Antibiotics Treatment Ameliorates TET2 Loss of Function Associated Hematological Malignancies

Abstract Background: The DNA methylation dioxygenase TET2 is frequently mutated in hematological malignancies[1,2]; however, TET2 mutations are also frequently detected in individuals with clonal hematopoiesis[3], suggesting additional factors might be required to promote the malignant transformation of TET2-defective hematopoietic cells. Indeed, microbia-triggered inflammation has been recently shown to promote the myeloproliferation of Tet2 deficient hematopoietic cells[4]. This finding points to a strong association between intestinal microbiota and epigenetic alterations in the hematopoietic system during disease progression. Gut microbiota can be affected by antibiotics treatment and antibiotics could alter murine hematopoiesis through the depletion of intestinal microbiota[5]. Antibiotic treatment is commonly used in hematological malignancy patients with infection[6], which is one of the major complications in these patients. Nonetheless, it is not clear how antibiotics might impinge on malignant blood cells with epigenetic defects (e.g., Tet2 deficiency). In this study, we aim to characterize the impact of antibiotics treatment on Tet2 deficient malignant cells in the hematological system. Methods: We established two sets of Tet2 knockout (KO) malignant cell lines derived from aged Tet2KO mice with T cell lymphoma (n = 6) or MDS/CMML (n =3). Flow cytometry analysis and molecular analysis confirmed the tumor phenotypes. Furthermore, these cells were transplantable in immune competent CD45.1 mice and underwent expansion without losing their tumor properties. To evaluate the impact of antibiotics treatment on tumor growth, we transplanted Tet2KO T cell lymphoma cells or CMML/MDS cells into CD45.1 recipient mice followed by antibiotic treatment (ampicillin, vancomycin, neomycin, and metronidazole) for 10 days. Then we monitored tumor cell growth in peripheral blood, spleens and bone marrows collected from CD45.1 mice with and without antibiotics cocktails treatment. We also collected the Tet2KO tumor cells 20 days after antibiotics treatment and will perform transcriptomic and 5hmC profiling to further investigate how antibiotics alters the molecular signatures in Tet2KO tumor cells. Results: Consistent with earlier reports, we observed a moderate bone marrow suppression during the antibiotics treatment in recipient mice but not after cessation of treatment[5]. Interestingly, we also observed that antibiotics treatment significantly suppressed Tet2KO tumor growth in the recipient mice. Mice treated with antibiotics cocktails showed a pronounced decrease of microbiota in collected feces. Furthermore, the antibiotics treated group displayed significantly longer survival compared with the untreated group. Flow cytometry analysis showed a significant decrease of CD45.2+ Tet2KO tumor cells in peripheral blood, spleens and bone marrow 20 days after antibiotics treatment. Detailed analysis in bone marrow collected from recipient mice transferred with CMML/MDS-like Tet2KO revealed up to 80% reduction of c-Kit+ cancer stem-like cells in the antibiotics treated group without significantly affecting endogenous hematopoietic cells in recipient mice 20 days after cessation of antibiotic treatment. Conclusion: In summary, we conclude that antibiotics treatment could partially suppress/delay Tet2KO tumor cells growth. Further RNA-seq and 5hmC mapping will be performed to gain detailed mechanistic insights. Furthermore, we will determine whether the suppressive effect is due to direct action on tumor cells or because of indirect effects from the removal of intestinal microbiota. Nonetheless, our data demonstrated the potential benefits of antibiotics treatment on hematological malignancies associated with Tet2 loss of function. References 1. Couronne, L., Bastard, C. & Bernard, O. A. (2012). N Engl J Med 366, 95-96. 2. Delhommeau, F., Dupont, S., Della Valle, V. et al. (2009). N Engl J Med 360, 2289-2301. 3. Busque, L., Patel, J. P., Figueroa, M. E. et al. (2012). Nat Genet 44, 1179-1181. 4. Meisel, M., Hinterleitner, R., Pacis, A. et al. (2018). Nature 557, 580-584. 5. Josefsdottir, K. S., Baldridge, M. T., Kadmon, C. S. et al. (2017). Blood 129, 729-739. 6. Safdar, A. & Armstrong, D. (2011). Clin Infect Dis 53, 798-806. Disclosures No relevant conflicts of interest to declare.

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Activated JAK2 Signaling in Primary Human Hematopoietic Cells Drives Erythropoietin-Independent Erythropoiesis and Myelofibrosis.

