While autologous hematopoietic stem/progenitor cell (HSPC) transplantation has been used successf... more While autologous hematopoietic stem/progenitor cell (HSPC) transplantation has been used successfully for many years, new approaches are being explored to boost the number of HSPC (CD34+ cells) collected by leukapheresis to permit more rapid hematopoietic recovery. Still, a considerable number of patients may be excluded from this procedure because a sufficient quantity of HSPC cannot be obtained by standard mobilization agents. Among factors regulating mobilization is stromal cell-derived factor (SDF)-1 constitutively produced by bone marrow (BM) stromal cells and which is known to retain HSPC in the BM. Responsiveness to an SDF-1 gradient can be measured in vitro using the chemotactic index, defined as the ratio of the number of cells that migrate towards SDF-1 and the number of cells that migrate towards media alone. We hypothesized that (i) a high chemotactic index would indicate a greater ability of the HSPC to be retained in their BM niches and therefore more difficulty in coaxing them out into the circulation, as could be the case in poor mobilizers; and that (ii) AMD3100, an antagonist of the SDF-1 receptor CXCR4, will have a greater disruptive effect on SDF-1-dependent signalling in poor mobilizers. Leukapheresis products were obtained from patients diagnosed with Hodgkin’s or non-Hodgkin’s lymphoma and CD34+ cells isolated by positive selection were loaded onto Boyden chambers and allowed to migrate across bare filters towards a gradient of SDF-1 (200 ng/mL). In some experiments cells were incubated with AMD3100 (10 mg/mL) for the duration of the assay. We observed broad inter-patient differences in in vitro migratory ability of CD34+ cells, ranging from 3.1 ± 0.6 to 21.7 ± 2.1 % for spontaneous or passive percentage migration; and from 11.1 ± 0.7 to 58.6 ± 10.7 % for SDF-1-directed chemotaxis, neither of which bore any correlation with the number of CD34+ cells/kg obtained from the leukapheresis product. However, a negative correlation (r=−0.7) was found between the chemotactic index and the percentage of CD34+ cells obtained from the leukapheresis product, i.e., the good mobilizers responded poorly to an SDF-1 gradient whereas the poor mobilizers responded better. Also a higher expression of CXCR4 was observed (by flow cytometry) in samples from poor mobilizers. Chemotaxis towards SDF-1 was reduced by 10 to 80% by AMD3100 and there was a positive correlation (r=0.6) between chemotactic index and percentage inhibition, i.e., chemotaxis of poor mobilizers was significantly more inhibited by this CXCR4 antagonist. Thus our results suggest that the chemotactic index could be employed as a predictor of good vs. poor mobilization; optimal mobilization especially in poor mobilizers may be achieved by a protocol with both G-CSF and AMD3100; for good mobilizers, including AMD3100 for mobilization could lead to a reduced requirement for volume of leukapheresis product and number of collections; and finally in an allogeneic transplant setting the chemotactic index could be used to predict whether a normal donor of HSPC will be a good or poor mobilizer.
The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem... more The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem/progenitor cells (LSPC) within the bone marrow (BM) niche, as well as in the high relapse rates of acute myeloid leukemia (AML). Down-regulating CD44 could be clinically relevant not only for suppression of the deregulated function of LSPC but also in LSPC response to chemotherapeutic agents. Small interfering RNA (siRNA) delivery is a promising approach for AML treatment, and we recently reported effective siRNA delivery into difficult-to-transfect AML cell lines using lipid-substituted polyethylenimine/siRNA complexes (polymeric nanoparticles). In this study, we investigated polymeric nanoparticle-mediated silencing of CD44 in CD34+ LSPC cell models (leukemic KG-1 and KG-1a cell lines) as well as primary AML cells. Polymeric nanoparticle-mediated silencing decreased surface CD44 levels in KG-1, KG-1a and primary AML cells by up to 27%, 30% and 20% at day 3, respectively. Moreover, CD44 silencing resulted in induction of apoptosis in KG-1 cells, reduced adhesion of KG-1 and KG-1a cells to hyaluronic acid-coated cell culture plates and BM-MSC, and decreased adhesion of primary AML cells to BM-MSC. Our results suggest that polymeric nanoparticle-mediated silencing of CD44 might be a useful technique for inhibiting LSPC interactions with their microenvironment, thereby prohibiting leukemia progression or sensitizing LSPC to chemotherapy.
The cell-surface proteolytic enzymes membrane-type (MT)-matrix metalloproteinases (MMPs) activate... more The cell-surface proteolytic enzymes membrane-type (MT)-matrix metalloproteinases (MMPs) activate secreted latent forms of MMPs and play a key role in cell migration and tumor cell invasion and metastasis. Previously we reported that mobilized peripheral blood (mPB) CD34+ cells as well as AML blasts secrete inactive (pro)MMP-2, in contrast to normal steady-state bone marrow (BM) CD34+ cells which do not. In this study we hypothesized that the egress from BM of normal hematopoietic stem/progenitor cells during G-CSF-induced mobilization or of AML blasts shares a common mechanism that involves activation of secreted proMMP-2 by MT-MMPs. We evaluated the expression of MT-MMPs (MT1-MMP, MT2-, MT3- MT4-, MT5-, MT6-) in normal and leukemic hematopoietic cells, as well as in BM stromal cells, and determined their role in proMMP-2 activation and migration. We found MT1-MMP expression (mRNA and protein) in mPB CD34+ cells and in the majority of AML samples (from 21 out of 26 patients) and upregulation of MT1-MMP by G-CSF in steady-state BM CD34+ cells. Moreover, we confirmed the secretion of proMMP-2 in media conditioned by hematopoietic (mPB CD34+, AML) cells and stromal cells. However, we detected the active form of MMP-2 in co-cultures of the hematopoietic (mPB CD34+, AML) cells with stromal cells, as well as in co-cultures of steady-state BM CD34+ cells stimulated with G-CSF with stroma. In cultures of stromal cells alone G-CSF had no effect on the expression of either MT1-MMP or MMP-2. To evaluate the role of MT-MMPs in proMMP activation, we examined the leukemic KG-1 cell line, which we found not to express any MT-MMPs, and did not detect active MMP-2 in co-cultures with stroma. On the other hand, primary AML samples that did not express MT1-MMP but expressed MT2-, MT4- or MT5-MMPs activated proMMP-2 in co-cultures with stroma. Moreover, the MT1-MMP inhibitor epigallocatechin-3-gallate significantly reduced trans-Matrigel migration of mPB CD34+ (by 50%) and AML cells (by 70–90%). Hence we suggest that in the BM microenvironment (i) MT1-MMP localized on the surface of hematopoietic cells activates MMP-2, inducing a highly proteolytic microenvironment, and (ii) MT1-MMP upregulation by G-CSF in BM CD34+ cells can result in CD34+ cell mobilization from BM. Similarly, the constitutive high expression of MT1-MMP and other MT-MMPs in AML blasts also contributes to MMP-2 activation in the BM microenvironment and may be conducive to the egress of AML blasts from the BM.
