Primary Hematopoietic Cells Research Articles

Failure of metabolic checkpoint control during late-stage granulopoiesis drives neutropenia in reticular dysgenesis

AbstractCellular metabolism is highly dynamic during hematopoiesis, yet the regulatory networks that maintain metabolic homeostasis during differentiation are incompletely understood. Herein, we have studied the grave immunodeficiency syndrome reticular dysgenesis caused by loss of mitochondrial adenylate kinase 2 (AK2) function. By coupling single-cell transcriptomics in samples from patients with reticular dysgenesis with a CRISPR model of this disorder in primary human hematopoietic stem cells, we found that the consequences of AK2 deficiency for the hematopoietic system are contingent on the effective engagement of metabolic checkpoints. In hematopoietic stem and progenitor cells, including early granulocyte precursors, AK2 deficiency reduced mechanistic target of rapamycin (mTOR) signaling and anabolic pathway activation. This conserved nutrient homeostasis and maintained cell survival and proliferation. In contrast, during late-stage granulopoiesis, metabolic checkpoints were ineffective, leading to a paradoxical upregulation of mTOR activity and energy-consuming anabolic pathways such as ribonucleoprotein synthesis in AK2-deficient cells. This caused nucleotide imbalance, including highly elevated adenosine monophosphate and inosine monophosphate levels, the depletion of essential substrates such as NAD+ and aspartate, and ultimately resulted in proliferation arrest and demise of the granulocyte lineage. Our findings suggest that even severe metabolic defects can be tolerated with the help of metabolic checkpoints but that the failure of such checkpoints in differentiated cells results in a catastrophic loss of homeostasis.

A millifluidic bioreactor allows the long term culture of primary lymphocytes or CD34+ hematopoietic cells while allowing the detection of tumorigenic expansion.

Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore, genetic engineering of the above-mentioned primary human cells has several safety needs, including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work, we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB, we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4+ T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34+ cells. A fraction of these cells is viable for up to 3months. Next, we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4+ or CD34+ primary cells, and their growth was followed. By this approach, we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment, whereas tumor cells, after an adaptation phase, expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells.

Selective degradation of mutant FMS-like tyrosine kinase-3 requires BIM-dependent depletion of heat shock proteins

AbstractInternal tandem duplications in the FMS-like tyrosine kinase-3 (FLT3-ITD) are common mutations in acute myeloid leukemia (AML). Proteolysis-targeting chimeras (PROTACs) that induce proteasomal degradation of mutated FLT3 emerge as innovative pharmacological approach. Molecular mechanisms that control targeted proteolysis beyond the ubiquitin-proteasome-system are undefined and PROTACs are the only known type of FLT3 degraders. We report that the von-Hippel-Lindau ubiquitin-ligase based FLT3 PROTAC MA49 (melotinib-49) and the FLT3 hydrophobic tagging molecule MA50 (halotinib-50) reduce endoplasmic reticulum-associated, oncogenic FLT3-ITD but spare FLT3. Nanomolar doses of MA49 and MA50 induce apoptosis of human leukemic cell lines and primary AML blasts with FLT3-ITD (p < 0.05-0.0001), but not of primary hematopoietic stem cells and differentiated immune cells, FLT3 wild-type cells, retinal cells, and c-KIT-dependent cells. In vivo activity of MA49 against FLT3-ITD-positive leukemia cells is verified in a Danio rerio model. The degrader-induced loss of FLT3-ITD involves the pro-apoptotic BH3-only protein BIM and a previously unidentified degrader-induced depletion of protein-folding chaperones. The expression levels of HSP90 and HSP110 correlate with reduced AML patient survival (p < 0.1) and HSP90, HSP110, and BIM are linked to the expression of FLT3 in primary AML cells (p < 0.01). HSP90 suppresses degrader-induced FLT3-ITD elimination and thereby establishes a mechanistically defined feed-back circuit.

ThymoSpheres culture: A model to study human polyclonal unconventional T cells.

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T-cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single-cell RNA and combined T-cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal αβ unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2 (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCR-α chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms.

DNA contamination within recombinant adeno-associated virus preparations correlates with decreased CD34+ cell clonogenic potential

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPC) but have many outstanding concerns. Specifically, their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging, a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally, this process results in packaging of complete viral particles only containing rAAV genomes; however, this is not the case. Through this study, we were able to leverage Single-Stranded Virus (SSV)-seq, an NGS-based method to quantify all DNA species present within rAAV preps. From this, it was determined that much of the DNA within some rAAV preps is not vector-genome derived, and there is wide variability in the contamination by DNA across various preps. Furthermore, we demonstrate that transducing CD34+ hematopoietic stem and progenitor cells (HSPC) with preps with higher contaminating DNA resulted in decreased clonogenic potential, altered transcriptomic profiles, and decreased genomic editing. Collectively, this study characterized the effects of DNA contamination within rAAV preps on CD34+ HSPC cellular potential.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.

