Nore1 inhibits age-associated myeloid lineage skewing and clonal hematopoiesis but facilitates termination of emergency (stress) granulopoiesis.

Age-associated bone marrow changes include myeloid skewing and mutations that lead to clonal hematopoiesis. Molecular mechanisms for these events are ill defined, but decreased expression of Irf8/Icsbp (interferon regulatory factor 8/interferon consensus sequence binding protein) in aging hematopoietic stem cells may contribute. Irf8 functions as a leukemia suppressor for chronic myeloid leukemia, and young Irf8-/- mice have neutrophilia with progression to acute myeloid leukemia (AML) with aging. Irf8 is also required to terminate emergency granulopoiesis during the innate immune response, suggesting this may be the physiologic counterpart to leukemia suppression by this transcription factor. Identifying Irf8 effectors may define mediators of both events and thus contributors to age-related bone marrow disorders. In this study, we identified RASSF5 (encoding Nore1) as an Irf8 target gene and investigated the role of Nore1 in hematopoiesis. We found Irf8 activates RASSF5 transcription and increases Nore1a expression during emergency granulopoiesis. Similar to Irf8-/- mice, we found that young Rassf5-/- mice had increased neutrophils and progressed to AML with aging. We identified enhanced DNA damage, excess clonal hematopoiesis, and a distinct mutation profile in hematopoietic stem cells from aging Rassf5-/- mice compared with wildtype. We found sustained emergency granulopoiesis in Rassf5-/- mice, with repeated episodes accelerating AML, also similar to Irf8-/- mice. Identifying Nore1a downstream from Irf8 defines a pathway involved in leukemia suppression and the innate immune response and suggests a novel molecular mechanism contributing to age-related clonal myeloid disorders.

The Interferon Consensus Sequence Binding Protein (Icsbp/Irf8) Is Required for Termination of Emergency Granulopoiesis.

Emergency granulopoiesis occurs in response to infectious or inflammatory challenge and is a component of the innate immune response. Some molecular events involved in initiating emergency granulopoiesis are known, but termination of this process is less well defined. In this study, we found that the interferon consensus sequence binding protein (Icsbp/Irf8) was required to terminate emergency granulopoiesis. Icsbp is an interferon regulatory transcription factor with leukemia suppressor activity. Expression of Icsbp is decreased in chronic myeloid leukemia, and Icsbp(-/-) mice exhibit progressive granulocytosis with evolution to blast crisis, similar to the course of human chronic myeloid leukemia. In this study, we found aberrantly sustained granulocyte production in Icsbp(-/-) mice after stimulation of an emergency granulopoiesis response. Icsbp represses transcription of the genes encoding Fas-associated phosphatase 1 (Fap1) and growth arrest-specific 2 (Gas2) and activates genes encoding Fanconi C and F. After stimulation of emergency granulopoiesis, we found increased and sustained expression of Fap1 and Gas2 in bone marrow myeloid progenitor cells from Icsbp(-/-) mice in comparison with the wild type. This was associated with resistance to Fas-induced apoptosis and increased ß-catenin activity in these cells. We also found that repeated episodes of emergency granulopoiesis accelerated progression to acute myeloid leukemia in Icsbp(-/-) mice. This was associated with impaired Fanconi C and F expression and increased sensitivity to DNA damage in bone marrow myeloid progenitors. Our results suggest that impaired Icsbp expression enhances leukemogenesis by deregulating processes that normally limit granulocyte expansion during the innate immune response.

The leukemia-associated fusion protein Tel-platelet-derived growth factor receptor ß (Tel-PdgfRß) inhibits transcriptional repression of PTPN13 gene by interferon consensus sequence binding protein (Icsbp).

Icsbp is an interferon regulatory transcription factor with leukemia suppressor activity. In previous studies, we identified the gene encoding Fas-associated phosphatase 1 (Fap1; the PTPN13 gene) as an Icsbp target. In the current study, we determine that repression of PTPN13 by Icsbp requires cooperation with Tel and histone deacetylase 3 (Hdac3). These factors form a multiprotein complex that requires pre-binding of Tel to the PTPN13 cis element with subsequent recruitment of Icsbp and Hdac3. We found that knockdown of Tel or Hdac3 in myeloid cells increases Fap1 expression and results in Fap1-dependent resistance to Fas-induced apoptosis. The TEL gene was initially identified due to involvement in leukemia-associated chromosomal translocations. The first identified TEL translocation partner was the gene encoding platelet-derived growth factor receptor ß (PdgfRß). The resulting Tel-PdgfRß fusion protein exhibits constitutive tyrosine kinase activity and influences cellular proliferation. In the current studies, we find that Tel-PdgfRß influences apoptosis in a manner that is independent of tyrosine kinase activity. We found that Tel-PdgfRß expressing myeloid cells have increased Fap1 expression and Fap1-dependent Fas resistance. We determined that interaction between Tel and Tel-PdgfRß decreases Tel/Icsbp/Hdac3 binding to the PTPN13 cis element, resulting in increased transcription. Therefore, these studies identify a novel mechanism by which the Tel-PdgfRß oncoprotein may contribute to leukemogenesis.