Membrane type-matrix metalloproteinases (MT-MMPs) play a key role in tumor cell progression and m... more Membrane type-matrix metalloproteinases (MT-MMPs) play a key role in tumor cell progression and metastasis but their role in hematological malignancy and leukemic cell dissemination is still unclear. To date, six MT-MMPs have been identified, of which four (MT1-, MT2-, MT3- and MT5-MMP) possess a trans-membrane domain that tethers the enzyme to the plasma membrane and two (MT4- and MT6-MMP) are anchored to the cell surface via a glycophosphatidyl inositol domain. MT-MMPs are known to activate pro-forms of MMPs. We examined the expression of all six MT-MMPs in 13 myeloid and lymphoid cell lines using RT-PCR, Western blotting and FACS. We found that MT1-MMP was expressed on most of the cell lines tested, MT2-MMP was expressed in myeloid cell lines such as THP-1, HEL, K562 and U937, and in T cell lines such as Jurkat and CEM, but not in any of the tested B cell lines, MT3-MMP was not expressed in any of the cell lines except for THP-1, MT4-MMP was strongly expressed in all myeloid but not lymphoid cell lines and MT5-MMP and MT6-MMP were expressed in most of the myeloid cell lines. Next, we examined the expression of all six MT-MMPs in primary AML samples and found that MT1-MMP is expressed in 20 out of 24 AML patient samples as detected by RT-PCR and Western blotting, MT2-MMP and MT4-MMP were expressed in 21 and 22 out of 24 samples, respectively, and MT6-MMP was expressed in 17 out of the 17 samples tested. In contrast, MT3- and MT5-MMPs were not found in the primary AML samples. Zymographic analysis showed that the pro form of MMP-2 was secreted in media conditioned by AML blasts and became activated only when these AML blasts were co-cultured with BM stromal cells. Since TNF-α is endogenously secreted by AML blasts we next stimulated these cells with human recombinant TNF-α and found strong upregulation of MT1-MMP and MT6-MMP. Moreover, zymography demonstrated that TNF-α-stimulated AML blasts are more potent in activation of pro-MMP-2 than unstimulated cells, indicating that activation may occur via upregulation of MT-MMPs. Furthermore, we found that TNF-α stimulation led to stronger upregulation/induction of MT1- and MT2-MMPs in CD34+ cells than in CD34- cells isolated from AML patients. In conclusion, we report here for the first time that MT1-, 2-, 4- and 6-MMPs are expressed in AML blasts and suggest that these MT-MMPs may contribute to the invasive phenotype of this malignancy.
Stromal-cell derived factor (SDF)-1α/CXCL12 and its cognate receptor, CXCR4, play a crucial role ... more Stromal-cell derived factor (SDF)-1α/CXCL12 and its cognate receptor, CXCR4, play a crucial role in the trafficking of normal hematopoietic stem/progenitor cells (HSPC) and their homing/retention in bone marrow. Consequently, modulation of CXCR4 expression in HSPC could be applied therapeutically to improve the efficiency of HSPC transplantation. It is known that gene expression can be regulated by chromatin remodelling. Two groups of histone modifying enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC) participate in the regulation of chromatin structure, and hence gene expression. Disruption of normal HAT or HDAC activities has been found in many human cancers. Recently, several structurally diverse and highly specific HDAC inhibitors (HDI) have been reported. They act as strong modulators of growth, differentiation and apoptosis in several types of cancer, particularly acute myeloid leukemia (AML). However, very little is known regarding the effects of HDI on HSPC. We have previously shown that a specific short-chain fatty acid HDI, valproic acid (VPA), enhances CXCR4 expression and function in normal HSPC (Blood2007: 110; 425a). In order to determine whether other structurally diverse classes of HDI are able to influence CXCR4 expression in HSPC through chromatin remodelling, we investigated the effect of potent hydroxamic acid HDI Trichostatin A (TSA) on CXCR4 in normal HSPC. We examined the effect of TSA on CXCR4 expression (by FACS and real-time RT-PCR), modulation of CXCR4 transcription (chromatin immunoprecipitation (X-ChIP) analysis) and on functional response towards an SDF-1α gradient (by chemotaxis assay) of HSPC (CD34+ cells from cord blood (CB) and the models of immature hematopoietic cells expressing CD34 antigen, namely AML cell lines KG-1a and KG-1). Cells were incubated for 24 h in IMDM supplemented with 20% FCS in the presence of TSA (0.1 μM). We found that TSA increases the percentage of CXCR4-expressing CB CD34+, KG-1a, KG-1 cells (2.5-, 8- and 3-fold, respectively). This effect was also confirmed at the mRNA level in CB CD34+, KG-1a and KG-1 cells (by about 2.5-, 5- and 2.5-fold up-regulation, respectively). Moreover, X-ChIP analysis showed a significant increase in association of acetyl-histone H4 binding to the CXCR4 promoter in CB CD34+ and KG-1 cells (2- and 1.7-fold, respectively). TSA was also shown to significantly increase the chemotaxis of KG-1a cells towards SDF-1α (20 ng/mL), which was inhibited by AMD3100, a potent antagonist of CXCR4. We conclude that other HDI such as TSA regulate CXCR4 expression in HSPC by chromatin remodelling and we suggest that priming of HSPC with HDI may improve their homing and engraftment into bone marrow, especially in CB transplantation.
