Reduction of RUNX1 transcription factor activity by a CBFA2T3-mimicking peptide: application to B cell precursor acute lymphoblastic leukemia

Abstract

Background: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia.

Methods: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models.

Results: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation.

Conclusions: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.

Keywords: AML1; CBFA2T3; Childhood leukemia; Driver loop; ETO2; Inhibitor; NHR2; RUNX1; Transcription factor.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

RUNX1 and CBFA2T3 mRNA level are positively correlated in ETV6-RUNX1 BCP-ALL. a and b The graphs represent the mRNA expression level of CBFA2T3 and RUNX1 in human pediatric ETV6-RUNX1 BCP-ALL bone marrow mononuclear cells and the other non-ETV6-RUNX1 BCP-ALL bone marrow mononuclear cells. Data of mRNA levels (expressed in Fragments Per Kilobase Million—FPKM) have been extracted from the St. Jude Children's Research Hospital RNA-Seq Pediatric Cancer Data Portal [31]. For a ****p < 0.0001 in t-test between both conditions, for b a Pearson correlation has been performed. ns, non significant. Mean data and numbers of samples (n) are indicated above each graph. c Correlation between RUNX1 and CBFA2T3 mRNA levels originating from REH cells and Nalm6 cells that overexpress or are depleted for RUNX1 or CBFA2T3. Statistical analysis has been performed using Pearson correlation. n, number of samples per condition

Fig. 2

RUNX1 and CBFA2T3 interplay for their transcription. a and b ChIP-Seq profiles across the human RUNX1 (a) and CBFA2T3 (b) genes. Genomic tracks display ChIP-Seq profiles of RUNX1 from REH cells, Nalm6 cells and BCP-ALL patients, and of the histones H3K27ac, H3K4me3, and H3K4me1 from REH and Nalm6 cells. RUNX1 ChIP-Seq from bone marrow mononuclear cells isolated from three pre-B acute lymphoblastic leukemia patients (BCP-ALL) are also displayed. ChIP-Seq reads were aligned to the reference human genome version GRCh37 (hg19). Each genomic regions of RUNX1 and CBFA2T3 genes that are subsequently studied are indicated by boxes. c–e Luciferase assays with a plasmid containing the RUNX1 promoter (chr21: 940,581–940,336(C), CBFA2T3 enhancer (chr16:89,045,181–89,045,538) (D) and a repetition of RUNX1-consensus motif (E) upstream a minimal promoter and a luciferase ORF, in presence of RUNX1 and CBFA2T3 expressing plasmids in HEK293 cells. Note that the HEK293 cells do not endogenously express RUNX1 and CBFA2T3. Luciferase levels (Firefly luciferase/Renilla luciferase) are represented using a scatter dot plot indicating the means and S.D. NS: non-significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 in Mann–Whitney tests compared to the control condition

Fig. 3

CBFA2T3 and RUNX1 colocalize. a, b, c, e and f Quantitation of protein co-localization per nucleus and visualized by Proximity Ligation Assay (PLA) dots in REH cells (a, e), Nalm6 cells (b, f) and BCP-ALL patient cells (c), presented with the mean values ± S.D. Antibodies used are indicated under each plot. Positive controls (total CBFA2T3, where primary antibodies against two different epitopes of CBFA2T3 were used) and negative controls (only one anti-CBFA2T3) were included. One representative experiment of at least two independent experiments is shown. The data are normalized against total CBFA2T3. The mean value is indicated above each plot. The positive threshold value is represented by the dotted line (set at two S.D over the background signal as described in [25]). NS: non-significant, * p < 0.05, **** p < 0.0001 in Fisher’s exact test compared to the negative control condition. d Co-immunoprecipitation (co-IP) using (left panel) IgG or RUNX1 antibody in REH cells, and (in right panel) anti-Flag antibody in HEK293 cells expressing RUNX1-Halotag and/or CBFA2T3-Flag plasmids. Western blots were performed with RUNX1 and CBFA2T3 antibodies. Molecular weights are indicated on the right. g Density plots of CBFA2T3 Chip-Seq signals into RUNX1-bound regions or random regions in REH and Nalm6 cells. h ChIP-Seq profiles across the human CBFA2T3 gene. Genomic tracks display ChIP-Seq profiles for RUNX1 and CBFA2T3 from REH cells. ChIP-Seq reads were aligned to the reference human genome version GRCh37 (hg19). Two enhancers at + 9 kb and − 2 kb have been focused on. i Logo corresponding to the RUNX1 enriched motif for CBFA2T3 regions in REH cells. REH and Nalm6 CBFA2T3 Chip-Seq have been analyzed with the Analysis of Motif Enrichment of the MEME suite [32]. The optimal enrichment p-value of the motif according to the Fisher’s exact test, adjusted for multiple tests using a Bonferroni correction is indicated

