Multiple Hematopoietic Lineages Research Articles

Caebv Disease Originates from Infected Hematopoietic Stem Cells

Background Chronic active Epstein-Barr Virus (EBV) disease (CAEBV) is lethal syndrome due to persistent EBV infection. When diagnosed as CAEBV, EBV infection was observed in multiple hematopoietic lineages, but the etiology of CAEBV is still elusive. Methods Bone marrow and peripheral cells derived from five CAEBV patients, one EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) patient, and two healthy controls were analyzed. Multiple assays were applied to identify and characterize EBV-infected cells, including quantitative PCR (qPCR), PrimeFlow and single-cell RNA-sequencing (scRNA-seq). Based on scRNA-seq data, alterations in gene expression of particular cell types were analyzed between CAEBV patients and controls, and between infected and uninfected cells. One CAEBV patient was treated with allogeneic hematopoietic stem cell transplantation (HSCT), and the samples derived from this patient were analyzed again six month after HSCT. Results EBV infected the full spectrum of the hematopoietic system including both lymphoid and myeloid lineages, so as hematopoietic stem cells (HSCs) in the CAEBV patients. EBV-infected HSCs exhibited a higher differentiation rate towards downstream lineages, and the EBV infection had an impact on both the innate and adaptive immunity, resulting in inflammatory symptoms. EBV infected cells were thoroughly removed from the hematopoietic system after HSCT. Conclusions Token together,multiple lines of evidence presented in this study suggest that CAEBV disease originates from the infected hematopoietic stem cells. Figure 1. EBV-infected HSCs were detected in CAEBV patients with multiple assays.

Chronic active Epstein-Barr virus disease originates from infected hematopoietic stem cells

AbstractChronic active Epstein-Barr virus (EBV) disease (CAEBV) is a lethal syndrome because of persistent EBV infection. When diagnosed as CAEBV, EBV infection was observed in multiple hematopoietic lineages, but the etiology of CAEBV is still elusive. Bone marrow and peripheral cells derived from 5 patients with CAEBV, 1 patient with EBV-associated hemophagocytic lymphohistiocytosis, and 2 healthy controls were analyzed. Multiple assays were applied to identify and characterize EBV-infected cells, including quantitative polymerase chain reaction, PrimeFlow, and single-cell RNA-sequencing (scRNA-seq). Based on scRNA-seq data, alterations in gene expression of particular cell types were analyzed between patients with CAEBV and controls, and between infected and uninfected cells. One patient with CAEBV was treated with allogeneic hematopoietic stem cell transplantation (HSCT), and the samples derived from this patient were analyzed again 6 months after HSCT. EBV infected the full spectrum of the hematopoietic system including both lymphoid and myeloid lineages, as well as the hematopoietic stem cells (HSCs) of the patients with CAEBV. EBV-infected HSCs exhibited a higher differentiation rate toward downstream lineages, and the EBV infection had an impact on both the innate and adaptive immunity, resulting in inflammatory symptoms. EBV-infected cells were thoroughly removed from the hematopoietic system after HSCT. Taken together, multiple lines of evidence presented in this study suggest that CAEBV disease originates from the infected HSCs, which might potentially lead to innovative therapy strategies for CAEBV.

Novel Approaches and Future Directions in Myelodysplastic Syndrome Treatment.

Myelodysplastic syndromes/neoplasms (MDSs) constitute a heterogeneous group of clonal disorders that are clinically characterized by dysplastic changes in multiple hematopoietic lineages, cytopenias, and a variable risk of progression to acute myeloid leukemia. Patients with MDS are classified as either lower- or higher-risk based on risk stratification tools such as the International Prognostic Scoring System and its revised version, which continue to be the basis for prognosis and treatment selection. Although anemic patients with lower-risk MDS are currently treated with an erythropoiesis-stimulating agent, luspatercept, and transfusions, the telomerase inhibitor imetelstat and the hypoxia-inducible factor α inhibitor roxadustat have shown encouraging early results and are now in phase III clinical trials. For higher-risk MDS patients, hypomethylating agent monotherapy continues to be the standard of care. However, with various novel hypomethylating agent-based combination therapies in advanced clinical testing and an increased emphasis on individualized biomarker-driven treatment decisions, the standard therapy paradigms might change in the future.

GATA1 controls numbers of hematopoietic progenitors and their response to autoimmune neuroinflammation

Abstract GATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b +myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge. This work was supported by a grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (T32 training grant T32AI134646).

