The Clinical, Molecular, and Mechanistic Basis of RUNX1 Mutations Identified in Hematological Malignancies

RUNX1 plays an important role in the regulation of normal hematopoiesis. RUNX1 mutations are frequently found and have been intensively studied in hematological malignancies. Germline mutations in RUNX1 cause familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). Somatic mutations of RUNX1 are observed in various types of hematological malignancies, such as AML, acute lymphoblastic leukemia (ALL), myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN), chronic myelomonocytic leukemia (CMML), and congenital bone marrow failure (CBMF). Here, we systematically review the clinical and molecular characteristics of RUNX1 mutations, the mechanisms of pathogenesis caused by RUNX1 mutations, and potential therapeutic strategies to target RUNX1-mutated cases of hematological malignancies.


INTRODUCTION
The RUNX1 transcription factor is a critical regulator of embryogenesis and definitive hematopoiesis in vertebrates. Since the somatic point mutation of RUNX1 was first identified two decades ago, RUNX1 has become known to be one of the most frequently mutated genes in a variety of hematological malignancies ( Fig. 1) (Deltcheva and Nimmo, 2017;Hayashi et al., 2017;Osato et al., 1999). Despite the improvement of technology for the detection of mutations and a deeper understanding of the diseases, there are still unanswered questions about the functional consequences of RUNX1 mutations in hematological malignancies, such as (1) the frequency of different RUNX1 mutations in various subgroups of hematological malignancies and their impact on prognosis; (2) the mechanisms of how RUNX1 mutations contribute to pathogenesis; and (3) the potential mechanism-based therapeutic strategies. In this review article, we describe the clinical and molecular characteristics of RUNX1 mutations, the mechanisms of pathogenesis caused by its mutations, and potential therapeutic strategies for those RUNX1-mutated cases.

GERMLINE MUTATION OF RUNX1 AND FPD/AML
Familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) is an autosomal dominant disorder characterized by quantitative and qualitative platelet abnor-malities and predisposition to AML (Online Mendelian Inheritance in Man [OMIM] No. #601399). To date, more than 70 families have been reported (Cavalcante de Andrade Silva et al., 2018;Latger-Cannard et al., 2016;Sood et al., 2017;Vormittag-Nocito et al., 2019). FPD/AML is caused by germline mutations of RUNX1, which is located at 21q22 and plays pivotal roles in the regulation of hematopoietic differentiation (Song et al., 1999). RUNX1 is essential for the development of hematopoietic stem cells (HSCs) in the embryonic stage. In adult hematopoiesis, however, it is dispensable for the maintenance of HSCs but required for megakaryocyte maturation and T lymphocyte-lineage differentiation (Ichikawa et al., 2004;Taniuchi et al., 2002). Loss-of-function or dominant-negative effect caused by mutated RUNX1 leads to the phenotype of FPD/AML (Cavalcante de Andrade Silva et al., 2018;Latger-Cannard et al., 2016;Vormittag-Nocito et al., 2019). Most of the mutations were clustered in the runt homology domain (RHD) and the c-terminal transactivation domain (TAD) with a few exceptions (Schlegelberger and Heller, 2017;Sood et al., 2017). FPD/AML was reported to transform to MDS/AML at a median onset age of 33 years old (Churpek et al., 2013). The median incidence rate of transformation is ranged from 35% to 44% in different studies (Godley, 2014;Owen et al., 2008a;2008b). A few cases transformed to other types of leukemia, such as T-ALL (Nishimoto et al., 2010) or CMML (Shiba et al., 2012). Compared with loss-of-function mutations, dominant-negative mutations of RUNX1 are correlated to a higher risk of developing hematological malignancies (Latger-Cannard et al., 2016). However, these RUNX1 mutations by themselves are not sufficient for the development of leukemias. Additional mutations in RUNX1 (a second mutation), CDC25C, epigenetic modifiers, splicing factors, and tumor suppressors were reported to coordinately induce myeloid malignancies (Antony-Debre et al., 2016;Preudhomme et al., 2009;Yoshimi et al., 2014). Mutations in ASXL1, TET2, IDH1, CEBPD, RB1, MLL2, FLT3-ITD, WT1, and SRSF2 have also been detected by next-generation sequencing (Schlegelberger and Heller, 2017).

RUNX1 MUTATION-RELATED ALL
RUNX1 mutations were detected in 15.5% to 18.3% of patients with T-ALL, 3.8% of patients with B-ALL (Grossmann et al., 2011a;, and in 9.2% of childhood patients with T-ALL. The incidence was higher in patients with early T-cell precursor (ETP) ALL, reaching 15.6%. RUNX1 mutations were reported to be associated with ETP ALL, which is a high-risk subtype of ALL lacking several T cell surface markers and exhibiting aberrant expression of myeloid and stem cell markers . Mutated RUNX1 in T-ALL was associated with older age and lower white blood cell count, but not platelet count, hemoglobin levels, gender or karyotype (Grossmann et al., 2011a). Mutated RUNX1 conferred a poor prognosis on early T-ALL patients with inferior OS (Grossmann et al., 2011a;.
In the sections above, we briefly introduced and summarized the clinical and molecular characteristics of RUNX1 mutations in each of hematological malignancies (Table 1).

