Abnormal Hematopoiesis Research Articles

Antibiotic‐associated neutropenia is marked by the depletion of intestinal Lachnospiraceae and associated metabolites in pediatric patients

AbstractProlonged antibiotic exposure causes dangerous hematologic side effects, including neutropenia, in up to 34% of patients. Murine studies established a link between the intestinal microbiota and hematopoiesis. To identify factors that predispose to neutropenia in pediatric patients, we evaluated changes in microbiota‐derived metabolites and intestinal microbiota composition after prolonged courses of antibiotics. In this multi‐center study, patients with infections requiring anticipated antibiotic treatment of two or more weeks were enrolled. Stool samples were obtained at the start and completion of antibiotics or at neutropenia onset (prospective arm). Some patients were enrolled in a retrospective arm in which a stool sample was collected at the time of neutropenia during antibiotic therapy and 2–4 weeks after completion of antibiotics with recovery of blood counts. We identified 10 patients who developed neutropenia on antibiotics and 29 controls matched for age, sex, race, and ethnicity. Clinical data demonstrated no association between neutropenia and the type of infection or antibiotic used; however, patients with neutropenia were admitted to the intensive care unit more often and received longer courses of antibiotics. Reduced intestinal microbiome richness and, specifically, decreased abundance of Lachnospiraceae family members correlated with neutropenia. Untargeted stool metabolomic profiling revealed several metabolites that were depleted exclusively in patients with neutropenia, including members of the urea cycle pathway, pyrimidine metabolism, and fatty acid metabolism that are known to be produced by Lachnospiraceae. Our study shows a relationship between intestinal microbiota disruption and abnormal hematopoiesis and identifies taxa and metabolites likely to contribute to microbiota‐sustained hematopoiesis.

Maternal NO2 exposure and fetal growth restriction: Hypoxia transmission and lncRNAs-proinflammation-mediated abnormal hematopoiesis

Epidemiological studies show a strong correlation between air pollution and fetal growth restriction (FGR), but existing results are controversial due to inherent limitations, such as causality of specific pollutants, developmental origin, and maternal-fetal transmission. To address this controversy, we first conducted a retrospective analysis of 28,796 newborns and revealed that maternal nitrogen dioxide (NO2) exposure during the second trimester was positively associated with FGR, with an adjusted odds ratio of 1.075 (95% confidence interval: 1.020-1.133) per 10 μg/m3 NO2 increase for small for gestational age. Then, by establishing an animal model of prenatal NO2 exposure, we confirmed its adverse effects on embryonic growth and hematopoiesis in the yolk sac and fetal liver, primarily affecting the differentiation of hematopoietic stem and progenitor cells and erythroid maturation. By applying internal exposure analyses coupled with 15N isotope tracing, we found that maternal NO2 inhalation induced acquired methemoglobinemia through its byproducts and placental hypoxia in pregnant mice. Importantly, by combining transcriptional profiling, bioinformatics analysis, and RNA binding protein immunoprecipitation (RIP)/chromatin immunoprecipitation (CHIP), we clarified that placental-fetal hypoxia transmission activated hypoxia-inducible factors, disturbed hematopoiesis through the hypoxia-inducible factor 1β-long noncoding RNAs-CCAAT/enhancer binding protein alpha-proinflammatory signaling pathway, ultimately contributing to FGR progression. These findings provide insights for risk prevention and clinical intervention to promote child well-being in NO2-polluted areas.

Unveiling immunological signatures and predictors of response to immunosuppressive therapy in acquired aplastic anemia.

Acquired aplastic anemia (AA) often results from immune destruction of hematopoietic stem and progenitor cells. However, only 60%-70% of patients with AA respond to immunosuppressive therapy (IST). There is a lack of strong predictive markers for response to IST which can help therapy. Our study sought to pinpoint unique immune markers in AA patients and validate established predictors for response to IST. We enrolled 51 severe AA patients and analyzed 57 immunological parameters via flow cytometry. Additionally, we measured paroxysmal nocturnal hemoglobinuria (PNH) clone, telomere length, and thrombopoietin (TPO) levels prior to IST. After a 6-month follow-up, a response was observed. Patients with AA had a distinct immunological signature characterized by absolute lymphopenia, skewed CD4/CD8 ratio with expansion of CD8 T cells with activated and senescent phenotype. Treg counts were reduced, while the proportion of Treg A and B was comparable to controls. Treatment response was correlated with elevated absolute neutrophil count (ANC), absolute reticulocyte count (ARC), and reduced CD57+ CD8+ naive cells and B cell % before therapy. However, predictors like TPO, telomere length, and PNH did not emerge as indicators of treatment response. Identifying predictors for treatment response in AA is challenging due to abnormal hematopoiesis, genetic mutations, and treatment variables.

Mechanism and clinical progression of solid tumors bone marrow metastasis.

The rich blood supply of the bone marrow provides favorable conditions for tumor cell proliferation and growth. In the disease's early stages, circulating tumor cells can escape to the bone marrow and form imperceptible micro metastases. These tumor cells may be reactivated to regain the ability to grow aggressively and eventually develop into visible metastases. Symptomatic bone marrow metastases with abnormal hematopoiesis solid tumor metastases are rare and have poor prognoses. Treatment options are carefully chosen because of the suppression of bone marrow function. In this review, we summarized the mechanisms involved in developing bone marrow metastases from tumor cells and the clinical features, treatment options, and prognosis of patients with symptomatic bone marrow metastases from different solid tumors reported in the literature.

MicroRNA target homeobox messenger RNA in HIV induced hematopoietic inhibition.