Blood ◽

10.1182/blood.v106.11.374.374 ◽

2005 ◽

Vol 106 (11) ◽

pp. 374-374

Author(s):

James A. Kennedy ◽

Frederic Barabe ◽

Dwayne L. Barber ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Lentiviral Vector ◽

Hematopoietic Cells ◽

In Vivo Studies ◽

Causative Role ◽

Post Transplant ◽

The Impact ◽

Insight Into

Abstract An activating mutation in the JAK2 tyrosine kinase has recently been identified in patients with polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). However, at this time, evidence of a causative role for activated JAK2 signaling in the pathogenesis of these myeloproliferative disorders is limited. Given that these are clonal stem cell diseases, the impact of deregulated JAK-STAT signaling must be considered in the context of primitive human hematopoietic cells. To this end, a lentiviral vector encoding the TEL-JAK2 (5–19) fusion was employed to infect lineage-depleted human cord blood, resulting in constitutive activation of STAT5 as well as the ERK and PI-3 kinase signaling pathways. Interestingly, when grown in culture under conditions that normally support the exclusive production of cells from the granulopoietic and monocytic lineages, cells expressing TEL-JAK2 underwent a burst of erythropoietin (EPO)-independent erythropoiesis; an outcome that could even occur in serum-free media in the absence of supportive cytokines. Furthermore, TEL-JAK2 cells exhibited erythroid colony-forming capacity in the absence of exogeneous EPO, a hallmark of PV. In order to gain insight into the in vivo effects of activated JAK2 signaling, transduced cord blood cells were injected intrafemorally into sublethally irradiated NOD/SCID mice. When animals were sacrificed 3 weeks post-transplant, the percentage of human engraftment was comparable between TEL-JAK2 and control animals; however, total cellularity was significantly reduced in the injected femur of TEL-JAK2 mice. At 9 weeks post-transplant, this reduction in cellularity was noted not only in the injected femur, but also in distant marrow cavities. Histological examination of the bone marrow from TEL-JAK2-transplanted mice showed a decrease in cellularity, the presence of an atypical megakaryocyte population, and patent sinusoids. Most notably, reticulin staining revealed the existence of a fibrous network in the bone marrow of TEL-JAK2 mice, indicative of myelofibrotic change. Taken together, these in vitro and in vivo studies provide functional evidence that in human hematopoietic cells, activated JAK2 signaling can initiate disease processes bearing similarity to both PV and IMF. Furthermore, they provide the first example of an experimental xenotransplant model of IMF which can be utilized to provide insight into both the pathogenesis of this disease and potential therapeutic targets.

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Direct Intra-Bone Marrow Transplant of Cord Blood Cells: A Way To Overcome Delayed Engraftment.

Blood ◽

10.1182/blood.v108.11.3190.3190 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3190-3190 ◽

Cited By ~ 3

Author(s):

Adalberto Ibatici ◽

Anna Maria Raiola ◽

Marina Podesta ◽

Sarah Pozzi ◽

Silvia Lucchetti ◽

...

Keyword(s):