Impact of Therapy on the Anamnestic Response to Seasonal Influenza Vaccination in Patients with Myeloid Malignancies

Myelodysplastic syndromes (MDS) are diseases of bone marrow failure that result in dysplasia of multiple hematopoietic lineages producing anemia, thrombocytopenia, and increased risk of infections. While progression to acute myeloid leukemia (AML) is a phenomenon that receives most of the focus in managing this disease, prior studies have shown that infection is the leading cause of mortality. This is true across all MDS risk groups and highlights the critical importance of research into the clinical management and prevention of infection in patients with MDS, especially in the age of emergent pandemics such as COVID-19. We have previously observed that patients with MDS exhibit decreased responses to vaccination against SARS-CoV-2 (Bellusci, et al., Blood, 2022). There remains a gap in our knowledge regarding the success of vaccination against common seasonal pathogens in patients with MDS, such as influenza. Although patients with MDS and AML are routinely offered yearly influenza vaccination during the flu season, the efficacy of vaccination in producing immunity in the context of the underlying disease biology and the impact of therapies used for treatment of the disease (azacitidine & decitabine (HMAs), venetoclax + HMA and cytarabine based intensive chemotherapy) remains understudied. We hypothesized that MDS/AML patients would exhibit a decreased response to vaccination against seasonal influenza compared to healthy age-matched controls. To test this hypothesis, we enrolled patients in an IRB-approved non-randomized study (I-54017) that spanned the 2017-2018 to 2020-2021 influenza seasons. This study examined 5 patient groups: Cohort 1: Patients with MDS on best supportive care (ESAs, growth factors, transfusions, antibiotics; no disease modifying therapy; n = 28); Cohort 2: Patients with MDS or AML early in HMA therapy (≤5 cycles; n = 26); Cohort 3: Patients with MDS or AML on longer term HMA (> 6 cycles; n = 28); Cohort 4: Patients with AML receiving cytarabine based intensive chemotherapy ("7+3", HIDAC, CLAG+/-M, FLAG+/-Ida, etc.). Patients in this arm should not have received an HMA in the 6 months prior to enrollment (n = 10); Cohort 5: Healthy volunteers of similar age profile without antecedent or concurrent hematologic disorders (n = 12). All subjects received the yearly Fluzone High-Dose vaccine (Sanofi). Serum samples were collected at 1) baseline; 2) days 25-95 after vaccination; and 3) days 115-185 after vaccination. Viral titers were measured using the viral microneutralization assay against seasonal influenza vaccine strains based upon the yearly vaccine product. Positive responses to vaccination were defined as a 4-fold increase in titer compared to baseline. For each subject, we calculated a strain score (range 0-1) based on the fraction of 4-fold changes across either 3 to 4 influenza strains depending upon the yearly vaccination product; a score of 1 indicates a 4-fold increase in titers against all tested strains. Strain score was modeled as a function of study year, disease cohort, and age; blood-type, gender, race, and IPSS-M were analyzed but were not statistically significant. The primary analysis was a generalized linear model (GLM). In concert with prior reports, we found that compared to healthy donors, AML patients receiving intensive chemotherapy showed decreased vaccine response (Fig.1). In contrast, those with MDS responded equivalently to vaccination compared with healthy donors. Overall, these results demonstrate that in contrast with vaccination against newly emergent diseases (COVID-19) against which MDS patients appear to have inferior immune response, anamnestic responses to vaccination against previously encountered pathogens (influenza) are normal. As reported previously, patients with AML receiving intensive chemotherapy exhibit reduced protection from vaccination against both emergent pathogens (COVID-19) and amnestic vaccines (influenza). These results indicate that vaccination boosters, such as those given yearly against influenza, against previously encountered pathogens are likely to provide protection in MDS patients irrespective of standard therapy, but the efficacy of vaccination against novel pathogens may be less effective. These data highlight the complex immune milieu in patients with MDS and emphasize the importance of careful individualized vaccination strategies in those with hematological malignancy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pharmacological Disruption of the Interplay between HDM2-p53 and NFκB Depletes Myelofibrosis Progenitor/Stem Cells and Dampens the Accompanying Inflammatory Milieu