Cytopenias are a common occurrence due to abnormal hematopoiesis persistent in patients suffering from and advancing with HIV/AIDS. In order to develop efficacious therapies against cytopenias, it is necessary to understand the mechanisms by which HIV infection affects the differentiation of hematopoietic stem-progenitor cells (HSPCs), causing hematopoietic inhibition, that leads to hematological disorders. Currently, only the antiretrovirals that are being used to treat HIV infection and indirectly lower the levels of virus replication also co-attenuate cytopenias. The evidence available suggests that this indirect efficacy may not prevail for the lifetime of the infected patients, and the acquired immunodeficiency can overtake the beneficial consequences of decreased virus replication. As cited in this article, we and our colleagues are the first to make a foray into the involvement of microRNAs and their use as potential interventional treatments for the cytopenias that occur with HIV/AIDS. Herein, we progressed further in the direction of the mechanisms of the involvement of homeobox gene regulation to cause cytopenias. We had previously shown that HIV-1 inhibits multi-lineage hematopoiesis of the CD34+ cells using SCID-hu Thy/Liv animals in vivo. Furthermore, we demonstrated that the virus-induced hematopoietic inhibition occurred despite the CD34+ cells being resistant to HIV-1 infection. We set out to search for the specific host factors secreted by CD4+ T-cells that likely participate in the inhibition of hematopoiesis of the HIV infection-resistant CD34+ cells. More recently, we reported the identification of virus-infected CD4+ thymocyte-secreted miRNA-15a and miRNA-24 and that their differential expression following HIV infection causes the indirect inhibition of hematopoiesis. We then hypothesized that the observed miRNA differential expression in the virus-infected T-cells causes the abnormal regulation of homeobox (HOX) gene-encoded transcriptomes in the CD34+ cells, affecting specific MAPK signaling and CD34+ cell fate, thereby disrupting normal hematopoiesis. We present that in HIV infection, miRNA-mediated post-transcriptional dysregulation of HOXB3 mRNA inhibits multi-lineage hematopoiesis, which translates into hematological disorders in virus-infected patients with HIV/AIDS. These observations portend specific microRNA candidates for potential efficacy against the virus-induced cytopenias that are otherwise not treatable by the existing HAART/ART regimens, which are primarily designed and applicable for the attenuation of virus replication.

Down syndrome-associated leukaemias: current evidence and challenges.

Children with Down syndrome (DS) are at increased risk of developing haematological malignancies, in particular acute megakaryoblastic leukaemia and acute lymphoblastic leukaemia. The microenvironment established by abnormal haematopoiesis driven by trisomy 21 is compounded by additional genetic and epigenetic changes that can drive leukaemogenesis in patients with DS. GATA-binding protein 1 (GATA1) somatic mutations are implicated in the development of transient abnormal myelopoiesis and the progression to myeloid leukaemia of DS (ML-DS) and provide a model of the multi-step process of leukaemogenesis in DS. This review summarises key genetic drivers for the development of leukaemia in patients with DS, the biology and treatment of ML-DS and DS-associated acute lymphoblastic leukaemia, late effects of treatments for DS-leukaemias and the focus for future targeted therapy.

Unhappy Balloon, Nitrous Oxide-induced Subacute Combined Degeneration of the Spinal Cord: A Review of Case Series

Background: The younger generation in Korea easily exposed to nitrous oxide (N<sub>2</sub>O) under the name “Happy Balloon” may abuse it. N<sub>2</sub>O can irreversibly oxidize vitamin B<sub>12</sub> and cause abnormal hematopoiesis or nervous system toxicity such as subacute combined degeneration (SCD). The objective of this study was to assist in early diagnosis of N<sub>2</sub>O-induced SCD of spinal cord by characterizing its clinical manifestations.Methods: Four patients with myelopathy after abusing N<sub>2</sub>O were enrolled. To characterize N<sub>2</sub>O-induced myelopathy, previously reported cases of N<sub>2</sub>O-induced SCD were searched through PubMed and KoreaMed. Collected cases and our four patients were analyzed.Results: A total of 30 patients with N<sub>2</sub>O-induced myelopathy (26 males and 4 females with mean age of 24 years) were analyzed. These patients took a median dose of 650 canisters for a median duration of 3.5 months. All patients presented sensory disturbances, which involved the lower extremities more frequently (100%) than the upper extremities (63.3%). Gait ataxia (76.7%), weakness in the upper (23.3%) and lower (36.7%) extremities, bladder symptoms (26.7%), Rhomberg sign (43.3%), and Lhermitte’s phenomenon (10.0%) were observed. Serum vitamin B12 levels were decreased in many (63.3%) patients and homocysteine levels were elevated in all. Of 20 patients who underwent magnetic resonance imaging (MRI) of the spine, 19 had abnormal findings. Three patients presented with contrast enhancement in lesions.Conclusions: We strongly recommend that history of N<sub>2</sub>O abuse should be asked for young patients with suspected myelopathy, especially those presenting with gait ataxia and sensory disturbances suggesting posterior column dysfunction and those presenting long-segment lesion involving the upper cervical cord on MRI.

Integrating AI and ML in Myelodysplastic Syndrome Diagnosis: State-of-the-Art and Future Prospects.

Myelodysplastic syndrome (MDS) is composed of diverse hematological malignancies caused by dysfunctional stem cells, leading to abnormal hematopoiesis and cytopenia. Approximately 30% of MDS cases progress to acute myeloid leukemia (AML), a more aggressive disease. Early detection is crucial to intervene before MDS progresses to AML. The current diagnostic process for MDS involves analyzing peripheral blood smear (PBS), bone marrow sample (BMS), and flow cytometry (FC) data, along with clinical patient information, which is labor-intensive and time-consuming. Recent advancements in machine learning offer an opportunity for faster, automated, and accurate diagnosis of MDS. In this review, we aim to provide an overview of the current applications of AI in the diagnosis of MDS and highlight their advantages, disadvantages, and performance metrics.

Single Cell Analysis and Functional Features of Circulating CD34 + Cells from Patients with Sickle Cell Anaemia Unveil a Defect in the Myeloid Lineage

