Phagocytosis-Mediated Acute Hemolytic Events Induce Distinct Responses By Inflammatory Monocytes

Abstract Introduction Red blood cell (RBC) transfusions are a common therapy, with ~15 million RBC units administered annually in the United States. Studies in mice and dogs identified increased circulating levels of multiple inflammatory cytokines after transfusions of refrigerator storage-damaged RBCs. One chemokine identified was monocyte chemoattractant protein 1 (MCP-1; also known as CCL2). MCP-1 is the ligand for CCR2, which is expressed by several cell types including Ly6Chi (i.e., inflammatory) monocytes. One reserve site of Ly6Chi monocytes is the bone marrow, from which they emigrate into the circulation in a CCR2-dependent manner to traffic to sites of inflammation. Because the spleen is a primary site of RBC clearance, we aimed to identify splenic cells responsible for producing MCP-1 following transfusions of storage-damaged or antibody-coated RBCs. Methods Leukoreduced murine RBCs were prepared from wild-type (WT) C57BL/6 donors and refrigerator stored for 12-13 days. MCP-1 transgenic reporter mice were transfused with fresh, or storage-damaged ("old"), or fresh anti-RBC antibody-coated RBCs. Spleen, bone marrow, and blood were collected post-transfusion and cells were analyzed by multi-parameter flow cytometry. WT C57BL/6 mice were also transfused with fresh or old RBCs; splenocytes isolated post-transfusion were flow sorted followed by RNA isolation and RT-PCR. Results Splenic red pulp macrophages (VCAM1hi, F4/80hi, CD11blo) were primarily responsible for erythrophagocytosis after transfusions with old or antibody-coated RBCs. However, in each case, Ly6C hi, CD11b+ splenic inflammatory monocytes in MCP-1 reporter mice expressed MCP-1. MCP-1 expression by these cells was also confirmed in WT recipients by RT-PCR after flow sorting. Interestingly, only a small percentage of inflammatory monocytes ingested transfused RBCs. Furthermore, circulating inflammatory monocyte levels increased following transfusion of old or antibody-coated RBCs, accompanied by reduced levels in the bone marrow. Conclusions Although red pulp macrophages were the major cell type responsible for clearing transfused refrigerator-damaged or antibody-coated RBCs, a different splenic cell population (i.e., inflammatory monocytes) produced MCP-1. Thus, the splenic reserve of inflammatory monocytes produced an inflammatory response following phagocytosis-mediated acute hemolytic events. Increased numbers of circulating inflammatory monocytes and reduced numbers in the bone marrow suggest that these cells emigrated from the bone marrow, perhaps in response to MCP-1 signaling. Thus, one possible mechanism explaining our results is that erythrophagocytosis in the spleen induces an inflammatory response, triggering splenic inflammatory monocytes to synthesize MCP-1 and release it into the circulation; MCP-1 then binds CCR2 on bone marrow inflammatory monocytes, causing their egress into the circulation. The additional signals involved in these phenomena, along with their clinical relevance to transfusion medicine, require additional investigation. Disclosures No relevant conflicts of interest to declare.

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Three Novel AML Cases Harboring the Semi-Cryptic t(7;21)(p22;q22) Translocation Expressing RUNX1-USP42 Fusion Transcripts Associated with Diploidy/Tetraploidy and/or 5q Alterations: a Probably Underestimated Combination

Blood ◽

10.1182/blood.v116.21.2708.2708 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2708-2708

Author(s):

Eric Jeandidier ◽

Carine Gervais ◽

Isabelle Radford-Weiss ◽

Catherine Gangneux ◽

Valerie Rimelen ◽

...

