Experimental Hematology

Loss and overexpression of FAT1 occurs among different cancers with these divergent states equated with tumor suppressor and oncogene activity, respectively. Regarding the latter, FAT1 is highly expressed in a high proportion of human acute leukemias relative to normal blood cells, with evidence pointing to an oncogenic role. We hypothesized that this occurrence represents legacy expression of FAT1 in undefined hematopoietic precursor subsets that is sustained following transformation, predicating a role for FAT1 during normal hematopoiesis. We explored this concept by using the Vavi-Cre strain to construct conditional knockout (cKO) mice where Fat1 expression was deleted at the hemopoietic stem cell stage. Extensive analysis of precursor and mature blood populations using multi-panel flow cytometry revealed no ostensible differences between Fat1 cKO mice and normal littermates. Further functional comparisons involving colony forming unit and competitive bone marrow transplantation assays support the conclusion that Fat1 is dispensable for normal murine haematopoiesis.

Mesenchymal stem/stromal cells (MSCs) within the bone marrow microenvironment (BMME) support normal hematopoietic stem and progenitor cells (HSPCs). However, the heterogeneity of human MSCs has limited the understanding of their contribution to clonal dynamics and evolution to myelodysplastic syndromes (MDS). We combined three MSC cell surface markers, CD271, VCAM-1 (Vascular Cell Adhesion Molecule-1) and CD146, to isolate distinct subsets of human MSCs from bone marrow aspirates of healthy controls (Control BM). Based on transcriptional and functional analysis, CD271+CD106+CD146+ (NGFR+/VCAM1+/MCAM+/Lin-; NVML) cells display stem cell characteristics, are compatible with murine BM- derived Leptin receptor positive MSCs and provide superior support for normal HSPCs. MSC subsets from 17 patients with MDS demonstrated shared transcriptional changes in spite of mutational heterogeneity in the MDS clones, with loss of preferential support of normal HSPCs by MDS-derived NVML cells. Our data provide a new approach to dissect microenvironment-dependent mechanisms regulating clonal dynamics and progression to MDS.

Excessive fetal growth is associated with DNA methylation alterations in human hematopoietic stem and progenitor cells (HSPC), but their functional impact remains elusive. We implemented an integrative analysis combining single-cell epigenomics, single-cell transcriptomics, and in vitro analyses to functionally link DNA methylation changes to putative alterations of HSPC functions. We showed in hematopoietic stem cells (HSC) from large for gestational age neonates that both DNA hypermethylation and chromatin rearrangement target a specific network of transcription factors known to sustain stem cell quiescence. In parallel, we found a decrease expression of key genes regulating HSC differentiation including EGR1, KLF2, SOCS3, and JUNB. Our functional analyses showed that this epigenetic programming was associated with a decreased ability for HSCs to stay quiescent. Taken together, our multimodal approach using single-cell (epi)genomics showed that human fetal overgrowth affects hematopoietic stem cells quiescence maintenance via epigenetic programming.

DOT1L is essential for early hematopoiesis but the precise mechanisms remain largely unclear. The only known function of DOT1L is histone H3 lysine 79 (H3K79) methylation. We generated two mouse models; a Dot1L-knockout (Dot1L-KO), and another possessing a point mutation in its methyltransferase domain (Dot1L-MM) to determine the role of its catalytic activity during early hematopoiesis. We observed that Dot1L-KO embryos suffered from severe anemia, while Dot1L-MM embryos showed minimal to no anemia. However, ex vivo culture of Dot1L-MM hematopoietic progenitors (HPCs) exhibited defective development of myeloid and mixed progenitors. DOT1L is a well-recognized, cell-type specific epigenetic regulator of gene expression. To elucidate the mechanisms underlying such diverse hematopoietic properties of Dot1L-KO and Dot1L-MM HPCs, we examined their whole transcriptomes. Extensively self-renewing erythroblast (ESRE) cultures were established using yolk sac (YS) cells collected on embryonic day 10.5 (E10.5). Dot1l-KO and Dot1l-MM cells expanded significantly less than the wildtype cells and showed slower progression through the cell cycle. Total RNA extracted from the wildtype and Dot1l-mutant ESRE cells were subjected to RNA-seq analyses. We observed that the majority (~82%) of the differentially expressed genes (DEGs) were upregulated in both of the Dot1L-mutants, which suggests that DOT1L predominantly acts as a transcriptional repressor in HPCs. We also observed that about ~40% of the DEGs were unique to either of the mutant group, suggesting that DOT1L possesses both methyltransferase domain-dependent and -independent functions. We further analyzed Gene Ontology and signaling pathways relevant to the DEGs common to both mutant groups and those that were unique to either group. Among the common DEGs, we observed upregulation of CDK inhibitors, which explains the cell cycle arrest in both of the Dot1L-mutant progenitors.