Polymorphonuclear neutrophils (PMN) have long been suspected to be involved in the pathophysiology of Sickle cell anemia (SCA) since early studies have reported a higher neutrophil count in patients at steady-state and its positive correlation with SCA severity. This high leukocyte count has long be considered as a peripheral phenomenon, driven by RBC hemolysis causing systemic inflammation and neutrophils recruitment. Nevertheless, several lines of evidence suggest that hematopoietic stem/progenitor cell (HSPC) function might be perturbed in SCA. Given that recent report showed an increased apoptosis during terminal erythroid maturation of SCA cells and that erythroid and myeloid differentiation derive from the same common myeloid progenitor (CMP), we investigated a possible enhanced myelopoiesis in SCA and its causes. First, to better understand when and how hyperleukocytosis and chronic inflammation begin in these patients, we analyzed longitudinal biological data from 1250 infants with SCA aged 1-24 months. We observed that the increase in neutrophils count began as early as the first months of life, and was statistically correlated with the drop in HbF rate and with the increase in reticulocytes count, much more than with the increase of the LDH level. These results seem to contradict the hypothesis of chronic inflammation driven by intravascular hemolysis. To assess whether the abnormal hematopoiesis is associated with a defect on lineage commitment of Hematopoietic Stem/Progenitor Cells (HSPCs), we evaluated the phenotype of myeloid and erythroid progenitors isolated from peripheral blood (PB) of untreated patients with SCA. While the portion of the common myeloid progenitor (CMP) was similar among both groups, we observed a significant increase of granulocyte-monocyte progenitors (GMP) in SCA patients (n=6) compared to health donors (HDs) (n=9), as well as a significant decrease of erythroid progenitors (MEP). Interestingly, HSPCs from SCA PB produced more myeloid colonies in vitro than those in HD HSPCs. In addition, colony-forming assay of sorted SCA CMP displayed an increase of granulo-monocytes colonies CFU-GM and decrease of erythroid colonies BFU-E compared to HDs, indicating an increased myeloid commitment of SCA HSPCs. To comprehensively characterize HSPC composition, we performed 10´ scRNA-seq on CD34+ HSPCs isolated from HDs (n=3) and patients with SCA (n=4). We confirmed that SCA HSPCs have a high proportion of neutrophil progenitors. More interestingly, MPP/HSC populations from SCA patients highly express several genes involved in myeloid differentiation like SELL (L-Selectin) and CSF3R (G-CSF receptor) genes, indicating an enhanced myeloid commitment of early progenitor in SCA. To better characterize the myeloid features of these progenitors, we investigated ex- vivo granulopoiesis of PB HSPCs from SCA patients (n=10) and HDs (n=9) in presence of G-CSF as CSF3R is highly expressed in early SCA progenitors. While cell proliferation was similar among both groups, unexpectedly SCA myeloid cells presented a significant delayed expression of CD11b (p<0.01). Interestingly, we also observed an overexpression of monocytic markers HLA-DR and CD36 in a subset of differentiated cells, suggesting a potential commitment of part of the myeloid progenitors to a monocyte lineage , confirmed by MGG staining. To further understand this defect, we performed RNAseq analysis of the GMP isolated from PB of SCA patients (n=4) and HD (n=4). We identified a total of 420 differentially expressed genes (DEGs). Among these DEGs, significantly downregulated genes in SCA included granulocyte differentiation program such as GFI1, C/EBPa, and JAK3. In return, significantly upregulated genes included KLF4, supporting the monocytic bias observed in the early myeloid progenitors. In conclusion, these data reveal the myeloid-primed signature in PB HSPCs of patients with SCA, likely responsible for the elevated PMN and monocyte count in these patients. This initial breakthrough in the understanding of the development of hyperleukocytosis and chronic inflammation in these patients will open up new early therapeutic options. Moreover, since CD34+ cells of the peripheral blood are the main source of HSPCs for the development of gene therapy in SCA, deciphering their functional feature and myelo-monocytic skewing could be of interest to guide the gene editing strategy.

The Mechanism of Setd2 in Mesenchymal Stromal Cell in MDS-Associated Ineffective Hematopoiety

Myelodysplastic syndrome (MDS) is a heterogeneous, clonal, hematopoietic stem progenitor cell malignant disease. Bone marrow microenvironment participates in the occurrence and development of MDS and plays a key role. Histone methyltransferase Setd2 is the only enzyme that can catalyze the methylation of mammalian histone H3K36Me3. Previous studies have confirmed that Setd2 is involved in the the transformation of MDS to leukemia, and the expression of S100A9 decreases after Setd2 knockout. This study is based on NHD13 mice with mesenchymal stem cell (MSC) targeted knockout of Setd2. Through experimental methods such as flow cytometry, peripheral blood routine, Wright staining, HE staining, and Gomori staining, it finds that Setd2 targeted knockout mice undergo faster leukemia transformation compared to NHD13 mice; Peripheral blood white blood cells increase, while hemoglobin and platelets decrease; The differentiation of hematopoietic stem progenitor cells in bone marrow is abnormal, with abnormal hematopoiesis and increased blast cells; Spleen fibrosis aggravates and megakaryocyte infiltrates; and Extramedullary hematopoiesis in liver. Through flow cytometry,β- Galactosidase staining and other experimental methods, we finds that compared with NHD13 mice, the MSC morphology of knockout mice is irregular, and the support function for hematopoietic cells is weakened. This study demonstrates that MSC targeted knockout of Setd2 has a promoting effect on the progression of MDS. We used mass spectrometry to identify proteins that are significantly upregulated in the targeted knockout mouse bone marrow supernatant compared to the NHD13 mouse bone marrow supernatant, and and select the protein Uba5 related to hematopoiesis from them. The expression of Uba5 in the bone marrow supernatant of knockout mice is significantly higher than that in the NHD13 group through WB validation. It is speculated that the hematopoietic changes in Setd2 targeted knockout mice may be related to the downstream target protein Uba5 of Setd2. This study takes the change of bone marrow microenvironment as an entry point to explore the possible mechanism of MDS ineffective hematopoiesis, hoping to provide microenvironment regulation therapy for some MDS patients with genes that hematopoietic stem cell transplantation that cannot cure(such as TP53, NRAS, KRAS), and provide new ideas for MDS treatment. There are still many limitations in this study, further clinical validation and vivo and vitro rescue experiments on downstream target genes and proteins are still needed in the future.

Interplay between Inflammatory Microenvironment and RUNX1-Mediated Transcriptomic Changes Drives Defective Hematopoiesis in Familial Platelet Disorder

