Targeting PIP4K2 Lipid Kinases through Thz-P1-2 Reveals Vulnerabilities Related to Mitochondrial Homeostasis and Autophagy in Acute Leukemia

Abstract

Mitochondrial metabolism plays an important role in the therapeutic response of acute myeloid leukemia (AML) cell subsets and affects treatment outcome. Of not, leukemia stem cells (LSC) were shown to have increased sensitivity to disruptions of the oxidative phosphorylation process. These vulnerabilities may be specifically targeted with the BCL2 inhibitor venetoclax, which suppresses the tricarboxylic acid cycle by inhibiting amino acid metabolism. Additionally, autophagy and lysosomal biosynthesis have emerged as potential targets to improve the therapeutic response to these diseases. Genetic models of phosphatidylinositol-5-phosphate 4-kinase type 2 proteins (PIP4K2s) inhibition demonstrated the relevance of these enzymes in mitochondrial homeostasis and autophagic flux. High PIP4K2A and PIP4K2C transcript levels were associated with adverse cytogenetic risk and poor clinical outcomes in AML. Taking together, these data suggest that PIP4K2s inhibitors may have antileukemic effects and may synergize with venetoclax. Here, we uncover the cellular and molecular effects of THZ-P1-2, a pan-inhibitor of PIP4K2s, in acute leukemia cells and tested aforementioned hypothesis. Using a panel of 21 commonly used human myeloid and lymphoid leukemic cell lines, THZ-P1-2 caused a dose-dependent decrease in viability in these leukemia cells. Our results indicated cytostatic effects associated with a reduction of cells in S and G2/M phases when THZ-P1-2 was administrated at low concentrations (1.6 or 3.2 µM). Conversely, when THZ-P1-2 was administrated at high concentrations (6.4 µM), we observed cytotoxic effects linked with an increased accumulation of cells in the subG1 (all p < 0.05). The presence of DNA damage after exposure to THZ-P1-2 in leukemia cells was confirmed by the comet assay (all p < 0.05). An increase in apoptosis was detected in all cellular models upon the treatment with THZ-P1-2 in leukemia cells (all p < 0.05). Notably, acidic vesicular organelles levels were substantially elevated upon THZ-P1-2 exposure, particularly at concentrations that induced lower levels of apoptosis. At the molecular level, THZ-P1-2 induced the expression of apoptosis markers (PARP1 cleavage) and DNA damage (γH2AX) at levels compatible with those observed in previous cellular assays. Furthermore, LC3BII and SQSTM1/p62 levels were increased, which indicates a failure to complete the autophagic flow. In addition, exposure to THZ-P1-2 reduced basal and maximal cellular respiration capacity as well as glycolytic flux. The combined treatment showed synergistic effects, reducing the IC50 of venetoclax and strongly increasing the induction of apoptosis in leukemia cells [apoptosis rates (mean, SD); vehicle: 3.8, 0.7%, 6.4 µM venetoclax: 33.3, 4.8%, 6.4 µM THZ-P1-2: 18.5, 2.8%, 12.5 µM THZ-P1-2: 64.3, 4.3%, 6.4 µM venetoclax plus 6.4 µM THZ-P1-2: 78.3, 1.2%, 6.4 µM venetoclax plus 12.5 µM THZ-P1-2: 97.4, 0.3%, all p < 0.05]. Similar to what was observed in the cell line model, THZ-P1-2 reduced the viability of primary leukemic cells (AML, n = 45; ALL, n = 20) in a dose-dependent manner, which was correlated with a more metabolically active phenotype (oxygen consumption and extracellular acidification) of the leukemic blasts. Proteome analysis was performed on samples from patients considered sensitive and resistant to THZ-P1-2, which presented distinct protein signatures. A gene enrichment analysis identified multiple processes and signaling pathways associated with mitochondrial metabolism, cell cycle stage, leukemic cell-of-origin, and TP53 pathway (all FDR q-value < 0.05). Minimal effects of THZ-P1-2 observed in healthy CD34+ cells suggested a favorable therapeutic window. Our study provides insight into pharmacological inhibition of PIP4Ks targeting mitochondrial homeostasis and autophagy shedding light on a new class of drugs for acute leukemias. Supported by FAPESP, CNPq and CAPES.