Engraftment, fate, and function of HoxB8-conditional neutrophil progenitors in the unconditioned murine host

The development and use of murine myeloid progenitor cell lines that are conditionally immortalized through expression of HoxB8 has provided a valuable tool for studies of neutrophil biology. Recent work has extended the utility of HoxB8-conditional progenitors to the in vivo setting via their transplantation into irradiated mice. Here, we describe the isolation of HoxB8-conditional progenitor cell lines that are unique in their ability to engraft in the naïve host in the absence of conditioning of the hematopoietic niche. Our results indicate that HoxB8-conditional progenitors engraft in a β1 integrin-dependent manner and transiently generate donor-derived mature neutrophils. Furthermore, we show that neutrophils derived in vivo from transplanted HoxB8-conditional progenitors are mobilized to the periphery and recruited to sites of inflammation in a manner that depends on the C-X-C chemokine receptor 2 and β2 integrins, the same mechanisms that have been described for recruitment of endogenous primary neutrophils. Together, our studies advance the understanding of HoxB8-conditional neutrophil progenitors and describe an innovative tool that, by virtue of its ability to engraft in the naïve host, will facilitate mechanistic in vivo experimentation on neutrophils.

Modeling human yolk sac hematopoiesis with pluripotent stem cells

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac–derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

Increased Expression of CD56 on Plasmacytoid Dendritic Cells in Myeloid Leukemia Associated with Down Syndrome (ML-DS) Is Normal Regeneration and Not Measurable Residual Disease

Introduction: It has long been known that Down syndrome is associated with an increased risk for hematologic malignancies. One such disease is myeloid leukemia associated with Down syndrome (ML-DS), a disease that almost always occurs during the first 5 years of life. As research on ML-DS has progressed, understanding has grown that after the initiation of therapy, the non-neoplastic myeloid progenitor cells in ML-DS patients have a characteristic immunophenotype with the expression of CD56 on a subset of the CD34+ myeloid progenitor cells being one of the most notable features. The discovery that this immunophenotype is normal in ML-DS patients post-therapy has been of the utmost importance as it has led to such patients being properly classified as being negative for measurable residual disease. Plasmacytoid dendritic cells (pDCs) are another cell type in which CD56 expression is often part of the neoplastic immunophenotype, and we hypothesized that CD56 may also be differentially expressed in ML-DS pDCs post-therapy. Herein, we investigated the immunophenotype of pDCs in ML-DS patients found to be negative for measurable residual disease. Methods: A total of 10 bone marrow specimens from ML-DS patients post-treatment initiation and 7 bone marrow specimens from patients that did not have DS, were aged 0-4 years (matching the age range of ML-DS), had a myeloid neoplasm and were post-treatment initiation (non-DS) were included in this study. All specimens were found to be negative for measurable residual disease by difference from normal (ΔN) flow cytometry (the gold standard for the determination of residual disease in the Children's Oncology Group 1531 study on ML-DS) and were evaluated for CD56 and CD303 expression on pDCs. pDCs were defined as HLA-DR+/CD123++ (high intensity). Results: As expected, the ML-DS patients had a significantly greater percentage of CD34+CD56+ myeloid progenitor cells than the non-DS group, both in terms of percent total non-erythroid cells (0.9% vs 0.006%, P<0.001) and percent total myeloid progenitors (38% vs 0.57%, P<0.001). There was not a significant difference between groups in terms of pDC percentage (ML-DS 0.63% of total non-erythroid cells vs non-DS 0.53%, P=0.9%). There were also no significant differences in CD303 expression between the groups, both in terms of percent positive (ML-DS 89% vs non-DS 92%, P=0.5) and mean fluorescence intensity (MFI, in PE) (ML-DS 190 vs non-DS 246, P=0.3). On the other hand, the ML-DS group had significantly greater CD56 expression than the non-DS group, both in percent positive (74% vs 25%, P=0.005) and MFI (PE) (122 vs 5.9, P=0.005). Nine of the 10 ML-DS specimens had CD56 expression on greater than 50% of pDCs, and 3 showed a CD56 MFI of over 200. Conclusions: The data from this preliminary study indicate that much like the myeloid progenitor cells, the pDCs in ML-DS patients after the initiation of therapy have an immunophenotype that could be mistaken as abnormal. Importantly, they show that the setting of cutoff values for the determination of abnormal pDC CD56 expression, even relatively high ones, could lead to false positive results in ML-DS specimens post-treatment initiation. The dissemination of this knowledge is of increased importance as more flow cytometry laboratories begin to increase their investigation of pDCs. Further studies are needed to delineate common mechanisms between the expression of CD56 in myeloid progenitor cells and pDCs in ML-DS patients post-treatment initiation. Disclosures Pardo: Hematologics, Inc.: Current Employment. Lott: Hematologics, Inc.: Current Employment. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership.