Fig. 4

The NHR2 domain of CBFA2T3 is involved in the interaction between RUNX1 and CBFA2T3. a Representation of the full-length and truncated CBFA2T3 and RUNX1 proteins used to the functional domain analysis. NHR, Nervy Homology Region; TD, Transactivation domain; ID, inhibitory domain. b, c Co-immunoprecipitation (IP) using anti-Flag antibody in HEK293 cells expressing various full-length and truncated proteins for CBFA2T3 (b) and RUNX1 (c) described in (a). Western blots were performed with RUNX1, Myc and CBFA2T3 antibodies. Molecular weights are indicated on the right. d Quantitation of protein co-localization per nucleus and visualized by Proximity Ligation Assay (PLA) dots in HEK293 cells expressing CBFA2T3NHR2-Myc with either RUNX1-Halotag or CBFA2T3-Flag, presented with the mean values ± S.D. Antibodies used are anti-RUNX1, anti-Myc and anti-Flag. Positive control (total RUNX1) and negative control were included. The data are normalized against total RUNX1. The mean value is indicated above each plot. The positive threshold value is represented by the dotted line as described in [25]. ****p < 0.0001 in Fisher’s exact test compared to the negative control condition

Fig. 5

Free NHR2 domain of CBFA2T3 disrupts the protein complex formed by RUNX1 and CBFA2T3. a and b Quantitation of RUNX1 and CBFA2T3 protein co-localization per nucleus and visualized by PLA dots in REH cells (a) and HEK293 cells (b) in presence or absence of CBFA2T3NHR2-Myc, presented with the mean values ± S.D. The mean value is indicated above each plot. The statistical analyses are run with parametric t-tests. *p < 0.1; ****p < 0.0001. The right panel shows a set of pictures of PLA between RUNX1 and CBFA2T3 for HEK293 cells in presence or absence of CBFA2T3NHR2-Myc. The PLA dots are in purple, nuclei are in blue (DAPI). Bar: 10 um. c Co-immunoprecipitation (IP) using anti-Flag antibody in HEK293 cells expressing RUNX1-Halotag, CBFA2T3-Flag or CBFA2T3NHR2-Myc. Western blots were performed with RUNX1, Myc and CBFA2T3 antibodies. Molecular weights are indicated on the right

Fig. 6

Free NHR2 domain of CBFA2T3 inhibits the cooperative effect of CBFA2T3 on RUNX1 activity. a–d Luciferase assays with the RUNX1-consensus motif repetition (a), the RUNX1 promoter (b), the CBFA2T3 enhancer (c) and the c-KIT enhancer (d) upstream a minimal promoter and a luciferase ORF, in presence of RUNX1 and CBFA2T3 or CBFA2T3NHR2 expressing plasmids in HEK293 cells. Luciferase levels (Firefly luciferase/Renilla luciferase) are represented using a scatter dot plot indicating the means and S.D. ns: non-significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 in Mann–Whitney tests compared to the control condition. e Relative mRNA expression of RUNX1, CBFA2T3 and c-KIT measured by RT-qPCR in REH cells, and REH expressing CBFA2T3NHR2 cells (REH+NHR2). Results are presented in-terms of a fold change after normalizing with ABL mRNA. f Schematic representation of the activation loop between RUNX1 and CBFA2T3. Expression of the free NHR2 domain interrupts this loop

Fig. 7

Expression of free NHR2 domain of CBFA2T3 delays BCP-ALL cell proliferation. a and b Proliferation curves from REHshcontrol, REHshRUNX1, REHshCBFA2T3−1, REHshCBFA2T3−2 (a), or REHshcontrol and REH+NHR2 cells (b). Three experiments are represented for each condition. For more readability, statistical analyses have been run only for the last day (day 7) and compared to the condition REHshcontrol. * p < 0.05. c, d Kaplan–Meier survival curves from immunodeficient NOD/scid IL2 Rg null mice xenografted with 100,000 cells of REH, REHshRUNX1, REHshCBFA2T3−1, REHshCBFA2T3−2 (C), or REH+NHR2 (D) (n = 7–8 per group). The general condition of mice was monitored daily until experiment ended. Mantel-Cox statistical tests compared to the condition REHshcontrol. **p < 0.01, ***p < 0.001. e We propose the model in which CBFA2T3 acts as an activator of RUNX1 transcription activity and sustains cell proliferation. Presence of free NHR2 domain disrupts this activation complex and slows down proliferation