Hematopoiesis plays a critical role in maintaining a diverse pool of blood cells throughout human life. Despite recent efforts with single-cell data analyses, the nature of the early cell fate decisions and compartmentalization of progenitors remains contentious due to the sparsity and noise of the data. Using publically available single-cell RNA-Seq hematopoietic data from bone marrow, with three different matrix factorization approaches to recover associated gene modules from cell clusters reveals a tri-directional and hierarchically-structured transcriptional landscape of hematopoietic differentiation. We also devised a bootstrap method, which in combination with the above can better characterize the progenitor compartments and retrieve cellular hierarchies.

The transcription factor GATA2 has pivotal roles in hematopoiesis. Germline GATA2 mutations in patients result in GATA2 haploinsufficiency syndrome characterized by immunodeficiency, bone marrow failure, and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Clinical symptoms in GATA2 patients are diverse and mechanisms driving GATA2 related phenotypes largely unknown. To explore the impact of GATA2 haploinsufficiency on hematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow (KM). single nucleus (sn)-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a +3.5-4.1kb enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression. This is suggestive of an auto-regulatory feedback mechanism, where gata2b expression remains at sufficient levels after the loss of a single allele to maintain the HSPC pool. As a result, gata2b+/- chromatin is also more accessible in the erythroid and myeloid lineage, causing several defects. scRNA-seq data revealed a differentiation delay in erythroid progenitors, hallmarked by downregulation of intrinsic signals like cytoskeletal transcripts, aberrant proliferative signatures, and downregulation of Gata1a, a master regulator of erythropoiesis, likely preceding erythroid dysplasia. This shows that the cell intrinsic compensatory mechanisms for the maintenance of normal levels of Gata2b to maintain HSPC integrity result in aberrant lineage differentiation and a preleukemia syndrome.

Induced pluripotent stem cells (iPSCs) generated from patients with chronic myeloid leukemia (CML) have the potential for disease modeling to study disease pathogenesis and screening therapeutic interventions. In this study, we aimed to generate iPSCs from CD34+ hematopoietic progenitors of CML patients with varying responses to tyrosine kinase inhibitor (TKI) therapy. The generated CML-CD34-iPSC colonies displayed atypical "dome-shaped" morphology and underwent spontaneous differentiation in a few days. However, supplementation with imatinib (IM), the most widely used TKI to treat CML patients, in the culture medium improved the stability and maintenance of all isolated CML-CD34-iPSC colonies, allowing them to be maintained for more than 20 passages without significant differentiation. In contrast to previous studies, our results indicate that suppressing the BCR::ABL1 oncogenic pathway is essential for efficiently generating stable CML-iPSC colonies. Furthermore, we successfully differentiated these iPSCs to CD34+ hematopoietic progenitors both in the presence and absence of IM. This robust protocol for generating CML-iPSCs provides a valuable resource for disease modelling. The generated iPSCs will be a valuable tool for investigating CML pathophysiology, drug resistance mechanisms, and drug screening to identify novel and effective therapies for this disease.

