Cereblon harnesses Myc-dependent bioenergetics and activity of CD8+ T lymphocytes.

Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known "neosubstrates," such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.

HDAC11 deficiency disrupts oncogene-induced hematopoiesis in myeloproliferative neoplasms.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

Polycythemia vera transforming to myelofibrosis and then biphenotype acute leukemia.

BACKGROUND: A case with 17-year detailed illness history including evolution of polycythemia vera (PV) to myelofibrosis (MF) and then biphenotype acute leukemia (BAL) was reported. Ten years of PV followed by seven years of MF and then BAL, the patient experienced a classical "complete course" of myeloproliferative neoplasm (MPN). High WBC counts as well as low Hb and platelet counts in MF phase, long disease course, older than 50 years age, and positive JAK2 were her high risk factors of transformation from MPN to leukemia. Pancytopenia in her secondary MF phase responded well to the therapy of corticosteroids, which indicated that the immune mechanism was involved in the pathogenesis of MF. Progression of PV to MF and then BAL might be related to discontinuation of interferon-alpha because of poor tolerance.

[Changes of natural kill cell in peripheral blood of patients with myelodysplastic syndrome].

OBJECTIVE: To analyze the percentage and functional changes of natural kill (NK) cell in peripheral blood of myelodysplastic syndrome (MDS) patients so as to evaluate the relationships between these changes and hematopoietic functions and explore the role of NK cell in the pathogenesis of MDS. METHODS: By flow cytometry, the percentage of NK cell (CD3⁻CD56⁺CD16⁺) in peripheral blood lymphocytes was detected in 20 MDS patients, 15 acute myelogenous leukemic (AML) and 15 normal controls from March 2013 to November 2013 at our hospital.NK cell activation receptors (NKG2D, NKp44), inhibitory receptors (CD158a, CD158b), perforin and granzyme-ß of patients and normal controls were also detected. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil granulocyte (ANC%), lymphocyte (Lym%), reticulocyte (RET%) and hemoglobin, thrombocyte in peripheral blood and the hematopoietic function in bone marrow (CD34⁺%) were evaluated. RESULTS: (1) The percentage of NK cell (3.87% ± 0.97%) in MDS patients was significantly lower than that of normal controls (6.08% ± 1.37%, P < 0.05) and higher than that of AML patients (2.58% ± 0.78%, P < 0.05).(2) The expression of NKG2D in MDS patients (52.83%) was significantly lower than that of normal controls (86.36%, P < 0.05) and higher than that of AML patients (42.00%, P < 0.05). The expression of NKp44 in MDS patients (2.41%) was significantly higher than that of normal controls (0.62%) and AML patients (0.92%) (both P < 0.05). (3) The expression of CD158a in MDS and AML patients (5.46% ± 2.40%, 4.05% ± 1.89%) were significantly both lower than those of normal controls (7.97% ± 2.85%, both P < 0.05). The expression of CD158b had no significant difference among the 3 groups. (4) The expression of perforin in MDS patients (17.83%) was significantly lower than that of normal controls (59.79%, P < 0.05) and higher than that of AML patients (13.06%, P < 0.05). The expressions of granzyme-ß in MDS and AML patients (21.54%, 30.65%) were significantly both lower than those of normal controls (83.42%, both P < 0.05). (5) The percentage of NK cell and the expression of NKG2D and NKp44 in MDS patients with low-risk group were in turn higher than those of high-risk group (4.06% ± 0.56% vs 3.59% ± 1.37%, 53.42% ± 6.85% vs 47.29% ± 8.08%, 2.46% vs 2.07%, all P < 0.05) , the expression of CD158a and CD158b were in turn lower than those of high-risk group, but both P > 0.05.(6) The percentage of NK was positively correlated with ANC% (r = 0.780, P < 0.05), but negatively with Lym% and the CD34⁺% in bone marrow (r = -0.543, -0.610, both P < 0.05). The expression of CD158a was positively correlated with CD34⁺% in bone marrow (r = 0.612, P < 0.05). The expressions of NKp44, CD158a, perforin and granzyme-ß of NK cells had no correlation with hematopoiesis (all P > 0.05). CONCLUSION: The lowered percentage and function of NK may cause the immunological dysfunction and lead to underkill of pathological hematopoietic cells in MDS.