Background: Familial platelet disorder (FPD) is caused by germline loss-of-function mutations in RUNX1 an essential transcription factor regulating hematopoiesis. The absence of functional RUNX1 leads to mild-to-moderate thrombocytopenia, platelet dysfunction, and increased bleeding in FPD patients. In ~40% of cases, the patients progress to overt leukemia at median age of 33 years through unknown mechanisms. FPD patients also present with skin allergy and eczema which are associated with systematic elevation of cytokines. Given the well-established connection between inflammation and leukemia initiation and progression, we sought to investigate whether inflammatory stress play a role in regulating abnormal hematopoiesis and eventually cause clonal evolution in FPD. Methods: We acquired bone marrow cells from FPD patients who had not developed leukemia yet. We then compared FPD bone marrow hematopoietic stem and progenitors (HSPCs) with healthy progenitors using in vitro differentiation and colony formation assays. For comprehensive mechanistic evaluation, we performed 10x single-cell RNA sequencing (scRNA-seq) on FPD (n=10) and healthy (n=4) bone marrow cells, analyzing >100,000 cells using Seruat package. We evaluated cytokine levels in bone marrow fluid and peripheral blood using 65-plex Luminex assay. The impact of the inflammatory cytokines and intervention strategies were tested using in vitro differentiation and colony formation assays. The results were validated in vivo using mice with germline Runx1 mutation. Results: We identified that FPD progenitors are impaired in megakaryocytic (>2.0-fold, p<0.05) and erythroid differentiation with increased myeloid differentiation (>1.7-fold, p<0.05) compared to healthy control. In addition, FPD HSPCs show increased myeloid colony formation (>1.6-fold, p<0.05) and serial replating ability. scRNA-seq identified 18 cell clusters and pseudotime analysis revealed higher percentage of FPD cells differentiating towards monocytes, supporting enhanced myeloid differentiation. The pathway enrichment analysis of differentially expressed genes in HSC cluster revealed upregulation of inflammatory (TNFα via NF-kB) and pro-survival pathways (PI3K/AKT/mTOR). Accordingly, cytokine profiling revealed upregulation of numerous cytokines and chemokines in FPD compared to healthy samples including TNFα, CXCL8, CCL24, CCL2, and IL1β (>2.5 to 1000-fold, p<0.05), secreted by progenitors, monocytes, and MSCs. Further, under inflammatory stress mediated by TNFα, CXCL8, CCL24, CCL2, and IL1β, FPD HSPCs exhibited resistant to growth suppression and have increased myeloid colony formation. However, pharmacological inhibition of individual cytokines only partially rescued the differentiation defects, possibly due to compensatory effects of each cytokine. Phospho-flow analysis revealed that increased cytokine levels leads to the activation of PI3K/mTOR and JAK signaling that we also found upregulated using scRNA-seq analysis. Interestingly, inhibition of mTORi (rapamycin and AZD2014), PI3Ki (idelalisib), and JAKi (ruxolitinib) showed an increase in percentage of megakaryocytes (up to 2-fold, p<0.05) and a decrease in monocytes (up to 0.6-fold change, p<0.05). Also, inhibition of these pathways in FPD reduced cytokines levels. In line with human FPD data, in vivo treatment of Runx1 R188Q/+ mouse with ruxolitinib suppressed myeloid differentiation with no effects on megakaryocytes, while rapamycin resulted in significantly increased platelet activation compared to untreated mice (>1.7-fold, p<0.05) and a reduction of monocytes (<0.5-fold, p<0.05), suggesting mTOR inhibition might be an effective intervention strategy. Conclusion: FPD HSPCs manifest defective megakaryopoiesis with increased myelopoiesis. The elevated autocrine and paracrine inflammatory stress is an early event in FPD, driving myeloid differentiation. This inflammatory stress mediates activation of PI3K/mTOR and JAK signaling, leading to augmented myeloid differentiation. Consequently, the observed FPD phenotypes are the result of combined transcriptional changes and inflammatory stress. Targeting inflammatory and pro-survival signaling at an early stage might mitigate hematopoietic defects and delay disease evolution in FPD. Our results advance mechanistic understanding and hold significant clinical implications.

Brd4 Inhibition Abrogates Inflammation and Self-Renewal in a Murine Model of Tet2 Mutated Clonal Hematopoiesis

Clonal hematopoiesis (CH) is characterized by the somatic acquisition of malignancy associated mutations in hematopoietic stem cells (HSCs). Loss-of-function mutations in TET2 occur in one third of CH cases and are highly associated with the subsequent development of myeloid malignancies. Tet2 KO mice demonstrate increased HSC self-renewal, expansion of myeloid precursors, elevated inflammation, and progression to leukemia with long latencies, similar to CH to AML progression in humans. Additionally, leukemia development in Tet2 KO mice is dependent on increased production of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α through hyperactive toll-like-receptor signaling (TLR). Despite extensive characterization of the adverse effects related to TET2 mutations, no effect intervention or pharmacologic therapy has been developed to slow its known progression. To investigate altered pathways in Tet2 mutant CH that might be amenable to pharmacologic targeting, we performed RNA-seq on sorted Lin -cKIT + (LK) myeloid progenitors and Lin -cKIT +Sca-1 + (LSK) stem cells from Tet2 KO vs WT mice. We found significant upregulation of hallmark JAK/STAT3 and TNF-α signaling pathways in Tet2 KO cells. The epigenetic reader protein bromo- and extra-terminal domain 4 (Brd4) interacts with both NF-κB and STAT3 to promote transcriptional activity and regulate inflammation. We hypothesized inhibition of Brd4 would reverse abnormal hematopoiesis in Tet2 KOs via reduction in inflammatory signaling. Therefore, we generated double KO mice by intercrossing Mx1-Cre mice with Tet2 conditional KO and Brd4 conditional KO mice. While deletion of Brd4 during fetal hematopoiesis is lethal, we have successfully induced deletion of Brd4 both alone and in combination with Tet2 in young adult animals without any observed lethality for 6 months. To study pharmacologic Brd4 inhibition in Tet2 KO mice, we utilized PLX51107, a structurally distinct BET inhibitor previously reported on by our laboratory. As expected, Tet2 KO marrow demonstrated increased colony formation and serial replating ability compared to WT controls. However, colony formation by Brd4 Tet2 DKO marrow was significantly decreased when compared to Tet2 KO ( p = 0.0003, n=5) or WT mice ( p = 0.0048, n=5) (Figure 1A). Similarly, PLX51107 treatment reduced Tet2 KO colony formation ( p = 0.0001, n=5) and abrogated serial replating ability ( p < 0.01, n=5). When Brd4 KO or Brd4 Tet2 DKO marrow was treated with PLX51107, which also inhibits Brd2 and Brd3, there was no significant change in colony formation compared to untreated marrow, suggesting Brd4 has a non-redundant role in colony formation. Collectively, these results suggest Brd4 plays a key role in self-renewal of Tet2 KO HSCs. To further characterize the role of Brd4 in Tet2 mutated CH, single cell RNA-seq analysis of Brd4 KO and Brd4 Tet2 DKO marrow populations is in progress. In murine models of Tet2 CH, disease progression is associated with emergence of an aberrant population of inflammatory macrophages. We therefore investigated functional differences in macrophages from WT, single, and double KO mice. LPS-stimulated bone marrow derived macrophages (BMDMs) from Brd4 Tet2 DKO mice produced significantly less TNF-α, IL-1β, IL12p40, and IL-18 when compared to single KO or WT controls ( p < 0.001, n=3) (Figure 1B). Similarly, we found mRNA expression of these cytokines to be decreased in LPS-stimulated Brd4 Tet2 DKO BMDMs when compared to single KO and WT controls ( p < 0.001, n=3). Similar results were observed in LPS-stimulated Tet2 KO and WT BMDMs treated with PLX51107 ( p < 0.005, n=3). Additionally, PLX51107 treatment reduced protein levels pSTAT3, NLRP3, and phosphorylated p65, key downstream targets of TLR signaling, in LPS-stimulated BMDMs. Together, these results suggest that Brd4 is required for the hyperinflammation characteristic of Tet2 mutated macrophages that drive progression to myeloid malignancies. In summary, we report that genetic or pharmacologic inhibition of Brd4 reduces the increased self-renewal and elevated inflammation in a murine model of Tet2 mutated CH. Our novel inducible double knockout mouse model of CH achieved simultaneous knockout of Brd4 and Tet2, which has never been investigated. Collectively, our data provides rationale for further pre-clinical investigation of BET inhibitors to prevent progression of Tet2 CH.