We and others have observed that myelofibrosis (MF) hematopoietic stem/progenitor cells (HSC/HPCs) are characterized by upregulation of HDM2 which promotes the degradation of wild type p53 (1-4). NFκB which serves as a transcription factor (TF) for many inflammatory cytokines and promotes resistance to programmed cell death, and HIF1α, the master TF for cellular and developmental responses to hypoxia, with implied roles in angiogenesis and MF progression (5) was also upregulated. Using RT-qPCR, we determined that a correlation existed between increased transcripts of HDM2, HIF1α and NFkB and decreased transcripts of p53. Furthermore, transcripts of cytokines (LCN2, IL8 and VEGF) that are known to be regulated by HIF1α and NFkB were elevated in MF CD34+ cells as compared to normal donor (ND) CD34+ cells (p-value <0.05 for each gene). These findings indicate that the interplay between HDM2/p53, HIF1α and NFκB pathways play a role in the resistance of MF CD34+ cells to apoptosis and the generation of MF pro-inflammatory cytokines. The p53 and NFκB target genes play crucial roles in cell proliferation and survival and cancer progression. BET (Bromodomain and Extra-Terminal domain) protein family inhibitor (BETi) studies have implicated the BET protein member BRD4 in NFκB-dependent promoter and super-enhancer modulation. In order to develop a therapeutic regimen that would deplete MF CD34+ and dampen the MF pro-inflammatory milieu, we evaluated the effects of a combination of an HDM2 antagonist (HDM2a), which results in increased p53 activity, with a BETi, which repressed NFκB activity, on depletion of MF HSPCs. Treatment with either an HDM2a or BETi alone increased apoptosis of MF CD34+ cells. Combination of these the two drugs (HDM2a+BETi) led to an even greater degree of apoptosis (p=0.01 and p=0.02, respectively). RT-qPCR data showed that treatment with HDM2a increased the transcripts of downstream p53 targets including p21 and PUMA in a dose dependent fashion while HDM2a+BETi increased both genes to an even greater extent (p<0.01). The ability of these drugs to eliminate MF HSPCs was evaluated with 10 MF and 5 ND samples using colony formation assays. Treatment with either drug alone or in combination displayed limited effects on colony formation by ND CD34+ cells. By contrast, treatment with HDM2a or BETi alone decreased MF colony formation by 35% and 50% respectively, while the HDM2a+BETi combination led to an 80% reduction in MF colony numbers. JAK2V617F genotyping revealed that treatment with either drug alone decreased JAK2V617F+ colony number by 21.3% and 47%, respectively, while the combination decreased JAK2V617F+ colonies by 64% as compared to control cells. To evaluate the ability of these drugs to eliminate MF HSCs, an immunocompromised NSGS mouse (expressing human IL3, GM-CSF and SCF) xenograft transplantation model was used. MF CD34+ cells were pretreated with vehicle, HDM2a or BETi alone or HDM2a+BETi for 3 days, followed by transplantation into NSGS mice. After 12 weeks, mice were sacrificed and the degree of human cells chimerism in the bone marrows (BM) and spleens of recipient mice evaluated. As compared to vehicle treatment, the mice receiving HDM2a treated grafts had on average 40% less hCD45+ cell chimerism in both organs. BETi treated grafts led to on average a 20% decrease in hCD45+ cells chimerism in the BM but surprisingly not the spleen (Figure 1A). Mice receiving grafts treated with HDM2a+BETi exhibited on average a 60% reduction in hCD45+ cell chimerism in the BM and spleen. Moreover, the grafts treated with HDM2a+BETi generated reduced numbers of cells belonging to multiple hematopoietic lineages (CD34+, CD33+ and CD41+ cells) in both organs of NSGS as compared to mice receiving grafts pretreated with either drug alone indicating that the HDM2a+BETi combination is effective in depleting MF HSCs (Figure 1). Furthermore, treatment of MF CD34+ cells with WT p53 with an HDM2a or BETi decreased activation of the NFkB pathway, and IL8 and VEGF transcript levels. In addition, treatment of MF CD34+ cells co-cultured with ND mesenchymal stem cells and endothelial cells with the HDM2a+BETi combination led to a 50% reduction in protein levels of IL8, VEGF in the culture supernatants. These results indicate that combinations of pharmacological agents that disrupt the interplay between HDM2/p53, HIF1α and N-κB pathways could serve as effective therapeutic strategies to treat MF patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GATA1 controls numbers of hematopoietic progenitors and their response to autoimmune neuroinflammation

AbstractGATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b+ myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge.

EE30 Economic Impact of Trilaciclib for Chemotherapy-Induced Myelosuppression (CIM) in Extensive-Stage Small Cell Lung Cancer (ES-SCLC): Economic Evaluation from the Provider and Patient-Caregiver Perspectives in the United States

Commonly manifested as neutropenia, anemia, and thrombocytopenia, CIM is a major complication of systemic chemotherapy. We evaluated the economic impact of trilaciclib, a first-in-class breakthrough therapy that protects multiple hematopoietic lineages simultaneously against CIM in adult patients with ES-SCLC receiving platinum/etoposide- or topotecan-containing chemotherapy from both US provider and patient-caregiver perspectives.

NuRD complex recruitment to Thpok mediates CD4+ T cell lineage differentiation.

Although BTB-zinc finger (BTB-ZF) transcription factors control the differentiation of multiple hematopoietic and immune lineages, how they function is poorly understood. The BTB-ZF factor Thpok controls intrathymic CD4+ T cell development and the expression of most CD4+ and CD8+ lineage genes. Here, we identify the nucleosome remodeling and deacetylase (NuRD) complex as a critical Thpok cofactor. Using mass spectrometry and coimmunoprecipitation in primary T cells, we show that Thpok binds NuRD components independently of DNA association. We locate three amino acid residues within the Thpok BTB domain that are required for both NuRD binding and Thpok functions. Conversely, a chimeric protein merging the NuRD component Mta2 to a BTB-less version of Thpok supports CD4+ T cell development, indicating that NuRD recruitment recapitulates the functions of the Thpok BTB domain. We found that NuRD mediates Thpok repression of CD8+ lineage genes, including the transcription factor Runx3, but is dispensable for Cd4 expression. We show that these functions cannot be performed by the BTB domain of the Thpok-related factor Bcl6, which fails to bind NuRD. Thus, cofactor binding critically contributes to the functional specificity of BTB-ZF factors, which control the differentiation of most hematopoietic subsets.