CRISPR Dependency Screens in Primary Hematopoietic Stem Cells Identify KDM3B as a Genotype-specific Vulnerability in IDH2- and TET2-mutant Cells.

Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs. Significance: Given the broad prevalence, comorbidities, and risk of malignant transformation associated with CH, there is an unmet need to identify therapeutic targets. We develop an ex vivo platform to perform CRISPR/Cas9 screens in primary HSPCs. We identify KDM3B and downstream signaling components as genotype-specific dependencies in CH and myeloid malignancies. See related commentary by Khabusheva and Goodell, p. 1768.

The Leukemic Isocitrate Dehydrogenase (IDH) 1/2 Mutations Impair Myeloid and Erythroid Cell Differentiation of Primary Human Hematopoietic Stem and Progenitor Cells (HSPCs).

How hematopoietic stem and progenitor cell (HSPC) fate decisions are affected by genetic alterations acquired during AML leukemogenesis is poorly understood and mainly explored in animal models. Here, we study isocitrate dehydrogenase (IDH) gene mutations in the human model of HSPC and discuss the available literature on this topic. IDH1/2 mutations occur in ~20% of AML cases, are recognized among the mutations earliest acquired during leukemogenesis, and are targets of specific inhibitors (ivosidenib and enasidenib, respectively). In order to investigate the direct effects of these mutations on HSPCs, we expressed IDH1-R132H or IDH2-R140Q mutants into human CD34+ healthy donor cells via lentiviral transduction and analyzed the colony-forming unit (CFU) ability. CFU ability was dramatically compromised with a complete trilineage block of differentiation. Strikingly, the block was reversed by specific inhibitors, confirming that it was a specific effect induced by the mutants. In line with this observation, the CD34+ leukemic precursors isolated from a patient with IDH2-mutated AML at baseline and during enasidenib treatment showed progressive and marked improvements in their fitness over time, in terms of CFU ability and propensity to differentiate. They attained clonal trilinear reconstitution of hematopoiesis and complete hematological remission.

Resolving the chromatin impact of mosaic variants with targeted Fiber-seq.

Accurately quantifying the functional consequences of non-coding mosaic variants requires the pairing of DNA sequence with both accessible and closed chromatin architectures along individual DNA molecules-a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic and epigenomic profiling across targeted >100 kilobase loci with ~10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the DMPK CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent. Furthermore, we reveal that therapeutic adenine base editing of the segmentally duplicated γ-globin (HBG1/HBG2) promoters in primary human hematopoietic cells induced towards an erythroblast lineage increases the accessibility of the HBG1 promoter as well as neighboring regulatory elements. Overall, we find that these non-protein coding mosaic variants can have complex impacts on chromatin architectures, including extending beyond the regulatory element harboring the variant.

Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells.

Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donors. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.

Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells

Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donors. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.

A JAGN1-associated severe congenital neutropenia zebrafish model revealed an altered G-CSFR signaling and UPR activation

AbstractA variety of autosomal recessive mutations in the JAGN1 gene cause severe congenital neutropenia (CN). However, the underlying pathomechanism remains poorly understood, mainly because of the limited availability of primary hematopoietic stem cells from JAGN1-CN patients and the absence of animal models. In this study, we aimed to address these limitations by establishing a zebrafish model of JAGN1-CN. We found 2 paralogs of the human JAGN1 gene, namely jagn1a and jagn1b, which play distinct roles during zebrafish hematopoiesis. Using various approaches such as morpholino-based knockdown, CRISPR/Cas9–based gene editing, and misexpression of a jagn1b harboring a specific human mutation, we successfully developed neutropenia while leaving other hematopoietic lineages unaffected. Further analysis of our model revealed significant upregulation of apoptosis and genes involved in the unfolded protein response (UPR). However, neither UPR nor apoptosis is the primary mechanism that leads to neutropenia in zebrafish. Instead, Jagn1b has a critical role in granulocyte colony-stimulating factor receptor signaling and steady-state granulopoiesis, shedding light on the pathogenesis of neutropenia associated with JAGN1 mutations. The establishment of a zebrafish model for JAGN1-CN represents a significant advancement in understanding the specific pathologic pathways underlying the disease. This model provides a valuable in vivo tool for further investigation and exploration of potential therapeutic strategies.