Abstract 4518 A 33 year old male initially diagnosed with myelodysplastic syndrome presented with... more Abstract 4518 A 33 year old male initially diagnosed with myelodysplastic syndrome presented with acute leukemia, his bone marrow demonstrating sheets of abnormal megakaryocytes. Cytogenetics revealed two cell lines, 44X –Y, –C and 46XY, both positive for the Philadelphia chromosome mutation. He underwent busulphan and cyclophosphamide conditioning and allogeneic bone marrow transplant in the absence of a course of induction chemotherapy. One year later he was free of leukemia. Ten years after the transplant he was discharged from hematology follow-up, declared cured. Twenty-two years later, the patient re-presented with significant anemia and circulating blasts. One year previous he had normal blood counts. Bone marrow biopsy revealed acute megakaroblastic leukemia (M7), with cytogenetics demonstrating Philadelphia chromosome positivity and two identified cell lines, one with deletion of the Y chromosome. The patient underwent induction chemotherapy with idarubicin and cytarabine in addition to imatinib. His recovery marrow revealed no residual leukemia, including normal cytogenetics. He remained BCR-ABL fusion gene transcript positive, and was maintained on single agent imatinib. Four hundred and forty one days after induction chemotherapy he received a second sibling matched allogeneic stem cell bone marrow transplant. One year later he remains free of leukemia with no detectable BCR-ABL fusion gene transcript. Acute megakaryoblastic leukemia is a rare entity representing…
MDS is characterized by ineffective hematopoiesis with increased intramedullary apoptosis which h... more MDS is characterized by ineffective hematopoiesis with increased intramedullary apoptosis which has been correlated in turn with high levels of tumor necrosis factor (TNF)- α. Previously, we showed that TNF-α strongly upregulates the secretion of the matrix metalloproteinases (MMP)-2 and -9 in normal CD34+ cells. Elevated expression of MMPs has been suggested to contribute to cancer progression and leukemic dissemination and MMPs also facilitate the secretion of TNF-α. In this work, we hypothesized that TNF-α-induced MMP production plays a role in the progression of MDS. We therefore examined the effects of recombinant human (rh) TNF-α on the expression and secretion of MMP-2, MMP-9 and membrane type (MT) 1-MMP (known activator of MMP-2), by MNC from MDS patients (using RT-PCR, zymography and Western blotting), as well as the effects of TNF-α on migration of MDS MNC across the reconstituted basement membrane Matrigel. We observed higher mean levels of TNF-α in media conditioned by mononuclear cells (MNC) obtained from 20 patients diagnosed with MDS (RAEB-T (4); RAEB (5), RA (10) and RARS (1)) in comparison to normal controls. We found that (i) MMP-9 is secreted by MDS MNC and this secretion is increased by TNF-α, (ii) MT1-MMP is expressed by the majority of MDS MNC and this expression is upregulated by TNF-α, and (iii) MMP-2 is detectable only in RAEB-T MNC samples and is activated by TNF-α. TNF-α also stimulated the migration of MDS MNC across Matrigel, which was inhibited by the inhibitor of MT1-MMP, epigallocatechin-3-gallate. Moreover, Enbrel (soluble TNF receptor fusion protein), which blocks TNF-α, also inhibited the expression of MT1-MMP and secretion of MMP-9 by MDS MNC as well as their migration across Matrigel. In addition, we observed increased activation of the latent form of MMP-2 in co-cultures of MDS MNC with bone marrow fibroblastic cells. We suggest that in MDS patients, TNF-α stimulates the expression of MMP-9 and MT1-MMP, inducing a highly proteolytic environment in bone marrow which could further increase processing and secretion of endogenous TNF-α. Therefore, we suggest that use of TNF-α inhibitors combined with MMP inhibitors could have therapeutic value in abrogating the progression of MDS.
Abstract 2338 Poster Board II-315 The addition of rituximab to fludarabine/cyclophosphamide chemo... more Abstract 2338 Poster Board II-315 The addition of rituximab to fludarabine/cyclophosphamide chemotherapy for the treatment of both previously untreated and relapsed/refractory patients with CLL has yielded a substantial increase in PFS as demonstrated in phase III trials (Hallek et al. ASH 2008, Robak et al. ASH 2008. Polymorphisms of the FC-g-R IIIa gene (SNP 158) results in a higher (VV genotype) or lower (FV and FF genotype) affinity for IgG1 monoclonal antibodies and subsequently modulate ADCC activity. In patients with follicular NHL treated with rituximab as monotherapy it has been shown that patients displaying the higher affinity FCGR3A variant had higher response rates compared to patients displaying the lower affinity variant (Cartron et al. Blood 2002; Weng et al. JCO 2003). Furthermore, the high affinity polymorphism of the FCGR2A gene (SNP 131) (HH genotype), has also demonstrated higher response rates compared to the lower affinity variant (RH and RR genotype) in this context (Weng et al. JCO 2003). The objective of this study was to evaluate the prognostic significance of FCGR polymorphisms in patients treated with FC vs R-FC within the REACH trial. REACH was an open-label, multicenter, randomized, phase III study to evaluate the efficacy and tolerability of R-FC versus FC in relapsed or refractory patients with CD20 positive CLL (N=552). The primary endpoint of the study was progression free survival. Results from allele specific PCR for FCGR2A (SNP 131, rs1801274) and FCGR3A (SNP 158, rs396991) genes were available from n= 419 of 546 unselected patients (FC: n=209 and R-FC: n=210). In the FC arm n=29 (14%) displayed the FCGR3A VV genotype, n=96 (46%) FV genotype, and n=84 (40%) FF genotype. ). In the R-FC arm n=20 (10%) displayed the FCGR3A VV genotype, n=106 (50%) FV genotype, and n=84 (40%) FF genotype. The incidences of FCGR2A for HH, RH, RR genotypes were 27%, 49%, 24%, respectively in the FC arm and 26%, 55%, 19%, respectively in the R-FC arm. Overall, the study demonstrated prolonged PFS for R-FC vs FC treatment (HR=0.65 (0.51,0.82); p=0.00022). With respect to PFS, FCGR3A and FCGR2A polymorphisms did not demonstrate prognostic significance in the FC arm (p=0.42 and p=0.64, respectively) or R-FC arm (p=0.41 and p=0.88, respectively). Subgroup analysis revealed that those patients with lower affinity genotypes (FCGR3A: FV/FF; FCGR2A: RH/RR) benefited