Hematopoietic Stem and Progenitor Cell Maintenance and Multiple Lineage Differentiation Is an Integral Function of NFATc1.

Hematopoietic stem and progenitor cell (HSPC) maintenance and the differentiation of various lineages is a highly complex but precisely regulated process. Multiple signaling pathways and an array of transcription factors influence HSPC maintenance and the differentiation of individual lineages to constitute a functional hematopoietic system. Nuclear factor of activated T cell (NFAT) family transcription factors have been studied in the context of development and function of multiple mature hematopoietic lineage cells. However, until now their contribution in HSPC physiology and HSPC differentiation to multiple hematopoietic lineages has remained poorly understood. Here, we show that NFAT proteins, specifically NFATc1, play an indispensable role in the maintenance of HSPCs. In the absence of NFATc1, very few HSPCs develop in the bone marrow, which are functionally defective. In addition to HSPC maintenance, NFATc1 also critically regulates differentiation of lymphoid, myeloid, and erythroid lineage cells from HSPCs. Deficiency of NFATc1 strongly impaired, while enhanced NFATc1 activity augmented, the differentiation of these lineages, which further attested to the vital involvement of NFATc1 in regulating hematopoiesis. Hematopoietic defects due to lack of NFATc1 activity can lead to severe pathologies such as lymphopenia, myelopenia, and a drastically reduced lifespan underlining the critical role NFATc1 plays in HSPC maintenance and in the differentaion of various lineages. Our findings suggest that NFATc1 is a critical component of the myriad signaling and transcriptional regulators that are essential to maintain normal hematopoiesis.

Peripheral Blood Single-Cell Genotyping and Phenotyping in Multiple Myeloma Reveals Shared Mutations across Multiple Hematopoietic Cell Lineages

Background:Multiple myeloma (MM) is a hematologic malignancy of terminally differentiated plasma cells. While most genomic studies of the disease have focused on characterizing isolated bone marrow plasma cells, few have addressed somatic mutations within the immune microenvironment. We previously demonstrated that somatic mutations are significantly more numerous in unsorted blood compared to bone marrow and that the number of mutations correlated with elevated markers of disease. We also reported that genes implicated in clonal hematopoiesis of indeterminate potential (CHIP) are commonly mutated in the blood from patients with MM.Aim:To integrate single-cell DNA mutation analysis and protein expression to map the clonal relationship between circulating plasma cells and non-plasma cells within the peripheral blood of patients with MM.Methods:Although bulk DNA sequencing can estimate clonal heterogeneity using analytic deconvolution techniques, it is unable to distinguish which mutations occur in the same clone(s) nor can it correlate genotype with immunophenotype from the same cell. To accurately describe the clonal architecture of the blood, we combined targeted single-cell DNA sequencing (scDNA-seq) coupled with protein expression to define mutations specific to cell phenotypes using the Tapestri Platform (Mission Bio). For this analysis, we developed a custom amplicon panel covering 22 frequently mutated genes in MM and/or CHIP to perform scDNA-seq and 4 antibodies targeting CD138, CD19, CD3, and CD33 for immunophenotyping or plasma cells, B cells, T cells, and myeloid cells respectively.Results:Peripheral blood mononuclear cells were isolated from 17 blood samples collected from 12 patients with MM. Three samples were collected at diagnosis before treatment, 8 were collected post-treatment during remission, and 6 were collected at relapse. Serial samples were obtained before and after autologous stem cell transplant from 4 patients. A total of 85,262 cells were sequenced uncovering 154 unique variants in 22 genes after quality filtering. There were 47 somatic variants, defined as those present in less than 40% of cells per sample. There were 52,136 total clones, defined as cells with identical genotypes, within this heterogenous population of cells. The most common non-synonymous somatic mutations were identified in genes SETD2, KMT2C, PPM1D. An immunophenotype was assigned to 82% of cells according to the protein with the most abundant number of reads. Dimensionality reduction of single-cell genotype using t-distributed stochastic neighbor embedding (t-SNE) effectively clustered cells according to sample and by patient in cases where there were multiple samples from the same individual. Patients with active myeloma and known circulating plasma cells detected by clinical flow cytometry were found to have the greatest proportion of CD138+ cells as well as the highest mutational burden. It was rare to detect the same clone in two separate samples collected from the same patient at different time points (median time be collection was 248 days) which may be due to under-sampling or clonal evolution. Surprisingly, all somatic variants detected in plasma cells, were also present in other cell types suggesting they were acquired from a common progenitor cell rather than after differentiation.Conclusions:scDNA-seq coupled with immunophenotyping of the peripheral blood of patients with MM reveals somatic variants are shared across multiple hematopoietic cell lineages including plasma cells indicating these mutational are acquired from a common progenitor. DisclosuresDurruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Wang: Mission Bio, Inc: Current Employment.

