Myeloid Precursors In Bone Marrow Research Articles

Septin-6 Regulates Murine and Human Hematopoiesis, and Its Dysregulation Is Associated with Pediatric Myelodysplasia

Septin family proteins are key regulators of cytoskeletal organization and cytokinesis although little is known about their function in normal hematopoiesis. Septins are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previously, we identified a non-syndromic newborn with severe neutropenia which progressed to myelodysplastic syndrome (MDS) (Renella et al. AJH, 2022). Next-generation sequencing revealed a novel X-linked germline mutation in the SEPTIN-6 gene associated with dysmyelopoiesis (SEPTIN6 c.1282T>C); and a second stop-gain somatic mutation hypothesized to rescue hematopoietic function, but the mechanism of MDS was not defined. Based on our initial publication, a second patient with neutropenia/MDS has since been identified in Seattle with a mutation in the same codon (SEPTIN6 c.1282T>A) suggesting an important new molecular mechanism for pediatric MDS. Both identified patients had a high degree of myeloid tetraploidy in the marrow and were refractory to G-CSF. To assess the pathobiology of the published patient's mutation, we replicated the germline T>C mutation using single base-pair gene editing in normal donor primary CD34+ cells. Editing rates were ~50-70%. Morphological studies demonstrated that SEPTIN-6 edited human CD34+ cells demonstrated enlarged, multi-nucleated and dysplastic nuclei. These findings were similar to the appearance of the patient's bone marrow myeloid precursor cells. These edited cells also demonstrated a decline in clonogenic activity notably with a selective reduction in granulocyte-macrophage (GM) colonies (Table 1). Both myeloid and erythroid colonies showed a reduction in colony size. These data directly confirm an important role for this mutated SEPTIN-6 in abnormal hematopoietic function. The septin-6 family of proteins consist of septin-6, 8, 10, 11 and 14 which may have overlapping functions in cells. Murine single cell mRNA expression data demonstrate that hematopoietic stem and progenitor cells (HSPC) express significant levels of Septin-6, Septin-11 and 8, with little to no expression of Septin-10 and 14. To better understand the functional role of Septin-6, we created lentivirus vectors tagged with a GFP reporter that contained shRNAs against Septin-6. Culture of Septin-6 knockdown (Septin-6 KD) murine HSPC in IMDM (10ng/ml IL-3, 25ng/ml of SCF, TPO, Flt-3 and IL-6) resulted in an average of 18-fold decrease in cell counts compared to controls (p=0.02). This was accompanied by an increase in cell cycle: S-phase: Septin-6 KD 55.7%; controls 42.5%, p=0.05 and a cumulative decrease in the G0/G1 and G2/M phase suggesting that Septin-6 is important in regulating cell cycle in HSPC. Controls were a non-targeting shRNA and an empty vector for all experiments described. Furthermore, knockdown of Septin-6 caused a decrease in total clonogenic activity with defects seen in both myeloid (GM) and erythroid (BFU-E) colonies (Table 1). Competitive repopulation assays, wherein transduced BoyJ HSPC (GFP+ Septin KD and BFP+ control were mixed 1:1) were injected into lethally irradiated C57Bl/6 mice, demonstrated a reduction in multilineage chimerism in the peripheral blood, bone marrow, spleen and thymus in primary and secondary transplant recipients after knockdown of Septin-6 (Table 2). To better define function in specific hematopoietic populations of HSPC, we performed knockdown in FACS-sorted purified cells. Engraftment studies demonstrated a significant reduction in bone marrow chimerism in primary transplant recipients after knockdown of Septin-6 most clearly in ST-HSC (Table 2) with a trend towards a decrease in LT-HSC and MPP. These data support a key role of Septin-6 in murine and human hematopoiesis via alterations in cell cycle and validate the pathogenic nature of a specific mutation in human SEPTIN-6. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 5626: Acquisition of lymphatic phenotype in bone marrow endothelial progenitors is regulated by immunosuppressive Th2 cytokines

Abstract Introduction: Myeloid-lymphatic endothelial progenitors (M-LECPs) significantly contribute to expansion of tumor lymphatics and metastasis to lymph nodes. Bone marrow (BM) generated M-LECPs are a subset of M2-type of tumor-associated macrophages (TAMs) that heavily infiltrate breast and other human cancers. The M2-type of TAMs is primarily induced by immunosuppressive Th2 cytokines IL-4, IL-13, and IL-10. The goal of this study was to determine whether Th2 factors also promote the lymphatic phenotype in precursors of M-LECPs. Methods: Mouse BM cells were differentiated into M-LECPs by priming with CSF-1 followed by activation of LPS-TLR4 pathway. Transcripts of IL-4, IL-13, and IL-10 were measured by qPCR, and their proteins secreted into conditioned medium were detected by ELISA. The levels of receptors for Th2 cytokines IL-4, IL-13, and IL-10 as well as for stem cells (CD117, Sca-1), M2-TAMs (CD204, CD206) and lymphatic endothelial cells (Lyve-1, podoplanin, integrin-9a, collectin-12 and stabilin-1) were quantified by qPCR and flow cytometry. Impact of TLR4 induced autocrine IL-10 pathway was determined by analyzing the surface markers and gene profiles of differentiated M-LECPs in the presence of IL-10 blocking or control antibodies. Tumor levels of Th2 cytokines and expression of Th2 receptors in Lyve-1+ progenitors were detected by ELISA and immunofluorescence, respectively. Results: More than 95% of myeloid precursors treated with CSF-1 and LPS upregulated all three Th2 receptors and IL-10 but not IL-4 or IL-13 ligands. All receptors were functional as indicated by upregulation of M2, immunosuppressive, lymphatic and stem markers in cells co-treated with exogenous Th2 ligands as compared with CSF-1 alone. Blocking autocrine IL-10 significantly suppressed both M2-specific and pro-lymphatic differentiation. Tumor-infiltrating M-LECPs expressed Th2 receptors as well as all corresponding ligands including IL-4 and IL-13 that were absent in the BM cells. Conclusion: These data present a novel evidence that activation of immunosuppressive Th2 pathways in BM myeloid precursors induces both the M2-type and lymphatic phenotype. Expansion of lymphatics can enhance tumor immunosuppression by physical removal of immunostimulatory cells. These findings suggest that targeting Th2 pathways can simultaneously relieve immunosuppression and inhibit differentiation of pro-lymphatic progenitors that promote metastasis. Citation Format: Maria Espinoza Gonzalez, Lisa Volk-Draper, Nihit Bhattarai, Sophia Ran. Acquisition of lymphatic phenotype in bone marrow endothelial progenitors is regulated by immunosuppressive Th2 cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5626.

