JAK2-Mediated Extrinsic Survival of CML Stem Cells: Exploring the Potential Combination of BCR-ABL and JAK2 Inhibitors In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 199-199
Author(s):  
Elie Traer ◽  
Ryan MacKenzie ◽  
Jennifer Snead ◽  
Brian J Druker ◽  
Michael W Deininger
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Median Survival ◽  
Conditioned Media ◽  
High Dose ◽  
Protective Effects ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Jak2 Inhibitors

Abstract Abstract 199 Background: The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib are very effective for the treatment of chronic phase CML. However, the majority of these patients continue to have persistence of CML cells despite continued therapy, suggesting that TKIs fail to target leukemic stem cells (LSCs). There is increasing evidence that the bone marrow microenvironment provides a sanctuary to LSCs, thereby contributing to persistence. Results: We used the human stromal cell lines (HS-5, HS-23, HS-27a) to model the microenvironment. Conditioned media from HS-5, but not HS-23 or HS-27a, reduced apoptosis of CML cell lines treated with TKIs (K562, LAMA-84, KBM-5 and KYO-1), consistent with previous reports. Similarly, CML CD34+ cells were protected from 5 μ M imatinib in a 4 day co-culture with HS-5 cells, as assessed by CFU-GM colony survival (23% vs 9% when compared to untreated controls, N=6, p=0.018). We were also able to demonstrate protection from TKIs with transwells over HS-5 and with HS-5 conditioned media, which suggests that factors secreted by HS-5 cells protect CML cells from TKIs. Cytokine analysis of conditioned media revealed relatively higher concentrations of IL6, IL-8, MCP-1, MCP-3, G-CSF and GM-CSF from HS-5 as compared to HS-23 and HS-27a. Since IL-6, G-CSF and GM-CSF are known to signal via JAK2, we tested combinations of imatinib and JAK2 inhibitors (TG101209 or CYT387) using our in vitro assay. Combination treatment with imatinib and CYT387 or TG101209 abrogated the protective effects of HS-5 conditioned media in CML cell lines. Combination treatment of CML CD34+ in HS-5 co-culture assays also abrogated the protective effects of stroma on colony formation. However, we observed that both normal CD34+ and CML CD34+ colony formation was dramatically reduced by JAK2 inhibitors using our HS-5 co-culture system, particularly at higher doses. Thus, it was unclear if a potential therapeutic window existed in vivo. To test the potential of combination therapy in vivo, we infected marrow from Balb/c mice with a retrovirus that simultaneously expresses BCR-ABL and GFP, followed by transplantation into lethally irradiated syngeneic recipients. The mice were separated into five cohorts: vehicle control, TG101209 (200mg/kg/d), nilotinib (75mg/kg/d), nilotinib + low-dose TG101209 (50mg/kg/d) and nilotinib + high-dose TG101209 (200mg/kg/d). The vehicle-treated control group died rapidly of myeloproliferative disease (MPD) with a median survival of 15.5 days. The median survival of mice treated with TG101209 was slightly prolonged at 20.5 days (p=0.06); however, these mice also died of MPD with enlarged spleens/livers and lung hemorrhage. The survival curves of mice treated with nilotinib monotherapy and nilotinib + low-dose TG101209 were similar (median survival not reached at termination of experiment). Mice treated with nilotinib + high-dose TG101209 initially had minimal mortality, however on day 26 the mice began to die without signs of MPD (no definitive cause of death at autopsy) and thus the remaining cohort was sacrificed on day 27 for analysis. In stark contrast to the other cohorts, the spleens of these mice were very small, leukopenic, and largely devoid of normal follicles, with decreased spleen weight compared to mice treated with nilotinib monotherapy (0.025 vs. 0.072 gm, p<0.01). The bone marrow was also profoundly hypocellular, suggesting that myelosuppresion (anemia and leukopenia in particular) may have been a factor in mortality. However, despite toxicity this combination may retain a degree of selectivity for BCR-ABL cells since disease burden, as measured by the percentage of GFP-positive cells, was reduced compared to nilotinib monotherapy (spleen: 4% vs 11.7%, p=0.047; bone marrow: 8.7% vs 13.8%, p=0.22). Conclusions: (1) Factors secreted by human bone marrow stromal cells attenuate the effects of imatinib in CML cell lines and primary CML CD34+ cells in a JAK2-dependent fashion. (2) Simultaneous in vivo inhibition of BCR-ABL and JAK2 dramatically reduces BCR-ABL expressing cells, but at the cost of marrow toxicity. We speculate that this limitation may be overcome by intermittent rather than continuous JAK2 inhibition, a strategy that might avoid toxicity while reducing persistent BCR-ABL disease burden. Disclosures: Deininger: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Genzyme: Research Funding; Ariad: Consultancy.

