Upping the antizyme: AZIN1 directs stem cell fate

In this issue of Blood, Wang et al¹ report that adenosine-to-inosine (A-to-I) RNA editing of antizyme inhibitor 1, Azin1, is a novel regulator of hematopoietic cell fate, capable of influencing self-renewal and differentiation at the stem cell level.

Hematopoietic stem cells have served as one of the main experimental models for decades.² The study of normal tissue-specific stem cells and the exploration of the capacity of cancer stem cells to self-renew and induce metastasis have been based on the biology of hematopoietic stem cells. Therefore, dissecting the regulatory networks that maintain stem cell function, such as quiescence, self-renewal, and differentiation, as Wang et al elegantly demonstrate, is both timely and impactful.

Enzymatic editing of RNA, and specifically A-to-I editing of AZIN1, has been implicated in the progression and therapeutic resistance of a wide range of malignancies.^3-8 Specifically, increased levels of RNA editing of AZIN1, mediated by adenosine deaminase acting on RNA-1 (ADAR1), have been shown to lead to disease progression and higher tumor-initiating potential in hepatocellular carcinoma.³ Similarly, both ADAR1 expression and AZIN1 RNA editing levels were found to be significantly elevated in colorectal and gastric cancers compared with healthy mucosa.^4,5 In addition, hyper-editing of AZIN1 has been demonstrated to be a prognostic factor for survival and metastasis in both colorectal and gastric cancers.^4,5 In non–small cell lung cancers, RNA editing of AZIN1 accelerated cell proliferation and promoted tumor cell migration.⁶ In malignant hematopoiesis, we and others discovered increased ADAR1-mediated editing of AZIN1 and other transcripts as part of an RNA editing cancer stem cell signature that was associated with disease progression in myeloproliferative neoplasms, chronic myeloid leukemia, and multiple myeloma.^7-10 In addition, deregulated RNA editing via ADAR1 promotes transformation of pre-leukemia stem cells into leukemia stem cells and increases malignant self-renewal capacity.¹⁰ Thus, RNA editing of AZIN1 has been implicated in conferring “stemness” to premalignant progenitor cells⁵ and warrants further investigation in its role in stem cell biology.

In the current study, Wang et al find translocation of Azin1 protein, AZI, from the cytoplasm into the nucleus on editing.¹ The authors identify the nuclear interaction of AZI with DEAD box polypeptide 1, DDX1, as key to the regulatory network governing hematopoietic stem cell maintenance. Moreover, they elucidate the A-to-I editomes for 12 murine adult hematopoietic cell populations, which is an important dataset and resource for future studies. After identifying Azin1 as a stage-specific editing site exclusive to hematopoietic stem and progenitor cells, the authors reveal the importance of RNA editing and Azin1 to hematopoietic stem cell regulatory gene expression and protein function through a series of in vitro and in vivo assays. Next, the authors perform immunoprecipitation and mass spectrometry, identifying DDX1 as one of the top 10 specific interacting proteins when comparing edited and nonedited Azin1. The interaction between AZI and DDX1 is specific to the nuclear location of edited AZI. Using chromatin immunoprecipitation, the authors show that AZI and DDX1 are involved in the transcriptional regulation of hematopoietic stem cells via genes bound by both proteins simultaneously, such as Plaur, Tlr2, and Plxnc1. In several in vitro knockdown and overexpression experiments, the functional importance of the AZI–DDX1 interaction to the self-renewal capacity of hematopoietic stem cells was shown. The editing of Azin1, resulting in nuclear translocation, enables binding to DDX1, which in turn induces further regulatory gene expression, leading to differentiation of hematopoietic stem cells and successful lineage reconstitution. In hematopoietic stem cells with nonedited Azin1, AZI remains in the cytoplasm and is therefore unable to bind to DDX1, resulting in failed reconstitution.

The main limitation of this study is the exclusive use of murine model systems, whereas ADAR1-mediated editing is predicated primarily on the presence of primate-specific Alu sequences. Future studies will need to confirm these data in human primary hematopoietic stem and progenitor cells. Moreover, there is no investigation into the effect of differential ADAR1 expression levels on A-to-I editing of Azin1 or effects of edited Azin1 on ADAR1 expression levels regarding a possible feedback loop mechanism. Nonetheless, the mechanistic insights into A-to-I edited Azin1 and its regulatory impact on stem cell function are novel and will facilitate future studies. Additionally, these data may inform the development of novel therapeutics targeting edited Azin1 or other downstream regulators. Future studies might focus on the downstream regulators the authors touch on in their last figure, such as Plaur and others, to investigate potentially druggable targets and to glean further understanding of major regulatory networks that AZI and DDX1 might govern.

Overall, this study demonstrates the vital role of ADAR1-mediated RNA editing of Azin1 in the maintenance of hematopoietic stem cell function, thereby setting the stage for developing RNA editing–targeted therapeutics for stem cell expansion and conversely preventing AZIN1-induced cancer stem cell generation by inhibiting this process when deregulated.