Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia–initiating CBFB::MYH11 oncofusion protein
Ryan B. Day, … , Christopher A. Miller, Timothy J. Ley
Ryan B. Day, … , Christopher A. Miller, Timothy J. Ley
Published December 7, 2023
Citation Information: J Clin Invest. 2024;134(4):e176311. https://doi.org/10.1172/JCI176311.
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Research Article Oncology

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia–initiating CBFB::MYH11 oncofusion protein

Abstract

Several canonical translocations produce oncofusion genes that can initiate acute myeloid leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase (TurboID) in-frame with 3 favorable-risk AML oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11) and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID–interacting proteins were largely cytoplasmic, probably because of an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.

Authors

Ryan B. Day, Julia A. Hickman, Ziheng Xu, Casey D.S. Katerndahl, Francesca Ferraro, Sai Mukund Ramakrishnan, Petra Erdmann-Gilmore, Robert W. Sprung, Yiling Mi, R. Reid Townsend, Christopher A. Miller, Timothy J. Ley

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Figure 8

RUNX1/CBFB expression ratio is disrupted in human AML.

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RUNX1/CBFB expression ratio is disrupted in human AML. (A) TCGA LAML CB...
(A) TCGA LAML CBFB RNA-Seq data for the indicated healthy donor cells or AMLs. Black dashed line, mean healthy donor expression; red dashed line, mean AML expression. CBFB mRNA levels are lower in all AMLs, relative to healthy donor CD34+ cells. Each point represents an individual sample, bar indicates mean, box indicates 95% confidence interval, whiskers indicate value range. (B) Ratio of normalized, length-scaled RUNX1/CBFB mRNA expression. Black dashed line, 1:1 ratio; red dashed line, mean AML ratio. All AMLs have elevated RUNX1/CBFB ratios relative to healthy donor samples; CBFB::MYH11, RUNX1, and CBFB-mutated AMLs have the highest ratios. (C) Representative Jess blot (total of 7 experiments) of human non-CBFB::MYH11 (n = 6 patients) or CBFB::MYH11 (n = 14 patients) AML protein lysates for CBFB. Upper band (red box) indicates CBFB::MYH11; lower band (blue box) indicates WT CBFB. (D) CBFB::MYH11 to CBFB ratio in CBFB::MYH11 AMLs (n = 14 patients). Each point represents 1 patient; for patients in whom a sample was assayed more than once, point indicates mean of all assays. Dotted line, 1:1 ratio. The average CBFB::MYH11/CBFB ratio was 4.5:1, indicating that CBFB::MYH11 protein is more abundant than CBFB protein in primary human AMLs. (E) CBFB mRNA read counts from the validation cohort (Figure 7E) normalized to healthy donor CD34+ expression mean, grouped by exons 1–5 (unaffected by CBFB::MYH11 translocation) or exon 6 (lost with CBFB::MYH11 translocation). CBFB exons 1–5 expression is similar in CBFB::MYH11, RUNX1::RUNX1T1, and NPM1c AMLs, suggesting that CBFB locus transcriptional activity is unaffected by CBFB::MYH11 translocation. CBFB exon 6 reads are decreased by approximately 50% relative to exons 1–5 in CBFB::MYH11 AML, consistent with translocation-induced loss of one CBFB exon 6 allele. Paired 2-tailed t test between exons 1–5 and exon 6 reads within each group, ***q < 0.001.