The AML1/ETO Target LAT2 Membrane Adaptor Molecule Is Regulated during Normal Monocytic Differentiation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2401-2401
Author(s):  
Jesus Duque-Afonso ◽  
Leticia Solari ◽  
Michael Luebbert
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Lines ◽  
U937 Cells ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Gm Csf ◽  
Adaptor Molecule ◽  
Cd34 Cells ◽  
Conditional Expression

Abstract LAT2 (NTAL/LAB/WBSCR5) is a 28 KDa membrane protein which acts as adaptor molecule in the signalling pathways of FcεR I, c-Kit, B cell and T cell receptor. Bone marrow-derived mast cells from knock-out (KO) mice are hyperresponsive to stimulation via FcεR I. Although LAT2 is highly expressed in B cells, no major changes were found in function or development of B cells from LAT2 KO mice. An autoimmunity syndrome in LAT2 KO mice is caused, at least in part, by hyperreactivity and higher proliferation of T cells. Previously, we showed that LAT2 mRNA is repressed in vivo by AML1/ETO which was confirmed by others in several large series of primary AML blasts. We wished to elucidate the possible role of LAT2 during the myelopoiesis. AML1/ETO was induced by Ponasterone A in an ecdysone-inducible system in U937 cells (9/14/18 cell line). AML bone marrow samples from 43 patients (pts) were analyzed for LAT2 expression. Several myeloid cell lines were treated either with ATRA, DMSO or PMA for 3 days. Normal CD34+ cells were differentiated ex vivo by G-CSF towards granulocytes and by GM-CSF plus IL-4 towards monocytes and dendritic cells. LAT2 expression was determined by Northern and Western blot. LAT2 protein was repressed not only in AML1/ETO positive primary AML blasts (6/6), but also in blasts from patients with deletions of chromosome 7 (3/4) and the t(15;17) (4/4); expression was moderate to high in AML blasts with normal karyotype (14/15). LAT2 was expressed in normal monocytes and even higher in alveolar macrophages but not in granulocytes of healthy donors. It was downregulated after ATRA-induced granulocytic differentiation of NB4, HL60 and U937 cells but upregulated after DMSO-induced granulocytic differentiation of HL60 cells and PMA-induced monocytic-macrophage differentiation of HL60, U937 and Kasumi-1 cells. In normal CD34+ cells, LAT2 was strongly induced 7 days after the addition of G-CSF and GM-CSF+IL4 respectively, but after 14 days it was downregulated (0.7 +/− 0.4-fold) by G-CSF-induced granulocytic differentiation and upregulated (5.8 +/− 2.8-fold) by GM-CSF+IL4-induced monocytic-DC differentiation. Conditional expression of AML1/ETO in 9/14/18-U937 cells partially inhibited the PMA- and vitamin D3-induced monocytic differentiation of these cells, as determined by FACS for CD11b and CD11c. In conclusion, LAT2 protein is strongly repressed by AML1/ETO in primary leukemias and is upregulated during the monocytic differentiation in several cell lines and normal CD34+ cells. Further studies in a LAT2 knock-down by shRNAs in U937 cells are warranted to functionally address its possible role in monocytic differentiation.

scholarly journals Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3079-3089
Author(s):  
J Mladenovic ◽  
SM Anderson
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conditioned Media ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf

The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.