significantly from the addition of rituximab (HR=0.68 (0.51,0.9); p=0.0063 and HR=0.68 (0.51,0.93); p=0.014, respectively). Similar levels of benefits were suggested for those patients with higher affinity genotype (HR=0.86 (0.4,1.84); p=0.7 and HR=0.7 (0.41,1.18); p=0.18, respectively) but not statistically significant, potentially due to the lower number of patients in the high affinity groups. Multivariate analysis including FCGR genotypes, treatment arm (FC vs R-FC), Binet stage (c vs a/b), age, del(17p), IgVH mutational status revealed age (HR=1.02 (1.01,1.04); p=0.0042), binet stage HR=1.81 (1.37,2.39); p=0.000027), del(17p) (HR=2.49 (1.66,3.74); p=0.000011), treatment arm (HR=0.71 (0.54,0.92); p=0.011), IgVH (HR=2.09 (1.55-2.81); p=0.0000014), as independent prognostic factors for PFS. In summary these data demonstrate that FCGR2A and FCGR3A polymorphisms do not significantly influence the outcome of relapsed or refractory CLL patients treated with FC with or without rituximab. Disclosures: Dornan: Genentech, Inc.: Employment, Equity Ownership. Off Label Use: Rituximab together with FC chemotherapy in relapsed/refractory CLL. Spleiss:Roche: Employment. Yeh:Genentech, Inc.: Employment, Equity Ownership. Duchateau-Nguyen:Roche: Employment. Robak:Celgene: Consultancy; Roche: Honoraria, Research Funding; Genmab: Research Funding; Cambridge Antibody Technology: Research Funding; GlaxoSmithKline: Honoraria. Solal-Celigny:Roche: Honoraria, Research Funding. Warzocha:BMS: Consultancy, Honoraria; Celgene: Consultancy; Roche: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Loscertales:Roche: Consultancy. Catalano:Roche: Honoraria, Research Funding, Travel grants. Larratt:Roche: Honoraria; Novartis: Honoraria. Bence-Bruckler:Roche: Consultancy. Geisler:Bayer Schering: Consultancy, Research Funding, Speakers Bureau; Santaris Pharma: Consultancy; Celgene: Consultancy; Fresenius: Consultancy. Montillo:Bayer Schering: Honoraria, Research Funding, Speakers Bureau. Wenger:Roche: Employment. Weisser:Roche: Employment.
BackgroundT cell exhaustion compromises antitumor immunity, and a sustained elevation of co-inhib... more BackgroundT cell exhaustion compromises antitumor immunity, and a sustained elevation of co-inhibitory receptors is a hallmark of T cell exhaustion in solid tumors. Similarly, upregulation of co-inhibitory receptors has been reported in T cells in hematological cancers such as chronic lymphocytic leukemia (CLL). However, the role of CD160, a glycosylphosphatidylinositol-anchored protein, as one of these co-inhibitory receptors has been contradictory in T cell function. Therefore, we decided to elucidate how CD160 expression and/or co-expression with other co-inhibitory receptors influence T cell effector functions in patients with CLL.MethodsWe studied 56 patients with CLL and 25 age-matched and sex-matched healthy controls in this study. The expression of different co-inhibitory receptors was analyzed in T cells obtained from the peripheral blood or the bone marrow. Also, we quantified the properties of extracellular vesicles (EVs) in the plasma of patients with CLL versus healthy ...
Matrix metalloproteinase (MMP)-14 expression correlates with progression and metastasis of multip... more Matrix metalloproteinase (MMP)-14 expression correlates with progression and metastasis of multiple tumor cell types and is a major mediator of cell migration and invasion. MMP-14 possesses a trans-membrane domain that tethers the enzyme to the plasma membrane and not only activates proMMP-2 but also degrades extracellular matrix (ECM) by pericellular proteolysis and cleaves several non-ECM molecules, including adhesion molecules and chemokines. To assess the role of MMP-14 in leukemic dissemination we evaluated its expression in myeloid cell lines and primary acute myelogenous leukemic (AML) samples. Using RT-PCR, flow cytometry and Western blotting we found that MMP-14 is highly expressed in leukemic myeloid cell lines THP-1, U937, HEL and K562; and primary samples from 37 out of 40 patients (pts) diagnosed with AML (WHO classification, AML with recurrent cytogenetic translocations: 9 pts; AML with multilineage dysplasia: 4 pts; AML not otherwise categorized: 27 pts). Moreover, pr...
MDS is characterized by ineffective hematopoiesis with increased intramedullary apoptosis which h... more MDS is characterized by ineffective hematopoiesis with increased intramedullary apoptosis which has been correlated in turn with high levels of tumor necrosis factor (TNF)- α. Previously, we showed that TNF-α strongly upregulates the secretion of the matrix metalloproteinases (MMP)-2 and -9 in normal CD34+ cells. Elevated expression of MMPs has been suggested to contribute to cancer progression and leukemic dissemination and MMPs also facilitate the secretion of TNF-α. In this work, we hypothesized that TNF-α-induced MMP production plays a role in the progression of MDS. We therefore examined the effects of recombinant human (rh) TNF-α on the expression and secretion of MMP-2, MMP-9 and membrane type (MT) 1-MMP (known activator of MMP-2), by MNC from MDS patients (using RT-PCR, zymography and Western blotting), as well as the effects of TNF-α on migration of MDS MNC across the reconstituted basement membrane Matrigel. We observed higher mean levels of TNF-α in media conditioned by m...
While autologous hematopoietic stem/progenitor cell (HSPC) transplantation has been used successf... more While autologous hematopoietic stem/progenitor cell (HSPC) transplantation has been used successfully for many years, new approaches are being explored to boost the number of HSPC (CD34+ cells) collected by leukapheresis to permit more rapid hematopoietic recovery. Still, a considerable number of patients may be excluded from this procedure because a sufficient quantity of HSPC cannot be obtained by standard mobilization agents. Among factors regulating mobilization is stromal cell-derived factor (SDF)-1 constitutively produced by bone marrow (BM) stromal cells and which is known to retain HSPC in the BM. Responsiveness to an SDF-1 gradient can be measured in vitro using the chemotactic index, defined as the ratio of the number of cells that migrate towards SDF-1 and the number of cells that migrate towards media alone. We hypothesized that (i) a high chemotactic index would indicate a greater ability of the HSPC to be retained in their BM niches and therefore more difficulty in coa...