Comparative Cost-Efficiency Analysis of Trilaciclib, a Novel CDK4/6 Inhibitor, in the Prophylaxis of Chemotherapy-Induced Myelosuppression

Introduction. Trilaciclib (Cosela TM), a first-in-class breakthrough therapy, is a novel kinase CDK4/6 inhibitor that protects multiple hematopoietic lineages simultaneously by transiently arresting hematopoietic stem and progenitor cells (HSPC) in the G1 phase of the cell cycle and thus protecting HSPCs from damage by cytotoxic chemotherapy. Administered intravenously prior to the start of chemotherapy, it proactively delivers multilineage myeloprotection and therefore has the potential to improve the 3 major clinical manifestations of chemotherapy-induced myelosuppression (CIM): neutropenia, anemia, and thrombocytopenia. Trilaciclib was approved by the FDA in February 2021 and included in the NCCN Guidelines® in March 2021 for managing CIM in patients with extensive-stage small cell lung cancer (ES-SCLC) treated with a platinum/etoposide-containing or topotecan-containing regimen. As an alternative to single lineage-specific interventions with biological agents for neutropenia ([peg]filgrastim, ), anemia ([darb]epoetin alfa, alfa), and thrombocytopenia (romiplostim), trilaciclib may offer economic value in the prophylaxis and/or management of these adverse events. Cost-efficiency analysis evaluates the costs of various scenarios of delivering treatments to identify the most efficient cost-structure contributing to the value equation. The objective of this cost-efficiency analysis was to determine the savings (losses) in drug costs of managing 1 patient at risk for concurrent neutropenia and anemia and thrombocytopenia (NAT), neutropenia and anemia (NA), neutropenia and thrombocytopenia (NT), or anemia and thrombocytopenia (AT) with trilaciclib as opposed to any combination of biologicals, including biosimilars.Methods. Drug cost inputs included the wholesale acquisition cost (WAC; per REDBOOK; all agents) and the average sales price (ASP; per Centers for Medicare and Medicaid Services; all agents except trilaciclib as no ASP is available yet). An average cost was calculated for biosimilars. Dosing schedules were based on the maximum possible dose therapy in the NCCN Guidelines®. An Excel model was developed to calculate, for the 4 scenarios of CIM and the 32 WAC and ASP cost inputs across all agents, the drug cost for every possible combination of originator and biosimilar agents for 1 patient. These estimates were compared to the WAC of trilaciclib to determine the differential cost-efficiency of trilaciclib in terms of savings ($) accrued or losses (-$) incurred. This analysis focused on treatment with biological agents recommended in the NCCN Guidelines®. It did not consider platelet and red blood cell transfusion for thrombocytopenia and hence is limited to patients for whom transfusion is contra-indicated, physician decision to use romiplostim, or patient preference.Results. Table 1 presents the lowest, highest, mean, and median WAC and ASP differentials by chemotherapy-induced myelosuppression scenario. Mean and median differentials indicated cost savings with trilaciclib use across all chemotherapy-induced myelosuppression scenarios, with the exception of the ASP (with trilaciclib WAC) NA scenarios where losses were noted. Depending on single agents needed, savings ranged from $485.57 (AT ASP) to $9,531.60 (NAT WAC) per patient; with mean/median savings ranging from $646.36/$533.81 (AT ASP) to $7,397.69/$7,358.31 per patient.Conclusions. Myeloprotection with trilaciclib is a cost-efficient intervention when compared to managing individual or multiple myelosuppressive events with single-lineage, supportive care therapies. Unsurprisingly, costing conclusions are affected by the selection of an appropriate price benchmark. ASP tends to fluctuate more than WAC and is typically lower hence an ASP-based cost model may likely underrepresent the cost-efficiency of trilaciclib, as may be the case in the neutropenia/anemia scenario.(Data will be updated by conference time with the trilaciclib ASP if available). [Display omitted] DisclosuresMcBride: BMS: Current Employment. Henry: CVS Health: Current Employment. MacDonald: Matrix45, LLC: Current holder of individual stocks in a privately-held company. Abraham: Matrix45, LLC: Current holder of individual stocks in a privately-held company. OffLabel Disclosure:This presentation will discuss the use of romiplostim for the management of chemotherapy induced thrombocytopenia, an indication for which it has not received FDA approval.