Th2 Cytokines IL-4, IL-13, and IL-10 Promote Differentiation of Pro-Lymphatic Progenitors Derived from Bone Marrow Myeloid Precursors.

Myeloid-lymphatic endothelial cell progenitors (M-LECP) are a subset of bone marrow (BM)-derived cells characterized by expression of M2-type macrophage markers. We previously showed significant contribution of M-LECP to tumor lymphatic formation and metastasis in human clinical breast tumors and corresponding mouse models. Since M2 type is induced in macrophages by immunosuppressive Th2 cytokines IL-4, IL-13, and IL-10, we hypothesized that these factors might promote pro-lymphatic specification of M-LECP during their differentiation from BM myeloid precursors. To test this hypothesis, we analyzed expression of Th2 cytokines and their receptors in mouse BM cells under conditions leading to M-LECP differentiation, namely, CSF-1 treatment followed by activation of TLR4. We found that under these conditions, all three Th2 receptors were strongly upregulated in >95% of the cells that also secrete endogenous IL-10, but not IL-4 or IL-13 ligands. However, addition of any of the Th2 factors to CSF-1 primed cells significantly increased generation of myeloid-lymphatic progenitors as indicated by co-induction of lymphatic-specific (eg, Lyve-1, integrin-a9, collectin-12, and stabilin-1) and M2-type markers (eg, CD163, CD204, CD206, and PD-L1). Antibody-mediated blockade of either IL-10 receptor (IL-10R) or IL-10 ligand significantly reduced both immunosuppressive and lymphatic phenotypes. Moreover, tumor-recruited Lyve-1+ lymphatic progenitors in vivo expressed all Th2 receptors as well as corresponding ligands, including IL-4 and IL-13, which were absent in BM cells. This study presents original evidence for the significant role of Th2 cytokines in co-development of immunosuppressive and lymphatic phenotypes in tumor-recruited M2-type myeloid cells. Progenitor-mediated increase in lymphatic vessels can enhance immunosuppression by physical removal of stimulatory immune cells. Thus, targeting Th2 pathways might simultaneously relieve immunosuppression and inhibit differentiation of pro-lymphatic progenitors that ultimately promote tumor spread.

The Effect of the MK0339 Neutrophil Elastase Inhibitor on Proliferation and Maturation of iPSC Derived from Patients with ELANE, TCIRG1 and Whim Syndrome Associated Neutropenia

Genetic neutropenias are heterogeneous group of hematological diseases characterized by impaired survival and maturation of myeloid precursors in bone marrow. G-CSF and HSCT are currently the only effective treatment options. MK-0339 is a potent, cell permeable, orally absorbed inhibitor of neutrophil elastase (NE), previously investigated in preclinical and clinical studies by Merck/DuPont as a potential anti-inflammatory drug. We have previously reported that MK-0339 increases neutrophil proliferation in cellular models of ELANE associated neutropenia (2016 [ASH Annual Meeting Abstracts] Blood. 2016;128:406; 2017 J Leuk Biol [in press]). To examine the specificity of these responses, we investigated the effects of MK-0339 on iPSCs from healthy volunteers and compared its effects on differentiation of iPSC derived from patients with ELANE, TCIRG-1 and CXCR4 associated neutropenia.We generated induced pluripotent stem cells (iPSC) from dermal fibroblasts from healthy volunteers and neutropenic patients harboring ELANE, TCIRG1 and CXCR4 mutations and developed a feeder free, reproducible and efficient protocol for myeloid differentiation.In our initial experiments, we observed that iPSC lines derived from patients with ELANE mutations (P139L and G214R) consistently grew much slower and formed much smaller colonies than the controls using both mTeSR1/matrigel and DEF-CS feeder free culture medium systems. We observed more than 3-fold growth reduction of patient derived lines compared to healthy volunteers.We next tested the effects of MK-0339 under the same culture conditions. Growth of control and ELANE patient cell lines was enhanced, and the cells appeared more robust, after addition of 1uM MK-0339. We were surprised to see a similar proliferative effect (~40% increase) with Ramos (human Burkitt's lymphoma cell line) cells cultured in the presence of 1uM MK-0339, although neither the Ramos cells nor the ELANE iPSCs had detectable levels of ELANE expression. The enhanced growth of control and patient iPSCs and the results with Ramos cells suggested that the effects of MK-0339 are not solely mediated via inhibition of NE.We next compared myeloid differentiation and CD34+ cell proliferation of iPSC from patients with ELANE, TCIRG1 and CXCR4 associated neutropenia in HPC expansion medium, using granulocytic differentiation markers and flow cytometry. Myeloid maturation was impaired and inefficient for both ELANE (P139L and G214R) patient derived cell lines. Myeloid differentiation of the TCIRG1 patient derived was also impaired. Introduction of MK-0339 restored the impaired myeloid differentiation capacity of both ELANE and TCIRG1 positive patient derived cell lines. An iPSC line derived from a WHIM (CXCR4 mutation) patient appeared to have normal maturation and proliferation. Western blot analysis revealed elevated levels of endoplasmic reticulum chaperone GRP78/BiP in ELANE and TCIRG1 mutant cell lines which was corrected by MK-0339 treatment.These studies demonstrate heretofore unreported proliferative effects of MK-0339 on cell lines not expressing neutrophil elastase. They suggest important mechanisms beyond enzyme inhibition to account for these findings. Although preliminary, we believe that this work suggests that MK-0339 may be a novel, oral therapy for both ELANE and non-ELANE associated neutropenia. DisclosuresDale:University of Washington: Employment, Research Funding; National Institutes of Health: Research Funding; WedMD/Medscape: Membership on an entity's Board of Directors or advisory committees; Wolter Kluwer: Other: Editor, Current Opinions in Hematology; Philips: Research Funding; X4Pharma: Research Funding; Shire: Other: Independent Review Board; Omeros: Other: DSMB; Coherus: Consultancy; Boheringer-Ingelheim: Consultancy; Prolong: Consultancy; Hospira: Consultancy; Genkyotex: Other: DSMB (work completed 6/2015); Genzyme (now owned by Sanofi-Aventis): Consultancy, Patents & Royalties, Research Funding; Cellerant: Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy, Editor, Current Opinions in Hematology, Honoraria; Amgen: Consultancy, Research Funding; American College of Physicians: Other: Editor and author; GlaxoSmithKline: Equity Ownership; Johnson&Johnson: Equity Ownership.

