Journal: bioRxiv

Article Title: The PIP4K2 inhibitor THZ-P1-2 exhibits antileukemia activity by disruption of mitochondrial homeostasis and autophagy

doi: 10.1101/2022.08.20.504641

Figure Lengend Snippet: (A) Dose-response cytotoxicity was analyzed using a methylthiazoletetrazolium (MTT) assay in samples from acute myeloid leukemia (AML) or T- and B-acute lymphoblastic leukemia (T- or B-ALL) patients treated with vehicle or increasing concentrations of THZ-P1-2 (1.6, 3.2, 6.4, 12.5, 25, 50, and 100 μM) for 72 h. Values are expressed as the percentage of viable cells for each condition relative to vehicle-treated cells. The IC 50 values for each patient sample are described. (B) Apoptosis was detected by flow cytometry in gated human Sca1 - CD45 + CD34 + (or CD117 + cells for NPM1 -mutant AMLs) of acute myeloid leukemia (AML) samples in a co-culture system using a FITC-annexin V/DAPI staining method. Cells were treated with vehicle, cytarabine (AraC, 250 and 500 nM), venetoclax (VEN, 100 and 500 nM), and/or THZ-P1-2 (3.2 or 6.4 μM) for 72 h. Bar graphs represent the mean ± SD of all the independent patients screened, each point represents a patient. The p values are indicated in the graphs; * p < 0.05, ** p < 0.01, *** p < 0.0001; ANOVA and Bonferroni post-test. (C) Correlation analysis of THZ-P1-2 response with clinical, laboratory, molecular, and metabolic characteristics of the samples. Note that THZ-P1-2 responsiveness (area under curve, AUC) was clusterized with markers of mitochondrial metabolic markers (Levels of MMP in the blasts – Blast_TMRE; Basal OCR and Max OCR). Data were processed using the Morpheus platform ( https://software.broadinstitute.org/morpheus/ ) (D) Association of THZ-P1-2 sensitivity (area under the curve, AUC), mutations in FLT3, NPM1 , and RUNX1 , and European LeukemiaNet (ELN) risk stratification. (E) Evaluation of long-term proliferation in neonatal cord blood (CB) CD34 + and primary AML mononuclear cells using a co-culture system. Data are expressed as mean ± SD of at least three independent experiments. The time points and p values are indicated in the graphs; * p < 0.05, ** p < 0.01, *** p < 0.0001; ANOVA and Bonferroni post-test. (F) Neonatal cord blood (CB) CD34 + cells were plated in cytokine-supplemented methylcellulose in the presence of vehicle or THZ-P1-2 (3.2 or 6.4 μM). Colonies were counted after 8-14 days of culture and are represented as the percent of vehicle-treated controls. Bars indicate the mean ± SD of at least three assays. (G-H) Mitochondrial membrane potential was detected by flow cytometry in gated human CD45 + CD34 + (or CD117 + for NPM1 -mutant AMLs)/annexin V - from samples from acute myeloid leukemia (AML) in a co-culture system using the TMRE staining method. Cells were treated with vehicle, cytarabine (AraC, 250 nM), venetoclax (VEN, 100 or 500 nM), and/or THZ-P1-2 (3.2 [alone or in combination with VEN 100 nM] or 6.4 μM) for 72 h. Bar graphs represent the mean ± SD of at least three independent experiments, each point represents a patient. The p values are indicated in the graphs; * p < 0.05, ** p < 0.01, *** p < 0.0001; ANOVA and Bonferroni post-test. (I) Differently expressed proteins obtained from THZ-P1-2 (THZ)-sensitive (n = 3) and THZ-resistant (n = 3) AML patients were included in the heatmap (all false discovery rate (FDR) q-values (FDR q) < 0.25). (J) The bar graph represents the normalized enrichment scores (NES) for Hallmark, Reactome, and Kegg gene sets with FDR q < 0.05. (K) GSEA plots for enriched molecular signatures in THZ-P1-2 (THZ) resistant vs . sensitive AML patient’s proteome are also shown. NES and FDR q are indicated.

Article Snippet: Venetoclax (ABT-199) was obtained from TargetMol (Target Molecule Corp., Boston, MA, USA) and diluted to a 50 mM stock solution in DMSO.

Techniques: MTT Assay, Flow Cytometry, Mutagenesis, Co-Culture Assay, Staining, Software