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Myelodysplastic syndrome (MDS) is a group of hematopoietic disorders with limited treatment options. Anemia is a common symptom in MDS, and although erythropoiesis-stimulating agents such as erythropoietin, lenalidomide, and luspatercept are available to treat anemia, many MDS patients do not respond to these first-line therapies. Therefore, alternative drug development strategies are needed to improve therapeutic efficacy. Splicing modulators to correct splicing-related defects have shown promising results in clinical trials. Targeting differentiation of early erythroid progenitors to increase the erythroid output in MDS is another novel approach, which has shown encouraging results at the pre-clinical stage. Together, these therapeutic strategies provide new avenues to target MDS symptoms untreatable previously.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis and frequent progression to leukemia. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually dominate the bone marrow space. Despite several studies implicating mesenchymal stromal or stem cells (MSCs), a principal component of the HSC niche, in the inhibition of normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. Here, we demonstrate that both human and mouse MDS cells perturb bone metabolism by suppressing the osteolineage differentiation of MSCs, which impairs the ability of MSCs to support normal HSCs. Enforced MSC differentiation rescues the suppressed normal hematopoiesis in both in vivo and in vitro MDS models. Intriguingly, the suppression effect is reversible and mediated by extracellular vesicles (EVs) derived from MDS cells. These findings shed light on the novel MDS EV-MSC axis in ineffective hematopoiesis.

A growing number of large-scale genomic studies of cancer have uncovered mutations that drive cancer by perturbing co- and post-transcriptional regulation of gene expression. These include alterations that impact each phase of RNA processing, including splicing, transport, editing, and decay of messenger RNA. The discovery of these events illuminates a number of novel therapeutic vulnerabilities generated by aberrant RNA processing in cancer, several of which have progressed to clinical development. Mutations in the spliceosomal genes SRSF2, U2AF1 and SF3B1 are commonly found in patients with leukemia and are among the most common class of genetic alterations in myelodysplastic syndromes (MDS), clonal hematopoiesis, and chronic lymphocytic leukemia (CLL). These mutations occur at highly restricted amino acid residues, are always heterozygous, and never co-occur with one another. Work from our group and others has identified that these mutations alter RNA splicing in a manner distinct from loss of function and drive development of aberrantly spliced mRNAs, a portion of which are required for disease maintenance. In contrast to splicing factors affected by hotspot mutations, functional alterations induced by loss of functions in the RNA splicing factor ZRSR2 are less understood. Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles for the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections between minor introns, hematopoiesis, and cancers are unclear. These events will be discussed and provide novel insights into the role of mutant splicing factors in the pathogenesis of leukemia.

Epigenetic regulators, ASXL1, DNMT3A, and TET2 are frequently mutated in clonal hematopoiesis (CH). Dnmt3a- or Tet2-deficient mice increase self-renewal of hematopoietic stem cells (HSCs), suggesting that mutations in DNMT3A and TET2 provoke clonal expansion of hematopoietic cells, leading to subsequent CH in humans. On the contrary, ASXL1-mutated mice show reduced numbers and functions of HSCs. Thus, how ASXL1 mutations drive CH are not understood. Here, we investigated the effects of ASXL1 mutations on physiological aging of HSCs using knockin (KI) mice expressing a C-terminally truncated form of ASXL1 (ASXL1-MT). We found that HSCs expressing ASXL1-MT exhibited a competitive disadvantage after transplantation. On the other hand, in a genetic mosaic mouse, they displayed a growth advantage to occupy the HSC compartment over time, recapitulating CH in humans. As a mechanism by which ASXL1-MT causes CH, we show that ASXL1-MT binds and stabilizes phosphorylated Akt to activate Akt/mTOR pathway in coordination with Bap1. Activated Akt/mTOR induces dysregulated cell cycle progression and proliferation of HSCs. Meanwhile, it also compromises HSC functions along with activated mitochondrial metabolism, ROS overproduction, and increased DNA damage. Treatment with an mTOR inhibitor rapamycin suppressed aberrant proliferation of HSCs and ameliorated dysregulated hematopoiesis in aged ASXL1-MT KI mice. Taken together, ASXL1-MT impairs function of HSCs, whereas it confers a clonal advantage on HSCs during aging specifically in native hematopoiesis. ASXL1 mutation-mediated expansion of HSCs associated with increased DNA damage can induce development of CH, resulting in malignant transformation with secondary mutations. Akt/mTOR signaling could be a therapeutic target to individuals with CH harboring ASXL1 mutations.

In this issue of Cancer Cell, Obeng et al. identify the consequences of expressing the most common mutation in the spliceosomal gene SF3B1 on hematopoiesis. The knockin mouse model described represents a valuable tool to dissect the effects of SF3B1 mutations on transformation, splicing, and less well-characterized functions of SF3B1.

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.

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.