Keyword(s):

Bone Marrow ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Fusion Transcript ◽

Fish Analysis ◽

Chimeric Transcript ◽

Rt Pcr ◽

Balanced Translocation ◽

Fusion Transcripts ◽

Cryptic Translocation

Abstract Abstract 2708 RUNX1 is implicated in numerous chromosomal abnormalities acquired in acute myeloid leukemia (AML). The most frequent one, the t(8;21) is associated with a particular morphology together with a favorable prognosis. This is not the case for other 21q abnormalities, that are much less frequent and for which the prognosis is quite different. Moreover, beside point mutations, conventional cytogenetics failed to detect some of chromosomal alterations involving RUNX1. Recently 3 cases of the rare and semi-cryptic t(7;21)(p22;q22) translocation expressing the RUNX1-USP42 fusion transcripts have been reported, demonstrating the recurrence of this abnormality in AML. We describe here 3 additional cases with the same translocation and fusion transcripts, associated to 5q alterations leading to EGR1 and CSF1R heterozygous losses. In all our patients, the t(7;21)(p22.1;q22.3) was initially detected by the systematic FISH evaluation of the blastic populations using ETO-AML1 Dual Fusion probe. Patient#1 bone marrow karyotype was characterized by a tetraploid clone (89,XXYY) with loss of chromosomes 15, 17 and 18 in addition to the t(7;21), and a unbalanced translocation der(5)t(5;13)(q23;q?) between long arms of chromosomes 5 and 13, resulting in a heterozygous loss of EGR1 and CSF1R. Patient #2 blood and bone marrow karyotypes revealed a diploid clone with a del(5)(q31q33) associated with the t(7;21). The FISH analysis confirmed EGR1 and CSF1R deletions. In patient #3, the bone marrow karyotype showed diploid/tetraploïd clones, both harboring the t(7;21)(p22;q22), confirmed by FISH experiments (WCP7, AML1 probes). In addition, a der(5)t(1;5)(q3?2;q21-23) was identified within the tetraploïd clone, resulting in the loss of EGR1 and CSF1R, confirmed by FISH. In all three cases a RUNX1-USP42 fusion transcript was detected using RT-PCR, as well as the reciprocal transcript. Sequence analysis of RT-PCR products showed that the breakpoints occurred exactly in the same introns of USP42 and RUNX1 as in the previously described cases. For patient #1 and #3 a chimeric transcript was found formed of the RUNX1 exon 7 fused to the USP42 exon 3. In patient #2, a shorter chimeric transcript arised from the fusion of the RUNX1 exon 5 to the exon 3 of USP42. As already noticed in the previous reports, an alternative splicing of the RUNX1 exon 6 has been detected in these three cases. The description of these 3 novel t(7;21) confirm the recurrence of this balanced translocation in AML, and shows that this chromosomal abnormality is often associated with diploid/tetraploid clones and/or 5q alterations. Special attention should be paid in karyotype analysis of AML with diploid or tetraploid clones harboring 5q alterations. In such cases RUNX1 rearrangements should be explored using FISH analysis, and RUNX1-USP42 fusion transcript should be searched by RT-PCR in positive cases. Prospective and retrospective studies of AML have now to be settled in order to assess the incidence and clinical relevance of this cryptic translocation. Disclosures: No relevant conflicts of interest to declare.

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Preliminary Study of the Autoantigens on the Membrane of Erythropoietic Cells of the Patients with BMMNC- Coomb's Test(+) Hemocytopenia

Blood ◽

10.1182/blood.v116.21.4435.4435 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4435-4435

Author(s):