[TET2 and DLK1 gene expression and their clinical significance in bone marrow CD(3)(+) T cells of patients with myelodysplastic syndrome].

OBJECTIVE: To investigate the expression of TET2 and DLK1 mRNA in bone marrow CD(3)(+) T cells of patients with myelodysplastic syndrome (MDS) and their clinical significance and to explore the potential mechanism of abnormal cell-mediated immunity. METHODS: CD(3)(+) T cells were sorted by magnetic activated cell-sorting system. The expressions of TET2 and DLK1 mRNA in bone marrow CD(3)(+) T cells from 26 MDS patients and 16 healthy controls were detected by fluorescence quantitative PCR. RESULTS: The expression of TET2 mRNA in CD(3)(+) T cells was down-regulated in the MDS patients by (0.16 ± 0.15) fold compared with the controls (P < 0.05). The expression of TET2 mRNA in CD(3)(+) T cells of MDS patients was positively correlated with serum complement C(3) (r = 0.404, P < 0.05). The expression of DLK1 mRNA in CD(3)(+) T cells was up-regulated in the MDS patients by (1.61 ± 0.88) folds compared with the controls (P < 0.05). Grouped by the chromosomes, the patients with chromosome abnormalities presented significantly higher DLK1 mRNA level than those with normal chromosomes [(1.45 ± 0.44) folds, P < 0.05]. The expression of DLK1 mRNA in CD(3)(+) T cells of MDS patients was positively correlated with the proportion of bone marrow blasts (r = 0.343, P < 0.05). CONCLUSIONS: The mRNA expression of TET2 in CD(3)(+) T cells of MDS patients was decreased while the mRNA expression of DLK1 was increased, which might decline the immune surveillance function. The findings would be useful for exploring the mechanism of immune tolerance.

[Differentiation and expression of membrane hemopoietic cytokine receptors on CD34+ bone marrow cells in patients with myelodysplastic syndromes].

OBJECTIVE: To detect the abnormalities of differentiation and expression of membrane hemopoietic cytokine receptors on CD34+ bone marrow cells in patients with myelodysplastic syndromes (MDS). METHODS: Forty-five newly diagnosed MDS cases from July 2008 to March 2010 in our hospital and 30 normal controls were enrolled. There were 17 low-risk and 28 high-risk patients. The CD34+CD38+ and CD34+CD38- bone marrow cells and the expressions of stem cell factor receptor (SCF-R), erythropoietin receptor (EpoR), granulocyte colony-stimulating factor receptors (G-CSFR) and thrombopoietin receptor (TpoR) on those cells were measured by flow cytometry. RESULTS: The mean percentage of CD34+ in karyocyte of MDS cases in high-risk patients [0.53% (0.10%-1.68%)] was significantly higher than that of control group [0.13% (0.08%-0.32%), P<0.01]. The mean percentages of CD34+CD38+ cells were significantly lower in low and high-risk groups (86.3%±8.5% and 82.6%±11.1%) than those in control group (92.3%±3.4%). And the percentage of CD34+CD38- cells was significantly higher in either low-risk or high-risk group (13.7%±8.5% and 17.4%±11.0%) than that in control group (7.7%±3.4%, both P<0.05). In control group, the mean percentage of antigen expression of EpoR was significantly lower in CD34+CD38+ cells than that in CD34+CD38- cells (18.7%±18.3% vs 63.6%±20.0%, P<0.01). The expressions of SCF-R, G-CSFR and TpoR were not significantly different between two cell populations. The expressions of EpoR on CD34+CD38+ cells of low and high-risk MDS groups [9.0% (1.4%-12.7%), 5.2% (1.1%-14.1%)] were significantly lower than those of control group [9.6% (5.1%-30.1%), both P<0.05]. The expressions of G-CSFR on CD34+CD38+ cells of low and high-risk MDS groups (29.8%±19.1%, 28.7%±21.1%) were significantly lower than those of control group (44.4%±23.4%, both P<0.05). The quantities of EpoR on CD34+CD38- cells of low and high-risk MDS groups (42.2%±21.9%, 25.7%±15.6%) were significantly lower than those of control group (63.6%±20.0%, both P<0.01). The expressions of TpoR on CD34+CD38- cells of low and high-risk MDS groups (5.4%±4.7%, 4.1%±4.0%) were significantly lower than those of control group (10.1%±8.3%, both P<0.05). The incidence of cytopenia with low expression rates of hemopoietic cytokine receptors on CD34+ cells was higher than that of MDS with high expression rates. CONCLUSION: The abnormalities of differentiation and membrane hemopoietic cytokine receptors expression of CD34+ bone marrow cells in MDS are associated with MDS cytopenia and may be useful for the diagnosis of MDS.