Bone Marrow ◽

Pilot Study ◽

Cord Blood ◽

Blood Cells ◽

Therapeutic Option ◽

Hematopoietic Cells ◽

Adult Patients ◽

Cord Blood Cells ◽

The Impact ◽

Seeding Efficiency

Abstract The major limit of cord blood (CB) transplant in adult patients is represented by the delayed engraftment. Newer approaches have been suggested including double cord blood transplant and infusion of CD34+ selected cells from a relative to achieve a transient engraftment until CB cells generate mature blood cells. Our initial pre-clinical studies showed that intra-bone injection of hematopoietic cells in the mouse was associated with an engraftment about 10 times more efficient with respect to the usual intravenous injection. Moreover, we had already demonstrated that the defect of cord blood cells in term of delayed engraftment was not related to the lower number of hematopoietic cells infused since the capacity to reconstitute the host stem cell reservoir was higher than adult bone marrow. Therefore, we set-up a pilot study to evaluate whether this type of transplant could shorten the time of engraftment. Five patients [4 male (M) and 1 female (F)] have been enrolled in this study. Clinical characteristics at transplant were: three patients had acute myeloid leukaemia: 2 refractory-relapsing and one in 1st CR (age 37 M, 50 M, 60 M) and 2 acute lymphoblastic leukaemia: one in 1st CR and one in 2nd CR (age 42 F, 18 M). Cord Blood grafts were matched as follows: 3/6 in one patients, 4/6 in three patients, and 5/6 in one patient for HLA-A, B, DRB1 (serology for HLA class I and allelic for class II). Patients were prepared with standard conditioning including TBI. Anti-Thymocyte-Globulin 3.75mg/kg was given on day −3,−2. GVHD Prophylaxis was cyclosporine from day -7 (therapeutic range 200–400 ng /ml) plus mycophenolate-mofetil 15mg/kg b.i.d. from day +1 to +28. The number of cord blood cells infused was 2.4, 1.6, 2.4, 3.0, 2.7 x 10^7/kg b.w. Cells were infused under very short general anaesthesia (propofol) of 5–10 minutes in the posterior iliac crest with a standard bone marrow aspiration needle (gauge n. 14). Before infusion, CB cells were washed to eliminate DMSO. Sustained engraftment of PMN (more than 3 consecutive days > 500 PMN x10^9/l) was recorded at day: + 20, 25, 18, 20, 23. Platelets sustained engraftment (more than 3 consecutive days > 20.000 Platelets x10^9/l) was recorded at day: +32, 42, 25, 29, 31.Acute GVHD was absent in these five patients. This is a pilot study with very short follow-up (2 to 5 months). Thus, no conclusive answer can be drawn on the impact of this technique on the final outcome of these patients. However, the time to PMN and Platelets engraftment is much shorter with respect to the available data obtained with intra-venous (IV) injection of the same amount of cells. Seeding efficiency experiments have shown that less than 10% of the injected cells via IV actually seed in the active hematopoietic sites. Cord blood cells, injected directly into the bone marrow, may undergo a better homing without loss, proliferate and finally colonise the rest of the bone marrow; thus, improving considerably the seeding efficiency. Alternatively, CB cells may undergo faster maturation if injected intra-bone marrow and, at the same time, colonise more efficiently the rest of active hematopoietic sites. If confirmed in larger studies, this approach may render cord blood cells transplant suitable for a greater number of adult patients who so far were excluded from this therapeutic option. This work was supported by AIRC, CARIGE; Compagnia di San Paolo.

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Limited Regeneration but Functional Preservation of Hematopoietic Stem Cells in the Mice Developing Leukemia via Constitutive Expression of Notch1.

Blood ◽

10.1182/blood.v110.11.204.204 ◽

2007 ◽

Vol 110 (11) ◽

pp. 204-204 ◽

Cited By ~ 1

Author(s):

Xiaoxia Hu ◽

Hongmei Shen ◽

Hui Yu ◽

Feng Xu ◽

Jianmin Wang ◽

...

Keyword(s):

Bone Marrow ◽

Molecular Mechanisms ◽

Lymphoblastic Leukemia ◽

Hematopoietic Cells ◽

Constitutive Expression ◽

Hematopoietic Progenitor Cell ◽

Control Group ◽

Extrinsic Factors ◽

Hematopoietic Stem ◽

The Impact

Abstract Leukemia development is a complex process involving both intrinsic and extrinsic factors. While many environmental factors have been studied, the impact of leukemic environment on normal hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) has not been definitively investigated. In this study, we have formally addressed this important issue by examining the potential functional alterations of HSC and HPC in the mice bearing Notch1-induced T acute lymphoblastic leukemia (T-ALL). The MSCV retrovirus vector containing cDNA encoding oncogenic intracellular domain of Notch1 (ICN1) pseudotyped with VSV-G was used to infect Lin−Sca-1+ cells in order to induce leukemic development. Normal hematopoietic cells from the B6.SJL strain (CD45.1+) were co-transplanted with Notch1 transduced Lin−Sca-1+ cells (CD45.2+) into lethally irradiated recipients. In this robust leukemia model with 100% penetrance, the normal hematopoietic cell compartment marked by CD45.1 in the leukemic marrow was sorted for phenotypic analyses and functional assays at different time points. Same numbers of the normal hematopoietic cells without Notch1-transduced cells were transplanted into the irradiated recipients as controls. As expected, progressive hematopoietic suppression was observed at both HSC and HPC levels in the leukemic mice. The frequency of HSC enriched population (Lin−c-Kit+Sca-1+, LKS) in the leukemic group was 7 times lower than that in the control at the 4th week of leukemogensis. When normalized to the bone marrow cellularity, the absolute yield of each population was 246 times lower in the leukemic group than that in the control group. These data were highly consistent with significantly lower yields of colony forming unit (CFU) and cobblestone area forming cell (CAFC). To measure the long-term engraftment of HSCs from leukemic environment, we performed the competitive bone marrow transplantation (cBMT), in which equal numbers of CD45.1+ cells isolated from leukemic or control mice and competitor cells (CD45.1/.2) at the 2nd week of leukemogenesis were co-transplanted into lethally irradiated C57BL/6J recipients. Unexpectedly, the multilineage engraftment of the hematopoietic cells isolated from the leukemic mice was 3 times more than that of the control group. Moreover, HSCs from the leukemic environment remained functional in serial transplant recipients. Finally, to explore the underlying molecular mechanisms for the enhanced function of normal HSC in the cBMT model, we examined a number of cell cycle and self-renewal regulators in HSC and HPC from leukemic marrow and control group at the time of harvest prior to transplantation by qRT-PCR. There was a significant decrease in p18 expression when compared with the control, whereas p21 expression was significantly increased. Notch1, Gfi1 and c-myc signalings were also elevated in the HSCs from leukemic environment. In summary, our current work provides the first definitive evidence for the reversible inhibition of normal HSC growth by the leukemic environment, thereby having important implications for HSC transplantation as well as leukemogenesis.

