Abstract
HOXA9 and MEIS1 are frequently upregulated in acute myeloid leukemia (AML), including those with MLL-rearrangement. Because of their pivotal role in hemostasis, HOXA9 and MEIS1 appear non-druggable. We, thus, interrogated gene expression data of pre-leukemic (overexpressing Hoxa9) and leukemogenic (overexpressing Hoxa9 and Meis1; H9M) murine cell lines to identify cancer vulnerabilities. Through gene expression analysis and gene set enrichment analyses, we compiled a list of 15 candidates for functional validation. Using a novel lentiviral multiplexing approach, we selected and tested highly active sgRNAs to knockout candidate genes by CRISPR/Cas9, and subsequently identified a H9M cell growth dependency on the cytosolic phospholipase A2 (PLA2G4A). Similar results were obtained by shRNA-mediated suppression of Pla2g4a. Remarkably, pharmacologic inhibition of PLA2G4A with arachidonyl trifluoromethyl ketone (AACOCF3) accelerated the loss of H9M cells in bulk cultures. Additionally, AACOCF3 treatment of H9M cells reduced colony numbers and colony sizes in methylcellulose. Moreover, AACOCF3 was highly active in human AML with MLL rearrangement, in which PLA2G4A was significantly higher expressed than in AML patients without MLL rearrangement, and is sufficient as an independent prognostic marker. Our work, thus, identifies PLA2G4A as a prognostic marker and potential therapeutic target for H9M-dependent AML with MLL-rearrangement.
Highlights
Acute myeloid leukemia (AML) is an aggressive neoplastic disorder characterized by reduced differentiation capacity, increased proliferation, and tissue infiltration of myeloid progenitor cells
This approach led to the identification of PLA2G4A as a potential target and prognostic marker for Hoxa9- and Meis1-dependent (H9M)-driven leukemia
Prostaglandin endoperoxide synthase (PTGS; aka cyclo-oxygenase (COX)) subsequently converts AA into a prostaglandin (PG) H2 intermediate, which is further processed by prostaglandin E synthase (PTGES) into PGE2 as well as into thromboxanes, prostacyclins, and additional prostaglandins by alternative tissue specific isomerases [42]
Summary
Acute myeloid leukemia (AML) is an aggressive neoplastic disorder characterized by reduced differentiation capacity, increased proliferation, and tissue infiltration of myeloid progenitor cells. MEIS1 mediates resistance against differentiation-inducing cues in malignant hematopoiesis [7,8,9] Both genes on their own harbor limited transforming potential in mouse studies, their co-expression triggers aggressive disease, which resembles human AML with rearranged MLL (MLLr) [10]. In flow cytometric singleplex and multiplex assays with 18 FGB vectors (15 candidate genes and three controls), cleavage efficiencies were calculated by the percentage of EBFP2 positive and negative cells in each color-coded population These experiments revealed cutting efficiencies of >50% for sgRNAs against F2rl (66.7%), Neto (52.2%), Itgav (66.1%), P2rx (61.3%), Pla2g4a (69.8%), F2r (56.6%), Leprel (69.9%), Cxxc (56.7%), and Oaf (63.6%), as well as for the Tet (70.9%) positive control (Figure 2D) and a high correlation between singleplex and multiplex determined recombination rates (Pearson’s r = 0.99, R2 = 0.99) regardless of the requirement to stain for surface CAAR epitopes (Figure 2D).
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