Non-Hematopoietic Stromal Cells Sense Toll-Like Receptor 4 Agonists and Consequently Enhance Myelopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2583-2583
Author(s):  
Steffen Boettcher ◽  
Patrick Ziegler ◽  
Michael A Schmid ◽  
Guido Garavaglia ◽  
Hitoshi Takizawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Cell ◽  
Cell Production ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Human Bmscs ◽  
Emergency Myelopoiesis ◽  
Stimulation Of

Abstract Abstract 2583 Hematopoiesis is tightly regulated by growth factors acting on stem and progenitor cells (HSPCs) in the bone marrow. During systemic infections cytokines are elevated in serum, myelopoiesis is enhanced, and myeloid CFUs and granulocytes increase in circulation. However, the underlying mechanisms of this “emergency” myelopoietic response have not been defined. Sensing of conserved pathogen-associated products by specialized pattern-recognition receptors such as Toll-like receptors (TLRs) is crucial for rapid responses to infection. Based on the well-known regulatory function of the BM microenvironment, we hypothesized that bone marrow stromal cells (BMSCs) express TLRs and possess all functional properties required to sense microbes and drive emergency myelopoiesis. Human BMSCs expressed Tlr1, Tlr5, and Tlr6 at similar levels and Tlr3 and Tlr4 mRNA at about 2-log higher compared to dendrititc cells (DCs). Stimulation of BMSCs with the TLR4 agonist LPS led to de novo expression of G-csf and Gm-csf, and increased M-csf, Il-6, and Il-11 expression. In line with this, LPS induced production of G-CSF and GM-CSF protein and significantly enhanced the secretion of M-CSF, IL-6, and IL-11. Using LPS-stimulated BMSC culture supernatant in myeloid CFU assays led to a 2.5-fold higher myeloid CFU activity compared to un-stimulated BMSC supernatant. This effect was partly mimicked by adding G-, M-, and GM-CSF to the methylcellulose cultures. Importantly, direct LPS stimulation of CB CD34+ cells had no effect. Furthermore, co-culture of BMSCs and CB CD34+ cells together with LPS for 12 days led to approximate 2-fold higher recovery of immuno-phenotypically primitive CD34+ cells, and retained up to 8-fold more CD34+ cells in divisions 0–3 as compared to LPS-free co-cultures as measured by CFSE dilution. When subjected to cytokine-supplemented myeloid CFU assays or transplanted into newborn RAG2-/- γc-/- mice to evaluate lymphoid differentiation, recovered CD34+ cells from LPS-stimulated BMSC cultures gave rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus proving maintenance of primitive hematopoietic progenitors. To elucidate the in vivo relevance of the findings and to clarify the contribution of stromal vs. hematopoietic cell expressed TLR4, we generated chimeras with TLR4-/- hematopoiesis in a wild-type (WT) background (hematopoietic-TLR4-/-) and WT hematopoiesis in a TLR4-/- background (non-hematopoietic-TLR4-/-). Chimeric, WT, and TLR4-/- mice were injected with LPS and hallmarks of myelopoietic responses such as G-CSF expression, myeloid cell mobilization from the BM, and increased myeloid cell production in the BM was evaluated. Significant G-csf mRNA induction could be observed in the BM of WT and hematopoietic-TLR4-/- mice. To a much lesser, non-significant extent, this effect could be observed also in non-hematopoietic-TLR4-/- mice, while no transcripts were detectable in TLR4-/- mice. Accordingly, serum G-CSF levels significantly increased 10-fold in WT and hematopoietic-TLR4-/- mice after LPS injection, but no increase was detectable in non-hematopoietic-TLR4-/- and TLR4-/- mice. LPS injection also resulted in a significant decrease in BM cellularity accompanied by an increase of spleen cell numbers only in WT and hematopoietic-TLR4-/- mice. Furthermore, Gr-1highCD11blow/+ mature myeloid cells were significantly reduced whereas Gr-1lowCD11blow/+ immature promyelocytes and myelocytes significantly increased (2.5-fold) in the BM of WT and hematopoietic-TLR4-/- mice. In contrast, similar changes in cellular composition could not be observed in TLR4-/- and non-hematopoietic-TLR4-/- mice, while a small, but still significant 1.25-fold increase in immature Gr-1lowCD11blow/+ cells was detectable in non-hematopoietic-TLR4-/- mice. Finally, inflammation-induced Sca-1 upregulation on HSPCs and increasing frequencies of GMPs were only observed in WT and hematopoietic-TLR4-/- mice. Collectively, our in vitro data demonstrate that human BMSCs are able to sense pathogens and stimulate emergency myelopoiesis but also prevent loss of HSPCs by enhancing their maintenance. Importantly, in vivo signaling via non-hematopoietic cell-expressed TLR4 is sufficient and is the main mechanism regulating both the release of mature myeloid cells from and the enhanced myeloid cell production in the bone marrow during systemic challenges. Disclosures: No relevant conflicts of interest to declare.

