Human Bone Marrow Stroma Research Articles

Chaperon Protein GRP78 Released from MPN Cells Increase Expression of Lysyl Oxidase in Human Stroma Cell Line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found an increased expression of the chaperone protein glucose-regulated protein (GRP)78, which is a central regulator of ER stress in megakaryocytes from primary myelofibrosis (PMF) (n=7) or post-essential thrombocythemia myelofibrosis(POST-ET-MF) patients (n=2) regardless of the type of driver mutation. In accord with these notions, we found that GRP78 was overexpressed in JAK2V617F-harboring cell lines (HEL and SET-2) and in UT-7/TPO cells overexpressing either type1 or type2 mutant form of calreticulin. To address the mechanism responsible for the induction of GRP78, we treated the HEL and SET-2 cells with 4-phenyl butyric acid and tauroursodeoxycholic acid both of which are well known inhibitors of ER stress, however, these inhibitors did not affect the expression levels of GRP78. These results indicated that overexpression of GRP78 shown in these cells were not a result of activation of ER stress. In contrast, ruxolitinib, an inhibitor of JAK2V617F, blocked GRP78 expression in these cells. We also found ruxolitinib blocked nuclear accumulation of the transcription factor 4 (ATF4), one of the main transcription factors which regulate GRP78. Furthermore, knockdown of ATF4 using siRNA also resulted in suppression of GRP78 levels in HEL and SET-2 cells. These results suggested that JAK2V617F induce GRP78 through activation of ATF4. We also confirmed that megakaryocytes from MF patients had statistically higher nuclear to cytoplasmic intensity of ATF4 compared to megakaryocytes found in control subjects. For the next step, we tried to find out the pathological action of GRP78 in MPN cells. Although the GRP78 usually works as chaperon protein in cytosol, some cancer cells secret GRP78 into tumor microenvironment and activates signal transduction molecules in surrounding cells. Therefore, we hypothesized that overexpressed GRP78 proteins in MPN cells may secret and modulate phenotype of bone marrow stroma cells. Initially, we confirmed the presence of GRP78 proteins in culture media from HEL and SET-2 cells. Next, we investigated whether GRP78 secreted by HEL and SET-2 cells affect phenotype of bone marrow stromal cells. For this purpose, we used human bone marrow stroma cell line, HS-5. HS-5 cells were co-cultured with HEL or SET-2 cells with trans-well system, and changes of several key molecules involved in development of fibrosis were analyzed. We found that co-culture with HEL or SET-2 cells clearly indued expression of lysyl oxidase (LOX) , which mediate cross-linking of collagen fiber and induce tissue fibrosis , in HS-5 cells. Anti-GRP78 neutralizing antibody abrogated LOX elevation, in contrast, recombinant form of GRP78 proteins induced LOX proteins in HS-5. According to the above results, we examined LOX expression in bone marrow specimens from MF patients. The areas of LOX expression cells were statistically higher in MF patients compared to control group. In addition, high power field observations detected not only in round cells but also in spindle like cells expressed LOX in MF patients, suggesting that bone marrow stromal cells in MF patients express LOX. In conclusion, we demonstrated that GRP78 was overexpressed both in MPN derived cell lines and also in primary megakaryocytes from MF patients. Our observations proposed that overexpressed GRP78 in MPN cells contribute development of myelofibrosis through secreted in microenvironment and induced expression of extracellular matrix modulating enzyme LOX in bone marrow stromal cells.

Nanostructured graphene oxide enriched with metallic nanoparticles as a biointerface to enhance cell adhesion through mechanosensory modifications.

Nanostructuring is a process involving surface manipulation at the nanometric level, which improves the mechanical and biological properties of biomaterials. Specifically, it affects the mechanotransductive perception of the microenvironment of cells. Mechanical force conversion into an electrical or chemical signal contributes to the induction of a specific cellular response. The relationship between the cells and growth surface induces a biointerface-modifying cytophysiology and consequently a therapeutic effect. In this study, we present the fabrication of graphene oxide (GO)-based nanofilms decorated with metallic nanoparticles (NPs) as potential coatings for biomaterials. Our investigation showed the effect of decorating GO with metallic NPs for the modification of the physicochemical properties of nanostructures in the form of nanoflakes and nanofilms. A comprehensive biocompatibility screening panel revealed no disturbance in the metabolic activity of human fibroblasts (HFFF2) and bone marrow stroma cells (HS-5) cultivated on the GO nanofilms decorated with gold and copper NPs, whereas a significant cytotoxic effect of the GO nanocomplex decorated with silver NPs was demonstrated. The GO nanofilm decorated with gold NPs beneficially managed early cell adhesion as a result of the transient upregulation of α1β5 integrin expression, acceleration of cellspreading, and formation of elongated filopodia. Additionally, the cells, sensing the substrate derived from the nanocomplex enriched with gold NPs, showed reduced elasticity and altered levels of vimentin expression. In the future, GO nanocomplexes decorated with gold NPs can be incorporated in the structure of architecturally designed biomimetic biomaterials as biocompatible nanostructuring agents with proadhesive properties.