Talen Mediated Therapeutic Gene Editing Strategy for ß-Hemoglobinopathies

Hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are the most common single-gene disorders in the world and represent a major global public health concern. The unifying principle of this heterogeneous mix of gene mutations is the decreased production of wild type hemoglobin molecules either due to structural defects in the case of SCD or insufficient production of β-hemoglobin subunits. Hemoglobinopathy patients with gene variants that induce expression of fetal (γ) hemoglobin often exhibit a less severe phenotype. Recent evidence has demonstrated that the in vitro introduction of a previously described naturally occurring 13bp deletion in the promoter of the Aγ globin gene can reconstitute the induction of fetal hemoglobin seen in patients with this mutation. As temporary expression of endonuclease is a key element of a clinically viable editing technique, we have generated a set of TALENs (Transcription Activator-Like Effector Nucleases) targeting the 13bp deletion site that are transfected as mRNA. In human CD34 cells, transfection with and expression of the TALEN pair results in INDEL generation at both the Aγ and Gγ globin loci confirmed by sequencing, T7 analysis and ddPCR. INDEL generation occurs in both the Aγ and Gγ globin promoters with slightly higher rates at the Aγ globin locus (~53%) compared to the Gγ globin locus (~45%) (Figure 3). Compared to control cells, edited CD34+ cells placed in erythroid differentiation media result in an increased expression of fetal hemoglobin with a near doubling in the frequency of ‘F-Cells’ by flow cytometry and a threefold increase in γ globin protein detected by HPLC (Figure 1 & 2). These findings support a model in which TALEN induced targeted INDELs in the γ globin promoter are capable of de-repressing fetal hemoglobin in hematopoietic progenitor cells. d13 TALEN edited CD34 cells have also been transplanted into NSG-W41 mice, a strain demonstrated to be more permissive of human erythropoiesis and are capable of high rates of engraftment with minimal conditioning (66-95% human chimerism at 14 weeks). An analysis of the INDEL rates retained by the engrafted cell populations 14 weeks post-transplantation demonstrates an average gene editing retention rate of approximately 50% (individual animals ranged from 15-77%) (Figure 4). These edits are retained in multiple hematopoietic cell lineages from each mouse suggesting that editing is occurring at least in the hematopoietic progenitor population and does not rule out the possibility of edited long term repopulating stem cells. A unique feature of targeted gene editing at this locus is that therapeutic benefit can be theoretically derived both from INDEL generation as well as the introduction of synthetic sequence via the homologous repair pathway. AAV transfected homologous recombination templates have been generated with a goal of further manipulating hemoglobin expression and allowing for the selection of edited cells. A basic GFP selection template has been successfully integrated at the target site (approximately 27% efficiency) resulting in erythroid and myeloid methylcellulose progenitor colonies detectable by fluorescence microscopy. Other repair templates that either introduce the specific 13bp deletion γ-hemoglobin promoter or elements of the β-hemoglobin promoter result in higher rates of HbF expression in GFP+ cells. A T87Q anti-sickling β hemoglobin construct has also been successfully integrated and expressed in human CD34 cells resulting in both an increase in HbF expression as well as the presence of T87Q hemoglobin making up a combined 60% of the total β hemoglobin proteins expressed in edited cells. Continued efforts are underway to optimize repair template designs, editing efficiencies and selection strategies to prepare this approach for clinical applications. [Display omitted] DisclosuresScharenberg:Casebia: Employment.

JAK inhibition in early-onset somatic, nonclonal STAT5B gain-of-function disease

Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathways mediate the signaling of multiple cytokines for a myriad of leukocyte functions.1 Somatic gain-of-function (GOF) mutations in STAT5B, especially N642H, are described in multiple neoplasms, in particular leukemia and lymphoma, and can be associated with significant eosinophilia.2-5 A different phenotype for somatic STAT5B N642H mutations has recently been observed whereby a nonmalignant process affects multiple hematopoietic lineages and presents with early-onset, extreme hypereosinophilia with urticaria, dermatitis, and diarrhea.

Loss of Parathyroid Hormone Receptor Signaling in Osteoprogenitors Is Associated With Accumulation of Multiple Hematopoietic Lineages in the Bone Marrow.

Osteoblasts and their progenitors play an important role in the support of hematopoiesis within the bone marrow (BM) microenvironment. We have previously reported that parathyroid hormone receptor (PTH1R) signaling in osteoprogenitors is required for normal B cell precursor differentiation, and for trafficking of maturing B cells out of the BM. Cells of the osteoblast lineage have been implicated in the regulation of several other hematopoietic cell populations, but the effects of PTH1R signaling in osteoprogenitors on other maturing hematopoietic populations have not been investigated. Here we report that numbers of maturing myeloid, T cell, and erythroid populations were increased in the BM of mice lacking PTH1R in Osx-expressing osteoprogenitors (PTH1R-OsxKO mice; knockout [KO]). This increase in maturing hematopoietic populations was not associated with an increase in progenitor populations or proliferation. The spleens of PTH1R-OsxKO mice were small with decreased numbers of all hematopoietic populations, suggesting that trafficking of mature hematopoietic populations between BM and spleen is impaired in the absence of PTH1R in osteoprogenitors. RNA sequencing (RNAseq) of osteoprogenitors and their descendants in bone and BM revealed increased expression of vascular cell adhesion protein 1 (VCAM-1) and C-X-C motif chemokine ligand 12 (CXCL12), factors that are involved in trafficking of several hematopoietic populations. © 2022 American Society for Bone and Mineral Research (ASBMR).

