Erythroid Colonies Research Articles

Childhood and adolescent essential thrombocythemia and prefibrotic primary myelofibrosis: insights into diagnosis, outcomes, and treatment from a large Chinese cohort.

The paucity of essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (pre-PMF) in individuals younger than 18 years highlights several unresolved issues in diagnosis, clinical outcomes, and treatment strategies. To address these knowledge gaps, we analyzed a large bidirectional cohort consisting of childhood and adolescent ET (CAA-ET, n = 156) and pre-PMF (CAA-preMF, n = 13), as well as adult ET (n = 349). We introduced immunophenotypic abnormalities as novel clonal markers in CAA-ET and CAA-preMF, establishing a comprehensive method for clonal marker detection that integrated driver and non-driver mutations, positive endogenous erythroid colony formation, immunophenotypic abnormalities, and chromosomal aberrations. Next-generation sequencing revealed distinct mutational profiles between CAA-ET and adult ET, along with different age-related trends in the distribution of driver mutations. Venous thrombosis was more prevalent in CAA-ET, with JAK2 V617F emerging as a potential risk factor (P = 0.018). Immunophenotypic abnormalities were identified as risk factors for disease progression (P = 0.027). Significant differences between expected and actual treatment practices were identified. Compared to CAA-ET, CAA-preMF demonstrated poorer progression-free survival (P < 0.001) and faster disease progression (P = 0.019). This study provides a critical foundation for refining diagnostic, prognostic, and therapeutic approaches for CAA-ET and CAA-preMF.

Aplastic anemia in association with multiple myeloma: clinical and pathophysiological insights

We investigated immune cytopenia in multiple myeloma (MM) patients with concurrent acquired aplastic anemia (AA), focusing on three clinical cases treated with plasma cell-directed therapy. All three patients achieved partial response in MM and one patient experienced complete resolution of AA. Two patients had partial improvement in transfusion requirement but continued to suffer from severe AA, leading to immunosuppressive therapy (IST) with improvement of transfusion requirement in both patients. In vitro serum testing of these patients demonstrated platelet mitochondrial dysfunction and platelet apoptosis but did not show sera-specific inhibition of erythroid colony formation in progenitor cells. The levels of IL8 and IL15 were elevated in MM patients with AA, implicating their potential roles in this co-occurrence. Response to IST points to the possibility of myeloma-dysregulated immune system leading to autoreactive T-cell destruction of hematopoietic stem and progenitor cells, offering insights for developing new treatment for cytopenia in MM.

Multiplex imaging reveals spatially resolved DNA-damage response neighborhoods in TP53-mutated myelodysplastic neoplasms.

While increased DNA damage is a well-described feature of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), it is unclear whether all lineages and all regions of the marrow are homogeneously affected. In this study, we performed immunohistochemistry on formalin-fixed, paraffin-embedded whole-section bone marrow biopsies using a well-established antibody to detect pH2A.X (phosphorylated histone variant H2A.X) that recognizes DNA double-strand breaks. Focusing on TP53-mutated and complex karyotype MDS/AML, we find a greater pH2A.X+ DNA damage burden compared to TP53 wild-type neoplastic cases and non-neoplastic controls. To understand how double-strand breaks vary between lineages and spatially in TP53-mutated specimens, we applied a low-multiplex immunofluorescence staining and spatial analysis protocol to visualize pH2A.X+ cells with p53 protein staining and lineage markers. pH2A.X marked predominantly mid- to late-stage erythroids, whereas early erythroids and CD34+ blasts were relatively spared. In a prototypical example, these pH2A.X+ erythroids were organized locally as distinct colonies, and each colony displayed pH2A.X+ puncta at a synchronous level. This highly coordinated immunophenotypic expression was also seen for p53 protein staining and among presumed early myeloid colonies. Neighborhood clustering analysis showed distinct marrow regions differentially enriched in pH2A.X+/p53+ erythroid or myeloid colonies, indicating spatial heterogeneity of DNA-damage response and p53 protein expression. The lineage and architectural context within which DNA damage phenotype and oncogenic protein are expressed is relevant to current therapeutic developments that leverage macrophage phagocytosis to remove leukemic cells in part due to irreparable DNA damage. © 2024 The Pathological Society of Great Britain and Ireland.

Identification and characterization of myeloid cells localized in the tadpole liver cortex in Xenopus laevis

In the present study, using transgenic frogs that express GFP specifically in myeloid cells under the myeloperoxidase enhancer sequence, we found that myeloperoxidase-positive cells are localized in the liver cortex at the late tadpole stages. Immunohistochemical analysis revealed that myelopoiesis in the liver cortex became evident after st. 50 and reached its peak by st. 56. Transplantation experiments indicated that cells with a high density at the liver cortex were derived from the dorso-lateral plate tissue in the neurula embryo. Analysis of smear samples of the cells isolated from collagenase-treated liver tissues of the transgenic tadpoles indicated that myeloid cells were the major population of blood cells in the larval liver and that, in addition to myeloid colonies, erythroid colonies expanded in entire liver after metamorphosis. Cells that were purified from the livers of transgenic tadpoles according to the GFP expression exhibited the multi-lobed nuclei. The results of present study provide evidence that the liver cortex of the Xenopus tadpole is a major site of granulopoiesis.

Abstract 4562: Activity of PARP1-selective inhibitor SNV-001 in models of HRD cancers as monotherapy and in combination

Abstract Current FDA approved poly (ADP-ribose) polymerase inhibitors (PARPi) inhibit both PARP1 and PARP2. Genetic studies indicate that loss of PARP1 is synthetic lethal with homologous recombination deficiency (HRD), including BRCA1/2 mutations, and PARP2 depletion may be linked to hematologic toxicity. To date, no first generation PARPi has been successfully combined with standard of care (SoC) agents in the clinic without significant reduction in dosing intensity because of overlapping toxicities. A selective inhibitor could potentially improve the therapeutic index and provide additional opportunities in combination strategies especially with drugs that are prone to myelosuppression. SNV-001 potently and selectively inhibits PARP1 in biochemical and cellular assays. SNV-001 inhibits PARylation in the low nanomolar range with over 500-fold selectivity against PARP2. SNV-001 did not inhibit any other PARP family member tested up to 10 uM in biochemical assays confirming its selectivity for PARP1. In human erythroid and myeloid colony formation assays, the IC50s of SNV-001 for both lineages were approximately 100-times higher than the target efficacious concentration based on the IC95 for inhibition of DLD1 (BRCA2 -/-) colony formation. In a subcutaneous MDA-MB-436 (BRCA1mut) xenograft model, oral administration of SNV-001 once daily resulted in a dose-dependent tumor regression. SNV-001 showed greater efficacy at lower doses compared to olaparib at 100 mg/kg or at equivalent doses of AZD9574. SNV-001 showed promising synergy with SoC chemotherapies or DDR inhibitors in cell proliferation assays. SNV-001 enhanced cytotoxicity of the DNA-damaging agent topotecan or carboplatin in Capan-1 (BRCA2mut) and HCC1395 (BRCA1mut) cells. Synergy was seen between SNV-001 and cell cycle check point inhibitors camonsertib (ATRi) or adavosertib (WEE1i) in DLD1 (BRCA2 -/-) and UWB1.289 (BRCA1mut) cells, as DNA damage induced by PARP1 trapping relies on cell cycle modulation for repair. Combination of SNV-001 with polymerase theta inhibitor ART558 or USP1 inhibitor KSQ4279 exhibited synergistic effects in DLD1 (BRCA2 -/-) or UWB1.289 (BRCA1mut) cells. These results suggest that inhibition of microhomology-mediated end-joining and translesion synthesis pathways may further exacerbate SNV-001-induced DDR in certain cellular contexts.In summary, the PARP1 selective inhibitor SNV-001 demonstrates potent antitumor activity and shows promising synergy between SoC chemotherapies or DDR inhibitors. Current data highlight a potential strategy for improving treatment efficacy and overcoming resistance. Further clinical investigations are warranted to validate this combination strategy. Citation Format: Zhentian Li, Qipeng Fan, Mingming Gao, Jun Pan, Liangxing Wu, Wenqing Yao, Phillip C. Liu. Activity of PARP1-selective inhibitor SNV-001 in models of HRD cancers as monotherapy and in combination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4562.