Early Growth Response (EGR)-1 Is a Key Regulator of Human Primary Bone Marrow Stroma Cells with a Dual Role in Proliferation and Hematopoietic Stroma Support Function

Hematopoietic stem cell (HSC) niches are specialized local tissue microenvironments that maintain HSCs and regulate HSC functions. Bone marrow mesenchymal stroma cells (BMSCs) are essential constituents of the HSC niche and they give rise to skeletal lineage cells as well as the hematopoietic stroma. Despite recent progress in the identification of the primary bone fide BMSC in human bone marrow, knowledge regarding the regulation of BMSC proliferation, differentiation and hematopoietic support function is incomplete.We have previously shown that the expression of Early Growth Response 1 (EGR1) in primary BMSCs is substantially higher compared to the non-colony-forming cells and cultured stromal cells, respectively, indicating a possible role of EGR1 in BMSC regulation (Li et al., Stem Cell Reports, 3(6):965-74, 2014). EGR1 encodes a zinc finger transcription factor that exemplifies a group of immediate early response genes, which have diverse functions in cell growth, development and stress response. EGR1 regulates murine HSC proliferation and localization (Min et al., Cell Stem Cell, 2(4):380-91, 2008), but its role in stroma cell regulation has thus far not been investigated. This study therefore aimed to investigate the functional role of EGR1 in human primary BMSCs.Quantitative gene expression analysis demonstrated that EGR1 expression was 126.9 ± 9.1-fold and 2.8 ± 0.2-fold higher in highly CFU-F (colony-forming unit, fibroblast) enriched human primary linneg/CD45neg/CD271pos/CD140aneg bone marrow cells compared to the non-colony forming CD45neg/CD271neg cell population and CD45neg/CD271pos/CD140apos cells with minimum CFU-F activity. Furthermore, we confirmed that EGR1 expression in cultured cells was down-regulated in comparison with primary BMSCs.Multi-lineage differentiation capacity and surface marker expression profiles of BMSCs were not affected by shRNA-induced EGR1 down-regulation. However, colony-forming capacity was enhanced (1.9 ± 0.1-fold) and population doubling times were reduced by EGR1 knockdown compared to scramble controls. Furthermore, the HSC stroma supporting function as measured by production of transplantable HSC in co-culture experiments (4-day serum-free culture supplemented with SCF 25 ng/ml, TPO 25 ng/ml, and FLT3L 25 ng/ml, 1×104 CD34pos seeded cord blood cells) was reduced when EGR1 knockdown cells were used as stroma layer.On the other hand, whereas CFU-F formation was virtually absent in EGR1 overexpressing cells and population doubling times increased by 2.4 ± 0.3-fold, the hematopoietic stroma support function was considerably improved by EGR1 overexpression as indicated by increased percentages and absolute numbers of transplantable CD34posCD90pos cells produced in co-cultures with EGR1 overexpressing BMSCs as stromal cells (12,496 ± 887 for EGR1 overexpression vs 6,867 ± 1242 for GFP control). Accordingly, CD34pos cells expanded on EGR1 overexpressing stroma exhibited improved in-vivo long-term reconstitution levels in NSG mice compared with cells grown on control stroma.To investigate how EGR1 regulates proliferation and hematopoiesis support in BMSCs, transwell experiments were performed showing that both soluble factors and cell-to-cell contact were important for EGR1-mediated hematopoiesis support. Furthermore, proteomic analysis revealed that a group of proteins related to oxidative-reduction processes was downregulated whereas reactive oxygen species (ROS) levels were elevated in EGR1 knockdown cells. In addition, gene expression profiling analysis identified a number of hematopoietic support genes that were upregulated in EGR1 overexpressing cells compared with control cells.In summary, our data indicate that EGR1 is an important BMSC regulator with a dual role on proliferation and hematopoietic stroma support function. EGR1 expression negatively regulated BMSC proliferation while positively regulating hematopoietic support function, which points to its possible role in coordinating the specific functions of BMSC in their different biological contexts. DisclosuresNo relevant conflicts of interest to declare.

The Secretome of Hypoxia Conditioned hMSC Loaded in a Central Depot Induces Chemotaxis and Angiogenesis in a Biomimetic Mineralized Collagen Bone Replacement Material.