Clonal hematopoiesis, myeloid disorders and BAX-mutated myelopoiesis in patients receiving venetoclax for CLL

The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia. As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment for CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (³ 1 of neutropenia, thrombocytopenia, anemia) lasting ³4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (tMNs). tMNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with tMNs. Single cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.

DDX41 is needed for pre-and post-natal hematopoietic stem cell differentiation in mice

DDX41 is a tumor suppressor frequently mutated in human myeloid neoplasms. DDX41 binds to DNA/RNA hybrids and interacts with spliceosome components. How it affects hematopoiesis is still unclear. Using a knockout mouse model, we demonstrate that DDX41 is required for mouse hematopoietic stem and progenitor cell (HSPC) survival and differentiation. Lack of DDX41 particularly affected myeloid progenitor development, starting at embryonic day 13.5. Transplantation of DDX41-deficient fetal liver and adult bone marrow (BM) cells were unable to rescue mice from lethal irradiation after transplantation. DDX41 knockout stem cells were also defective in ex vivo colony forming assays. RNASeq analysis of lineage-negative, cKit+Sca1+ cells isolated from fetal liver demonstrated that the expression of many genes associated with hematopoietic differentiation were altered in DDX41 knockout cells. Furthermore, altered splicing of genes involved in key biological processes were observed in cells lacking DDX41. Our data reveal a critical role for DDX41 in HSPC differentiation and myeloid progenitor development, likely through its regulation of gene expression programs and splicing.

An abundant myeloid progenitor shapes neonatal hematopoiesis of naked mole-rats

The naked mole-rat became a significant animal model for its exceptional longevity and resistance mechanisms to cancers, however the developmental properties of their native stem and progenitor compartments are poorly understood. Here we report a high frequency bone marrow progenitor exclusively present in neonatal animals. The heterotypic developmental stage was marked by an immunophenotype resembling adult naked mole-rat CMP-like progenitors with mature neutrophilic Thy1.1-antigen levels and showed a clear differentiation bias and transcriptome signature towards myelopoiesis. Importantly, myeloid progenitors overexpressed both CXCL12 and its alternative receptor CXCR7, thereby abrogating their homing capacity and produced multiple secreted matrix components related to neonatal bone marrow niche formation. These results indicate a transient ontogenic cell stage during postnatal hematopoiesis unique to naked mole-rats and further advances our understanding of the developmental steps involved in creating the regenerative cell pool and its environment in a long-lived species.

Open-Label Phase II Prospective, Randomized, Controlled Study of Romyelocel-L Myeloid Progenitor Cells to Reduce Infection During Induction Chemotherapy for Acute Myeloid Leukemia