While autologous hematopoietic stem/progenitor cell (HSPC) transplantation has been used successf... more While autologous hematopoietic stem/progenitor cell (HSPC) transplantation has been used successfully for many years, new approaches are being explored to boost the number of HSPC (CD34+ cells) collected by leukapheresis to permit more rapid hematopoietic recovery. Still, a considerable number of patients may be excluded from this procedure because a sufficient quantity of HSPC cannot be obtained by standard mobilization agents. Among factors regulating mobilization is stromal cell-derived factor (SDF)-1 constitutively produced by bone marrow (BM) stromal cells and which is known to retain HSPC in the BM. Responsiveness to an SDF-1 gradient can be measured in vitro using the chemotactic index, defined as the ratio of the number of cells that migrate towards SDF-1 and the number of cells that migrate towards media alone. We hypothesized that (i) a high chemotactic index would indicate a greater ability of the HSPC to be retained in their BM niches and therefore more difficulty in coaxing them out into the circulation, as could be the case in poor mobilizers; and that (ii) AMD3100, an antagonist of the SDF-1 receptor CXCR4, will have a greater disruptive effect on SDF-1-dependent signalling in poor mobilizers. Leukapheresis products were obtained from patients diagnosed with Hodgkin’s or non-Hodgkin’s lymphoma and CD34+ cells isolated by positive selection were loaded onto Boyden chambers and allowed to migrate across bare filters towards a gradient of SDF-1 (200 ng/mL). In some experiments cells were incubated with AMD3100 (10 mg/mL) for the duration of the assay. We observed broad inter-patient differences in in vitro migratory ability of CD34+ cells, ranging from 3.1 ± 0.6 to 21.7 ± 2.1 % for spontaneous or passive percentage migration; and from 11.1 ± 0.7 to 58.6 ± 10.7 % for SDF-1-directed chemotaxis, neither of which bore any correlation with the number of CD34+ cells/kg obtained from the leukapheresis product. However, a negative correlation (r=−0.7) was found between the chemotactic index and the percentage of CD34+ cells obtained from the leukapheresis product, i.e., the good mobilizers responded poorly to an SDF-1 gradient whereas the poor mobilizers responded better. Also a higher expression of CXCR4 was observed (by flow cytometry) in samples from poor mobilizers. Chemotaxis towards SDF-1 was reduced by 10 to 80% by AMD3100 and there was a positive correlation (r=0.6) between chemotactic index and percentage inhibition, i.e., chemotaxis of poor mobilizers was significantly more inhibited by this CXCR4 antagonist. Thus our results suggest that the chemotactic index could be employed as a predictor of good vs. poor mobilization; optimal mobilization especially in poor mobilizers may be achieved by a protocol with both G-CSF and AMD3100; for good mobilizers, including AMD3100 for mobilization could lead to a reduced requirement for volume of leukapheresis product and number of collections; and finally in an allogeneic transplant setting the chemotactic index could be used to predict whether a normal donor of HSPC will be a good or poor mobilizer.
The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem... more The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem/progenitor cells (LSPC) within the bone marrow (BM) niche, as well as in the high relapse rates of acute myeloid leukemia (AML). Down-regulating CD44 could be clinically relevant not only for suppression of the deregulated function of LSPC but also in LSPC response to chemotherapeutic agents. Small interfering RNA (siRNA) delivery is a promising approach for AML treatment, and we recently reported effective siRNA delivery into difficult-to-transfect AML cell lines using lipid-substituted polyethylenimine/siRNA complexes (polymeric nanoparticles). In this study, we investigated polymeric nanoparticle-mediated silencing of CD44 in CD34+ LSPC cell models (leukemic KG-1 and KG-1a cell lines) as well as primary AML cells. Polymeric nanoparticle-mediated silencing decreased surface CD44 levels in KG-1, KG-1a and primary AML cells by up to 27%, 30% and 20% at day 3, respectively. Moreover, CD44 silencing resulted in induction of apoptosis in KG-1 cells, reduced adhesion of KG-1 and KG-1a cells to hyaluronic acid-coated cell culture plates and BM-MSC, and decreased adhesion of primary AML cells to BM-MSC. Our results suggest that polymeric nanoparticle-mediated silencing of CD44 might be a useful technique for inhibiting LSPC interactions with their microenvironment, thereby prohibiting leukemia progression or sensitizing LSPC to chemotherapy.
The cell-surface proteolytic enzymes membrane-type (MT)-matrix metalloproteinases (MMPs) activate... more The cell-surface proteolytic enzymes membrane-type (MT)-matrix metalloproteinases (MMPs) activate secreted latent forms of MMPs and play a key role in cell migration and tumor cell invasion and metastasis. Previously we reported that mobilized peripheral blood (mPB) CD34+ cells as well as AML blasts secrete inactive (pro)MMP-2, in contrast to normal steady-state bone marrow (BM) CD34+ cells which do not. In this study we hypothesized that the egress from BM of normal hematopoietic stem/progenitor cells during G-CSF-induced mobilization or of AML blasts shares a common mechanism that involves activation of secreted proMMP-2 by MT-MMPs. We evaluated the expression of MT-MMPs (MT1-MMP, MT2-, MT3- MT4-, MT5-, MT6-) in normal and leukemic hematopoietic cells, as well as in BM stromal cells, and determined their role in proMMP-2 activation and migration. We found MT1-MMP expression (mRNA and protein) in mPB CD34+ cells and in the majority of AML samples (from 21 out of 26 patients) and upregulation of MT1-MMP by G-CSF in steady-state BM CD34+ cells. Moreover, we confirmed the secretion of proMMP-2 in media conditioned by hematopoietic (mPB CD34+, AML) cells and stromal cells. However, we detected the active form of MMP-2 in co-cultures of the hematopoietic (mPB CD34+, AML) cells with stromal cells, as well as in co-cultures of steady-state BM CD34+ cells stimulated with G-CSF with stroma. In cultures of stromal cells alone G-CSF had no effect on the expression of either MT1-MMP or MMP-2. To evaluate the role of MT-MMPs in proMMP activation, we examined the leukemic KG-1 cell line, which we found not to express any MT-MMPs, and did not detect active MMP-2 in co-cultures with stroma. On the other hand, primary AML samples that did not express MT1-MMP but expressed MT2-, MT4- or MT5-MMPs activated proMMP-2 in co-cultures with stroma. Moreover, the MT1-MMP inhibitor epigallocatechin-3-gallate significantly reduced trans-Matrigel migration of mPB CD34+ (by 50%) and AML cells (by 70–90%). Hence we suggest that in the BM microenvironment (i) MT1-MMP localized on the surface of hematopoietic cells activates MMP-2, inducing a highly proteolytic microenvironment, and (ii) MT1-MMP upregulation by G-CSF in BM CD34+ cells can result in CD34+ cell mobilization from BM. Similarly, the constitutive high expression of MT1-MMP and other MT-MMPs in AML blasts also contributes to MMP-2 activation in the BM microenvironment and may be conducive to the egress of AML blasts from the BM.
