The epigenetic regulators CBP and p300 facilitate leukemogenesis and represent therapeutic targets in acute myeloid leukemia

Abstract

Growing evidence links abnormal epigenetic control to the development of hematological malignancies. Accordingly, inhibition of epigenetic regulators is emerging as a promising therapeutic strategy. The acetylation status of lysine residues in histone tails is one of a number of epigenetic post-translational modifications that alter DNA-templated processes, such as transcription, to facilitate malignant transformation. Although histone deacetylases are already being clinically targeted, the role of histone lysine acetyltransferases (KAT) in malignancy is less well characterized. We chose to study this question in the context of acute myeloid leukemia (AML), where, using in vitro and in vivo genetic ablation and knockdown experiments in murine models, we demonstrate a role for the epigenetic regulators CBP and p300 in the induction and maintenance of AML. Furthermore, using selective small molecule inhibitors of their lysine acetyltransferase activity, we validate CBP/p300 as therapeutic targets in vitro across a wide range of human AML subtypes. We proceed to show that growth retardation occurs through the induction of transcriptional changes that induce apoptosis and cell-cycle arrest in leukemia cells and finally demonstrate the efficacy of the KAT inhibitors in decreasing clonogenic growth of primary AML patient samples. Taken together, these data suggest that CBP/p300 are promising therapeutic targets across multiple subtypes in AML.

Figure 1

Cbp^−/− cells are rapidly outcompeted by Cbp wt cells, under selective in vitro conditions, in MT2- and NHA9-driven AML. (a) Serial replating assays of MT2- and NHA9-driven leukemias demonstrate no difference in colony number or serial replating activity between transduced Cbp wt and Cbp^−/− progenitor cells. (b) Similar replating assays demonstrate no differences in the in vitro self-renewal potential of MT2 and NHA9 AML murine cell lines generated from Cbpfl/fl progenitors following expression of either Cre-puro or an empty puro vector, as both cell lines retained serial replating potential post-Cbp excision. (c) Genotyping of pooled colonies at the end of each round of replating revealed serial re-emergence of the un-excised Cbp allele, in the NHA9 and MT2, but not in the ME immortalized murine cell lines. *P < 0.05.

Figure 2

Cbp confers a selective advantage during initiation/progression of MT2-driven AML, under selective in vivo conditions. (a) Cbp wt and Cbp^−/− progenitor cells were transduced with MT2 and transplanted into lethally irradiated recipients. Genotyping of the Cbp wt and Cbp^−/− progenitor cells post-transduction confirmed almost complete excision of the Cbp allele. However, genotyping of leukemic cells revealed reemergence and clonal expansion of the un-excised Cbp allele. Furthermore, enrichment of leukemic cells via GFP sorting demonstrated only non-recombined cells, suggesting that Cbp loss confers a significant growth disadvantage during leukemia induction. All recipient mice succumbed to AML within 4 months post-transplantation. (b) c-kit⁺ Cbpfl/fl;Mx1-Cre⁺ BM cells were transduced with MT2 and transplanted into lethally irradiated recipients. The leukemias that arose in the primary recipients were transplanted into secondary animals. Excision of the Cbp allele in the secondary recipient animals resulted in decreased penetration of disease (only 40% of animals developed AML). Representative flow cytometry demonstrates that no GFP⁺ cells could be detected in the peripheral blood of non-diseased animals at the end of the experiment (d120).

Figure 3

Functional redundancy of Cbp and p300 during myeloid transformation. (a) p300 expression following lentiviral transduction of Cbp^−/− cells, using two different shRNAs. Expression was assessed by q-PCR using beta-actin as a reference gene. (b) sh-mediated knockdown of p300 decreases clonogenic potential of MT2 and NHA9 AML cell lines, particularly on a Cbp^−/− background. **P < 0.01.

Figure 4

Pharmacological inhibition of CBP and P300 suppresses the growth and decreases clonogenic potential of multiple AML cell lines in vitro. (a) C646 treatment of normal murine BM cells (n = 3) does not lead to significant changes of the number, or the types of colonies produced in serial replating assays. (b) C646 suppresses the growth of the NHA9 immortalized murine cell line in liquid culture and in methylcellulose assays. (c) Ten different human AML cell lines were tested against C646, over a period of 12 days in liquid culture conditions. The majority of these were responsive to C646 treatment (e.g., Kasumi-1), with 7/10 cell lines demonstrating a >50% decrease in cell numbers on day 12, compared with DMSO control. Two cell lines showed no response to CBP/p300 KAT inhibition (K562 and MOLM13). (d) Similarly, methylcellulose assays revealed a significant decrease in clonogenic potential in 8/10 human AML cell lines treated with C646. (e) Treatment with C646 induces apoptosis in the sensitive (Kasumi-1) but not in the resistant (K562) cell lines, as measured by Annexin/7-AAD staining. (f) Induction of apoptosis in the sensitive cell lines is accompanied by a modest G1 cell-cycle arrest (assessed by PI staining), however this is lacking in the resistant K562 cell line. *P < 0.05; **P < 0.01; ***P < 0.0001.

Figure 5

C646 treatment results in distinct transcriptional and chromatin acetylation changes associated with genomic integrity in sensitive AML cell lines. (a) Volcano plots for C646 vs DMSO treated samples (KG-1, Kasumi-1 and K562), showing fold-change (log₂) and P-value significance level (log₁₀) for all genes. A larger number of genes are differentially regulated in the sensitive (KG-1 and Kasumi-1) versus the resistant (K562) cell line. (b) Log fold change correlation plot for all genes between the sensitive KG-1 and Kasumi-1 following treatment, demonstrating a high degree of similarity in the transcriptional changes induced by C646. (c) Venn diagram of genes significantly differentially regulated (1.5 FC; < 0.05 adj. P-value) upon C646 treatment, between the different cell lines (left panel). Heatmap for the common list of genes (87 genes) found to be deregulated upon treatment in KG-1 and Kasumi-1 but not in K562 cells (right panel). (d) The ’common’ gene list was subjected to a Gene Ontology (Molecular Function) over-representation analysis. The significant results were displayed using a heatmap to highlight the percentage of shared gene between the categories, and demonstrates enrichment for processes including cell-cycle control, mitosis and DNA replication and repair. (e) ChIP-PCR analysis of H3K18 acetylation levels (24 h post-treatment) in the promoter regions of selected candidates from the list of the 87 downregulated genes. *P < 0.05; **P < 0.01.

Figure 6

C646 treatment decreases growth of primary human AML samples. (a) C646 treatment of normal human CD43+cells (n = 2) did not alter their clonogenic potential. (b) No induction of apoptosis was observed following treatment of normal CD34+ cells with C646, as measured by Annexin/7-AAD staining. (c) By contrast, a significant reduction in colony numbers was seen in 19/29 primary AML samples exposed to the C646 compound. (d) A number of primary AML samples (n = 29), covering a range of molecular subtypes, with variable karyotypic mutational and prognostic status, as shown in this table were assessed for sensitivity to C646 (see text and Supplementary Figure 6).