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

Identifying an AML Prognostic Model Using 10 Marker Genes from Single-Cell Transcriptome and Bulk Transcriptome Analysis.

Fanconi anemia (FA) is the predominant hereditary syndrome of bone marrow failure (BMF), distinguished by impairments in DNA repair mechanisms. The deficiency in the FANC pathway, which governs DNA repair and replication rescue, results in aberrant responses to DNA damage in individuals with FA. The objective of this study is to examine the involvement of the FANC core complex in BMF and ascertain nucleolar homeostasis-related genes by conducting transcriptome analysis on primary hematopoietic stem cells obtained from FA patients with FANCA and FANCC variants. In the present study, we analyzed scRNA-seq data obtained from both healthy donors and individuals diagnosed with FA in order to investigate the phenomenon of cell-cell communication. Through the implementation of trajectory analysis, the differentiation pathways of several progenitor cell types, such as HSC cells transitioning into LMPP, N, M, B-prog, and E cells, were elucidated. Moreover, by scrutinizing the inferred interactions, notable disparities in cell-cell communication were observed between FA patients and their healthy counterparts. Our analysis has unveiled heightened interactions involving TNFSF13B, MIF, IL16, and COL4A2 in patients with FA. Furthermore, we have developed a prognostic model for predicting the outcome of acute myeloid leukemia (AML) which has exhibited remarkable predictive precision, with an AUC exceeding 0.83 at the 3- and 5-year intervals following the baseline assessment. In summary, the prognostic model that has been developed provides a valuable instrument for forecasting outcomes of AML by utilizing the genes identified through both single-cell and bulk transcriptome analyses.

Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of β-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference.

Therapy via the gene addition of the anti-sickling βAS3-globin transgene is potentially curative for all β-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for βAS3-globin addition and evaluates strategies for an increased β-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-βAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior β-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBBIVSI-110(G>A) thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBBIVSI-110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent βAS3-globin expression. LVs were initially compared for their ability to achieve high β-like globin expression in HBBIVSI-110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI-110(G>A)-homozygous HSPCs. The latter revealed that β-globin promoter-driven designs for monotherapy with HBBIVSI-110(G>A)-specific shRNAmiRs only marginally increased β-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with βAS3-globin expression showed disease correction similar to that achieved by the parental GLV2-βAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI-110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-βAS3 for the treatment of severe β-hemoglobinopathies.

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia-initiating CBFB::MYH11 oncofusion protein.

Several canonical translocations produce oncofusion genes that can initiate acute myeloid leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase (TurboID) in-frame with 3 favorable-risk AML oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11) and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID-interacting proteins were largely cytoplasmic, probably because of an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.

