Marrow Stromal Cell Line Research Articles

Spleen tyrosine kinase influences the early stages of multilineage differentiation of bone marrow stromal cell lines by regulating phospholipase C gamma activities.

Bone marrow stromal cells (BMSCs) are multipotent cells that can differentiate into adipocytes and osteoblasts. Inadequate BMSC differentiation is occasionally implicated in chronic bone metabolic disorders. However, specific signaling pathways directing BMSC differentiation have not been elucidated. Here, we explored the roles of spleen tyrosine kinase (Syk) in BMSC differentiation into adipocytes and osteoblasts. We found that Syk phosphorylation was increased in the early stage, whereas its protein expression was gradually decreased during the adipogenic and osteogenic differentiation of two mouse mesenchymal stromal cell lines, ST2 and 10T(1/2), and a human BMSC line, UE6E-7-16. Syk inactivation with either a pharmacological inhibitor or Syk-specific siRNA suppressed adipogenic differentiation, characterized by decreased lipid droplet appearance and the gene expression of fatty acid protein 4 (Fabp4), peroxisome proliferator-activated receptor γ2 (Pparg2), CCAAT/enhancer binding proteins α (C/EBPα), and C/EBPβ. In contrast, Syk inhibition promoted osteogenic differentiation, represented by increase in matrix mineralization and alkaline phosphatase (ALP) activity, as well as the expression levels of osteocalcin, runt-related transcription factor 2 (Runx2), and distal-less homeobox 5 (Dlx5) mRNAs. We also found that Syk-induced signals are mediated by phospholipase C γ1 (PLCγ1) in osteogenesis and PLCγ2 in adipogenesis. Notably, Syk-activated PLCγ2 signaling was partly modulated through B-cell linker protein (BLNK) in adipogenic differentiation. On the other hand, growth factor receptor-binding protein 2 (Grb2) was involved in Syk-PLCγ1 axis in osteogenic differentiation. Taken together, these results indicate that Syk-PLCγ signaling has a dual role in regulating the initial stage of adipogenic and osteogenic differentiation of BMSCs.

Secretion of IL-1β from imatinib-resistant chronic myeloid leukemia cells contributes to BCR–ABL mutation-independent imatinib resistance

Abstract Some cases of chronic myelogenous leukemia are resistant to tyrosine kinase inhibitors (TKIs) independently of mutation in BCR–ABL, but the detailed mechanism underlying this resistance has not yet been elucidated. In this study, we generated a TKI-resistant CML cell line, K562R, that lacks a mutation in BCR–ABL. Interleukin-1β(IL-1β) was upregulated in imatinib-resistant K562R than in the parental cell line K562S, and higher levels of IL-1β contributed to the imatinib resistance of K562R and to cell survival. In addition, IL-1β secreted by K562R cells could stimulate a bone marrow stromal cell line to produce chemokines, such as CXCL11, that lead to the migration of the CML cells into the stroma. Thus, elevated IL-1β production from TKI-resistant K562R cells may contribute to TKI resistance by increasing cell viability and promoting cell migration.

ADP-ribosylation factor 1 (ARF1) takes part in cell proliferation and cell adhesion-mediated drug resistance (CAM-DR).

Cell adhesion-mediated drug resistance (CAM-DR) remains the primary obstacle in human multiple myeloma (MM) therapy. In this study, we aimed at investigating the expression and biologic function of ARF1 in MM. We determined that ARF1 expression was positively correlated with cell proliferation and knockdown of ARF1 contributed to CAM-DR. The enhancement in the adhesion of MM cells to fibronectin (FN) or the bone marrow stroma cell line HS-5 cells translated to an increased CAM-DR phenotype. Importantly, we showed that this CAM-DR phenotype was correlated with the phosphorylation of Akt and ERK in MM cells. Moreover, we sought to determine whether ARF1 could interact with p27 in RPMI8226 cells. Knockdown of ARF1 also significantly decreased pT157-p27 protein expression in RPMI8226 cells. Our research shows ARF1 may reverse CAM-DR by regulating phosphorylation of p27 at T157 in MM. Taken together, our data shed new light on the molecular mechanism of CAM-DR in MM, and targeting ARF1 may be a novel therapeutic approach for improving the effectiveness of chemotherapy in MM.

Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway

Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH)2D3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH)2D3 and DEX. Moreover, pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH)2D3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells.

Reelin promotes adhesion of multiple myeloma cells to bone marrow stromal cells via integrin β1 signaling.

