Bone Marrow Colony Research Articles

Myeloid-derived β-hexosaminidase is essential for neuronal health and lysosome function: implications for Sandhoff disease.

Lysosomal storage disorders (LSDs) are a large disease class involving lysosomal dysfunction, often resulting in neurodegeneration. Sandhoff disease (SD) is an LSD caused by a deficiency in the β subunit of the β-hexosaminidase enzyme ( Hexb ). Although Hexb expression in the brain is specific to microglia, SD primarily affects neurons. To understand how a microglial gene is involved in maintaining neuronal homeostasis, we demonstrated that β-hexosaminidase is secreted by microglia and integrated into the neuronal lysosomal compartment. To assess therapeutic relevance, we treated SD mice with bone marrow transplant and colony stimulating factor 1 receptor inhibition, which broadly replaced Hexb -/- microglia with Hexb -sufficient cells. This intervention reversed apoptotic gene signatures, improved behavior, restored enzymatic activity and Hexb expression, ameliorated substrate accumulation, and normalized neuronal lysosomal phenotypes. These results underscore the critical role of myeloid-derived β- hexosaminidase in neuronal lysosomal function and establish microglial replacement as a potential LSD therapy.

Effects of radiation mitigating amino acid mixture on mice of different sexes.

To date, few FDA-approved medical countermeasures are available for addressing hematopoietic acute radiation syndrome (H-ARS). In this study, we present our latest research findings focusing on the evaluation of a novel radiation mitigator known as the mitigating amino acid mixture (MAAM). MAAM is composed of five amino acids as the recently reported amino acid-based oral rehydration solution for mitigating gastrointestinal (GI)-ARS. CD2F1 male and female mice were exposed to 60Co-γ total body irradiation (TBI) at 9.0 or 9.5 Gy. Following irradiation, mice were orally administered with MAAM or a saline vehicle control once daily for a duration of 14 days, commencing 24 h after TBI. Mouse survival and body weight change were monitored for 30 days after irradiation. Complete blood counts (CBCs), bone marrow (BM) stem and progenitor cell survival (clonogenicity), and a serum cytokine antibody array were analyzed using samples from day 30 surviving mice. Our data revealed that MAAM treatment significantly enhanced survival rates in irradiated male CD2F1 mice, and the survival rate increased from 25% in the vehicle control group to 60% in the MAAM-treated group (p < 0.05) after 9.0 Gy TBI. The number of BM colonies significantly increased from 41.8 ± 6.4 /104 cells (in the vehicle group) to 78.5 ± 17.0 /104 cells (in the MAAM group) following 9.0 Gy TBI. Furthermore, MAAM treatment led to a decrease in the levels of six cytokines/proteins [cluster of differentiation 40 (CD40), interleukin (IL)-17A, C-X-C motif chemokine 10 (CXCL10/CRG-2), cutaneous T cell-attracting chemokine (CTACK), macrophage inflammatory protein (MIP)-3β, and IL-1β] and an increase in the levels of five other cytokines/proteins [IL-3Rβ, IL-5, leptin, IL-6, and stem cell factor (SCF)] in mouse serum compared to the vehicle group after 9.0 Gy TBI. However, similar alleviating effects of MAAM were not observed in the irradiated CD2F1 female mice. The serum cytokine profile in the irradiated female mice was different compared to the irradiated male mice. In summary, our data suggest that the beneficial effects of the mitigative amino acid combination treatment after radiation exposure may depend on sex.

Umbilical Cord Blood-Derived Cells Can Reconstruct Hematopoiesis in an Aplastic Anemia Animal Model.

To explore the efficacy and the mechanism of the umbilical cord-derived cells combined with cyclosporine A (CsA) in treating aplastic anemia (AA) in mice. Immune-mediated AA model mice were treated with CsA + UC mesenchymal stem cells (UC-MSC), CsA + umbilical cord blood regulatory T cells (UCB-Treg), UC-MSC, UCB-Treg, CsA alone, or blank control, respectively (n = 9 mice/group). CsA and the cell infusion was administered on d0. Routine peripheral blood testing was performed once weekly; bone marrow colony culture, bone marrow cell flow cytometry, peripheral blood T cell subsets, and serum inflammatory cytokines tests were performed on d14. Transcriptome sequencing was performed for cells from CsA + UC-MSC, CsA + UCB-Treg, and CsA groups to detect the possible related genes. Gene function cluster and signal pathway enrichment analysis were also performed. Blank control mice died due to pancytopenia within 21 days, whereas mice in other groups survived for >28 days. On d14, the CsA + UC-MSC and CsA + UCB-Treg groups had higher white blood cell (WBC) counts than the other groups (p < 0.05), along with higher burst-forming unit (BFU) and colony-forming unit-granulocyte, macrophage (CFU-GM) counts (p < 0.01). The CsA + UC-MSC group had the highest BFU count (p < 0.01). The CsA + UC-MSC and CsA + UCB-Treg groups exhibited the highest bone marrow CD34+ cell proportion (9.68% ± 1.35% and 8.17% ± 0.53%, respectively; p < 0.01). Tumor necrosis factor (TNF)-α and interleukin (IL)-2 levels in the CsA + UC-MSC group (p < 0.05) and TNF-α, interleukin-2, and interferon (INF)-γ levels in the CsA + UC-Treg group (p < 0.01) were lower than those in the CsA group. Compared with CsA treatment, CsA + UC-MSC significantly downregulated the histone methylation pathway (p < 0.05), whereas CsA + UCB-Treg significantly upregulated energy metabolism processes (p < 0.05). Treatment with CsA + UC-MSC upregulated superoxide dismutase activity compared with CsA + UCB-Treg treatment. Adding UC-MSC or UCB-Treg to CsA markedly enhanced the reconstruction of hematopoiesis in AA mice, with UC-MSC eliciting greater efficiency than UCB-Treg. Accordingly, the addition of these cells could further improve immune abnormalities.