Methods to Elucidate Hematopoietic Clonal Organization in Bone Marrow

Every tissue in the human body becomes a mosaic of somatic mutations throughout life. Each cell in the body contains a record of the genetic alterations occurring from conception until that individual cell came into existence through cell division. Most of these changes are benign, but cells with alterations that endow a selective advantage can preferentially divide and expand. Already, this process has been described in a diverse set of phenotypically normal tissues including skin, esophagus and colon (Colom et al., 2021; Lee-Six et al., 2019; Martincorena et al., 2015). In these solid organs, clonal mutations are geographically constrained within contiguous regions of tissue. In the blood, these somatic mutations are termed clonal hematopoiesis (CH) (Steensma et al., 2015). Previous work has extensively characterized the burden of CH in humans, showing an increased prevalence with age and increased risk for hematologic malignancy and all-cause mortality (Genovese et al., 2014; Jaiswal et al., 2014; Young et al., 2016; Young et al., 2019). Despite our detailed understanding of CH in the blood, little is known about how these hematopoietic clones exist in the bone marrow (BM) and how they interact with the BM niche. We hypothesize that the spatial restriction of clones observed in solid organs also exists in the BM. To characterize the spatial and clonal organization within the BM niche, we combined single cell proteogenomics with novel methods for spatially aware variant detection. We characterized the BM clonal organization of a patient with polycythemia vera (PV)-a myeloproliferative neoplasm (MPN). MPNs are blood disorders characterized by abnormal hematopoiesis and an increased risk of transformation to aggressive leukemia. Studying a patient with PV was ideal as this is an oligoclonal disease, and we had prior knowledge that the patient's disease was driven by a large JAK2 V617F mutated clone and a presumed DNMT3A R882Cmutated subclone. Single BM cells were prepared for proteogenomics using the Tapestri platform (Mission Bio). From the BM aspirate, 1,486 individual live BM cells were assayed using the TotalSeq-D Heme Oncology Cocktail V1.0 (BioLegend) for immunophenotyping and the 45 gene myeloid panel (Mission Bio) for genomic characterization. Analysis identified the known JAK2 V617F and DNMT3A R882C mutations, and previously unknown TET2 L1081* and GNAS R844H mutations. The single cell assay disentangled the clonal complexity of this patient's disease-a large JAK2 V617F homozygous clone, a small TET2 L1081* heterozygous subclone, a small JAK2 V617F heterozygous clone, and independent GNAS R844H heterozygous and DNMT3A R882C heterozygous clones. Proteomic analysis revealed that the cells clustered into four groups including; B-lymphocytes marked by CD19, T-lymphocytes marked by CD3, the presumptive MPN cells highly expressing CD141 (thrombomodulin) and CD71 (transferrin receptor 1), and a mixed population of largely CD38 positive hematopoietic progenitors. To further characterize the spatial organization of these hematopoietic clones in the BM, the patient's femur head was sliced into 2mm sections using a band-saw and then further partitioned with hand tools into approximately 400 BM fragments, of which 96 were selected for droplet digital PCR (ddPCR) targeting the mutations identified by single-cell and bulk sequencing. The ddPCR assay identified each of these mutations in all assayed BM fragments, albeit at a wide range of variant allele fractions (VAFs). Interestingly, this suggested a heterogeneous distribution of somatically mutated clones spatially in the BM. Clustering of these bone marrow fragments identified regions marked by high JAK2 V617F and TET2 L1081* VAF (cluster 2), high JAK2 V617F VAF alone (cluster 3), and lower VAF for any mutations (clusters 1 and 2; Figure 1). These findings provide a comprehensive characterization of spatial organization and clonal hierarchy within the BM. Surprisingly, the clonal mutations were not well mixed in the BM with regions of high clonal diversity millimeters away from regions with a paucity of these mutated clones. Future work will expand this platform to describe BM clonal architecture in a larger set of bone marrow specimens and a wide variety clinical scenarios to help further explain the role of the BM niche in CH, MPNs and leukemia.