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BCL2A1 Is A Survival and Immortalization Factor for Primitive Myeloid Hematopoietic Cells.

Blood ◽

10.1182/blood.v110.11.3365.3365 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3365-3365

Author(s):

Jean-Yves Metais ◽

Ashley E. Dunfee ◽

Rodrigo T. Calado ◽

Cynthia E. Dunbar

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Gene Product ◽

Myeloid Leukemia ◽

Hematopoietic Cells ◽

Marker Genes ◽

Empty Vector ◽

Myeloid Progenitor ◽

The Impact

Abstract We recently reported development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2005). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene in the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on myeloid cell behavior. To investigate the impact of the BCL2A1 gene product on hematopoietic cells, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentivirus vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins on hematopoiesis. We confirmed overexpression of the proteins in the producer cell line as well as in transduced cells by western blot using an anti-HA monoclonal antibody. BaF3 cell proliferation and survival are dependant on IL-3, and under IL-3 replete conditions overexpression of murine or human BCL2A1 did alter proliferation compared with untransduced cells or cells transduced with an empty vector. Removal of IL-3 from the cell culture media leads to rapid apoptosis of BaF3 cells, with cell cycle arrest in the G1 and an apoptotic subpopulation appearing within 24 hours of IL-3 removal. 45% untransduced or empty vector cells were apoptotic, and this fraction decreased to 30% and 15% respectively for BaF3 cells expressing murine or human BCL2A1. These results were confirmed by direct analysis of apoptosis. Only BaF3 cells over-expressing human BCL2A1 were still alive and arrested in G1 after 3 days of culture without IL-3. The murine BCL2A1 had similar but less striking effects. Gene expression analyses on the BaF3 cell populations are ongoing, to identify potential downstream targets of the BCL2A1 protein. The BCL2A1 and empty vectors were also utilized in murine bone marrow cell immortalization assay, previously utilized to identify genes impacting on the survival and expansion of primary myeloid progenitor cells (Du, 2005). In an initial set of experiments, clonal clonal expansion was obtained with marrow cells expressing murine (4 clones) and human (5 clones) BCL2A1 but not for empty vector or untransduced murine marrow. Mice have also been transplanted with primary bone marrow cells transduced with the BCL2A1 and control vectors, and are being followed for in vivo expansion of transduced clones and development of leukemia. In conclusion, we have confirmed the role of BCL2A1 as an anti-apoptotic protein, now in myeloid hematopoietic cells, and will continue to investigate the role of this gene product in hematopoiesis and leukemogenesis.

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Irf4-dependent CD103+CD11b+dendritic cells and the intestinal microbiome regulate monocyte and macrophage activation and intestinal peristalsis in postoperative ileus

Gut ◽

10.1136/gutjnl-2017-313856 ◽

2017 ◽

Vol 66 (12) ◽

pp. 2110-2120 ◽

Cited By ~ 18

Author(s):

Judith-Mira Pohl ◽

Sebastian Gutweiler ◽

Stephanie Thiebes ◽

Julia K Volke ◽

Ludger Klein-Hitpass ◽

...