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Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem cells (HSC) that are most frequently caused by acquired somatic mutations in JAK2. A number of conditional mouse models of JAK2-V617F-driven MPN have been generated that rely on Cre-LoxP mediated activation, resulting in polyclonal disease. To more closely mimic the monoclonal origin of human MPN, transplantations of single purified JAK2-mutant HSCs or bone marrow (BM) at limiting dilutions into lethally irradiated recipient mice have been previously performed. However, irradiation is known to alter the BM microenvironment and also to induce transient aplasia accompanied by elevated cytokine levels that promotes the expansion of the mutant clone. To overcome these limitations, we examined whether JAK2-V617F-mutant HSCs are able to engraft and initiate MPN in non-conditioned recipients. We found that BM from two different MPN models, one expressing the human JAK2-V617F, and another expressing the mouse Jak2-V617F, efficiently engrafted and initiated MPN in non-irradiated immunocompromised Rag2-/- recipients. MPN evolved even in transplantations at limiting dilutions, showing high competitiveness of single JAK2-mutant HSCs. Thus, JAK2-V617F mutant HSCs can outcompete resident non-mutated HSCs in the absence of elevated cytokine levels and without the need of emptying stem cell niches by irradiation. However, only BM from mice expressing the mouse Jak2-V617F engrafted and initiated disease in non-conditioned C57BL/6 mice, while BM from mice expressing the human JAK2-V617F was rejected, indicating that mouse Jak2-V617F is ignored by the immune surveillance. These results provide a possible explanation why JAK2-V617F is so frequently found in healthy individuals with clonal hematopoiesis.

Sickle cell disease (SCD) is caused by a single nucleotide transversion in exon 1 of the HBB gene that changes the hydrophobicity of adult globin ({beta}A), leading to substantial morbidity and reduced lifespan. Ex vivo autologous gene editing utilizing co-delivery of a designer nuclease along with a DNA donor template allows for precise homology-directed repair (HDR). These gene corrected cells when engrafted into the bone marrow (BM) can prove to be therapeutic and serves as an alternative to HLA-matched BM transplantation. In the current study, we extensively explored the role of single stranded oligonucleotide (ssODN) and recombinant adeno-associated 6 (rAAV6) donor template delivery to introduce a codon-optimized change (E6optE) or a sickle mutation (E6V) change following Crispr/Cas9-mediated cleavage of HBB in healthy human mobilized peripheral blood stem cells (mPBSCs). We achieved efficient HDR in vitro in edited cells and observed robust human CD45+ engraftment in the BM of NBSGW mice at 16-17 weeks. Notably, recipients of ssODN-modified HSC exhibited a significantly higher proportion of HDR-modified cells within individual BM, CD34+ and CD235+ compartments of both E6optE and E6V cohorts. We further assessed key functional outcomes including RNA transcripts analysis and globin sub-type expression. Our combined findings demonstrate the capacity to achieve clinically relevant HDR in vitro and in vivo using both donor template delivery method. The use of ssODN donor template-delivery is consistently associated with higher levels of gene correction in vivo as demonstrated by sustained engraftment of HDR-modified HSC and erythroid progeny. Finally, the HDR-based globin protein expression was significantly higher in the E6V ssODN-modified animals compared to the rAAV6-modified animals confirming that the ssODN donor template delivery outperforms rAAV6-donor template delivery.

The development of committed erythroid progenitors and their continued maturation into mature erythrocytes requires the cytokine erythropoietin (Epo). Here, we describe the immunophenotypic identification of a unique Epo-dependent colony-forming unit-erythroid (CFU-E) cell subtype that forms during early erythropoiesis (EE). This previously undescribed CFU-E subtype, termed late-CFU-E (lateC), lacks surface expression of the characteristic erythroid marker CD235a (glycophorin A) but has high levels of CD71 and CD105. LateCs could be prospectively detected in human bone marrow (BM) cells and, upon isolation and reculture, exhibited the potential to form CFU-E colonies in medium containing only Epo (no other cytokines) and continued differentiation along the erythroid trajectory. Analysis of ex vivo cultures of BM CD34+ cells showed that acquisition of the CD7hiCD105hi phenotype in lateCs is gradual and occurs through the formation of four EE cell subtypes. Of these, two are CD34+ burst-forming unit-erythroid (BFU-E) cells, distinguishable as CD7loCD105lo early BFU-E and CD7hiCD105lo late BFU-E, and two are CD34- CFU-Es, also distinguishable as CD71loCD105lo early CFU-E and CD7hiCD105lo mid-CFU-E. The transition of these EE populations is accompanied by a rise in CD36 expression, such that all lateCs are CD36+. Single cell RNA-sequencing analysis confirmed Epo-dependent formation of a CFU-E cluster that exhibits high coexpression of CD71, CD105, and CD36 transcripts. Gene set enrichment analysis revealed the involvement of genes specific to fatty acid and cholesterol metabolism in lateC formation. Overall, in addition to identifying a key Epo-dependent EE cell stage, this study provides a framework for investigation into mechanisms underlying other erythropoiesis-stimulating agents.

