The recurrent AML-associated CBFBD87GDSY mutation has hypomorphic function and is mechanistically distinct from CBFB:MYH11

Abstract Core Binding Factor (CBF) is a heterodimeric transcription factor composed of one alpha subunit (predominantly RUNX1 in hematopoietic cells), and one beta subunit (CBFB). RUNX1 is the DNA-binding subunit of the complex, while CBFB stabilizes the RUNX1-DNA interaction and protects the complex from ubiquitination and degradation. CBF is critical for hematopoiesis, and mice deficient in either CBFB or RUNX1 have severe hematopoietic defects. Dysregulation of CBF, through CBFB or RUNX1 mutation/rearrangement or transcriptional dysregulation is essentially universal in AML (Day et al. JCI 2023). We and others recently described a novel insertional CBFB mutation, CBFBD87GDSY, that is recurrent in human AML (Ryland et al. Blood 2022). While this is the only recurrent CBFB mutation in AML described to date, its mechanism of action is unknown; a better understanding of how CBFBD87GDSY promotes leukemogenesis may provide insight into how CBF dysregulation promotes leukemia development and maintenance. We recently showed that the CBFB::MYH11 oncofusion protein sequesters RUNX1 in the cytoplasm, leading to compensatory upregulation of RUNX1 mRNA (Day et al. JCI 2023). To determine if CBFBD87GDSY is also associated with RUNX1 upregulation, we analyzed publicly available RNA-Seq data from primary human AML CBFBD87GDSY cases, which similarly showed marked RUNX1 upregulation. To determine whether CBFBD87GDSY also alters CBFB or RUNX1 localization, we overexpressed CBFB::MYH11, wildtype CBFB (CBFBWT), or CBFBD87GDSY, fused to GFP, in the erythroleukemia cell line K562 and performed immunofluorescence for CBFB and RUNX1. While CBFB::MYH11 was predominantly cytoplasmic and mislocalized a pool of RUNX1 to the cytoplasm, both CBFBWT and CBFBD87GDSY were localized to the nucleus, and did not mislocalize RUNX1. We next utilized the Cbfbflox x Vav1-Cre conditional deletion model to delete Cbfb in all hematopoietic cells, resulting in profound hematopoietic defects and transcriptional dysregulation, including compensatory upregulation of Runx1 mRNA, exactly as seen in human AML. We retrovirally overexpressed cDNAs encoding CBFBWT, CBFBD87GDSY, or CBFBN104A (which disrupts RUNX1 binding), or an empty vector (EV) in wildtype and Cbfb-deficient murine HSPCs, and performed RNA-Seq 4 and 7 days after CBFB expression. While CBFBWT resulted in the normalization of Runx1 mRNA, CBFBD87GDSY did not; it caused only partial rescue of Runx1 expression, consistent with hypomorphic function. While CBFB::MYH11 expression in murine HSPCs results in compensatory Runx1 upregulation, CBFBD87GDSY expression in wildtype murine HSPC does not affect Runx1 mRNA expression, suggesting that CBFBD87GDSY is not a dominant negative. Finally, to test the functional consequences of CBFBD87GDSY in human cells, we used CRISPR/Cas9 to delete CBFB or AAVS1 (as a control) in K562 cells using two distinct CBFB CRISPR guides. We then retrovirally expressed CBFBWT, CBFBD87GDSY, or CBFBN104A, and measured cell growth using the Incucyte platform. Inactivation of CBFB resulted in severely impaired growth of K562 cells, relative to cells edited with AAVS1 CRISPR. Expression of CBFBWT in CBFB-deficient K562 cells rescued the growth defect caused by CBFB inactivation, while CBFBD87GDSY and CBFBN104A expression did not, suggesting that they are hypomorphic mutations. Consistent with the lack of a transcriptional effect of CBFBD87GDSY in wildtype murine HSPCs, expression of CBFBD87GDSY in K562 cells with AAVS1 CRISPR did not impair cell growth, suggesting that CBFBD87GDSY does not act as a dominant negative. In summary, CBFBD87GDSY acts as a hypomorphic allele in both a human AML cell line and primary murine HSPCs, and is mechanistically distinct from CBFB::MYH11. A conditional CbfbD87GDSY x Vav1-Cre knock-in mouse has been generated and is viable. CbfbD87GDSY mRNA is expressed at similar levels to CbfbWT mRNA. As expected, no abnormalities were observed in baseline hematopoiesis. Additional studies are ongoing to determine how CBFBD87GDSY affects the CBFB-RUNX1 interaction, RUNX1 protein expression, CBF transcriptional activity, and leukemogenic potential, are in process.