ALBERT

Description: Genes, Vol. 9, Pages 56: Identification of a Common Different Gene Expression Signature in Ischemic Cardiomyopathy Genes doi: 10.3390/genes9010056 Authors: Yana Li Qiu Jiang Zhiwen Ding Guijian Liu Peng Yu Guoliang Jiang Ziqing Yu Chunjie Yang Juying Qian Hong Jiang Yunzeng Zou The molecular mechanisms underlying the development of ischemic cardiomyopathy (ICM) remain poorly understood. Gene expression profiling is helpful to discover the molecular changes taking place in ICM. The aim of this study was to identify the genes that are significantly changed during the development of heart failure caused by ICM. The differentially expressed genes (DEGs) were identified from 162 control samples and 227 ICM patients. PANTHER was used to perform gene ontology (GO), and Reactome for pathway enrichment analysis. A protein–protein interaction network was established using STRING and Cytoscape. A further validation was performed by real-time polymerase chain reaction (RT-PCR). A total of 255 common DEGs was found. Gene ontology, pathway enrichment, and protein–protein interaction analysis showed that nucleic acid-binding proteins, enzymes, and transcription factors accounted for a great part of the DEGs, while immune system signaling and cytokine signaling displayed the most significant changes. Furthermore, seven hub genes and nine transcription factors were identified. Interestingly, the top five upregulated DEGs were located on chromosome Y, and four of the top five downregulated DEGs were involved in immune and inflammation signaling. Further, the top DEGs were validated by RT-PCR in human samples. Our study explored the possible molecular mechanisms of heart failure caused by ischemic heart disease.

Description: Hematopoiesis is an orchestrated process in which hematopoietic stem cells (HSCs) can self-renew and produce all lineages of blood cells. Majority of HSCs are in a quiescent state with a low growth rate. However, some genetic mutations that occur in HSCs impel HSCs to exit the quiescent state and to proliferate excessively, which enables mutant HSCs to outcompete normal HSCs and leads to clonal expansion of mutant HSCs. Myelodysplastic syndromes (MDSs) as a clonal disease, arise from the expansion of mutant HSCs and are characterized by morphologic dysplasia, ineffective hematopoiesis and an increased risk of transformation to acute myeloid leukemia. FoxM1 is one of transcription factors in the family of Fox ('Forkhead box') proteins. Analysis of public database revealed that the expression level of FOXM1 was decreased significantly in CD34 + cells from a subset of patients with MDS as compared to healthy individuals. Thus, we sought to determine whether haploinsufficiency of FOXM1 contributes to the development of MDS in mice. Our study showed that haploinsufficiency of Foxm1 led to an expansion of hematopoietic stem/progenitor cells in mice. Since FoxM1 has previously been implicated in regulation of cell cycle, we determined the cell cycle status of Foxm1 heterozygous HSCs. By BrdU incorporation assay, we showed that Foxm1 heterozygous HSCs have an increased S phase and G2/M phase as compared to control HSCs from wildtype mice. Additional analysis with Hochest33342/Pyronin-Y staining and Ki67/DAPI staining showed a significant decrease in the number of quiescent (G0) cells in Foxm1 heterozygous HSCs as compared to control HSCs. These results suggest that FoxM1 haploinsufficiency promotes HSCs to exit quiescence and to enter cell cycle, thereby leading to exhaustion of HSCs. To further assess the function of Foxm1 