Membrane type-matrix metalloproteinases (MT-MMPs) play a key role in tumor cell progression and m... more Membrane type-matrix metalloproteinases (MT-MMPs) play a key role in tumor cell progression and metastasis but their role in hematological malignancy and leukemic cell dissemination is still unclear. To date, six MT-MMPs have been identified, of which four (MT1-, MT2-, MT3- and MT5-MMP) possess a trans-membrane domain that tethers the enzyme to the plasma membrane and two (MT4- and MT6-MMP) are anchored to the cell surface via a glycophosphatidyl inositol domain. MT-MMPs are known to activate pro-forms of MMPs. We examined the expression of all six MT-MMPs in 13 myeloid and lymphoid cell lines using RT-PCR, Western blotting and FACS. We found that MT1-MMP was expressed on most of the cell lines tested, MT2-MMP was expressed in myeloid cell lines such as THP-1, HEL, K562 and U937, and in T cell lines such as Jurkat and CEM, but not in any of the tested B cell lines, MT3-MMP was not expressed in any of the cell lines except for THP-1, MT4-MMP was strongly expressed in all myeloid but not lymphoid cell lines and MT5-MMP and MT6-MMP were expressed in most of the myeloid cell lines. Next, we examined the expression of all six MT-MMPs in primary AML samples and found that MT1-MMP is expressed in 20 out of 24 AML patient samples as detected by RT-PCR and Western blotting, MT2-MMP and MT4-MMP were expressed in 21 and 22 out of 24 samples, respectively, and MT6-MMP was expressed in 17 out of the 17 samples tested. In contrast, MT3- and MT5-MMPs were not found in the primary AML samples. Zymographic analysis showed that the pro form of MMP-2 was secreted in media conditioned by AML blasts and became activated only when these AML blasts were co-cultured with BM stromal cells. Since TNF-α is endogenously secreted by AML blasts we next stimulated these cells with human recombinant TNF-α and found strong upregulation of MT1-MMP and MT6-MMP. Moreover, zymography demonstrated that TNF-α-stimulated AML blasts are more potent in activation of pro-MMP-2 than unstimulated cells, indicating that activation may occur via upregulation of MT-MMPs. Furthermore, we found that TNF-α stimulation led to stronger upregulation/induction of MT1- and MT2-MMPs in CD34+ cells than in CD34- cells isolated from AML patients. In conclusion, we report here for the first time that MT1-, 2-, 4- and 6-MMPs are expressed in AML blasts and suggest that these MT-MMPs may contribute to the invasive phenotype of this malignancy.
Stromal-cell derived factor (SDF)-1α/CXCL12 and its cognate receptor, CXCR4, play a crucial role ... more Stromal-cell derived factor (SDF)-1α/CXCL12 and its cognate receptor, CXCR4, play a crucial role in the trafficking of normal hematopoietic stem/progenitor cells (HSPC) and their homing/retention in bone marrow. Consequently, modulation of CXCR4 expression in HSPC could be applied therapeutically to improve the efficiency of HSPC transplantation. It is known that gene expression can be regulated by chromatin remodelling. Two groups of histone modifying enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC) participate in the regulation of chromatin structure, and hence gene expression. Disruption of normal HAT or HDAC activities has been found in many human cancers. Recently, several structurally diverse and highly specific HDAC inhibitors (HDI) have been reported. They act as strong modulators of growth, differentiation and apoptosis in several types of cancer, particularly acute myeloid leukemia (AML). However, very little is known regarding the effects of HDI on HSPC. We have previously shown that a specific short-chain fatty acid HDI, valproic acid (VPA), enhances CXCR4 expression and function in normal HSPC (Blood2007: 110; 425a). In order to determine whether other structurally diverse classes of HDI are able to influence CXCR4 expression in HSPC through chromatin remodelling, we investigated the effect of potent hydroxamic acid HDI Trichostatin A (TSA) on CXCR4 in normal HSPC. We examined the effect of TSA on CXCR4 expression (by FACS and real-time RT-PCR), modulation of CXCR4 transcription (chromatin immunoprecipitation (X-ChIP) analysis) and on functional response towards an SDF-1α gradient (by chemotaxis assay) of HSPC (CD34+ cells from cord blood (CB) and the models of immature hematopoietic cells expressing CD34 antigen, namely AML cell lines KG-1a and KG-1). Cells were incubated for 24 h in IMDM supplemented with 20% FCS in the presence of TSA (0.1 μM). We found that TSA increases the percentage of CXCR4-expressing CB CD34+, KG-1a, KG-1 cells (2.5-, 8- and 3-fold, respectively). This effect was also confirmed at the mRNA level in CB CD34+, KG-1a and KG-1 cells (by about 2.5-, 5- and 2.5-fold up-regulation, respectively). Moreover, X-ChIP analysis showed a significant increase in association of acetyl-histone H4 binding to the CXCR4 promoter in CB CD34+ and KG-1 cells (2- and 1.7-fold, respectively). TSA was also shown to significantly increase the chemotaxis of KG-1a cells towards SDF-1α (20 ng/mL), which was inhibited by AMD3100, a potent antagonist of CXCR4. We conclude that other HDI such as TSA regulate CXCR4 expression in HSPC by chromatin remodelling and we suggest that priming of HSPC with HDI may improve their homing and engraftment into bone marrow, especially in CB transplantation.