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.

Long-Term Hematopoietic Dysfunction in Patients with Single Large-Scale Mitochondrial DNA Deletion Syndromes

Pearson syndrome (PS) is the only single large-scale mitochondrial DNA deletion syndrome (SLSMDs) presenting substantial hematologic manifestations, usually a severe though transient cytopenia of childhood. Recently, progression to myeloid malignancies has been described among PS patients. Older PS patients, as well as patients with SLSMDs presenting with Kearns-Sayre syndrome (KSS) or with progressive external ophthalmoplegia (PEO), do not display hematologic abnormalities. Since the underlying mitochondrial DNA deletion is similar in all SLSMDs, we hypothesized that bone marrow (BM) dysfunction is a common and persistent manifestation in these patients. We studied 16 patients with molecular-confirmed SLSMDs: 13 with PS, and three with KSS. The median age at diagnosis was 25 months (range 2-136 months). Seventy-five percent of patients presented with cytopenia in infancy, and 56% required blood products. Resolution of cytopenia occurred in 92% of this subgroup, at a median age of 24 months (range 12-39 months). BM studies were performed in all patients, and 56% of patients had multiple BM evaluations. All BM biopsies examined showed signs of marrow dysfunction, including in patients without a history of cytopenia. Median BM cellularity was 50% (range 30-80%) at a median age of 5 years. BM samples showed a paucity of precursor myeloid cells, signs of dyserythropoiesis, and abnormal or missing megakaryocytes. We report low hematopoietic stem and progenitor cell functionality in patients with SLSMDs: The median CD34+ count was 0.68% (range 0.27-1.92%, expected range 1-2%). BM colony forming unit (CFU) capacity was reduced in SLSMD patients: the median CFU in patients was 280 per 10 6 cells compared to 1090 per 10 6 cells in healthy controls (p=0.004). Conventional cytogenetic analysis was done on 10 patients (62%), and FISH analysis was done on 15 patients (93%). Monosomy 7 was identified in seven patients (44%): in one patient the monosomy 7 resolved in a follow-up BM assay. Another patient with signs of myelodysplasia and 30% of the BM cells having monosomy 7 was referred to an allogeneic bone marrow transplantation. To conclude, long-term BM dysfunction is evident in all patients with SLSMD and includes a risk of developing clonal evolution and monosomy 7. This data, combined with other recent publications, highlights the ongoing marrow failure and suggest that SLSMD syndromes should be included under inherited BM failure syndromes.

Abstract 16084: Impairment of Cardio-Spleno-Bone Marrow Axis Following Myocardial Infarction in Diabetes Mellitus

Introduction: Precise comprehension of the cardio-spleen-bone-marrow immune cells axis is crucial to understanding the changes occurring during diabetes (DM) pathogenesis. This study aims to investigate the alterations in immune cell kinetics in DM after myocardial infarction (MI). Methods: MI was induced in DM and healthy control using C57BL/N6 mice. Animals were sacrificed at D1, D3, D7, &amp; D28 post-MI to obtain their heart, peripheral blood (PB), spleen, and bone marrow (BM) and then each organ cell suspension was isolated, and analyzed by flow cytometry. EPC-colony-forming assay (EPC-CFA) was performed using BM, PB, and spleen-derived MNCs. Results: Findings demonstrated that despite the normal production of BM, CD45+ cells are decreased at D1-D3 in spleen and PB of DM’s. Further analysis depicted that total and pro-inflammatory neutrophil (N1) decreased at D1-D3 in PB and spleen at D3-D7 in DM mice, suggesting that DM causes spleen's neutrophil alterations that cannot compensate for PB and ischemia tissue demand. Infiltrated macrophages (total, M1, M2) decreased at D3 in DM-ischemic heart. Total and M1 (D1-D3) and M2 (D3-D7) notably decreased in DM-PB than control group, showing impaired macrophage recruitment/polarization in DM. Moreover, heart myeloid dendritic cells (mDCs) increased from D1-D7, the latter enhanced recruitment of CD8 cells increase from D1 to D28 in DM-infarcted-myocardium. The total CD4 cells decreased at D1-D3 in DM-PB, indicating a delayed adaptive immune response to MI. The B-cells diminished at D1-D3 in PB, at D3 in myocardium, and D7 in spleen, indicating impaired mobilization of those cells from bone marrow to compensate. EPC-CFA demonstrated dramatically decrease of definitive EPC colonies in spleen and BM from D1-D28 in DM mice vs control in vitro , indicating that DM significantly impairs EPC colony-forming bioactivity by limiting EPCs' differentiation from primitive to definitive. Conclusion: Our study highlights significant alterations of cardio-spleno-bone-marrow immune cells axis following MI in DM animals, revealing delayed innate/adaptive immune responses and impaired differentiation of EPCs.