heterozygous HSCs in vivo, we performed competitive repopulation assay. We found that Foxm1 haploinsufficiency HSCs exhibited competitive repopulation advantage in the first and secondary recipient mice, but displayed significantly decreased capacity of repopulation in tertiary recipient mice as compared to control HSCs, suggesting that Foxm1 haploinsufficiency promoted clonal expansion of HSCs, which leads to an exhaustion of HSCs eventually. HSC proliferation can be induced by some specific immune effectors such as Toll-like receptor 4 (TLR4). Lipopolysaccharide (LPS) stimulates HSC proliferation by activating TLR4 signaling pathway. Low dose of LPS treatment over time accelerated the development of MDS in mice. Interestingly, low dose of LPS injection chronically induced defects in hematopoiesis in Foxm1 haploinsufficient mice but not the control wildtype mice. Recipient mice transplanted with Foxm1 heterozygous BM cells but not the control BM cells developed MDS-like disease with cytopenia and a decreased number of hematopoietic stem/progenitor cells after LPS stimulation. Moreover, we found that nearly half of aged Foxm1 haploinsufficient mice (20 months) developed splenomegaly. Analysis of histologic sections in Foxm1 haploinsufficient mice showed that the mice developed hematopoietic dysplasia including dysplastic megakaryocytes with bizarre-shaped nuclei in bone marrow and extramedullary hematopoiesis with accumulation of myeloid cells in spleen. RNA-seq analysis indicated that haploinsufficiency of Foxm1 perturbed multiple stem cell-maintenance mechanisms especially in metabolic processes. Taken together, our studies suggest that Foxm1 haploinsufficiency in mice causes clonal expansion of HSCs and promotes MDS-like disease, which underscores the significant role of FOXM1 downregulation in the initiation and development of human MDS. Disclosures No relevant conflicts of interest to declare.

Description: Here we report a dosage effect of c-Myc on hematopoiesis and its distinct role in mediating the Wnt/b-catenin pathway in hematopoietic stem cell (HSC) and bone marrow niche cells. We showed that c-Myc haploinsufficiency led to ineffective hematopoiesis by inhibiting HSC self-renewal and quiescence, and promoting its apoptosis. We have identified Nr4a1, Nr4a2 and Jmjd3, which are critical for the maintenance of HSC functions, as previously unrecognized downstream targets of c-Myc in HSCs. c-Myc directly binds to the promoter regions of Nr4a1, Nr4a2 and Jmjd3 and regulates their expression. Our results revealed that Nr4a1 and Nr4a2 mediated the function of c-Myc in regulating HSC quiescence while all three genes contribute to the function of c-Myc in the maintenance of HSC survival. Adenomatous polyposis coli (Apc) is a negative regulator of the Wnt/b-catenin pathway. We have provided the first evidence that Apc haploinsufficiency induced a blockage of erythroid lineage differentiation through promoting expression and secretion of IL6 in bone marrow endothelial cells. We found that c-Myc haploinsufficiency failed to rescue defective function of Apc-deficient HSCs in vivo but it was sufficient to prevent the development of severe anemia in Apc heterozygous mice, and to significantly prolong survival of such mice. Furthermore, we showed that c-Myc mediated Apc loss-induced IL6 secretion in endothelial cells, and c-Myc haploinsufficiency reversed the negative effect of Apc deficient endothelial cells on erythroid cell differentiation. Our studies indicate that c-Myc has a context-dependent role in mediating the function of Apc in hematopoiesis.