The development of biomaterials with intrinsic potential to stimulate endogenous tissue regeneration at the site of injury is a main demand on future implants in regenerative medicine. For critical-sized bone defects, an in situ tissue engineering concept is devised based on biomimetic mineralized collagen scaffolds. These scaffolds are functionalized with a central depot loaded with a signaling factor cocktail, obtained from secretome of hypoxia-conditioned human mesenchymal stem cells (MSC). Therefore, hypoxia-conditioned medium (HCM)-production is standardized and adapted to achieve high signaling factor-yields; a concentration protocol based on dialysis and freeze-drying is established to enable the integration of sufficient and defined amounts into the depot. In humid milieu-as after implantation-signaling factors are released by forming a chemotactic gradient, inducing a directed migration of human bone marrow stroma cells (hBMSC) into the scaffold. Angiogenic potential, determined by coculturing human umbilical vein endothelial cells (HUVEC) with osteogenically induced hBMSC shows prevascular structures, which sprout throughout the interconnected pores in a HCM-concentration-dependent manner. Retarded release by alginate-based (1 vol%) depots, significantly improves sprouting-depth and morphology of tubular structures. With the intrinsic potential to supply attracted cells with oxygen and nutrients, this bioactive material system has great potential for clinical translation.

A Novel Combination Regimen of BET and FLT3 Inhibition for FLT3-ITD Acute Myeloid Leukemia

Background: Acute myeloid leukemia patients with FLT3-ITD mutations have a high risk of relapse and death. FLT3 tyrosine kinase inhibitors such as quizartinib and gilteritinib improve overall survival in relapsed patients, but their efficacy is limited and most such patients die of the disease. This is because even with potent FLT3 inhibition, the disease persists within the bone marrow microenvironment, mainly due to bone marrow stroma activating parallel signaling pathways that maintain pro-survival factors. BET inhibitors suppress pro-survival factors such as c-Myc and Bcl2, but these drugs thus far have shown only limited single-agent clinical potential. PLX51107 is a novel BET inhibitor designed to inhibit BET activity in intermittent daily fashion to allow for greater tolerability. We investigated whether the addition of PLX51107 to potent FLT3 inhibition with quizartinib could overcome the protective effect of the bone marrow stroma through inhibition of c-Myc expression. Methods: We developed a plasma inhibitory activity assay for c-Myc (c-Myc PIA) to assess in vivo efficacy of the PLX51107 in patients and to identify c-Myc-inhibitory doses of the drug. We tested PLX51107 alone and in combination with quizartinib in murine models of AML and against primary FLT3-ITD AML cells co-cultured with human bone marrow stroma. In addition, we analyzed gene expression patterns in the treatment models to explore the basis of any observed synergistic cytotoxic effect. Results: In a murine xenograft model of AML using MV4-11 cells, PLX51107 alone induced suppression of tumor growth in association with a 90% decrease in c-Myc gene expression. The combination of PLX51107 and quizartinib induced complete tumor regression in 5 out of 7 animals after 14 days of treatment (Figure 1). Animals treated with a 5-day course of quizartinib alone displayed tumor regression persisting until day 26 of treatment, while the addition of PLX51107 resulted in tumor regression until day 39. In patients treated with a dose of 120 mg/day of PLX51107, the c-Myc PIA demonstrated a robust suppression of c-Myc expression for roughly 6 hours, returning to baseline between 7 and 9 hours post-treatment (Figure 2). The mean plasma concentration to achieve this inhibition was 3.3 uM, which, accounting for the difference in protein drug binding between plasma and culture medium with 10% FBS, corresponded to a concentration of 250 nM PLX51107 in culture medium. With this same concentration and schedule (i.e., at concentrations and exposure times equivalent to what human patients would experience taking 120 mg daily of PLX51107 and 60 mg daily of quizartinib), the combination induced synergistic cytotoxicity in a series of 10 different FLT3-ITD AML patient blast samples co-cultured with bone marrow stroma (Figure 3). C-Myc RNA and protein were directly suppressed in these primary samples, and ingenuity pathway analysis of RNA expression confirmed that c-Myc associated genes displayed the highest level of down-regulation. Conclusions: These studies suggest that combination therapy with approximately 120 mg/day PLX51107 and 60 mg/day quizartinib will be a more effective therapy for relapsed FLT3-ITD AML than 60 mg/day of quizartinib alone. The combination of FLT3 inhibition and BET inhibition may represent an attractive therapeutic option for FLT3-ITD AML. Disclosures Hizukuri: Daiichi Sankyo Co, Ltd: Employment. Severson:Plexxikon Inc.: Employment. Powell:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Ma:Plexxikon Inc: Employment. Narahara:Daiichi Sankyo Co, Ltd: Employment. Sumi:Daiichi Sankyo, Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Levis:FUJIFILM: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Menarini: Consultancy, Honoraria.