Efficient and Stable Makassar Base Editing in Nonhuman Primates

Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin chain. This nucleotide can be modified using adenine base editors (ABEs) to convert the defective SCD β-globin gene (HBB S) into the non-pathogenic Makassar β-globin (HBB M) variant. Proof-of-concept for this editing strategy in human and mouse hematopoietic stem cells (HSCs) has been previously demonstrated by transplantation after ex vivo modification as well as by in vivo adenoviral editing followed by selection. Here, we evaluated the feasibility, long-term efficiency, and safety of autologous ex vivo Makassar base editing in the preclinical nonhuman primate (NHP) large animal model to pave the way towards clinical applications in humans. As NHPs don't carry the sickle mutation (position A7 in the protospacer), bystander editing at position A9 was monitored as a surrogate. NHP CD34 + hematopoietic stem and progenitor cells (HSPCs) were enriched, CD34 +CD90 + HSCs FACS-purified as previously reported to increase the editing efficiency of short- and long-term engrafting cells, primed over-night, and ABE8e-NRCH base editor mRNA was delivered by electroporation. Base-edited HSCs were cultured overnight and then combined with unmodified CD34 +CD90 - cells and infused into animals after myeloablative total body irradiation. The infusion product was quality controlled by flow-cytometry, next generation sequencing (NGS), and assessing the mono-/bi-allelic base editing efficiency on a single cell level from colony-forming cell (CFC) assays. Animals were followed for up to 200 days analyzing the editing efficiency in peripheral blood (PB) white blood cells (WBCs), FACS-purified lineages from the PB, bone marrow (BM) WBCs, as well as FACS-purified BM HSPCs. Comprehensive bioinformatics analysis was performed on the generated NGS data to determine the frequency of editing over time as well as the on- and off-target editing within the protospacer at all four target locations. Finally, chromosomal integrity after base editing was confirmed performing Kromatid karyotyping assays on gene-edited BM-derived HSPCs. Ex vivo base editing of CD34 +CD90 + was highly reproducible at the Makassar locus reaching more than 60% efficiency within bulk HSCs. No impact of base editing on the myeloid and erythroid differentiation of HSCs was seen in CFC assays. Clonal analysis of colonies revealed that more than 90% of HSCs in the infusion product were edited with &amp;gt; 20% of cells showing biallelic editing. Gene-edited HSCs combined with unmodified progenitors rapidly engrafted in myeloablated animals without any noticeable delay in the recovery of neutrophils, red blood cells, as well as platelets and multilineage reconstitution was established within 3-6 months. Most importantly, stable editing of &amp;gt;20% was observed in PB WBCs and across all lineages throughout the entire follow-up. Similarly, the BM stem cell compartment fully recovered to baseline within 6-month post-transplant. Engrafted HSPCs demonstrated normal erythro-myeloid colony-formation potential. Assessment of editing in colonies showed persistence of HSCs with mono- and bi-allelic Makassar editing 6-month post-transplant. Closely matching the editing efficiency in the PB, all BM lineages, HSPC subsets, and colonies demonstrated &amp;gt;20% editing efficiency confirming successful editing of long-term persisting multipotent HSCs. Finally, comprehensive assessment of NGS data from all tissues in conjunction with the Kromatid assay revealed no unwanted off-target effects or chromosomal rearrangements. Editing was primarily observed at positions A9 and A12 confirming highly specific on-target editing. In summary, we demonstrate efficient, persistent, and safe base editing of NHP HSCs for the treatment of SCD. The editing efficiency of the bystander adenine in NHP HSCs is almost identical to the previously observed editing in human patient HSCs to generate the Makassar HBB. Base edited HSCs showed no impairment in homing, long-term engraftment, or multilineage differentiation in the NHP. These findings have major implications for the application of base editing in patients with hematological disease and disorders.

Novel CD123xCD3 Bispecific Igm Antibody, Igm-2537, Potently Induces T-Cell Mediated Cytotoxicity of Acute Myeloid Leukemia Cells In Vivo and in Vitro with Minimal Cytokine Release

Introduction: New therapeutics are needed for the effective treatment of acute myeloid leukemia (AML), as standard chemotherapeutics are poorly tolerated and ~50% of patients relapse primarily due to the incomplete elimination of leukemia stem cells (LSCs). CD123, the IL-3Rα chain, is highly expressed on leukemic blasts and LSCs, and is further increased in patients with poor prognostic factors. Bispecific T-cell engager (TCE) antibodies and CAR-T cells targeting CD123 have shown promising clinical efficacy in AML patients, but cytokine release syndrome limits their therapeutic window and remains a major safety concern. IGM-2537 is a novel pentameric IgM bispecific TCE antibody engineered with ten anti-CD123 binding sites, and an anti-CD3ε single chain Fv domain fused to the joining chain to engage T-cells. Here, we report that IGM-2537 not only potently induces T-cell mediated cytolytic killing of AML cells in vitro and in vivo but also demonstrates minimal cytokine release, and little to no effect on normal hematopoietic progenitor cells and human vascular endothelial cells (HUVECs) with in vitro and ex vivo studies. Additionally, an improved tolerability, excellent pharmacodynamic (PD) response and lower cytokine release was observed with a cynomolgus cross-reactive IgM TCE in monkeys. Materials and Methods:Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity and epitope mapping, respectively. T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 target cytotoxicity, T cell activation and cytokine release. Ex vivo AML colony formation assays were used to assess the activity on AML blasts. Human MV-4-11 and MOLM-13 xenograft tumor models in humanized NSG dKO mice were utilized to determine in vivo anti-tumor activity of IGM-2537. Healthy human PBMCs and bone marrow (BM) samples as well as HUVECs were used in ex vivo assays to address potential safety concerns of IGM-2537 mediated effects on hematopoietic progenitor cells and HUVECs, respectively. Lastly, to confirm that a CD123 directed IgM-based TCE molecule can potentially provide better safety profile in vivo, a cynomolgus-cross reactive IgM TCE comparator was assessed in monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound with high affinity and avidity to CD123. In vitro, IGM-2537 engaged both CD123 and CD3 to induce potent T-cell activation and T-cell mediated cytotoxicity of CD123 + AML cells, and autologous CD123 + basophils and plasmacytoid dendritic cells (pDCs). Though IGM-2537 demonstrated comparable maximal killing activity to a comparator IgG TCE, IGM-2537 demonstrated minimal cytokine release. In ex vivo patient-derived AML or normal BM colony formation assays, IGM-2537 eliminated AML colony forming cells at physiologically relevant effector/target (E/T) ratios but had little or no effect on normal hematopoietic progenitors. In addition, IGM-2537 demonstrated no cytotoxicity against HUVECs regardless of their CD123 expression at basal levels or at maximal levels upregulated by TNF-a and IFN-g, in contrast to a comparator IgG TCE. In vivo, IGM-2537 completely inhibited tumor growth in two AML xenograft tumor models, a subcutaneous MV-4-11 model, and a systemic MOLM-13 model in humanized NSG dKO mice. To further evaluate the potency and safety of the CD123xCD3 IgM bispecific TCE format in vivo, a cynomolgus cross-reactive CD123xCD3 IgM bispecific TCE was evaluated for tolerability and PD responses in cynomolgus monkeys using single doses. All animals tolerated the cross-reactive bispecific IgM TCE well at doses up to 10 mg/kg (the maximal dose level evaluated), a dose 100-fold greater than published doses of a comparator IgG TCE. Complete depletion of CD123 + basophils and substantial reductions of pDCs were seen in blood and BM with minimal to no cytokine induction. In summary, IGM-2537 demonstrated potent in vitro and in vivo T-cell mediated cytotoxicity of AML cell lines with minimal cytokine induction. The encouraging preclinical safety profiles were further supported by little/no effect on normal hematopoietic progenitor cells and HUVECs. These preclinical data support the clinical development of IGM-2537 for the treatment of AML and other hematological malignancies.