MECHANISMS
The RUNX1 transcription factor is a key regulator of normal hematopoiesis and its functional disruption by point mutations is one of the major factors for developing hematological malignancies (Deltcheva and Nimmo, 2017). There are two major subtypes of RUNX1 mutations in hematological malignancies: (1) the RHD, in which many mutations have been identified and are involved in residues at the DNA binding interface; (2) the TAD, in which most mutations result in production of the proteins lacking all or part of the TAD. Most of the RUNX1 mutations are mono-allelic, and different mutation types contribute to different biological properties of RUNX1 protein and presumably to disease phenotype as well (Mangan and Speck, 2011). We will describe the mechanisms of pathogenesis caused by RUNX1 mutations according to the biological function of RUNX1.

RUNX1 mutations on stem cells
RUNX1 is required for the emergence of adult HSCs during embryonic development and for the maturation of different lineages from HSCs in adult BM (Hong et al., 2017). Loss of RUNX1 function is associated with a pre-leukemic state, probably owing to the expansion of HSCs and progenitor cells, as well as differentiation defects. To rescue RUNX1 mutations in HSCs, genome editing technologies such as CRISPR-Cas9 will hopefully accelerate the studies of the mutations, leading to a better understanding of the pathogenesis of leukemia and novel targeted treatments (Sood et al., 2017).

RUNX1 mutations on cell cycle and genomic instability
RUNX1 levels are increased at the G1-S phase and decreased during G2/M transition in hematopoietic cells. RUNX1 mutations may cause enhanced proliferation, attenuated mitotic checkpoint, and cell-cycle arrest. RUNX1 mutations can also cause genomic instability including increased DNA damage and impaired DNA repair. The potential therapeutic options for mutated RUNX1-associated abnormalities of cell cycle and genomic instability may come down to check point inhibitors and DNA repair inhibitors, which can bypass cells with DNA damage/impaired DNA repair to M phase (Goyama et al., 2015;Ito et al., 2015).

RUNX1 mutations on oncogenic signaling pathways
Mutations in RUNX1 are associated with alterations of various signaling pathways, such as WNT, BMP, TGF-β, RAS-ERK, Hippo-YAP1, and Notch, most of which have been described in cases of solid tumors. Notably, the possible involvement of RUNX1 mutations in WNT signaling has been shown in AML. WNT signaling controls cellular proliferation and differentiation and aberrant activation of WNT signaling has been reported in various tumors. RUNX1 mutations were closely associated with hypermethylation of the promoter of one of the WNT inhibitor gene, SFRP2, in AML. It is suggested that the WNT inhibitor hypermethylation might lead to aberrant activation of WNT signaling and interact with genetic alterations in the leukemogenesis (Hou et al., 2011).

RUNX1 mutation in ribosomal biogenesis
Loss-of-function mutations of RUNX1 were found to exhibit reduced ribosomal biogenesis in HSCs. RUNX1 directly binds to promoters of the genes encoding ribosomal RNA/proteins and regulates their transcription. Thus, RUNX1 mutations may cause low biosynthetic activity and confer stress resistance on HSCs, which provides a proliferative advantage to HSCs at the preleukemic stage (Cai et al., 2015;Deltcheva and Nimmo, 2017). In clinical trials, L-leucine is administrated to patients with Diamond-Blackfan anemia (DBA), which is caused by loss-of-function mutations in ribosomal protein genes. It has been shown that the treatment can improve anemia in the genetic DBA mouse models as well as DBA patients, possibly through mTOR activation, resulting in stimulation of protein translation (Ruggero and Shimamura, 2014). Thus, L-leucine might be a possible therapeutic option for RUNX1-mutated cases as well.

Hypoxic microenvironment
It has been reported that RUNX1 suppresses transactivation activity of hypoxia-inducible factor 1α (HIF-1α), while HIF-1α increases the activity of RUNX1 (Peng et al., 2008). HIF-1α is critical for cellular response to hypoxia and facilitates glycolysis but suppresses the TCA cycle. As most of the RUNX1 mutations cause loss of its function, RUNX1-mutated HSCs may have more activated HIF-1α pathway and glycolysis-biased metabolism. Metabolic rewiring to a hypoxia-like status is a hallmark of cancer as well as MDS and maintains stemness of tumor initiating cells (Hayashi et al., 2019). Thus, HIF-1α inhibitors or metabolic pathway modulators could be potential therapeutic strategies.
In conclusion, we summarized the mechanisms of pathogenesis caused by RUNX1 mutations and potential therapeutic strategies for RUNX1-mutated cases (Fig. 2).

CONCLUSIONS AND PERSPECTIVES
In this review, we briefly described the impact of RUNX1 mutations on clinical disease phenotypes and prognosis in hematological malignancies and the mechanisms of how RUNX1 mutations contribute to pathophysiology. RUNX1 mutations are frequently observed in various types of hematological malignancies and contribute to poor prognosis. RUNX1 is one of the most extensively studied molecules in hematopoiesis and leukemogenesis, and it functions in a variety of biological processes including cell differentiation, proliferation, cell cycle, DNA repair, apoptosis, ribosomal biogenesis, and metabolism. There are still many unknowns, such as how mutations affect this diverse function of RUNX1 and their clinical outcomes. Further elucidation is needed for a deeper understanding of RUNX1-associated hematological malignancies and for the development of better therapeutic strategies.

Disclosure
The authors have no potential conflicts of interest to disclose.

ACKNOWLEDGMENTS
This review work was supported partly by grants from Taub Foundation (to G.H.), National Institutes Health (NIH) (R01 We apologize to all scientists in the field whose important work could not be cited in this report due to space limitation.