Case Report: Sirolimus Alleviates Persistent Cytopenia After CD19 CAR-T-Cell Therapy.

Chimeric antigen receptor T (CAR-T) cells show good efficacy in the treatment of relapsed and refractory B-cell tumors, such as acute B-cell leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). The main toxicities of CAR-T include cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, cytopenia, and severe infection. It is still very difficult for CAR-T to kill tumor cells to the maximum extent and avoid damaging normal organs. Here, we report a case of DLBCL with persistent grade 4 thrombocytopenia and severe platelet transfusion dependence treated with CD19 CAR-T cells. We used sirolimus to inhibit the sustained activation of CAR-T cells and restore normal bone marrow hematopoiesis and peripheral blood cells. Moreover, sirolimus treatment did not affect the short-term efficacy of CAR-T cells, and DLBCL was in complete remission at the end of follow-up. In conclusion, sirolimus can represent a new strategy for the management of CAR-T cell therapy-related toxicity, including but not limited to hematotoxicity. However, further controlled clinical studies are required to confirm these findings.

[Abnormalities of CD34+ cells differentiation and bone marrow cell cycle in myelodysplastic syndrome].

OBJECTIVES: To detect the abnormalities of CD(34)(+) cells differentiation and bone marrow cell cycle in myelodysplastic syndrome (MDS). METHODS: Fifty newly diagnosed MDS (17 in low risk and 33 in high risk), 8 acute myeloid leukemia preceded by MDS (MDS-AML) and 25 normal controls were enrolled into this study. Their CD(34)(+)CD(38)(+), CD(34)(+)CD(38)(-) bone marrow cells and bone marrow cell cycle were measured with flow cytometry. RESULTS: The mean percentages of CD(34)(+) cells in bone marrow karyocyte of high risk [(2.29 ± 2.17)%] and MDS-AML groups [(18.69 ± 17.47)%] were significantly higher than that of control group [(0.36 ± 0.49)%, P < 0.05]. The mean percentages of CD(34)(+)CD(38)(+) cells were significantly lower in low risk, high risk and MDS-AML groups [(86.09 ± 7.79)%, (81.68 ± 11.82)% and (82.88 ± 2.60)%, respectively] than that in control group [(92.21 ± 3.85)%, P < 0.05], thus the percentages of CD(34)(+)CD(38)(-) cells were significantly higher in either MDS (low risk and high risk) or MDS-AML groups [(13.91 ± 7.79)%, (18.32 ± 11.82)% or (17.13 ± 2.60)%, respectively] than that in control group [(7.79 ± 3.85)%, P < 0.05]. The percentages of CD(34)(+)CD(38)(-) cells of MDS cases correlated directly with their International Prognostic Scoring System (IPSS) (r = 0.493, P = 0.001) and WHO Adapted Prognostic Scoring System (WPSS) (r = 0.586, P = 0.000) scores. The percentages of bone marrow mononuclear cells (BMMNCs) in G(0)/G(1) phase of in low risk, high risk and MDS-AML groups [(94.52 ± 4.32)%, (96.07 ± 3.88)% and (94.65 ± 4.55)%, respectively] were significantly higher than that in control group [(88.94 ± 7.30)%, P < 0.01], thus the percentages of BMMNCs in S and G(2)/M phase were significantly lower in either MDS (low risk and high risk) or MDS-AML groups than that in control group (P < 0.05). MDS patients with low percentages of CD(34)(+)CD(38)(-) cells presented higher therapeutic efficacy than those with high percentages of CD(34)(+)CD(38)(-) cells, while without significant differences (P > 0.05). CONCLUSIONS: There are abnormalities of differentiation of CD(34)(+) bone marrow cells and high proportion of G(0)/G(1) cells which indicates a G(1) phase arrest in MDS that might be involved in the pathogenesis of MDS. So the examination of CD(34)(+) bone marrow cells and cell cycle might be helpful for MDS diagnosis and assessment of prognosis and therapeutic effects.