Pre-Clinical Efficacy of the Novel Kinase Inhibitor Nintedanib on PAX5 Fusion Genes in Pediatric Ph-like B-Cell Precursor Acute Lymphoblastic Leukemia

Despite the current risk-based stratification protocol, 15% of pediatric patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) still experience relapse. In the large subset of 'B-other' patients (negative for common fusion transcripts, non-high hyperdiploid and non-Down Syndrome), 'Ph-like' is a high risk subgroup with high incidence of relapses, which represents 30% of B-others or 10-15% of BCP-ALL patients. The PAX5 gene, encoding for a B-cell related transcription factor, is frequently involved in several translocations in Ph-like patients, determining the formation of fusion genes encoding for aberrant proteins. Our previous studies showed that PAX5 fusions sustain survival of leukemic cells by Lymphocyte kinase (LCK) hyperactivation, which can be targeted by the kinase inhibitor Nintedanib/BIBF1120. This study aims (i) to unravel the functional features of PAX5 fusion genes, elucidating the involved signaling pathways; (ii) to develop new pre-clinical strategies to target PAX5 fusion genes, testing the efficacy of the LCK inhibitor BIBF1120. We selected a cohort of 138 B-other cases among 400 childhood BCP-ALL cases enrolled in Italy in AIEOP-BFM ALL2000/R2006 protocols; by gene expression analysis (HG-U133Plus2.0 Affymetrix array), 59/138 presented as Ph-like, and by NGS, a driver fusion gene was identified in 34/59 (58%). Beside known genetic lesions, such as P2RY8/CLRF2 (N=9), EBF1 fusions (N=3), TCF3/HLF (N=1), IKZF1-fusion (N=1) and BCL9/MEF2D (N=1), and single cases with novel fusions, seven cases were carrying a PAX5 fusion gene, representing the most frequent rearrangement. Ex-vivo inhibition with BIBF1120 was setup on primary cells from 5 PAX5-fusion BCP-ALL samples, demonstrating significant efficacy in inducing leukemic cell apoptosis, both as single agent and in combination with standard chemotherapeutic agents (Annexin V viability assay of leukemic cells in co-culture on human bone marrow stroma). Strikingly, in 3/5 cases dexamethasone and BIBF1120 had a synergistic effect and were further tested in in vivo assays. A daily treatment was performed at bulk disease detection (BM aspiration, mean engraftment 20% CD10/CD19+) in patient-derived xenotransplanted NSG mice from 2 different PAX5 fusion cases. At the endpoint (after two weeks), in the PAX5/AUTS2 PDX mice we detected a mild effect in the BM by BIBF1120 alone (disease reduction 24%, p=0.057), further enhanced in combination with dexamethasone (-49%, p=0.005, with a mean engraftment in vehicle mice of 82.6%). In the spleen, the efficacy was highly significant both for BIBF1120 (-52%, p=0.025) and the combination (-91%, p=0.015, mean engraftment vehicle 69.5%). A similar statistical significant effect was observed also in peripheral blood, whilst, BIBF1120 alone showed a specific significant efficacy in CNS meninges. Analogous results have been observed in PDX from the PAX5/DACH2 PDX mice; BM leukemia decreased 47% using BIBF1120 alone (p=0.004), further diminished by the combination (-65%, p=0.0004, with a mean engraftment in vehicle mice of 65%). In the spleen, the efficacy was highly significant both with BIBF1120 (-45.6%, p=0.04) and for the combination (-96.3%, p=0.0008, mean engraftment vehicle 72.4%). Strikingly, BIBF1120 treatment alone showed analogous efficacy in PB and CNS, with leukemia decrease as low as -45% (p=0.04) and -76% (p=0.007), respectively, and the combination nearly achieved remission in PB (-94%, p=0.0001) and it was significant in CNS (-81.2%, p=0.03). Dexamethasone alone was not effective in the BM and spleen, whereas it decreased the leukemia bulk both in PB (-65%, p=0.0004) and CNS (-52.8%, p=0.03). Overall, BIBF1120 treatment was more effective than dexamethasone. Interestingly, phosphoflow analysis showed a marked inhibition by BIBF1120 of pAkt-Thr308 and its downstream effectors, such as pS6 and 4pEBP1, in ex vivo BM and spleen cells. In conclusions, PAX5 fusion genes are highly recurrent among Ph-like patients and can potentially be targeted by Nintendanib/BIBF1120, that showed a significant effect ex vivo and in vivo, even as a single agent. Disclosures No relevant conflicts of interest to declare.

Immortalized Human Bone Marrow Derived Stromal Cells in Treatment of Transient Cerebral Ischemia in Rats.