Wnt/β-Catenin-Signaling Modulates Megakaryopoiesis at the Megakaryocyte-Erythrocyte Progenitor Stage in the Hematopoietic System.

The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Many steps of the cell-fate decision remain to be elucidated, being important for cancer treatment. To explore the role of Wnt/β-catenin for MK and RBC differentiation, we activated β-catenin signaling in platelet-derived growth factor b (Pdgfb)-expressing cells of the HS using a Cre-lox approach (Ctnnb1BM-GOF). FACS analysis revealed that Pdgfb is mainly expressed by megakaryocytic progenitors (MKPs), MKs and platelets. Recombination resulted in a lethal phenotype in mutants (Ctnnb1BM-GOFwt/fl, Ctnnb1BM-GOFfl/fl) 3 weeks after tamoxifen injection, showing an increase in MKs in the BM and spleen, but no pronounced anemia despite reduced erythrocyte counts. BM transplantation (BMT) of Ctnnb1BM-GOF BM into lethally irradiated wildtype recipients (BMT-Ctnnb1BM-GOF) confirmed the megakaryocytic, but not the lethal phenotype. CFU-MK assays in vitro with BM cells of Ctnnb1BM-GOF mice supported MK skewing at the expense of erythroid colonies. Molecularly, the runt-related transcription factor 1 (RUNX1) mRNA, known to suppress erythropoiesis, was upregulated in Ctnnb1BM-GOF BM cells. In conclusion, β-catenin activation plays a key role in cell-fate decision favoring MK development at the expense of erythroid production.

Roles of Ribosomal S6 Kinases in Acute Leukemia and Normal Hematopoiesis

Ribosomal S6 kinases (RSKs) are serine-threonine kinases that are activated by the Ras/mitogen activated protein kinase signaling pathways and regulate cell proliferation and survival. There are 4 isoforms of RSK (RSK1, 2, 3, 4), and there is evidence that the isoforms mediate tissue-specific cellular functions. We hypothesized that RSK isoforms have distinct roles in hematopoiesisand evaluated RSK as a target for the treatment of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). To investigate isoform expression in hematopoiesis, we sorted cord blood mononuclear cells into hematopoietic progenitor stages and quantified RSK isoform mRNA by RT-qPCR. RSK1 expression remained consistent throughout hematopoiesis, whereas RSK2 expression decreased in the more differentiated populations. RSK3 was elevated in the lymphoid-primed multipotential progenitor and common myeloid progenitor stages and was greatly reduced in the more mature megakaryocyte-erythroid progenitor population. These results suggest that RSK proteins have isoform-specific roles in hematopoiesis. To analyze the importance of each isoform on hematopoiesis, we transduced cord blood CD34+ cells with lentiviral constructs expressing isoform-specific or control shRNAs. After liquid culture for 5 days, the cells were plated in methylcellulose media containing SCF, IL-3, GM-CSF, and EPO to determine the colony-forming potential. RSK1 and RSK2 knockdown decreased the colony-forming unit - erythroid colonies and increased the granulocyte/macrophage colony numbers. RSK3 knockdown increased the burst-forming unit - erythroid colonies and total colony numbers. After liquid culture days 8, 11, and 14, the erythroid and myeloid populations were analyzed by flow cytometry. RSK1 knockdown increased the CD11b+ and CD14+ myeloid populations and decreased the CD71+ erythroid population. RSK3 knockdown increased the erythroid population and decreased the myeloid populations. These data demonstrate that RSK1 and RSK3 have opposing effects on hematopoietic lineage determination. Overexpression of RSK proteins leads to aberrant cell survival and proliferation in many cancers including leukemia. We have previously reported that RSK and phosphorylated RSK are elevated in pediatric AML and are associated with a worse prognosis. There are only two orally bioavailable RSK inhibitors that have moved to phase I clinical trials: TAS0612 and PMD-026. TAS0612 is a multi-kinase inhibitor of RSK, AKT, and S6K. PMD-026 has high specificity to all four RSK isoforms. We evaluated the effects of both compounds in AML and ALL. To determine the effects of the RSK inhibitors on AML, we treated AML cell lines and patient samples with a range of doses of PMD-026, TAS0612, or 0.1% DMSO. After 3 days, we performed CellTiter Glo assays to determine the relative viable cell number and calculate the concentration of drug that inhibits cell growth by 50% (IC50). TAS0612 had an IC50 of 0.096-0.64 µM in AML cell lines Molm13, U937, AML193, and MV4;11. To test the cytotoxicity of TAS0612 on normal hematopoiesis, we plated healthy bone marrow mononuclear cells with TAS0612 in methylcellulose media. Colony formation was not adversely affected by TAS0612 up to 1 µM. PMD-026 was effective in Molm13, Kasumi-1, and MV4;11 with an IC50 of 0.34-0.55 µM and in AML patient samples 3123 and 4192 with an IC50 of 1.5-2.3 µM. PMD-026 did not affect healthy bone marrow colony formation up to 5 µM for a 2.5- to 10-fold therapeutic window. Furthermore, we tested the effects of RSK inhibitors in ALL. While PMD-026 was not effective, we found that B-ALL cell lines (REH, Nalm-6) and T-ALL cell lines (Loucy, Jurkat) were sensitive to RSK inhibition by TAS0612 with an IC50 of 0.056-0.19 µM. Western blot analysis of RSK protein revealed that RSK1, RSK2, and phospho-RSK proteins are elevated in ALL cell lines compared to healthy peripheral blood mononuclear cells. We are investigating the role of RSK and RSK isoforms in ALL. We found that RSK isoform expression is modified during hematopoiesis. Isoform expression influences early-fate decisions to differentiate into erythroid and myeloid lineages. RSK inhibitors PMD-026 and TAS0612 are effective at targeting AML and ALL cells with limited cytotoxicity to healthy cells, making them promising candidates for translation into clinical trials for AML and ALL.