Abstract 2933: Novel CD123xCD3 bispecific IgM antibody, IGM-2537, potently induces T-cell mediated cytotoxicity of acute myeloid leukemia cells with minimal cytokine release

Abstract Novel therapeutics are needed for the effective treatment of acute myeloid leukemia (AML), as standard chemotherapeutics are poorly tolerated and ~50% of patients relapse primarily due to the incomplete elimination of leukemia stem cells (LSCs). CD123, the IL-3Rα chain, is highly expressed on leukemic blasts, LSCs, and is further increased in patients with poor prognostic factors. Bispecific T-cell engager (TCE) antibodies and CAR-T cells targeting CD123 have shown promising clinical efficacy in AML patients, but cytokine release syndrome limits their therapeutic window and remains a major safety concern. IGM-2537 is a novel pentameric IgM bispecific TCE antibody engineered with ten anti-CD123 binding sites, and an anti-CD3ε single chain Fv domain fused to the joining chain to engage T-cells. Here, we report the functional characterization of IGM-2537 using in vitro, ex vivo and in vivo anti-tumor efficacy studies with preliminary safety evaluation of this novel class of IgM TCE. IGM-2537 bound with high selectivity, affinity and avidity to CD123 through an epitope distinct from IL-3. In vitro, IGM-2537 co-engaged with both CD123 and CD3 to induce potent T-cell activation and T-cell mediated cytotoxicity of AML cell lines. Though IGM-2537 demonstrated comparable maximal killing activity to a comparator IgG TCE, IGM-2537 demonstrated minimal cytokine release. In ex vivo patient-derived AML or normal bone marrow colony formation assays, IGM-2537 eliminated AML colony forming cells at physiologically relevant effector/target (E/T) ratios but spared normal progenitors. In addition, IGM-2537 potently depleted CD123+ basophils and plasmacytoid dendritic cells (pDC) in normal human peripheral blood mononuclear cells. In vivo, IGM-2537 completely inhibited tumor growth in the AML xenograft tumor model, MV4-11 in humanized NSGdKO mice at doses as low as 1 mg/kg. To further evaluate the potency and safety of the CD123xCD3 IgM bispecific TCE format in vivo, a cynomolgus cross-reactive CD123xCD3 IgM bispecific TCE was evaluated for tolerability and pharmacodynamic responses in cynomolgus monkeys. All animals tolerated this bispecific IgM TCE well at doses up to 10 mg/kg (the maximal dose level evaluated), a dose 100-fold greater than published doses of a comparator IgG TCE. Complete depletion of CD123+ basophils and substantial reductions of pDCs were seen in blood and bone marrow with minimal to no cytokine induction. In summary, IGM-2537 demonstrated potent in vitro and in vivo T-cell mediated cytotoxicity of AML cell lines with minimal cytokine induction. These preclinical data further support the clinical development of IGM-2537 for the treatment of AML and more broadly substantiate the use of IgM antibodies as a framework for TCEs to provide an improved therapeutic window for T-cell redirected therapeutics. Citation Format: Gene Li, Ling Wang, Poonam Yakkundi, Paul Hinton, Deepal Pandya, Keerthana Sekar, Rodine Rosete, Zhongde Ye, Nardeen Hanna, Maya F. Kotturi, Liz Bogaert, Jiyoung Hong, Christina Tsai, Thomas Manley, Bruce A. Keyt, Angus M. Sinclair, Liqin Liu. Novel CD123xCD3 bispecific IgM antibody, IGM-2537, potently induces T-cell mediated cytotoxicity of acute myeloid leukemia cells with minimal cytokine release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2933.

Aligning the Cord Blood–Bone Marrow Differences and the Post-transplantation Clinical Manifestations by Single-Cell RNA-Seq

Cord blood (CB) transplantation is a promising treatment for hematologic malignancies due to its strong graft-versus-leukemia effect and a low incidence of graft-versus-host disease. However, the risk of infection caused by delayed engraftment has limited its clinical application. In this study, we compared the single-cell RNA-seq of CB, bone marrow (BM), and granulocyte colony–stimulating factor primed BM to understand the differences between these grafts from a comprehensive view, and verified some differences in our clinical data of patients receiving transplantation. We focused on the biological features of key cell types involving the hematopoietic reconstitution and immune reconstitution. Based on the comparison of homing signal and differentiation potential of hematopoietic stem/progenitor cells (HSPCs), CB exhibited a lower content of HSPCs with weaker homing ability but higher stemness than BM. In addition, CB had a higher proportion of naïve T cells, while BM had a higher abundance of effector and memory T cells. Notably, the CD4+ naïve T cells in CB were prone to differentiate into Tregs. In response to neoantigens, the immune activation interactions between T cells and antigen-presenting cells were strong in CB, including CD40_CD40LG, IL16_CD4, and so on. In our clinical data, the subpopulation variations of T cells and the status of monocytes after transplantation were consistent with the results of the single-cell RNA-seq study above. CB, as a new birth system, is immature and active; several mechanisms contribute to its good anti-tumor effect, which can be introduced to other grafts. These findings provide insights into the development of new strategies for hematologic malignancies treatment.