Mechanisms of PML::Rara and GATA2 Protein Interactions in Hematopoietic Cells

Despite excellent outcomes for Acute Promyelocytic Leukemia (APL) patients with combined ATRA and Arsenic Trioxide therapy, the underlying molecular mechanisms by which the PML::RARA fusion oncoprotein initiates APL are not yet clear. We recently found that the ability of PML::RARA to initiate aberrant serial replating (a surrogate for self-renewal) is dependent upon its ability to bind to DNA, since PML::RARA with a missense mutation (C88A) that prevents the binding of RARA to DNA eliminates aberrant replating. Currently, however, there is no consensus regarding the genomic binding sites of PML::RARA in myeloid progenitor cells and APL cells. Also, the transcriptional and epigenetic consequences of PML::RARA expression in primary hematopoietic cells remain unclear. To define the genomic binding sites of PML::RARA, we generated V5 epitope tagged PML::RARA constructs in MSCV-based retroviral vectors, and transduced mouse hematopoietic stem progenitor cells (HSPCs) with retroviruses containing V5-PML::RARA WT, V5-PML::RARA C88A, or an empty vector. Anti-V5 ChIP-seq revealed that PML::RARA binds to 2,677 distinct loci in primary mouse hematopoietic cells, and 2,064 sites in primary human hematopoietic cells. We previously demonstrated that Gata2 expression is induced with PML::RARA, and indeed, we identified strong PML::RARA binding to the distal Gata2 enhancer in both human and mouse HSPCs. Surprisingly, ATAC-seq revealed that the vast majority of PML::RARA binding sites are found in regions that are chromatin-accessible even in WT promyelocytes, suggesting that these regions are “open” prior to PML::RARA expression, perhaps because of the normal binding of “pioneer” transcription factors to these regions in WT cells. Indeed, evaluation of transcription factor binding motifs in the regions occupied by PML::RARA revealed significant enrichment for RARA binding motifs, and also GATA motifs. We therefore performed ChIP-seq on GATA2 in HSPCs, which confirmed enrichment in binding at the same sites. These data suggested that PML::RARA and GATA2 may bind in close proximity at hundreds of loci in the genome. To test this, we performed proximity labeling studies followed by mass spectrometry using PML::RARA WT vs. PML::RARA C88A fused to the biotin ligase TurboID in primary HSPCs. This approach revealed that the interactions of several proteins with PML::RARA (including GATA2) are dependent on the binding of PML::RARA to DNA; this suggests that DNA may in fact act as a scaffold to stabilize these protein:protein interactions ( Figure 1). To determine whether Gata2 may induce increased chromatin accessibility following PML::RARA expression, we performed ATAC-seq following Gata2 or PML::RARA overexpression in WT hematopoietic progenitor cells. 3,157 regions shared increased accessibility with either PML::RARA or Gata2 overexpression. Moreover, at a transcriptional level, we found that 535 (27.4%) of PML::RARA differentially expressed genes were coordinately regulated by Gata2 in WT cells. This suggests that PML::RARA indirectly regulates many of its gene targets via the upregulation of Gata2, and that Gata2 may be required for PML::RARA to initiate its transcriptional program. To test this, we performed serial replating assays using cells where Gata2 was knocked out either before or after PML::RARA expression. Consistent with our previous study, knock out of Gata2 following PML::RARA expression led to increased self-renewal capacity. However, knock out of Gata2 before PML::RARA expression prevented self-renewal. These data suggest a model in which GATA2 “opens” specific chromatin regions in early myeloid cells, which are then accessible to RARA or PML::RARA. The two proteins interact at these sites, tethered by their mutual interactions to their respective DNA binding motifs. PML::RARA binding promotes a pre-leukemic transcriptional program mediated in part by positively regulating Gata2. Gata2 is therefore initially required for PML::RARA to establish its transcriptional program. However, once that program is established, Gata2 paradoxically acts as a tumor suppressor, performing a normal role to limit proliferative excess in hematopoietic stem and progenitor cells.

Primary U2AF1 S34F Mutated Hematopoietic Cells Are Sensitive to Nonsense-Mediated RNA Decay Disruption In Vivo