The close interaction between tumor cells and bone marrow stromal cells plays a crucial role in the tumorigenesis of multiple myeloma (MM). Reelin, an extracellular matrix protein, is found expressed in myeloma cells and is negatively associated with prognosis. We examined the role of Reelin in myeloma cell adhesion to bone marrow stromal cells and the signaling pathways involved. The results revealed that Reelin promoted the adhesion of myeloma cells to HS-5, a bone marrow stromal cell line, via the activation of β1 integrin. The resulting phosphorylation of focal adhesion kinase (FAK) led to the activation of Syk/STAT3 and Akt. Reelin's high affinity receptor ApoER2 indirectly modulated the adhesion of myeloma cells by promoting Reelin expression via Sp1. These findings indicate an important role for Reelin/integrin-β1-induced myeloma cell adhesion to bone marrow stromal cells and highlight the therapeutic potential of targeting Reelin/integrin/FAK axis.

Radiation Resistance of Double Knockout (DKO) Smad3-/- Fancd2-/- (129/Sv) Mouse Bone Marrow Stromal Cell Lines

Introduction: Bone marrow progenitor cells from Fanconi Anemia (FA) patients have a hyperactive TGF-β signaling pathway which may explain hematopoietic stem cell depletion leading to bone marrow failure (Zhang, et al, Cell Stem Cell, 18:668-681, 2016). To determine whether blockade of TGF-B signaling by knockout of Smad3 normalized hematopoiesis in Fancd2-/- mice, we bred two double knockout mouse strains. The first (DKO#1) was derived by crossing C57BL/6 Fancd2 +/- mice with 129/Sv Smad3 +/- mice. The second (DKO#2) was derived by mating 129/Sv Fancd2 +/- mice with the same 129/Sv Smad3 +/- mice.Materials and Methods: Long term bone marrow cultures (LTBMCs) were established from DKO#1, DKO#2, parental mouse strains and F1 (129/Sv X C57Bl/6) control mice for DKO#1, 129/Sv Smad3 -/-, and C57BL/6 Smad3 -/- mice . The cultures were scored for weekly numbers of cobblestone islands as an indicator of stem cells in the adherent layer, weekly production of nonadherent cells, and cells producing Day 7 and Day 14 CFU-GEMM .Bone marrow stromal cell lines were derived from the adherent layer of LTBMCs and radiosensitivity measured in clonogenic radiation survival curves. Cells were irradiated to doses of 0 to 8 Gy, plated in 4 well linbro plates, incubated for 7 days at 37oC, stained with crystal violet and colonies of greater than 50 cells counted. Western analysis of the cell lines quantitated expression levels of proteins involved in the TGF-β signaling pathway, and DNA double strand break repair by homologous recombination and nonhomologous end-joining.Results: Both DKO1 and DKO2 LTBMCs showed decreased duration and magnitude of hematopoiesis, thus being similar to that observed with both C57BL/6 Fancd2-/- and 129/Sv Fancd2-/- mouse marrow cultures. Both were significantly lower than that for control mouse or Smad3-/- (129/Sv) marrow cultures. As expected, radiation survival curves showed that both C57Bl/6 Fancd2-/- and 129/Sv Fancd2-/- marrow stromal cell lines were radiosensitive compared to control cell lines including: F1 control, C57BL/6 control, and 129/Sv control (Berhane, et al, Rad Res. 181:76-89, 2014, Berhane et.al, Rad Res 182:35-49, 2014). In contrast marrow stromal cell lines from Smad3-/- (129/Sv) marrow cultures were radioresistant (Epperly, et al, Rad Res 165:671-677, 2006). Fresh marrow CFU-GEMM from both DKO#1 and DKO#2 mice showed resistance to abrogation of colony formation by increasing concentrations of TGF-B, (similar to the 129/Sv Smad3-/- cell line). In contrast, cell lines from all controls and both Fancd2-/- mice showed clear TGF-B mediated inhibition of hemopoietic colony formation. In striking contrast to the above similarities between DKO#1 and DKO#2 mice, marrow stromal cell lines from DKO#1 were radiosensitive (like theirFancd2-/- parent) while those from DKO#2 were radioresistant (like their Smad3-/- parent). Thus, DKO#1 retained the C57Bl/6 Fancd2-/- genotype cell line radiosensitivity: Do of 1.41 ± 0.03 Gy and 1.45 ± 0.05 Gy respectively, and were more radiosensitive than the control F1 bone marrow stromal cell line (p = 0.0230 and 0.0418, respectively) and DKO#2 retained the 129/Sv Smad3-/- cell line genotype radioresistance (Do = 2.15 ± 0.13 Gy) compared to control 129/Sv stromal cells (Do = 1.86 ± 0.04, p = 0.0054). Western analysis revealed that p21 was elevated in DKO#2 but not DKO#1 marrow stromal cell lines.Conclusions: While marrow from both DKO#1 and DKO#2 mice showed resistance to TGF-B signaling consistent with their smad3-/- genotype, only DKO#1 stromal cells retained the radiosensitivity of their Fancd2-/- genotype. Reduced p21 in irradiated DKO#1 marrow stromal cells may have allowed procession through G to S phase causing reduced time for DNA repair and radiosensitivity. The irradiated DKO#2 cells may have been blocked by p21 from passing through the G1 checkpoint and may have allowed DNA strand break repair and radioresistance. These DKO mice and derived cell lines should be valuable for analysis of the interaction of TGF-B signaling and FA pathways.Supported by NIAID/NIH U19-AI068021 DisclosuresNo relevant conflicts of interest to declare.

