Cell-Autonomous Feedback Loop of RUNX1-p53-CBFB in Acute Myeloid Leukemia Cells

Abstract

Although runt-related transcription factor 1 (RUNX1) and its associating core binding factor-β (CBFB) play pivotal roles in leukemogenesis and inhibition of RUNX1 has now been widely recognized as a novel strategy in anti-leukemic therapies, it remains elusive how leukemic cells acquire resistance to RUNX1 inhibition therapies. Here, we show evidence that p53 (TP53) and CBFB are sequentially up-regulated in response to RUNX1 depletion in acute myeloid leukemia (AML) cells. The up-regulated CBFB expression in turn contributes to the stabilization of RUNX1, thereby constituting the RUNX1-p53-CBFB auto-regulatory feedback loop. To address RUNX1 depletion-mediated cellular responses in AML cells, we first performed shRNA-mediated knockdown experiments in human AML-derived MV4-11 and MOLM-13 cells, and investigated the expression of other CBF family proteins such as RUNX2, RUNX3 and CBFB. As we have previously reported, the expression of RUNX2 and RUNX3 were consistently elevated upon RUNX1 depletion. It has been known that CBFB stabilizes the RUNX transcription complex and enhances its DNA-binding capability. Based on this knowledge, we had expected that depletion of RUNX1 possibly disrupts the stable RUNX1/CBFB complex and thus down-regulates CBFB protein expression by destabilizing it. Contrary to our expectations, however, the expression of CBFB was also significantly up-regulated in response to RUNX1 -silencing in AML cells. Knockdown of RUNX2 and/or RUNX3 in addition to RUNX1 further induced CBFB expressions. Considering that the anti-tumor potency of RUNX gene silencing is highly dependent on functional p53 with the sequence-specific transactivation capability, we have sought to examine a possible involvement of p53 in the transcriptional regulation of CBFB. Indeed, series of RUNX family knockdown experiments elucidated that the expression levels of CBFB induced are proportional to the extent of p53 induction. In addition, we found that the expression levels of p53 and CBFB are positively-correlated in de novo AML patients from two independent studies (GSE22845; n = 154, GSE21261; n = 96). In a good agreement with these observations, knockdown of p53 in AML cells caused obvious reduction in CBFB relative to the control. Furthermore, ChIP-qPCR assay validated the actual binding of p53 at the promoter regions of CBFB. Given these information, RUNX1 depletion-mediated up-regulation of CBFB is under the control of the accumulated p53, and thus creating an autonomous RUNX1-p53-CBFB feedback regulatory system in AML cells. Moreover, AML cells derived from relapsed cases exhibited higher CBFB expression levels compared to those from primary AML cells at diagnosis (GSE17855 and GSE52891, n = 23). Consistent with these observations, RUNX1 inhibition treatment-resistant MV4-11 cells established from 4-month in vitro selection with the presence of specific RUNX inhibitors expressed higher CBFB relative to RUNX1 inhibition-sensitive ones. Besides, the ectopic expression of CBFB conferred resistance to RUNX1 inhibition therapy. Although the majority of primary de novo AML cases harbor wild-type p53, a small number of AML patients carry p53 mutations. According to our RUNX1-p53-CBFB loop working model, the stabilized mutant p53 possibly augments this feedback regulatory loop through the direct transactivation of CBFB and potentially contributes to the acquired resistance to RUNX1-inhibition therapy. We thus asked whether p53 mutants could up-regulate CBFB expression. To this end, we have prepared 3 representative p53 mutants (R175H, R248W, R273C) and transfected them into HEK293T cells. Intriguingly, these mutants significantly induced CBFB expressions. In addition, deep sequencing of genomic DNA extracted from the above-mentioned RUNX1 inhibition-resistant AML cells revealed that the majority of them acquire p53-stabilizing point mutations during treatment, possibly contributing to the enhanced transactivation of CBFB . These observations strongly suggest the RUNX inhibition-mediated treatment permits the selective proliferation of p53 -mutated AML cells and these cells acquire the enhanced tumorigenicity through the RUNX1-p53-CBFB axis. Collectively, our present results underscore the importance of RUNX1-p53-CBFB regulatory loop in the development and/or maintenance of AML cells, which could be targeted in strategizing anti-leukemia therapies. DisclosuresNo relevant conflicts of interest to declare.