Pp90RSK Isoforms Play Distinct Roles during Hematopoiesis

Ribosomal S6 Kinases (RSKs) include a family of serine/threonine kinases that regulate cell proliferation and survival. In humans, four RSK isoforms (RSK1-4) have been identified. Although they are 73-80% conserved in sequence homology, RSK isoforms are reported to have distinct functions. RSKs phosphorylate cyclic AMP response element binding protein (CREB) which is a regulator of hematopoietic proliferation and differentiation. RSK1 is hyperactivated in AML and is therefore a potential therapeutic target for acute myeloid leukemia (AML). Therefore, inhibition of RSK requires understanding of its role during normal hematopoiesis. Furthermore, the role of RSK isoforms during hematopoiesis and leukemogenesis has not been well characterized. To study the expression of RSK isoforms in hematopoiesis, we cultured human cord blood CD34+ (CBCD34+) hematopoietic stem and progenitor cells (HSPCs) in differentiating media containing SCF, Flt3L, TOP, IL-3, IL-6, GM-CSF, and EPO and performed qPCR with mRNA of the RSK isoforms at different stages of maturation up to 14 days. RPS6KA1 (RSK1) was expressed throughout hematopoiesis, but RPS6KA3 (RSK2) and RPS6KA2 (RSK3) showed higher expression by 2-fold (p<0.05) in the earlier stages of normal hematopoiesis. Moreover, RPS6KA3 is highly expressed in CD38-CD34+ HSCs compared with other progenitor populations (LMPP, MPP, CMP, GMP, and MEP) isolated from human cord blood. RPS6KA2 expression was higher in LMPP by 7-fold (p<0.05) and CMP by 3-fold (p<0.05) compared with HSCs but was barely detectable in MEPs. RPS6KA1 was ubiquitously expressed in all progenitor populations. These results suggest that RSK2 and RSK3 have distinct functions during early myelopoiesis. To study the requirement of RSK isoforms during hematopoiesis, we knocked down RSK expression by transducing lentivirus expressing isoform-specific shRNAs or scramble controls into human CBCD34+ cells. On day 5 following transduction, cells were sorted and plated in methylcellulose media and assessed for the colony-forming activity of RSK knockdown in HSPCs. While RSK3 knockdown increased BFU-E colonies by 1.5-fold (p<0.05) compared with control cells, RSK1 knockdown decreased BFU-E and CFU-E erythroid colonies by 2-fold (p<0.05) but increased CFU-GM colonies by 2-fold (p<0.05) compared with control cells. However, RSK2 knockdown did not show any significant effects on colony-forming activity of HSPCs. We confirmed the isoform-specific effect of RSKs in normal hematopoiesis using liquid culture assays. We collected transduced cells at day 8, 11, and 14 after transduction and analyzed cell populations by flow cytometry. Similar to colony formation assay results, RSK3 knockdown increased the CD71+ erythroid population by 1.5-fold (p<0.001), but suppressed production of the CD14+ monocyte population. RSK1 knockdown enhanced production of the CD11b+/CD14+ myeloid population, but inhibited the CD71+ erythroid production by 1.5-fold (p<0.001) compared with control HSPCs. There was no significant change in blood cell differentiation in RSK2 knockdown cells. These data demonstrate that RSK1 and RSK3 exert opposite functions during erythroid and myeloid differentiation of HSPCs, suggesting a novel role for RSK isoforms as a determinant of early fate decisions of HSCs. Disclosures No relevant conflicts of interest to declare.

Impact of Intrauterine Growth Restriction Diseases on The Umbilical Cord Blood CD34+ Cell Counts

Introduction: Different diseases in obstetrics and gynecology can affect the number of CD34+ cells in the umbilical cord blood. Objectives: This study aimed to evaluate the effect of Gestational Diabetes Mellitus (GDM), Gestational Hypertension (GHT) and Morbidly Adherent Placenta (MAP) on the content of CD34+ cells of umbilical cord blood and to compare the effectiveness of Sysmex XN20 analyzer to the flow cytometry method, which is the gold standard in CD34+ cells. Materials abd Methods: The umbilical cord blood (15 ml) was collected after the birth of the newborns. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus. The cells were stained with Procount ™ Progenitor Cell Count Kit with PE Labeled monoclonal anti-CD34+ antibody then analyzed with Flow Cytometry or Sysmex XN20 without staining, to identify CD34+ cells named as hematopoietic progenitor cells (HPC), respectively. Results: Flow cytometric evaluation revealed a significantly elevated (p<0.05) number of cord blood CD34+ cells in GDM and GHT groups compared with healthy controls. MAP patients had comparable CD34+ cells compared with healthy controls. A significant increase in lymphocyte counts was also observed in GDM and GHT groups compared with healthy controls. Sysmex analysis however only revealed an increase in lymphocyte numbers in GHT but picked no differences across groups in HPC. Correlation between Sysmex and flow cytometry results was weak in control, GHT, GDM and MAP groups r: 0570/p<0.01, r: 0.5727/p: 0.0708, r:0.2149/p: 0.4779, r: 0.111/p: 0.779, respectively. Conclusions: CD34+ cells were significantly higher in the GHT and GDM groups compared with healthy control cord blood. The correlations between Flow cytometry and Sysmex were not strong.