Abstract 4518 A 33 year old male initially diagnosed with myelodysplastic syndrome presented with... more Abstract 4518 A 33 year old male initially diagnosed with myelodysplastic syndrome presented with acute leukemia, his bone marrow demonstrating sheets of abnormal megakaryocytes. Cytogenetics revealed two cell lines, 44X –Y, –C and 46XY, both positive for the Philadelphia chromosome mutation. He underwent busulphan and cyclophosphamide conditioning and allogeneic bone marrow transplant in the absence of a course of induction chemotherapy. One year later he was free of leukemia. Ten years after the transplant he was discharged from hematology follow-up, declared cured. Twenty-two years later, the patient re-presented with significant anemia and circulating blasts. One year previous he had normal blood counts. Bone marrow biopsy revealed acute megakaroblastic leukemia (M7), with cytogenetics demonstrating Philadelphia chromosome positivity and two identified cell lines, one with deletion of the Y chromosome. The patient underwent induction chemotherapy with idarubicin and cytarabine in addition to imatinib. His recovery marrow revealed no residual leukemia, including normal cytogenetics. He remained BCR-ABL fusion gene transcript positive, and was maintained on single agent imatinib. Four hundred and forty one days after induction chemotherapy he received a second sibling matched allogeneic stem cell bone marrow transplant. One year later he remains free of leukemia with no detectable BCR-ABL fusion gene transcript. Acute megakaryoblastic leukemia is a rare entity representing…
MDS is characterized by ineffective hematopoiesis with increased intramedullary apoptosis which h... more MDS is characterized by ineffective hematopoiesis with increased intramedullary apoptosis which has been correlated in turn with high levels of tumor necrosis factor (TNF)- α. Previously, we showed that TNF-α strongly upregulates the secretion of the matrix metalloproteinases (MMP)-2 and -9 in normal CD34+ cells. Elevated expression of MMPs has been suggested to contribute to cancer progression and leukemic dissemination and MMPs also facilitate the secretion of TNF-α. In this work, we hypothesized that TNF-α-induced MMP production plays a role in the progression of MDS. We therefore examined the effects of recombinant human (rh) TNF-α on the expression and secretion of MMP-2, MMP-9 and membrane type (MT) 1-MMP (known activator of MMP-2), by MNC from MDS patients (using RT-PCR, zymography and Western blotting), as well as the effects of TNF-α on migration of MDS MNC across the reconstituted basement membrane Matrigel. We observed higher mean levels of TNF-α in media conditioned by mononuclear cells (MNC) obtained from 20 patients diagnosed with MDS (RAEB-T (4); RAEB (5), RA (10) and RARS (1)) in comparison to normal controls. We found that (i) MMP-9 is secreted by MDS MNC and this secretion is increased by TNF-α, (ii) MT1-MMP is expressed by the majority of MDS MNC and this expression is upregulated by TNF-α, and (iii) MMP-2 is detectable only in RAEB-T MNC samples and is activated by TNF-α. TNF-α also stimulated the migration of MDS MNC across Matrigel, which was inhibited by the inhibitor of MT1-MMP, epigallocatechin-3-gallate. Moreover, Enbrel (soluble TNF receptor fusion protein), which blocks TNF-α, also inhibited the expression of MT1-MMP and secretion of MMP-9 by MDS MNC as well as their migration across Matrigel. In addition, we observed increased activation of the latent form of MMP-2 in co-cultures of MDS MNC with bone marrow fibroblastic cells. We suggest that in MDS patients, TNF-α stimulates the expression of MMP-9 and MT1-MMP, inducing a highly proteolytic environment in bone marrow which could further increase processing and secretion of endogenous TNF-α. Therefore, we suggest that use of TNF-α inhibitors combined with MMP inhibitors could have therapeutic value in abrogating the progression of MDS.
Abstract 2338 Poster Board II-315 The addition of rituximab to fludarabine/cyclophosphamide chemo... more Abstract 2338 Poster Board II-315 The addition of rituximab to fludarabine/cyclophosphamide chemotherapy for the treatment of both previously untreated and relapsed/refractory patients with CLL has yielded a substantial increase in PFS as demonstrated in phase III trials (Hallek et al. ASH 2008, Robak et al. ASH 2008. Polymorphisms of the FC-g-R IIIa gene (SNP 158) results in a higher (VV genotype) or lower (FV and FF genotype) affinity for IgG1 monoclonal antibodies and subsequently modulate ADCC activity. In patients with follicular NHL treated with rituximab as monotherapy it has been shown that patients displaying the higher affinity FCGR3A variant had higher response rates compared to patients displaying the lower affinity variant (Cartron et al. Blood 2002; Weng et al. JCO 2003). Furthermore, the high affinity polymorphism of the FCGR2A gene (SNP 131) (HH genotype), has also demonstrated higher response rates compared to the lower affinity variant (RH and RR genotype) in this context (Weng et al. JCO 2003). The objective of this study was to evaluate the prognostic significance of FCGR polymorphisms in patients treated with FC vs R-FC within the REACH trial. REACH was an open-label, multicenter, randomized, phase III study to evaluate the efficacy and tolerability of R-FC versus FC in relapsed or refractory patients with CD20 positive CLL (N=552). The primary endpoint of the study was progression free survival. Results from allele specific PCR for FCGR2A (SNP 131, rs1801274) and FCGR3A (SNP 158, rs396991) genes were available from n= 419 of 546 unselected patients (FC: n=209 and R-FC: n=210). In the FC arm n=29 (14%) displayed the FCGR3A VV genotype, n=96 (46%) FV genotype, and n=84 (40%) FF genotype. ). In the R-FC arm n=20 (10%) displayed the FCGR3A VV genotype, n=106 (50%) FV genotype, and n=84 (40%) FF genotype. The incidences of FCGR2A for HH, RH, RR genotypes were 27%, 49%, 24%, respectively in the FC arm and 26%, 55%, 19%, respectively in the R-FC arm. Overall, the study demonstrated prolonged PFS for R-FC vs FC treatment (HR=0.65 (0.51,0.82); p=0.00022). With respect to PFS, FCGR3A and FCGR2A polymorphisms did not demonstrate prognostic significance in the FC arm (p=0.42 and p=0.64, respectively) or R-FC arm (p=0.41 and p=0.88, respectively). Subgroup analysis revealed that those patients with lower affinity genotypes (FCGR3A: FV/FF; FCGR2A: RH/RR) benefited significantly from