Base Edited HSPCs Are Shielded from Targeted CD33 Therapy but Preserve CD33 Expression

Targeted therapy for AML remains challenging as there is broad overlap between targetable surface proteins on transformed cells and healthy hematopoietic stem and progenitor cells (HSPC). CD33 is expressed in about 90% of patients with AML but also on healthy myeloid cells with strong expression on progenitor cells and only weak expression on mature neutrophils. Given the higher expression on leukemic blasts compared to their healthy counterparts, CD33 is an attractive target in AML. However, targeting CD33 e.g. with the antibody-drug conjugate (ADC) gemtuzumab ozogamicin often leads to prolonged cytopenia due to suppression of normal myelopoiesis. Transplantation of genetically engineered CD33 knock-out allogenic HSPCs protects reconstituting hematopoietic cells from targeted immunotherapy administered for disease control after transplantation. Early clinical trials explore this option but the long-term effect of loss-of-function of CD33 remains unknown. We recently demonstrated that single amino acid substitutions engineered into CD45, CD117 or CD123 protect HSPCs from targeted therapies against these antigens while preserving their function. Here, we describe the identification of CD33 shielding amino acid substitutions that can be engineered by base editing and preserve CD33 expression on HSPCs. We identified binding sites of two CD33 antibodies by alanine scanning of the entire extracellular domain. The most favorable amino acid substitutions at key residues were analyzed computationally and binding reduction was subsequently validated by flow cytometry. 23 individual amino acid substitutions at 3 positions were tested. Eleven retained similar expression, purity and biophysical properties as wildtype CD33. Those variants are shielded from antibody 1 even at high concentrations when assessed by biolayer interferometry. In addition, three variants are shielded from antibody 2 binding. We used the ABE8e_SpRY base editor to engineer HSPCs and tiled 21 sgRNAs centered around the intended edit. Five days after electroporation, HSPCs were evaluated for editing efficiency (Sanger sequencing) and binding to antibody 1 (flow cytometry). sgRNA4 showed high editing efficiency of about 80% as well as loss of binding to antibody 1 (Figure A) while binding to the control antibody that binds a different epitope was maintained. Importantly, Sanger sequencing did not show off-target editing of the closely related SIGLEC22P pseudogene. Edited (sgRNA4) and non-edited control (NTC) HSPCs formed comparable numbers of myeloid and erythroid colonies (Figure B). Furthermore, in vitro differentiation showed no difference in frequency of CD14, CD15, CD19, CD33, CD41 and GlyA positive cells indicating that the edited HSPCs did not display major functional alterations. In summary, CD33 variants that can be engineered into HSPCs with high editing efficiency by base editing protected from binding to antibody 1 but preserved CD33 expression. These edited HSPCs could be therapeutically useful, in particular for continued immunotherapy after allogenic HSC transplantation. Similar to HSPCs devoid of CD33 these engineered cells could enable tumor-selective immunotherapy using ADCs or CAR T cells but with preserved CD33 expression and function. Furthermore, since base editing is suitable for multiplexing, they may in the future be multiplexed to protect CD33 and other targets for combination immunotherapy.

BCOR Loss Confers Increased Stemness and Partially Rescues RUNX1-Deficient Phenotypes in Human Hematopoietic Stem and Progenitor Cells

Introduction: Loss of function mutations in BCL-6 corepressor (BCOR) commonly occur in de novo acute myeloid leukemia (AML) and germline hematopoietic malignancies, most notably in those arising from RUNX1 familial platelet disorder (RUNX1-FPD). Second only to loss of heterozygosity of RUNX1, mutations in BCOR are the most commonly acquired mutations upon progression to myeloid malignancy in RUNX1-FPD. Additionally, RUNX1 and BCOR mutations commonly co-occur in de novo AML, and both are classified as “adverse risk” by the European LeukemiaNet if found at AML diagnosis. However, the role of BCOR in leukemogenesis is poorly understood, and there are limited studies investigating co-loss of BCOR and RUNX1 in hematopoietic stem and progenitor cells (HSPCs). Methods: We developed a model of BCOR loss in primary human CD34+ HSPCs using CRISPR/Cas9 and AAV6-mediated homology directed repair to knock-in a fluorescent reporter expression cassette into the BCOR locus. We used this system to investigate how BCOR knockout (KO) perturbs normal HSPC function. We combined this approach with a previously developed analogous system to disrupt the RUNX1 locus to investigatehow BCOR loss in the setting of RUNX1 KO modulates the effects of RUNX1 loss on HSPC function. Results: Here, we found that BCOR loss increased capacity for serial replating and conferred a competitive advantage over control HSPCs. In serial replating assays, BCOR-deficient HSPCs had a 5-fold increase in colony formation after one passage with a striking majority of colonies being erythroid. In liquid differentiation assays, BCOR-deficient HSPCs consistently had a 2-fold increase in CD34+ cells over a 21 day time course. When co-cultured with control HSPCs in a 1:1 ratio in stem expansion media (base media + SCF, FLT3-L, TPO, IL-6, UM-171), BCOR-deficient HSPCs expanded to comprise 70% of the culture at 14 days. This competitive advantage was recapitulated in vivo. At the 12-week timepoint of a competitive transplantation assay in NSG mice using HSPCs from 3 different donors, BCOR KO cells (marked by BFP) on average made up 75% of the engrafted human CD45+ population, while control cells (expressing GFP from the AAVS1 safe harbor locus) made up only 21%. Additionally, BCOR loss partially rescued RUNX1-induced stem cell dysfunction in vitro. Compared to RUNX1 deficiency alone, HSPCs deficient in both RUNX1 and BCOR had a 4-fold increase in colony formation after two passages in serial replating assays. Our lab has previously shown that RUNX1 loss ablates erythroid colony formation and skews towards monocytic colonies. Despite BCOR loss alone having a strong skew towards erythroid colonies, BCOR loss in RUNX1-deficient HSPCs did not induce erythroid colony formation. This suggests that in double mutant HSPCs, RUNX1 loss drives lineage decisions while BCOR loss drives the increase in serial replating potential. Ongoing efforts are aimed at characterizing combinatorial effects of BCOR and RUNX1 loss on hematopoiesis and stem cell function through in vitro liquid differentiation assays and in vivo transplantations, as well as investigating the molecular underpinnings of these phenotypes through RNA-seq. Conclusion: In summary, we have established a model of BCOR loss in primary human HSPCs to evaluate not only the role of BCOR in hematopoiesis, but also to characterize how BCOR is involved in leukemic transformation when combined with RUNX1. We show that BCOR deficiency enhances several attributes associated with stem cell function, including increased potential for serial replating, increased CD34 positive population, and increased engraftment compared to control HSPCs. When combined with RUNX1 loss, BCOR loss increases the serial replating potential of RUNX1-deficient cells, suggesting some rescue of RUNX1 KO-induced stem cell dysfunction. Ultimately, this model will enable us to elucidate the molecular mechanisms underlying increased stemness and competitive advantage in BCOR-deficient HSPCs, interrogate RUNX1-FPD disease progression associated with BCOR mutation, and validate potential therapeutics.

