Presence Of Thrombopoietin Research Articles

Abstract 5336: Establishment and characterization of a novel inducible Apoc2 knockout mouse model to study the roles of Apoc2 in normal hematopoiesis

Abstract Apolipoprotein C2 (Apoc2) is upregulated in acute myeloid leukemia and presents a viable therapeutic target. Whether this gene plays a role in normal hematopoiesis remains unknown. To investigate the impact of targeting Apoc2 on normal hematopoiesis, we established and characterized an inducible Apoc2 knockout mouse model. Homozygous floxed Apoc2 mice (Apoc2fl/fl) were obtained from the NIH (Dr. Remaley’s lab) and were crossed with homozygous R26-CreERT2 mice. After three generations, Gt(ROSA)26SorCre/Cre, Apoc2fl/fl mice were generated and confirmed by genotyping. Male (M) and female (F) mice were injected with tamoxifen (TAM: 20mg/mL dissolved in corn oil) intraperitoneally (75mg/kg per day for 5 days) to induce Apoc2 knockout (Apoc2-KO). The control wild type (WT) group included Gt(ROSA)26Sorwt/wt, Apoc2fl/fl mice, and CD45.2+ WT mice injected with TAM, or Gt(ROSA)26SorCre/Cre, Apoc2fl/fl mice injected with corn oil.No obvious abnormal phenotypes were observed in Apoc2-KO mice. The average increase of body weight 3 weeks post TAM or corn oil injections of 5-week-old Gt(ROSA)26SorCre/Cre, Apoc2fl/fl siblings was not statistically significantly different (Apoc2-KO (n = 2M + 2F) vs WT (n = 2M + 2F) = 2.450g vs 2.125g, P = 0.63). Analyses of Apoc2-KO (n = 5M + 3F) and WT (n = 4M + 2F showed no significant difference in livers and spleens weight when normalized to mice age. The percentage of knockdown in liver (99.96%), spleen (77.32%), bone marrow (BM, 83.56%) and blood (82.02%) was confirmed by qPCR. In-vitro cell expansion was performed in the presence of thrombopoietin and stem cell factor for BM cells and IL-2 and PHA for T cells. Apoc2-KO BM cells exhibited similar in vitro expansion to that of control mice (Apoc2-KO vs WT: Day 10/Day 6: 1.22 fold vs 1.57 fold; Day 10/Day 1: 57.26% vs 72.34%; P > 0.05; and Cell Trace assay: mean fluorescence intensity (MFI) of Day 9 normalized to Day1: Apoc2-KO vs WT = 0.37 vs 0.39; P > 0.05). Also, the colony forming ability was not significantly different between Apoc2-KO and WT BM cells (Apoc2-KO vs WT = 79.09 units vs 81.47 units, P = 0.62). In-vitro T cell expansions of splenocytes were not significantly different between Apoc2-KO and WT mice (Apoc2-KO vs WT: Day 10/Day 4: 1.73 fold vs 1.83 fold; Cell Trace: mean MFI of Day 9 normalized to Day1: Apoc2-KO vs WT = 0.6208 vs 0.7088; P > 0.05). Hematology analysis assessing white blood cell count, red blood cell count, hemoglobin count, neutrophil, lymphocytes, showed no significant difference between the groups.Here we report the first inducible Apoc2 knockout mouse model and demonstrated that Apoc2 knockout has limited impact on normal hematopoietic cells in mice. On-going research using this model will assess the impact of Apoc2 knockout on ageing related phenotypes and leukemia progression. Citation Format: Xiaochen Zhang, Ritu Chandwani, Mateusz Pospiech, Yiting Meng, Kanaka Dhuri, Bhakti Thakker, Atham Ali, Shephali M. Kadam, Yu-Chun Wei, Houda Alachkar. Establishment and characterization of a novel inducible Apoc2 knockout mouse model to study the roles of Apoc2 in normal hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5336.

Bone Marrow CD8 + Tissue-Resident Memory T Cell Promote B Cell Differentiation and Inhibit Platelet Production in Patients with Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease in which enhanced anti-platelet T cell activity and dysfunction of CD8 + T cells are involved(Audia et al., 2013; Semple & Freedman, 1991). Persistent secretion of antibodies by long-lived plasma cells and memory B cells in the bone marrow contributes to ITP pathogenesis (Roeser, Lazarus, & Mahevas, 2023). However, the role of memory T cells, especially tissue-resident memory T cells (TRM), in ITP remains underexplored. TRM develop in specific tissues and play crucial role in local immunity. TRM exert their effects through mechanisms including the induction of local humoral responses, contributing to autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Chang et al., 2021; Gu, Song, Roh, Jung, & Kim, 2021). Bone marrow contains one of the largest pools of memory CD8 + T cells. However, the involvement of bone marrow TRM in ITP pathogenesis remains poorly understood. In this study, we observed a significantly higher proportion of CD8 + TRM (CD8 +CD45RA -CD45RO +CD69 +) in the bone marrow of ITP patients compared to healthy controls. However, no significant differences were observed in CD4 + TRM, CD8 + TCM and CD8 + TEM between the two groups. CD8 + TRM from ITP patients exhibited increased levels of IFN-γ, NLRP3, IL-1β, TNF-α, and decreased levels of IL-10 compared to those from healthy controls. Furthermore, CD8 + TRM proportions were significantly higher in glucocorticoid responders than non-responders, suggesting a potential role of CD8 + TRMs in mediating glucocorticoid therapeutic effects. Transcriptome sequencing revealed upregulated genes, including IGLC2, ASAP2, HIST2H2AA4, IGLV2-11, C1QA, ATP5MC1, IL5RA, and CYC1, and downregulated genes, including CCR9, HSPE1P18, RGCC, and ITGAE, in TRM cells from ITP patients compared to controls. GO/KEGG analysis of the top 500 upregulated genes highlighted pathways associated with B cell-associated immunity. To explore the effect of TRM on B cells, bone marrow CD19 + B cells were co-cultured with autologous TRM or memory T cells without TRM (TM) in the presence of IgM, and the proportion of memory B cells (CD19 +CD27 +CD38 -) and plasma cells (CD19 -CD24 -CD38 +) were measured by flow cytometry. The supernatants were collected, and cytokines and IgG level were tested by ELISA. Results showed that TRMs from ITP patients promoted the differentiation of memory B cells and IgG antibody production compared with those from healthy controls. However, no significant difference was observed in the proportion of naive B cells and plasma cells between the two groups. TRM, but not TM augmented B cell differentiation into memory B cells or plasma cells both in ITP patients and healthy controls. Additionally, TRM-primed B cells showed a stronger effect on antibody production and resulted in a higher level of IgG in supernatant compared with TM primed B cells. In line with these findings, the levels of B cell activating factor (BAFF), a proliferation inducing ligand (APRIL), and sCD40L, which were critical cytokines for B cell survival and differentiation, were significantly higher in supernatants from ITP patients than those from healthy controls. Moreover, the levels of BAFF, APRIL and CD40L in co-culture system with TRM were significantly increased compared to the system with TM, providing insights into the impact of TRM on B cell differentiation and function. To further investigate the role of TRM in regulating platelet production in ITP patients, we cocultured TRM with CD34 +cells in the presence of thrombopoietin (TPO), stem cell factor (SCF) and interleukin (IL)-3. Results showed that TRM did not suppress the production of CD41 + megakaryocytes from CD34 + cells, but reduced megakaryocyte apoptosis and increased the percentage of polyploidy (≥4N) megakaryocytes compared with TM. Additionally, TRM inhibited reticulated platelet formation and platelet production. Notably, dexamethasone (DXM) modulation alleviated certain suppressive effects on platelet production. In summary, our findings reveal the involvement of impaired bone marrow CD8 + TRM in the pathogenesis of ITP through the promotion of B cell differentiation and inhibition of platelet production. These results shed new light on the potential of immune therapy targeting TRM in the management of ITP.

