Cluster Regulation of RUNX Family By "Gene Switch" Triggers a Profound Tumor Regression of Diverse Origins
Authors: Morita Ken , Suzuki Kensho , Maeda Shintaro , Mitsuda Yoshihide , Yano Ayaka , Yamada Yoshimi , Kiyose Hiroki , Tokushige Chieko , Liu Paul P. , Adachi Souichi , Sugiyama Hiroshi , Kamikubo Yasuhiko 
Published: October 2019

Abstract
Although Runt-related transcription factor 1 (RUNX1), a member of RUNX family and a distant relative of p53, has been generally considered to be a tumor suppressor, a growing body of evidence strongly suggests its pro-oncogenic property in acute myeloid leukemia (AML). Here we demonstrate that switching off RUNX cluster utilizing the newly synthesized compound, which specifically bound to a particular base sequence of DNA, was highly effective against leukemia as well as dismal-prognostic solid tumors arising from diverse origins in vivo. Firstly, to assess the RUNX1 loss in AML cells, we performed shRNA-mediated RUNX1 knockdown experiments. Silencing of RUNX1 stimulated cell cycle arrest at G0/G1 phase and simultaneously induced apoptosis in AML cells bearing wild-type p53. RUNX1 depletion induced remarkable induction of p53 as well as its target gene products and additive knockdown of p53 in these cell lines reverted the phenotype of RUNX1-depletion, indicating that RUNX1 is functionally dependent on proficient p53 pathway. In addition, cycloheximide chase assay revealed that RUNX1 negatively regulates p53 protein in AML cells. In silico data analysis of clinical gene expression array data sets and ChIP-seq experiments using anti-RUNX1 antibody identified 32 candidate genes potentially required for RUNX1-dependent degradation of p53. Among them, we focused on BCL11A and TRIM24, both of which are established mediators of p53 degradation. In accordance with these observations, knockdown of RUNX1 resulted in a significant down-regulation of BCL11A and TRIM24 both at mRNA and protein levels. ChIP-qPCR assay further validated the actual binding of RUNX1 at the promoter regions of these genes, and reintroduction of BCL11A or TRIM24 into RUNX1-silenced AML cells restored their proliferation speed to the control levels. These data suggests that RUNX1 depletion-mediated growth inhibitory effect on leukemia cells depends on p53 activation via transcriptional regulation of BCL11A and TRIM24. Though RUNX1 depletion was highly effective on proliferation of AML cells, a small sub-population of leukemia cells retained the proliferation potential even after the silencing of RUNX1. Since it has been shown that RUNX family member has a redundant function, we next examined the other RUNX family members such as RUNX2 and RUNX3 in RUNX1-knocked down AML cells. Under our tetracycline-inducible shRNA expression system, the expression levels of RUNX1-target genes were decreased at 24 h after RUNX1 knockdown, however, their expression levels were reciprocally increased at 48 h accompanied by increment of RUNX2 and RUNX3 expressions, suggesting that RUNX2 and RUNX3 might compensate for the loss of RUNX1 functions. ChIP-qPCR assay and luciferase reporter experiments confirmed that individual RUNX family member consistently suppressed the promoter activity of the other RUNX members. In accordance with these findings, additional knockdown of RUNX2, RUNX3 or both of them in RUNX1-depleted AML cells effectively repressed RUNX1-target gene expressions and completely suppressed their proliferation. Thus the simultaneous targeting of all RUNX family members as a cluster achieves more stringent control of leukemia cells. Since sequencing analysis of the functional gene alterations of RUNX family members revealed the existence of mutations in a mutual-exclusive manner not only in AML cells but also in various cancers, their functional redundancy in the maintenance of AML cells might be generally accepted. To achieve cluster regulations of RUNX, we conducted a synthesized molecule library screening and succeeded in extracting agents that could irreversibly block the RUNX cluster genes expression profiling through dismantling protein-DNA interactions sequence-specifically. These reagents were highly effective against leukemia as well as dismal-prognostic solid tumors arising from diverse origins in vitro. Furthermore, these reagents were exceptionally well-tolerated in mice and exerted excellent efficacy against xenograft mice models of AML, acute lymphoblastc leukemia, lung and gastric cancers, extending their overall survival periods in vivo. Together, this work identifies the crucial role of RUNX cluster in the maintenance and the progression of cancer cells, and the indicated gene switch technology-dependent its modulation would be a novel strategy to control malignancies.

Disclosures
No relevant conflicts of interest to declare.