Severe Congenital Neutropenia-Type 5: Impaired T Cell Proliferation, Aberrant Th1 Cytokine Production, Abnormal Megakaryocytes, and Impaired Platelet Granule Formation in a Patient with VPS45 Deficiency Caused By Uniparental Isodisomy

Introduction VPS45 is essential in early endosome formation with rabenosyn-5 and syntaxin 16. VPS45 deficiency is one of the inborn errors of immunity characterized by severe congenital neutropenia and myelofibrosis. Impaired production and function of neutrophils, increased cell death, and abnormal hematopoiesis have been reported, whereas there are several undetermined pathophysiologies, such as T and/or NK lymphopenia or myelofibrosis. Here, we experienced a girl with VPS45 deficiency presenting with neutropenia at birth, progressive T and NK lymphopenia, hepatosplenomegaly, and myelofibrosis in infancy. We examined the pathophysiology of VPS45 deficiency focusing on hematologic disorder, T cell function, and myelofibrosis. Methods We sequentially evaluated peripheral blood and bone marrow cell counts and morphologic features. Genetic analysis was performed using DNA from peripheral blood. We evaluated T cell proliferation using the CFDA-SE cell tracer kit with the stimulation of PHA-L or anti-CD3/CD28 beads. Serum cytokine analysis was performed by beads array. Protein expression was examined in platelets from the patient and the healthy control by western blot analysis. Results Case: The patient was born to nonconsanguineous Japanese parents. She received a blood examination for mild respiratory distress at birth. This examination demonstrated neutropenia, the emergence of myeloid cells, and nucleated red blood cells in the peripheral blood, whereas mature neutrophils were confirmed in the bone marrow. Genetic analysis demonstrated homozygous pathogenic variants of p.E238K in VPS45. The same heterozygous variant was confirmed in her father, but not in her mother. SNP array analysis demonstrated that she inherited chromosome 1 only from her father and developed VPS45 deficiency by paternal uniparental isodisomy. At 7 months, she demonstrated T and NK lymphocytopenia, macrothrombocytopenia, and hepatosplenomegaly. Bone marrow examination demonstrated the presence of blast cells, micro or separated megakaryocytes, and pseudo-Pelger-Huet anomaly. Electronic microscopy of peripheral blood demonstrated abnormal granules in neutrophils and macro-thrombocytes with reduced alpha granules and open channel enlargement. Elevated interferon-gamma, IL-12, and IL-23 levels, representing T helper 1 (Th1) activation were found. We initiated ruxolitinib at 7 months to suppress aberrant Th1 activation and extra-medullar hematopoiesis which could be associated with massive hepatosplenomegaly. Ruxolitinib was effective for hepatosplenomegaly and reduced Th1 cytokine levels without significant complications. She received hematopoietic cell transplantation with bone marrow from an HLA-matched unrelated donor 30 days after the initiation of ruxolitinib. T cell function: With anti-CD3/CD28 beads stimulation, the patient's CD8-positive cells were significantly less proliferative. Either CD4-positive or CD8-positive cells did not proliferate with PHA-L stimulation. Platelet analysis: Western blot analysis of platelet demonstrated a reduced amount of alpha granule proteins (von Willebrand factor and PF4), not of p-selectin. Discussion VPS45 deficiency was characterized by severe congenital neutropenia and myelofibrosis. Furthermore, T cell deficiency, especially CD8-positive T lymphopenia was observed in some patients with VPS45 deficiency. Our study suggested that T cell lymphopenia would be associated with impaired TCR signaling and mitogen response, although the detailed mechanism remains to be elucidated. The impaired platelet alpha granule formation confirmed by electron microscopy and western blot analysis in our patients was compatible with the previous VPS45 deficiency patient. A similar manifestation is observed in gray platelet syndrome which is also associated with affected endosomal transport. Affected endosomal transport would be responsible for myelofibrosis. We hypothesized that massive hepatosplenomegaly was attributable to the hyper-activated Th1 cells and extra-medullar hematopoiesis associated with JAK1 and JAK2 signaling. Indeed, JAK1/ JAK2 inhibitor, ruxolitinib improved hepatosplenomegaly in this patient. Conclusion VPS45 deficiency could broadly affect not only neutropenia and myelofibrosis but T cell, NK cell, megakaryocyte, and platelet function.

Integrating AI and ML in Myelodysplastic Syndrome Diagnosis: State of the Art and Future Prospects

Introduction: Myelodysplastic syndrome (MDS) is a diverse group of hematological malignancies characterized by dysfunctional pluripotent stem cells, leading to abnormal hematopoiesis and cytopenia. Early diagnosis is crucial for improving patient outcomes. Artificial Intelligence (AI) and Machine Learning (ML) have emerged as powerful tools in healthcare for disease diagnosis and monitoring. This review aims to summarize the current state of AI application in the diagnosis of MDS, discussing various ML models, their advantages, disadvantages, and performance metrics. Methods: A comprehensive search strategy was developed using Medical Subject Headings (MeSH) terms and relevant keywords related to MDS and AI/ML. Pubmed, Embase, and scopus were searched, and duplicates were removed. Only original research articles studying ML algorithms in the diagnosis of MDS in human subjects were included. Studies on animals, reviews, non-original articles, and non-English papers were excluded. For each article, data on the area under the receiver operating curve, sensitivity, specificity, and accuracy for the best preforming model were collected. Results: The search yielded 313 articles, and after screening, 16 articles met the inclusion criteria. These articles were grouped based on the data utilized in the training of their models. Five articles utilized bone marrow smears for the training of their data. Their models aimed to detect dysplastic cells and blasts in bone marrow smears. The majority of these studies utilized convolutional neural network (CNN), a ML algorithm, for image recognition. One study utilized decision tree models. The AUC of these models ranged between 0.8 and 0.996. All the models were internally validated but were not externally validates. Three studies utilized peripheral blood smears for the training of their ML models. Two of these studies also utilized CNN for the detection of dysplasia and subsequent diagnosis of MDS. One study utilized CART to create a decision tree algorithm. The AUC for these models ranged between 0.982-0.99. Four studies utilized flowcytometry data from bone marrow samples for the training of their AI models. These models achieved AUC ranging between 0.935-0.97. Two of these models were both internally and externally validated, one of which was on a prospective cohort. Conclusion: The integration of AI and ML in MDS diagnosis holds promise for early detection and improved patient care. The reviewed studies showed encouraging results, indicating that AI-based approaches can complement traditional diagnostic methods and aid in making more informed clinical decisions especially when it comes to analysing blood/bone smears. However, many of these models need to be externally validated in large prospective cohorts to establish AI's role in routine clinical practice.