MED16 Negatively Regulates Erythropoiesis and Myelopoiesis through Modulation of Chromatin Accessibility

Tissue-specific transcription factors work in conjunction with numerous coregulators to modulate transcription networks during hematopoiesis; disruptions in this process may lead to hematopoietic disorders, including myelodysplastic syndromes (MDS). While the Mediator complex is known to regulate various aspects of hematopoiesis, the mechanisms underlying subunit-specific functions have not been fully understood. Here, we uncovered that MED16, a core component of Mediator tail module, serves as a crucial negative regulator of erythropoiesis and myelopoiesis by controlling chromatin accessibility at specific target genes. To investigate the function of MED16 in vivo, we transplanted control (shSCR) or MED16-deficient (shMED16) human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) into immunodeficient mice. Mice receiving shMED16 HSPCs showed a higher proportion of human erythroid and myeloid cells in the bone marrow (BM), while B cell and T cell reconstitution remained unaffected. MED16 deficiency led to an increased number of myeloid colonies and a more pronounced increase in erythroid colonies in the colony-forming assay. During erythroid differentiation, MED16 deficiency resulted in an elevated ratio of CD235a+ cells and increased enucleation. Furthermore, hematopoietic-specific Med16 knockout ( Mx1-Cre × Med16 -/- Med16 KO)mice showed normal BM and spleen cellularity, with an increased frequency of common myeloid progenitors (CMPs). Med16 KO mice exhibited a higher frequency of CD11b+ myeloid cells and Ter119+ erythroid cells in the BM and spleen. A higher percentage of enucleated erythrocytes were observed in the BM of Med16 KO mice. Additionally, in the model of phenylhydrazine-induced hemolytic anemia, Med16 KO mice showed accelerated red blood cell recovery and pronounced splenomegaly, highlighting the crucial role of Med16 in stress erythropoiesis. Our findings demonstrate that MED16 plays a repressive role in hematopoietic development. To elucidate how MED16 regulates gene expression, we conducted single-cell RNA-seq and ATAC-seq of cultured CD34+ cells after MED16 knockdown in primary human CD34+ HSPCs. MED16 deficiency resulted in upregulation of erythroid and innate immunity genes in erythroid and myeloid cells, respectively. Interestingly, MED16 deficiency led to significantly increased chromatin accessibility around the transcription start sites of upregulated genes. Considering the Mediator complex often interacts with transcription factors to convey information, we conducted MED16 chromatin IP sequencing (ChIP-seq). The majority of MED16 occupancy peaks were located at promoters (59.68%). Colocalization analysis of MED16 with chromatin occupancy of hematopoietic-related transcription factors revealed significant overlap with RUNX1. To unveil the mechanism of MED16-mediated transcriptional repression, we conducted immunoprecipitation coupled with mass spectrometry on cultured CD34+ cells to identify MED16-interacting nuclear proteins. We identified the FAcilitates Chromatin Transcription (FACT) complex as a potential candidate. ChIP-seq of the FACT complex subunit SPT16 showed weakened chromatin occupancy signals upon MED16 deficiency. Similar results were observed in mouse BM Ter119+ erythroblasts. These findings indicate that MED16 plays a crucial role in modulating chromatin accessibility and transcriptional repression, potentially through cooperation with RUNX1 and the FACT complex. Furthermore, we found that MED16 overexpression impeded erythroid cell enucleation, similar to MDS erythroid dysplasia. We observed higher MED16 expression in BM-derived CD34+ cells from 183 MDS patients across different subtypes compared to healthy individuals. Interestingly, knocking down MED16 in bone marrow mononuclear cells from MDS patients restored normal erythropoiesis. Transcriptome analysis revealed that MED16 deficiency upregulated innate immunity and erythroid genes. Together, these findings shed light on the previously unknown role of MED16 in hematopoiesis and its association with MDS. Our study provides mechanistic insights into the lineage commitment and developmental progression of hematopoiesis governed by Mediator and may have implications for the treatment of hematological disorders.

Septin11 and Septin 8 Play Critical and Overlapping Roles Murine Stem Cell Function

Septins are highly conserved GTP binding proteins which regulate a variety of cellular functions including cytokinesis, polarity, cell cycle, vesicle trafficking, and exocytosis and are now considered the fourth component of the cytoskeleton. Septins are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previous data in our laboratory led to the discovery of a small molecular inhibitor, DW0254, which inhibits RAS function and its downstream target RhoGTPase, Rac, including the hematopoietic-specific Rac2 GTPase, also implicated in MLL leukemias. The inhibitor leads to apoptosis of leukemia cell lines. Using biophysical methods, we discovered that DW0254 binds both the RAS chaperone, PDE6D, and Septin11, which is a member of the Septin6 family (Sara CN, et al., Blood Cancer 2022). Although Septins have been reported to be involved in function of the RhoGTPase Cdc42 and hematopoietic stem progenitor cell (HSPC) polarity, the role of Septin11 in hematopoiesis has not been reported. Septin11 is a member of the Septin6 family of proteins which consists of Septin6, 8, 10, 11 and 14 with significant homology between family members. Previous data in yeast has hypothesized functional overlap within each family of Septin proteins. To better understand the functional role of Septin11, we created lentivirus vectors tagged with a GFP reporter that contained shmiRs against Septin11. Culture of Septin11 knockdown (Septin11 KD) murine HSPC in IMDM (10ng/ml IL-3, 25ng/ml of SCF, TPO, Flt-3 and IL-6) resulted in an average of 13-fold decrease in cell counts compared to controls (p=0.02) and a decrease in total clonogenic activity (Figure 1) with defects seen in both myeloid (GM) and erythroid (BFU-E) colonies. Reduced clonogenic numbers were accompanied by a significant increase in early apoptotic cells suggesting that Septin11 is important in regulating apoptosis in HSPC. To determine the potential role of Septin11 in more primitive hematopoietic cells, we utilized competitive repopulation assays. Transduced BoyJ HSPC (GFP+ Septin11 KD and BFP+ control, mixed 1:1) were injected into lethally irradiated C57Bl/6 mice. We observed a reduction in multilineage chimerism in the peripheral blood, bone marrow, spleen and thymus in primary transplant recipients after knockdown of Septin11 (Table 1). To better define function in specific hematopoietic populations of HSPC, we performed knockdown in FACS-sorted purified cells. Engraftment studies demonstrated a significant reduction in bone marrow chimerism in primary transplant recipients after knockdown of Septin11 most clearly in LT-HSC and ST-HSC (Table 1) with no statistically significant change in MPP. Therefore, to better study the hematopoietic stem cell compartment, we performed secondary transplants utilizing the cells obtained from primary competitive repopulation assays. We observed an unexpected recovery in engraftment in secondary transplant recipients that was accompanied by an increase in the mRNA expression of Septin8. Since Septin8 and Septin11 are closely related we hypothesized that induction of Septin8 was subserving key functions of Septin11. To test this hypothesis, we created a lentivirus vector that expressed both a shmiR against Septin11 and the Septin8 cDNA. Concurrent overexpression of Septin8 with Septin11 knockdown HSPC caused a significant increase in the total number of colonies compared to controls (Figure 1). These data indicate an important role of Septin11 in murine hematopoiesis and a potential redundant role for Septin8.