PURPOSE Standard cytotoxic induction chemotherapy for acute myeloid leukemia (AML) results in prolonged neutropenia and risk of infection. Romyelocel-L is a universal, allogeneic myeloid progenitor cell product being studied to reduce infection during induction chemotherapy. PATIENTS AND METHODS One hundred sixty-three patients with de novo AML (age ≥ 55 years) receiving induction chemotherapy were randomly assigned on day 0 (d0), of whom 120 were evaluable. Subjects received either romyelocel-L infusion on d9 with granulocyte colony-stimulating factor (G-CSF) starting daily d14 (treatment group) or G-CSF daily alone on d14 (control) until absolute neutrophil count recovery to 500/µL. End points included days in febrile episode, microbiologically defined infections, clinically diagnosed infection, and days in hospital. RESULTS Mean days in febrile episode was shorter in the treatment arm from d15 through d28 (2.36 v 3.90; P = .02). Similarly, a trend toward decreased microbiologically defined infections and clinically diagnosed infection in the treatment arm was observed from d9 to d28 (35.6% v 47.5%; P = .09), reaching a statistically significant difference from d15 to d28 (6.8% v 27.9%; P = .002). Because of this, antibacterial or antifungal use for treatment of an infection was significantly less in the treatment group (d9-d28: 44.1% v 63.9%; P = .01). Significantly fewer patients in the treatment arm received empiric antifungals from d9 tod28 (42.4% v 63.9%; P = .02) and d15-d28 (42.4% v 62.3%; P = .02). Patients in the treatment arm also had 3.2 fewer hospital days compared with control (25.5 v 28.7; P = .001). Remission rates and days to absolute neutrophil count recovery were similar in the two groups. No patients in the romyelocel-L plus G-CSF group died because of infection compared with two patients in the control arm. No graft-versus-host disease was observed. CONCLUSION Subjects receiving romyelocel-L showed a decreased incidence of infections, antimicrobial use, and hospitalization, suggesting that romyelocel-L may provide a new option to reduce infections in patients with AML undergoing induction therapy.

Clastogenicity and Aneugenicity of 1,4-Benzoquinone in Different Lineages of Mouse Hematopoietic Stem/Progenitor Cells

Previous reports on hematotoxicity and leukemogenicity related to benzene exposure highlighted its adverse effects on hematopoiesis. Despite the reported findings, studies concerning the mechanism of benzene affecting chromosomal integrity in lineage-committed hematopoietic stem/progenitor cells (HSPCs) remain unclear. Here, we studied the clastogenicity and aneugenicity of benzene in lineage-committed HSPCs via karyotyping. Isolated mouse bone marrow cells (MBMCs) were exposed to the benzene metabolite 1,4-benzoquinone (1,4-BQ) at 1.25, 2.5, 5, 7, and 12 μM for 24 h, followed by karyotyping. Then, the chromosomal aberration (CA) in 1,4-BQ-exposed hematopoietic progenitor cells (HPCs) comprising myeloid, Pre-B lymphoid, and erythroid lineages were evaluated following colony-forming cell (CFC) assay. Percentage of CA, predominantly via Robertsonian translocation (Rb), was increased significantly (p < 0.05) in MBMCs and all progenitors at all concentrations. As a comparison, Pre-B lymphoid progenitor demonstrated a significantly higher percentage of CA (p < 0.05) than erythroid progenitor at 1.25, 2.5, and 7 μM as well as a significantly higher percentage (p < 0.05) than myeloid progenitor at 7 μM of 1,4-BQ. In conclusion, 1,4-BQ induced CA, particularly via Rb in both MBMCs and HPCs, notably via a lineage-dependent response. The role of lineage specificity in governing the clastogenicity and aneugenicity of 1,4-BQ deserves further investigation.

The genetic abnormalities investigation of leukemia in past few decades

Cytogenetic and molecular analysis of each patient’s leukemia cells has become an essential component of diagnosis prior to treatment. It has furthered our understanding of leukemogenesis at a molecular level. Specific and well-characterized recurring chromosomal abnormalities facilitate diagnosis, confirm subtype classification, and have major prognostic value for treatment planning. Conventional chromosome analysis is a basic way for diagnosis and treatment. In addition in this way evaluation of disease progression is important and so it is the only method that can identify the presence of clonal evolution, particularly in accelerated and relapse phase in the disease. Also conventional cytogenetic can detect chromosomal abnormality associated with its advanced phase. Anyhow, the value of translocation rates in interphase and metaphase nuclei in monitoring leukemia is at the time of diagnosis and after treatment additionally. Genomic profiling transformed our understanding of the genetic basis of leukemia particularly in acute leukemia, which is a malignant clonal proliferation in lymphoid stem cells or myeloid progenitor cells.