The Transcriptional Impact of 5q Deletion in MDS at Single Cell Resolution

Introduction: Myelodysplastic syndromes with 5q deletion (del(5q) MDS) are characterized by two commonly deleted regions (CDRs): 5q31 and 5q32-33. Although several genes within these CDRs have been associated with disease pathophysiology, the molecular basis underlying its origin is still unknown. Lenalidomide represents the first therapeutic approach for del(5q) MDS, but patients experience loss of responsiveness within 2-3 years, highlighting the need of characterizing the mechanisms impairing treatment response. While previous studies have identified dysregulated genes and pathways in the disease, they overlooked that the hematopoietic system of these patients is composed of a mixture of cells with and without the deletion (del(5q) and non-del(5q) cells), which hampers the ability to unravel the real transcriptional impact of the deletion. Methods: We isolated by FACS and performed single-cell RNA sequencing (scRNAseq, 10XGenomics Chromium platform) of CD34+ cells from 4 untreated patients, 2 patients treated with lenalidomide (partial (PCR) and complete (CCR) cytogenetic responders), and 3 healthy elderly donors. To identify del(5q) and non-del(5q) cells, the copy number alteration (CNA) inference methods CopyKat and CaSpER were applied. To unveil the transcriptional alterations prompted by the deletion, differential expression (DE), gene regulatory network (GRN) and cell-to-cell communication (CCC) analyses were performed (Figure 1). Results: CNA methods robustly identified del(5q) cells and non-del(5q) cells, which was consistent with the del(5q) percentage obtained by karyotyping. At diagnosis, del(5q) cells were detected in all hematopoietic progenitors, including the hematopoietic stem cells (HSC), suggesting that the deletion occurs in the earliest stages of hematopoietic differentiation. Although the distribution of del(5q) cells was highly heterogeneous among patients (Figure 2), a positive enrichment of del(5q) cells was detected in erythroid progenitors, underscoring the role of this genetic lesion in the anemia characterizing del(5q) MDS. To delve into the transcriptional mechanisms differentiating del(5q) and non-del(5q) cells, we performed DE and GRN analyses. Only 7 genes were downregulated in del(5q) cells; however, most play a key role in MDS and other tumors, such as PRSS21, MAP3K7CL, and CCL5. GRN analyses identified regulons JARID2, IRF1 and KAT6B, known to be key for proper hematopoietic differentiation, showing less activity in del(5q) cells. Likewise, the regulons RERE and KDM2A, which downregulated genes involved in ribosomal processes, were more active in del(5q) cells, supporting the concept that cells harboring the deletion may have a more prominent role in promoting altered hematopoiesis. However, comparisons against healthy donor cells suggested that non-del(5q) cells could share similar, albeit attenuated, abnormal behavior to del(5q) cells. In line with these results, CCC analyses identified minimal differential interactions in del(5q) cells with respect to non-del(5q) cells, indicating that the deletion does not confer a differential communicative potential to the cells. Importantly, several interactions present in MDS cells were absent in healthy cells, such as the AGTRAP-RACK1, whose members have been postulated as potential therapeutic targets for promoting proliferation in other cancers. After lenalidomide, del(5q) and non-del(5q) cells from PCR and CCR patients showed higher protein degradation and erythropoietin signaling, as well as higher activity of the PD-L1/PD-1 pathway, suggesting a potential immunosuppressive mechanism of these cells after treatment. Even if several transcriptional alterations detected at diagnosis were reverted after lenalidomide, ribosomal translation processes were still altered, even at time of CCR. These results evidence that, after lenalidomide, non-del(5q) progenitor cells revert some of the transcriptional alterations that present at diagnosis, while maintaining other lesions that could be relevant for abnormal hematopoiesis, and potentially, for the future relapse of the patients. Conclusions: This study provides the first characterization of del(5q) and non-del(5q) cells at the single-cell resolution and reveals previously unknown transcriptional alterations that could contribute to disease pathogenesis or less responsiveness to lenalidomide.

STAG2 Somatic Mutations Are Associated with Specific Dysplastic Megakaryocytic and Myeloid Cell Features in Myelodysplastic Syndrome

Introduction: Although myelodysplastic neoplasms/myelodysplastic syndromes (MDS) are driven by genetic mutations, their diagnosis relies on morphologic evaluation of abnormal hematopoiesis in the bone marrow. Only a small number of genetic abnormalities define bone marrow morphologic features in MDS, such as those harboring SF3B1 mutations and deletions of chromosome 5q. We hypothesized that there are additional genetic alterations and dysplastic morphologic features in MDS that have not been well-described in the literature. We assessed genetic morphologic associations between 50 commonly mutated genes and 10 morphologic features in a cohort of MDS bone marrows with a high degree of dysplasia. We validated and extended these findings in an independent cohort of myeloid neoplasms. STAG2 mutations were strongly associated with specific dysplastic megakaryocytic and myeloid cell features in both cohorts. Methods: We reviewed bone marrow core biopsies and aspirates from 272 MDS clinical cases (2013-2014) and selected 89 for further study based on the presence of prominent dysplastic features. Each case was independently evaluated by two pathologists for the following dysplastic features: small hypolobated megakaryocytes, widely separated megakaryocyte nuclei; abnormal myeloid nuclear segmentation, myeloid hypogranulation, Auer rods; ring sideroblasts, erythroid binucleation, irregular erythrocyte nuclei, erythroid nuclear cytoplasmic dyssynchrony, and erythroid karyorrhexis. Targeted next generation sequencing was performed using a 50 gene panel comprising the most common somatic myeloid mutations. For each dysplastic feature, univariate risk analysis was used to identify genetic mutations for use in multivariable analysis. Significant associations were validated through blinded morphologic review in an independent cohort of 155 bone marrow biopsies of myeloid neoplasms, comprised of 83 myelodysplastic syndromes, 33 myelodysplastic/myeloproliferative neoplasms and 39 acute myeloid leukemias, diagnosed between 2014 and 2019 and selected for mutations identified in the initial discovery cohort ( STAG2, RUNX1, SRSF2, ASXL1 and SETBP1) . Results: Pairwise analyses demonstrated broad patterns of genetic associations with myeloid/megakaryocytic dysplasia versus erythroid dysplasia (Panel 1). In multivariable analysis, STAG2 and SRSF2 mutations were significantly associated with megakaryocytes with widely separated nuclei ( STAG2, OR = 25.5, 95% CI [4.17, 493.5], P = 0.003; SRSF2, OR = 10.1, 95% CI [2.07, 75.0], P = 0.008). Mutations in STAG2 and SETBP1 were significantly associated with abnormal myeloid nuclear segmentation ( STAG2, OR = 7.08, 95% CI [1.94, 28.0], P = 0.003; SETBP1, OR = 12.2, 95% CI [1.29, 267.4], P = 0.04). Furthermore, STAG2 mutations were significantly associated with myeloid cell hypogranulation (OR = 12.7, 95% CI [3.10, 86.3], P = 0.002). Because this initial cohort was relatively small, which led to wide confidence intervals suggesting estimation instability, we validated and extended these findings in an independent cohort of myeloid neoplasms. In the validation cohort (enriched for mutations in STAG2, SRSF2, and SETBP1), STAG2 mutations were significantly associated with separated megakaryocyte nuclei (univariate OR = 4.18, 95% CI [1.98, 8.80], P = 0.0002), abnormal myeloid nuclear segmentation (univariate OR = 3.47, 95% CI [1.47, 8.22], P = 0.008), and myeloid cell hypogranulation (multivariable OR = 5.28, 95% CI [2.18, 13.4], P = 0.0003) (Panel 2). In contrast, morphologic associations with SRSF2 and SETBP1 mutations were not validated. Conclusion: STAG2 genetic alterations are strongly associated with the presence of separated megakaryocyte nuclei, abnormal myeloid nuclear segmentation and myeloid cell hypogranulation in MDS. Our data suggest that STAG2 may affect pathways involved in nuclear shape control and secondary granule formation. Panel 1: Pairwise associations between gene mutation and bone marrow morphology in MDS. N, number. Panel 2: Multivariable (black) and univariate (white) analysis in two independent cohorts showing significant gene associations with megakaryocytes with separated nuclei, abnormal myeloid nuclear segmentation, and myeloid cell hypogranulation. CI, confidence interval.