Description: The somatic mutation MPL W515L constitutively activates signaling pathways including JAK/STATs, MAPK, and PI3K/AKT, leading to myeloproliferative neoplasm (MPN). How each of these signaling pathways contributes to MPL W515L-induced MPN has not been well defined. Moreover, the requirement for the tyrosine residues 599 and 604, which have been shown to be critical for normal MPL signaling, in MPL W515L-induced disease remains uncharacterized. Thus, we created W515L/Y599F double mutant (abbreviated as MPL 515/599), W515L/Y604F double mutant (MPL 515/604), and the MPL W515L/Y599F/Y604F triple mutant (MPL 515/599/604) and tested their activities in the TPO-dependent G1ME cell line. Interestingly, MPL 515/604 supported TPO-independent proliferation similar to MPL W515L whereas MPL 515/599 exhibited significantly impaired ability to do so. In contrast, the triple mutant completely lost the ability to support TPO-independent proliferation. Consistent with their effect on cell proliferation, MPL W515L and MPL 515/604 significantly increased CFU-Mk in murine bone marrow while MPL 515/599 and the triple mutant did not. Moreover, MPL 515/599 exhibited impaired phosphorylation of Shc, STAT3, STAT5, AKT, and ERK while MPL 515/604 barely had any effect on their phosphorylation. Notably, MPL 515/599/604 further reduced STAT5 phosphorylation although it retained STAT3 phosphorylation as compared to MPL 515/599. Together, these results reveal that Y599 is critical in mediating MPL W515L-induced megakaryocyte hyperproliferation and signaling. To further test which downstream signaling pathway is critical for MPL W515L-induced megakaryocyte hyperproliferation, we restored each signaling in MPL 515/599/604-transduced G1ME cells by overexpressing constitutively active forms of downstream signaling molecules and GFP. Cells overexpressing constitutively active STAT5 or AKT showed an increased GFP percentage over time with TPO depletion. However, constitutively active STAT3 or Ras did not affect cell growth. We verified the requirement for STAT5 in MPL W515L-induced megakaryocyte hyperproliferation by overexpressing MPL W515L in murine bone marrow progenitors from STAT5-null and littermate control mice. Deletion of STAT5 significantly reduced CFU-Mk and impaired CD41 expression. Notably, MPL W515L-induced expansion of CFU-Mk observed in littermate control cells was dramatically impaired in STAT5-null cells. These findings suggest that STAT5 is critical in MPL W515L-induced megakaryocyte hyperproliferation. To further test the in vivo role of Y599 and Y604 in MPL W515L-induced MPN, we performed bone marrow transplantation. Consistent with the in vitro phenotype, MPL W515L and MPL 515/604 caused MPN characterized by hypercellularity of bone marrow, leukocytosis, thrombocytosis, splenomegaly, hepatomegaly, and infiltration of blood cells to liver and lung. Interestingly, the disease burden of MPL 515/604 was more severe, with higher WBC and platelet counts than that seen with MPL W515L. Surprisingly, MPL 515/599 and MPL 515/599/604 caused hypercellularity of bone marrow, although they did not cause any other significant features of MPN. MPL 515/599 also caused splenomegaly without other symptoms. Unexpectedly, all MPL mutant alleles supported expansion of LSK cells in bone marrow. Despite the finding that MPL W515L and MPL 515/604 caused thrombocytosis, they did not increase MEP numbers. In fact, all mutant MPL alleles except the triple mutant increased HPC, CMP, and GMP cells. In addition, mature myeloid and megakaryocyte lineages were expanded, possibly at the expense of erythrocytes. In the spleen, we also observed expansion of megakaryocytes in the double mutant alleles and a huge expansion of myeloid lineage cells, but a decrease of erythrocytes in MPL 515/604. Thus our study has revealed distinct features of Y599 and Y604 in mediating MPL W515L-induced signaling, megakaryocyte hyperproliferation, and MPN. Y599 plays a critical role and cooperates with Y604. Our study also suggests that MPL cytosolic phosphorylated Y599 and flanking sequences could become targets for pharmacologic inhibition in MPNs. Disclosures No relevant conflicts of interest to declare.