Targeting PLK1 As a Novel Strategy for Acute Myeloid Leukemias with Fanconi Anemia Pathway Mutations

The Fanconi anemia (FA) signaling network is critical to maintaining genomic integrity during interphase and mitosis. Biallelic germline mutation of any of the 22 genes that constitute this pathway (FANCA-FANCW) results in the disorder FA, which is characterized by congenital malformations, a predisposition to bone marrow failure, and pediatric acute myeloid leukemias (AMLs). Among the general population, acquired genetic disruptions of the FA pathway are present in 18% of sporadic pediatric AMLs, underscoring the importance of this pathway in the prevention of hematological malignancy. FANCA is the gene most commonly mutated in FA and is one of the most frequently disrupted FA pathway genes in sporadic AML. Therefore, the identification of precision therapies for FANCA-deficient AML is a high clinical priority. To identify synthetic lethal targets of FANCA, we previously identified mitotic phosphosignaling pathways required for the survival of FANCA-/- patient-derived fibroblasts through a kinome-wide shRNA screen. This screen identified the mitotic kinases CHEK1, PLK1, and BUB1 as hits. These findings corroborate work by others who have identified synthetic lethal interactions between PLK1 and the FA pathway components FANCG and BRCA1, suggesting inactivation of the FA pathway may sensitize cancers to PLK1 inhibition. The clinical success of PLK1 inhibitors, such as volasertib and onvansertib, has been limited by the absence of predictive biomarkers of response and adverse side effects. To test our hypothesis that FA gene mutations may predict sensitivity to PLK1 inhibition we generated an isogenic panel of human AML cells with a stable knockout of the FANCA gene. Utilizing in vitro cell viability assays, we have demonstrated the selective killing of FA-deficient AMLs by pharmacological PLK1 inhibition through low doses of volasertib and onvansertib. We also found that PLK1-inhibition impairs the hematopoietic potential of Fanca-/- murine hematopoietic stem and progenitor cells by performing in vitro bone marrow colony formation assays. Similarly, we demonstrated that primary human AMLs with multiple disruptions within the FA-pathway were hypersensitive to low doses of volasertib. To investigate the mechanism of synthetic lethality, we performed quantitative proteomic analysis of FANCA immunoprecipitates, which identified PLK1 as a novel FANCA-binding partner during mitosis. We validated this finding through high-resolution immunofluorescence microscopy and proximity ligation assays, which revealed a direct interaction between FANCA and PLK1 at kinetochores. Additionally, we demonstrated that volasertib treatment induces monoubiquitination of FANCD2 and colocalization of FANCA with FANCD2 at gamma-H2AX foci. Together, these findings indicate that PLK1 inhibition causes damage to mitotic chromosomes and subsequent activation of the FA pathway. Our work identifies a mitosis-specific vulnerability of FA-deficient cells and suggests that genetic disruptions of the FA pathway may be predictive of sensitivity to PLK1 inhibition, providing a preclinical rationale for the development of precision therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Peculiarities of functioning of hematopoietic progenitor cells of bone marrow of patients with myelodysplastic syndrome in culture in vitro and in cell diffusion chambers in vivo

Myelodysplastic syndrome is a clonal disease with changes that occur due to mutations that lead to cytogenetic disorders and involve the hematopoietic stem cell. Researchers have shown that colony growth types in leukemic cell culture with acute and chronic myeloid leukaemia are of a prognostic value to determine the likelihood of remission or disease progression. However, there is no single point of view on the prognostic value of clonal culture research methods in MDS. The aim of the study was to determine the role of hematopoietic progenitor cells, the closest descendants of stem cells, in the formation of the pathological process in MDS, and identify the features of colony formation and morphological composition of bone marrow colonies in patients in vitro and in vivo in diffusion chambers implanted in the abdominal cavity of linear animals to determine their possible prognostic value. Bone marrow samples from patients with MDS with an excess of blasts-1 were examined by haematological, cultural, cytological and statistical methods. The features of colony formation in culture with semi-liquid agar of bone marrow of patients with MDS with an excess of blasts-1 were revealed. It has been established that in patients with the disease detection, the colony-forming ability in the semi-liquid agar of progenitor cells is reduced, the number of cells in the colonies decreases, and the appearance of capricious forms of cell aggregates with changes in culture differentiation were detected. The ability of bone marrow in MDS with an excess of blasts-1 to form colony clones by hematopoietic progenitor cells when cultured in semi-liquid agar both in vitro and in vivo was evaluated. Informative indicators of morphofunctional characteristics of stem cells and their descendants in culture, such as colony-forming ability and cell content of colonies, were determined. It is proved that the results of cultivation in diffusion chambers in vivo are comparable with the data obtained in vitro, and can become, along with traditional methods, an additional sign of detection and progression of MDS.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Mitigation of total body irradiation-induced mortality and hematopoietic injury of mice by a thrombopoietin mimetic (JNJ-26366821)