Abnormal adipogenic signaling in the bone marrow mesenchymal stem cells contributes to supportive microenvironment for leukemia development

BackgroundAcute myeloid leukemia (AML) is an aggressive hematological malignancy, associated with unfavorable patient outcome, primarily due to disease relapse. Mesenchymal stem cells (MSCs) residing in the bone marrow (BM) niche are the source of mesenchyma-derived subpopulations, including adipocytes, and osteocytes, that are critical for normal hematopoiesis. This study aimed to characterize BM-derived adipocyte/osteocyte fractions and their crosstalk with AML cells as a potential mechanism underlying leukemogenesis.MethodsBM cell subpopulations derived from primary AML patients were evaluated using humanized ex-vivo and in-vivo models, established for this study. The models comprised AML blasts, normal hematopoietic stem and progenitor cells and mesenchymal stromal subpopulations. ELISA, FACS analysis, colony forming unit assay, whole exome sequencing and real-time qPCR were employed to assess the differentiation capacity, genetic status, gene expression and function of these cell fractions. To explore communication pathways between AML cells and BM subpopulations, levels of signaling mediators, including cytokines and chemokines, were measured using the ProcartaPlex multiplex immunoassay.ResultsThe study revealed deficiencies in adipogenic/osteogenic differentiation of BM-MSCs derived from AML patients, with adipocytes directly promoting survival and clonogenicity of AML cells in-vitro. In whole exome sequencing of BM-MSC/stromal cells, the AHNAK2 gene, associated with the stimulation of adipocyte differentiation, was found to be mutated and significantly under-expressed, implying its abnormal function in AML. The evaluation of communication pathways between AML cells and BM subpopulations demonstrated pronounced alterations in the crosstalk between these cell fractions. This was reflected by significantly elevated levels of signaling mediators cytokines/chemokines, in AML-induced adipocytes/osteocytes compared to non-induced MSCs, indicating abnormal hematopoiesis. Furthermore, in-vivo experiments using a fully humanized 3D scaffold model, showed that AML-induced adipocytes were the dominant component of the tumor microenvironment, providing preferential support to leukemia cell survival and proliferation.ConclusionsThis study has disclosed direct contribution of impaired functional, genetic and molecular properties of AML patient-derived adipocytes to effective protection of AML blasts from apoptosis and to stimulation of their growth in vitro and in vivo, which overall leads to disease propagation and relapse. The detected AHNAK2 gene mutations in AML-MSCs point to their involvement in the mechanism underlying abnormal adipogenesis.AaFEVfq6u-1YqH7bnQ85uaVideo

Understanding blood development and leukemia using sequencing-based technologies and human cell systems.

Our current understanding of human hematopoiesis has undergone significant transformation throughout the years, challenging conventional views. The evolution of high-throughput technologies has enabled the accumulation of diverse data types, offering new avenues for investigating key regulatory processes in blood cell production and disease. In this review, we will explore the opportunities presented by these advancements for unraveling the molecular mechanisms underlying normal and abnormal hematopoiesis. Specifically, we will focus on the importance of enhancer-associated regulatory networks and highlight the crucial role of enhancer-derived transcription regulation. Additionally, we will discuss the unprecedented power of single-cell methods and the progression in using in vitro human blood differentiation system, in particular induced pluripotent stem cell models, in dissecting hematopoietic processes. Furthermore, we will explore the potential of ever more nuanced patient profiling to allow precision medicine approaches. Ultimately, we advocate for a multiparameter, regulatory network-based approach for providing a more holistic understanding of normal hematopoiesis and blood disorders.

A systematic approach identifies p53-DREAM pathway target genes associated with blood or brain abnormalities.

p53 (encoded by Trp53) is a tumor suppressor, but mouse models have revealed that increased p53 activity may cause bone marrow failure, likely through dimerization partner, RB-like, E2F4/E2F5 and MuvB (DREAM) complex-mediated gene repression. Here, we designed a systematic approach to identify p53-DREAM pathway targets, the repression of which might contribute to abnormal hematopoiesis. We used Gene Ontology analysis to study transcriptomic changes associated with bone marrow cell differentiation, then chromatin immunoprecipitation-sequencing (ChIP-seq) data to identify DREAM-bound promoters. We next created positional frequency matrices to identify evolutionary conserved sequence elements potentially bound by DREAM. The same approach was developed to find p53-DREAM targets associated with brain abnormalities, also observed in mice with increased p53 activity. Putative DREAM-binding sites were found for 151 candidate target genes, of which 106 are mutated in a blood or brain genetic disorder. Twenty-one DREAM-binding sites were tested and found to impact gene expression in luciferase assays, to notably regulate genes mutated in dyskeratosis congenita (Rtel1), Fanconi anemia (Fanca), Diamond-Blackfan anemia (Tsr2), primary microcephaly [Casc5 (or Knl1), Ncaph and Wdr62] and pontocerebellar hypoplasia (Toe1). These results provide clues on the role of the p53-DREAM pathway in regulating hematopoiesis and brain development, with implications for tumorigenesis.