The Impact of the Bone Marrow Microenvironment on T Cell Redirection Therapeutics

Redirecting T cells to specifically target and kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of CD19xCD3 BiTE (Blincyto) for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. For instance, the bone marrow (BM) niche is appreciated to be a site of immune privilege at steady state to allow for normal hematopoiesis and immune cell generation. Additionally, in hematological malignancies, the BM niche is protective to leukemic stem cells, a phenomenon that has minimized the efficacy of several anti-cancer drugs including chemotherapy, targeted small molecule inhibitors and antibody based therapies.In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using antibodies made with the Genmab DuoBody® technology targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia (AML; KG-1, MOLM-13 and OCI-AML5) or multiple myeloma (MM; H929 and RPMI8226) cell lines with bone marrow stromal cell lines (HS-5 and HS-27a) significantly protected leukemic cells from DuoBody®-T cell mediated lysis in vitro. Specifically, co-culture of bone marrow stromal cells in killing assays led to a 20-50% decrease in the maximum observed cytotoxicity and a 3-10 fold weaker EC50, reflecting an impact on both the efficacy and potency of bispecific DuoBody® antibodies. Similar results were also observed with primary stromal cells obtained from healthy donors. Furthermore, presence of stromal cells in a humanized xenograft AML model attenuated tumor growth inhibition (TGI) observed with DuoBody® treatment (78% TGI with MOLM-13 vs 15% TGI with MOLM-13+HS-5). Impaired TGI correlated with reduced T cell activation (7 fold decrease in CD25 upregulation) and production of granzyme B (8 fold reduction), providing one potential mechanism to explain loss of activity of the DuoBody® antibody. In vitro trans-well redirection assays revealed that cell-cell contact with stromal cells was crucial for reduced T cell activation and target cell survival relative to controls. Additionally, leukemic cells not killed by T cells were observed to preferentially cluster around stromal cells. We propose that target cells can evade T cell death by a stromal cell dependent mechanism involving activation of multiple pro-survival and anti-apoptotic pathways in leukemic cells in addition to suppressed activation of T cells. We are currently studying pathways mediating the cross-talk between cancer, immune and stromal cells. A better understanding of the mechanisms underlying the protective and immunosuppressive nature of the BM microenvironment will be instrumental to the design of more effective CD3 redirected therapeutics or novel combinatorial regimens for robust anti-cancer responses. DisclosuresNair-Gupta:Janssen: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Smith:Janssen Research & Development, LLC: Employment, Equity Ownership. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Pillarisetti:Janssen Research and Development, LLC: Employment. Baldwin:Janssen: Employment. Packman:Janssen: Employment. Elsayed:Janssen: Employment. Attar:Janssen: Employment. Gaudet:Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued.

Evolution of malignant plasmacytoma cell lines from K14E7 Fancd2-/- mouse long-term bone marrow cultures.