Rong Fu ◽

Hui Liu ◽

Zonghong Shao ◽

Jun Wang ◽

Lijuan Li

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Mononuclear Cell ◽

Rt Pcr ◽

Glycine Buffer ◽

Healthy Donors ◽

Preliminary Study ◽

The Relationship ◽

Epor Expression ◽

Normal Controls

Abstract Abstract 4435 Objective To observe the relationship between EPO receptor(EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cell Coomb's (BMMNC-Coomb's) test(+) immuno-related pancytopenia(IRP), and then explore the probable autoantigens of auto-Ab in IRP. Methods 46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without auto-Ab on erythropoietic cells) and 18 healthy donors as controls were enrolled in this study. EPOR expression on their nuclear erythrocytes were tested with flow cytometry to observe the relationship between EPOR and auto-Ab; After sorting erythropoietic cells in bone marrow, EPOR mRNA and protein Stat5,P-Stat5 were investigated by RT-PCR and Western blot to observe the production of EPOR and EPO/EPOR signal transduction; Finally, EPOR expression on the membrane were tested again after stripping auto-Ab with glycine buffer. Results (1) EPOR of auto-Ab(+) arm(1.59±0.87)% was significantly lower than that of auto-Ab (-) arm(4.58±4.09)%(P<0.01), and the latter was significantly higher than that of normal controls (2.27±1.76)%(P<0.05); EPOR of IRP patients was negatively correlated with their auto-Ab (r=-0.543,P=0.000) and its regression equation was Y(EPOR)=0.040-0.335X(auto-Ab);(2)EPOR mRNA of auto-Ab(+) arm(0.685±0.136)was significantly higher than that of auto-Ab (-) arm(0.554±0.116)(P<0.01)and normal controls (0.580±0.119)(P<0.05);(3)Protein Stat5 of auto-Ab(+) arm(1.45±0.94) was significantly higher than that of normal controls (0.54±0.36)(P<0.05); While P-Stat5 of auto-Ab(+) arm(0.42±0.18) was significantly lower than that of normal controls (0.85±0.38)(P<0.05); (4) EPOR expression became higher while auto-Ab became lower after stripping with glycine buffer. Conclusion The auto-Ab of some IRP patients might block or competitively inhibit the EPOR on the membrane of erythropoietic cells. EPOR was one of autoantigens in IRP. Disclosures: No relevant conflicts of interest to declare.

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Bortezomib In Combination with Dexamethasone and Liposome- Adriamycin Chemotherapy Regimen for Relapsed and Refractory Hodgkin Lymphoma: A Case Report

Blood ◽

10.1182/blood.v116.21.4829.4829 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4829-4829

Author(s):

Yi Li ◽

Gaofeng Zheng ◽

Xiaoyan Han ◽

Jimin Shi ◽

Jingsong He ◽

...

Keyword(s):

Bone Marrow ◽

Hodgkin Lymphoma ◽

Radiation Treatment ◽

Conflicts Of Interest ◽

Single Agent ◽

Bone Marrow Aspiration ◽

Salvage Chemotherapy ◽

New Combination ◽

Dependent Manner ◽

Line Therapy

Abstract Abstract 4829 Bortezomib is effective in multiple myeloma, but also in other malignancies, particularly in lymphoma. Here we present a-23-year-old-male patient who complained of a progressive chest pain for 2 years. The CT scan of local hospital showed a mass that measured 5×14cm in the anterior mediastinum. He also had multiple lymphadenopathies in other areas. Tissue biopsy verified Classical Hodgkin lymphoma, nodular sclerosis (NDHL) IIIA. Then he had 12 cycles of ABVD, 1 cycle of MINE and CHOPE, 2 cycles of Hyper-CVAD regimen as prior treatments. Initial therapies induced good responses, but his disease progressed before he was transferred to hospital on December 29, 2009. His blood counts showed that WBC 30×10E9/L, with 89% neutrophils, Hb 99g/L, platelet 452 ×10E9/L. Bone marrow aspiration with immunophenotyping, analysis of TCR/IgH and the chromosome were normal. Biopsy reconfirmed the diagnosis of Hodgkin lymphoma. Lymphoma masses in his right neck, and severe edema with his right arm. He was treated with 2 cycle of IGVP regimen (ifosfamide, gemzar, vindesine, prednsone) and 2 cycles of GIP regimen (ifosfamide, gemzar, prednsone). With more progression, he subsequently received methotrexate and Ara-c with minimal response. Two weeks later, CT showed lung infection and pleural effusion, multiple lymphadenopathy. Lymph nodes decreased mildly and edema disappeared temporary after 3 cycles of R-CHOP. He was then treated with Bortezomib, dexamethasone and L-ADM. Four days after the first cycle, lymphoma masses decreased and significantly and his edema resolved. Due to the severe periphery neuropathy, he declined bortezomib, so 2 weeks later, the symptoms back again, and the FMD regimen (fludarabine, mitoxantrone, dexamethasone) was given. He then chose palliative radiation treatment and eventually died of pneumonia. Nearly 30%-40% of Hodgkin lymphoma relapsed after first line therapy even responds well at the initial stage. To these relapsed ones, the second line therapy including salvage chemotherapy and ASCT (autologous stem cell transplantation) act as the major treatment, but only a minority patients benefit from it. Treatment is limited to relapse and refractory ones hence they deserved more focus. The Bortezomib + L-ADM regimen exhibiting its efficacious after two cycles and the condition changed obviously. Our case is showing more antitumor activity of the new combination relative to either single agent alone. The new regimen responded well and with milder bone marrow suppression. Further study is still needed to demonstrate the relationship between the effective duration and treatment cycles. Some results showed that bortezomib inhibited cell proliferation and induced apoptosis in HL cell lines in a dose-dependent manner. At the same time, it is imperative to be aware of the adverse effects, such as peripheral neuropathy, thrombocytopenia and herpes zoster. As the patient mentioned above, the new regimen may work more optimally if more cycles were carried out, and he would have eventually responded if the complication was well controlled. Disclosures: No relevant conflicts of interest to declare.