Expression of mutant splicing factors alters RNA splicing in myeloid malignancies, including increased production of nonsense transcripts that rely on nonsense-mediated RNA decay (NMD) for clearance. Cell lines expressing spliceosome mutants are more sensitive to NMD inhibition than wild-type cells, and the differential sensitivity is partially dependent on R-loop formation. However, the impact of NMD disruption on the viability of primary hematopoietic cells with splicing factor mutations has not been tested in vivo. We engineered mouse MLL-AF9 acute myeloid leukemia (AML) cells to inducibly express a wild type ( U2AF1 WT) or mutant ( U2AF1 S34F) transgene of the U2AF1 splicing factor, and tested the effects of inhibiting the NMD pathway protein kinase, SMG1, in vivo. We injected U2AF1 WT and U2AF1 S34F mouse AML cells (B6, CD45.2) into congenic, irradiated secondary recipient mice (B6, CD45.1), induced transgene expression, and then treated the mice for 2 weeks with either a SMG1 inhibitor (SMG1i) at 60 mg/kg/day or vehicle. SMG1i treatment increased the overall survival (OS) of mice bearing U2AF1S34F AML cells (median OS of 53 days compared to 42 days for mice treated with vehicle; p =0.0003), but did not impact the OS of mice with U2AF1WT AML cells (median OS of 33 days for SMG1i-treated mice compared to median OS of 32 days for vehicle-treated mice, p=0.360). We extended these findings to a second cohort of mice, where we transplanted one primary U2AF1 S34F banked mouse AML sample into 48 secondary recipient mice. To allow direct comparison of isogenic cells with or without U2AF1 S34F expression, half the mice were treated with doxycycline to induce U2AF1 S34F expression, and half of the mice did not receive doxycycline. SMG1i treatment increased the OS of mice expressing the U2AF1S34F mutant allele (median OS of 66 days, compared to median OS of 54 days for mice treated with vehicle; p<0.0001), but had no impact on survival of mice bearing AML cells where U2AF1 S34F was not expressed (median OS of 50 days, compared to median OS of 52 days for mice treated with vehicle; p=0.95). To explore the underlying mechanism for the sensitivity of spliceosome mutant cells to NMD inhibition, we first examined the effects of SMG1i treatment on cell cycle progression and cell death in primary cells. We observed that primary U2AF1 S34F-expressing AML cells treated with SMG1i ex vivo (24 hours) had an increased fraction of cells in G2/M phase compared to U2AF1 WT-treated cells (mean 48% vs. 11% at 100nM, respectively, p<0.001). In addition, the fraction of Annexin V + cells was increased in U2AF1 S34F-expressing cells treated with SMG1i ex vivo compared to U2AF1 WT treated cells (mean fold-change at 500nM relative to DMSO 4.0 vs. 2.3, respectively, p=0.027). We then isolated primary mouse AML cells after 7 days of in vivo SMG1i treatment and analyzed R-loops (S9.6 antibody) and DNA damage (gH2AX) by immunofluorescence. SMG1i-treated U2AF1 S34F cells had a marked increase in R-loop levels compared to U2AF1 WT-treated cells (mean S9.6 nuclear intensity, 306.9 vs 182.6, respectively, p<0.0001). In addition, H2AX phosphorylation was increased in SMG1i-treated U2AF1 S34F compared to U2AF1 WT cells (mean gH2AX nuclear intensity, 136.7 vs 87.8, respectively p<0.0001).Thus, disrupting NMD in splicing factor mutant AML cells alters cell cycle progression and causes R-loop accumulation, DNA damage, and apoptotic cell death. The NMD pathway has been implicated in the regulation of the unfolded protein response (UPR) that controls protein homeostasis and impacts cell viability. We generated K562 cell lines expressing doxycycline-inducible U2AF1 S34F or U2AF1 WT constructs and confirmed increased sensitivity of U2AF1 S34F cells treated with SMG1i(IC50 128.1nM vs 578.5nM at 72 hours, respectively, p<0.0001). Next, we performed RNA-sequencing on U2AF1 WT and U2AF1 S34F cells that were treated with vehicle or SMG1i for 24 hours (n=3) and observed upregulation of endoplasmic reticulum and UPR pathway genes in SMG1i-treated U2AF1 S34F compared to U2AF1 WT cells (GSEA; FDR<0.01). Validation of these findings using primary mouse AML cells is ongoing, including the analysis of key regulators and protein biomarkers of the UPR pathway. The vulnerability of primary hematopoietic cancer cells with spliceosome mutations to NMD inhibition suggests the possibility for therapeutic targeting of NMD to treat myeloid malignancies with aberrant splicing.

Modulating Nucleotide Metabolism Enhances Prime Editing in Hematopoietic Stem and Progenitor Cells

Therapeutic prime editing (PE) of hematopoietic stem cells (HSCs) holds great potential to correct pathogenic mutations causative of severe blood disorders. While recent advances have improved the technology, efficient prime editing remains challenging in primary hematopoietic cells. Since SAMHD1 has been identified as an antiviral factor restricting the nucleotide pool available for reverse transcription and virus replication, we hypothesized that modulating this metabolic pathway could enhance reverse transcription and thus prime editing in HSCs. We devised strategies to modulate nucleotide metabolism by supplementing deoxynucleosides (dNs) and delivering the viral accessory protein Vpx using virus-like particles (VLPs). Exogenous dNs supplementation and Vpx-mediated proteasomal degradation of SAMHD1 markedly increased PE efficiency in HSPCs from different healthy donors from an average of 14% to 32% editing with the PE2max system, and from 51% to 70% editing with the PE3max system. Supplementing individual dNs or all dNs minus one also boosted prime editing, but all four dNs were needed for maximal enhancement of PE from an average of 41% to 64% editing at ATP1A1. Using a prime editing guide RNA designed to correct the HBB-E6V mutation causative of sickle cell disease, we achieved an average of 58% and 44% editing with the PE3max and PE3bmax systems, respectively. Modulation of nucleotide metabolism also ameliorated prime editing in both resting and activated CD3 + T cells, although to a lesser extent than in HSPCs. Of relevance, our strategies had little to no impact on PE in K562 and Jurkat cells, suggesting that nucleotide metabolism does not restrict PE in cancer cell lines. Altogether, modulation of nucleotide metabolism should enhance prime editing to facilitate the clinical translation of gene therapies for severe blood disorders. This work highlights a key metabolic restriction for therapeutic prime editing of primary cells and could help design future approaches to engineer quiescent HSCs in vivo.