We tested the effect of expression of the Human Papilloma Virus (HPV E7) oncogene on hematopoiesis in long-term bone marrow cultures (LTBMCs) derived from K14E7 (FVB) Fancd2−/− (129/Sv), K14E7 Fancd2+/+, Fancd2−/−, and control (FVB X 129/Sv) Fl mice. K14E7 Fancd2−/− and Fancd2−/− LTBMCs showed decreased duration of production of total nonadherent hematopoietic cells and progenitors forming day 7 and day 14 multilineage CFU-GEMM colonies in secondary cultures (7 wks and 8 wks respectively) compared to cultures from K14E7 Fancd2+/+ (17 wks) or control mice (18 wks) p < 0.0001. Marrow stromal cell lines derived from both K14E7 Fancd2−/− and Fancd2−/− cultures were radiosensitive, as were IL-3 dependent hematopoietic progenitor cell lines derived from K14E7 Fancd2−/− cultures. In contrast, Fancd2−/− mouse hematopoietic progenitor cell lines and fresh marrow were radioresistant. K14E7 Fancd2−/− mouse freshly explanted bone marrow expressed no detectable K14 or E7; however, LTBMCs produced K14 positive factor-independent (FI) clonal malignant plasmacytoma forming cell lines in which E7 was detected in the nucleus with p53 and Rb. Transfection of an E6/E7 plasmid into Fancd2−/−, but not control Fancd2+/+ IL-3 dependent hematopoietic progenitor cell lines, increased cloning efficiency, cell growth, and induced malignant cell lines. Therefore, the altered radiobiology of hematopoietic progenitor cells and malignant transformation in vitro by K14E7 expression in cells of the Fancd2−/− genotype suggests a potential role of HPV in hematopoietic malignancies in FA patients.

Quantitative proteomic analysis and comparison of two bone marrow stromal cell lines using the SILAC method.

Two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells, have frequently been used in studies of cross talk between cells in the bone marrow microenvironment and hematopoietic cells. Altered expression of proteins is typically associated with cell-cell signal transduction and regulation of cellular functions. Many studies have focused on key proteins that contribute to functional differences in cell co-culture models, but global quantitative proteome analysis of HS5 and HS27a has not been performed. We employed the stable isotope labeling by amino acids in cell culture (SILAC) method using two stable isotopes each of arginine and lysine to label proteins in the two cell lines. Labeled proteins were analyzed by 2-D ultrahigh-resolution liquid chromatography- LTQ/Orbitrap mass spectrometry. Among 4,213 unique identified and annotated proteins in the cell lines, 1,462 were detected in two independent experiments. Of these, 69 exhibited significant upregulation and 48 significant downregulation (>95% confidence) in HS27a relative to HS5 cells. Gene ontology term and pathway analysis indicated that the differentially regulated proteins were involved in cellular movement, cell-to-cell signaling and interaction, and hematologic system development and function. A total of 55 items were identified in both genomic and proteomic databases. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed on 7 proteins randomly selected from 28 differentially expressed proteins that were identified in both databases and were involved in the top networks/pathways. We observed a decrease in apoptosis in co-cultured KG1a cells when integrin αV was inhibited in HS27a cells, which suggested the functional role of integrin αV in the co-culture system. The integrated genomic/proteomic approach described here, and the identified proteins, will provide a useful basis for further elucidation of molecular mechanisms in the bone marrow microenvironment and for ongoing studies of cross talk among stromal cells and myeloma cells in co-culture systems.

Induction of Multidrug Resistance of Acute Myeloid Leukemia Cells by Cocultured Stromal Cells via Upregulation of the PI3K/Akt Signaling Pathway.

This study aimed to investigate the role of the PI3K/Akt signaling pathway in multidrug resistance of acute myeloid leukemia (AML) cells induced by cocultured stromal cells. Human AML cell lines HL-60 and U937 were adhesion cocultured with human bone marrow stromal cell line HS-5 cells. Such coculturing induced HL-60 and U937 cells resistant to chemotherapeutic drugs including daunorubicin (DNR), homoharringtonine (HHT), and cytosine arabinoside (Ara-C). The coculturing-induced resistance of AML cells to DNR, HHT, and Ara-C can be partially reversed by inhibition of the PI3K/Akt signaling pathway. Clinically, AML patients with a low level of PTEN and a high level of CCND1 had high relapse rates within 1 year, and newly diagnosed AML patients with extramedullary infiltration had a low level of PTEN. This study confirms the involvement of the PI3K/Akt signaling pathway in multidrug resistance in AML cells induced by stroma and suggests that the expression of PTEN and CCND1 may be a prognostic indicator for AML.