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Expression of dlk1 in the Bone Marrow Cells of Patients with Myelodysplastic Syndrome and Its Clinical Significance

Blood ◽

10.1182/blood.v118.21.5042.5042 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5042-5042

Author(s):

Zonghong Shao ◽

Lanzhu Yue ◽

Rong Fu ◽

Lijuan Li ◽

Erbao Ruan ◽

...

Keyword(s):

Bone Marrow ◽

Early Diagnosis ◽

Myelodysplastic Syndrome ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Rt Pcr ◽

Normal Controls ◽

Lower Expression ◽

Bone Marrow Blasts ◽

Marrow Cells

Abstract Abstract 5042 Objective To investigate the expression of dlk1 gene (delta-like 1) in the bone marrow cells of patients with Myelodysplastic syndrome (MDS), and explore the molecular marker for early diagnosis of MDS. Methods The expression of dlk1 mRNA in the bone marrow cells of cases with MDS, AML and normal controls were measured by RT-PCR, aiming to search for the cytogenetic marker of MDS malignant clone. Results The expression of dlk1 mRNA in bone marrow cells of MDS patients (0.7342±0.3652) was significantly higher than that of normal controls (0.4801±0.1759) (P<0.05), and was significantly positively correlated with the proportion of bone marrow blasts(r=0.467,P<0.05). The expression of dlk1 mRNA significantly increased as the subtype of MDS advanced (P<0.05). Patients with abnormal karyotypes displayed significantly higher expression of dlk1 mRNA (0.9007±0.4334) than those with normal karyotypes (0.6411±0.2630) (P<0.05). Patients with higher expression of dlk1(≥0.8) presented significantly higher malignant clone burden (0.4134±0.3999) than those with lower expression (<0.8) of dlk1 (0.1517±0.3109) (P<0.05). Conclusion dlk1 gene was highly expressed in MDS patients, which increased as the subtype of MDS advanced. The expression of dlk1 mRNA was significantly positively correlated with the proportion of bone marrow blasts. High expression of dlk1 gene suggests high malignant clone burden of MDS. Disclosures: No relevant conflicts of interest to declare.

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Differential Expression and Functions of NOTCH Family Members and Involvement of NR4A1 in the Regulation of Apoptosis in CLL

Blood ◽

10.1182/blood.v120.21.3919.3919 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3919-3919

Author(s):

Rainer Hubmann ◽

Martin Hilgarth ◽

Susanne Schnabl ◽

Elena Ponath ◽

Dita Demirtas ◽

...