Keyword(s):

Dendritic Cells ◽

Antibiotic Treatment ◽

Intestinal Microbiota ◽

Postoperative Ileus ◽

Intestinal Microbiome ◽

Oral Antibiotic ◽

Monocytes And Macrophages ◽

Intestinal Peristalsis ◽

The Impact

ObjectivePostoperative ileus (POI), the most frequent complication after intestinal surgery, depends on dendritic cells (DCs) and macrophages. Here, we have investigated the mechanism that activates these cells and the contribution of the intestinal microbiota for POI induction.DesignPOI was induced by manipulating the intestine of mice, which selectively lack DCs, monocytes or macrophages. The disease severity in the small and large intestine was analysed by determining the distribution of orally applied fluorescein isothiocyanate-dextran and by measuring the excretion time of a retrogradely inserted glass ball. The impact of the microbiota on intestinal peristalsis was evaluated after oral antibiotic treatment.ResultsWe found thatCd11c-Cre+Irf4flox/floxmice lack CD103+CD11b+DCs, a DC subset unique to the intestine whose function is poorly understood. Their absence in the intestinal muscularis reduced pathogenic inducible nitric oxide synthase (iNOS) production by monocytes and macrophages and ameliorated POI. Pathogenic iNOS was produced in the jejunum by resident Ly6C–macrophages and infiltrating chemokine receptor 2-dependent Ly6C+monocytes, but in the colon only by the latter demonstrating differential tolerance mechanisms along the intestinal tract. Consistently, depletion of both cell subsets reduced small intestinal POI, whereas the depletion of Ly6C+monocytes alone was sufficient to prevent large intestinal POI. The differential role of monocytes and macrophages in small and large intestinal POI suggested a potential role of the intestinal microbiota. Indeed, antibiotic treatment reduced iNOS levels and ameliorated POI.ConclusionsOur findings reveal that CD103+CD11b+DCs and the intestinal microbiome are a prerequisite for the activation of intestinal monocytes and macrophages and for dysregulating intestinal motility in POI.

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Antibiotic Intervention Affects Maternal Immunity During Gestation in Mice

Frontiers in Immunology ◽

10.3389/fimmu.2021.685742 ◽

2021 ◽

Vol 12 ◽

Author(s):

Marilen Benner ◽

Alejandro Lopez-Rincon ◽

Suzan Thijssen ◽

Johan Garssen ◽

Gerben Ferwerda ◽

...

Keyword(s):

Antibiotic Treatment ◽

Treated Group ◽

Mesenteric Lymph Nodes ◽

Negative Effects ◽

T Helper 17 ◽

Immunological Parameters ◽

Maternal Immunity ◽

Immune Parameters ◽

Ensemble Strategy ◽

The Impact

BackgroundPregnancy is a portentous stage in life, during which countless events are precisely orchestrated to ensure a healthy offspring. Maternal microbial communities are thought to have a profound impact on development. Although antibiotic drugs may interfere in these processes, they constitute the most frequently prescribed medication during pregnancy to prohibit detrimental consequences of infections. Gestational antibiotic intervention is linked to preeclampsia and negative effects on neonatal immunity. Even though perturbations in the immune system of the mother can affect reproductive health, the impact of microbial manipulation on maternal immunity is still unknown.AimTo assess whether antibiotic treatment influences maternal immunity during pregnancy.MethodsPregnant mice were treated with broad-spectrum antibiotics. The maternal gut microbiome was assessed. Numerous immune parameters throughout the maternal body, including placenta and amniotic fluid were investigated and a novel machine-learning ensemble strategy was used to identify immunological parameters that allow distinction between the control and antibiotic-treated group.ResultsAntibiotic treatment reduced diversity of maternal microbiota, but litter sizes remained unaffected. Effects of antibiotic treatment on immunity reached as far as the placenta. Four immunological features were identified by recursive feature selection to contribute to the most robust classification (splenic T helper 17 cells and CD5+ B cells, CD4+ T cells in mesenteric lymph nodes and RORγT mRNA expression in placenta).ConclusionIn the present study, antibiotic treatment was able to affect the carefully coordinated immunity during pregnancy. These findings highlight the importance of inclusion of immunological parameters when studying the effects of medication used during gestation.

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The PAF1c Subunit Cdc73 Is Essential for Hematopoiesis and Displays Differential Gene Regulation in MLL-AF9 Driven Leukemia

Blood ◽

10.1182/blood-2018-99-118703 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1280-1280

Author(s):

Nirmalya SAHA ◽

James Ropa ◽

Lili Chen ◽

Hsiang-Yu Hu ◽

Maria Mysliwski ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Hematopoietic Cells ◽