Abstract 1655: Expression of the HPV E7 oncogene in K14E7Fancd2-/- mouse long term bone marrow culture derived hematopoietic cells produces malignant plasmacytomas

Abstract Introduction: Transgenic FVB/n mice with the HPV E7 oncogene under the control of the CK14 promoter were bred with Fancd2+/- (129/Sv background) mice to obtain K14E7 Fancd2-/- mice Park, et al. Cancer Research, 70(23): 9959, 2010), in which delivery of 4-nitroquinoline N-oxide in drinking water produces oral and esophageal adenomas. The hematopoietic system, of these mice was studied, using long term bone marrow cultures (LTBMCs) from Fancd2-/- (SV129), K14E7 transgenic (FVB/n), and K14E7 Fancd2-/- mice. Methods: LTBMCs were established by flushing bone marrow into T-25 flasks from which bone marrow stromal cell lines and IL3-dependent hematopoietic cell lines were developed. The cell lines were analyzed using irradiation survival curves, Western analysis for protein expression. K14E7 Fancd2-/- IL3 dependent clonal cell lines of the IL3-dependent cell lines were established by flow cytometry. Clonal lines were expanded and tested for tumorigenicity by injecting 1 × 106 cells of each of four clones into flanks of K14E7 Fancd2+/+ mice. Results: K14E7 Fancd2-/- LTBMCs were similar to those from Sv/129 Fancd2-/-: 1) hematopoiesis was shortened when compared to K14E7 Fancd2+/+ or Fancd2+/+ LTBMCs, respectively. Stromal cell line irradiation survival curves with K14E7 Fancd2+/+ and Fancd2-/- cell lines showed (Do = 1.48 ± 0.05 and 1.52 ± 0.15 Gy) they were radiosensitive compared to those from K14E7 Fancd2+/+ or Sv/129 Fancd2+/+ cells (Do = 2.33 ± 0.11 and 2.23 ± 0.01 Gy, p = 0.0043 and 0.0368, respectively). Fancd2-/- stromal cells were more radiosensitive than Fancd2+/+ cells (decreased shoulder on the survival curve of n = 1.5 ± 0.5 compared to 4.7 ± 0.3 (p = 0.0126). K14E7Fancd2+/+ cells had an n of 1.9 ± 0.3 compared to 3.5 ± 0.1 (p = 0.0309) for Sv/129 cells. In contrast, hematopoietic IL-3 dependent K14E7Fancd2+/+ and Sv/129 Fancd2+/+ cell lines were similar (Do = 2.15 ± 0.4 and 1.92 ± 0.06, p = 0.6469, respectively). Confirming prior data, (Berhane et. al, Rad Res 182: 35, 2014) Sv/129 Fancd2-/- IL-3 dependent cell lines were radioresistant compared to Fancd2+/+ cell lines (Do = 2.12 ± 0.12 and 1.92 ± 0.06, respectively, p = 0.0236). In contrast, K14E7 Fancd2-/- cell lines were radiosensitive compared to K14E7 Fancd2+/+ cell lines (1.44 ± 0.13 and 2.15 ± 0.28, respectively, p = 0.0498). Hematopoietic and marrow stromal cell lines from K14E7Fancd2-/- marrow expressed cytokeratin 14 and E7 oncogene by Western analysis. K14E7 Fancd2-/- (but no other genotype derived) IL-3 dependent cell lines transformed in vitro to dense tumor cells lines. Cloned sublines were injected into K14E7 Fancd2+/- mice producing plasmacytomas. Conclusions: Expression of the E7 oncogene in the K14/E7 Fancd2-/- mouse marrow derived IL-3 dependent cell lines leads to radiosensitivity and tumorgenicity. Supported by NIAID/NIH U19-A 1068021 and the Fanconi Anemia Research Foundation. Citation Format: Joel S. Greenberger, Lora Rigatti, Aranee Sivanathan, Shaonan Cao, Xichen Zhang, Donna Shields, Darcy Franicola, Michael Epperly. Expression of the HPV E7 oncogene in K14E7Fancd2-/- mouse long term bone marrow culture derived hematopoietic cells produces malignant plasmacytomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1655.

Abstract 3291: The expression of CD20 on malignant B cells is regulated by chemokine signaling through the CXCR4/SDF-1 axis: implications for targeting the microenvironmental interactions