Hypoxia Inducible Factor (HIF)-2α Enhances Proliferation Of Malignant Hematopoietic Cells In The Hypoxic Malignant Bone Marrow

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2895-2895
Author(s):  
Catherine E Forristal ◽  
Falak Helwani ◽  
Sally Martin ◽  
Bianca Nowlan ◽  
Ingrid G Winkler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Lines ◽  
Median Survival ◽  
Hematopoietic Cells ◽  
Hazard Ratio ◽  
Leukemic Cells ◽  
Gm Csf ◽  
Empty Vector ◽  
Proliferative Advantage

Abstract Hypoxia and hypoxia-inducible factors (HIFs) are implicated in the regulation of normal and malignant hematopoiesis. HIF-1α stabilization makes leukemia stem cells and normal HSC dormant and is necessary to maintain their self-renewal potential. In sharp contrast, HIF-2α, which shares 60% homology with HIF-1a, promotes proliferation of renal clear carcinoma and embryonic stem cells by enhancing expression of oct-4, sox2 and activating c-myc. In this study, we investigated the role of hypoxia and HIF-2α in leukemia. In normal mouse and human bone marrow (BM), HIF-2α mRNA expression was observed predominantly in non-hematopoietic stromal cells, while hematopoietic cells displayed low to undetectable levels. In contrast, HIF-2α mRNA and protein were detected in the BM of moribund NOD/SCID mice engrafted with 3 different human ALL, and in cultured human ALL and AML cell lines, suggesting that HIF-2α is abnormally expressed in leukemic cells. To investigate the potential roles of HIF-2α in leukemic cells, we cloned human HIF-2α cDNA into the MXIE retroviral vector. In a 1st model the GM-CSF-dependent mouse pre-leukemic cell line FDCP1, which does not express HIF-2α, was retrovirally transduced with HIF-2α. HIF-2α provided a significant proliferative advantage to FDCP1 cells in hypoxic or normoxic cultures and reduced GM-CSF dependency. We next transplanted retrovirally transduced FDCP1 cells into non-irradiated syngeneic DBA/2 mice. All recipients of FDCP1 transduced with HIF-2α-MXIE vector succumbed to leukemia by week 28 post-transplantation. In sharp contrast, mice receiving FDCP1 transduced with empty MXIE vector, displayed a leukemia penetrance of only 15% by week 45 (Fig. 1a; p=0.0001 log rank, hazard ratio = 12.28).Fig. 1Percent survival of recipients of (a) FDCP1 cells retrovirally transduced with HIF-2α-MXIE vector or MXIE control empty vector, (b) vavBcl2 HSC transduced with HIF-2α-MXIE vector or MXIE empty vector, and (c) HL60 cells transduced with HIF-2α knocked-down or scrambled control lentiviral vectors.Fig. 1. Percent survival of recipients of (a) FDCP1 cells retrovirally transduced with HIF-2α-MXIE vector or MXIE control empty vector, (b) vavBcl2 HSC transduced with HIF-2α-MXIE vector or MXIE empty vector, and (c) HL60 cells transduced with HIF-2α knocked-down or scrambled control lentiviral vectors. In a 2nd model, HSC from vavBcl2 transgenic mice were transduced with human HIF-2α-containing or empty MXIE retroviral vectors and subsequently transplanted into lethally irradiated wild-type recipients. Transduction of vavBcl2 HSC with HIF-2α resulted in the outgrowth of HIF-2α-expressing B cells which was not observed in recipients of vavBcl2 HSC transduced with empty vector. Consequently recipients of HIF-2α transduced vavBcl2 HSC succumbed more rapidly to spontaneous lymphoma compared to controls (Fig. 1b; p=0.036 log rank, hazard ratio = 2.971, MXIE median survival = 56 weeks, HIF2α median survival = 41 weeks). Finally, HIF-2α was knocked-down in human leukemia cell lines U937 and HL60 using a shRNA lentiviral vector. HIF-2α knock-down resulted in a 2-fold decrease in proliferation in vitro. We next transplanted HL60-HIF-2a shRNA and HL60-scrambled shRNA cells into NOD/SCID/ IL2Rγ-/- (NSG) mice for each group. Notably, all recipients of HL60-HIF-2a shRNA cells succumbed to leukemia significantly later than recipients of HL60-scrambled shRNA cells (Fig. 1c; p=<0.027 Log-rank, hazard ratio = 0.1918, Scrambled median survival = 5 weeks, HIF-2α knock down median survival = 6 weeks). Together these data suggest that expression of HIF-2α in malignant hematopoietic cells provides a proliferative advantage in the hypoxic malignant BM enabling them to proliferate in the hypoxic leukemic BM while the proliferation of normal HSC, which do not express HIF-2α, is blocked by hypoxia-stabilized HIF-1α. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3079-3089 ◽  
Author(s):  
J Mladenovic ◽  
SM Anderson
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conditioned Media ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf

Abstract The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.

The Neurexin I Alpha/Neurexophilin Axis as An Anti-Proliferative Factor In Human Cord Blood and Murine Bone Marrow Hematopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2627-2627
Author(s):  
John Kinzfogl ◽  
Giao Hangoc ◽  
Hal E. Broxmeyer
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
High Concentration ◽  
Blocking Antibody ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Signaling Axis ◽  
Single Cell Culture

Abstract Abstract 2627 The membrane-bound receptors Neurexin I alpha (NRXN1α) and Dystroglycan (DAG1) serve as ligands for each other, and the Neurexophilins (NXPHs) are thought to function as natural antagonists for this interaction. This signaling axis centered on NRXN1α, DAG1 and the three highly homologous NXPHs, plays a well-defined role in neuronal development and synapse development. The failure of DAG1 knock-out mice to develop a mesoderm led us to hypothesize that this axis plays a role in hematopoiesis. Consistent with previous research, DAG1 is heavily (>95%) expressed in human (hu) umbilical cord blood (CB) CD34+ cells, including the primitive CD38- subpopulation. NRXN1α is expressed at similar levels in these populations as well. Hematopoietic cell populations are more precisely defined in the murine (mu) system, and adult mu bone marrow (BM) exhibits a similar trend to hu CB with NRXN1α expression being higher in the more primitive cells and decreasing as the cells differentiate. A high concentration of Neurexophilins is present in the hu CB plasma (900ng/ml), though it is absent in adult mu blood plasma and fetal bovine serum. Neither blocking DAG1 with a well-described blocking antibody, nor stimulation with recombinant NRXN1α has any observable effect on stimulation of hematopoietic progenitor cell (HPC) proliferation in vitro. This led us to investigate the other receptor present in the axis, NRXN1α. We first used alpha-latrotoxin, an excitatory toxin for NRXN1α. While no stimulatory or inhibitory proliferation effect was observed under normal growth conditions, alpha-latrotoxin did promote HPC survival under growth factor starved conditions as effectively as SDF1α/CXCL12 (a known HPC survival enhancing factor), suggesting a possible protective role for the DAG1-NRXN1α axis during stress. In contrast, we identified the Neurexophilins as anti-proliferative agents for immature hematopoietic cells. When injected in vivo, recombinant NXPH1 acts in a myelo- and lympho-suppressive manner. Absolute numbers of mu HPCs were suppressed in a dose- and time-dependent manner, with the maximal dosage decreasing HPC numbers by over 50%. The decrease in absolute colony number was mirrored by a decrease in the cycling population of HPCs in mice treated with NXPH1, as measured by the high specificity activated tritiated thymidine kill assay. Peripheral blood neutrophils, lymphocytes, monocytes and platelets, were also decreased in a time-dependant fashion after in vivo exposure to NXPH1. Intriguingly, the most primitive LTR-HSCs behave in the opposite manner, with a three-fold increase in cycling status induced via the Rapamycin sensitive mTOR pathway. During in vitro culture NXPH1 is only able to suppress colony forming ability of unseparated HPCs treated with both GM-CSF and SCF in combination in comparison to stimulation by other cytokines. This response is most likely due to increased NRXN1α expression in response to stimulation by GM-CSF and SCF. Given the high level of DAG1 expression on colony forming hematopoietic cells, we hypothesized that endogenous DAG1 may be competing with NXPH1 for NRXN1α binding. We thus exposed cells to a well studied DAG1 blocking antibody or plated CD34+ cells in single cell culture. The in vitro results were then able to recapitulate the results observed in vivo and a 50% decrease in colony forming ability is observed in combination with the following cytokines: GM-CSF, IL-3, GM-CSF+SCF, and IL-3+SCF as well as under maximally stimulatory conditions (IL-3, GM-CSF, SCF, and Epo for hu cells and SCF, PWMSCM, and Epo for mu cell). The ability of NXPH1 to inhibit proliferation of CD34+ cells in single cell culture suggests that NXPH1 acts directly on HPCs. The anti-proliferative function of the NXPHs are consistent with previous literature correlating NXPH expression in cancers with negative prognoses and these data may provide a mechanism for the immune system evasion of cancer. Additionally, the high concentration of NXPH in CB plasma may help explain the relatively long time to engraftment of transplanted CB cells. More broadly, these results demonstrate the presence and function of a tightly regulated signaling axis centered on NRXN1α in hematopoiesis. Disclosures: Broxmeyer: CordUse: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The Ets-related transcription factor PU.1 immortalizes erythroblasts