BAFF Produced by Neutrophils and Dendritic Cells Is Regulated Differently and Has Distinct Roles in Antibody Responses and Protective Immunity against West Nile Virus.

B cell activating factor (BAFF) is essential for B cells to develop and respond to Ags. Dysregulation of BAFF contributes to the development of some autoimmune diseases and malignancies. Little is known about when, where, and how BAFF is produced in vivo and about which BAFF-producing cells contribute to B cell responses. To better understand BAFF functions, we created BAFF reporter (BAFF-RFP) mice and Baff floxed (Bafffl/fl ) mice. Splenic and bone marrow neutrophils (Nphs) from BAFF-RFP mice expressed the highest constitutive levels of BAFF; other myeloid subsets, including conventional dendritic cells (cDCs) and monocyte (MO) subsets, expressed lower levels. Treatment of BAFF-RFP mice with polyinosinic:polycytidylic acid increased BAFF expression in splenic Ly6Chi inflammatory MOs, CD11bhi activated NK subset, and in bone marrow myeloid precursors. Postinfection with West Nile virus (WNV), BAFF increased in CD8- cDCs and Nphs, and BAFF+ CD11bhi NK cells expanded in draining lymph nodes. The cell- and tissue-specific increases in BAFF expression were dependent on type I IFN signaling. MAVS also was required or contributed to BAFF expression in dendritic cell and MO subsets, respectively. Mice with deletion of Baff in either cDCs or Nphs had reduced Ab responses after NP-Ficoll immunization; thus, BAFF produced by both cDCs and Nphs contributes to T cell-independent Ab responses. Conversely, mice with a cDC Baff deficiency had increased mortality after WNV infection and decreased WNV-specific IgG and neutralizing Ab responses. BAFF produced by Nphs and cDCs is regulated differently and has key roles in Ab responses and protective immunity.

CRISPR/Cas9 Mediated ELANE Knock-out Restores Survival and Granulocytic Differentiation of HL60 Cells Expressing Mutant Neutrophil Elastase: Is Neutrophil Elastase a Dispensible Granulocyte Protease?

Background : Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease expressed primarily in neutrophils. Expression of the mutant protein impairs survival and maturation of myeloid precursors in bone marrow. More than 130 different ELANE mutations have been found in patients with cyclic and congenital neutropenia, and genotype-phenotype studies suggest that specific mutations cause more severe disease. (Curr Op Hematol. 2015;22:3-11) Mutant NE is also implicated as the primary cause of ELANE associated neutropenia by studies showing that cell permeable inhibitors of NE correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). Hypothesis: If inhibition of NE can correct the cellular defect, deletion of ELANE will have similar effects and permit studies to see if NE is an essential neutrophil protease. Methods: We used CRISPR/Cas9 mediated gene editing to create a cellular model of congenital neutropenia in the commercially available human promyelocytic cell line, HL60. We used CRISPR/Cas9 editing technology to create engineered HL60 cell lines with knock-in (KI) ELANE P139L, C151Y and G214R single point heterozygous mutations. Using the same technology, we then performed ELANE gene complete knock out (KO) of ELANE in each mutant cell line. All engineered cell lines as well as wild-type HL60 cells were cultured for 5-7 days in complete RPMI supplemented with 2uM all-trans retinoic acid (ATRA) to trigger myeloid differentiation. Survival of these cell lines was investigated using Annexin V-PE staining and flow cytometric analysis. Granulocytic differentiation was evaluated using CD11b surface marker staining and flow cytometry and by performing manual differential cell counts. We measured the unfolded protein response (UPR) by western blotting using UPR specific antibodies, phagocytosis with E. coli particles using fluorescence detection, IL-8 stimulated chemotaxis in trans-well system and PMA activated respiratory burst by flow cytometry. Results: Cells expressing the P139L and C151Y mutant ELANE had increased Annexin V staining more than a 2-fold increase in apoptotic cells at 7 days in culture. Granulocytic differentiation, measured by surface CD11b expression, was significantly impaired. (p<0.0001). Cytospins stained with Diff-Quik showed a typical block of myeloid differentiation and a significant deficiency of mature neutrophils in 7-day cultures. Western blot analysis using antibodies to GRP78/BiP and ATF6 showed a typical UPR signature in both ELANE mutant cell lines compared to wild type. Chemotaxis to the IL8 chemo-attractant was severely impaired in cells expressing mutant NE. Respiratory burst and phagocytosis was also altered in cells expressing mutant NE. ELANE KO corrected all these cellular and functional abnormalities, reverting these functions toward the wild phenotype. Studies are in progress for the third cell line expressing the G214R mutation. Conclusions: CRISPR/Cas9 engineered HL60 cell lines expressing mutant NE are a highly reproducible and reliable cellular model for investigating ELANE associated neutropenia. The presence of mutant NE severely impairs neutrophil functions, and its deletion through KO of ELANE corrects the cellular abnormalities and reverses the induced abnormalities. These results suggest that CRISPR/Cas9 mediated genetic knock down of NE is a novel therapeutic approach for treatment of this disorder. This data also suggests that total absence of NE does not alter basic functions of granulocytes. Disclosures Dale: Sanofi Aventis: Consultancy, Honoraria; Athelas: Equity Ownership; Amgen: Consultancy, Research Funding; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer/Ingelheim: Consultancy; Coherus: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding.