The Role of Gpibα in Control of Megakaryocyte Reactivity to Thrombopoietin

Thrombopoietin (TPO) was first described in 1958 as a humoral factor that regulates platelet production. TPO binds to c-Mpl and stimulates the expansion and differentiation of megakaryocyte precursors and the maturation of megakaryocytes. Notably, TPO has no significant effect on the formation stage of megakaryocyte boundary membrane system and platelet release. Studies investigating GPIbα deficiency have demonstrated its substantial effects on megakaryocyte proliferation, ploidy maturation, and resultant platelet count reduction. Furthermore, our previous study demonstrated that GPIbα deficiency in mice and patients with Bernard-Soulier syndrome (BSS) results in reduced circulating TPO levels. Surprisingly, this decrease in TPO levels is not due to increased TPO clearance by platelets lacking GPIbα but rather a decrease in TPO production, which is regulated by GPIbα through the interaction with liver cells. Based on these findings, we hypothesize that augmenting exogenous TPO levels may effectively promote platelet production in GPIbα deficiency. We first generated C57/BL6 background GPIbα deficiency mice and measured their platelet levels using an automatic blood routine counter, confirming previous findings of a lower platelet count in GPIbα deficiency mice. Subsequently, both GPIbα deficiency and wild-type mice were intraperitoneally injected with recombinant mouse TPO (rmTPO) simultaneously. Surprisingly, we observed that the GPIbα deficiency mice exhibited reduced reactivity to TPO, resulting in a significant decrease in platelet production compared to wild-type mice. To elucidate it mechanism, we conducted comprehensive studies in vitro, isolating bone marrow-derived lin- cells from both GPIbα-deficient and wild-type mice using magnetic beads. These cells were then cultured in the presence of rmTPO, and the results revealed that GPIbα deficiency led to the inhibition of megakaryocyte cell ploidy maturation and platelet production compared to wild-type mice. Furthermore, we designed RNA small interference sequences targeting GPIbα. By conducting in vitro interference experiment using wild-type mice bone marrow-derived lin- cells with TPO, the knockdown of GPIbα resulted in inhibited megakaryocyte cell ploidy maturation and platelet production, consistent with the findings from the deficiency mice. In addition to our prior experiments investigating the GPIbα mediated TPO generation, we have also explored the impact of GPIbα antibodies on TPO production, revealing a significant reduction in TPO levels among GPIbα-positive immune thrombocytopenia (ITP) patients. These data raise our curiosity regarding the responsiveness to TPO in ITP, particularly among GPIbα-positive ITP patients. To verify our hypothesis, a retrospective analysis was performed to assess the response to TPO-related treatments in ITP patients, including eltrombopag, romiplostim, avatrombopag and recombinant human thrombopoietin (rhTPO). The outcomes observed in ITP patients positive for GPIbα antibodies revealed noteworthy patterns. The retrospective analysis revealed that ITP patients exhibiting GPIbα positivity displayed a worse response to thrombopoietin receptor agonists (TPO-RA) and rhTPO treatment. To gain deeper insights into the aforementioned findings, we conducted in vitro investigations on antibody-bound megakaryocytes. Specifically, we cultured wild-type mice bone marrow-derived lin- cells in the presence of TPO, with or without anti-GPIbα antibody intervention. The antibody group exhibited inhibition of megakaryocyte ploidy maturation and platelet production compared to the negative control group. In summary, our findings suggest that GPIbα plays a critical role in control of TPO reactivity, and its absence or interference can lead to impaired megakaryocyte ploidy maturation and platelet production. Moreover, ITP patients with anti-GPIbα antibody is associated with decreased TPO responsiveness, probably contributing to the observed poor response to TPO-based treatments. These findings have significant clinical implications, shedding light on platelet-related disorders and potential therapeutic strategies. Specifically, understanding the influence of GPIbα on megakaryocyte TPO reactivity will offer avenues for novel therapeutic interventions targeting platelet-related disorders.