The threat of a nuclear attack has increased in recent years highlighting the benefit of developing additional therapies for the treatment of victims suffering from Acute Radiation Syndrome (ARS). In this work, we evaluated the impact of a PEGylated thrombopoietin mimetic peptide, JNJ-26366821, on the mortality and hematopoietic effects associated with ARS in mice exposed to lethal doses of total body irradiation (TBI). JNJ-26366821 was efficacious as a mitigator of mortality and thrombocytopenia associated with ARS in both CD2F1 and C57BL/6 mice exposed to TBI from a cobalt-60 gamma-ray source. Single administration of doses ranging from 0.3 to 1 mg/kg, given 4, 8, 12 or 24 h post-TBI (LD70 dose) increased survival by 30–90% as compared to saline control treatment. At the conclusion of the 30-day study, significant increases in bone marrow colony forming units and megakaryocytes were observed in animals administered JNJ-26366821 compared to those administered saline. In addition, enhanced recovery of FLT3-L levels was observed in JNJ-26366821-treated animals. Probit analysis of survival in the JNJ-26366821- and saline-treated cohorts revealed a dose reduction factor of 1.113 and significant increases in survival for up to 6 months following irradiation. These results support the potential use of JNJ-26366821 as a medical countermeasure for treatment of acute TBI exposure in case of a radiological/nuclear event when administered from 4 to 24 h post-TBI.

Panaxdiol saponins component promotes hematopoiesis by regulating GATA transcription factors of intracellular signaling pathway in mouse bone marrow.

BackgroundOur research team has identified a biological active component, panaxadiol saponins component (PDS-C) isolated from total saponins of panax ginseng as a potential targeted drug for treating hemocytopenia. PDS-C possesses dual activities, namely that of promoting hematopoiesis and regulating immune function. Our study is to observe effects of PDS-C on promoting hematopoiesis in normal and aplastic anemia (AA) mice, furthermore, to explore its possible mechanism.MethodsBone marrow nucleated cells were cultured for colony forming assay of CFU-GM, CFU-E and CFU-MK in the presence of PDS-C at different concentration. The proliferation and differentiation-related genes expression profile was analyzed with DNA membrane microarray. The mRNA expression levels and protein phosphorylated state of GATA-1, GATA-2 transcription factors and AKT-1, MAPK14 protein kinases were detected by RT-qPCR and Western blot, the DNA binding activity and components of GATA-DNA complex were analyzed by EMSA and antibody gel supershift assay.ResultsIn response to PDS-C at 10, 25 and 50 mg/L, the bone marrow colony numbers of CFU-GM, CFU-E and CFU-MK increased significantly by 25.7%±3.1% to 42.4%±4.5% respectively in normal mice, and 29.7%±3.7% to 53.2%±7.1% in AA mice. The gene microarray profile initiated by PDS-C provided the up-regulated genes by more than 3 times, which can be classified into 11 categories according to their functions, including GATA-1, GATA-2, and AKT-1, MAPK14. The mRNA expression levels of GATA-1, GATA-2 were consistent with their gene microarray profile in PDS-C treated erythroid and megakaryocytic hematopoietic cells. Meanwhile, PDS-C could not only up-regulate expression levels of GATA-1, GATA-2 proteins, but also enhance phosphorylated activity state. Furthermore, PDS-C obviously enhanced binding activity of GATA protein with DNA in erythroid and megakaryocytic cells, and the main composition of GATA-DNA complex was GATA-2 and GATA-1.ConclusionsPDS-C displays the role to promote proliferation and induce differentiation for hematopoietic cells. Its action mechanism may involve in GATA-1, GATA-2 transcription factors, including up-regulating mRNA and protein expression, enhancing DNA binding activity, phosphorylated functional activity and up-regulating AKT-1, MAPK14 protein kinases as the upstream signaling molecule for activation GATA-1, GATA-2 respectively in hematopoietic cells.

Gene Therapy for Fanconi Anemia [Group A]: Interim Results of RP-L102 Clinical Trials

Gene Therapy for Fanconi Anemia [Group A]: Interim Results of RP-L102 Clinical Trials

Novel Polyethylene Glycol-Conjugated Triazole Derivative with High Thyrointegrin αvβ3 Affinity in Acute Myeloid Leukemia Management.

Simple SummaryThe integrin αvβ3 antagonist (P-bi-TAT) is associated with the downregulation of signaling pathways, especially NF-κB pathway, which play an important role in leukemogenesis. Our results provide a rationale for further evaluation of the αvβ3 antagonists as providing potential therapeutic utility not only in AML but also in different malignancies.(1) Background: Acute myeloid leukemia (AML) accounts for up to one-third of more than 60,000 leukemia cases diagnosed annually in the U.S. Primary AML cells express membrane αvβ3 integrin, which is associated with adverse prognosis and resistance to chemotherapies. A novel anticancer compound Polyethylene glycol-conjugated bi-TriAzole Tetraiodothyroacetic acid (P-bi-TAT) interacts with high affinity (Ki 0.3 nM) and specificity with the thyrointegrin αvβ3. We evaluated P-bi-TAT activities in two different AML models representing monocytic and myelocytic forms of acute leukemia. (2) Methods and Results: The in vivo AML models were established prior to initiation of treatment protocols by grafting human leukemia cells in immunocompromised mice. IVIS imaging scans revealed that leukemic colonies were extensively established throughout the bone marrow, liver, and lung of the untreated animals. In animals treated with P-bi-TAT at daily doses ranging from 1–10 mg/kg, subcutaneously for 2–3 weeks, IVIS imaging scans revealed 95% reduction in bone marrow colonies and leukemic colonies in liver and lung. Also, the leukemic cells were not detected in bone marrow samples of P-bi-TAT-treated animals. The anti-neoplastic effect of P-bi-TAT administration on leukemic cells was associated with marked inhibition of NF-κB activity. We conclude that experimental P-bi-TAT therapy in vivo appears extraordinarily effective against the two forms of human AML models in mice. Because the P-bi-TAT molecular target, thyrointegrin αvβ3, is consistently expressed in many, if not all, clinical AML samples, P-bi-TAT-based therapy seems to have significant clinical potential in treating most AML sub-types. Hence, P-bi-TAT represents a promising targeted therapeutic agent for AML patients.