Obesity-Driven Acceleration of Acute Promyelocytic Leukemia Development in Mice with Inserted PML-RARa

While a number of solid tumors are identified as obesity-associated cancers, multiple myeloma and acute promyelocytic leukemia (APL) are the only hematologic malignancies included in this category. APL is a highly treatable form of AML characterized by maturation arrest at the promyelocyte stage, and frequent cures with combined ATRA and Arsenic Trioxide therapy. APL is initiated by a chromosomal translocation that causes the fusion of thePMLandRARAgenes, leading to the production of PML-RARA protein. To determine if obesity accelerates the development of APL, we used a genetically-engineered knock-in model in which human PML-RARA cDNA was inserted into the 5' untranslated region of the murine cathepsin G gene (Ctsg), which targets expression to early myeloid progenitor cells. This knock-in transgene was extensively backcrossed (&amp;gt;20 generations) into the C57BL/6J (B6) genetic background, which has been consistently shown to render mice susceptible to diet-induced obesity.Ctsg-PML-RARA mice developed on the B6 background generate a lethal APL-like leukemia with a latency of 8-12 months, and a penetrance of about 60%, as cooperating mutations occur and cause clinical disease. We placed cohorts of B6.Ctsg-PML-RARA heterozygous (mCG+/PR) and wild-type (WT) mice, genotyped by PCR, on high-fat (HFD) [57% coconut oil fat] and low-fat (LFD) [10% coconut oil fat] diets, collecting peripheral blood via the retro- orbital sinus at 30 day intervals and performing automated blood counts to observe temporal associations in disease development. HFD feeding significantly accelerated the development of APL in mCG+/PR mice, leading to anemia, thrombocytopenia, leukocytosis of the granulocyte series, and splenomegaly. Survival rate reduction demonstrated sexual-dimorphism, with male HFD-fed mCG+/PR mice displaying a median leukemia-free survival time of 211 days compared to 266 days in the female HFD-fed mCG+/PR cohort. In contrast, LFD-fed mCG+/PR male mice showed a median survival time of 318 days, whereas none of LFD-fed mCG+/PR female group nor any of the WT groups on HFD or LFD have reached median survival as of 325 days. Moreover, no signs of leukemia were observed in either HFD- and LFD-fed WT groups, indicating that adiposity alone had no leukemogenic effect. These studies clearly demonstrate that obesity accelerates leukemogenesis in theCtsg-PML-RARA mouse model of acute promyelocytic leukemia. High density flow cytometry will be applied in the bone marrow of the different groups to evaluate the modulation of multiple hematopoietic cell lineages and cell surface markers. Disclosures No relevant conflicts of interest to declare.

Putative Mechanisms Underlying Cardiovascular Disease Associated with Clonal Hematopoiesis of Indeterminate Potential.

Characterized by the expansion of somatic mutations in the hematopoietic lineages of aging individuals, clonal hematopoiesis of indeterminate potential (CHIP) is a common condition that increases the risk of developing hematological malignancies and cardiovascular disease (CVD). The presence of CHIP-associated mutations in hematopoietic stem and progenitor cells (HSPCs) suggests that these mutations may alter the functions of the diverse hematopoietic lineages, many of which influence the pathogenesis of CVD. Inflammation may be a potential pathogenic mechanism, linking both CVD and hematological malignancy. However, it remains unknown whether CHIP-associated CVD and hematological malignancy are features of a common disease spectrum. The contributions of CHIP-associated mutations to both CVD and hematological malignancy underscore the importance of stem cell biology in pathogenesis and treatment. This review discusses possible mechanisms underlying the contributions of multiple hematopoietic lineages to CHIP-associated CVD and the putative pathogenic links between CHIP-associated CVD and hematological malignancy.

An in vitro quantitative systems pharmacology approach for deconvolving mechanisms of drug-induced, multilineage cytopenias.

