Heterozygous Mutations in DDX41 Cause Erythroid Progenitor Cell Defects

Germline mutations in the RNA Helicase gene DDX41 cause inherited susceptibility to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). These mutations are always heterozygous and are typically frameshifts, causing loss of protein expression. We recently reported that at least one functional copy of DDX41 is essential for hematopoiesis, and that DDX41 is required for ribosome biogenesis. While biallelic DDX41 mutations cause dramatic defects in hematopoiesis, the role of heterozygous mutations in Myelodysplastic Syndrome pathogenesis is not yet understood. Recent clinical studies have pointed out that some patients bearing germline DDX41 mutations have idiopathic cytopenias of unknown significance (ICUS) prior to MDS onset, suggesting that underlying hematopoietic defects precede and potentially contribute to the onset of MDS/AML (Choi et al., Haemotologica 2021). It has also been noted that the majority of DDX41-mutant MDS patients have refractory anemia, indicating that the erythroid lineage is particularly effected in these patients (Sebert et al., Blood 2019). Since ribosome defects are a common cause of inherited anemias and also contribute to MDS pathogenesis, we characterized the effect of heterozygous DDX41 mutations on erythropoiesis in murine and human models. Mice that have been transplanted with Ddx41 +/- bone marrow develop anemia at 12-15 months post-transplant, indicating that detection of erythroid defects in vivo is aging-dependent. We characterized the effect of heterozygosity of Ddx41 on erythroid progenitor function in vitro and found that Ddx41 +/- bone marrow from young mice yields fewer BFU-E in colony assays but comparable numbers of myeloid colonies. Liquid culture erythroid differentiation of Ddx41 +/- bone marrow produces fewer CD71+ Ter119+ progenitors than controls. To characterize the effect of heterozygous DDX41 mutations on human erythropoiesis, we generated induced pluripotent stem cells bearing heterozygous frameshift mutations in DDX41 using CRISPR. We found that these DDX41 +/- iPSC lines produced CD43+/CD34+ hematopoietic progenitor cells (HPC) with equal efficiency as unmodified control iPSC. However, once these HPC were induced to differentiate down the erythroid lineage in liquid culture, they made fewer CD71+ GLYA+ erythroid progenitors and fewer hemoglobinized cells. The DDX41 +/- HPC also produced fewer BFU-E in colony assays. Mechanistically, we found that the in vitro-derived erythroid progenitors from both mice and human iPSC had decreased protein translation, suggesting that ribosome defects underlie the observed erythroid differentiation defects. In diseases such as Diamond Blackfan Anemia and Dyskeratosis Congenita, ribosome defects lead to p53 activation which reduces cell cycle progression in erythroid progenitors. To test the role of p53 in the erythroid defects caused by Ddx41 heterozygosity, we crossed Ddx41 +/- mice with p53-knockout mice and found that loss of p53 fully rescued the BFU-E colony formation of Ddx41 +/- bone marrow HPC. We confirmed this finding using CRISPR-mediated knockout of p53 in Ddx41 +/- BM HPC. Collectively, these results suggest that a mild ribosome defect in DDX41 +/- HPC causes a deficit in erythropoiesis that results in anemia with aging. It is likely that this anemia causes stress in the bone marrow and a selective environment in which malignant hematopoietic stem and progenitor cells arise, leading to MDS and AML. Disclosures Starczynowski: kurome Inc: Consultancy.

Effect of ultrasound and microbubbles on PEG coated gold nanorod thermal therapy

The effectiveness of PEG coated gold nanorod and laser thermal therapy (AuNR+L) depends on gold nanoparticle delivery. The application of ultrasound and microbubbles (USMB) has been shown to enhance drug delivery across cell membranes. This study investigated the effect of the combined treatment of ultrasound and microbubbles with PEG coated gold nanorod thermal therapy on cancer cells. Cells in suspension were exposed to combinations of AuNR, laser, and USMB. Following the treatment, cell viability was assessed with propidium iodide marker and flow cytometry, and with colony assays. Cell death significantly increased when USMB was combined with AuNR+L during laser treatment compared to either treatment on its own, whereas, in the absence of AuNR, NIR laser light had a protective effect on cells exposed to USMB. Generally, USMB induced an additive therapeutic effect on cell viability when combined with AuNR+L.