The impact of spliceosome mutations in MDS.

The impact of spliceosome mutations in MDS.

Abstract ES10-3: Blockade of estrogen signaling boosts antitumor immunity by inhibiting tumor-induced pathological myelopoiesis

Abstract Although the role of estrogen signaling in the progression of a subset of breast tumors has been underscored by the clinical use of Estrogen Receptor antagonists, how estrogens impact protective anti-tumor immunity remains poorly investigated. Here we show that estrogen signaling accelerates the progression of different estrogen insensitive tumor models by contributing to tumor-promoting emergency myelopoiesis. This results in increased mobilization of myeloid-derived suppressor cells (MDSCs), and also increases their intrinsic immunosuppressive activity on a per cell basis, in vivo in the bone marrow of tumor-bearing hosts. Accordingly, estrogens have a profound effect on T-cell-dependent anti-tumor immunity and tumor-promoting inflammation, even independently of the sensitivity of tumor cells to estrogen signaling. Differences in tumor growth in the presence of estrogen-insensitive tumor cells are dependent on blunted anti-tumor immunity and, correspondingly, disappear in immunodeficient hosts and also upon MDSC depletion. Mechanistically, estrogen receptor alpha activates the STAT3 pathway in human and mouse bone marrow myeloid precursors by enhancing JAK2 and SRC activity. Consequently, selective antagonists of estrogen receptor alpha (but not estrogen receptor beta) could have significant effects in delaying the progression of estrogen-sensitive and estrogen-insensitive tumors, providing a rationale for blocking estrogen signals to boost the effectiveness of anti-cancer immunotherapies. Our data therefore provide novel mechanistic insight into how enhanced estrogenic activity contributes to malignant progression in established tumors by driving pathological, tumor-promoting myelopoiesis. Our work suggests that new anti-estrogen drugs without agonistic effects (different from tamoxifen) could have benefits to boost protective immunity in a wide range of cancers, while augmented estrogenic activity could contribute to tumor initiation and/or malignant progression. Citation Format: Conejo-Garcia J. Blockade of estrogen signaling boosts antitumor immunity by inhibiting tumor-induced pathological myelopoiesis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES10-3.

CRISPR/Cas9 Knock-in HL60 Cells Closely Simulate Cellular and Functional Abnormalities of ELANE associated Neutropenia; Phenotype Rescue with MK-0339 Neutrophil Elastase Inhibitor

Background: Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. Congenital neutropenia is characterized by low neutrophil counts in peripheral blood and impaired survival and maturation of myeloid precursors in bone marrow. G-CSF and HSCT are currently the only effective treatment options. To date, more than 100 different ELANE mutations have been reported. We previously described that some mutations (e.g. G214R and C151Y) are associated with more severe outcomes, while others (e.g. P139L and R220Q) result in relatively mild clinical phenotypes. (Curr Op Hematol. 2015;22:3-11) To explain the biological effects of various ELANE mutants we have created CRISPR/Cas9 edited ELANE mutants using the commercially available human promyelocytic cell line HL60.Methods: We used CRISPR/Cas9 editing technology to create engineered HL60 cell lines with ELANE P139L and C151Y single point heterozygous mutations. Both ELANE mutant cell lines as well as wild-type HL60 cells were cultured for 7 days in complete RPMI supplemented with 2uM all-trans retinoic acid (ATRA) to trigger myeloid differentiation. Survival of these cell lines was investigated using Annexin V-PE staining and flow cytometric analysis. Granulocytic differentiation was evaluated using CD11b surface marker staining and flow cytometry and by performing manual differential cell counts. Unfolded protein response (UPR) was measured by western blotting using UPR specific antibodies.MK-0339 is a potent, cell permeable, orally absorbed inhibitor of neutrophil elastase (NE), previously investigated in preclinical and clinical studies by Merck/DuPont as a potential anti-inflammatory drug. We have recently reported that MK-0339 increases cell survival and myeloid differentiation in cellular models of ELANE associated neutropenia. (Makaryan, et al, J Leukoc Biol. 2017;102(4):1143-1151). We examined the effects of MK-0339 on these cell lines.Results: Annexin V staining showed more than 2-fold increase in apoptotic cells in both mutant cell lines compared to wild-type.Granulocytic differentiation measured by surface CD11b expression was significantly impaired in both mutant cell lines (p-values <0.0001). Cytospins stained with Diff-Quik showed a typical block of myeloid differentiation and a significant deficiency of mature myelocytes.Western blot analysis using antibodies to GRP78/BiP and ATF6 showed a typical UPR signature in both ELANE mutant cell lines compared to wild type. It is important to note that the C151Y mutant, the mutant clinically associated with more severe disease, shows more severe impairment compared to P139L. Addition of 1uM MK-0339 to the culture completely restored normal survival and myeloid differentiation of both mutant cell lines.Conclusions: We believe CRISPR/Cas9 engineered HL60 cell lines expressing mutant NE are a highly reproducible and reliable cellular model for investigating genetic neutropenias. These results suggest that a panel of different mutant ELANE HL60 cell lines will help to elucidate the molecular and biochemical origin of phenotypic variability in ELANE associated neutropenia. DisclosuresDale:Athelas, Inc.: Equity Ownership; Amgen: Consultancy, Research Funding; Sanofi-Aventi: Consultancy, Honoraria; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer-Ingelheim: Consultancy; Coherus: Consultancy.