The interferon consensus sequence binding protein (ICSBP/IRF8) activates transcription of the FANCF gene during myeloid differentiation.

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor with leukemia-suppressor activity. ICSBP regulates genes that are involved in phagocyte function, proliferation, and apoptosis. In murine models ICSBP deficiency results in a myeloproliferative disorder (MPD) with increased mature neutrophils. Over time this MPD progresses to acute myeloid leukemia (AML), suggesting that ICSBP deficiency is adequate for MPD, but additional genetic lesions are required for AML. The hypothesis of these studies is that dysregulation of key target genes predisposes to disease progression under conditions of decreased ICSBP expression. To investigate this hypothesis, we used chromatin co-immunoprecipitation to identify genes involved the ICSBP-leukemia suppressor effect. In the current studies, we identify the gene encoding Fanconi F (FANCF) as an ICSBP target gene. FancF participates in a repair of cross-linked DNA. We identify a FANCF promoter cis element, which is activated by ICSBP in differentiating myeloid cells. We also determine that DNA cross-link repair is impaired in ICSBP-deficient myeloid cells in a FancF-dependent manner. This effect is observed in differentiating cells, suggesting that ICSBP protects against the genotoxic stress of myelopoiesis. Decreased ICSBP expression is found in human AML and chronic myeloid leukemia during blast crisis (CML-BC). Our studies suggest that ICSBP deficiency may be functionally important for accumulation of chromosomal abnormalities during disease progression in these myeloid malignancies.

Toxoplasma gondii actively remodels the microtubule network in host cells.

Toxoplasma gondii infection triggers host microtubule rearrangement and organelle recruitment around the parasite vacuole. Factors affecting initial stages of microtubule remodeling are unknown. To illuminate the mechanism, we tested the hypothesis that the parasite actively remodels host microtubules. Utilizing heat-killed parasites and time-lapse analysis, we determined microtubule rearrangement requires living parasites and is time dependent. We discovered a novel aster of microtubules (MTs) associates with the vacuole within 1h of infection. This aster lacks the concentrated foci of gamma (gamma)-tubulin normally associated with MT nucleation sites. Unexpectedly, vacuole enlargement does not correlate with an increase in MT staining around the vacuole. We conclude microtubule remodeling does not result from steric constraints. Using nocodazole washout studies, we demonstrate the vacuole nucleates host microtubule growth in-vivo via gamma-tubulin-associated sites. Moreover, superinfected host cells display multiple gamma-tubulin foci. Microtubule dynamics are critical for cell cycle control in uninfected cells. Using non-confluent monolayers, we show host cells commonly fail to finish cytokinesis resulting in larger, multinucleated cells. Our data suggest intimate interactions between T. gondii and host microtubules result in suppression of cell division and/or cause a mitotic defect, thus providing a larger space for parasite duplication.

Decreased calpain activity in chronic myeloid leukemia impairs apoptosis by increasing survivin in myeloid progenitors and xiap1 in differentiating granulocytes.

Chronic Myeloid Leukemia (CML) is characterized by translocations between chromosomes 9 and 22, resulting in expression of Bcr-abl oncogenes. Although the clinical course of CML was revolutionized by development of Bcr-abl-directed tyrosine kinase inhibitors (TKIs), CML is not cured by these agents. Specifically, the majority of subjects relapsed in clinical trials attempting TKI discontinuation, suggesting persistence of leukemia stem cells (LSCs) even in molecular remission. Identifying mechanisms of CML-LSC persistence may suggest rationale therapeutic targets to augment TKI efficacy and lead to cure. Apoptosis resistance is one proposed mechanism. In prior studies, we identified increased expression of Growth Arrest Specific 2 (Gas2; a Calpain inhibitor) in Bcr-abl+ bone marrow progenitor cells. A number of previously described Calpain substrates might influence apoptosis in CML, including ßcatenin and the X-linked Inhibitor of Apoptosis Protein 1 (Xiap1). We previously found Gas2/Calpain dependent stabilization of ßcatenin in CML, and increased expression of ßcatenin target genes, including Survivin (also an IAP). In the current work, we investigate contributions of Survivin and Xiap1 to Fas-resistance in Bcr-abl+ bone marrow cells. Inhibitors of these proteins are currently in clinical trials for other malignancies, but a role for either IAP in CML-LSC persistence is unknown.

Bcr-abl regulates Stat5 through Shp2, the interferon consensus sequence binding protein (Icsbp/Irf8), growth arrest specific 2 (Gas2) and calpain.