Keyword(s):

Cell Viability ◽

Mrna Expression ◽

Target Gene ◽

Conflicts Of Interest ◽

Family Members ◽

Dependent Manner ◽

Rt Pcr ◽

Activated State ◽

Induced Apoptosis ◽

The Individual

Abstract Abstract 3919 Chronic lymphocytic leukemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC) dependent manner linking NOTCH2 to the activated state of the leukemic cells. The transcriptional activity of NOTCH2 is associated with the expression of CD23 and enhanced CLL cell viability. However, the regulation and possible functions of the individual NOTCH family members (NOTCH1–4) in CLL cells remain to be clarified. We took advantage of targeting nuclear NOTCH2 using the recently identified NOTCH2 transactivation inhibitor gliotoxin (WO 2006/135949). We also analysed the regulation and possible function of NOTCH1–4 in PKC stimulated CLL cells using a PMA model (Hubmann et al., BJH 2010) and a microenvironment model where CLL lymphocytes were co-cultured with primary bone marrow stromal cells (BMSC) (Shehata et al., BLOOD 2010). Electrophoretic mobility shift assays (EMSA) demonstrated that gliotoxin inhibited DNA-bound NOTCH2 complexes in PMA stimulated CLL cells in parallel to increasing the rate of apoptosis (mean±SD: 67±31% in gliotoxin treated cells versus 13±14% in the untreated controls, n=21). This was associated with downregulation of CD23A mRNA expression and CD23 surface expression (mean±SD: 42±32% versus 83±17%, n=21) as assessed by RT-PCR and FACS analysis. Exceptionally, one CLL case with a recently described NOTCH1 gain of function mutation appeared to be less sensitive to gliotoxin and had a persistent high expression of CD23. We next tested whether NOTCH2 inhibition by gliotoxin is a selective process or indirectly mediated by effects on proteasome regulated apoptosis. Proteasome assays showed that gliotoxin had a minimal or no effect on the chymotrypsin like activity of the proteasome in CLL cells. In addition, the activity of the proteasome regulated transcription factor NFκB and the expression of its target genes like BCL2 and MCL1 were also not influenced by gliotoxin. These data point to the selectivity of targeting NOTCH2 signaling by gliotoxin rather than indirectly through the regulation of proteasome activity. Short term (4 hours) exposure of CLL cells revealed that NOTCH1 was equally transcribed in unstimulated and in PMA activated CLL cells. NOTCH2 was upregulated in PMA activated CLL lymphocytes whereas NOTCH4 was only weakly detectable in unstimulated CLL cells. Gliotoxin treatment resulted in the downregulation of NOTCH1, NOTCH2 and NOTCH4 mRNA expression. Interestingly, the inhibition of NOTCH2 activity by gliotoxin was associated with the concomitant induction of NOTCH3 signaling especially in the presence of PMA. This was indicated by the induced mRNA expression of NOTCH3 and its preferred target gene HEY1. Moreover, the induced transcription of HEY1 correlated with the upregulation of NR4A1, a key regulator of apoptosis in activated lymphocytes. These data may thus point to a pro-apoptotic role for NOTCH3/HEY1/NR4A1 signaling in CLL cells. The data also suggest that gliotoxin induced apoptosis is associated with differential regulation of the anti-apoptotic and pro-apoptotic arms of NOTCH signalling in CLL cells. RT-PCR revealed that NOTCH1 and NOTCH2 are the main NOTCH family members which are expressed in CLL cells under co-culture conditions with BMSC and in freshly isolated CLL cells. Exposure to gliotoxin in co-culture selectively induced apoptosis in CLL cells and led to downregulation of NOTCH1 and NOTCH2 together with upregulation of NOTCH3 mRNA expression. In summary, the data suggest that nuclear NOTCH2 activity might protect activated CLL cells from apoptosis by modulating the expression of NR4A1. The induced expression of NOTCH3 and its target gene HEY1 by gliotoxin reveals the complex role of different NOTCH family members in the regulation of apoptosis in CLL cells. Therefore, the individual NOTCH receptors may have opposite effects on CLL cell viability which should be considered in therapeutic approaches aimed to target NOTCH signaling in CLL. Disclosures: No relevant conflicts of interest to declare.

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Leukocyte Factor XII Mediates Inflammation and Its Deficiency Promotes Wound Healing

Blood ◽

10.1182/blood.v120.21.616.616 ◽

2012 ◽

Vol 120 (21) ◽

pp. 616-616

Author(s):

Evi Stavrou ◽

Matthew J. Fullana ◽

Gretchen LaRusch ◽

Cindy Yushin Chang ◽

Chao Fang ◽

...