Abstract SY01-02: Blockade of estrogen signaling boosts antitumor immunity by dwindling cancer-promoting myelopoiesis

Abstract Although the role of estrogen signaling in the progression of breast tumors and a subset of ovarian cancer patients has been underscored by the clinical use of ER antagonists, how estrogens impact the orchestration and maintenance of protective antitumor immunity remains elusive. Here we show that estrogen signaling accelerates the progression of different estrogen-insensitive tumor models by contributing to tumor-promoting, expanded myelopoiesis. This results in increased mobilization of myeloid-derived suppressor cells (MDSCs) and also the augmentation of their intrinsic immunosuppressive activity on a per cell basis, in vivo in the bone marrow of tumor-bearing hosts. Accordingly, estrogens had a profound effect on T cell-dependent antitumor immunity and tumor-promoting inflammation, independently of the sensitivity of tumor cells to estrogen signaling. Differences in tumor growth in the presence of estrogen-insensitive tumor cells are dependent on blunted antitumor immunity and, correspondingly, disappear in immunodeficient hosts and also upon MDSC depletion. Mechanistically, estrogen receptor alpha activates the STAT3 pathway in human and mouse bone marrow myeloid precursors by enhancing JAK2 and SRC activity. Consequently, fulvestrant had significant effects in delaying the progression of estrogen-insensitive tumors, providing a rationale for blocking estrogen signals to boost the effectiveness of anticancer immunotherapies, which is currently being tested at our institution in breast cancer patients. Our data therefore provide novel mechanistic insight into how enhanced estrogenic activity contributes to malignant progression in established tumors by driving pathologic, tumor-promoting myelopoiesis. Our work suggests that new antiestrogen drugs that have no agonistic effects (different from tamoxifen) could have benefits to boost protective immunity in a wide range of cancers, while augmented estrogenic activity could contribute to tumor initiation and/or malignant progression. Citation Format: Nikolaos Svoronos, Alfredo Perales-Puchalt, Jose R. Conejo-Garcia. Blockade of estrogen signaling boosts antitumor immunity by dwindling cancer-promoting myelopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY01-02.

Early TNFα signaling results in pulmonary DC1 polarization and programing of murine myeloid precursor cells in the bone marrow towards DC1 polarization throughout Cryptococcus neoformans infection

Abstract TNFα is required for protective Th1/Th17 immunity to Cryptococcus neoformans (CN), an opportunistic pulmonary fungal pathogen. The effects of TNFα are linked to the stable, early classical activation of dendritic cells (DC1), preventing alternative (DC2) activation. We hypothesized that TNFα signaling facilitates epigenetic modification of key DC genes during CN infection. We tested this hypothesis using CBA/J mice infected intratracheally with CN and injected once on day 0 intraperitoneally with control or a TNFα-depleting antibody (αTNFα). αTNFα mice had no difference in pulmonary CFU at 7 days post-infection (dpi), but had significantly higher fungal burden and extrapulmonary dissemination at 14 and 28 dpi. DCs from the lungs of control mice at 7 dpi had increased association of iNOS and IL-12b promoters with the activating modification histone 3 lysine 4 trimethylation (H3K4me3), while DCs from αTNFα mice did not. Because DCs have a relatively short half-life during infection, we assessed the bone marrow (BM) myeloid precursor cells (MPCs) from infected animals with and without TNFα. Intranuclear flow cytometry for H3K4me3 showed distinct patterns of global trimethylation between infected control and αTNFα mice as early as 7 dpi. We next tested whether BM-derived DCs (BMDCs) matured ex vivo from infected animals would yield DC1 or DC2 cells. BMDCs from control animals became DC1, and resisted changes to polarization when challenged with the DC2-skewing IL-4, while BMDCs matured from TNFα-depleted mice maintained DC2 polarization when challenged with IFNγ. We conclude that TNFα epigenetically modifies key DC1 genes in MPCs, thereby sustaining lasting DC1 programing of DCs required for clearance of CN from the infected lungs.

Silberline & Shinemax sign technical collaboration agreement

Osteoclasts are components of hematopoietic stem cell (HSC) niches, but their role as contributors to the HSC homeostasis and release are still controversial. We aimed to investigate whether an acute blood loss of 10% of total blood content, along with the consequent intense hematopoiesis, would affect osteoclast differentiation and activity. Isolated peripheral blood, spleen, and bone marrow (BM) cells from bones of hind limbs were investigated for the presence of specific subpopulations of osteoclast precursors: B220-CD3-NK1.1-CD11b-/lowCD115+CD117+ cells in BM, and B220-CD3-NK1.1-Gr-1-CD11b+CD115+ cells in peripheral blood and spleen as well as the receptor activator of nuclear factor κ-B+ cycle-arrested quiescent osteoclast precursors. Expression of osteoclastogenesis-related genes CD115, receptor activator of nuclear factor κ-B, and cathepsin K, the potential of BM cells to form osteoclast-like cells in vitro, and osteoclast activity in vivo were also evaluated. We observed an increase in spleen cellularity and myelopoiesis during week 1 following blood loss, without any significant effects on BM cellularity or BM myeloid precursors, including cells with high osteoclastogenic potential. However, at 1 week postbleeding, hematopoiesis significantly promoted the expression of cathepsin K, interleukin-34, and bone morphogenetic protein-6. Quiescent osteoclast precursors increased significantly in spleen 2 days following bleeding, whereas osteoclast activity remained unchanged up to 2 weeks postbleeding. Osteoclast-dependent B-cell differentiation was affected at the pre-B stage of maturation in BM, whereas the Lin-Sca-1+c-kit+ population expanded in BM and spleen after 2 days postbleeding. Our data demonstrate that an acute blood loss promotes differentiation and maturation of osteoclasts at 1 week but does not enhance osteoresorption at 2 weeks postbleeding. Our data also identify osteoclast differentiation as a consequent and important event in establishing HSC homeostasis following hematopoietic stress.