[Expression of differentiation antigens on bone marrow myeloid cells of the patients with myelodysplastic syndromes and its clinical significances].

OBJECTIVE: To detect the abnormal differentiation of bone marrow myeloid cells in myelodysplastic syndromes (MDS) and its correlation with the prognosis of MDS patients. METHODS: Quantitative assessment of CD11b, CD13, CD16 and HLA-DR expression on the membrane of bone marrow granulocytes, and CD71 and glycophorin A on erythroblasts of 12 MDS patients in low-risk, 22 in high-risk and 31 normal controls was conducted with flow cytometry. The correlation between the abnormality and the prognosis of MDS cases were analyzed. RESULTS: The granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The "right hook", "sickle" and "retroflex 7" shape expressions were found in normal controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+ (0.39 +/- 0.34) and CD16-/CD16+ (1.33 +/- 0.77) were significantly higher in high-risk MDS group than those of control group (0.07 +/- 0.05 and 0.39 +/- 0.31 respectively) (P < 0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of MDS groups was lower while MFI of CD13 was higher. The mean percentages of CD11b-HLA-DR+ 3.88% +/- 3.07%, CD11b-HLA-DR- 16.23% +/- 15.59%, CD16-HLA-DR- 41.12% +/- 24.53%, CD11b+CD16- 33.53% +/- 17.26% and CD13+CD16- 44.51% +/- 21.99% granulocytes of high-risk MDS group were significantly higher than those of low-risk and control groups (P < 0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double positive expression was found in all controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentage of double positive cells in CD45- and glycophorin A+ cell population was significantly lower in low-risk and high-risk MDS groups. The abnormal numbers and patterns of the antigen expression of MDS cases per case correlated directly with their IPSS (international prognostic scoring system) (r = 0.690, P = 0.000) and WPSS (WHO adapted prognostic scoring system) (r = 0.651, P = 0.000) scores. CONCLUSION: There is an abnormal expression of differentiation antigens on bone marrow myeloid cells of MDS patients. And the severity is correlated with the prognosis. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS.

Abnormal platelet phenotypes in patients with myelodysplastic syndromes.