Myelosuppression is one of the most common and severe adverse events associated with anti-cancer therapies and can be a source of drug attrition. Current mathematical modeling methods for assessing cytopenia risk rely on indirect measurements of drug effects and primarily focus on single lineage responses to drugs. However, anti-cancer therapies have diverse mechanisms with varying degrees of effect across hematopoietic lineages. To improve predictive understanding of drug-induced myelosuppression, we developed a quantitative systems pharmacology (QSP) model of hematopoiesis in vitro for quantifying the effects of anti-cancer agents on multiple hematopoietic cell lineages. We calibrated the system parameters of the model to cell kinetics data without treatment and then validated the model by showing that the inferred mechanisms of anti-proliferation and/or cell-killing are consistent with the published mechanisms for three classes of drugs with different mechanisms of action. Using a set of compounds as a reference set, we then analyzed novel compounds to predict their mechanisms and magnitude of myelosuppression. Further, these quantitative mechanisms are valuable for the development of translational in vivo models to predict clinical cytopenia effects.

E protein support of Zeb2 expression regulates development of multiple hematopoietic lineages

Abstract Zeb2 is an E-box binding transcriptional repressor that were first discovered to be important for epithelial to mesenchymal transition. We recently discovered a genetic circuit involving Zeb2, Nfil3, Id2 that governs dendritic cell (DC) specification in the multipotent common dendritic cell progenitor (CDP). Nfil3 expression is required for the transition from a Zeb2hiand Id2lo stage into a Zeb2lo and Id2hi stage, which has exclusively cDC1 potential. This circuit blocks E protein activity to exclude pDC potential within the progenitor. However how E proteins regulate pDC development is still unclear. Here, we identified an enhancer element 165kb downstream of Zeb2 transcriptional starting site that can be bound by E proteins. Deletion of this enhancer completely abrogates pDC development. Interestingly, the activity of this enhancer is also required for monocytes and B cell development, while tissue resident macrophages used a different enhancer. A circuit involving Zeb2, Id2 and E proteins, where E proteins binding at this cis-element supports Zeb2 expression, is essential for lineage specification at multiple bifurcation point in the hematopoietic tree.

Elucidating an Essential Role of Promoting Endothelial-to-Hematopoietic Transition By a Key EMT-Regulator ZEB2 Using a Human iPSC-Based Model

ZEB2 (Zinc finger E-box binding homeobox 2; also known as SMAD interacting protein-1 or SIP1) is a DNA-binding transcriptional regulator associated with epithelial-to-mesenchymal-transition (EMT). It has been reported that it is important in many other developmental processes, including mesoderm formation during gastrulation and neural crest formation. More recently, a study reported that inactivation of ZEB2 in mice results in differentiation defects in multiple hematopoietic cell lineages (1). In order to examine the roles of ZEB2 in human developmental process and hematopoiesis, we decided to apply human induced pluripotent stem cells (iPSCs) as a model with a pair of isogenic iPSCs with or without functional ZEB2 expression. We used a validated iPSC-based hematopoietic differentiation under a feeder- and xeno-free culture condition was used to study the role of ZEB2 in hematopoietic cell formation and to avoid other EMT-inducing factors in serum. We found that ZEB2 expression is abundant in either cord blood cells or human iPSCs-derived hematopoietic stem/progenitor cells (HSPCs), indicating that ZEB2 deficiency may hinder human hematopoietic differentiation. To knock out the ZEB2 gene and generate ZEB2 deficient human iPSCs, we used two different CRISPR guide RNAs as well as SpCas9 in a human iPSC line BC1. An engineered isogenic BC1 iPSC line that has both alleles 155-bp deletion in ZEB2 exon 1 (ZEB2 -/-) was successfully isolated and expanded, resulting in the frameshift and the consequent disappearance of ZEB2 protein expression. The complete ZEB2 knockout had little effects on human iPSCs expansion. However, we observed that the differentiation of ZEB-/- BC1 into CD34+CD45+ HSPCs was decreased. The number of ZEB-/- BC1-derived HSPCs was 5-fold lower than that from wide type BC1 control. Moreover, ZEB2-deleted HSPCs were defected in hematopoietic colony formation. To figure out at which hematopoietic differentiating stage the defects occurred, we applied inducible iPSC's endothelium-hematopoietic transformation (EHT) system to mimic hemogenic endothelial cells (ECs) giving rise to HSPCs. By using this established EHT system, ZEB2 deficient iPSCs were differentiated in order into CD34+CD31+CD144+ ECs, and then to HSPCs (2). We found that ZEB2 depletion did not adversely affect the formation of ECs from human iPSCs in this EHT experimental system but did impact on HSPCs generation from ECs. This study is unique in several ways to elucidate the roles of ZEB2 in human hematopoietic development. This prospective genetic model allowed us to pinpoint ZEB2 is critical at the stage of EHT during human hematopoietic cell formation. Our results indicate that contribution of ZEB2 deficiency in blood disorders might result from the inhibition of HSPC formation. The human iPSC-based hematopoietic differentiation system coupled with precisely edited isogenic iPSC lines also provide a more controlled genetic model for studying functions of others gene in their regulation of human hematopoiesis.