Huber buys Martinswerk (ATH & magnesium hydroxide) from Albemarle

Osteoclasts are components of hematopoietic stem cell (HSC) niches, but their role as contributors to the HSC homeostasis and release are still controversial. We aimed to investigate whether an acute blood loss of 10% of total blood content, along with the consequent intense hematopoiesis, would affect osteoclast differentiation and activity. Isolated peripheral blood, spleen, and bone marrow (BM) cells from bones of hind limbs were investigated for the presence of specific subpopulations of osteoclast precursors: B220-CD3-NK1.1-CD11b-/lowCD115+CD117+ cells in BM, and B220-CD3-NK1.1-Gr-1-CD11b+CD115+ cells in peripheral blood and spleen as well as the receptor activator of nuclear factor κ-B+ cycle-arrested quiescent osteoclast precursors. Expression of osteoclastogenesis-related genes CD115, receptor activator of nuclear factor κ-B, and cathepsin K, the potential of BM cells to form osteoclast-like cells in vitro, and osteoclast activity in vivo were also evaluated. We observed an increase in spleen cellularity and myelopoiesis during week 1 following blood loss, without any significant effects on BM cellularity or BM myeloid precursors, including cells with high osteoclastogenic potential. However, at 1 week postbleeding, hematopoiesis significantly promoted the expression of cathepsin K, interleukin-34, and bone morphogenetic protein-6. Quiescent osteoclast precursors increased significantly in spleen 2 days following bleeding, whereas osteoclast activity remained unchanged up to 2 weeks postbleeding. Osteoclast-dependent B-cell differentiation was affected at the pre-B stage of maturation in BM, whereas the Lin-Sca-1+c-kit+ population expanded in BM and spleen after 2 days postbleeding. Our data demonstrate that an acute blood loss promotes differentiation and maturation of osteoclasts at 1 week but does not enhance osteoresorption at 2 weeks postbleeding. Our data also identify osteoclast differentiation as a consequent and important event in establishing HSC homeostasis following hematopoietic stress.

Osteoclasts in the interface with electrospun hydroxyapatite

Electrospinning is a method to produce lightweight, resorbable and bioinspired scaffolds for tissue engineering. Here we investigated the influence of electrospun hydroxyapatite fibers (HA) on macrophages and osteoclasts. A mouse macrophage cell line (RAW 264.7) and human bone marrow derived primary osteoclasts (hOC) were cultured with electrospun HA fibers embedded in Matrigel. Cell morphology and the secretion of pro-inflammatory cytokines (IL-6 and TNF-α) were analyzed using macrophages. Both fluorescent microscopy and scanning electron microscopy indicated that the cell morphology differed on the various materials (HA fibers on Matrigel, pure Matrigel and a glass control). Control macrophages were activated with bacterial lipopolysaccharide (LPS) but electrospun HA did not provoke an inflammatory response. Cytokine secretion detected with enzyme-linked immunosorbent assay (ELISA) also supported this observation. LPS, but not HA fibers, stimulated TNF-α and IL-6 secretion by macrophages at the 2 day time point. After 4 days in culture there was an increasing trend in cytokine secretion in the HA fiber samples. Human bone marrow myeloid precursor cells were able to fuse and differentiate on the fibrous mineral scaffold to form functional multinuclear osteoclasts that were able to resorb the HA nanofibers. This indicates that osteoclasts do not necessarily need a continuous bone surface but osteoclast ruffled border membranes can form a resorption interface with a fibrous mineral scaffold.

Preclinical Mechanistic Studies Investigating Neutrophil and Lymphoid Cell Depletion By IMGN529, a CD37-Targeting Antibody-Drug Conjugate (ADC)