Cerebral ischemic stroke may cause a number of adverse neurological complications or behavioral disorders. Moreover, cerebral ischemic stroke is a prevalent disease with limited treatment strategies and may increase chances of developing neurodegenerative diseases, e.g., Alzheimer's disease, in the future. Therefore, alternative strategies are highly needed for improving the therapy for ischemic stroke. Human bone marrow stroma stem cells, also known as mesenchymal stem cells (hMSC), have a demonstrative role in treating transient cerebral ischemia, yet whether immortalized hMSC may have a better effect than hMSC is unknown. Here, we addressed this question. A rat cerebral ischemia reperfusion (IR) model was used to compare the effects of hMSC and an immortalized hMSC by human telomerase reverse transcriptase (hMSC-TERT). We found that both hMSC and hMSC-TERT similarly attenuated the elevation of serum neuron specific enolase levels after IR. Transplantation with hMSC alleviated cerebral infarction, alleviated brain edema, improved neural function, and reduced brain cell apoptosis, in a significantly better degree than transplantation with hMSC. Thus, these data suggest that immortalized hMSC may have an advantage over hMSC in treatment of transient cerebral ischemia.

Abstract 4486: Reexpression of LSAMP, a gene frequently deleted in African American prostate cancers, alters adhesive qualities of prostate tumor cells and inhibits Akt, ERK1/2, and β-Catenin signaling axis

Abstract INTRODUCTION: Although African American (AA) men have the highest prostate cancer (CaP) incidence and mortality rates in the US, most studies of CaP genome have been performed using tumor specimens from Caucasian Americans (CA). To address the underrepresentation of AA CaP in these studies, we performed whole genome sequencing, IHC and FISH validation on tissue microarrays, and analysis of TCGA data, on a total of 438 CaPs. We noted less frequent alterations of two common CaP driver genes, PTEN and ERG, in CaP genomes of AA compared to CA men, confirming earlier observations by others and us. We reported a higher frequency of a genomic deletion of the Limbic System Associated Membrane Protein (LSAMP) gene, located on 3q13.31 locus in AA CaP, which was also associated with rapid biochemical recurrence. A recent study of CaP genome in a Chinese cohort detected LSAMP alterations at a frequency similar to that in our AA cases. LSAMP loss in other cancers have been associated with aggressive disease. We hypothesize that the loss of LSAMP contributes to CaP progression. Here, we report further biological evaluations of LSAMP modulation in CaP cells. METHODS: The copy number and expression of LSAMP in CaP cell lines were assessed. We established stable doxycycline inducible LSAMP expression in LNCaP and in MDA PCa 2b cells (AA patient derived cell line with monoallelic LSAMP deletion), and constitutive expression in DU145 cells. We assessed the effect of LSAMP expression on cell-cell adhesion by monitoring the binding of LSAMP expressing and control DU145 cells to mouse fibroblast (NIH-3T3), normal human prostate stroma (WPMY-1), human endothelial (HUVEC), and human bone marrow stroma (HS-5) cells. We analyzed LSAMP modulated signaling pathways in the inducible and constitutively LSAMP expressing cell lines by immunoblot and immunofluorescence assays. RESULTS: Overexpression of LSAMP resulted in attenuated cell proliferation and increased adhesion of cells to extracellular matrix proteins. LSAMP expression in DU145 cells increased their adhesion to fibroblast and prostate stroma cell lines, and concomitantly decreased their adhesion to endothelial, and bone marrow stroma cells. LSAMP expression resulted in decreased expression of FGFR2, FGFR4, and EphA3 receptor tyrosine kinases, and decreased phosphorylation of their downstream targets in Akt and ERK1/2 signaling pathways, leading to the inactivation of β-catenin. LSAMP expression also alters the levels of some Integrins. CONCLUSION: Cell biologic features of LSAMP reported here, along with observations of genomic inactivations of LSAMP in CaP support its role in CaP progression through altered cell adhesion. These data provide the first insight into LSAMP function in CaP, corroborating with studies that suggest bona fide tumor suppressor function for LSAMP in cancers. Citation Format: Kevin Babcock, Inger L. Rosner, Shiv Srivastava, Albert Dobi, Shyh-Han Tan. Reexpression of LSAMP, a gene frequently deleted in African American prostate cancers, alters adhesive qualities of prostate tumor cells and inhibits Akt, ERK1/2, and β-Catenin signaling axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4486.

Identification and Clinical Exploration of Individualized Targeted Therapeutic Approaches in Acute Myeloid Leukemia Patients By Integrating Drug Response and Deep Molecular Profiles