1993 ◽  
Vol 13 (9) ◽  
pp. 5670-5678
Author(s):  
S Schuetze ◽  
P E Stenberg ◽  
D Kabat
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Cell Lines ◽  
Cultured Cells ◽  
Low Frequency ◽  
In Vivo Studies ◽  
Bone Marrow Cultures ◽  
Clonal Cell Lines ◽  
Related Transcription Factor

In vivo studies of Friend virus erythroleukemia have implied that proviral integrations adjacent to the gene for the Ets-related transcription factor PU.1 may inhibit the commitment of erythroblasts to differentiate and cause their capability for indefinite transplantation (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 62:4129-4135, 1988; R. Paul, S. Schuetze, S. L. Kozak, C. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test this hypothesis, we ligated PU.1 cDNA into a retroviral vector and studied its effects on cultured cells. Infection of fibroblasts with PU.1-encoding retrovirus resulted in PU.1 synthesis followed by nuclear pyknosis, cell rounding, and degeneration. In contrast, in long-term bone marrow cultures, erythroblasts were efficiently and rapidly immortalized. The resulting cell lines were polyclonal populations that contained PU.1, were morphologically blast-like, required erythropoietin and bone marrow stromal cells for survival and proliferation, and spontaneously differentiated at low frequency to synthesize hemoglobin. After 9 months in culture, erythroblasts became stroma independent, and they then grew as clonal cell lines. We conclude that PU.1 perturbs the pathway(s) that controls potential for indefinite proliferation and that it can be used to generate permanent erythroblast cell lines.

scholarly journals A comparison of hematopoiesis in normal and splenectomized mice treated with granulocyte colony-stimulating factor

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 563-569 ◽  
Author(s):  
G Molineux ◽  
Z Pojda ◽  
TM Dexter
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Extramedullary Hematopoiesis ◽  
High Dose ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Response Data ◽  
Hematopoietic Precursor ◽  
Stimulating Factor

Abstract Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induces leukocytosis in vivo in both intact and splenectomized mice. Full dose response data showed a plateau in this effect at doses over 500 micrograms rhG-CSF/kg body weight/d in intact mice. The effect is magnified in splenectomized mice, where leukocyte numbers reach 100 x 10(6) mL after 4 days' treatment at 250 micrograms/kg/d. Further hematopoietic precursor populations are also affected in both marrow and the spleen; in general, marrow parameters were depressed, while splenic populations were enlarged. In splenectomized mice, both blood- borne stem cells were enhanced, and foci of extramedullary hematopoiesis were enlarged in addition to the effects seen in intact mice. In the marrow of splenectomized and intact mice treated with a high dose of G-CSF, erythroid suppression in the marrow was confirmed with radioactive iron. Our studies confirm and extend previous work on the mode of action of G-CSF, and indicate that side effects of high dose G-CSF therapy might include erythroid suppression in the bone marrow.