Hematopoietic stem cell (HSC) transplantation (HST) is a curative treatment for many hematopoietic cancers and bone marrow (BM) disorders but is currently limited by numerous complications including a lengthy recovery period, prolonged neutropenia resulting in severe infections and bleeding, and a high incidence of graft vs. host disease (GVHD). While clinical studies have demonstrated that sex mismatch, notably male recipients with female donor cells, results in increased risk of GVHD (likely due to male recipient minor histocompatibility antigens targeted by donor female T-cells 1), increased non-relapse mortality, and decreased overall survival, the mechanisms underlying sex-determinants on hematopoiesis and post-transplant recovery are not clear. In this manuscript we have identified: 1) unique expression of hematopoietic niche factors in the BM and spleens of male and female mice, 2) altered kinetics of hematopoietic reconstitution following transplantation when male vs. female BM is used as the donor cell source, 3) a sex-specific role for the recipient niche in promoting post HST recovery, and 4) a dose-dependent role for exogenous sex hormones in maintaining hematopoietic stem and progenitor cells (HSPCs). Taken together, these data demonstrate that sex-specific cellular and molecular signaling occurs during hematopoietic regeneration. Further identifying novel sex-dependent determinants of regeneration following transplantation will not only enhance understanding of steady state versus regeneration hematopoiesis but may also reveal unique (and potentially sex-specific) therapeutic targets to accelerate hematologic recovery. Key PointsO_LIMale and female mice display altered kinetics of regeneration following HST due to unique niche factors in hematopoietic compartments. C_LIO_LIExogenous steroid sex hormones uniquely regulate the pool of hematopoietic stem and progenitor cells and may impact transplantation outcomes. C_LI

Clonal hematopoiesis (CH) is an early indicator of hematologic malignancies, driven by mutations in hematopoietic stem cells (HSCs) such as TET2 or TP53. Mutations in ZBTB33 have been implicated in MDS and suggested as a potential driver of CH. However, the role of ZBTB33 in hematopoiesis and its involvement in CH remains unclear. We generated a Zbtb33-knockout mouse strain to elucidate its role in hematopoiesis and the immune system. Our findings indicate that hematopoiesis in Zbtb33-defecient mice appeared grossly normal, and competitive bone marrow transplantation assays demonstrated that loss of Zbtb33 in HSCs did not confer expansional advantage. Introducing the Zbtb33 mutation into Tet2- or Tp53-mutation background yielded no synergistical effects. Tumor challenging assays suggested that Zbtb33 influences cancer immunity response, rather than directly driving CH or myeloid malignancies. In summary, ZBTB33 deficiency was insufficient to induce clonal hematopoiesis but may have a regulatory role in tumor microenvironment. Statement of significanceClonal hematopoiesis (CH) is linked to mutations in hematopoietic stem cells, but the role of Zbtb33 in CH remains unclear. To investigate this, we examined the function of Zbtb33 under physiological conditions and in response to external stimuli. Additionally, we explored whether Zbtb33 mutations cooperate with other genetic mutations to drive clonal hematopoiesis. Key pointsO_LILoss of Zbtb33 fails to induce clonal hematopoiesis and does not synergize with Tet or Tp53 mutations. C_LIO_LIHowever, it plays a significant role in regulating cancer immunity and the tumor microenvironment. C_LI