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&amp;lt;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.

Oxidative Stress Damage of Iron Overload on Bone Marrow Erythropoiesis, Heart and Liver in Non-Transfusion Dependent Thalassemia

Background: Non-transfusion-dependent thalassemia (NTDT) characterized by ineffective hematopoiesis and increased intestinal iron absorption, and excessive iron deposits in the bone marrow, heart, and liver, causing progressive organ damage. It has been reported that oxidative stress damage caused by iron overload could induce apoptosis or ferroptosis. However, the related studies and mechanisms are not well unknown in NTDT disorders. Aims: To investigate the effects of iron overload on bone marrow erythropoiesis, heart and liver in NTDT patients and Hbb th3(th3/+) mice. Methods: Patients (19-47 y of age) and 12-month-old Hbb th3(th3/+) mice with NTDT were selected as thalassemia group, healthy human and normal mice were selected as control group. Iron overload related indexes, bone marrow erythropoiesis, oxidative stress, apoptosis and ferroptosis of each sample were detected by Elisa, flow cytometry, RT-PCR and Weston blot methods, respectively. Results: Compared with the control group, Serum ferritin is significantly increased in NTDT group, and there is imbalance of iron metabolism. And in NTDT patients, liver and myocardial iron deposition was assessed by MRI analysis, resonance results suggest that the degree of iron overload in liver is more significant than that in heart. The damage of erythropoiesis in NTDT group was more severe than that in control group, which showed active erythropoiesis, and the differentiation of erythroid precursor cells stagnated in early stage of erythroid precursor cells, and there were maturation disorders. The oxidative stress-related indexes reactive oxygen species (ROS), lipid peroxidation and malondialdehyde (MDA) induced by iron overload were also significantly increased in the NTDT group. However, significant signs of apoptosis were observed in the bone marrow of the NTDT group, including increased proportion of apoptotic cells, signs of apoptosis were mainly observed in the mitochondria of erythroid colony electron microscopy, and the level of GPX4 that is a key indicator of ferroptosis, was increased. But the expression of GPX4, VDAC2 and SLC7A11 of ferroptosis was decreased in the heart and liver with heavy iron deposition, and the signs of ferroptosis were also observed under electron microscopy. Summary/Conclusion: Our study found that there are differences in iron overload and injury performance among different organs in NTDT. Oxidative stress caused by iron overload in the bone marrow impairs erythropoiesis mainly through apoptosis, whereas signs of ferroptosis were observed in the liver and heart of Hbb th3(th3/+) mice.These results suggest that with the gradual accumulation of iron circulation, the damage is a dynamic process, and different organs show different signs of damage, which may be related to the different mechanisms of damage, tolerance and compensation of organs, but further studies are needed to confirm.

A STAT3 Degrader Demonstrates Pre-Clinical Efficacy in Venetoclax Resistant Acute Myeloid Leukemia

Introduction: Acute Myeloid leukemia (AML) is an aggressive hematological malignancy resulting from the transformation of immature myeloid progenitor cells followed by an uncontrolled clonal proliferation and accumulation of the transformed cells. AML is the most common myeloid malignancy in the elderly [1]. Venetoclax (Ven) is a selective inhibitor of the anti-apoptotic BCL2 protein. Ven is FDA approved in combination with hypomethylating agents (HMA's) or low dose cytarabine for the treatment of de-novo AML in patients &amp;gt; 75 years or those ineligible for standard induction therapies. Despite the recent FDA approval of HMA/Ven and other novel therapies, 5-year survival in newly diagnosed AML remain less than 30% and disease relapse is inevitable without a bridge to allogeneic stem cell transplantation [2]. Signal transducer and activator of transcription 3 (STAT3) belonging to the STAT family of transcription factors is well known to be inappropriately activated in several malignancies [3]. On stimulation by cytokines such as IL-6, STAT3 undergoes dimerization and phosphorylation. Phosphorylation at Tyr 705 leads to its translocation to the nucleus whereas phosphorylation at Ser 727 translocates it to the mitochondria [4]. Previous data from our lab demonstrated de-methylation and overexpression of STAT3 in MDS &amp; AML stem cells, that is associated with an adverse prognosis [5]. We have also reported that STAT3 controls several important leukemic drivers such as the anti-apoptotic protein myeloid cell leukemia-1 (MCL1). MCL1 overexpression is the central mechanism of resistance to BCL2 inhibition (Ven) in AML [5]. While MCL1 is a well-known direct transcriptional target of STAT3, the role of STAT3 in venetoclax resistance (Ven-res) is unknown. Methods &amp; Results: To understand the role of STAT3 in Venetoclax resistance (Ven-res), Ven-res AML cell lines (MOLM-13, MV-4-11) as well as a Ven-res large cell lymphoma cell line (SU-DHL-1) were generated, which exhibited increased levels of both total STAT3, phospho-STAT3 and its downstream effector, MCL1, when compared to their parental cell lines (Fig. 1A). Data from &amp;gt; 90 AML patients treated with prior Ven also showed high expression of total STAT3 along with enhanced phosphorylation for p Ser727 and p Tyr-705 STAT3, that strongly correlated with worse overall survival (OS) and reduced remission duration (RemDur) (p&amp;lt;0.05). A highly specific potent heterobifunctional degrader of STAT3 (degrader) resulted in a dose dependent and selective degradation of STAT3 in both parental and Ven-res hematological malignancy cell lines. Treatment with degrader showed a significant (&amp;gt;60%) decrease in p Tyr-705 as wellas p Ser-727 STAT3 levels in MOLM-13 parental as well as MOLM-13 Ven-res cells. STAT3 degradation also led to induction of apoptosis in both parental and Ven-res AML cell lines (p &amp;lt; 0.001). Importantly, colony assay of Ven-res AML patient samples showed effective degradation of STAT3 (&amp;gt;90%) together with the increased erythroid and myeloid differentiation (~ 2 fold) on treatment with degrader. Interestingly, no differentiation was observed in healthy samples, suggesting the specificity of the degrader to AML stem and progenitor cells. In patients with Ven-res AML, erythroid colony counts were seen to increase (~1.5 fold) with a concomitant decrease in myeloid colony counts (&amp;gt;2.5 fold), on treatment with degrader. This observation supports the clinical significance of our studies in AML patients with anemia. Although BH3 profiling of Ven-res MOLM-13 supports an increased dependency on MCL1, we have observed that treatment with degrader led to ~20% reduction in the MCL1 dependent mitochondrial depolarization. Additionally, cell derived xenograft (CDX) models of Ven-res showed significant reduction of p Tyr-705 STAT3(~60%), total STAT3 (&amp;gt;90%) and MCL1 (~70%), on treatment with STAT3 degrader - KT-333 (currently in an early phase clinical trial:NCT05225584), as early as week 2 (Fig 1B). Significant decrease in intracellular STAT3 in the spleen cells was also observed (p &amp;lt; 0.05), with ongoing survival analysis. Conclusion: Our study suggests that targeting STAT3 and the downstream MCL1 represents a novel and effective strategy for Ven-resistant AML patients in clinic, with strong mechanistic rationales that can spur further clinical development of STAT3 degraders especially given the significant side effects of direct MCL1 inhibitors.