Preclinical Evaluation of a Bone-Marrow Autograft Culture Procedure for Generating Lymphokine-Activated Killer Cells in Vitro

1992 ◽  
Vol 3 (suppl b) ◽  
pp. 123-127 ◽  
Author(s):  
Hans-Georg Klingemann ◽  
Heather Deal ◽  
Dianne Reid ◽  
Connie J Eaves
Keyword(s):  
Bone Marrow ◽  
Calf Serum ◽  
Cell Activity ◽  
Hematopoietic Cells ◽  
Lak Cells ◽  
High Dose ◽  
Lymphokine Activated Killer Cells ◽  
Lak Cell

Despite the use of high dose chemoradiotherapy for the treatment of acute leukemia. relapse continues to be a major cause of death in patients given an autologous bone marrow transplant. Further augmentation of pretransplant chemotherapy causes life threatening toxicity to nonhematopoietic tissues and the effectiveness of currently available ex vivo purging methods in reducing the relapse rate is unclear. Recently, data from experimental models have suggested that bone marrow-derived lymphokine (IL-2)-activated killer (BM-LAK) cells might be used to eliminate residual leukemic cells both in vivo and in vitro. To evaluate this possibility clinically, a procedure was developed for culturing whole marrow harvests with IL-2 prior to use as autografts, and a number of variables examined that might affect either the generation of BM-LAK cells or the recovery of the primitive hematopoietic cells. The use of Dexter long term culture (LTC) conditions, which expose the cells to horse serum and hydrocortisone. supported LAK cell generation as effectively as fetal calf serum (FCS) -containing medium in seven-day cultures. Maintenance of BM-LAK cell activity after a further seven days of culture in the presence of IL-2 was also tested. As in the clinical setting. patients would receive IL-2 in vivo for an additional week immediately following infusion of the cultured marrow autograft. Generation ofBM-LAK activity was dependent on the presence of IL-2 and could be sustained by further incubation in medium containing IL-2. Primitive hematopoietic cells were quantitated by measuring the number of in vitro colony-forming progenitors produced after five weeks in secondary Dexter-type LTC. Maintenance of these 'LTC-initiating cells' was unaffected by lL-2 in the culture medium. These results suggest that LAK cells can be generated efficien tly in seven-day marrow autograft cultures containing IL-2 under conditions that allow the most primitive human hematopoietic cells currently detectable to be maintained.

scholarly journals Low-risk intensive therapy for multiple myeloma with combined autologous bone marrow and blood stem cell support

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1666-1672
Author(s):  
S Jagannath ◽  
DH Vesole ◽  
L Glenn ◽  
J Crowley ◽  
B Barlogie
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mononuclear Cells ◽  
Intensive Therapy ◽  
Autologous Bone Marrow ◽  
Blood Stem Cell ◽  
High Dose ◽  
Platelet Recovery ◽  
Gm Csf ◽  
Autologous Bone

To improve the safety of autotransplantation for myeloma, peripheral blood stem cell (PBSC) collection was attempted in 75 previously treated patients after the administration of high-dose cyclophosphamide (HD-CTX; 6 g/m2) with or without granulocyte-macrophage colony- stimulating factor (GM-CSF). Sixty patients subsequently received melphalan 200 mg/m2 (57 patients) or melphalan 140 mg/m2 and total body irradiation (850 cGy) (3 patients) supported by both autologous bone marrow and PBSC; 38 patients received GM-CSF posttransplantation. Among 72 patients undergoing PBSC apheresis, “good” mobilization (greater than 50 colony-forming units granulocyte-macrophage [CFU-GM] per 10(5) mononuclear cells) was achieved when prior chemotherapy did not exceed 1 year and when GM-CSF was used post-HD-CTX; similarly, rapid platelet recovery to 50,000/microL within 2 weeks was associated with “good” PBSC mobilization. These same variables also predicted for rapid engraftment after autotransplantation, so that hematologic recovery (granulocytes greater than 500/microL and platelets greater than 50,000/microL) proceeded within 2 weeks among the 37 patients with “good” PBSC collection. As a result of rapid neutrophil recovery (greater than 500/microL) within a median of 2 weeks, infectious complications both post-HD-CTX and posttransplant were readily manageable, resulting in only one treatment-related death post-HD-CTX. The cumulative response rate (greater than or equal to 75% cytoreduction) for all 75 patients was 68%, with 12-month event-free and overall survival projections of about 85%. Using both bone marrow and PBSC together with GM-CSF, autotransplants are safe and appear effective in myeloma, especially when prior therapy had been limited to less than 1 year. More than 80% of transplanted patients achieved complete hematologic recovery within a median of 1 month posttransplant (granulocytes greater than 1,500/microL; platelets greater than 100,000/microL; hemoglobin greater than 10 g%), thus providing sufficient hematopoietic reserve for further chemotherapy in the event of posttransplant relapse.

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