IntroductionMost patients with acute myeloid leukemia (AML) are still missing effective options for targeted treatments. Here, we applied individualized systems medicine (ISM) concept1 by integrating deep molecular profiles (genomics, transcriptomics) and ex vivo drug response profiles with 521 oncology drugs in 154 AML patient samples. The aim was to identify new treatment opportunities for molecular subsets of AML patients. When feasible, ISM guided treatment opportunities were applied clinically for AML patient treatment. Serial samples were available to identify molecular alterations in response to targeted drug treatment and to monitor therapeutic success or failure. We also aimed at testing the impact of bone marrow stromal cell conditioned media on drug response profiles in AML patients2.MethodsSamples from bone marrow or blood of 122 AML patients and 17 healthy donors were obtained with written consent and ethical approval (239/13/03/00/2010 and 303/13/03/01/2011) from the Hematology Clinic, Comprehensive Cancer Center, Helsinki University Hospital. The ex vivo drug sensitivity and resistance testing (DSRT) assay was performed with 521 approved oncology drugs and investigational oncology compounds as described earlier1. In this study, freshly isolated mononuclear cells were randomly resuspended either in standard mononuclear cell medium (MCM, PromoCell) or in human bone marrow stroma derived conditioned medium (CM) for drug testing. DNA samples from same mononuclear cells were subjected to whole exome and transcriptome sequencing and data were analyzed as described previsously2. Hierarchical clustering and non-parametric rank correlation were performed with drugs and samples. Wilcoxon sign ranked test was applied between wild type and mutated samples to identify significant mutation-drug associations.ResultsHierarchical clustering was largely independent of clinical features such as disease status or risk class. A strong drug sub-cluster with a unique response profile was composed of that of the MDM2 antagonist idasanutlin along with BCL-2 inhibitors navitoclax and venetoclax (Figure). BET inhibitors (JQ1, I-BET151, birabresib) and MEK inhibitors (trametinib, selumetinib) were positively correlated with each other suggesting an association between bromodomain mediated epigenetic deregulation and up-regulation of the MEK pathway in a subset of patients. Comparison between patient samples profiled in CM (n=77) vs MCM medium (n=77) indicated higher efficacy of MDM2 modulator idasanutlin in MCM while BET inhibitors responded more strongly in CM. Other differences observed earlier by Karjalainen et al1 between the two media types were also validated. Furthermore, 16 chemorefractory and one diagnostic stage patients were treated with the targeted drugs suggested by this ISM approach. We observed complete remission or leukemia free state in 35% (6/17) of the AML patients given tailored treatment in an observational study. The targeted drugs used for clinical translation included ruxolitinib (in n=4 patients), temsirolimus (n=5), trametinib (n=4), sunitinib (n=7), dasatinib (n=7), sorafeninb (n=4), omacetaxine (n=3) and dexamethasone (n=5).SummaryThis study highlights the potential of individualized systems medicine (ISM) approach in the identification of effective treatment opportunities for individual patients with AML. Identifying molecular markers for ex vivo drug responses can help to assign treatments to the patient subgroups most likely to respond in clinical trials. [Display omitted] DisclosuresHeckman:Orion Pharma: Research Funding; Novartis: Research Funding; IMI2 project HARMONY: Research Funding; Pfizer: Research Funding; Celgene: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

LPS-stimulated human bone marrow stroma cells support myeloid cell development and progenitor cell maintenance.

The nonhematopoietic bone marrow (BM) microenvironment provides a functional niche for hematopoietic cell maintenance, recruitment, and differentiation. It consists of multiple cell types including vasculature, bone, adipose tissue, and fibroblast-like bone marrow stromal cells (BMSC), which can be summarized under the generic term niche cells. BMSC express Toll-like receptors (TLRs) and are capable to respond to TLR-agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis. Here, we show that in addition to enhanced myeloid colony formation from human CD34+ cells, lipopolysaccharide (LPS) stimulation retains overall higher numbers of CD34+ cells in co-culture assays using BMSC, with eightfold more CD34+ cells that underwent up to three divisions as compared to non-stimulated assays. When subjected to cytokine-supplemented myeloid colony-forming unit (CFU) assays or transplanted into newborn RAG2(-/-) γc (-/-) mice, CD34(+) cells from LPS-stimulated BMSC cultures give rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus supporting maintenance of myeloid and lymphoid primed hematopoietic progenitor cells (HPCs) under inflammatory conditions. Collectively, we suggest that BMSC enhance hematopoiesis during inflammatory conditions to support the replenishment of innate immune effector cells and to prevent the exhaustion of the hematopoietic stem and progenitor cell (HSPC) pool.

Abstract 3295: A novel patient-derived xenograft model for evaluating the role of TSLP in CRLF2 B-ALL

