Therapeutic Targeting of RUNX-NFATC2 Axis for Acute Promyelocytic Leukemia and T Cell Immunity
Authors: Kubota Hirohito , Furuichi Kana , Masuda Tatsuya , Tatsuta Toshiya , Hiramatsu Hidefumi , Takita Junko , Hiroshi Sugiyama , Adachi Souichi , Kamikubo Yasuhiko 
Published: November 2019

Runt-related transcription factor (RUNX) play pivotal roles in leukemogenesis and inhibition of RUNX has now been widely recognized as a novel strategy in anti-leukemic therapies. However, the role of RUNX remains elusive in acute promyelocytic leukemia (APL). Here, we demonstrate that targeting RUNX1-NFATC2 axis is effective strategy to suppress drug-resistant (DR)-APL cells. Considering the well-established roles of RUNX and NFATC2 in T cell immunity, we also apply targeting RUNX-NFATC2 strategy to suppress T cell activation and xenogeneic graft-versus-host disease (GVHD).
Firstly, to investigate whether RUNX1 is essential for APL proliferation and maintenance, we examined cell proliferation of all-trans retinoic acid (ATRA) resistant APL cell line, NB4, using shRNA-mediated knockdown of RUNX1. Profound knockdown of RUNX1 in NB4 cells led to growth suppression and apoptotic cell death. We found that NFATC2 is one of the most consistently up-regulated genes in RUNX1-high expressing APL cells derived from previously reported human clinical samples (GSE2550, GSE13159, GSE61804), and also has RUNX1-binding regions in the promoter from chromatin-immunoprecipitation and sequencing (ChIP-Seq) data (GSE22178, GSE31221). ChIP qPCR assay confirmed the actual binding of RUNX1 in the NFATC2 promotor region of NB4. In addition, luciferase reporter experiments showed NFATC2 promoter significantly increased its reporter activity by RUNX1 over-expression. These results confirmed RUNX1 directly upregulated NFATC2 transcriptional activity. Consistent with these findings, silencing of RUNX1 suppressed the expression of NFATC2. Besides, silencing of NFATC2 in NB4 cells suppressed cell growth and induced apoptotic cell death.
Next, the efficacy of our novel RUNX inhibitor: chlorambucil-conjugated pyrrole-imidazole polyamide (Chb-M'), which specifically binds to the consensus RUNX-binding sequence, was examined for NB4. Chb-M' was remarkably more effective against NB4 (IC50 value at nM level) than ATRA and arsenic trioxide. Consistent with association between RUNX1 and NFATC2, Chb-M' suppressed the expression of NFATC2 of NB4. Furthermore, Chb-M′ suppressed NB4 proliferation and NFATC2 expression in xenograft tumor model.
Since RUNX and NFATC2 are key regulators of T cell function, we next investigate whether targeting RUNX is also effective for T cell mediated diseases including GVHD. Previous reports showed that RUNX1 directly regulate Th1 cytokine genes such as IL2 and IFNG (Ono M et. al. Nature 2007). From public ChIp-Seq data (ENCODE project. Nature 2012), RUNX1 also binds to NFATC2 promotor region in mouse and human primary CD4 T cells and T-ALL cell line, Jurkat.
To study the efficacy of Chb-M' for allo-reactive T-cell activation and proliferation, we performed mixed lymphocyte reaction from different healthy donors using thymidine uptake assay. Compared to control, Chb-M' significantly reduced T cell proliferation. To examine the effect of Chb-M' for T cell cytokine expression, we stimulated peripheral blood mononuclear cells (PBMC) with PMA-ionomycin and measured cytokine expression by quantitative PCR. Results showed that Chb-M'moderately but significantly reduced IL2 , TNF , IFNG and NFATC2 mRNA expression. To investigate whether RUNX is essential for cytokine and NFATC2 expression, we silenced RUNX family by shRNA-knockdown of Jurkat E6.1 cell line. Knockdown of RUNX family reduced IL2 , TNF and NFATC2 expression. These results indicated targeting RUNX-NFATC2 axis is also effective for T cell activation and cytokine expression.
Finally, we examined in vivo effect of Chb-M' for xenogeneic GVHD mouse model by transplanting human PBMC onto immunodeficient mice. Compared to control, mice injected by Chb-M' showed almost no sign of GVHD assessed by clinical score and pathological score of lung and liver. Analysing peripheral blood of GVHD mice showed that especially CD4 T cell was decreased and GVHD-associated cytokines including TNF-α and GM-CSF were reduced in Chb-M' injected mice.
Taken together, we have shown that RUNX transcriptionally upregulated NFATC2, which is essential for APL proliferation and T cell activation. RUNX-NFATC2 axis can be a novel therapeutic target against DR-APL and GVHD.

Disclosures
No relevant conflicts of interest to declare.