Keyword(s):

Bone Marrow ◽

Wound Healing ◽

Inflammatory Response ◽

Wound Repair ◽

Conflicts Of Interest ◽

Marrow Cell ◽

Inflammatory Cells ◽

Leukocyte Migration ◽

Factor Xii ◽

High Power Field

Abstract Abstract 616 Background: Older investigations suggest that Factor XII (FXII) influences the inflammatory response. FXII deficient patients have reduced leukocyte migration into skin windows. In addition to bradykinin generation, FXII regulates the expression of monocyte FcγII receptor and stimulates monocytes and macrophages to release interleukin (IL)-1 and IL-6. In vitro, purified FXIIa corrects neutrophil aggregation and degranulation defects in FXII-deficient plasma. Our laboratory demonstrated that FXII signals through uPAR, β1 integrin and the EGFR to stimulate ERK1/2 and Akt phosphorylation (Blood 115:5111, 2010). The downstream consequences of this pathway are FXII-induced cell proliferation, growth and angiogenesis. Since FXII modulates wound angiogenesis, we examined the role for FXII in the inflammatory response and wound repair. Investigations: Exon 3 to 8–deleted FXII knockout mice (FXII−/−) were wounded by creating full thickness (5 mm) punch biopsies on their backs. The total surface area of inflammatory cells recruited to the injury site per 20X high power field (HPF) was determined and analyzed by Image J (NIH). We observed that on Day 1, FXII−/− mice exhibit significantly decreased recruitment of CD11 b–labeled inflammatory cells to injury sites compared with wild type mice (WT) [p=0.0136]. Similar results are observed when uPAR KO mice were compared to WT [p=0.0001], suggesting that leukocyte migration in part is mediated through this receptor. Next we determined the nature of cells in the wound sites. FXII−/− mice have reduced wound neutrophils (Gr-1 staining) and macrophages (F4-80 staining). On the thioglycolate (TG)-induced peritoneal inflammation assay, the number of peritoneal exudate cells (PECs) was measured in WT and FXII−/− mice 1 and 7 days following instillation. FXII−/− mice exhibit significantly decreased number of PEC's on days 1 and 7. Giemsa-Wright stain of peritoneal lavage fluid with manual differential counts or flow cytometry shows that there is a disproportionate decrease in neutrophil recruitment in peritoneal fluid of FXII−/− mice. Also, FXII−/− macrophages have reduced adherence to plastic. Identical assays performed on uPAR and bradykinin B2 receptor (B2R) KO mice did not reveal the same inflammatory defects; both uPAR and B2R KO mice have mild decreases in PEC cell numbers following TG instillation, but overall their inflammatory response remained intact. Investigations next determined if the leukocyte migration defect seen in FXII−/− mice is related to plasma FXII or an intrinsic leukocyte defect. On adoptive bone marrow (BM) transplantation experiments, WT bone marrow transplanted to FXII−/− mice corrects the TG-induced PEC migration defect after both 1 and 7 days from instillation. Alternatively, transplantation of FXII−/− bone marrow into WT mice produced a leukocyte migration defect. In order to further discriminate the contribution of FXII in leukocyte migration, we produced plasma FXII deficiency in WT mice using FXII siRNA. WT mice treated with FXII siRNA reduced plasma FXII activity to <10% at 24 h (T1/2= 6 h). These mice did not have a defect in PEC recruitment. In vivo infusion of purified FXII did not correct the leukocyte peritoneal migration defect in FXII−/− mice. Next we harvested BM from WT mice and isolated leukocytes to prepare bone marrow cell-derived mRNA. The mRNA was reverse-transcribed to cDNA and it demonstrates XII cDNA that shares sequence homology to hepatic XII from exons 2 through 14. Immunofluorescence staining of BM-derived leukocytes demonstrates XII-positive leukocytes with nuclear morphology resembling monocytes and neutrophils (Fig. 1). Finally although leukocyte FXII promotes their migration into wounds, FXII deficiency in leukocytes paradoxically is associated with improved rates of wound healing after punch biopsy. Conclusions: These data indicate that there is a unique pool of FXII in leukocytes distinct from plasma (hepatic) FXII. Leukocyte FXII, not plasma FXII, is responsible for leukocyte function in wounds and inflammation sites. FXII−/− mice have attenuated wound injury and, paradoxically, improved wound healing rates. Modulation of leukocyte FXII is a target for promotion of wound healing. Disclosures: No relevant conflicts of interest to declare.