Abstract B-cell acute lymphoblastic leukemia (B-ALL) with genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL) is associated with poor outcomes. CRLF2 B-ALL is 5 times more common in Hispanic children than others making it a significant biological component of pediatric cancer health disparities. CRLF2 is a component of the receptor complex that is activated by the cytokine, TSLP. Receptor signaling induces Jak/STAT5 and PI3/AKT/mTOR pathway activation and plays a role in the proliferation and differentiation B cell precursors. We found that primary human bone marrow (BM) stroma express TSLP providing an in vivo source of TSLP to stimulate CRLF2 B-ALL cells. Our goal was to develop patient-derived xenograft (PDX) models of CRLF2 B-ALL for studies to understand disease mechanisms and identify therapies to treat CRLF2 B-ALL and reduce the health disparities for Hispanic children with this disease. PDX models are possible because many cytokines produced in the mouse show cross species activity on human cells. However, available data suggests that mouse TSLP does not activate human CRLF2-mediated signals. Using phospho flow cytometry we show that mouse TSLP was unable to induce increases in pSTAT5, pAKT and pS6 observed in CRLF2 B-ALL cells stimulated with human TSLP. We developed a human TSLP +/- PDX model system by transplanting immune deficient NSG mice with HS-27 stroma transduced to express human hTSLP (hTSLP+ mice) or with control vector (hTSLP- mice). Human TSLP was present at normal human serum levels in hTSLP+ mice but undetectable in hTSLP- mice. To identify genes targeted by TSLP in CRLF2 B-ALL and verify pathway activation, we transplanted primary leukemia cells from a Hispanic patient into hTSLP+ and hTSLP- mice. Whole genome microarray was performed on CRLF2 B-ALL cells isolated from the BM of the hTSLP+ and hTSLP- PDX mice. Microarray identified 280 genes that are upregulated and 281 genes that are downregulated in vivo in leukemia cells from hTSLP+ as compared to hTSLP- PDX mice. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in hTSLP+ as compared to hTSLP- mice, confirming hTSLP activity in the hTSLP+ PDX mice. Our next question was whether cells expanded in hTSLP+ vs. hTSLP- mice would exhibit changes in their ability to respond to TSLP. When we subjected PDX-expanded primary CRLF2 B-ALL cells to ex vivo TSLP stimulation ∼1/3 fewer gene targets were up- and downregulated in the leukemia cells expanded in hTSLP- mice as compared to cells from hTSLP+ mice. This suggests that CRLF2 B-ALL cells expanded in xenograft without TSLP lose some of their ability to respond to TSLP. The hTSLP+ CRLF2 B-ALL PDX mice described here provide a novel preclinical model for studying disease mechanisms and identifying therapies to target signaling pathways activated by TSLP in CRLF2 B-ALL and reduce cancer health disparities for this disease. Citation Format: Olivia L. Francis, Parveen Shiraz, Terry-Ann Milford, Ineavely Baez, Jacqueline S. Coats, Karina Mayagoitia, Elizabeth Ginelli, Katherine R. Salcedo-Concepcion, Shannalee Martinez, Xiaobing Zhang, Valeri Filippov, Ruijun J. Su, Ross Fisher, Christopher L. Morris, Sinisa Dovat, Kimberly J. Payne. A novel patient-derived xenograft model for evaluating the role of TSLP in CRLF2 B-ALL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3295. doi:10.1158/1538-7445.AM2015-3295

MP21-08 SKELETAL MYOGENIC DIFFERENTIATION OF HUMAN URINE-DERIVED CELLS AS A POTENTIAL SOURCE OF CELL THERAPY FOR URETHRAL SPHINCTER MUSCLE DYSFUNCTION

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology II1 Apr 2015MP21-08 SKELETAL MYOGENIC DIFFERENTIATION OF HUMAN URINE-DERIVED CELLS AS A POTENTIAL SOURCE OF CELL THERAPY FOR URETHRAL SPHINCTER MUSCLE DYSFUNCTION Wei Chen, M Xie, Bin Yang, S Bharadwaj, Song Li, Guihua Liu, Anthony Atala, and Yuanyuan Zhang Wei ChenWei Chen More articles by this author , M XieM Xie More articles by this author , Bin YangBin Yang More articles by this author , S BharadwajS Bharadwaj More articles by this author , Song LiSong Li More articles by this author , Guihua LiuGuihua Liu More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Yuanyuan ZhangYuanyuan Zhang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.960AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Adult stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration in treatment of urethral sphincter dysfunction. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle lineage cells, and potentially be used for skeletal muscle regeneration when treating urinary incontinence due to urethral sphincter dysfunction. METHODS USCs were harvested from six healthy individuals aged 25–55 years old. Expression profiles of cell-surface markers were assessed by flow cytometry. Growth factors and cytokines secreted from USCs and human bone marrow stroma cells (BMSC, as a control) culture supernatant were measure by specific ELISAs. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. RESULTS Human USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. A series of growth factors and cytokines (such as IGF1, HGF, FGF, VEGF, and PDGF) were identified at higher levels in the culture supernatant of USCs than in BMSCs. After myogenic differentiation, morphology of USCs changed from ‘rice-grain’-like cells to spindle-shaped cells and then to myotube-like structures. The USCs expressed specific skeletal muscle transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed skeletal muscle markers stably in vivo. CONCLUSIONS Our findings suggest that USCs can differentiate into a skeletal muscle lineage cells in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy to induce urethral sphincter skeletal muscle regeneration and thereby treat urinary incontinence. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e235 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wei Chen More articles by this author M Xie More articles by this author Bin Yang More articles by this author S Bharadwaj More articles by this author Song Li More articles by this author Guihua Liu More articles by this author Anthony Atala More articles by this author Yuanyuan Zhang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Metabolic Reprogramming of Acute Myeloid Leukemia Blasts By Bone Marrow Stroma Cells