Effect of ultrasound and microbubbles on PEG coated gold nanorod thermal therapy

The effectiveness of PEG coated gold nanorod and laser thermal therapy (AuNR+L) depends on gold nanoparticle delivery. The application of ultrasound and microbubbles (USMB) has been shown to enhance drug delivery across cell membranes. This study investigated the effect of the combined treatment of ultrasound and microbubbles with PEG coated gold nanorod thermal therapy on cancer cells. Cells in suspension were exposed to combinations of AuNR, laser, and USMB. Following the treatment, cell viability was assessed with propidium iodide marker and flow cytometry, and with colony assays. Cell death significantly increased when USMB was combined with AuNR+L during laser treatment compared to either treatment on its own, whereas, in the absence of AuNR, NIR laser light had a protective effect on cells exposed to USMB. Generally, USMB induced an additive therapeutic effect on cell viability when combined with AuNR+L.

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.

RAS mutations drive proliferative chronic myelomonocytic leukemia via a KMT2A-PLK1 axis

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

Erythropoiesis Failure in Diamond-Blackfan Anemia Starts at the Oligopotent Common Myeloid and Megakaryocyte-Erythroid Progenitor Stage

Diamond-Blackfan anemia (DBA) is a hereditary bone marrow failure disorder that is characterized by erythropoiesis failure and chronic anemia and is frequently associated with physical malformations. Considered a ribosomopathy, most patients harbor pathogenic mutations in one of at least 22 large or small ribosomal protein genes in an autosomal dominant manner, however rarer cases involving mutations in GATA1, TSR2 and EPO have also been described. A recent study of the architecture of the human hematopoietic hierarchy throughout development (Notta et al, Science 2016) showed that, after birth, unipotent progenitors can derive directly from multipotent progenitors without intermediate differentiation into oligopotent progenitors. Critically, this work identified novel progenitors for which the abundance, clonal capacity and progenitor function is currently unknown in many benign and malignant hematological disorders. Specifically, multipotent, common myeloid and megakaryocyte-erythroid progenitors (MPP, CMP and MEP) were found to be functionally heterogeneous and could be subdivided (F1, F2 and F3 subtypes) based on the combination of CD71 and BAH1 expression. We hypothesized that in-depth analysis of these novel and previously reported progenitors will better elucidate hematopoiesis in DBA, providing insights in to DBA pathogenesis and erythropoietic failure. Using flow cytometry, we quantified the frequencies of 11 hematopoietic stem and progenitor cell (HSPC) populations: HSC, MPP F1-F3, CMP F1-F3, MEP F1-F3 and granulocyte-monocyte progenitors (GMP), from the BM specimens of 8 DBA patients harboring ribosomal protein mutations and 6 healthy age-matched control donors. To characterize the function of HSPCs in DBA we then utilized a highly sensitive and quantitative single-cell in-vitro stromal feeder-based differentiation assay that supports the expansion and lineage commitment of single HSCPs towards mature cell types (myeloid, erythroid and megakaryocytes) with progeny immunophenotyping and quantification by flow cytometry. Our refined quantification of HSPCs by flow cytometry showed that the defect of erythropoiesis in DBA patients starts at the CMP/MEP compartment. Namely, CMP-F2 and CMP-F3 as well as MEP-F2 and MEP-F3 sub-populations that are predicted to form erythroid cells were significantly reduced in DBA patients compared to healthy aged-matched individuals (Figure 1A-B). In contrast, CMP/MEP-F1 sub-populations that are predicted to form only myeloid cells (BAH1-/CD71-) showed no significant difference compared to healthy controls. Importantly, the multipotent compartment (HSC and MPP) did not significantly differ from that of healthy controls. Functional analysis of F2/F3 CMP/MEP HSCP sub-populations in single-cell colony assays showed that they have a high propensity to produce erythroid progeny in healthy control samples (Figure 1C). In contrast, in DBA patients these HSCPs had limited erythroid forming potential, but maintain a high propensity to develop myeloid colonies (Figure 1D). To our knowledge this is the first study that the HSCP sub-populations F1/F2/F3 HSCP fractions have been analyzed in DBA. Collectively, these data indicate that the hematopoietic defect in DBA occurs in erythroid-producing CMP/MEP populations. Our data show that these sub-populations are both reduced and dysfunctional in DBA patients and we suggest these abnormalities lead to the pure red cell aplasia seen in these patients. Figure 1: DBA patients have reduced CMP and MEP sub-populations that behave dysfunctional in-vitro. A-B) DBA patients have reduced CMP-F2/3 (A) and MEP-F2/3 (B) sub-populations in their bone marrow. C) CMP F2-F3 and MEP F2-F3 have a high propensity to produce erythroid cell types in healthy controls. D) Single-cells isolated from the CMP-F2/3 and MEP-F2 population of DBA patients produced proportionally fewer erythroid and greater myeloid colonies in in-vitro colony assays (plot shows the mean respective proportions from all healthy controls and DBA patients tested, each N=5). Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