INTRODUCTION: Hemorrhage and infection are two main causes of death in patients with myelodysplastic syndromes (MDS), and it is becoming increasingly clear that platelet dysfunction can also affect the process of hemostasis and anti-infection. The aim of this study was to evaluate activation function and immune-related function of platelets in MDS. METHODS: We included 29 MDS patients and divided them into different subgroups (low-risk group and high-risk group; untreated group and treated group; pretransfusion group and post-transfusion group) according to IPSS-R score, hypomethylating agents (HMAs) therapy, and platelet transfusion history. Platelet light scatter properties, expression of CD41a, activation-associated phenotypes (CD62p and CD63), and immune-associated phenotypes (CD154 and TLR4) were detected by multiparameter flow cytometry. RESULTS: Expression of CD41a was decreased (P < .05), and no difference was found in platelet light scatter properties between MDS patients and healthy subjects (P > .05). Significantly decreased expression frequency and intensity of activation phenotype CD63 were found in patients with MDS (P < .05). Low-risk MDS showed lower expression frequency while high-risk MDS showed reduced mean fluorescence intensity (MFI) of CD63. Decreased expression of CD154 and TLR4 was found in MDS patients (P < .05) which was significantly elevated after HMAs therapy (P < .05). Particularly, MFI of CD154 and TLR4 reduced in high-risk MDS patients (P < .05). CONCLUSION: Myelodysplastic syndromes patients displayed defective expression of both activation- and immune-associated platelet phenotypes, with differential mechanisms between low-risk and high-risk groups regarding phenotype alterations. The findings confirmed impaired platelet phenotypes in MDS which may assist in the diagnosis and identification of MDS patients.

Abnormal Ferroptosis in Myelodysplastic Syndrome.

BACKGROUND: Ferroptosis is a form of iron-dependent non-apoptotic cell death, with characteristics of loss of the activity of the lipid repair enzyme, glutathione (GSH) peroxidase 4 (GPX4), and accumulation of lethal reactive lipid oxygen species. The mechanism of ferroptosis in myelodysplastic syndrome (MDS) is unclear. METHODS: Cell viability assay, reactive oxygen species (ROS) assay, GSH assay, and GPX activity assay were performed to study the regulation of ferroptosis in MDS cells obtained from MDS patients, the iron overload model mice, and cell lines. RESULTS: The growth-inhibitory effect of decitabine could be partially reversed by ferrostatin-1 and iron-chelating agent [desferrioxamine (DFO)] in MDS cell lines. Erastin could increase the cytotoxicity of decitabine on MDS cells. The level of GSH and the activity of GPX4 decreased, whereas the ROS level increased in MDS cells upon treatment with decitabine, which could be reversed by ferrostatin-1. The concentration of hemoglobin in peripheral blood of iron overload mice was negatively correlated with intracellular Fe2+ level and ferritin concentration. Iron overload (IO) led to decreased viability of bone marrow mononuclear cells (BMMNCs), which was negatively correlated with intracellular Fe2+ level. Ferrostatin-1 partially reversed the decline of cell viability in IO groups. The level of GSH and the activity of GPX4 decreased, whereas the ROS level increased in BMMNCs of IO mice. DFO could increase the level of GSH. Ferrostatin-1 and DFO could increase the GPX4 activity of BMMNCs in IO mice. Ferrostatin-1 could significantly reverse the growth-inhibitory effect of decitabine in MDS patients. Decitabine could significantly increase the ROS level in MDS groups, which could be inhibited by ferrostatin-1 or promoted by erastin. Ferrostatin-1 could significantly reverse the inhibitory effect of decitabine on GSH levels in MDS patients. Erastin combined with decitabine could further reduce the GSH level. Erastin could further decrease the activity of GPX4 compared with the decitabine group. CONCLUSION: Ferroptosis may account for the main mechanisms of how decitabine induced death of MDS cells. Decitabine-induced ROS raise leads to ferroptosis in MDS cells by decreasing GSH level and GPX4 activity.