Abstract The introduction of anti-CD20 antibodies has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin lymphomas. The aim of this study was to analyze molecular pathways that influence expression of CD20 since this is largely unknown. This is of a great clinical interest, as combinatorial therapy of novel BCR-signaling inhibitors ibrutinib and idelalisib currently focuses mainly on the use with anti-CD20 antibodies. Firstly, we analyzed samples obtained from CLL patients treated with ibrutinib and showed that administration of ibrutinib in vivo leads to CD20 down-modulation (P&amp;lt;0.0001). That implies that CD20 expression might be regulated by a yet unknown mechanism in the context of immune niches. Therefore, we focused on determining the effect of microenvironmental interactions on CD20 expression on malignant B cells. We co-cultured primary CLL cells with the bone marrow stromal cell line HS-5. This revealed that CLL cells co-cultured with HS-5 had higher surface CD20 expression compared to control cells cultured on plastic (P&amp;lt;0.01). Moreover, CLL cells that have been recently released from the lymph node microenvironment to the peripheral blood (CXCR4dimCD5bright) had ∼2-fold higher surface CD20 expression when compared to CLL cells circulating in blood of the same patient for a longer time (CXCR4brightCD5dim cells; P&amp;lt;0.0001). Further, sorted CXCR4dimCD5bright CLL cells had also ∼2 times higher CD20 mRNA expression (P = 0.002) suggesting that changes in CD20 expression were rather due to changes in its mRNA levels than surface modulation. As CD20 expression was decreasing with the transition from CXCR4dim to CXCR4bright CLL cells, we hypothesized that the CXCR4/SDF-1 axis is directly implicated in CD20 regulation. Indeed, CLL cells treated with SDF-1α (CXCL12), a ligand for CXCR4 produced by stromal cells, significantly up-regulated surface CD20 (P&amp;lt;0.05). On the contrary, the treatment with plerixafor, CXCR4 antagonist, inhibited this SDF-1α mediated up-regulation of CD20 which proves that SDF-1α binding to CXCR4 is directly involved in CD20 regulation. Altogether, the CD20 levels are up-regulated within the context of microenvironmental interactions through the CXCR4/SDF-1 axis, and the impairment of microenvironmental interactions mediated by ibrutinib down-regulates CD20 expression. This study reveals a novel regulation of CD20 expression in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684; NGS-PTL(306242); EHA Fellowship award; Ministry of Health of CR (16-29622A); Academy of Sciences of CR (16-13334Y); Ministry of Education, Youth and Sports, grant LD15144 (COST CZ); MUNI/A/1028/2015; CZ.1.05/1.1.00/02.0068; G.P. is supported by Ostrava city. Citation Format: Gabriela Pavlasova, Marek Borsky, Vaclav Seda, Katerina Cerna, Jitka Osickova, Michael Doubek, Yvona Brychtova, Jiri Mayer, Sarka Pospisilova, Matthew S. Davids, Jennifer R. Brown, Marek Mraz. The expression of CD20 on malignant B cells is regulated by chemokine signaling through the CXCR4/SDF-1 axis: implications for targeting the microenvironmental interactions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3291.

Abstract 4100: Stromalcell suppression of estrogen receptor in growth-arrested breast cancer cellsand promotion of outgrowth of estrogen-independent clones

Abstract Breast cancers have a poorer prognosis if estrogen receptor expression was lost during recurrence. It is unclear whether this conversion is cell autonomous or whether it can be promoted by the microenvironment during cancer dormancy. Bone marrow is the most common site for luminal breast cancer recurrence. We explored whether the HS5 bone marrow stromal cell line that is known to arrest co-cultured breast cancer cells could suppress estrogen receptor alpha (ER) expression during arrest, and whether it could prime the emergence of estrogen-independent breast cancer clones. We demonstrated that paracrine signaling from HS5 cells downregulated ER at the mRNA level and also decreased ER protein stability in T47D and MCF7 breast cancer cells. Conditioned medium (CM) from HS5 arrested the cells in G0/G1 in part through interleukin-1 (IL1), inhibiting mammosphere formation and 2D-growth despite activation of proliferative pathways (Erk and AKT) by the CM. Reversal of stromal-induced growth arrest by blockade of signaling by IL1 in HS5-CM enabled the outgrowth of ER-negative breast cancer cells that were fulvestrant-resistant and estrogen-independent. Outgrowth depended on AKT signaling. Our results describe pro- and anti-proliferative paracrine signals arising from a bone marrow stromal cell line. Blockade of the growth suppressive signaling unmasked stromal facilitation of ER conversion and anti-estrogen resistance in breast cancer cells. Citation Format: Richard Steinman, Jingjing Huang, Paul Woods, Daniel Normolle, Julie Goff, Christine Stehle. Stromalcell suppression of estrogen receptor in growth-arrested breast cancer cellsand promotion of outgrowth of estrogen-independent clones. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4100.