Distinctive Roles of Nitric Oxide Synthase Isoforms in Hydroxyurea-Mediated Cytostasis of Erythroid Progenitors

Chemotherapeutic hydroxyurea (HU) is used in the therapy of sickle cell anemia, thalassemia and myeloproliferative neoplasms according to its stimulation of fetal hemoglobin and myelosuppression. We reported that most nitric oxide (NO) in mammals is produced by the reduction of nitrate and nitrite. The NO properties of HU have been widely explored, while its enzymatic NO synthase (NOS) dependence is not completely defined. We have already demonstrated concomitant inhibition of erythroid cell proliferation by HU and NO donors. In this study, we investigated the NOS-mediation in HU regulation of proliferation, differentiation and cell cycle in HEL92.1.7 human erythroleukemic cell line and / or erythroid progenitors of Nos2- or Nos3-null mice. HU induced a dose-dependent increase in NOS2 and NOS3 protein levels and their enzymatic activity by increasing production of NO and citrulline in erythroleukemic cells. Inhibition of the NOS enzymes by pan-specific L-NAME, NOS2 inhibitor 1400W, or NOS2/NOS3 inhibitor DPI was sufficient to prevent the HU-mediated decrease in HEL92.1.7 proliferation assessed by Ki67 immunofluorescence. HU increased the frequency of cells in S-phase (66.58±2.44 vs. 55.18±1,06%) of the cycle at the expense of G0/G1 (17.07±1.67 vs. 28.55±1.01%) due to blocked DNA synthesis. The combined treatment of HU with 10μM DPI decreased the number of cells in G0/G1 (3.34±0.98 vs. 17.07±1.67%) and increased in S (72.3±0.96 vs. 66.58±%) and G2/M (24.36±0.19 vs. 16.35±2.28%) compared with HU alone. Moreover, NOS3 knock-down by shRNA decreased the percentage of cells in S phase from 69,19±2.68 to 34.23±1.07% (p&amp;lt;0.001) and increased in G2/M from 15.62±2.17 to 34.71±3.76 (p&amp;lt;0.001). HU-induced apoptosis was prevented by NOS2 (1400W) inhibition, determined by annexin V, decreasing the number of cells in early and late apoptosis in erythroleukemic cells. In accordance, NOS3 knock-down was not able to rescue HU-induced apoptosis. Furthermore, by performing in vitro enzymatic assay we showed that regulation of NOS2 activity seems to be achieved by direct interaction with HU leading to a dose- and time-dependent increase in NO and citrulline products. HU increased the expression of Nos1/2 isoforms by 20%, and Nos3 by 10% in mouse erythroid progenitors (mERP) of wild-type mice. Bone marrow cells isolated from Nos2- or Nos3-null mice treated orally with HU showed decreased protein nitrosylation in bone marrow compared to HU-treated wild-type mice. In ex vivo colony formation assay, both Nos2-/- and Nos3-/- bone marrow cells formed significantly more late erythroid colonies (CFU-E, 34- and 24-fold, respectively, p&amp;lt;0.001), and Nos2-/- also yielded more of the early erythroid (BFU-E, 9- and 7-fold, p&amp;lt;0.01) and granulocyte/macrophage (CFU-GM, 14- and 15-fold, p&amp;lt;0.01) colonies compared to wild-type mice untreated or HU-treated. HU treatment of mERP, of Nos2-/- and Nos3-/- origin, demonstrated decreased frequency of S-phase cells to levels comparable to untreated wild-type mice. Nos2 and Nos3 deficiency was able to rescue Caspase-3 expression in the presence of HU in mERP. Nevertheless, HU increased the quantity of late apoptotic cells in both Nos2 and Nos3 deficient mice. Our results demonstrated that HU induces the enzymatic activity of NOS proteins that mediate the cytostatic effect. NOS2/3 enzymes are involved in HU-inhibition of proliferation of human erythroleukemic cells and ex vivo differentiation of mERP. The observed NOS enzymes were fully and partially involved in HU-induced apoptosis of HEL92.1.7 cells and mERP, respectively. Uncovering all aspects of HU mechanism of action may help to increase the responsiveness or reduce the negative effects of cytotoxic chemotherapy.

