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

Abstract

The treatment of acute leukemia is challenging because of the genetic heterogeneity between and within patients. Leukemic stem cells (LSCs) are relatively drug-resistant and frequently relapse. Their plasticity and capacity to adapt to extracellular stress, in which mitochondrial metabolism and autophagy play important roles, further complicates treatment. Genetic models of phosphatidylinositol-5-phosphate 4-kinase type 2 protein (PIP4K2s) inhibition have demonstrated the relevance of these enzymes in mitochondrial homeostasis and autophagic flux. Here, we uncovered the cellular and molecular effects of THZ-P1-2, a pan-inhibitor of PIP4K2s, in acute leukemia cells. THZ-P1-2 reduced cell viability and induced DNA damage, apoptosis, loss of mitochondrial membrane potential, and the accumulation of acidic vesicular organelles. Protein expression analysis revealed that THZ-P1-2 impaired autophagic flux. In addition, THZ-P1-2 induced cell differentiation and showed synergistic effects with venetoclax. In primary leukemia cells, LC-MS/MS-based proteome analysis revealed that sensitivity to THZ-P1-2 is associated with mitochondrial metabolism, cell cycle, cell-of-origin (hematopoietic stem cell and myeloid progenitor), and the TP53 pathway. The minimal effects of THZ-P1-2 observed in healthy CD34⁺ cells suggest a favorable therapeutic window. Our study provides insights into the pharmacological inhibition of PIP4K2s targeting mitochondrial homeostasis and autophagy, shedding light on a new class of drugs for acute leukemia.

Fig. 1. Pharmacological PIP4K2s inhibition reduces cell viability and induces DNA damage in leukemia cells.

A Dose-response cytotoxicity was analyzed using a methylthiazoletetrazolium (MTT) assay in a panel of myeloid and lymphoid leukemic cell lines 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 IC₅₀ values and leukemia cell lines used are described in the figure. B Dose- and time-response cytotoxicity was evaluated by methylthiazoletetrazolium (MTT) in MV4-11, OCI-AML3, Jurkat, and NALM6 cells treated with vehicle or with increasing concentrations of THZ-P1-2 (1.6, 3.2, 6.4, 12.5, 25, and 50 μM) for 24, 48, and 72 h. Bar graphs represent values expressed as a percentage for each condition relative to vehicle-treated controls. Results are presented as the mean ± SD of at least three independent experiments. The p values and cell lines are indicated in the graphs; *p < 0.05; **p < 0.01; *** p < 0.001, ANOVA and Bonferroni post-test. C Phases of the cell cycle were determined by analyzing the DNA content by staining with propidium iodide and acquiring the data by flow cytometry following exposure of MV4-11, OCI-AML3, Jurkat, and NALM6 cells to the vehicle or THZ-P1-2 (1.6, 3.2, and 6.4 μM) for 24 h. A representative histogram for each condition is illustrated. The mean ± SD of at least three independent experiments is represented in the bar graph. The p values and cell lines are indicated in the graphs; *p < 0.05; **p < 0.01; ***p < 0.001, ANOVA and Bonferroni post-test. D DNA damage was evaluated by the single-cell comet assay in MV4-11 and Jurkat treated with vehicle or THZ-P1-2 (3.2 or 6.4 µM) for 48 h. Scatter plots represent the tail moment values (head/tail DNA ratio) obtained using the LUCIA Comet Assay™ software. **p < 0.01; ***p < 0.001, ANOVA and Bonferroni post-test.

Fig. 2. THZ-P1-2 induces apoptosis and dysfunction in mitochondria and autophagic flux.

A Apoptosis was detected by flow cytometry in MV4-11, OCI-AML3, Jurkat, and NALM6 cells treated with vehicle or with increasing concentrations of THZ-P1-2 (1.6, 3.2, and 6.4 μM) for 24 h using an APC-annexin V/PI staining method. Representative dot plots are shown for each condition. The upper and lower right quadrants (Q2 plus Q3) cumulatively contain the apoptotic cell population (annexin V⁺ cells). Bar graphs represent the mean ± SD of at least three independent experiments. The p values and cell lines are indicated in the graphs; *p < 0.05, **p < 0.01, ***p < 0.0001; ANOVA and Bonferroni post-test. B Mitochondrial membrane potential (ΔΨ_M) analysis was evaluated using the JC-1 staining method and flow cytometry. Leukemic cells were treated with vehicle or THZ-P1-2 (1.6, 3.2, and 6.4 μM) for 24 h. Representative dot plots are shown for each condition; the gate FL-2 contains cells with intact mitochondria and the gate FL-2/FL-1 contains cells with damaged mitochondria. Bar graphs represent the mean ± SD of at least three independent experiments and the p values are indicated; **p < 0.01, ***p < 0.0001; ANOVA and Bonferroni post-test. C The evaluation of acidic vesicular organelles (AVOs) was investigated through acridine orange (AO) labeling and flow cytometry in AML and ALL cell lines treated with vehicle or THZ-P1-2 (1.6, 3.2, and 6.4 μM) for 24 h. Bar graphs represent the mean ± SD of at least three independent experiments and the p values are indicated; *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA and Bonferroni post-test. D Alternatively, the presence of AVOs was confirmed by immunofluorescence on OCI-AML3 and NALM6 cell lines treated with vehicle or THZ-P1-2 (3.2 and 6.4 μM) for 72 h on a Lionheart FX automated microscope at magnification, ×400. The overlapping GFP and RFP channels are shown.