Description: Relapse after initial achievement of complete remission remains a major issue in the treatment of Acute Myeloid Leukemia (AML). Emerging evidence suggest a critical role of Leukemia Stem Cells (LSCs) during AML relapse. FOXM1 is a member of the forkhead family of transcription factors. Here we report a novel role of Foxm1 as a key regulator of LSCs. MLL-rearranged AML patients have a very poor prognosis and are more resistant to traditional chemotherapy. Recently, we found that high FOXM1 expression is associated with MLL-rearranged AMLs and AMLs with a complex karyotype. We also found that loss of Foxm1 significantly reduced serial replating capacity of MLL-AF9 (MA9)-induced myeloid progenitor cells and increased apoptosis of MA9-induced leukemia stem cell (MA9-LSC)-enriched cells but not mature leukemia cells in vitro. In addition, we showed that Foxm1 loss in mice markedly delayed the initiation and progression of MLL-AF9-induced AML. Notably, Foxm1 loss reduced the number of MA9-LSCs as well as quiescence of MA9-LSCs. However, Foxm1 loss significantly increased apoptosis of LSCs but not normal HSCs in vivo. Our RNA-seq data revealed that expression of both Bcl2, a survival factor and p21Cip1, known as cyclin-dependent inhibitor 1, are significantly decreased as a consequence of Foxm1 deletion in MA9-LSCs. In addition, Foxm1 loss led to down-regulation of Itga1. Of interest, Chip-PCR revealed that Foxm1 regulates Itga1 expression by directly binding to its promoter. Collectively, these data suggest that Foxm1 is required for the maintenance of quiescence and survival of MA9-LSCs. Mechanistically, we found that loss of Foxm1 inhibited leukemogenic function of MA9-LSCs, at least partially through down-regulating the a1b1-mediated integrin pathway. We next demonstrated that conditional deletion of single or both alleles of Foxm1 significantly delayed the progression of MA9-induced AML after initiation of disease in mice, and that pharmacological inhibition of Foxm1 prolonged disease latency of MA9-induced AML in mice. It has been reported that MA9-induced mouse leukemia cells are resistant to chemotherapeutic drugs. Notably, our data showed that deletion of Foxm1 or loss of a single allele of Foxm1 significantly increases the sensitivity of MA9-induced leukemia cells to chemotherapeutic drugs in mice. Furthermore, we found that human AML cell lines with expression of MA9, the MA9-transduced primary human CD34+ cells as well as primary bone marrow cells from patients with MA9-induced AML, are more sensitive to FOXM1 inhibition in vitro than the control human CD34+ cells. Moreover, inhibition of FOXM1 significantly prolonged the survival of xenografted mice with MA9-tranduced human CD34+ cells as well as primary bone marrow cells from a MLL leukemia patient. Of note, we found that FOXM1 inhibition also significantly induced the apoptosis of human CD34+ LSCs in vivo in xenografted mice. Our studies strongly suggest that inhibition of FOXM1 may benefit MLL leukemia patients by eliminating LSCs, thereby reducing the frequency of relapse in these patients after treatment. Disclosures No relevant conflicts of interest to declare.

Description: It has been shown that loss of c-Myc leads to accumulation of hematopoietic stem cells (HSCs) and severe cytopenia as a consequence of a blockage of HSC differentiation. Here we report a role of c-Myc haploinsufficiency in regulating HSC quiescence and self-renewal. We showed that c-Myc haploinsufficient mice displayed decreased white blood count and number of lymphocytes with normal myeloid cell differentiation. The number of HSCs and hematopoietic progenitor cells (HPCs) were all decreased significantly in c-Myc haploinsufficient mice as compared with control mice. We found that c-Myc haploinsufficiency inhibited HSC self-renewal capacity, increased proliferation and decreased quiescence of HSCs in vivo. By transplantation assays, we showed that c-Myc haploinsufficiency has extrinsic and intrinsic effects on the maintenance of HSCs in vivo. Our study suggests that loss of c-Myc activity and reduced dosage of c-Myc have distinct effects on HSC functions. c-Myc is a critical downstream mediator of the Wnt/b-catenin pathway. We showed that c-Myc haploinsufficiency is sufficient to prevent severe anemia in Apc heterozygous mice, and to significantly prolong the survival of Apc heterozygous mice. In addition, treatment of Apc haploinsufficient mice by a c-Myc inhibitor significantly reversed anemia in Apc-deficient mice. By transplantation assay, we further demonstrated that reduced expression of c-Myc in the bone marrow niche is responsible for prevention of severe anemia in Apc-deficient mice. However, we found that reduction of c-Myc by loss of a single allele of c-Myc did not rescue defective self-renewal capacity of Apc haploinsufficient HSCs. Taken together, our studies indicate that c-Myc mediates the function of the Wnt/b-catenin signaling pathway in bone marrow niche but not in HSCs. Disclosures No relevant conflicts of interest to declare.