Logical modelling of myelofibrotic microenvironment predicts dysregulated progenitor stem cell crosstalk

Primary myelofibrosis is an untreatable age-related disorder of haematopoiesis in which a break in the crosstalk between progenitor Haematopoietic Stem Cells (HSCs) and neighbouring mesenchymal stem cells causes HSCs to rapidly proliferate and migrate out of the bone marrow. Around 90% of patients harbour mutations in driver genes that all converge to overactivate haematopoietic JAK-STAT signalling, which is thought to be critical for disease progression, as well as microenvironment modification induced by chronic inflammation. The trigger to the initial event is unknown but dysregulated thrombopoietin (TPO) and Toll-Like Receptor (TLR) signalling are hypothesised to initiate chronic inflammation which then disrupts stem cell crosstalk. Using a systems biology approach, we have constructed an intercellular logical model that captures JAK-STAT signalling and key crosstalk channels between haematopoietic and mesenchymal stem cells. The aim of the model is to decipher how TPO and TLR stimulation can perturb the bone marrow microenvironment and dysregulate stem cell crosstalk. The model predicted conditions in which the disease was averted and established for both wildtype and ectopically JAK mutated simulations. The presence of TPO and TLR are both required to disturb stem cell crosstalk and result in the disease for wildtype. TLR signalling alone was sufficient to perturb the crosstalk and drive disease progression for JAK mutated simulations. Furthermore, the model predicts probabilities of disease onset for wildtype simulations that match clinical data. These predictions might explain why patients who test negative for the JAK mutation can still be diagnosed with PMF, in which continual exposure to TPO and TLR receptor activation may trigger the initial inflammatory event that perturbs the bone marrow microenvironment and induce disease onset.

Regulation of megakaryo/thrombopoiesis by endosomal toll-like receptor 7 and 8 activation of CD34+ cells in a viral infection model

BackgroundCD34+ cells, megakaryocytes (MKs), and platelets express toll-like receptors (TLRs) that enable these cells to amplify the host innate immune response. However, the role of TLR7/TLR8 activation in megakaryopoiesis has not yet been investigated. ObjectivesWe evaluated the effect of coxsackievirus B3 (CVB3) and synthetic TLR7/TLR8 agonists on the development of human MKs and production of platelets. MethodsCD34+ cells from human umbilical cord were inoculated with CVB3 or stimulated with synthetic TLR7/TLR8 agonists and then cultured in the presence of thrombopoietin. ResultsCD34+ cells, MK progenitor cells, and mature MKs expressed TLR7 and TLR8, and exposure to CVB3 resulted in productive infection, as determined by the presence of viral infectious particles in culture supernatants. Cell expansion, differentiation into MKs, MK maturation, and platelet biogenesis were significantly reduced in CD34+-infected cultures. The reduction in MK growth was not due to an alteration in cellular proliferation but was accompanied by an increase in cellular apoptosis and pyroptosis. Impairment of MK generation and maturation of viable cells were also associated with decreased expression of transcription factors involved in these processes. These effects were completely abrogated by TLR7 but not TLR8 antagonists and mimicked by TLR7 but not TLR8 agonists. CVB3 infection of CD34+ cells increased the immunophenotype of MKs characterized as CD148+/CD48+ or CD41+/CD53+ cells. ConclusionThese data suggest a novel role of TLR7 in megakaryo/thrombopoiesis that may contribute to a better understanding of the molecular basis underlying thrombocytopenia and the immunologic role of MKs in viral infection processes.

Abstract 13784: Thrombopoietin Restores Megakaryopoiesis By Protecting Bone Marrow Endothelial Progenitor Cells Of Patients With Hematologic Malignancies Undergoing Chemotherapy

Introduction and Objective: Thrombocytopenia is the most common side effect of patients with hematologic malignancies undergoing chemotherapy. Platelet production depends on a good bone marrow (BM) microenvironment, especially the endothelial progenitor cells (EPC), which play an important role in angiogenesis and thus supporting megakaryopoiesis. However, little is known regarding the function of EPC in patients with hematologic malignancies. Therefore, the present study aimed to examine the role of EPC in angiogenesis and megakaryopoiesis, and the effect of thrombopoietin (TPO) on EPC in patients with hematologic malignancies. Methods: Twenty-three patients with ALL or AML, and ten age-matched healthy donors were recruited in the present study. EPCs were isolated from BM mononuclear cells. The cell count, proliferation, migration, and tube formation assays were conducted. Megakaryocytes were quantified by flow cytometry after coculturing with EPC in the absence and presence of TPO (100 ng/ml). In addition, transgenic zebrafish with vascular endothelial cells expressing EGFP [Tg(fli-1:EGFP)] were used to examine the angiogenic effect of TPO. Simvastatin was used to induce vascular defects in zebrafish embryos. The embryos were then treated with or without TPO (10 -3 U/μl) by intravenous injection before fluorescent images were captured. Results: The number of BM EPC was significantly smaller in patients with ALL and AML than in healthy donors. The BM EPCs from patients showed significantly reduced proliferation, migration and tube formation when compared to those from healthy donors. TPO (100 ng/ml) significantly improved those functions of EPC, and significantly increased the number of megakaryocytes after coculture. In Tg(fli-1:EGFP) zebrafish embryos, TPO (10 -3 U/μl) significantly increased the SIV vascularized area in the presence of simvastatin. Conclusion: The present study shows that dysfunctional BM EPC contributes to the pathogenesis of thrombocytopenia in patients with hematologic malignancies undergoing chemotherapy; and such impaired functions of BM EPC could be restored by TPO. In addition, TPO increases the number of megakaryocytes after coculture with EPC in vitro and promotes angiogenesis in zebrafish embryos.

