD34+ cells from patients with AML versus normal donors. G, GSEA for complement and DNA repair pathways in AML versus normal samples.
Fig 3: Osimertinib selectively kills CD34+ AML and CML cells. A, Primary CD34− cells were treated with 2 μmol/L osimertinib for 72 hours. Cell apoptosis was measured by Annexin V/PI staining. Representative flow cytometry plots, cell viability measured by trypan blue, and quantification of Annexin V+% are shown. B, BMMCs of patients with AML (n = 17) were treated with 2 μmol/L osimertinib for 48 hours and cell apoptosis was measured by Annexin V/PI assay co-staining with CD34-APC. Flow cytometry plots of two representative patients and quantification of Annexin V+% are shown. C, Leukemia cell lines KG-1, Kasumi-1, U937, THP1, Molm13, and K562 were treated with 2 μmol/L osimertinib for 72 hours and Annexin V+% was measured by Annexin V/PI assay co-staining with CD34-APC. Right, quantification of Annexin V+PI− and Annexin V+PI+ cells. *, P < 0.01; ****, P < 0.0001, between the line-pointed group.
Fig 4: Osimertinib downregulates level of tyrosine phosphorylation of CD34. A, Heat map of levels of the indicated phosphorylated tyrosine sites in osimertinib-treated and untreated (ctrl) CD34+ cells. The CD34+ cells were purified from three AML individuals and pooled, followed by treatment with 2 μmol/L osimertinib for 48 hours. Tyrosine-phosphorylated peptides were enriched and analyzed by MS. The colors in the map represent the quantitative value (normalized total spectra) according to the Scaffold_4.3.3. B, Phosphoproteome changes observed in osimertinib-treated CD34+ cells analyzed by String database. The edge indicates known interaction between two proteins. Sequence logo represents the motif clustering from the tyrosine phosphorylation data set. C and D, The osimertinib treated or untreated CD34+ cells were collected and the cell lysates were immunoprecipitated with anti-CD34 or IgG antibodies, followed by PRM-MS analysis. Intensity histogram of nonphosphorylated (C) and phosphorylated Y329-containing peptide (D) of CD34 protein was quantified. E, Level of Y329-phosphorylation of CD34 quantified by PRM-MS assays in AML cell lines and 5 primary AML CD34+ samples. Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01, against untreated group.
Fig 5: Osimertinib impairs interaction between CD34 and the Src protein kinase. A–C, IP assays were conducted using antibodies against Src or CD34 in KG-1, Kasumi-1, and primary CD34+ AML cells. *, the heavy chain of IgG. D, Immunofluorescent staining of Src and CD34 in KG-1 and Kasumi-1. E, IP assay was conducted using antibody against CD34 in primary CD34+ AML cells treated with/without osimertinib. F and G, Kasumi-1 cells were infected with flag-tagged WT (F) or C199S mutant (G) CD34 and then treated with/without osimertinib. IP assay was conducted using anti-Flag antibody. H, Recombinant CD34 peptide (aa324–352) was incubated with Src (middle) or Lyn (bottom), followed by MS/MS analysis. Total ion chromatogram (TIC, sum of MS1 intensities over time) and fragment-ion spectra (MS2, MS/MS) for the major peaks in the TIC-MS1 spectrum of nonphosphorylated peptides (left) and phosphorylated peptides (right) are shown. The b ions and y ions are labeled in blue and red, respectively. I, KG-1 cells were infected with CD34-targeting CRISPR-Cas9 to deplete CD34 expression, followed by overexpression with flag-tagged WT or Y329F mutant, and cell proliferation was evaluated. J and K, Immunoblot analysis showing activity of the STAT3, AKT, ERK, Src in response to osimertinib (1 μmol/L, 72 hours) in primary CD34+ or CD34− cells (J) or KG-1 and Kasumi-1 cells (2 μmol/L, 72 hours; K). L, Primary CD34+ cells purified from patients with AML (n = 3) were treated with 1 μmol/L osimertinib, 2 μmol/L saracatinib, or osimertinib plus saracatinib (combo), and apoptosis was measured by Annexin V/PI staining. M, Primary CD34+ AML cells were treated with 1 μmol/L osimertinib, 1 μmol/L stattic, or osimertinib plus stattic (combo), and apoptosis was measured by Annexin V/PI staining. Quantified apoptotic cells are shown. **, P < 0.005 between the line-pointed group.
Supplier Page from Abcam for Recombinant Human CD34 protein