BackgroundMixed phenotype acute leukemia (MPAL) is a rare subgroup of leukemia characterized by blast cells that display both myeloid and lymphoid lineage features, making this cancer difficult to diagnose and treat. A deeper characterization of MPAL at the molecular level is essential to better understand similarities/differences to the more common and better-studied leukemias, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on MPAL bone marrow (BM) samples in an attempt to develop a more granular map of the MPAL microenvironment landscape. MethodsWe analyzed [~]16,000 cells from five pediatric MPAL BM samples collected at diagnosis to generate a single-cell transcriptomic landscape of B/Myeloid (B/My) and T/Myeloid (T/My) MPAL blasts and associated microenvironment cells. Cell clusters were identified using principal component analysis and uniform manifold approximation and projection (UMAP). Unsupervised analysis was performed to determine the overall relationship among B/My MPAL, T/My MPAL, and other acute leukemias - B-ALL, T-ALL, and AML. Supervised differentially expressed gene (DEG) analysis was performed to identify B/My and T/My MPAL blast-specific signatures. MPAL sample transcriptome profiles were compared with normal BM stem and immune cells to identify MPAL-specific dysregulation. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. Comparative analysis was performed on diagnostic samples based on their future minimal residual disease (MRD) and relapse status. ResultsB/My MPAL and T/My MPAL blasts displayed distinct subtype-specific blast signatures. UMAP analysis revealed that B/My MPAL samples had greater overlap with B-ALL samples, while T/My MPAL samples clustered separately from other acute leukemia subtypes. Genes overexpressed in both MPAL subtypes blasts compared to other leukemias and healthy controls included PLIN2, CD81, and UBE2S. B/My MPAL blast-specific genes included IRS2, SMIM3, and HBEGF, whereas T/My MPAL blast-overexpressed genes included IER5, BOD1L1, and HPGD. Sirtuin signaling, p38 MPAK signaling, and PI3K signaling pathways were upregulated in B/My MPAL blasts while oxidative phosphorylation and Rho family GTPases signaling pathways were upregulated in T/My MPAL blasts. Transcriptomic, pathways, and cell communication level differences were observed in the MPAL samples based on future MRD and clinical outcome status. ConclusionsWe have for the first time described the single-cell landscape of pediatric MPAL and demonstrate that B/My and T/My MPAL have unique scRNAseq profiles distinct from each other as well as from ALL and AML.

Sickle cell disease (SCD) is caused by the production of an abnormal adult hemoglobin that generates sickle-shaped red blood cells (RBCs). Transplantation of autologous genetically corrected hematopoietic stem/progenitor cells (HSPCs) represents a promising therapy. Persistent fetal hemoglobin expression improves SCD. Here, we engineered the fetal HBG1/2 promoters by replacing the BCL11A repressor binding site (BS) with a TAL1:GATA1 motif recognized by transcriptional activators. We exploited the prime editing nuclease (PEn) that efficiently installed the TAL1:GATA1 motif in K562 cells, outperforming the original PE. Non-homologous end joining (NHEJ) and/or alternative-end joining (alt-EJ) pathway inhibition enhanced precise editing. However, this strategy was poorly efficient in patients HSPCs. Alternatively, we used CRISPR/Cas9 nuclease to either disrupt the BCL11A BS via NHEJ and/or alt-EJ or to replace it with the TAL1:GATA1 motif via homology-directed repair (HDR) using a donor ssODN template. NHEJ and alt-EJ inhibition improved product purity, reducing InDels and achieving superior precise editing efficiency compared to PEn in K562 and HSPCs. HDR-edited HSPCs preserved clonogenic capacity and differentiated into RBCs showing elevated HBG expression and correction of the sickling phenotype. These results demonstrate that replacing the BCL11A BS with a TAL1:GATA1 motif is a potent strategy for reactivating HBG1/2 to treat SCD.