β1,4-Galactosyltransferase 1 Is a Key Regulator of Thrombopoiesis

Sialylated derivatives of N-Acetyl Lactosamine (LacNAc) regulate platelet lifespan, hepatic thrombopoietin (TPO) production, and thus thrombopoiesis. A major enzyme that synthesizes LacNAc is β1,4-galactosyltransferase 1 (β4GalT1), the gene of which is highly conserved through evolution. Long considered a housekeeping gene, the transcriptional regulation of B 4GALT1 remained widely understudied, and recent observations have shown that itspromoter region is rich in enhancer sequences.Both TPO and the chemokine CXCL12 regulate thrombopoiesis. Here we report that TPO and CXCL12 regulate megakaryocyte (MK) function by increasing β 4galt1 transcription . TPO and CXCL12 induced a two-wave expression of LacNAc in MKs. TPO was required for LacNAc production in wild type (WT) MKs, and β4GalT1 deletion in mice (β4GalT1-/-) resulted in severe thrombocytopenia due to increased integrin activity in MKs, and defective MK localization within the bone marrow (BM).Detailed analysis of β4GalT1-/- MKs and platelets showed that β4GalT1-/- MKs are almost completely devoid of LacNAc expression. Complete lack of galactosyltransferase activity in β4GalT1-/- MKs and platelets was demonstrated using in vitro enzymatic assays, showing that LacNAc synthesis in MKs and platelets depends primarily on β4GalT1. In WT MKs cultured in the presence of TPO alone, and in isolated circulating platelets, LacNAc expression was particularly evident in polypeptides with molecular weight ranging between 100-150 kDa, which were identified as αIIbβ3, GPIbα and β1 integrin. Addition of CXCL12 in vitro increased LacNAc expression specifically on a 130-kDa polypeptide, identified as the β1 integrin subunit. Further detailed investigation of receptor expression and function showed that WT MKs expressed two β1 integrin glycoforms of 130 and 100 kDa, and WT platelets expressed only the 130-kDa glycoform. β4GalT1-/- MKs and platelets expressed only the immature 100-kDa β1 integrin glycoform. While the subcellular distribution β1 integrin was indistinguishable between WT and β4GalT1-/- MKs and platelets, lack of LacNAc synthesis resulted in increased of β1 integrin surface expression, as determined by flow cytometry. β4GalT1-/- platelet adhesion to collagen and laminin was significantly increased and was unaffected by addition of RGDS. By contrast, GPIbα and αIIbβ3 expression and function were not affected by β4GalT1 loss.Together, the results suggest that TPO and CXCL12 upregulate β4GalT1-dependent LacNAc synthesis to promote thrombopoiesis, likely regulating MK β1 integrin expression, glycosylation and subsequent interaction with components of the extracellular matrix. Investigating the mechanisms regulating posttranslational modifications induced by various hematopoietic stimulating chemokines and cytokines will contribute to better understand thrombopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Concomitant Occurrence of Polyclonal Hematopoiesis and Cell-Autonomous Megakaryopoiesis in Triple-Negative Essential Thrombocythemia

Somatic mutations in JAK2, MPL, and CALR are found in approximately 80% of patients with essential thrombocythemia (ET), whereas the remaining patients are negative for disease-defining mutations and are defined as triple-negative (TN). Studies have shown that some patients with TN-ET harbor non-canonical mutations in JAK2 and MPL; however, the failure to identify recurrent mutations in most patients has made the pathogenesis of TN-ET ambiguous (Milosevic Feenstra et al. Blood 2016, Cabagnols et al. Blood 2016). In this study, we screened 483 patients suspected as having ET in a single center, performed mutation analysis for JAK2 V617F, CALR exon 9, and MPL exon 10, and centrally reviewed bone marrow specimens. We identified 23 patients with TN-ET based on the WHO 2016 criteria. Sequencing analysis of these patients revealed non-canonical mutations in JAK2 and MPL in 4 cases. Whole exome-sequencing analysis of genomic DNA from peripheral blood and CD3-positive cells from 9 patients revealed that 2 patients harbored somatic mutations in other genes; 7 patients showed no detectable somatic mutation. A STAT5 reporter assay revealed that unlike JAK2 V617F and MPL W515L, all non-canonical mutants of JAK2 or MPL activated STAT5 similar to wild-type proteins, suggesting that these mutations did not drive the disease. Statistical analysis of clinical records revealed that patients with TN-ET were mostly young (median age of 36.0 years), female (18/23, 78.3%), and had neither a history of thrombosis nor progression to secondary myelofibrosis and leukemia, demonstrating the unique characteristics of TN-ET. The presence of clonal hematopoiesis, analyzed using genomic DNA purified from granulocytes of peripheral blood from female patients in a human androgen receptor assay, revealed that only 1 out of 15 patients was clonal. Hypothesizing that TN-ET was reactive thrombocytosis, the concentrations of cytokines promoting platelet production such as thrombopoietin (TPO) and interleukin-6 (IL-6) in the serum were analyzed. However, no significant differences in concentrations were observed among ET with driver mutations, TN-ET, and healthy individuals. We next examined the capacity of hematopoietic stem cells from patients with TN-ET to form megakaryocytic colonies. CD34-positive cells purified from cryopreserved bone marrow cells were cultured in the absence or presence of TPO. CD34-positive cells derived from patients with TN-ET exhibited an equivalent capacity to form megakaryocytic colonies compared to those from patients with ET harboring a driver mutation, even in the absence of TPO (Figure 1). Thus, in TN-ET, megakaryopoiesis may have been induced in a cell-autonomous manner. In 10 patients with TN-ET with available blood count data, no sign of thrombocytosis was observed before ET development, indicating that thrombocytosis was not hereditary but rather occurred via an alternate mechanism, such as aberrations in epigenomic regulation that induced cellular transformation (Ohnishi et al, Cell 2015). Taken together, TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis. Figure 1 Disclosures Komatsu: Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy.