CD37 is a surface antigen widely expressed on malignant B cells in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). In normal tissues, CD37 expression is restricted to lymphoid tissues and blood cells, with high levels of expression on B lymphocytes and low levels on non-B lymphoid and myeloid cells. IMGN529 is a CD37-targeting ADC currently in a Phase I clinical study in adult patients with relapsed or refractory NHL (NCT01534715). This ADC uniquely combines the intrinsic pro-apoptotic and immune effector activities of its anti-CD37 antibody component with the potent cytotoxic mechanism provided by targeted delivery of its maytansinoid payload, DM1. In the Phase I study, IMGN529 has demonstrated early evidence of clinical activity. A reduction in lymphocyte counts was also observed in the majority of patients after dosing, consistent with the proposed mechanism of action of a CD37-targeted therapy. However, in the initial dose-escalation phase, some patients experienced transient, early-onset neutropenia.To investigate the potential mechanisms of this transient neutropenia observed in patients, different pre-clinical models were considered and utilized to recapitulate clinical findings. In vitro studies with peripheral blood cells from normal human donors demonstrated that incubation with IMGN529 for 1 hour or 24 hours resulted in significant B-cell depletion with no apparent neutrophil depletion detected, similar to observations after rituximab treatment. In contrast, alemtuzumab treatment in vitro resulted in both B-cell and neutrophil depletion. This is consistent with the high level of CD37 expression on target B cells and the relatively low CD37 expression level on other blood cells. Analysis of cytokine release by normal human donor peripheral blood cells incubated with IMGN529 revealed increased levels of IL-8, CCL2 (MCP-1) and CCL4 (MIP-1β), but not IL-6 or TNF, to a similar extent as rituximab but less pronounced than alemtuzumab. An anti-murine CD37 antibody was identified to enable in vivo studies in a murine model and characterize CD37 expression on murine blood cells. Similar to the expression profile of CD37 in human peripheral blood cells, CD37 expression on murine peripheral blood cells was highest in B cells, with much lower expression seen on T cells and granulocytes. In vivo activity of the anti-muCD37 antibody and the corresponding ADC, with the same SMCC-DM1 linker-payload combination as IMGN529, was evaluated to discern antibody and payload-mediated events in comparison to the classic cytotoxic cyclophosphamide (CPA). Treatment of C57/B6 mice with 1-10 mg/kg of anti-muCD37 antibody or anti-muCD37 ADC resulted in a significant decrease in absolute lymphocyte counts (ALC) lasting greater than 7 days and a transient decrease in absolute neutrophil counts (ANC) lasting 1-2 days. A non-targeted control SMCC-DM1 ADC had no effect on ALC or ANC counts, suggesting the decrease is a CD37-mediated effect. In contrast, treatment with CPA resulted in an ALC decrease with similar kinetics but a more pronounced ANC decline. No impact on bone marrow lymphocyte, myeloid or erythroid precursor cell counts was observed in response to the anti-muCD37 antibody or anti-muCD37 ADC, whereas CPA treatment caused reduced cellularity with a decrease in the percentage of mature myeloid precursors and neutrophils in bone marrow. Elevated levels of CCL2 and CCL4 chemokines were detected in mouse plasma after anti-muCD37 ADC treatment, which may contribute to a redistribution of circulating neutrophils into peripheral tissues. Studies are currently underway to assess neutrophil distribution in murine tissues post anti-muCD37 ADC treatment.Current preclinical studies provide no clear evidence for direct IMGN529-mediated depletion of normal human neutrophils in the context of B-cell depletion in vitro. In vivo studies with an anti-muCD37 ADC recapitulate transient peripheral lymphopenia and neutropenia with no impact on bone marrow precursors observed, indicative of a different mechanism than classic chemotherapy-induced bone marrow myelosuppression. These preliminary results suggest a role for chemokine-mediated neutrophil redistribution following CD37 engagement, which is the subject of further studies. DisclosuresDeckert:ImmunoGen, Inc.: Employment, Equity Ownership. Ponte:ImmunoGen, Inc.: Employment, Equity Ownership. Coccia:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Chicklas:ImmunoGen, Inc.: Employment, Equity Ownership. Yi:ImmunoGen, Inc.: Employment, Equity Ownership. Watkins:ImmunoGen, Inc.: Employment, Equity Ownership. Ruiz-Soto:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Lutz:ImmunoGen, Inc.: Employment, Equity Ownership.

Abstract A37: MerTK receptor tyrosine kinase is a therapeutic target in pediatric acute leukemia

Abstract Acute myeloid leukemia (AML) and relapsed or refractory acute lymphoblastic leukemia (ALL) continue to be extremely difficult to treat and unacceptably low overall survival rates mandate the exploration of new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation has been associated with poor prognosis in patients with AML and ALL, providing strategic targets for novel therapy development. The MerTK receptor tyrosine kinase is overexpressed in a majority of leukemia cell lines and pediatric patient samples at the time of diagnosis and expression is often upregulated at relapse. In contrast, normal bone marrow myeloid and lymphoid precursors express little or no MerTK. Stimulation of leukemia cell lines with Gas6, a MerTK ligand, activates prosurvival and proliferative signaling pathways known to contribute to leukemogenesis, including ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. Inhibition of MerTK using shRNA significantly increased myeloblast apoptosis in response to serum starvation in the Nomo-1 and Kasumi cell lines and decreased colony forming potential. Importantly, these phenotypes were reversed by exogenous expression of wild-type MerTK protein but not by expression of a mutant MerTK that lacks kinase activity, indicating that oncogenic roles for MerTK are mediated its kinase activity. Additionally, NOD-scid-gamma mice transplanted with Nomo-1 myeloblasts or 697 lymphoblasts with reduced levels of MerTK have a significantly prolonged median survival time compared to mice transplanted with control cell lines. These data validate Mer as a potential therapeutic target in acute leukemias. We have recently developed first-in-class MerTK-selective small molecule tyrosine kinase inhibitors (MerTKIs). Treatment with MerTKI phenocopied the effects of shRNA, including inhibition of prosurvival signaling through ERK1/2, Akt, and STAT6. Flow cytometric analysis of myeloblast cell lines following treatment with MerTKI and staining with YoPro-1-iodide and propidium iodide dyes revealed induction of apoptosis in Mer-positive leukemia cell lines (mean of 66±10% apoptotic and dead cells verses 20±10% after treatment with vehicle, p&amp;lt;0.01). In an orthotopic B-ALL murine xenograft model of minimal residual disease, treatment with MerTKI significantly inhibited leukemogenesis and increased median survival from 25 days after inoculation with tumor cells to 65 days (p &amp;lt; 0.0001). In a similar model in which leukemia was established for 11 days prior to initiation of treatment, median survival increased from 25 to 47 days in response to treatment with MerTKI (p &amp;lt; 0.0001). In both models, tumor burden measured by bioluminescent imaging was significantly decreased in mice treated with MerTKI relative to mice treated with vehicle, even after the development of advanced disease in the control animals (0.27 +/- 0.04 x107 photons/second in the minimal residual disease model and 0.98 +/- 0.23 x107 photons/second in the established disease model verses 26.75 +/- 4.54 x107 photons/sec in vehicle-treated mice, p &amp;lt; 0.0001). These data indicate roles for MerTK in pediatric acute lymphoid and myeloid leukemias and validate targeted inhibition of MerTK as a potential therapeutic strategy. Citation Format: Douglas K. Graham, Deborah DeRyckere, Alisa Lee-Sherick, Amanda A. Hill, Katherine Minson, Wnag Xiaodong, Weihe Zhang, Jing Liu, Stephen Frye, H. Shelton Earp. MerTK receptor tyrosine kinase is a therapeutic target in pediatric acute leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A37.