Interferon-Alpha Impairs Neoplastic Vasculogenesis in a 3D iPSC-Based Model of JAK2V617F+ Myeloproliferative Neoplasms

Overt Myelofibrosis (MF) is an end-stage subtype of BCR-ABL1 negative myeloproliferative neoplasms (MPN), which is characterized by progressive bone marrow (BM) fibrosis resulting in loss of normal hematopoietic support as well as inflammatory and vascular complications. While aberrant neoangiogenesis is known to participate in the pathogenesis of MF, Erba et al. (Am J Pathol, 2017) suggested the occurrence of endothelial-to-mesenchymal transition (EndMT) in BM and spleen of MF patients (pts). Given the relevance of endothelial cells (EC) for MF development and the occurrence of JAK2V617F-mutated EC in MPN pts, targeting of the vascular niche has emerged as a promising therapeutic strategy. To untangle the impact of the JAK2V617F mutation on human vasculogenesis, we leveraged high-purity and clonal patient-specific induced pluripotent stem cell (iPSC)-derived EC (iEC) harboring either the JAK2V617F mutation in hetero- (JAK2V617F HET) or homozygosis (JAK2V617F HOM). JAK2 WT and CRISPR-repaired isogenic JAK2 WT (both referred to as JAK2 WT) were used as controls. In a first set of in vitro experiments, JAK2V617F mutated iEC showed a significantly increased proliferation rate in comparison to JAK2 WT iEC. We next evaluated the effect of selected drugs on iEC viability to assess their vascular toxicity. While treatment with 1 µM each of fedratinib, anagrelide or venetoclax led to a decrease in iEC viability (all **p ≤ .01), no significant alterations were observed with 1 µM ruxolitinib (RUX) or 1000 U/ml interferon-alpha (IFNa). Overall, no differences between the 3 genotypes were observed, pointing towards a genotype-independent mode of action. Given the favorable vascular toxicity profile of IFNa, as shown above, its anti-angiogenic potential was then assessed via endothelial tube formation assays. Pre-incubation with 1000 U/ml IFNa prior to seeding of the cells onto a reconstituted basement membrane matrix led to a significant inhibition of capillary-like structures formation in JAK2 WT and JAK2V617F HET or HOM iEC. One of the limitations of 2D culture assays is the lack of 3D homotypic cellular organization, compromising its translational value. Therefore, we established an adaptive 3D fibrin-based model to recapitulate, in vitro, the critical EC-pericyte crosstalk required for formation and maturation of blood vessels. Recent studies highlighted the capacity of BM mesenchymal stem cells (MSC) to differentiate into pericytes. Therefore, we seeded JAK2 WT and JAK2V617F HETor HOM iEC with or without MSC in fibrin hydrogels. MSC and iEC were visualized using different PKH dyes as fluorescent cell linkers to enable live tracking of vascular tree formation. Although self-assembly of JAK2 WT and JAK2V617F HET or HOM iEC into vessel-like structures was observed in the generated monocultures, the presence of MSC in the cocultures led to a significant increase of the formed network area (Fig. 1), hence recapitulating the critical support of pericytes. While acute IFNa exposure did not alter vascular tree formation (Fig. 1), a separate pre-treatment of iEC and MSC prior to cocultivation, significantly impaired neoplastic vasculogenesis (Fig. 2). Interestingly, interactive microscopy image analysis revealed an increased MSC/iEC interaction in JAK2V617F HOM pre-treated cocultures (*p ≤ .05). Bulk RNA-sequencing of JAK2 WT and JAK2V617F HET or HOM iEC treated for 24h with 1000 U/ml showed a strong response to IFNa. Future analysis may unravel a JAK2V617F specific transcription signature. Moreover, we induced EndMT in our 3D iEC-MSC coculture model by treatment with pro-inflammatory cytokines, reflected by the loss of EC markers (e.g. Claudin 5) and upregulation of mesenchymal markers (e.g. aSMA) by immunofluorescence. We are currently evaluating the capacity of RUX vs IFNa to inhibit EndMT. In summary, we show that IFNa impairs neoplastic vasculogenesis in a robust in vitro 3D coculture system that enables precise control of cellularity and genotype. Our model is a valuable tool to decipher the poorly investigated impact of IFNa on the BM microenvironment. In addition, its translational and scalable properties (e.g. by integrating primary or iPSC-derived hematopoietic cells) will allow us to study the impact of additional drugs on MPN-associated fibrotic transformation of the BM.