Altered follicular helper T cell impaired antibody production in a murine model of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic diseases which have a high risk of progressing to acute myeloid leukemia. MDS patients have immunologic deficiency, including T and B cells dysfunction. Follicular T helper cells (Tfh, CD4+CXCR5+) are an important subset of helper T cells which help to the formation of germinal centers and B cells differentiation. In this study, we investigated the proportion and function of Tfh using NUP98-HOXD13 transgenic (NHD13) mice model with MDS phenotype. The proportion of Tfh from bone marrow and spleen of NHD13 mice decreased compared with wild type (WT) mice tested by flow cytometry. In NHD13 mice spleens, there were decreased CXCR5+ cells and increased PD-1+ cells using immunohistochemistry. The active markers (ICOS, CD40L and OX40) expressed on Tfh of NHD13 mice were decreased. In contrast, PD-1 expression on Tfh of NHD13 mice was higher than that of WT mice. After coculture with Tfh from NHD13 mice, IgG and IgM production of B cells were decreased. In conclusion, the proportion and function of Tfh in the MDS mice model were altered. The dysfunction and reduction of Tfh may inhibit B cells differentiation and antibody production. Abnormal Tfh might contribute to the immune tolerance promoting the progression of MDS.

Mesenchymal Cell Reprogramming in Experimental MPLW515L Mouse Model of Myelofibrosis.

Myelofibrosis is an indicator of poor prognosis in myeloproliferative neoplasms (MPNs), but the precise mechanism(s) contributing to extracellular matrix remodeling and collagen deposition in the bone marrow (BM) niche remains unanswered. In this study, we isolated mesenchymal stromal cells (MSCs) from mice transplanted with wild-type thrombopoietin receptor (MPLWT) and MPLW515L retroviral-transduced bone marrow. Using MSCs derived from MPLW515-transplant recipients, excessive collagen deposition was maintained in the absence of the virus and neoplastic hematopoietic cells suggested that the MSCs were reprogrammed in vivo. TGFß production by malignant megakaryocytes plays a definitive role promoting myelofibrosis in MPNs. However, TGFß was equally expressed by MSCs derived from MPLWT and MPLW515L expressing mice and the addition of neutralizing anti-TGFß antibody only partially reduced collagen secretion in vitro. Interestingly, profibrotic MSCs displayed increased levels of pSmad3 and pSTAT3 suggesting that inflammatory mediators cooperating with the TGFß-receptor signaling may maintain the aberrant phenotype ex vivo. FGFb is a known suppressor of TGFß signaling. Reduced collagen deposition by FGFb-treated MSCs derived from MPLW515L mice suggests that the activating pathway is vulnerable to this suppressive mediator. Therefore, our findings have implications for the future investigation of therapies to reverse fibrosis in MPNs.

Efficacy of ALK5 inhibition in myelofibrosis.

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-ß family members are profibrotic cytokines and we observed significant TGF-ß1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-ß1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-ß1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-ß receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-ß/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

Th17 Cells Exhibit Antitumor Effects in MDS Possibly through Augmenting Functions of CD8+ T Cells.

Th17 cells are a newly found subset of distinct CD4+ Th effector cells' family and are found to play an important role in cancers. Myelodysplastic syndromes (MDS) are a common malignant hematological disease. Here, we showed that both the percentage and the function of Th17 cells were elevated in low-risk MDS while being decreased in high-risk MDS. Levels of upstream molecules of Th17 cells, IL-6 and IL-23, were higher in low-risk MDS but lower in high-risk MDS patients. The abnormal percentage of Th17 cells was closely related to clinical parameters including karyotype, morphologic blast percentage of bone marrow, peripheral absolute neutrophil count, and hemoglobin concentration. Furthermore, expression rates of perforin and granzyme B in BM CD3+CD8+ cells (cytotoxic T lymphocyte, CTL) positively correlated with levels of IL-17 but negatively correlated with BM blast percentage and could be significantly increased after stimulation with human recombinant IL-17 (rhIL-17). Our results suggested that Th17 cells might play an antitumor effect in the pathogenesis of MDS through IL-17/CTL pathway.

Elevated TIM3+ hematopoietic stem cells in untreated myelodysplastic syndrome displayed aberrant differentiation, overproliferation and decreased apoptosis.

