A novel Menin-MLL1 inhibitor, DS-1594a, prevents the progression of acute leukemia with rearranged MLL1 or mutated NPM1.

BACKGROUND: Mixed lineage leukemia 1-rearranged (MLL1-r) acute leukemia patients respond poorly to currently available treatments and there is a need to develop more effective therapies directly disrupting the Menin‒MLL1 complex. Small-molecule-mediated inhibition of the protein‒protein interaction between Menin and MLL1 fusion proteins is a potential therapeutic strategy for patients with MLL1-r or mutated-nucleophosmin 1 (NPM1c) acute leukemia. In this study, we preclinically evaluated the new compound DS-1594a and its salts. METHODS: We evaluated the preclinical efficacy of DS-1594a as well as DS-1594a·HCl (the HCl salt of DS-1594a) and DS-1594a·succinate (the succinic acid salt of DS-1594a, DS-1594b) in vitro and in vivo using acute myeloid leukemia (AML)/acute lymphoblastic leukemia (ALL) models. RESULTS: Our results showed that MLL1-r or NPM1c human leukemic cell lines were selectively and highly sensitive to DS-1594a·HCl, with 50% growth inhibition values < 30 nM. Compared with cytrabine, the standard chemotherapy drug as AML therapy, both DS-1594a·HCl and DS-1594a·succinate mediated the eradication of potential leukemia-initiating cells by enhancing differentiation and reducing serial colony-forming potential in MLL1-r AML cells in vitro. The results were confirmed by flow cytometry, RNA sequencing, RT‒qPCR and chromatin immunoprecipitation sequencing analyses. DS-1594a·HCl and DS-1594a·succinate exhibited significant antitumor efficacy and survival benefit in MOLM-13 cell and patient-derived xenograft models of MLL1-r or NPM1c acute leukemia in vivo. CONCLUSION: We have generated a novel, potent, orally available small-molecule inhibitor of the Menin-MLL1 interaction, DS-1594a. Our results suggest that DS-1594a has medicinal properties distinct from those of cytarabine and that DS-1594a has the potential to be a new anticancer therapy and support oral dosing regimen for clinical studies (NCT04752163).

ASXL1 and SETBP1 mutations promote leukaemogenesis by repressing TGFß pathway genes through histone deacetylation.

Mutations in ASXL1 and SETBP1 genes have been frequently detected and often coexist in myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We previously showed that coexpression of mutant ASXL1 and SETBP1 in hematopoietic progenitor cells induced downregulation of TGFß pathway genes and promoted the development of MDS/AML in a mouse model of bone marrow transplantation. However, whether the repression of TGFß pathway in fact contributes to leukaemogenesis remains unclear. Moreover, mechanisms for the repression of TGFß pathway genes in ASXL1/SETBP1-mutated MDS/AML cells have not been fully understood. In this study, we showed that expression of a constitutively active TGFß type I receptor (ALK5-TD) inhibited leukaemic proliferation of MDS/AML cells expressing mutant ASXL1/SETBP1. We also found aberrantly reduced acetylation of several lysine residues on histone H3 and H4 around the promoter regions of multiple TGFß pathway genes. The histone deacetylase (HDAC) inhibitor vorinostat reversed histone acetylation at these promoter regions, and induced transcriptional derepression of the TGFß pathway genes. Furthermore, vorinostat showed robust growth-inhibitory effect in cells expressing mutant ASXL1, whereas it showed only a marginal effect in normal bone marrow cells. These data indicate that HDAC inhibitors will be promising therapeutic drugs for MDS and AML with ASXL1 and SETBP1 mutations.

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.

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.

Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia.

The DDX41 gene, encoding a DEAD-box type ATP-dependent RNA helicase, is rarely but reproducibly mutated in myeloid diseases. The acquired mutation in DDX41 is highly concentrated at c.G1574A (p.R525H) in the conserved motif VI located at the C-terminus of the helicase core domain where ATP interacts and is hydrolyzed. Therefore, it is likely that the p.R525H mutation perturbs ATPase activity in a dominant-negative manner. In this study, we screened for the DDX41 mutation of CD34-positive tumor cells based on mRNA sequencing and identified the p.R525H mutation in three cases among 23 patients. Intriguingly, these patients commonly exhibited acute myeloid leukemia (AML) with peripheral blood cytopenias and low blast counts, suggesting that the mutation inhibits the growth and differentiation of hematopoietic cells. Data from cord blood cells and leukemia cell lines suggest a role for DDX41 in preribosomal RNA processing, in which the expression of the p.R525H mutant causes a certain ribosomopathy phenotype in hematopoietic cells by suppressing MDM2-mediated RB degradation, thus triggering the inhibition of E2F activity. This study uncovered a pathogenic role of p.R525H DDX41 in the slow growth rate of tumor cells. Age-dependent epigenetic alterations or other somatic changes might collaborate with the mutation to cause AML.

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.

Truncation mutants of ASXL1 observed in myeloid malignancies are expressed at detectable protein levels.

Recent progress in deep sequencing technologies has revealed many novel mutations in a variety of genes in patients with myelodysplastic syndromes (MDS). Most of these mutations are thought to be loss-of-function mutations, with some exceptions, such as the gain-of-function IDH1/2 and SRSF2 mutations. Among the mutations, ASXL1 mutations attract much attention; the ASXL1 mutations are identified in a variety of hematologic malignancies and always predicts poor prognosis. It was found that the C-terminal truncating mutants of the ASXL1 or ASXL1 deletion induced MDS-like diseases in mouse. In addition, it has recently been reported that ASXL1 mutations are frequently found in clonal hematopoiesis in healthy elderly people, who frequently progress to hematologic malignancies. However, the underlying molecular mechanisms by which ASXL1 mutations induce hematologic malignancies are not fully understood. Moreover, whether ASXL1 mutations are loss-of-function mutations or dominant-negative or gain-of-function mutations remains a matter of controversy. We here present solid evidence indicating that the C-terminal truncating ASXL1 protein is indeed expressed in cells harboring homozygous mutations of ASXL1, indicating the ASXL1 mutations are dominant-negative or gain-of-function mutations; for the first time, we detected the truncated ASXL1 proteins in two cell lines lacking the intact ASXL1 gene by mass spectrometry and Western blot analyses. Thus, together with our previous results, the present results indicate that the truncating ASXL1 mutant is indeed expressed in MDS cells and may play a role in MDS pathogenesis not previously considered.

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.

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.