Megakaryocyte-Derived TGF-β1 Negatively Regulates Megakaryopoiesis in Mice

Megakaryocytes (MK) and platelets contain a high concentration of transforming growth factor β1 (TGFβ1). Mice with conditional deletion of Tgfb1 in megakaryocytes (PF4Cre/Tgfb1flox/flox) resulted in >90% reduction of TGFβ1 in platelets and 50% reduction of TGFβ1 in plasma. TGFβ1 has been shown to play an inhibitory role in megakaryopoiesis in vitro, and inhibiting TGFβ1 increased megakaryopoiesis in vivo. However, the source of TGFβ1 in megakaryopoiesis is unknown. In this study, we tested whether megakaryocyte-derived TGFβ1 contributes to megakaryopoiesis in bone marrow (BM) by comparing three groups of mice: PF4Cre/Tgfb1flox/flox, littermate control Tgfb1flox/flox, and WTC57Bl/6 mice. Bones (femurs) from these mice (n=12) (age 15-30 weeks, males 60% and females 40%) were harvested, fixed, decalcified, sectioned, and H&E stained. Whole stained BM areas of the sectioned femurs were scanned with an Aperio slide scanner to quantify the number of megakaryocytes and the demarcation membrane system (DMS) and ploidy (nucleus size) of the megakaryocytes were quantified by manually counting megakaryocytes and tracing their DMS and nucleus. The percentage of MK among total BM cells was calculated by dividing total numbers of BM cells in the total area of a BM section with the number of MK in the section. Freshly isolated BM cells were cultured in vitro in culture medium (DMEM+10%FBS) in the presence of thrombopoietin (TPO, 100 ng/ml) with and without TGFβ1 (20 ng/ml) or with a neutralized antibody against the active form of TGFβ1 (AF-101; 2 ug/ml). TGFβ1 and TPO levels in plasma, BM exudates, and cells were measured by ELISA. PF4Cre/Tgfb1flox/floxmice had >50% reduction in TGFβ1 levels in BM cells and exudates (TGFβ1 levels in BM exudates were 1.4 ± 0.033 ng in WT and 0.68 ± 0.065 ng in PF4CreTgfb1flox/floxmice, p<0.01; and in BM cells 50 ± 9 ng/ml in WT and 22 ± 4.2 ng/ml in PF4CreTgfb1flox/flox; p<0.001). MK numbers were ~25% higher in PF4Cre/Tgfb1flox/floxmice (n=6) compared to combined littermate controls (n=3) and WT (n=3) (MK was 0.30 ± 0.02% in PF4Cre/Tgfb1flox/flox and 0.23 ± 0.16% in combined controls; p<0.001 (n=6), whereas blood platelet counts were only marginally higher in PF4Cre/Tgfb1flox/flox (1114 ± 300) vs. controls (806 ± 116; p<0.05). There was a ~2-fold higher plasma TPO levels in PF4CreTgfb1flox/floxmice vs. WT (p=0.04, n=4). Increased DMS and nucleus areas in MK have been shown to correlate with proplatelets formation and platelet production. However, DMS and nuclear areas remained unchanged between genotypes [(DMS area was 197 ± 46 in PF4CreTgfb1flox/flox and 228 ± 50 um2 in combined WT and littermate controls (p=0.3), and nucleus size was 154 ± 23 in PF4CreTgfb1flox/flox and 160 ± 33 um2 in controls (p=0.7)], indicating that the role of TGFβ1 is limited to megakaryopoiesis. To test whether the in vivo phenotype was recapitulated, we cultured whole BM isolated from WT and PF4Cre/Tgfb1flox/flox mice, which showed a ~2.5-fold increase in MK numbers vs. WT when cultured for 5 days in TPO-supplemented medium. The addition of recombinant TGFβ1 in culture medium inhibited MK numbers, and a neutralizing antibody against TGFβ1 resulted in increased MK numbers. We conclude that MK-derived TGFβ1 negatively regulates megakaryopoiesis in mice. Further investigation is needed to determine the mechanism by which TGFβ1 regulates TPO-induced megakaryopoiesis. Our study may be important in megakaryocyte generation in vitro and may have important implications in vivo under normal and stress-inducing conditions where variable megakaryopoiesis is observed, such as essential thrombocythemia and primary myelofibrosis. Disclosures No relevant conflicts of interest to declare.

Augmentation of NK Cell Proliferation and Anti-tumor Immunity by Transgenic Expression of Receptors for EPO or TPO

Many investigational adoptive immunotherapy regimens utilizing natural killer (NK) cells require the administration of interleukin-2 (IL-2) or IL-15, but these cytokines cause serious dose-dependent toxicities. To reduce or preclude the necessity for IL-2 use, we investigated whether genetic engineering of NK cells to express the erythropoietin (EPO) receptor (EPOR) or thrombopoietin (TPO) receptor (c-MPL) could be used as a method to improve NK cell survival and function. Viral transduction of NK-92 cells to express EPOR or c-MPL receptors conveyed signaling via appropriate pathways, protected cells from apoptosis, augmented cellular proliferation, and increased cell cytotoxic function in response to EPO or TPO ligands invitro. In the presence of TPO, viral transduction of primary human NK cells to express c-MPL enhanced cellular proliferation and increased degranulation and cytokine production toward target cells invitro. In contrast, transgenic expression of EPOR did not augment the proliferation of primary NK cells. In immunodeficient mice receiving TPO, invivo persistence of primary human NK cells genetically modified to express c-MPL was higher compared with control NK cells. These data support the concept that genetic manipulation of NK cells to express hematopoietic growth factor receptors could be used as a strategy to augment NK cell proliferation and antitumor immunity.

Activation of toll‐like receptors 2 and 4 on CD34+ cells increases human megakaryo/thrombopoiesis induced by thrombopoietin