Polycythemia Vera with Elevated Erythropoietin Level: A Review

According to the 2016 World Health Organization (WHO) recommendations, polycythemia vera (PV) should be suspected at hemoglobin levels of 16.5 gm/dl in males and 16 gm/dl in females. Depending on the underlying driving factor, polycythemia is classified to primary and secondary. Secondary polycythemia is driven by erythropoietin (EPO), which can be released due to hypoxemia, as seen in people living at high altitudes, those with chronic lung diseases, obstructive sleep apnea (OSA) and smoking. It can also be directly released by an underlying EPO secreting tumor, such as renal cell carcinoma. On the other hand, primary polycythemia or polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) in which erythrocytosis is driven by an acquired activating mutation that increases sensitivity to EPO or causes EPO independent erythroid colony formation. Manifestations of PV include fatigue, excessive itching, abdominal pain, weight loss, and fever. However, the most critical complications of PV, which impact survival, are thrombotic complications such as stroke and venous thromboembolism and leukemic transformation. The risk for leukemic transformation in patients with PV is about 2-5% over 15 years and is more common in individuals older than 60. History and physical examination should focus on smoking history and signs and symptoms of cardiopulmonary diseases causing chronic or intermittent hypoxemia, as seen in OSA. On the other hand, findings of abdominal pain, early satiety, weight loss, erythromelalgia, and hepatosplenomegaly would be suggestive of PV. Laboratory workup classically starts with measuring EPO level which further directs workup toward PV or secondary polycythemia. If workup is suggestive of PV, the following steps would be testing for activating Janus Kinase 2 (JAK2) mutation (V617F), which has a sensitivity of 97% for PV, and bone marrow biopsy which shows hypercellularity with trilineage growth (panmyelosis) in cases of PV. EPO measurement constitutes an essential step in polycythemia workup. It is widely accepted that elevated EPO levels are associated with secondary polycythemia; and suppressed EPO level was considered a criterion to diagnose PV, according to the WHO 2016 diagnostic criteria for PV. We conducted a systematic review searching the entity of PV with elevated EPO to study the clinical significance and outcomes. Our search yielded 4 cases summarized in Table 1. The cases were of three males and one female, with a mean age of 50. Two of them were healthy, with two with comorbid diseases, and as seen in the table; only one was a smoker. The most common symptom was headache, followed by erythromelalgia. Thrombotic phenomena happened in a single case in the form of Budd Chiari syndrome. Hepatosplenomegaly was found in a single case, and splenomegaly was found in two cases. The mean Hb level was 20.2 gm/dl, and the mean EPO level was 213. EPO is a glycoprotein hormone that is mainly produced by the kidneys. However, it is also produced in small amounts in the liver, spleen, bone marrow, lungs, and brain. The primary regulator of EPO production is the partial pressure of oxygen (PaO2). Multiple case reports and case series suggested an association between PV with elevated EPO and Budd Chiari Syndrome. Thumures et al. described four patients that were diagnosed with Budd Chiari Syndrome with elevated red cell mass and were found to have elevated serum erythropoietin levels. PV was confirmed with JAK2 mutation assay and bone marrow biopsy examination. It is speculated that liver hypoxia with subsequent hepatocyte injury and necrosis caused by Budd Chiari Syndrome leads to EPO level elevation. Accordingly, we assume that patients with PV and elevated EPO have some degree of organ injury that results in the release of EPO into circulation. This highlights the importance of a systematic approach in medicine, where signs and symptoms remain the cornerstone in diagnosis and where lab tests should be taken in their appropriate clinical context. Thus, we recommend testing for JAK2 mutation in the proper clinical scenario regardless of the EPO levels. It still remains to be clarified whether there are any prognostic importance of the elevated EPO level in these patients and whether it would alter the management.

Hematopoietic Stem Cells Expressing Engineered CD45 Enable a Near Universal Targeted Therapy for Hematologic Diseases

Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment for a broad range of hematological malignancies. Unfortunately, the current standard of care relies on untargeted toxic conditioning regimens associated with severe morbidity and mortality curtailing HCT's wider use. In the last decade, antigen-specific cell depleting therapies have revolutionized clinical practice in hematology. Although target selection remains complex, the pan-hematopoietic marker CD45, a protein tyrosine phosphatase which is exclusively expressed on all nucleated hematopoietic cells, could enable targeted depletion of the entire hematopoietic system including HSCs. Combined with HSCs engineered to be shielded from a CD45-targeting antibody-drug conjugate (ADC), this approach could create a near-universal strategy to replace a diseased hematopoietic system, irrespective of disease etiology or cell type. We performed alanine (Ala) scanning of the entire extracellular domain of CD45 to identify specific residues forming the epitopes for three different monoclonal antibodies (mAbs). Next, we screened base editors (BE) to generate amino acid substitutions that reduced or abolished the binding of specific mAbs, purified the first two extracellular regions of the most promising protein variants (CD45 D1-D2) and measured their binding to the mAbs by biolayer interferometry (BLI). Two antibodies showed drastically reduced or no binding to some of the CD45 variants. Some variants only reduced the binding of the mAb of the corresponding epitope while others affected the binding of mAbs from multiple epitopes located in more distant regions. These results suggested that several CD45 amino acid exchanges altered the receptor structure in a way that spanned at least two structural domains. This interpretation is supported by a substantial decrease in protein thermal stability, calling for caution when selecting suitable variants. Nevertheless, through a series of experiments and optimizations of the sgRNAs we identified a BE (ABE8e-SpRY + sgRNA49.3) that resulted in biophysically stable protein variants with editing of about 50% of hematopoietic stem and progenitor cells (HSPCs). These base edited HSPCs formed equal numbers of colonies as well as relative numbers of myeloid and erythroid colonies comparable to control HSPCs. Furthermore, they engrafted and supported multi-lineage cell differentiation in NBSGW host mice comparable to non-edited control HSPCs. Secondary transplantation experiments confirmed successful editing of long-term reconstituting HSCs (LT-HSCs). Collectively, these results indicate that the edited HSPCs were functionally intact. We further developed a humanized, Fc silent CD45-ADC designated CIM053-ADC. A single dose of 0.5mg/kg was very well tolerated by NBSGW mice engrafted with hCD34 + HSPCs and effectively depleted human hematopoietic cells including LT-HSCs. Next, we injected luciferase marked MOLM-14 cells, an aggressive myeloid tumor cell line, in mice priorly xenografted with edited or control human HSPCs. CIM053-ADC treatment was initiated 12 days after tumor injection when mice demonstrated clearly detectable, extensive and multilocular disease ( Fig. A). We deliberately assigned the mice with the most advanced disease to the ADC group. Mice then obtained saline or two doses of CIM053-ADC i.v.. Tumors were cleared in all CIM053-ADC treated mice whereas in all the mice that obtained saline the MOLM-14 cells rapidly grew and mice had to be euthanized 21-23 days post tumor injection ( Fig. B). Importantly, CIM053-ADC eliminated unedited hCD45 + non-tumor cells but base edited cells persisted in all tested organs. In summary, we developed a novel, highly potent tumor-selective therapy that spares a shielded hematopoietic system. Since CIM053-ADC targets the pan-hematopoietic marker CD45 this approach may not only be applicable to treat AML but most hematologic malignancies and possibly other diseases requiring HCT.