Acute hematopoietic stress in mice is followed by enhanced osteoclast maturation in the bone marrow microenvironment

Osteoclasts are components of hematopoietic stem cell (HSC) niches, but their role as contributors to the HSC homeostasis and release are still controversial. We aimed to investigate whether an acute blood loss of 10% of total blood content, along with the consequent intense hematopoiesis, would affect osteoclast differentiation and activity. Isolated peripheral blood, spleen, and bone marrow (BM) cells from bones of hind limbs were investigated for the presence of specific subpopulations of osteoclast precursors: B220(-)CD3(-)NK1.1(-)CD11b(-/low)CD115(+)CD117(+) cells in BM, and B220(-)CD3(-)NK1.1(-)Gr-1(-)CD11b(+)CD115(+) cells in peripheral blood and spleen as well as the receptor activator of nuclear factor κ-B(+) cycle-arrested quiescent osteoclast precursors. Expression of osteoclastogenesis-related genes CD115, receptor activator of nuclear factor κ-B, and cathepsin K, the potential of BM cells to form osteoclast-like cells in vitro, and osteoclast activity in vivo were also evaluated. We observed an increase in spleen cellularity and myelopoiesis during week 1 following blood loss, without any significant effects on BM cellularity or BM myeloid precursors, including cells with high osteoclastogenic potential. However, at 1 week postbleeding, hematopoiesis significantly promoted the expression of cathepsin K, interleukin-34, and bone morphogenetic protein-6. Quiescent osteoclast precursors increased significantly in spleen 2 days following bleeding, whereas osteoclast activity remained unchanged up to 2 weeks postbleeding. Osteoclast-dependent B-cell differentiation was affected at the pre-B stage of maturation in BM, whereas the Lin(-)Sca-1(+)c-kit(+) population expanded in BM and spleen after 2 days postbleeding. Our data demonstrate that an acute blood loss promotes differentiation and maturation of osteoclasts at 1 week but does not enhance osteoresorption at 2 weeks postbleeding. Our data also identify osteoclast differentiation as a consequent and important event in establishing HSC homeostasis following hematopoietic stress.

Familial Mediterranean fever gene mutations as a risk factor for early coronary artery disease

Cardiovascular diseases (CVD) are very common in the general population. Atherosclerosis is the main pathogenesis. Familial Mediterranean fever (FMF) is an autosomal recessive disease. The gene causing FMF, designated MEFV, encodes a protein called pyrin or marenostrin that is expressed mainly in myeloid bone marrow precursors, neutrophils and monocytes. We herein aimed to determine the prevalence of MEFV mutations (all exon 2, 10 mutations) in patients with early coronary heart disease (early CHD) and coronary heart disease (CHD) with multiple risk factors and among the healthy subjects as controls. A total of 197 patients and 119 healthy subjects were recruited and enrolled into three groups in terms of inclusion criteria. Ninety-one patients diagnosed with early CHD enrolled into group one (men <45years of age, women <40years of age), 106 patients with CHD (men >50years of age) to group two and 119 healthy controls enrolled into group three. None of patients was diagnosed with FMF. The diagnosis of CHD was established on electrocardiographic changes, echocardiography and coronary angiography. Thirty-eight patients (41.8%) with early CHD, 17 patients (16%) with CHD and 24 healthy controls (20.2%) carried at least one mutated MEFV allele. Young patients with CHD have different risk factor profiles, clinical presentations and prognoses than older patients. Young patients with CHD usually have multiple risk factors. This study suggests that MEFV mutations in early CHD patients had significantly increased in contrast to CHD patients and healthy controls.

AB0008 Familial mediterranean fever (FMF) gene mutations (MEFV): are they a risk factor for coronary artery disease?

BackgroundFamilial Mediterranean fever (FMF) is an autosomal recessive disease which predominantly affects certain ethnic groups mainly Sephardic Jews, Turks, Arabs and Armenians. The gene causing FMF, designated MEFV, encodes a...

Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia

Acute myeloid leukemia (AML) continues to be extremely difficult to treat successfully, and the unacceptably low overall survival rates mandate that we assess new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation in AML has been associated with poor prognosis and provides strategic targets for novel therapy development. We found that Mer receptor tyrosine kinase was over-expressed in a majority of pediatric (29/36, 80%) and adult (10/10, 100%) primary AML patient blasts at the time of diagnosis, and 100% of patient samples at the time of relapse. Mer was also found to be expressed in 12 of 14 AML cell lines (86%). In contrast, normal bone marrow myeloid precursors expressed little to no Mer. Following AML cell line stimulation with Gas6, a Mer ligand, we observed activation of prosurvival and proliferative signaling pathways, including phosphorylation of ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. To assess the phenotypic role of Mer in AML, two independent short-hairpin RNA (shRNA) constructs were used to decrease Mer expression in the AML cell lines Nomo-1 and Kasumi-1. Reduction of Mer protein levels significantly increased rates of myeloblast apoptosis two to threefold in response to serum starvation. Furthermore, myeloblasts with knocked-down Mer demonstrated decreased colony formation by 67–87%, relative to control cell lines (P<0.01). NOD-SCID-gamma mice transplanted with Nomo-1 myeloblasts with reduced levels of Mer had a significant prolongation in survival compared with mice transplanted with the parental or control cell lines (median survival 17 days in parental and control cell lines, versus 32–36 days in Mer knockdown cell lines, P<0.0001). These data suggest a role for Mer in acute myeloid leukemogenesis and indicate that targeted inhibition of Mer may be an effective therapeutic strategy in pediatric and adult AML.