Pharmacological inhibition of the transcription factor PU.1 in leukemia
Iléana Antony-Debré, Ananya Paul, Joana Leite, Kelly Mitchell, Hye Mi Kim, Luis A. Carvajal, Tihomira I. Todorova, Kenneth Huang, Arvind Kumar, Abdelbasset A. Farahat, Boris Bartholdy, Swathi-Rao Narayanagari, Jiahao Chen, Alberto Ambesi-Impiombato, Adolfo A. Ferrando, Ioannis Mantzaris, Evripidis Gavathiotis, Amit Verma, Britta Will, David W. Boykin, W. David Wilson, Gregory M.K. Poon, Ulrich Steidl
Iléana Antony-Debré, Ananya Paul, Joana Leite, Kelly Mitchell, Hye Mi Kim, Luis A. Carvajal, Tihomira I. Todorova, Kenneth Huang, Arvind Kumar, Abdelbasset A. Farahat, Boris Bartholdy, Swathi-Rao Narayanagari, Jiahao Chen, Alberto Ambesi-Impiombato, Adolfo A. Ferrando, Ioannis Mantzaris, Evripidis Gavathiotis, Amit Verma, Britta Will, David W. Boykin, W. David Wilson, Gregory M.K. Poon, Ulrich Steidl
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Research Article Hematology

Pharmacological inhibition of the transcription factor PU.1 in leukemia

Abstract

The transcription factor PU.1 is often impaired in patients with acute myeloid leukemia (AML). Here, we used AML cells that already had low PU.1 levels and further inhibited PU.1 using either RNA interference or, to our knowledge, first-in-class small-molecule inhibitors of PU.1 that we developed specifically to allosterically interfere with PU.1-chromatin binding through interaction with the DNA minor groove that flanks PU.1-binding motifs. These small molecules of the heterocyclic diamidine family disrupted the interaction of PU.1 with target gene promoters and led to downregulation of canonical PU.1 transcriptional targets. shRNA or small-molecule inhibition of PU.1 in AML cells from either PU.1lo mutant mice or human patients with AML-inhibited cell growth and clonogenicity and induced apoptosis. In murine and human AML (xeno)transplantation models, treatment with our PU.1 inhibitors decreased tumor burden and resulted in increased survival. Thus, our study provides proof of concept that PU.1 inhibition has potential as a therapeutic strategy for the treatment of AML and for the development of small-molecule inhibitors of PU.1.

Authors

Iléana Antony-Debré, Ananya Paul, Joana Leite, Kelly Mitchell, Hye Mi Kim, Luis A. Carvajal, Tihomira I. Todorova, Kenneth Huang, Arvind Kumar, Abdelbasset A. Farahat, Boris Bartholdy, Swathi-Rao Narayanagari, Jiahao Chen, Alberto Ambesi-Impiombato, Adolfo A. Ferrando, Ioannis Mantzaris, Evripidis Gavathiotis, Amit Verma, Britta Will, David W. Boykin, W. David Wilson, Gregory M.K. Poon, Ulrich Steidl

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Figure 5

Inhibitors show on-target PU.1 inhibitory activity in AML cells.

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Inhibitors show on-target PU.1 inhibitory activity in AML cells. (A) qRT...
(A) qRT-PCR analysis of PU.1 target genes after PU.1 URE–/– AML cell treatment (n = 3–7), normalized to Gapdh. Fold change compared with vehicle is shown. (B) Mean fluorescence intensity (MFI) of BM MNCs isolated from PU.1/GFP-knockin mice (38) after treatment (n = 5). Fold change compared with vehicle is shown. (C) Quantitative ChIP assays of PU.1 occupancy after treatment of PU.1 URE+/–Msh2–/– AML cells (n = 5). Myogenin was used as a negative control. (D–I) Transcriptome analysis of PU.1 URE–/– AML cells after a 24-hour treatment with DB2313 (n = 3) versus vehicle (n = 3). (D) Differentially expressed genes upon treatment were tested for enrichment of genes directly regulated by PU.1, or regulated by the other ETS transcription factors using Ingenuity Knowledge Base (generated with the use of IPA). Dotted line represents the significance threshold (–log P value >1.3). (E and F) Comparative analysis of deregulated genes in PU.1 URE–/– AML cells after treatment and in PUER cells after PU.1 induction (GEO GSE13125). (E) Venn diagram shows significant overlap between the 2 data sets. (F) Deregulated canonical pathways between the data sets. Colored squares indicate the activation Z score. (G) GSEA enrichment plot of PU.1 positively regulated genes (regulon) in AML cells (from the MILE AML network, as determined by the ARACNe algorithm) against the global list of differentially expressed genes upon treatment, ranked by the drug response (as measured by t score of DB2313 vs. vehicle). (H) Heatmap of leading-edge genes showing row-normalized relative expression. (I) Enrichment of PU.1 binding at promoters of deregulated genes in PU.1 URE–/– AML cells upon treatment. Publicly available PU.1 ChIP-seq data from PUER cells (GSE63317) were used for this analysis. Up, upregulated; Down, downregulated. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA (A–C), hypergeometric test (E), Fisher’s exact test (I), or according to ref. 57 (G).