Abstract 762: The interleukin-1 inflammatory cytokine family modulates p62/SQSTM1 accumulation in breast cancer cell lines

Abstract Tumors are likened to wounds that do not heal. Thus, not surprisingly, inflammatory cytokines are present in the primary and metastatic tumor microenvironments due, in part, to infiltrating bone-derived cells (e.g., macrophages). We previously discovered that a bone marrow stromal cell line that secretes the inflammatory cytokine, interleukin-1 beta (IL-1â), upregulates p62 mRNA and protein in prostate cancer cell lines and we showed that p62 can be cytoprotective for prostate cancer cells. p62/Sequestome-1 is a multifunctional scaffold protein that can be seen microscopically as subcellular “speckles” which serve as organizing centers for multiple different signaling cascades, ranging from autophagy-mediated degradation of ubiquitinated proteins to KEAP1-NRF2 antioxidant response. To determine if our previous discoveries might be cancer-type specific and to test our hypothesis that p62 promotes cancer cell survival in response to inflammatory cytokines, we expanded our studies to various breast cancer cell lines of varying receptor status, including ZR57-1, T47D, and MDA-MB-468 and determined cell response two interleukin-1 family members, alpha (á) and beta (â). We treated the breast cancer cells with IL-1á and/or IL-1â and analyzed p62 mRNA and protein levels, subcellular localization, and protein-protein interactions. IL-1á and IL-1â can induce p62 speckle accumulation that can be seen to co-localize with the autophagosome membrane protein, LC3B, suggesting an autophagy-dependent regulation and/or function for p62. In addition, p62 co-immunoprecipitates with KEAP1, suggesting a KEAP1-NRF2-dependent antioxidant function for p62. Taken together, our data implies that cancer cells usurp inflammatory cytokines, such as IL-1, present in the primary or metastatic tumor microenvironment to promote cell survival through proteins such as p62. Citation Format: Afshan Fathima Nawas, Janani Ramachandran, Shayna Thomas, Jananisree Ravichandran, Nikki Delk. The interleukin-1 inflammatory cytokine family modulates p62/SQSTM1 accumulation in breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 762.

Cell adhesion induces overexpression of chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) and contributes to cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma cells

Previous studies have shown that chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) exerts its anti-apoptotic function in many solid cancers. However, its role in human multiple myeloma (MM) has not been thoroughly elucidated. In this study, we investigate the role of CHD1L in MM. Preliminarily, up-regulation and down-regulation assay verified that CHD1L exerts its anti-apoptotic role through the apoptotic pathway involving caspase-9–caspase-3 apoptotic pathway in MM cells. In addition, we determined that CHD1L expression is increased when MM cells were adhered to fibronectin (FN) or bone marrow stromal cell line HS-5 cells and cell adhesion assay indicated that CHD1L siRNA reversed the high cell adhesion rate. Consistent with the reduced adhesion rate, the cells translated to a compromised cell adhesion-mediated drug resistance (CAM-DR) phenotype in MM. In summary, we will propose strategies for developing a CHD1L inhibitor for potential treatment of MM.

Sprouty2 regulates proliferation and survival of multiple myeloma byinhibiting activation of the ERK1/2 pathway invitro and invivo.

Multiple myeloma (MM) is an incurable disease, and its pathogenesis remains unclear. MicroRNA (miR)-21 was detected at a high level in MM and plays a key role in the pathogenesis of MM. However, Sprouty2 (spry2), a downstream target of miR-21, has low expression, and its mechanism in MM is unknown. We investigated whether spry2 could exert an antimyeloma effect and further studied the potential pathogenesis and progression of MM. To address the functional consequences of spry2, we assessed the expression levels of spry2 in several myeloma cell lines and detected low expression levels in MMcells. Overexpression of spry2 suppressed growth and colony formation ability and decreased the phosphorylation of extracellular signal-regulated kinases 1 and 2. Spry2 also decreased secretion of vascular endothelial growth factor and partially enhanced the sensitivity of MMcells to an inhibitor of mitogen-activated protein kinases 1 and 2. Additionally, spry2 inhibited the tumorigenesis and angiogenesis of MMcells invivo. In summary, we report for the first time that spry2 can inhibit MMcell growth and survival with a concomitant reduction in phosphorylation of extracellular signal-regulated kinases 1 and 2 invitro and invivo.

