Sayuri Horikawa

SETBP1 Mutations Drive Leukemic Transformation in ASXL1-Mutated MDS

Mutations in ASXL1 are frequent in patients with myelodysplastic syndrome (MDS) and are associated with adverse survival, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) is not fully understood. Recently, it has been found that deletion of Asxl1 or expression of C-terminal-truncating ASXL1-MTs inhibit myeloid differentiation and induce MDS-like disease in mice. Here, we find that SET-binding protein 1 (SETBP1) mutations (SETBP1-MT) are enriched among ASXL1-mutated MDS patients and associated with increased incidence of leukemic transformation, as well as shorter survival, suggesting that SETBP1-MT play a critical role in leukemic transformation of MDS. We identify that SETBP1-MT inhibit ubiquitination and subsequent degradation of SETBP1, resulting in increased expression. Expression of SETBP1-MT, in turn, inhibited protein phosphatase 2A activity, leading to Akt activation and enhanced expression of posterior Hoxa genes in ASXL1-mutant cells. Biologically, SETBP1-MT augmented ASXL1-MT-induced differentiation block, inhibited apoptosis and enhanced myeloid colony output. SETBP1-MT collaborated with ASXL1-MT in inducing acute myeloid leukemia in vivo. The combination of ASXL1-MT and SETBP1-MT activated a stem cell signature and repressed the tumor growth factor-β signaling pathway, in contrast to the ASXL1-MT-induced MDS model. These data reveal that SETBP1-MT are critical drivers of ASXL1-mutated MDS and identify several deregulated pathways as potential therapeutic targets in high-risk MDS.

Human NAT10 is an ATP-dependent RNA acetyltransferase responsible for N4-acetylcytidine formation in 18 S ribosomal RNA (rRNA).

Background: Post-transcriptional modifications of rRNAs play important roles in biogenesis and function of ribosome. Results: NAT10 is an ATP-dependent RNA acetyltransferase responsible for N4-acetylcytidine formation of 18 S rRNA. Conclusion: NAT10 and ac4C1842 are required for pre-18 S rRNA processing. Significance: 40 S subunit formation is regulated by a single acetylation of 18 S rRNA, implying a regulatory mechanism for ribosome biogenesis by sensing the cellular energy budget. Human N-acetyltransferase 10 (NAT10) is known to be a lysine acetyltransferase that targets microtubules and histones and plays an important role in cell division. NAT10 is highly expressed in malignant tumors, and is also a promising target for therapies against laminopathies and premature aging. Here we report that NAT10 is an ATP-dependent RNA acetyltransferase responsible for formation of N4-acetylcytidine (ac4C) at position 1842 in the terminal helix of mammalian 18 S rRNA. RNAi-mediated knockdown of NAT10 resulted in growth retardation of human cells, and this was accompanied by high-level accumulation of the 30 S precursor of 18 S rRNA, suggesting that ac4C1842 formation catalyzed by NAT10 is involved in rRNA processing and ribosome biogenesis.

The molecular basis of myeloid malignancies

Myeloid malignancies consist of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and myeloproliferative neoplasm (MPN). The latter two diseases have preleukemic features and frequently evolve to AML. As with solid tumors, multiple mutations are required for leukemogenesis. A decade ago, these gene alterations were subdivided into two categories: class I mutations stimulating cell growth or inhibiting apoptosis; and class II mutations that hamper differentiation of hematopoietic cells. In mouse models, class I mutations such as the Bcr-Abl fusion kinase induce MPN by themselves and some class II mutations such as Runx1 mutations induce MDS. Combinations of class I and class II mutations induce AML in a variety of mouse models. Thus, it was postulated that hematopoietic cells whose differentiation is blocked by class II mutations would autonomously proliferate with class I mutations leading to the development of leukemia. Recent progress in high-speed sequencing has enabled efficient identification of novel mutations in a variety of molecules including epigenetic factors, splicing factors, signaling molecules and proteins in the cohesin complex; most of these are not categorized as either class I or class II mutations. The functional consequences of these mutations are now being extensively investigated. In this article, we will review the molecular basis of hematological malignancies, focusing on mouse models and the interfaces between these models and clinical findings, and revisit the classical class I/II hypothesis.

A C-terminal mutant of CCAAT-enhancer-binding protein α (C/EBPα-Cm) downregulates Csf1r, a potent accelerator in the progression of acute myeloid leukemia with C/EBPα-Cm.