Role of Plasma Gelsolin Protein in the Final Stage of Erythropoiesis and in Correction of Erythroid Dysplasia In Vitro

Gelsolin, an actin-remodeling protein, is involved in cell motility, cytoskeletal remodeling, and cytokinesis and is abnormally expressed in many cancers. Recently, human recombinant plasma gelsolin protein (pGSN) was reported to have important roles in cell cycle and maturation of primary erythroblasts. However, the role of human plasma gelsolin in late stage erythroblasts prior to enucleation and putative clinical relevance in patients with myelodysplastic syndrome (MDS) and hemato-oncologic diseases have not been reported. Polychromatic and orthochromatic erythroblasts differentiated from human cord blood CD34+ cells, and human bone marrow (BM) cells derived from patients with MDS, were cultured in serum-free medium containing pGSN. Effects of pGSN on mitochondria, erythroid dysplasia, and enucleation were assessed in cellular and transcriptional levels. With pGSN treatment, terminal maturation at the stage of poly- and ortho-chromatic erythroblasts was enhanced, with higher numbers of orthochromatic erythroblasts and enucleated red blood cells (RBCs). pGSN also significantly decreased dysplastic features of cell morphology. Moreover, we found that patients with MDS with multi-lineage dysplasia or with excess blasts-1 showed significantly decreased expression of gelsolin mRNA (GSN) in their peripheral blood. When BM erythroblasts of MDS patients were cultured with pGSN, levels of mRNA transcripts related to terminal erythropoiesis and enucleation were markedly increased, with significantly decreased erythroid dysplasia. Moreover, pGSN treatment enhanced mitochondrial transmembrane potential that is unregulated in MDS and cultured cells. Our findings demonstrate a key role for plasma gelsolin in erythropoiesis and in gelsolin-depleted MDS patients, and raises the possibility that pGSN administration may promote erythropoiesis in erythroid dysplasia.

Ibrutinib Suppresses Early Megakaryopoiesis but Enhances Proplatelet Formation

Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase, has a favorable safety profile in patients with B cell-related malignancies. A primary adverse effect of ibrutinib is thrombocytopenia in the early stages of treatment, but platelet counts increase or recover as treatment continues. Currently, the effects of ibrutinib on megakaryopoiesis remain unclear. In this study, we investigated the mechanism by which ibrutinib induces thrombocytopenia using cord blood CD34+ hematopoietic stem cells (HSCs), a human megakaryoblastic cell line (SET-2), and C57BL/6 mice. We show that treatment with ibrutinib can suppress CD34+ HSC differentiation into megakaryocytes (MKs) and decrease the number of colony-forming unit-MKs (CFU-MKs). The ibrutinib-dependent inhibition of early megakaryopoiesis seems to mainly involve impaired proliferation of progenitor cells without induction of apoptosis. The effects of ibrutinib on late-stage megakaryopoiesis, in contrast to early-stage megakaryopoiesis, include enhanced MK differentiation, ploidy, and proplatelet formation in CD34+ HSC-derived MKs and SET-2 cells. We also demonstrated that MK adhesion and spreading, but not migration, were inhibited by ibrutinib. Furthermore, we revealed that integrin αIIbβ3 outside-in signaling in MKs was inhibited by ibrutinib. Consistent with previous clinical observations, in C57BL/6 mice treated with ibrutinib, platelet counts decreased by days 2 to 7 and recovered to normal levels by day 15. Together, these results reveal the pathogenesis of ibrutinib-induced transient thrombocytopenia. In conclusion, ibrutinib suppresses early megakaryopoiesis, as evidenced by inhibition of MK progenitor cell proliferation and CFU-MK formation. Ibrutinib enhances MK differentiation, ploidy, and proplatelet formation, while it impairs integrin αIIbβ3 outside-in signaling.

Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations

Somatic mutations in genes coding for splicing factors, e.g. SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with Myelodysplastic Syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease therefore making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation-associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association of R-loops and ATR sensitivity with the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.