Haploinsufficiency of Multiple Genes on Del(5q) Cooperate to Induce B Cell Abnormalities in Mice

Interstitial deletions on chromosome 5q [del(5q)] are the most common cytogenetic alteration in patients with myelodysplastic syndrome (MDS), affecting up to 25% of patients. The size of the deletion and the breakpoints vary among patients and include two commonly deleted regions (CDR). The distal CDR is located between 5q32-5q33 and the proximal CDR is located on 5q31. Several genes in the CDRs have been implicated in del(5q)-associated disease pathogenesis through haploinsufficiency, including RPS14, SPARC, miR-145 and miR-146 in the distal CDR, and EGR1, CTNNA1, and HSPA9 in the proximal CDR. Most patients have deletions that span both CDRs, therefore, we asked whether simultaneous deletion of genes on both CDRs cooperate to contribute to altered hematopoiesis.In this study, we crossed Hspa9+/- mice (a gene on the proximal CDR) with Cd74-Nid67fl/+ mice (loxP sites flanking 8 distal CDR genes , Nid67, Dctn4, Rbm22, Myoz3, Synpo, Ndst1, Rps14 and Cd74) . Cd74-Nid67fl/+ mice also express the Lmo2-Cre transgene that is expressed during embryonic hematopoiesis. We comprehensively characterized the hematopoiesis in (1) wild type control mice, (2) Hspa9+/- mice, (3) Lmo2-Cre+ mice, (4) Cd74-Nid67fl/+ ; Lmo2-Cre+ mice, and (5) double mutant Hspa9+/- ; Cd74-Nid67fl/+ ; Lmo2-Cre+ mice. We observed the previously reported reduction in bone marrow colony forming unit-preB (CFU-preB) colony formation in Hspa9+/- single-mutant mice. We also observed the reported anemia with decreased RBC maturation (n=4-8, P<0.05) and increased apoptosis in bulk bone marrow cells (n=4-8, P<0.05) in Cd74-Nid67fl/+ ; Lmo2-Cre+ (Cd74-Nid67+/-) mice. At 18 months of age, Cd74-Nid67+/- mice also developed reduction in B cell frequency in the bone marrow (BM) (≥2.1-fold, n=3-5, P<0.01), spleen (≥1.4 -fold, n=3-5, P<0.05) and peripheral blood (≥2.0-fold, n=3-5, P<0.05) (Figure 1a) that was associated with reduction in CFU-preB methylcellulose colonies (≥8.4-fold, n=3-5, P<0.001) compared to wild type and Lmo2-Cre+ control mice (Figure 1b). Hspa9+/- ; Cd74-Nid67fl/+ ; Lmo2-Cre+ double mutant mice showed a reduction of CFU-PreB methylcellulose colonies compared to wild type and Lmo2-Cre+ control mice (≥42.4-fold, n=3-5, P<0.001) (Figure 1b). Hspa9+/- ; Cd74-Nid67fl/+ ; Lmo2-Cre+ mice also developed a reduction in common lymphoid progenitors (≥1.6-fold, n=3-5, P<0.05) and Hardy B cell fractions A-E (≥3.4-fold, n=3-5, P<0.05) in the BM, with at least a 2-fold reduction in mature B cell frequencies in the BM, spleen and peripheral blood (n=3-5, P<0.05) compared to wild type, Lmo2-Cre+ and Hspa9+/- control mice (Figure 1a). These data suggest that haploinsufficiency of Hspa9 cooperates with haploinsufficiency of one or more genes deleted in Cd74-Nid67+/- mice to reduce B cells. We next asked which of the 8 genes on the Cd74-Nid67 deleted interval contributed to the B cell defect.CD74 is a transmembrane protein expressed on antigen presenting cells and mediates major histocompatibility (MHC) class II assembly and trafficking. It has been suggested to regulate mature B cell survival through macrophage migration inhibitory factor (MIF) signaling. To better understand the role of CD74 in hematopoiesis, we characterized B cells in Cd74 knockout mice. We observed that Cd74 homozygous and heterozygous knockoutmice (2-3 months old) have decreased CFU-PreB colonies in BM compared to wild type control mice (4.4-fold, n=4, P<0.01). The Cd74-/- mice, but not Cd74+/- mice, also have decreased peripheral blood B cells (1.5-fold, n=4, P<0.01) at 3 months. This result indicates that loss of Cd74 likely contributes to the B cell phenotypes observed in the Cd74-Nid67+/- mice. We predict that heterozygous loss of Cd74 and Hspa9 will cooperate to induce similar B cell phenotypes observed in Hspa9+/-; Cd74-Nid67+/- double mutant mice. These studies are ongoing by intercrossing Cd74+/- and Hspa9+/- mice, as well as mechanistic studies to understand how Hspa9 and Cd74 haploinsufficiency regulate B cell development. Collectively, these studies may provide insight into B cell alterations that occur in MDS patients with del(5q). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CSF3R Mutations Synergize with CEBPA and SETBP1 Mutations in Acute Myeloid Leukemia and Chronic Neutrophilic Leukemia