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

Background: Stanozolol and danazol are widely used in the treatment of aplastic anemia (AA), however, they may have different effects on the recovery of hematopoiesis with the detail mechanism unclear. Methods: Bone marrow mononuclear cells from 5 newly diagnosed AA patients and 5 healthy volunteers were collected for marrow colony assays and cultured together with stanozolol, danazol or blank control, separately. After incubated for 14 days, colonies of different lineage were calculated, and erythroid or megakaryocytic differentiation was also identified by the mean fluorescence intensity (MFI) of CD235a or CD41 expressed on the harvest cells. Meanwhile, CB6F1/Crl mice were injected with 1×106 C57BL/6 donor originated lymphocytes after irradiated with 5Gy total body irradiation to setup a model for immune-mediated bone marrow failure (AA mice model). AA mice were treated with CsA monotherapy, CsA combined with stanozolol, CsA combined with danazol for 30 days, respectively. Peripheral blood cell counts once a week, bone marrow colony assays at the end of one month were performed. Proportion of T cell subsets, level of inflammatory factors, EPO and TPO were detected before and after treatment. Level of EPO receptor on the progenitor cells after treatment were tested by western blot. Results: For ex vivo experiment, although the number of BFU-E, CFU-GM and CFU-GEMM colonies of AA patients were significantly lower than that of the normal controls (P<0.05), the number of colonies and MFI of CD235a or CD41 expression of the harvested cultured cells had no significant difference among different treatment groups, either in AA patients or in normal controls, showing no direct hematopoietic stimulating effects of stanozolol and danazol on progenitor cells. However, in the in vivo experiment, AA mice treated with CsA and danazol showed the most rapid recovery of megakaryopoiesis, with the platelet count returned to normal level after three weeks' treatment, at least one week earlier than the other groups. Whereas mice treated with CsA and stanozolol had the best hemoglobin level at the end of treatment (P<0.05). Bone marrow colony assays at the 30 days showed that the number of BFU-E was the highest in mice treated with CsA and stanozolol while the number of CFU-GM was the highest in those with CsA and danazol. Compared to CsA monotherapy, additional stanozolol and danazol can both increase the level of regulatory T cells and up-regulate interleukin-10 (P<0.05). But interferon-α and tumor necrosis factor-α were more effectively reduced by danazol than stanozolol (P<0.05). CsA and stanozolol- treated mice showed higher serum EPO (corrected by HGB level)and higher EPO receptor(EPOR) level on the hematopoietic precursor cells compared with other groups (P<0.05). Conclusions: Neither stanozolol or danazol directly stimulated hematopoiesis in vitro. But in vivo, stanozolol may have advantage in improving erythropoiesis while danazol may has more effects on white cells or platelet. Danazol had more comprehensive immunosuppressive roles compared with stanozolol. Stanozolol can enhance the expression of EPO, probably by increasing EPOR expression on hematopoietic precursor cells. Disclosures No relevant conflicts of interest to declare.