Icsbp/Irf8 is an interferon regulatory transcription factor that functions as a suppressor of myeloid leukemias. Consistent with this activity, Icsbp represses a set of genes encoding proteins that promote cell proliferation/survival. One such gene encodes Gas2, a calpain inhibitor. We previously found that increased Gas2-expression in Bcr-abl+ cells stabilized ßcatenin; a Calpain substrate. This was of interest, because ßcatenin contributes to disease progression in chronic myeloid leukemia (CML). Calpain has additional substrates implicated in leukemogenesis, including Stat5. In the current study, we hypothesized that Stat5 activity in CML is regulated by Gas2/Calpain. We found that Bcr-abl-induced, Shp2-dependent dephosphorylation of Icsbp impaired repression of GAS2 by this transcription factor. The consequent decrease in Calpain activity stabilized Stat5 protein; increasing the absolute abundance of both phospho and total Stat5. This enhanced repression of the IRF8 promoter by Stat5 in a manner dependent on Icsbp, Gas2 and Calpain, but not Stat5 tyrosine phosphorylation. During normal myelopoiesis, increased expression and phosphorylation of Icsbp inhibits Calpain. In contrast, constitutive activation of Shp2 in Bcr-abl+ cells impairs regulation of Gas2/Calpain by Icsbp, aberrantly stabilizing Stat5 and enhancing IRF8 repression. This novel feedback mechanism enhances leukemogenesis by increasing Stat5 and decreasing Icsbp. Bcr-abl targeted tyrosine kinase inhibitors (TKIs) provide long term disease control, but CML is not cured by these agents. Our studies suggest targeting Calpain might be a rational therapeutic approach to decrease persistent leukemia stem cells (LSCs) during TKI-treatment.

Increased Fanconi C expression contributes to the emergency granulopoiesis response.

Emergency granulopoiesis is a component of the innate immune response that is induced in response to infectious or inflammatory challenge. It is characterized by the rapid expansion and differentiation of granulocyte/monocyte progenitor (GMP) populations, which is due in part to a shortened S-phase of the cell cycle. We found that IRF8 (also known as ICSBP), an interferon regulatory transcription factor that activates phagocyte effector genes during the innate immune response, activates the gene encoding Fanconi C (Fancc) in murine myeloid progenitor cells. Moreover, IRF8-induced Fancc transcription was augmented by treatment with IL-1ß, an essential cytokine for emergency granulopoiesis. The Fanconi pathway participates in repair of stalled or collapsed replication forks during DNA replication, leading us to hypothesize that the Fanconi pathway contributes to genomic stability during emergency granulopoiesis. In support of this hypothesis, Fancc(-/-) mice developed anemia and neutropenia during repeated, failed episodes of emergency granulopoiesis. Failed emergency granulopoiesis in Fancc(-/-) mice was associated with excess apoptosis of HSCs and progenitor cells in the bone marrow and impaired HSC function. These studies have implications for understanding the pathogenesis of bone marrow failure in Fanconi anemia and suggest possible therapeutic approaches.

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells.

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB knockdown with two independent siRNAs was shown to impair PRL-induced reporter expression in breast cancer cell line. cDNA microarray analysis was performed on these cells to assess the effect of CypB reduction, and revealed a significant decrease in PRL-induced endogenous gene expression in two breast cancer cell lines. Parallel functional assays revealed corresponding alterations of both anchorage-independent cell growth and cell motility of breast cancer cells. Our results demonstrate that CypB expression levels significantly modulate PRL-induced function in breast cancer cells ultimately resulting in enhanced levels of PRL-responsive gene expression, cell growth, and migration. Given the increasingly appreciated role of PRL in the pathogenesis of breast cancer, the actions of CypB detailed here are of biological significance.

Molecular evolution of the vesicle coat component betaCOP in Toxoplasma gondii.

Coatomer coated (COPI) vesicles play a pivotal role for multiple membrane trafficking steps throughout the eukaryotic cell. Our focus is on betaCOP, one of the most well known components of the COPI multi-protein complex. Amino acid differences in betaCOP may dictate functional divergence across species during the course of evolution, especially with regards to the evolutionary pressures on obligate intracellular parasites. A bioinformatic analysis of betaCOP amino acid sequences was conducted for 49 eukaryotic species. Cloning and sequence analysis of the Toxoplasma gondii betaCOP homologue revealed several amino acid insertions unique to T. gondii and one C-terminal insertion that is unique to apicomplexan parasites. These findings led us to investigate the possibility that betaCOP experienced functional divergence during the course of its evolution. Bayesian phylogenetic analysis revealed a tree consistent with pan eukaryote distribution and long-branch lengths were observed among the apicomplexans. Further analysis revealed that kinetoplast betaCOP underwent the most amount of change, leading to perhaps an overall change of function. In comparison, T. gondii exhibited subtle yet specific amino acid changes. The amino acid substitutions did not occur in the same places as other lineages, suggesting that TgbetaCOP has a role specific to the apicomplexans. Our work identifies 48 residues that are likely to be functionally important when comparing apicomplexan, kinetoplastid, and fungal betaCOP.