Two types of CCAAT-enhancer-binding protein α (C/EBPα) mutants are found in acute myeloid leukemia (AML) patients: N-terminal frame-shift mutants (C/EBPα-N(m)) generating p30 as a dominant form and C-terminal basic leucine zipper domain mutants (C/EBPα-C(m)). We have previously shown that C/EBPα-K304_R323dup belonging to C/EBPα-C(m), but not C/EBPα-T60fsX159 belonging to C/EBPα-N(m), alone induced AML in mouse bone marrow transplantation (BMT) models. Here we show that various C/EBPα-C(m) mutations have a similar, but not identical, potential in myeloid leukemogenesis. Notably, like C/EBPα-K304_R323dup, any type of C/EBPα-C(m) tested (C/EBPα-S299_K304dup, K313dup, or N321D) by itself induced AML, albeit with different latencies after BMT; C/EBPα-N321D induced AML with the shortest latency. By analyzing the gene expression profiles of C/EBPα-N321D- and mock-transduced c-kit(+)Sca-1(+)Lin(-) cells, we identified Csf1r as a gene downregulated by C/EBPα-N321D. In addition, leukemic cells expressing C/EBPα-C(m) exhibited low levels of colony stimulating factor 1 receptor in mice. On the other hand, transduction with C/EBPα-N(m) did not influence Csf1r expression in c-kit(+)Sca-1(+)Lin(-) cells, implying a unique role for C/EBPα-C(m) in downregulating Csf1r. Importantly, Csf1r overexpression collaborated with C/EBPα-N321D to induce fulminant AML with leukocytosis in mouse BMT models to a greater extent than did C/EBPα-N321D alone. Collectively, these results suggest that C/EBPα-C(m)-mediated downregulation of Csf1r has a negative, rather than a positive, impact on the progression of AML involving C/EBPα-C(m), which might possibly be accelerated by additional genetic and/or epigenetic alterations inducing Csf1r upregulation.

A novel ASXL1–OGT axis plays roles in H3K4 methylation and tumor suppression in myeloid malignancies

ASXL1 plays key roles in epigenetic regulation of gene expression through methylation of histone H3K27, and disruption of ASXL1 drives myeloid malignancies, at least in part, via derepression of posterior HOXA loci. However, little is known about the identity of proteins that interact with ASXL1 and about the functions of ASXL1 in modulation of the active histone mark, such as H3K4 methylation. In this study, we demonstrate that ASXL1 is a part of a protein complex containing HCFC1 and OGT; OGT directly stabilizes ASXL1 by O-GlcNAcylation. Disruption of this novel axis inhibited myeloid differentiation and H3K4 methylation as well as H2B glycosylation and impaired transcription of genes involved in myeloid differentiation, splicing, and ribosomal functions; this has implications for myelodysplastic syndrome (MDS) pathogenesis, as each of these processes are perturbed in the disease. This axis is responsible for tumor suppression in the myeloid compartment, as reactivation of OGT induced myeloid differentiation and reduced leukemogenecity both in vivo and in vitro. Our data also suggest that MLL5, a known HCFC1/OGT-interacting protein, is responsible for gene activation by the ASXL1–OGT axis. These data shed light on the novel roles of the ASXL1–OGT axis in H3K4 methylation and activation of transcription.

Expression of mutant Asxl1 perturbs hematopoiesis and promotes susceptibility to leukemic transformation.

Additional sex combs like 1 (ASXL1) is frequently mutated in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP). Although loss of ASXL1 promotes hematopoietic transformation, there is growing evidence that ASXL1 mutations might confer an alteration of function. In this study, we identify that physiological expression of a C-terminal truncated Asxl1 mutant in vivo using conditional knock-in (KI) results in myeloid skewing, age-dependent anemia, thrombocytosis, and morphological dysplasia. Although expression of mutant Asxl1 altered the functions of hematopoietic stem cells (HSCs), it maintained their survival in competitive transplantation assays and increased susceptibility to leukemic transformation by co-occurring RUNX1 mutation or viral insertional mutagenesis. KI mice displayed substantial reductions in H3K4me3 and H2AK119Ub without significant reductions in H3K27me3, distinct from the effects of Asxl1 loss. Chromatin immunoprecipitation followed by next-generation sequencing analysis demonstrated opposing effects of wild-type and mutant Asxl1 on H3K4me3. These findings reveal that ASXL1 mutations confer HSCs with an altered epigenome and increase susceptibility for leukemic transformation, presenting a novel model for CHIP.

Novel working hypothesis for pathogenesis of hematological malignancies: combination of mutations-induced cellular phenotypes determines the disease (cMIP-DD).

Recent progress in high-speed sequencing technology has revealed that tumors harbor novel mutations in a variety of genes including those for molecules involved in epigenetics and splicing, some of which were not categorized to previously thought malignancy-related genes. However, despite thorough identification of mutations in solid tumors and hematological malignancies, how these mutations induce cell transformation still remains elusive. In addition, each tumor usually contains multiple mutations or sometimes consists of multiple clones, which makes functional analysis difficult. Fifteen years ago, it was proposed that combination of two types of mutations induce acute leukemia; Class I mutations induce cell growth or inhibit apoptosis while class II mutations block differentiation, co-operating in inducing acute leukemia. This notion has been proven using a variety of mouse models, however most of recently found mutations are not typical class I/II mutations. Although some novel mutations have been found to functionally work as class I or II mutation in leukemogenesis, the classical class I/II theory seems to be too simple to explain the whole story. We here overview the molecular basis of hematological malignancies based on clinical and experimental results, and propose a new working hypothesis for leukemogenesis.