Fig. 3. THZ-P1-2 induces markers of apoptosis, DNA damage, and blockage of autophagic flow.

A Western blot analysis for PARP1, γH2AX, p62/SQSTM1, and LC3B in total extracts from MV4-11, OCI-AML3, Jurkat, and NALM6 cells treated with vehicle or with increasing doses of THZ-P1-2 (1.6, 3.2, and 6.4 µM) for 24 h. Membranes were reincubated with α-tubulin antibody and developed with the SuperSignal™ West Dura Extended Duration Substrate system and GBox. Band intensities of cleaved-PARP1, γH2AX, p62/SQSTM1, and LC3B were corrected by α-tubulin expression and normalized by vehicle-treated cells. B Heatmap depicting the gene expression profile of leukemic cell lines treated with vehicle or THZ-P1-2 (6.4 μM) for 24 h. The blue color in the heatmap indicates decreased mRNA levels, while red indicates induced mRNA levels, which were normalized by vehicle-treated cells (n = 4). Leukemia cells that showed the lowest rates of THZ-P1-2-induced apoptosis were considered resistant. C Network for THZ-P1-2 modulated genes constructed using the GeneMANIA database (https://genemania.org/). A total of seven genes (BBC3, ATG5, MAP1LC3B, MCL1, CDKN1B, BAX, and BCL211) were significantly modulated in all cell lines tested and are illustrated as crosshatched circles; the interacting genes included by modeling the software are indicated by circles without crosshatched. The main biological interactions and associated functions are described in the literature.

Fig. 4. THZ-P1-2 reduces cellular metabolism in leukemic cells.

A Mitochondrial membrane potential analysis was evaluated using the tetramethylrhodamine, ethyl ester, perchlorate (TMRE) staining method and flow cytometry. OCI-AML3 and MV4-11 cells were treated with vehicle or THZ-P1-2 (3.2, and 6.4 μM) for 24 h. Values are expressed as the fold-change of vehicle-treated cells. Bar graphs represent the mean ± SD of at least three independent experiments and the p values are indicated; *p < 0.05, ***p < 0.0001; ANOVA and Bonferroni post-test. B Oxygen consumption rate (OCR) was determined in vehicle- or THZ-P1-2-treated (6.4 μM) OCI-AML3 and MV4-11 cells using a high-resolution respirometry. A representative line graph containing OCR upon sequential addition of oligomycin (Oligo A), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone plus antimycin (Rot + Anti A) are illustrated; OCR was measured over time. Values are expressed as the fold-change of vehicle-treated cells. Bar graphs represent the mean ± SD of OCR at baseline (C) and maximum state (upon FCCP) (D) of at least three independent experiments. The p values and cell lines are indicated in the graphs; ***p < 0.0001; Student t-test. E Extracellular acidification rate (ECAR) was evaluated in vehicle- or THZ-P1-2-treated (6.4 μM) OCI-AML3 and MV4-11 cells by Seahorse XF96 analyzer. A representative line graph containing ECAR upon sequential addition of glucose (Gluco), oligomycin (Oligo A), and 2-deoxy-d-glucose (2-DG) is illustrated; ECAR was measured over time. F Values are expressed as the fold-change of vehicle-treated cells. Bar graphs represent the mean ± SD of at least three independent experiments. The p values and cell lines are indicated in the graphs; ***p < 0.0001; Student t-test.

Fig. 5. Pharmacological PIP4K2s inhibition selectively reduces cell viability of primary leukemic blasts.

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 IC₅₀ 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 were 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). AML patients with higher AUC were considered resistant to THZ-P1-2. 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.