The Integrator Complex Is Essential for Erythropoiesis

Erythropoiesis is a finely orchestrated process that involves the generation of a ~2.5 million red blood cells per second to maintain homeostasis and prevent anemia. During terminal maturation, erythroid precursors upregulate erythroid-specific genes while silencing non-erythroid genes in the setting of a cell that is rapidly dividing, and nucleus that is condensing in preparation for enucleation. Our group has shown that regulation of RNAPII activity is an essential determinant of erythroid cell function. During terminal erythroid maturation, RNA polymerase II (RNAPII) levels decline dramatically, becoming a scarce resource allocated to erythroid genes, such as alpha and beta globin (Murphy Blood 2021). The mechanism(s) by which RNAPII is removed from genes unnecessary to erythroid differentiation and shunted to produce mRNA essential for red blood cell function are unknown. The Integrator Complex (INT) is a modular and multi-subunit machinery that binds to paused RNAPII and promotes promoter-proximal termination. INT is highly expressed in maturing erythroid cells, although little is known about the function of INT during erythropoiesis. We hypothesize that INT facilitates promoter-proximal termination at non-erythroid genes to maintain RNAPII availability during terminal erythroid maturation. The balance between kinase and phosphatase activity is a key regulator of RNAPII activity. Phosphorylation of the c-terminal domain of RNAPII by pTEFb facilitates RNAPII pause release, and promotes active transcription. Alternatively, subunit 8 of INT (INTS8) can dephosphorylate the c-terminal domain of RNAPII through association with protein phosphatase 2a (PP2A) (Huang Mol Cell 2020), enforcing RNAPII pausing and antagonizing pTEFb activity. Integrator subunit 11 then cleaves the nascent RNA causing promoter-proximal transcriptional termination (Wu PNAS 2017). To gain insights into the function of INT in erythropoiesis, we utilized the RNP method of Crispr/Cas 9 genome editing (Gundry Cell Reports 2016) to delete INTS8 from CD34+ HSPCs, and then subjected them to erythroid differentiation and colony forming assays. Disruption of INTS8 significantly impaired erythroid proliferation and viability. It also delayed erythroid maturation, evident by delayed GPA expression, less mature morphology, and lower rates of benzidine positivity. Moreover, INTS8 disruption led to a decline in erythroid colony-forming ability and decreased expression of alpha and beta globin. Together these data indicate that INT function is essential for erythropoiesis. Integrator subunit 1 (INTS1) serves as a molecular scaffold for the complex, and can be used as an indicator of INT levels and occupancy. Western blotting demonstrated that INTS1 protein increased after CD36 selection and remained high throughout terminal erythroid maturation. To delineate the genomic targets of INT we performed CUT&amp;RUN for INTS1 on day 7 and day 9 of erythroid culture following CD36 selection (as described in Gautier, Cell Reports 2016), corresponding to the basophilic and polychromatic stages of erythroid maturation. Approximately 50% of sites of INT occupancy were located at transcription start sites at both time points. Motif analyses of sites of INTS1 occupancy were enriched for transcription factors involved in signaling, including STAT5 (p1e-189) and SMAD2 (p1e-170), and INTS1 was present at several known STAT5 target genes including MYC and CCNB1. Interestingly, ~74% of genes with stably paused RNAPII, as indicated by a pausing index (PI) &amp;gt;4, were occupied by INTS1. Further, the level of INTS1 occupancy was significantly higher at genes with a PI &amp;gt;4 than genes with PI &amp;lt;4. The genomic occupancy of INTS increased from day 7 to day 9. Transcription start sites that gained INTS1 occupancy during maturation lost RNPII, suggesting a role for INT in promoting RNAPII eviction at these loci. Collectively, our findings identify a critical role for INT in governing erythroid maturation and gene expression, and provide novel insights into the transcriptional regulation of erythropoiesis.

Preclinical Activity of Novel TGF Beta Receptor I Kinase Inhibitors IOA-359 and IOA-360 for Treatment of Anemia in MDS/AML

Background: Overactivation of the Transforming growth factor beta (TGF-β) superfamily has been associated with bone marrow failure in MDS. TGF-β binds to set of receptors that include the TGF-β receptor I kinase (also known as ALK5), that in turn phosphorylates and activates the downstream SMAD2/3 proteins. Activation of SMAD2/3 transcription factors has been shown to occur in MDS and is associated with anemia. Thus, we wanted to evaluate the preclinical efficacy of novel, clinic-ready, TGF-b receptor I kinase small molecule inhibitors IOA-359 and IOA-360 in MDS models. Results: Using the TREEspot™ SCAN Max Kinase Panel of 403 kinases, IOA-359 and IOA-360 were found to be highly selective for ALK5. Both, IOA-359 and IOA-360 have a blocking activity against ALK5 similar to clinical stage ALK5 inhibitors. To evaluate the efficacy of IOA-359 and IOA-360, we first used HEK 293- Blue-TGF-β cells that stably overexpresses a SMAD inducible secreted embryonic alkaline phosphatase (SEAP) reporter. Both IOA-359 and IOA-360 were able to significantly inhibit TGF-β driven reporter at nanomolar doses ( Fig1A). Next, we showed that both ALK5 inhibitors were able to block TGF-β-mediated SMAD2/3 downstream phosphorylation/activation in a dose dependent manner in MDS and leukemic cell lines. Functionally, the ALK5 inhibitors did not lead to any increase in viability or proliferation in leukemic cell lines. We next tested the efficacy of the novel ALK5 inhibitors in primary patient samples from MDS and AML (N=8). Treatment with both ALK5 inhibitors led to increase in erythroid colony formation in majority of MDS samples in clonogenic assays. Colonies were picked up and examined for erythroid and myeloid differentiation by FACS analysis. We observed that ALK5 inhibition led to increase in Glycophorin A and CD71 expression suggesting enhanced erythroid differentiation ( Fig1B). Myeloid differentiation effects were modest in comparison. Furthermore, in selected samples, addition of Luspatercept led to greater maturation of erythrocytes. Treatment of healthy CD34 + hematopoietic stem and progenitors did not lead to significant changes in differentiation. Lastly, to evaluate downstream effects of ALK5 inhibition, we performed RNAseq analysis in MDS cells. Inhibition with ALK5 inhibitors led to numerous changes in genes affecting cellular differentiation, validating functional changes observed by us. Conclusion: Our current results support the preclinical in vitro efficacy of ALK5 inhibitors IOA-359 and IOA-360 alone and in combination with Luspartercept, highlighting their potential for further development and clinical testing in MDS/AML.

Physiological and regenerative functions of sterile-α motif protein-14 in hematopoiesis

Sterile α-motif domain-14 (Samd14) protein expression increases the regenerative capacity of the erythroid system. Samd14 is transcriptionally upregulated and promotes cell signaling via the receptor tyrosine kinase Kit in a critical window of acute erythroid regeneration. We generated a hematopoietic-specific conditional Samd14 knockout mouse model (Samd14-CKO) to study the role of Samd14 in hematopoiesis. The Samd14-CKO mouse was viable and exhibited no steady-state hematopoietic phenotype. Samd14-CKO mice were hypersensitive to 5-fluorouracil, resulting in more severe anemia during recovery and impaired erythroid progenitor colony formation. Ex vivo, Samd14-CKO hematopoietic progenitors were defective in their ability to form mast cells. Samd14-CKO mast cells exhibited altered Kit/stem cell factor (SCF), IL-3/IL-3R signaling, and less granularity than Samd14-FL/FL cells. Our findings indicate that Samd14 promotes both erythroid and mast cell functions. The Samd14-CKO mouse phenotype exhibits striking similarities to the KitW/W-v mice, which carry Kit mutations resulting in reduced tyrosine kinase-dependent signaling, causing mast cell and erythroid abnormalities. The Samd14-CKO mouse model is a new tool for studying hematologic pathologies involving Kit signaling.