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) are crucial for maintaining lifelong hematopoiesis. Developing methods to distinguish stem cells from other progenitors and evaluate stem cell functions has been a central task in stem cell research. Deep learning has been demonstrated as a powerful tool in cell image analysis and classification. In this study, we explored the possibility of using deep learning to differentiate HSCs and MPPs based on their light microscopy (DIC) images. After extensive training and validation with large image data sets, we successfully develop a three-class classifier (we named it the LSM model) that reliably differentiate long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs), and MPPs. Importantly, we demonstrated that our LSM model achieved its differentiating capability by learning the intrinsic morphological features from cell images. Furthermore, we showed that the performance of our LSM model was not affected by how these cells were identified and isolated, i.e., sorted by surface markers or intracellular GFP markers. Prospective identification of HSCs and MPPs in Evi1GFP transgenic mice by LSM model suggested that the cells with the highest GFP expression were LT-HSCs, and this prediction was substantiated later by a long-term competitive reconstitution assay. Moreover, based on DIC image data sets, we also successfully built another two-class classifier that can effectively distinguish aged HSCs from young HSCs, which both express the same surface markers but are functionally different. This finding is of particular interest since it may provide a novel quick and efficient approach, obviating the need for a time-consuming transplantation experiment, to evaluate the functional states of HSCs. Together, our study provides evidence for the first time that HSCs and MPPs can be differentiated by deep learning based on cell morphology. This novel and robust deep learning-based platform will provide a basis for the future development of a new generation stem cell identification and separation system. It may also provide new insight into molecular mechanisms underlying the self-renewal feature of stem cells.

Casein Kinase 2 (CK2) is a constitutively active kinase regulating proliferation and immune signaling and is frequently dysregulated in cancer, including acute myeloid leukemia (AML), making it a therapeutic target. CK2 comprises two catalytic subunits, CK2 or CK2, with two regulatory {beta} subunits. The role of CK2, the predominant catalytic subunit and principal mediator of CK2 kinase activity in hematopoietic cells, in steady-state hematopoiesis remains undefined. To define how CK2 shapes hematopoietic cells, we used bone marrow and spleen tissue samples of wild type control and conditional knock out (KO) of CK2 (Csnk2a1) in the hematopoietic compartment of transgenic mice. Using single-cell RNA sequencing, we profiled the transcriptomic changes associated with CK2 loss. Although HSC abundance was comparable between the control and CK2-deficient samples, HSCs experienced the largest transcriptional response to CK2 loss among all cell types. CK2-deficient HSCs displayed transcriptional remodeling for inflammatory and immune-associated programs, interferon signaling, and antigen presentation. Expression of inflammatory genes such as S100a8 and S100a9, changed in opposite directions in bone marrow and spleen HSCs, demonstrating the transcriptional consequences of CK2 loss shaped by tissue context. Using a network-based approach, we identified immune-associated transcription factors Nfkb1, Rfx5, Hes1, and AP-1 family members as regulatory hubs driving these inflammatory transcriptional states in CK2-deficient HSCs. Cell-cell communication profiling revealed multiple gains and losses in ligand-receptor communication between the HSCs and their immune microenvironment in KO. Our findings identify CK2 as a regulator of immune transcriptional programs in HSCs and suggest that disruption of CK2 signaling influences stem cell behavior and immune activation in contexts relevant to hematologic malignancies and CK2-targeted cancer therapies. Statement of significanceThis study reveals that inhibiting the protein CK2 forces blood stem cells into a stressed, immune-primed state. These tissue-specific findings highlight potential side effects for cancer therapies targeting this essential regulatory kinase.