Identification Of Bipotential Lin-CD34+CD38- Integrin α2- Erythrocyte-Megakaryocyte Progenitors In Human Bone Marrow

Human hematopoietic progenitor cells with megakaryocyte and erythroid commitment have been defined as the Lin-CD34+CD38+CD123-CD45RA- and in addition, as CD110+ (Manz et al. 2002; Edvardsson et al. 2006). However, previous colony assays have shown that bipotential megakaryocyte-erythroid progenitors (MEPs) also reside in the stem cell-enriched CD34+CD38- fraction in bone marrow (BM) (Debili et al., 1996). So far, the phenotype of this MEP population has remained obscure. We have here studied the expression of integrin α2 chain in normal human bone marrow CD34+CD38- stem and progenitors. Integrin α2 chain was expressed in most BM CD34+CD38- cells (96.3±3.8; mean±SD), in contrast to a more restricted expression in cord blood CD34+CD38- cells (Wong, et al. 2013). In order to investigate the potential functional differences associated with the expression of integrin α2 on BM CD34+CD38- cells, we characterized the integrin α2+ and α2- cell populations by in vitro LTC-IC, single cell colony assays and gene expression analysis. The LTC-IC progenitors were exclusively integrin α2+. In contrast, the integrin α2- fraction was highly enriched in erythroid BFU-E progenitors. In single cell colony assay in methylcellulose, 21.96% of single Lin-CD34+CD38- integrin α2- cells gave rise to BFU-E, as compared to 0.65% of integrin α2+ cells. Likewise, the frequency of megakaryocyte colony forming cells, analyzed by a serum free collagen based culture system, was significantly higher in the integrin α2- fraction, suggesting an existence of the bipotential MEP in the integrin α2- fraction. To confirm the existence of bipotential MEPs in the Lin-CD34+CD38-CD45RA- integrin α2- cell population, we performed single cell clonogenic assay using serum free medium with cytokines (SCF, TPO, IL-3 and EPO) in Terasaki plates. At day 12-14 of culture 30-40% of the cells showed clonal growth. The erythroid and megakaryocytic differentiation of the single cell derived clones was assessed by their immunophenotype and morphology. Interestingly, FACS analysis of the 32 clones indicated that all of them contained erythroid cells (CD235+CD41-). Most importantly, 22 of the 32 clones contained both erythroid and megakaryocytic cells (CD235-CD41+), showing a high degree of bipotential MEP activity in the integrin α2- population. Minimal myeloid differentiation (CD15/CD33/CD66b+ cells <0.5%) was seen in only 3 of the 32 clones. During the differentiation the integrin α2 receptor was upregulated in CD41+ megakaryocytic cells but not in the CD235+ erythroid cells. Furthermore, 83 clones derived from single cells, including clones with very limited number of cells, were individually transferred to cytospin slides for evaluation of their morphology. The small clones invariably consisted of megakaryocytic cells. 48.19% of all clones analyzed were identified as bipotent MEPs. This finding was further supported by upregulation of HBD and downregulation of GATA3, HLF in the integrin α2- cells, revealed by gene expression analysis. Taken together, our data demonstrate a high frequency of bipotential MEP progenitors in adult BM Lin-CD34+CD38-CD45RA- integrin α2- cell fraction and suggest that BM Lin-CD34+CD38- integrin α2- cells are transcriptionally primed towards development into erythroid and megakaryocytic cells. The identification of this novel bipotential MEP population provides a means for further analysis on lineage fate decisions of primitive erythroid and megakaryocytic cells and may facilitate studies aiming to expand and differentiate these lineages for clinical transplantation and transfusion trials. Disclosures: Ekblom: Novartis and BMS: Honoraria.