the addition of rituximab (HR=0.68 (0.51,0.9); p=0.0063 and HR=0.68 (0.51,0.93); p=0.014, respectively). Similar levels of benefits were suggested for those patients with higher affinity genotype (HR=0.86 (0.4,1.84); p=0.7 and HR=0.7 (0.41,1.18); p=0.18, respectively) but not statistically significant, potentially due to the lower number of patients in the high affinity groups. Multivariate analysis including FCGR genotypes, treatment arm (FC vs R-FC), Binet stage (c vs a/b), age, del(17p), IgVH mutational status revealed age (HR=1.02 (1.01,1.04); p=0.0042), binet stage HR=1.81 (1.37,2.39); p=0.000027), del(17p) (HR=2.49 (1.66,3.74); p=0.000011), treatment arm (HR=0.71 (0.54,0.92); p=0.011), IgVH (HR=2.09 (1.55-2.81); p=0.0000014), as independent prognostic factors for PFS. In summary these data demonstrate that FCGR2A and FCGR3A polymorphisms do not significantly influence the outcome of relapsed or refractory CLL patients treated with FC with or without rituximab. Disclosures: Dornan: Genentech, Inc.: Employment, Equity Ownership. Off Label Use: Rituximab together with FC chemotherapy in relapsed/refractory CLL. Spleiss:Roche: Employment. Yeh:Genentech, Inc.: Employment, Equity Ownership. Duchateau-Nguyen:Roche: Employment. Robak:Celgene: Consultancy; Roche: Honoraria, Research Funding; Genmab: Research Funding; Cambridge Antibody Technology: Research Funding; GlaxoSmithKline: Honoraria. Solal-Celigny:Roche: Honoraria, Research Funding. Warzocha:BMS: Consultancy, Honoraria; Celgene: Consultancy; Roche: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Loscertales:Roche: Consultancy. Catalano:Roche: Honoraria, Research Funding, Travel grants. Larratt:Roche: Honoraria; Novartis: Honoraria. Bence-Bruckler:Roche: Consultancy. Geisler:Bayer Schering: Consultancy, Research Funding, Speakers Bureau; Santaris Pharma: Consultancy; Celgene: Consultancy; Fresenius: Consultancy. Montillo:Bayer Schering: Honoraria, Research Funding, Speakers Bureau. Wenger:Roche: Employment. Weisser:Roche: Employment.
BackgroundT cell exhaustion compromises antitumor immunity, and a sustained elevation of co-inhib... more BackgroundT cell exhaustion compromises antitumor immunity, and a sustained elevation of co-inhibitory receptors is a hallmark of T cell exhaustion in solid tumors. Similarly, upregulation of co-inhibitory receptors has been reported in T cells in hematological cancers such as chronic lymphocytic leukemia (CLL). However, the role of CD160, a glycosylphosphatidylinositol-anchored protein, as one of these co-inhibitory receptors has been contradictory in T cell function. Therefore, we decided to elucidate how CD160 expression and/or co-expression with other co-inhibitory receptors influence T cell effector functions in patients with CLL.MethodsWe studied 56 patients with CLL and 25 age-matched and sex-matched healthy controls in this study. The expression of different co-inhibitory receptors was analyzed in T cells obtained from the peripheral blood or the bone marrow. Also, we quantified the properties of extracellular vesicles (EVs) in the plasma of patients with CLL versus healthy ...
Matrix metalloproteinase (MMP)-14 expression correlates with progression and metastasis of multip... more Matrix metalloproteinase (MMP)-14 expression correlates with progression and metastasis of multiple tumor cell types and is a major mediator of cell migration and invasion. MMP-14 possesses a trans-membrane domain that tethers the enzyme to the plasma membrane and not only activates proMMP-2 but also degrades extracellular matrix (ECM) by pericellular proteolysis and cleaves several non-ECM molecules, including adhesion molecules and chemokines. To assess the role of MMP-14 in leukemic dissemination we evaluated its expression in myeloid cell lines and primary acute myelogenous leukemic (AML) samples. Using RT-PCR, flow cytometry and Western blotting we found that MMP-14 is highly expressed in leukemic myeloid cell lines THP-1, U937, HEL and K562; and primary samples from 37 out of 40 patients (pts) diagnosed with AML (WHO classification, AML with recurrent cytogenetic translocations: 9 pts; AML with multilineage dysplasia: 4 pts; AML not otherwise categorized: 27 pts). Moreover, pr...
MDS is characterized by ineffective hematopoiesis with increased intramedullary apoptosis which h... more MDS is characterized by ineffective hematopoiesis with increased intramedullary apoptosis which has been correlated in turn with high levels of tumor necrosis factor (TNF)- α. Previously, we showed that TNF-α strongly upregulates the secretion of the matrix metalloproteinases (MMP)-2 and -9 in normal CD34+ cells. Elevated expression of MMPs has been suggested to contribute to cancer progression and leukemic dissemination and MMPs also facilitate the secretion of TNF-α. In this work, we hypothesized that TNF-α-induced MMP production plays a role in the progression of MDS. We therefore examined the effects of recombinant human (rh) TNF-α on the expression and secretion of MMP-2, MMP-9 and membrane type (MT) 1-MMP (known activator of MMP-2), by MNC from MDS patients (using RT-PCR, zymography and Western blotting), as well as the effects of TNF-α on migration of MDS MNC across the reconstituted basement membrane Matrigel. We observed higher mean levels of TNF-α in media conditioned by m...
While autologous hematopoietic stem/progenitor cell (HSPC) transplantation has been used successf... more While autologous hematopoietic stem/progenitor cell (HSPC) transplantation has been used successfully for many years, new approaches are being explored to boost the number of HSPC (CD34+ cells) collected by leukapheresis to permit more rapid hematopoietic recovery. Still, a considerable number of patients may be excluded from this procedure because a sufficient quantity of HSPC cannot be obtained by standard mobilization agents. Among factors regulating mobilization is stromal cell-derived factor (SDF)-1 constitutively produced by bone marrow (BM) stromal cells and which is known to retain HSPC in the BM. Responsiveness to an SDF-1 gradient can be measured in vitro using the chemotactic index, defined as the ratio of the number of cells that migrate towards SDF-1 and the number of cells that migrate towards media alone. We hypothesized that (i) a high chemotactic index would indicate a greater ability of the HSPC to be retained in their BM niches and therefore more difficulty in coa...
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