The microRNA cluster-221/222 is expressed in hematopoietic stem cells (HSC) and multipotent progenitors (MPP). To study its function in hematopoiesis, we generated mice, in which this cluster is selectively deleted by Vav-cre in HSC and, thus, in all hematopoietic cells. Fluorescence-activated cell sorting analyses of the lineage-negative HSC and MPP compartments in bone marrow at unperturbed, steady state hematopoiesis detect strong activation of HSC to MPP, as well as increased granulocytes in the periphery, induced by miR-221/222-deficiency. Short-term social stress on mice also activates HSC to MPP, but the time of stress is too short to detect further increases in granulocyte numbers. Single cell deep mRNA sequencing identifies Fos as direct, and Jun as well as six other immediate early genes (IEG) as indirect targets of miR-221/222 at unperturbed hematopoiesis. Three of these IEG - Klf6, Nr4a1 and Zfp36 - have previously been found to influence myelo/granulopoiesis. Short stress induces higher levels of the same, and an even larger number of IEGs, now also in MPP, indicating, that stress and miR-221/222 both activate HSC to MPP by IEG upregulation in perturbed hematopoiesis. Furthermore, combined stress and miR-221/222-deficiency rapidly increase numbers of myelo/granulocyte progenitors (MEP, GMP) in bone marrow. Additional indirect miR-221/222-targets become detectable in MPP, of which H3f3b has previously been found to influence myelopoiesis. In serial transplantations, miR-221/222-deficient HSC retain their capacity to home to, and become resident in bone marrow, but they loose their lymphopoietic capacities, thus their multipotency. Our results suggest, that miR-221/222-expression in HSC and MPP safeguards their quiescence and multipotency by downregulating the expression of IEG and of myelo/granulopoiesis-enhancing target genes. Since miR-221/222 is also expressed in human HSC and MPP, its expression should improve clinical settings of human bone marrow transplantations.

Mutations in RNA splicing factor SF3B1 are among the most common in MDS and are strongly associated with MDS with ring sideroblasts (MDS-RS). While aberrant splicing of terminal erythroid regulators has been implicated in MDS pathogenesis, the impact of SF3B1 mutations on early hematopoietic progenitor function remains unclear. Here, we identify CDK8, a key kinase of the mediator complex involved in transcriptional regulation, as a recurrent mis-spliced target in SF3B1-mutant MDS. Mutant SF3B1 induces cryptic 3' splice site selection in CDK8, leading to loss of CDK8 mRNA and protein. Using primary human HSPCs, our study identifies CDK8 as an important regulator of HSPC homeostasis and cell fate determination. CDK8 depletion results in expansion of HSPCs and shifts differentiation toward the erythroid and myeloid lineages, mirroring phenotypes observed in SF3B1-mutant MDS. Lastly, functional restoration of CDK8 rescues early erythroid phenotypes in SF3B1-mutant cells. These findings implicate CDK8 mis-splicing as a mechanistic driver of altered progenitor fate and dysplasia in SF3B1-mutant MDS, linking aberrant splicing to transcriptional dysregulation and hematopoietic lineage commitment.

Early mammalian erythropoiesis requires the DOT1L methyltransferase. We demonstrated that loss of DOT1L in mutant mice resulted in lethal anemia during midgestation. The molecular mechanisms by which DOT1L regulates embryonic erythropoiesis have not yet been elucidated. In this study, a methyltransferase mutant mouse line (Dot1L-MM) was generated to determine whether the methyltransferase activity of DOT1L is essential for erythropoiesis. Dot1L-MM mice displayed embryonic lethality between embryonic days 10.5 and 13.5, similar to Dot1lL knockout (Dot1L-KO) mice. However, when examined at E10.5, unlike the Dot1L-KO, Dot1L-MM embryos did not exhibit evidence of anemia. In ex vivo hematopoietic differentiation cultures, Dot1L-KO and Dot1L-MM yolk sac (YS) cells both formed reduced numbers of myeloid, and mixed hematopoietic colonies. Erythroid colonies were able to be formed in numbers equal to wildtype embryos. Extensively self-renewing erythroblast (ESRE) cultures were established using YS cells from E10.5 embryos. Dot1L-KO and Dot1L-MM cells expanded significantly less than wild-type cells and exhibited increased cell death. Strikingly, Dot1L-KO and Dot1L-MM cells of YS origin exhibited profound genomic instability, implicating DOT1L methyltransferase activity in maintenance of the genome as well as viability of hematopoietic progenitors. Our results indicate that the methyltransferase activity of DOT1L plays an important role early murine hematopoiesis.