Leukemic Cells ◽

Flow Cytometry Analysis ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

A Cell

Abstract The Polymerase Associated Factor 1 complex (PAF1c) functions at the interface of epigenetics and gene transcription. The PAF1c is a multi-protein complex composed of Paf1, Cdc73, Leo1, Ctr9, Rtf1 and WDR61, which have all been shown to play a role in disease progression and different types of cancer. Previous reports demonstrated that the PAF1c is required for MLL-fusion driven acute myeloid leukemia. This is due, in part, to a direct interaction between the PAF1c and wild type MLL or MLL fusion proteins. Importantly, targeted disruption of the PAF1c-MLL interaction impairs the growth of MLL-fusion leukemic cells but is tolerated by normal hematopoietic stem cells. These data point to differential functions for the PAF1c in normal and malignant hematopoietic cells that may be exploited for therapeutic purposes. However, a detailed exploration of the PAF1c in normal hematopoiesis is currently lacking. Here, we utilize a mouse genetic model to interrogate the role of the PAF1c subunit, Cdc73, in the development and sustenance of normal hematopoiesis. Using hematopoietic-specific constitutive and conditional drivers to express Cre recombinase, we efficiently excise floxed alleles of Cdc73 in hematopoietic cells. VavCre mediated excision of Cdc73 results in embryonic lethality due to hematopoietic failure. Characterization of the hematopoietic system demonstrated that cKit+ hematopoietic stem and progenitor cells (HSPC) are depleted due to Cdc73 knockout. We next investigated the role of Cdc73 in adult hematopoiesis using Mx1Cre mediated excision. Conditional knockout of Cdc73 in the adult hematopoietic system leads to lethality within 15 days of Cdc73 excision while no phenotype was observed in heterozygous Cdc73fl/wt controls. Pathological examination of bones in these mice showed extensive bone marrow failure. Flow cytometry analysis revealed that cKit+ HSPCs in adult mice are ablated following loss of Cdc73. Bone marrow transplantation assays demonstrated a cell autonomous requirement of Cdc73 for HSC function in vivo. To perform cellular characterization of HSPCs upon Cdc73 KO, we optimized excision conditions to capture cKit+ HSPCs with excised Cdc73 but before their exhaustion. Flow cytometry analysis demonstrated that Cdc73 KO leads to a cell cycle defect. Cdc73 excision leads to a 2.5 fold increase in the accumulation of HSPCs in the G0 phase of cell cycle with a reduction in the proliferative phases. This is accompanied with an increase in cellular death as indicated by Annexin V staining. Together, these data indicate that Cdc73 is required for cell cycle progression and HSPC survival. To understand the molecular function of Cdc73, we performed RNAseq analysis to identify genes regulated by Cdc73 in HSPCs. We observed 390 genes are upregulated and 433 genes are downregulated upon loss of Cdc73. Specifically, Cdc73 excision results in upregulation of cell cycle inhibitor genes such as p21 and p57, consistent with the cell cycle defect observed following Cdc73 excision. Further, when comparing our results to leukemic cells, we uncovered key differences in Cdc73 gene program regulation between ckit+ hematopoietic cells and MLL-AF9 AML cells. Loss of Cdc73 in leukemic cells leads to downregulation of genes associated with early hematopoietic progenitors and upregulation of myeloid differentiation genes consistent with previous studies. Interestingly, we observed a more even distribution of expression changes (non-directional) within these gene programs following Cdc73 inactivation in HSPCs. Most importantly, while loss of Cdc73 in MLL-AF9 AML cells leads to a profound downregulation of the Hoxa9/Meis1 gene program, excision of Cdc73 in HSPCs results in a modest non-directional change in expression of the Hoxa9/Meis1 gene program. This was attributed to no change in Hoxa9 and Meis1 expression in HSPCs following excision of Cdc73, in contrast to MLL-AF9 cells where these pro leukemic targets are significantly downregulated. Together, these data indicate an essential role for the PAF1c subunit Cdc73 in normal hematopoiesis but differential roles and context specific functions in normal and malignant hematopoiesis, which may be of therapeutic value for patients with AMLs expressing Hoxa9/Meis1 gene programs. Disclosures No relevant conflicts of interest to declare.