BackgroundPlatelet Toll‐like receptor (TLR)2/4 are key players in amplifying the host immune response; however, their role in human megakaryo/thrombopoiesis has not yet been defined. ObjectivesWe evaluated whether Pam3CSK4 or lipopolysaccharide (LPS), TLR2/4 ligands respectively, modulate human megakaryocyte development and platelet production. MethodsCD34+ cells from human umbilical cord were stimulated with LPS or Pam3CSK4 with or without thrombopoietin (TPO). ResultsCD34+ cells and megakaryocytes express TLR2 and TLR4 at both RNA and protein level; however, direct stimulation of CD34+ cells with LPS or Pam3CSK4 had no effect on cell growth. Interestingly, both TLR ligands markedly increased TPO‐induced CD34+ cell proliferation, megakaryocyte number and maturity, proplatelet and platelet production when added at day 0. In contrast, this synergism was not observed when TLR agonists were added 7 days after TPO addition. Interleukin‐6 (IL‐6) release was observed upon CD34+ or megakaryocyte stimulation with LPS or Pam3CSK4 but not with TPO and this effect was potentiated in combination with TPO. The increased proliferation and IL‐6 production induced by TPO + LPS or Pam3CSK4 were suppressed by TLR2/4 or IL‐6 neutralizing antibodies, as well as by PI3K/AKT and nuclear factor‐κB inhibitors. Additionally, increased proplatelet and platelet production were associated with enhanced nuclear translocation of nuclear factor‐E2. Finally, the supernatants of CD34+ cells stimulated with TPO+LPS‐induced CFU‐M colonies. ConclusionsOur data suggest that the activation of TLR2 and TLR4 in CD34+ cells and megakaryocytes in the presence of TPO may contribute to warrant platelet provision during infection episodes by an autocrine IL‐6 loop triggered by PI3K/NF‐κB axes.

Rap1 signal modulators control the maintenance of hematopoietic progenitors in bone marrow and adult long-term hematopoiesis.

Adult long‐term hematopoiesis depends on sustaining hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) niches, where their balance of quiescence, self‐renewal, and hematopoietic differentiation is tightly regulated. Although various BM stroma cells that produce niche factors have been identified, regulation of the intrinsic responsiveness of HSPC to the niche factors remains elusive. We previously reported that mice deficient for Sipa1, a Rap1 GTPase‐activating protein, develop diverse hematopoietic disorders of late onset. Here we showed that transplantation of BM cells expressing membrane‐targeted C3G (C3G‐F), a Rap1 GTP/GDP exchanger, resulted in the progressive decline of the numbers of HSPC repopulated in BM with time and impaired long‐term hematopoiesis of all cell lineages. C3G‐F/HSPC were sustained for months in spleen retaining hematopoietic potential, but these cells inefficiently contributed to overall hematopoietic reconstitution. C3G‐F/HSPC showed enhanced proliferation and differentiation with accelerated progenitor cell exhaustion in response to stem cell factor (SCF). Using a Ba/F3 cell line, we confirmed that the increased basal Rap1GTP levels with C3G‐F expression caused a markedly prolonged activation of c‐Kit receptor and downstream signaling through SCF ligation. A minor population of C3G‐F/HSPC also showed enhanced proliferation in the presence of thrombopoietin (TPO) compared to Vect/HSPC. Current results suggest an important role of basal Rap1 activation status of HSPC in their maintenance in BM for sustaining long‐term adult hematopoiesis.

CD110 promotes pancreatic cancer progression and its expression is correlated with poor prognosis.

This study aimed at investigating the function and significance of CD110 expression in pancreatic cancer. We performed immunohistochemical staining for CD110 expression in tumor samples from 86 patients with pancreatic cancer. We evaluated clinical outcomes and other clinicopathological factors to determine the significance of CD110 on survival and liver metastasis. We examine thrombopoietin-CD110 signaling in cancer cell extravasation in vitro and in vivo. We investigated the effects of CD110 knockdown on liver metastasis in a splenic xenograft mouse model. CD110 expression in cancer cells was associated with low-histological-grade invasive ductal carcinoma, and patients with high CD110 expression had poorer prognosis (P = 0.0003). High CD110 expression was an independent predictor of liver metastasis (P = 0.0422). Knockdown of CD110 expression significantly attenuated cell migration and invasion. Treatment with thrombopoietin promoted pancreatic cancer cell extravasation. In the presence of thrombopoietin, CD110 increased cell viability through the activation of the ERK-MYC signaling pathway. Knockdown of CD110 expression inhibited liver metastases in the mouse model. CD110 promotes pancreatic cancer progression and it may serve as a predictive factor for liver metastasis.

N‐methyl‐d‐aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

Unlabelled Box BackgroundN‐methyl‐d‐aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro‐ and anti‐differentiating effects have been shown in different contexts. ObjectivesThe aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells. MethodsMeg‐01, Set‐2, and K‐562 leukemic cell lines were differentiated using phorbol‐12‐myristate‐13‐acetate (PMA, 10 nmol L−1) or valproic acid (VPA, 500 μmol L−1). Normal megakaryocytes were grown from mouse marrow‐derived hematopoietic progenitors (lineage‐negative and CD41a‐enriched) in the presence of thrombopoietin (30‐40 nmol L−1). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK‐801 (100 μmol L−1); their effects compared against appropriate controls. ResultsThe most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK‐801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg‐01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves. ConclusionsNMDAR‐mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

Lnk Deficiency Leads to TPO-Mediated Osteoclastogenesis and Increased Bone Mass Phenotype.

The Lnk adapter protein negatively regulates the signaling of thrombopoietin (TPO), the main megakaryocyte (MK) growth factor. Lnk-deficient (-/-) mice have increased TPO signaling and increased MK number. Interestingly, several mouse models exist in which increased MK number leads to a high bone mass phenotype. Here we report the bone phenotype of these mice. MicroCT and static histomorphometric analyses at 20 weeks showed the distal femur of Lnk-/- mice to have significantly higher bone volume fraction and trabecular number compared to wild-type (WT) mice. Notably, despite a significant increase in the number of osteoclasts (OC), and decreased bone formation rate in Lnk-/- mice compared to WT mice, Lnk-/- mice demonstrated a 2.5-fold greater BV/TV suggesting impaired OC function in vivo. Additionally, Lnk-/- mouse femurs exhibited non-significant increases in mid-shaft cross-sectional area, yet increased periosteal BFR compared to WT femurs was observed. Lnk-/- femurs also had non-significant increases in polar moment of inertia and decreased cortical bone area and thickness, resulting in reduced bone stiffness, modulus, and strength compared to WT femurs. Of note, Lnk is expressed by OC lineage cells and when Lnk-/- OC progenitors are cultured in the presence of TPO, significantly more OC are observed than in WT cultures. Lnk is also expressed in osteoblast (OB) cells and in vitro reduced alkaline phosphatase activity was observed in Lnk-/- cultures. These data suggest that both direct effects on OB and OC as well as indirect effects of MK in regulating OB contributes to the observed high bone mass. J. Cell. Biochem. 118: 2231-2240, 2017. © 2017 Wiley Periodicals, Inc.