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Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v122.21.3169.3169 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3169-3169

Author(s):

Hugh Kikuchi ◽

Amofa Eunice ◽

Maeve McEnery ◽

Farzin Farzaneh ◽

Stephen A Schey ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Proliferation ◽

Bone Resorption ◽

Cell Lines ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Pi3k Pathway ◽

Dependent Manner ◽

Dose Dependent

Abstract Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM. Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation. GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone. We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone. Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941. GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting. We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures: No relevant conflicts of interest to declare.

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Nonhematopoietic Cells Control the Outcome of Infection with Listeria monocytogenes in a Nucleotide Oligomerization Domain 1-Dependent Manner

Infection and Immunity ◽

10.1128/iai.01068-08 ◽

2009 ◽

Vol 77 (7) ◽

pp. 2908-2918 ◽

Cited By ~ 22

Author(s):

Ahmed Mosa ◽

Christian Trumstedt ◽

Emma Eriksson ◽

Oliver Soehnlein ◽

Frank Heuts ◽

...

Keyword(s):

Bone Marrow ◽

Listeria Monocytogenes ◽

Bacterial Load ◽

Dependent Manner ◽

Inflammatory Monocytes ◽

Defensive Role ◽

Nucleotide Oligomerization ◽

Increased Susceptibility ◽

Nucleotide Oligomerization Domain ◽

Oligomerization Domain

ABSTRACT We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection with Listeria monocytogenes. Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses of L. monocytogenes, as measured by the bacterial load and survival. NOD1 also controlled dissemination of L. monocytogenes into the brain. The increased susceptibility to reinfection of NOD1−/− mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480+ Gr1+ inflammatory monocytes and lower numbers of F480− Gr1+ neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1−/− mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance to L. monocytogenes in bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection with L. monocytogenes or stimulation with different Toll-like receptor ligands. NOD1−/− BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth of L monocytogenes compared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition of L. monocytogenes growth were hampered in NOD1−/− BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection with L. monocytogenes and controls intracellular bacterial growth in different cell populations in vitro.

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Oxymatrine Induces Apoptosis in Human Myeloma Cells By Cell Cycle Arrest and Activation of Caspase-Dependent Apoptosis

Blood ◽

10.1182/blood.v124.21.5727.5727 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5727-5727

Author(s):