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Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

Cells ◽

10.3390/cells9051303 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1303 ◽

Cited By ~ 2

Author(s):

Alexandra Pritchard ◽

Sultan Tousif ◽

Yong Wang ◽

Kenneth Hough ◽

Saad Khan ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Cells ◽

Bone Marrow Cells ◽

Lung Tumor ◽

Macrophage Polarization ◽

M2 Macrophage ◽

M2 Macrophages ◽

M2 Macrophage Polarization ◽

The Impact ◽

M2 Phenotype

Cellular cross-talk within the tumor microenvironment (TME) by exosomes is known to promote tumor progression. Tumor promoting macrophages with an M2 phenotype are suppressors of anti-tumor immunity. However, the impact of tumor-derived exosomes in modulating macrophage polarization in the lung TME is largely unknown. Herein, we investigated if lung tumor-derived exosomes alter transcriptional and bioenergetic signatures of M0 macrophages and polarize them to an M2 phenotype. The concentration of exosomes produced by p53 null H358 lung tumor cells was significantly reduced compared to A549 (p53 wild-type) lung tumor cells, consistent with p53-mediated regulation of exosome production. In co-culture studies, M0 macrophages internalized tumor-derived exosomes, and differentiated into M2 phenotype. Importantly, we demonstrate that tumor-derived exosomes enhance the oxygen consumption rate of macrophages, altering their bioenergetic state consistent with that of M2 macrophages. In vitro co-cultures of M0 macrophages with H358 exosomes demonstrated that exosome-induced M2 polarization may be p53 independent. Murine bone marrow cells and bone marrow-derived myeloid-derived suppressor cells (MDSCs) co-cultured with lewis lung carcinoma (LLC)-derived exosomes differentiated to M2 macrophages. Collectively, these studies provide evidence for a novel role for lung tumor-exosomes in M2 macrophage polarization, which then offers new therapeutic targets for immunotherapy of lung cancer.

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Clinical and Prognostic Impact of (11;14)(q13;q32) Translocation on Patients with Multiple Myeloma

Blood ◽

10.1182/blood-2019-130809 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5507-5507

Author(s):

Daisuke Miura ◽

Kentaro Narita ◽

Ayumi Kuzume ◽

Rikako Tabata ◽

Toshiki Terao ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Cells ◽

Clinical Characteristics ◽

Mononuclear Cells ◽

Plasma Cells ◽

Stage Iii ◽

Prognostic Impact ◽

Cd20 Expression ◽

The Impact

Introduction. Translocations involving chromosome 14 at band q32, the immunoglobulin heavy chain (IgH) locus, are considered to be the most important initiating events for the development of multiple myeloma (MM). Among the IgH translocations in MM, t(11;14)(q13;q32) is the most frequently reported, and associated with a lymphoplasmacytic morphology. This translocation have been traditionally considered as standard-risk chromosomal abnormality compared to other translocations such as t(4;14) or t(14;16), although some controversies on the prognostic impact of this translocation still remain. This study aimed to clarify the clinical and prognostic impact of t(11;14) in Japanese patients in relation to other clinical variables such as immunophenotype of the tumor cells, other cytogenetic abnormalities, and use of stem cell transplantation (SCT). Patients and methods. Among the 244 consecutive patients with newly diagnosed MM, treated at Kameda Medical Center between April 2009 and July 2019, 234 patients, having cytogenetic analysis data (including t(11;14), t(4;14), t(14;16), and del(17p) by interphase fluorescence in situ hybridization (iFISH)) fully available, were included in this study. Data regarding the patients' clinical and laboratory characteristics, including the International Staging System (ISS), immunophenotype of the tumor cells, baseline circulating plasma cells (CPCs), treatment responses, disease progression, and survival status, were collected. iFISH was performed with CD138-purified bone marrow plasma cells, and the cut off values for translocation were ≥ 10% and for del(17p) ≥ 20%. Using multicolor flow cytometry, surface marker analysis of bone marrow samples and quantification of pre-treatment CPCs on peripheral blood mononuclear cells were simultaneously performed. CPCs were reported as the percentage of total mononuclear cells. Patients were considered to be negative for clonal CPCs at a sensitivity of 10−4 (0.01%) clonal plasma cells for all events evaluated. Results. The incidence of patients harboring t(11;14) was 24.4% (n = 57); t(11;14) was detected significantly high in light-chain-only subtypes (P < 0.001). We compared clinical characteristics of patients carrying t(11;14) with others. Myeloma cells with t(11;14) were associated with negative expression of CD56 (P < 0.001), CD117 (P = 0.046), and CD200 (P = 0.006), and positive expression of CD20 (P = 0.01) and CD81 (P = 0.035). Patients with t(11;14) were associated with positive CPCs (P = 0.011). In order to focus on the impact of t(11;14), we divided the patients into 4 groups: (A) no specific cytogenetic abnormality listed above (n = 137), (B) t(11;14) group (n = 57), (C) t(4;14) or t(14;16) group (n = 29), and (D) del(17p) only (n = 10), and the clinical characteristics and survival of the patients were compared across the three groups (A), (B), and (C). Almost all the patients (> 95%) in this cohort received bortezomib-based therapy. Median progression-free survival (PFS) and overall survival (OS) of patients in (A), (B), and (C) groups were 55.6, 34.2, and 30.2 months (m) (A vs. B, P = 0.036, and A vs. C, P = 0.031), and not reached, 51.2, and 79.8 m (A vs. B, P = 0.11, and A vs. C, P = 1.00), respectively. However, patients harboring t(11;14) were further divided into CD20-positive and -negative groups, the latter having poor prognosis (36.1 vs. 26.7, P = 1.0 for median PFS, and not reached vs. 44.2, P = 0.029 for median OS). Compared to other groups, patients without CD20 expression had significantly shorter OS (vs. A, vs. B, P = 0.024, 0.035, respectively), whereas those with CD20 expression tended to have longer OS, without statistical significance (Figure 1).Univariate analysis revealed ISS stage III, creatinine > 2.0 mg/dL, use of SCT, t(11;14) without CD20 expression, and age ≥ 70 years to be associated with shorter OS, whereas multivariate analysis demonstrated ISS stage III, use of SCT, and t(11;14) without expression CD20 (HR 1.88; 95% CI 1.10-3.21; P = 0.021) to be independent prognostic factors for poor OS. Conclusions. Our findings demonstrated that patients harboring t(11;14) had distinct clinical and immunophenotypic characteristics, two subsets of the disease entities with a clearly different survival according to CD20 expression. Disclosures Matsue: Ono Pharmaceutical: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Novartis Pharma K.K: Honoraria; Celgene: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria.

