Fig. 1.

a For 72 h treatment, QL47 inhibited FLT3-ITD-positive AML proliferation more potently compared to BTK inhibitor AVL292 and CGI1746 measured by CellTiter-Glo assay. b QL47 induced FLT3-ITD decrease in a dose-and time-dependent manner. c QL47 preincubated MV-4-11 cell lysis were exposed to 47biotin for 4 h, then precipitated by streptavidin agarose. After PBS washing, heat shock proteins binding with 47biotin were detected by western blotting, using HSP70 and HSP90 antibodies. d LC-MS and MS/MS for the identification of QL47 modification site in HSP70. Shown on the left are the selected-ion chromatograms (SICs) for monitoring the [M + 2H]²⁺ ion (m/z 513.7125) of the tryptic peptide TACER with Cys267 being modified by QL47 in the tryptic digestion mixture of HSP70 without (top) or with (bottom) QL47 treatment. Displayed on the right are the MS/MS for the [M + 2H]²⁺ ions of the tryptic peptide TACER with Cys267 being carbamidomethylated (CAM, top) or covalently modified with QL47 (bottom). e HEK293T cells were transient transfected with FLAG tagged HSP70/HSC70 WT and C267S mutation plasmids for 48 h, cell lysis was incubated with gradient-diluted 47biotin, the combined HSP70 WT or C267S mutation were precipitated by streptavidin agarose and detected with FLAG antibody. f HSP70 ATPase activity was carried out with the co-chaperon protein HSP40 in presence of QL47 and VER155008 or DMSO for 1 h, the ADP produced were measured with ADP-Glo assay. Data represent a mean of triplicate ± SD. g In vivo firefly luciferase assay were carried out on HEK293T cells by transient-transfected pcDNA3.1-luciferase with pcDNA3.1-HSP70 or vehicle vectors. After 48 h, cells were heat shocked at 45 °C for 1 h, and then recovered at 37 °C for 0/0.5/1 h, luminescence in cell suspension were measured and presented as percentage of initial luminescence activity. With HSP70 overexpression, luciferase recovered to a higher level in 1 h, and the refolding of luciferase significantly slowed down upon 10 μM QL47 exposure in a time-dependent manner. Data represent a mean of triplicate ± SD. h Constitutive knockdown MOLM13/MV-4-11 cells were obtained by infection with lentivirus containing double-stranded shRNA hairpin DNA sequences targeting HSP70 or HSC70. The knockdown efficacy and the effect on FLT3 protein were detected by western blotting, using specific antibody. i The capacity of colony formation was detected in shSCR MOLM13, KD HSP70, and KD HSC70 MOLM13 cells. The colonies were counted in three fields, which selected randomly under the microscope, data represent a mean of triplicates ± SD. j MOLM13 cells with HSP70 or HSC70 knockdown were injected into nu/nu mice, five mice per group, in vivo tumor genesis, and growth ability were tested after cells inoculation for 14 days. The data are presented as a mean of tumor volume (mm³) in each group ± SD. k Analysis of HSP70 expression and survival of AML patients from the TCGA database by GEPIA. Low HSPA1B but not HSPA8 is highly related to the longer survival of AML patients. l Cell viability was measured by CellTiter-Glo assay after 72 h treatments with DMSO or gradient-diluted QL47 in two FLT3-ITD-positive AML patients’ derived cells P1 and P2. m FLT3-ITD proteins were degraded and apoptosis was induced by 12 h treatment of QL47 in P1 patient cells. n Antitumor efficacy of QL47 in a bone marrow engrafted mouse model using MV-4-11 cells. Survival curves are shown. o Body weight monitoring in the MV-4-11 cell bone marrow engrafted tumor model. *P value < 0.05, **P value < 0.01, ***P value < 0.001, and ****P < 0.0001 by unpaired t test