Wenjun Wu ◽

Cai Wu ◽

Fuming Zi ◽

Yi Li ◽

Li Yang ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Growth Inhibition ◽

Signaling Pathway ◽

Cell Lines ◽

Western Blotting ◽

Cell Apoptosis ◽

Conflicts Of Interest ◽

Dependent Manner ◽

Rt Pcr

Abstract Background : Multiple myeloma (MM) is a B cell malignant hematologic cancer. Despite the introduction of new drugs and improvement of chemotherapy, MM is still an incurable disease. Oxymatrine (OMT), the active ingredients of traditional Chinese herbal medicine sophora, has been reported to have antitumor activity. This study was to estimate the therapeutic efficacy of OMT in MM. Methods: The growth inhibition of myeloma cell lines (RPMI8226, U266, ARP-1) or primary cells by OMT was assessed by MTT assay. Apoptosis of MM cells was examined by annexin V-FITC using flow cytometry analysis. DNA content was analyzed by flow cytometry. RT-PCR and western-blot analysis were used to assess the expression of Bcl-2 family proteins and the IAP family proteins. Western blotting was also used to elucidate the signaling pathway that may mediate OMT-induced apoptosis of MM cells. Results: OMT treatment resulted in cell growth inhibition and apoptosis in primary MM cells and all tested MM cell lines in a dose-dependent manner (P <0.05). To elucidate OMT -induced MM cell apoptosis, MM cell lines were treated with or without OMT for 24h and assessed for caspase activation and signaling pathway by Western blotting. The results showed the cleavage of PARP, caspase-3, and caspase-9, and p-AKT were down-regulated after OMT treatment. The mRNA expression of survivin and HIAP by RT-PCR was down-regulated. OMT treatment at 5mM for 48h resulted in increased G-phase cells and decreased S-phase cells in MM cell lines (P <0.05). Cell cycle repressor P21 protein was up-regulated while CDK4, CDK6 and CyclinD1 expression was down-regulated. Our finding also showed a synergistic anti-MM activity of OMT and dexamethasone or adriamycin at a low does (CI<1). In addition, LC3-II expression was significantly increased both in RPMI8226 and U266 cells after treatment with OMT. However, treatment with different doses of OMT and 5 mM autophagy inhibitor 3-MA, significant increased cell apoptosis (P <0.05). Conclusion: Our findings demonstrate the anti-MM activity of OMT and indicate that OMT alone or together with other MM chemotherapeutics may be a prospective treatment for MM. Disclosures No relevant conflicts of interest to declare.

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Constitutive Expression of CD40L on Bone Marrow Mast Cells Supports the Growth of Lymphoplasmacytic Cells in Patients with Waldenstrom’s Macroglobulinemia.

Blood ◽

10.1182/blood.v104.11.2350.2350 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2350-2350 ◽

Cited By ~ 1

Author(s):

Olivier Tournilhac ◽

Daniel Ditzel Santos ◽

Lian Xu ◽

Jeffery Kutok ◽

Yu Tsu Tai ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Mast Cells ◽

Cell Surface ◽

Tumor Cells ◽

Thymidine Uptake ◽

Dependent Manner ◽

Rt Pcr ◽

Potent Inducer ◽

Dose Dependent

Abstract CD40 ligand (CD40L) is a potent inducer of normal and malignant B-cell proliferation through interaction with CD40. We and others have observed excess mast cells (MC) in bone marrow (BM) biopsies of WM patients, which are commonly found admixed with tumor aggregates. (Tournilhac et al, JCO 2004, 22:571S). We therefore sought to clarify the role of MC in WM. Co-culture of 0.5% paraformaldehyde fixed, or sublethally irradiated HMC-1, LAD, and KU mast or basophilic cell lines and sorted BM lymphoplasmacytic cells (LPC) from 10 WM patients resulted in MC dose-dependent tumor colony formation and/or proliferation as assessed by 3H-thymidine uptake studies. As demonstrated by immunohistochemical, multicolor flow cytometric (CD117+FceRI+) and/or RT-PCR analysis, CD40L was expressed on BM MC from 29 of 31 (94%), 11 of 13 (85%), and 7 of 9 (78%) of WM patients, respectively. In contrast, cell surface CD40L expression was not detected by immunohistochemistry (p=0.00005) and flow cytometry (p=0.003) in 5 normal donors, and only faint expression for 1 of 5 normal donors by RT-PCR (p=0.09). Moreover, by multicolor flow cytometry, CD40 was expressed on BM tumor cells from 14/17 (83%) patients. CD40 functionality was confirmed either by the G28.5 CD40 agonistic antibody which induced dose dependent proliferation or by the rh-CD40L which partly prevented serum starvation-induced-apoptosis of WM LPC from 4/4 and 3/4 patients respectively. Importantly, expansion of tumor cells from 3 of 4 patients in mixed cultures with paraformaldehyde fixed MC was blocked in a dose dependent manner by use of a CD40L blocking protein (CD40:Fc). These studies demonstrate that CD40L is constitutively expressed on the cell surface of BM MC in WM and support the growth of WM tumor cells, and therefore provide the framework for therapeutic targeting of MC and MC-WM cell interactions in WM.

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