TIM3, as a negative regulator of anti-tumor immunity, is highly expressed on LSCs, but not on normal HSCs. TIM3 on HSCs in MDS patients has not been clarified. Here, both the percentage of TIM3 on HSCs and the MFI of TIM3+ HSCs were higher in untreated MDS than control and were closed to AML, and excessive TIM3+ HSCs was closely related to clinical parameters: WPSS score, karyotype analysis, morphologic blasts, the number of cytopenia involving hematopoietic lineages, anemia and granulocytopenia. TIM3+ HSCs expressed lower CD11b, TpoR, EpoR, G-CSFR and Annexin V, and higher CD71 and GATA2. TIM3+ HSCs displayed aberrant differentiation, overproliferation and decreased apoptosis. TIM3 might be a promising marker for identifying malignant clone cells in MDS and a candidate for targeted therapy.

Effect of DLK1 on tumorigenesis in CD34+CD38- bone marrow cells in myelodysplastic syndromes.

The myelodysplastic syndromes (MDSs) are a group of clonal stem cell disorders resulting from aberrations within hematopoietic stem cells (HSCs), which may lead to the onset of a number of diseases, including acute myeloid leukemia (AML). Recent studies have demonstrated that the expression levels of the DLK1 gene are increased in MDS. In order to determine whether the addition of DLK1 affects tumorigenesis, small interfering (si)RNAs were designed to target DLK1 in order to knockdown its expression in CD34+CD38- bone marrow cells in MDS. A lower proliferative rate was observed in the CD34+CD38- bone marrow cells following this knockdown of DLK1 expression. The suppression of DLK1 expression resulted in a less aggressive MDS phenotype, which suggests that the upregulation of DLK1 expression may play an oncogenic role in CD34+CD38- bone marrow cells.

Research progress on the red cell diseases in China.

In recent years, there have been lots of progresses in the studies on red cell diseases in China, especially bone marrow failure diseases including immuno-related pancytopenia, aplastic anemia, myelodysplastic syndrome, and paroxymal nocturnal hemoglobinuria. Numerous laboratory experiments as well as clinical researches have been carried out by Chinese hematologists, which brought about much clearer pathogenesis, more rational diagnosis methods and more effective therapies for red cell diseases.

Expression of DLK1 Gene in the Bone Marrow Cells of Patients with Myelodysplastic Syndromes and Its Clinical Significance.

OBJECTIVE: This study aims to investigate the expression of delta-like 1 (DLK1) gene in the bone marrow cells of patients with myelodysplastic syndromes (MDS) and to explore its molecular characteristics for the early diagnosis of MDS. METHODS: The expression of DLK1 mRNA in the bone marrow cells of cases with MDS, acute myeloid leukemia (AML), and normal control groups were measured by real-time polymerase chain reaction and were analyzed for clinical significance. RESULTS: Significantly higher expression of DLK1 mRNA was observed in the bone marrow cells of MDS patients (0.7342±0.3652) compared with the normal control group (0.4801±0.1759) (P<0.05). The expression of DLK1 mRNA had a positive correlation with the proportion of bone marrow blasts (r=0.467, P<0.05). Moreover, DLK1 mRNA expression was significantly increased as MDS progressed (P<0.05). Patients with abnormal karyotypes exhibited significantly higher expression of DLK1 mRNA (0.9007±0.4334) than those with normal karyotypes (0.6411±0.2630) (P<0.05). Subsequently, patients with highly expressed DLK1 (≥0.8) presented significantly higher malignant clone burden (0.4134±0.3999) than those with lower DLK1 expression (<0.8),(0.1517±0.3109), (P<0.05). CONCLUSIONS: The DLK1 gene was highly expressed in MDS patients, and was increased as MDS progressed. The expression of DLK1 mRNA was positively correlated with the proportion of the bone marrow blasts. A high expression of DLK1 gene suggested a higher malignant clone burden of MDS.