CSF3R is the receptor for granulocyte colony stimulating factor and plays a critical role in the proliferation and maturation of granulocytes. Although rare in most hematologic malignancies, we have discovered CSF3R mutations in ~80% of patients with Chronic Neutrophilic Leukemia (CNL) (Maxson et al, NEJM 2013). These mutations render the receptor constitutively active, leading to unrestrained neutrophil production. Although initially felt to be specific to CNL, through a collaboration with the National Cancer Institute/Children's Oncology Group TARGET Initiative, we identified a small fraction of patients with pediatric Acute Myeloid Leukemia (AML) that also have CSF3R mutations (Maxson et al, Blood, 2016). To interrogate this observation, we looked at co-occurring genetic lesions for the two malignancies. Interestingly, we found that the cooperating lesions are very different between the CNL and pediatric AML patient cohorts. For instance, ~25% of patients with CNL also have a mutation in SETBP1, and nearly half of patients with CSF3R-mutated pediatric AML have mutations in CEBPA. Furthermore, Lavallee et al identified CSF3R mutations as being specifically enriched within the cohort of AML patients with bi-allelic CEBPA mutations (Blood, 2016).We therefore explored whether these co-occurring mutations might synergize with CSF3R mutations in primary mouse bone marrow cells. We found that when WT CEBPA is co-expressed with the CSF3R T618I mutation, it strongly suppressed mouse bone marrow colony formation, while a CEBPA mutant enhanced colony formation. In addition, mutated SETBP1, which leads to protein overexpression, was co-expressed with CSF3R T618I. Interesting, WT SETBP1 enhanced colony formation, with a further increase in colony number with the SETBP1 G870S mutant. These studies suggest that there is a synergistic relationship between the CSF3R T618I mutation and two commonly co-mutated genes in CNL and pediatric AML. Since the CSF3R T618I mutation is very strongly activating, we wanted to use a model in which combinations of mutations could be expressed at endogenous levels to further study their interactions at the transcriptional level. To this end we are using CRISPR-Cas9 to generate myeloid cellular models. Two CRISPR-Cas9 gene-editing techniques are being employed to generate our mutations of interest following a Cas9-generated double-stranded break: (1) homology directed repair to insert point mutations into the CSF3R and SETBP1 loci, and (2) non-homologous end joining which results in frameshift mutations in CEBPA. We have completed validation of guide RNAs and HDR templates in 293T17 cells, with successful generation of both frameshift and specific point mutations. We have applied these tools to HL-60 cells, a human myeloid leukemia cell line, with successful isolation of clones with frameshift mutations. Investigation of these combinations of mutations in cell lines will allow us to determine how they cooperate to regulate transcriptional programs and produce distinct cellular identities. Our hope is that the principles learned from these well-defined diseases will provide a conceptual framework that will help us understand other myeloid malignancies with more complex clonal architecture and mutation combinations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Stanozolol and Danazol Have Different Effects on Hematopoiesis in the Murine Model of Immune-Mediated Bone Marrow Failure.

Background: Stanozolol and danazol are widely used in the treatment of aplastic anemia; however, their mechanisms of action are unclear.Methods: Bone marrow mononuclear cells from 10 patients newly diagnosed with aplastic anemia and 10 healthy volunteers were collected and cultured together with stanozolol, danazol, or blank control separately for marrow colony assays. K562 cell lines that had been incubated with stanozolol, danazol, or blank control were tested for erythroid or megakaryocytic differentiation. Meanwhile, CB6F1/Crl mice were injected with 1 × 106 C57BL/6 donor-originated lymphocytes after irradiation with 5 Gy total body irradiation to establish a model for immune-mediated bone marrow failure (aplastic anemia mouse model). Mice with aplastic anemia were treated with cyclosporin A monotherapy, cyclosporin A in combination with stanozolol, and cyclosporin A in combination with danazol for 30 days. Peripheral blood cell counts once a week and bone marrow colony assays at the end of 1 month were performed. The proportion of T cell subsets, level of inflammatory factors, erythropoietin, and thrombopoietin were detected before and after treatment. The levels of erythropoietin receptors on bone marrow mononuclear cells after treatment were tested using western blotting.Results: In the ex vivo experiments, the number of burst-forming units-erythroid; colony-forming units-granulocyte and macrophage; and colony-forming units-granulocyte, erythrocyte, monocyte, and megakaryocyte in the patients with aplastic anemia were significantly lower than that in the normal controls (P < 0.05). However, the number of colonies and mean fluorescence intensity of CD235a or CD41 expression in the harvested cultured cells were not significantly different among the different treatment groups in the patients with aplastic anemia, normal controls, and K562 cell lines. These results show that stanozolol and danazol produce no direct hematopoiesis-stimulating effects on progenitor cells. In the in vivo experiment, the mice with aplastic anemia treated with cyclosporin A and danazol exhibited the most rapid recovery of platelet; the platelet count returned to normal levels after 3 weeks of treatment, which was at least 1 week earlier than in the other groups. In contrast, mice treated with cyclosporin A and stanozolol exhibited the highest hemoglobin level at the end of treatment (P < 0.05). Bone marrow colony assays at 30 days showed that the number of burst-forming units-erythroid was the highest in mice treated with cyclosporin A and stanozolol, while the number of colony-forming units-granulocyte and macrophage was the highest in those treated with cyclosporin A and danazol. Compared to cyclosporin A monotherapy, additional stanozolol and danazol can both increase the level of regulatory T cells and upregulate interleukin-10, inhibiting the expression of tumor necrosis factor-α (P < 0.05). However, IL-2 was more effectively reduced by danazol than by stanozolol (P < 0.05). The cyclosporin A- and stanozolol-treated mice showed higher serum erythropoietin (corrected by hemoglobin level) and higher erythropoietin receptor levels in bone marrow mononuclear cells than the other groups (P < 0.05).Conclusions: Neither stanozolol nor danazol directly stimulated hematopoiesis in vitro. However, in vivo, stanozolol may exhibit an advantage in improving erythropoiesis, while danazol may induce stronger effects on platelets. Both danazol and stanozolol exhibited immunosuppressive roles. Stanozolol could enhance the secretion of erythropoietin and expression of erythropoietin receptor in bone marrow mononuclear cells.