Thrombopoietin contributes to the formation and the maintenance of hematopoietic progenitor-containing cell clusters in the aorta-gonad-mesonephros region

In the midgestation mouse embryo, hematopoietic cell clusters containing hematopoietic stem/progenitor cells arise in the aorta-gonad-mesonephros (AGM) region. We have previously reported that forced expression of the Sox17 transcription factor in CD45lowc-Kithigh AGM cells, which are the hematopoietic cellular component of the cell clusters, and subsequent coculture with OP9 stromal cells in the presence of three cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and thrombopoietin (TPO), led to the formation and the maintenance of cell clusters with cells at an undifferentiated state in vitro. In this study, we investigated the role of each cytokine in the formation of hematopoietic cell clusters. We cultured Sox17-transduced AGM cells with each of the 7 possible combinations of the three cytokines. The size and the number of Sox17-transduced cell clusters in the presence of TPO, either alone or in combination, were comparable to that observed with the complete set of the three cytokines. Expression of TPO receptor, c-Mpl was almost ubiquitously expressed and maintained in Sox17-transduced hematopoietic cell clusters. In addition, the expression level of c-Mpl was highest in the CD45lowc-Kithigh cells among the Sox17-transduced cell clusters. Moreover, c-Mpl protein was highly expressed in the intra-aortic hematopoietic cell clusters in comparison with endothelial cells of dorsal aorta. Finally, stimulation of the endothelial cells prepared from the AGM region by TPO induced the production of hematopoietic cells. These results suggest that TPO contributes to the formation and the maintenance of hematopoietic cell clusters in the AGM region.

Thrombopoietin Secretion by Human Ovarian Cancer Cells.

The thrombopoietin (TPO) gene expression in human ovary and cancer cells from patients with ovarian carcinomatosis, as well as several cancer cell lines including MDA-MB231 (breast cancer), K562 and HL60 (Leukemic cells), OVCAR-3NIH and SKOV-3 (ovarian cancer), was performed using RT PCR, real-time PCR, and gene sequencing. Human liver tissues are used as controls. The presence of TPO in the cells and its regulation by activated protein C were explored by flow cytometry. TPO content of cell extract as well as plasma of a patient with ovarian cancer was evaluated by ELISA. The functionality of TPO was performed in coculture on the basis of the viability of a TPO-dependent cell line (Ba/F3), MTT assay, and Annexin-V labeling. As in liver, ovarian tissues and all cancer cells lines except the MDA-MB231 express the three TPO-1 (full length TPO), TPO-2 (12 bp deletion), and TPO-3 (116 pb deletion) variants. Primary ovarian cancer cells as well as cancer cell lines produce TPO. The thrombopoietin production by OVCAR-3 increased when cells are stimulated by aPC. OVCAR-3 cell's supernatant can replace exogenous TPO and inhibited TPO-dependent cell line (Ba/F3) apoptosis. The thrombopoietin produced by tumor may have a direct effect on thrombocytosis/thrombosis occurrence in patients with ovarian cancer.

Interferon-γ Perturbs Key Signaling Pathways Induced By Thrombopoietin, but Not Eltrombopag, in Human Hematopoietic Stem/Progenitor Cells

▪Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling, through JAK-STAT and other pathways, which is tightly balanced by negative regulatory signaling processes. Recent studies indicate that chronic exposure of HSPCs to IFNγ, as exemplified in subjects with severe aplastic anemia (SAA), impairs self-renewal by perturbing TPO signaling pathways. Despite elevated levels of TPO in subjects with SAA, the TPO receptor agonist Eltrombopag (Epag) improves trilineage hematopoiesis in refractory SAA, suggesting that it may activate signaling within HSPC in a way that is distinct from TPO under inflammatory conditions. To address the paradox of Epag efficacy despite high endogenous TPO levels in bone marrow failure, G-CSF mobilized human CD34+ cells from 6 healthy donors were cultured in the presence of SCF, FLT3 and either TPO 5 ng/ml (TPO5) or Epag 3 μg/ml (Epag), with or without IFNγ 100 ng/ml. After 7 days in culture, cells were characterized via flow cytometry, CFU assay and transplantation in immunodeficient (NSG) mice. The percentages of CD34+ cells in cultures containing TPO5 or Epag alone were similar (83.3 ± 9.7% and 87.6 ± 7.1%, respectively), but were better preserved with Epag than TPO5 in the presence of IFNγ (46.7 ± 16.1% and 24.6 ± 15.0% respectively, p<0.05). Accordingly, when comparing 7-day cultures with and without IFNγ, the absolute numbers of CD34+ cells were markedly reduced with TPO5 (average 7.6-fold, p<0.005) but only minimally decreased with Epag (average 1.6-fold, p = n.s.). The adjusted numbers of CFUs after 7 days in the presence of IFNγ similarly decreased 2.7-fold with TPO5 but remained unchanged with Epag compared to cultures without IFNγ. When the 7-day expanded progeny of an equal starting number of CD34+ cells was transplanted in NSG mice, human cell engraftment was superior with Epag (34 ± 3.8% human CD45+ cells) than with TPO5 (21 ± 1.8% human CD45+ cells, p<0.05) cultures in the presence of IFNγ, suggesting an impact of Epag on the most primitive long-term repopulating HSPCs. To investigate potential mechanisms by which Epag positively affects maintenance of HSPCs under inflammatory conditions, we examined cell signaling pathways induced upon binding of TPO, Epag and IFNγ to their respective receptors in human CD34+ cells. At a concentration of 5ng/mL, TPO induced a rapid (peak < 1 hour) and high potency rise in STAT5 phosphorylation followed by a rapid (< 2 hours) decay in signal. In contrast, Epag induced a slow (peak 4 hours) low potency rise in STAT5 phosphorylation, and the signal persisted for at least 10 hours. The difference in cell signaling potency and kinetics between TPO and Epag is likely related to their binding to distinct regions of c-MPL, resulting in alternate receptor conformational changes. We next investigated the impact of IFNγ on TPO and Epag-induced STAT5 phosphorylation at the signal peak (<1 and 4 hours, respectively). As previously shown in murine HSPCs, IFNγ impaired TPO signaling in human HSPCs (Figure, panels A, C). In contrast, Epag-induced STAT5 phosphorylation was preserved or increased in the presence of IFNγ (Figure, panels B, C). When Epag and TPO were combined, inhibition of TPO signaling by IFNγ was partially restored (Figure, panel D). By reducing the dose of TPO from 5 to 1ng/mL, and therefore reducing the potency of signaling to levels similar to Epag, the inhibitory effect of IFNγ on TPO signaling was abolished (Figure, panel E). Activation of IFNγ receptor by its ligand induces phosphorylation of STAT1 and subsequent expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of both IFNγ and c-MPL receptors via inhibition of STAT1 and STAT5 phosphorylation, respectively. We found that IFNγ-induced phosphorylation of STAT1 was increased in the presence of TPO 5ng/mL (1.5-fold increase, p<0.05) but unaffected by Epag (1.1-fold increase, p = n.s.) or TPO 1ng/mL (1.1-fold increase, p = n.s.). Our data indicate that Epag counters IFNγ-induced perturbation of TPO signaling in human HSPCs. Epag produces an unopposed low potency, slow kinetic positive signal and activates c-Mpl above a threshold level critical for HSPC self-renewal. Epag’s evasion of IFN blockade of a critical pathway of growth factor cell signaling may explain its efficacy in improving hematopoiesis in SAA. [Display omitted] DisclosuresCheng:Novartis: Research Funding. Cheruku:Novartis: Research Funding. Alvarado:Novartis: Research Funding. Cash:Novartis: Research Funding. Dunbar:Novartis: Research Funding. Young:Novartis: Research Funding. Larochelle:Novartis: Research Funding.

Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown.In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+ mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/- (single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred.Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation.To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling.Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Identification of a Novel MPL Loss of Function Mutation in a Patient with Cyclic Thrombocytopenia and Characterization of This Syndrome

Introduction: Cyclic thrombocytopenia (CT) is a rare disease characterized by periodic fluctuations in platelet counts. The etiology of this disease has not been fully elucidated. Here, we present molecular characterization of CT in a 53-year-old male patient who has platelet counts ranging between 1 x 109/L to >400 x 109/L during a cycle period of 40 days which has occurred over several years.Methods: Blood transcriptome profiles and plasma cytokine levels were investigated in 24 samples that were sequentially collected every 3-4 days to cover two complete cycles. Total RNA was extracted after lysing red blood cells. 3SEQ (3′-end RNA sequencing for expression quantification) was performed for transcriptome profiling. SAMSeq quantitative analyses were conducted to identify differentially expressed genes. Three screening criteria were applied to this data set to identify a panel of exclusive platelet-specific genes. Plasma TPO (thrombopoietin)/testosterone/ estradiol/CD41a/CD42b levels were detected by ELISA. A total of 62 cytokines were screened using a luminex immunoassay. MPL (TPO receptor) gene was Sanger-sequenced using blood DNA and mRNA templates. DNA was extracted from the patient’s hair follicles to exam the presence of the identified MPL mutation, and in-silico predictions were conducted to evaluate the MPL mutation. The wild-type (WT) and c.1210G>A mutant MPL expression constructs were generated and transfected into Ba/F3 cells for stable MPL expression. TPO-stimulated, IL-3 independent growth of MPL-expressing Ba/F3 cells were assessed to determine the function of MPL proteins.Results: Plasma TPO levels cycled between 6 to 2745 pg/mL during the sampling period; TPO levels inversely mirrored the fluctuation of platelet counts and preceded the latter by 3-4 days. Additional plasma cytokines demonstrated either an inverse correlation (e.g. FasL, GM-CSF, and TNF-β, etc.), or well synchronized correlation (e.g. BDNF, PDGF-BB, and RANTES etc.), with platelet counts. The patient was negative for platelet autoantibodies. Transcriptome analysis revealed 977 genes with expression that positively correlated with platelet count changes. Unsupervised clustering stratified these genes into subgroups, including: i) platelet-specific genes, which precede platelet count changes by 3-4 days; ii) platelet-modulated genes, which follow platelet count changes and are regulated by coagulation factors (e.g. F2) and platelet-contained growth factors (e.g. PDGF-BB and EGF); and iii) neutrophil-specific genes, which precede platelet count changes by 7-10 days. Among platelet-specific genes, 34 genes were identified to have >50-fold induction over a cycle, and are present in the platelet transcriptome [Rowley JW, 2011]. A heat map of these exclusive platelet genes is shown in Figure 1A.A novel MPL c.1210G>A heterozygous mutation, which leads to a p.Gly404Arg substitution, was identified in both blood and hair follicle DNA samples of the patient. This substitution resides in a highly conserved site and is predicted to be deleterious. In addition, the mutation creates a novel splice site, which results in the insertion of a 36 bp intron fragment in approximately 15% of MPL transcripts. In vitro analysis confirmed that MPL c.1210G>A is a loss-of-function (LOF) variant. Ba/F3 cells that express WT MPL exhibited IL-3 independent growth in the presence of TPO; however, the Gly404Arg mutant failed to support TPO-stimulated growth (Figure 1B).Conclusion: We describe a patient with CT who exhibits fluctuations of multiple cytokines including an inverse correlation between TPO and platelet counts, and profound gene expression changes in neutrophil- and platelet-specific gene expressions which precede platelet count fluctuations. These data suggest that the cyclic megakaryopoiesis and thrombopoiesis underlies pathogenesis of the disease. Moreover, the identified novel LOF MPL allele may contribute to the disease pathogenesis. The biologic and molecular annotation of this case presents a unique opportunity to examine the transcriptional regulation of platelet homeostasis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.