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MDS1-EVI1 and EVI1 Overexpression Results in Changes in the Behavior of Murine Hematopoietic Cells.

Blood ◽

10.1182/blood.v108.11.1452.1452 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1452-1452

Author(s):

Jean-Yves Metais ◽

Rotraud Wieser ◽

Cynthia E. Dunbar

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Gene Products ◽

Gene Complex ◽

Murine Bone Marrow ◽

And Control ◽

The Impact ◽

Marrow Cells

Abstract We have found non-random patterns of retroviral integration in long-term hematopoietic repopulating cells in the rhesus macaque, with frequent integration events of MLV vectors into the MDS1-EVI1 gene complex. These findings, along with reports regarding frequent integration events in the same gene complex in patients with chronic granulomatous disease receiving MLV-transduced hematopoietic cells in a clinical trial and the ability of MLV vectors activating expression of this gene via integration to immortalize primary murine bone marrow cells, suggests these gene products could have important roles in normal and leukemic hematopoiesis. Expression from this gene complex can result in translation of at least three distinct proteins: MDS1, EVI1, and MDS1-EVI1. EVI1 has been the most studied protein of this locus. Its overexpression, as a consequence of chromosomal rearrangement or viral integration, is associated with leukemia. MDS1-EVI1 contains a PR domain that is lacking in EVI1 and is thought to possibly be antagonistic to EVI1, however the location of the integrations in our prior rhesus studies would indicate that overexpression of either gene product could be immortalizing. Both proteins share the same expression profile in normal tissues as well as most reports of myeloid leukemias. To investigate the impact of the three gene products on hematopoietic cells, we cloned murine mds1, evi1, and mds1-evi1 into the pMIEV-GFP retroviral vector and produced ecotropic vector particles. These were used to transduce the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these various gene products. Gene expression analysis using Afflymetrix arrays demonstrated that both EVI1 and MDS1-EVI1 expression produced dramatic changes in gene expression profiles of these cells, compared to MDS1 and control vector. For instance, EVI1 transduced cells overexpressed oncogenes such as small G proteins belonging to the RAS family. There was modulation of genes implied in hematopoiesis, apoptosis, TGF beta signaling, and cell cycle. To assess changes in cell cycling of transduced BaF3 cells we used a flow cytometric assay, which unraveled an arrest in G1 phase only when EVI1 was overexpressed. These changes were concomitant to an increased metabolic activity as measured by an MTT assay. Further studies of these different pathways have to be performed in order to confirm the results obtained by the DNA chips analysis. Primary murine bone marrow cells could be immortalized after transduction by both EVI1 and MDS1-EVI1 vectors, compared to MDS1 and control vectors. Mice have been transplanted with primary bone marrow cells transduced with all vectors, and are being followed for hematopoietic changes or leukemia. In conclusion, both MDS1-EVI1 and EVI1 overexpression appear to result in marked changes in the behavior of primitive hematopoietic cells.

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