Abstract B09: Differential effect of microenvironment on tumor phenotype using an immature T cell lymphoma mouse model

Abstract Human T cell lymphomas of an immature phenotype with mediastinal origin are classified as T cell lymphoblastic lymphomas (T-LBL) and comprise 85-90% of all lymphoblastic lymphomas. Treatment of T-LBL can be challenging because of the metastasizing nature of the disease, which commonly localizes to the bone marrow and can be found in other organs such as liver and spleen. We have developed a mouse T-LBL model by establishing a cell line from a spontaneous mediastinal tumor found in a T cell receptor (TCR) transgenic mouse strain. The resulting T-LBL like cell line, JMR4, has a known TCR (2C), known alloreactivity (H-2Ld), and known stimulating self-peptide (SIYR). JMR4 has an immature T cell phenotype, which includes a mixture of CD4+/CD8+, CD4lo/CD8+, CD4+/CD8lo and CD4+ cells. Other T cell markers, such as CD5, CD24 and CD69 are expressed at various levels depending on the phenotypic subset. The 2C TCR, which can be detected using the 1B2 idiotypic mAb, has been shown to be functional using flow cytometric analysis following TCR stimulation using peptide loaded thymic stromal feeder cells. In addition, JMR4 can be phenotypically altered (CD4/CD8/CD5/CD69 expression levels) differentially in vitro using the bone marrow stromal cell line OP9 and OP9DL, suggesting that it has pluripotency. Finally, when JMR4 is injected into sub-lethally irradiated mice, it develops into a multi-organ lymphoma. Interestingly, the phenotype of the JMR4 cells, based on CD4 and CD8 expression levels, is differentially regulated depending on organ type and MHC of the host, making JMR4 an ideal model to study the complex interactions of a tumor with its microenvironment. Understanding the mechanisms involved in tumor microenvironment (TME) induced changes to tumor cells should be important in understanding metastatic tumor progression and in designing new treatment regimens. Citation Format: Philip J. Lucas, Ronald E. Gress. Differential effect of microenvironment on tumor phenotype using an immature T cell lymphoma mouse model. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B09.

Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis.

F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin− BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo. Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80+ macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80+ macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro. Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo.

Transformed Phenotype of Bone Marrow Stromal Cell Lines Derived from K14E7 Fancd2-/- mice

To determine whether cytokeratin 14 promoter linked expression of the Human Papilloma Virus (HPV) oncogene detectably influenced biologic parameters of cell phenotypes other than squamous epithelium, continuous bone marrow cultures were derived from K14E7 Fancd2-/- mice (Park, et al., Cancer Research, 70(23): 9959-9968, 2010). Long-term bone marrow cultures derived from K14E7 Fancd2-/-, control Fancd2-/- (129/Sv), K14E7 (FVB/N), and wild type 129/Sv X FVB/N F1 mice were evaluated for longevity of hematopoiesis in long-term cultures and stromal cell lines were derived from each. Similar to Fancd2-/- mouse, long-term marrow cultures, K14E7 Fancd2-/- marrow cultures demonstrated decreased longevity of hematopoiesis with cessation of production of multi-lineage colony forming progenitor cells after 14 weeks. In contrast, wild type F1 and K14E7 long-term marrow cultures continued to produce hematopoietic cells for a significantly longer duration 25 weeks (p=0.0257). Bone marrow stromal and IL-3 dependent hematopoietic cell lines were derived from each genotype marrow culture. K14E7 Fancd2-/- hematopoietic cells showed reversal of the radiation resistance of Fancd2-/- IL-3 dependent cell lines (D0 of 1.34 ± 0.197Gy, ñ 4.0 ± 0.9 compared to D02.213 ± 0.124 Gy (p = 0.0284), ñ 3.3 ± 0.8. Thus, one phenotypic difference associated with K14E7 oncogene expression was reversal of radioresistance of Fancd2-/- hematopoietic cells. In contrast, bone marrow stromal cell lines from K14E7 Fancd2-/- remained radiosensitive similar to those from Fancd2-/- mice. K14E7 and wild type F1 marrow stromal cell lines showed intermediate radioresistance (p= 0.1759). To determine whether the E6/E7 oncogene had a biological effect in tissues other than squamous epithelium, tissue analysis for cytokeratins 13, 14, 6, and 10 was carried out. Cytokeratin 14 was detected only in squamous cells of the esophagus and oral cavity, not in bone marrow. E6/E7 oncogene was detected only in squamous cell lines expressing cytokeratin 14. However, bone marrow stromal cell lines from K14E7 Fancd2-/- marrow cultures demonstrated a unique phenomenon of cellular density, piling up and formation of tumors in vitro. Each of 22 single cell derived clonal sub-lines of K14E7 Fancd2-/- stromal cell lines demonstrated the same transformed phenotype. These data provide support for indirect effects of the E7 oncogene linked to the K14 promoter in Fancd2-/- hematopoietic and mesenchymal stem cell tissues.Supported by research grant NIAID/NIH, U19A168021. DisclosuresNo relevant conflicts of interest to declare.