Introduction: Acute myeloid leukemia (AML) represents the most common form of acute leukemia in adults. Despite the enormous efforts during the last decades treatment resistance is still observed at a high rate. Previous studies have shown that bone marrow stroma promotes an increased resilience of AML blasts towards chemotherapeutics. Furthermore, current data suggest that alterations of the malignant cells’ metabolism could represent a strong determinant for the disease’s (including AML) course and/or treatment resistance. In fact, a deregulated metabolism could lead to a reduced sensitivity towards therapy and it remains to be elucidated whether this is a mechanism contributing to the blast-protective effects elicited by the bone marrow stroma. Here, we sought out to characterize the impact of stroma cells on the AML blasts’ metabolism.Methods and Results: The human bone marrow stroma cell-line HS-5 was utilized for establishing the in vitro niche model. We compared in our assays AML cell lines (THP-1, OCI-AML, and KG-1) as well as primary blasts cultured on a HS-5 monolayer or alone. In line with previous observations we could detect an increased proportion of AML cells in the S-phase of the cell cycle upon co-culture with HS-5. When evaluating the cells’ metabolism we observed a shift towards glycolysis despite presence of oxygen, i.e. aerobic glycolysis or the “Warburg”-effect. Basal glycolysis as well as maximal glycolytic capacity upon blocking ATP production in the mitochondrial respiratory chain was increased. Respiration (including basal respiration, coupling efficacy, and maximal respiratory capacity) was not significantly affected. However, mitochondrial biogenesis appeared reduced.Increased glycolysis was accompanied by an increased up-take of fluorescently labeled glucose as well as an increased expression of glucose transporters. The expression of several glycolytic molecules found to be increased upon HS-5 co-culture. Noticeably, cell-to-cell contact was not a pre-requisite for the metabolic shift. Our data was further corroborated by direct observations from AML patients: AML blasts isolated from the bone marrow exhibited an up-regulated glycolysis as compared to their counterparts from the periphery collected at the same time point.Conclusion: Taken together, our data indicates a stromal cell-mediated metabolic shift in AML blasts towards aerobic glycolysis. This metabolic phenotype is linked to an unfavorable prognosis and increased chemo resistance. The underlying molecular pathways remain to be elucidated and could represent a promising target for future interventions. DisclosuresNo relevant conflicts of interest to declare.

Bone marrow stromal cells protect acute myeloid leukemia cells from anti-CD44 therapy partly through regulating PI3K/Akt-p27(Kip1) axis.

The anti-CD44 monoclonal antibody (mAb) A3D8 induces differentiation or apoptosis in vitro in various subtypes of acute myeloid leukemia (AML) via p27(Kip1) upregulation. Bone marrow (BM) stromal cells play a vital role in the development of chemoresistance in AML cells attached to the stroma. To investigate the effect of BM stroma adhesion induced AML resistance to A3D8, we developed a co-culture system composed of an AML-derived cell line (NB4) cultured with either a human BM stroma cell line (HS-5) or mesenchymal stem cells (MSCs). We found that NB4 cells adhered to HS-5 cells or MSCs developed resistance against the anti-proliferative effects of A3D8, and this action is caused by the activation of PI3K/Akt signaling following p27(Kip1) down-regulation and cytoplasmic re-localization. The stromal co-culture-induced resistance can be partially abolished by inhibiting the PI3K/Akt signaling pathway. Such findings were confirmed in two additional AML-derived cell lines as well as in primary AML cells. Our results suggest that BM stroma can induce A3D8 resistance in part via the PI3K/Akt-p27(Kip1) axis, and blocking PI3K/Akt pathway maybe necessary for anti-CD44 treatment on AML in BM microenvironment.

Abstract 4842: The stem cell niche detoxifies chemotherapy and protects malignant hematopoietic cells via expression of cytochrome P450 enzymes

Abstract The bone marrow (BM) microenvironment plays a critical role in drug resistance of malignant cells. Although both, direct contact as well as soluble factors have been proposed, the mechanisms responsible for this microenvironment-mediated protection remain unclear. We have recently shown that BM stroma protects human hematopoietic stem cells from the pro-differentiating effects of retinoid via cytochrome P450 (CYP) 26 mediated degradation (Ghiaur G et al., PNAS 2013). Here, we hypothesized that drug metabolism by BM stroma via CYP enzymes is a general cell-extrinsic mechanism of protection of malignant cells from chemotherapy. First, we have designed a qPCR expression array platform and found that human BM stroma expresses a variety of drug metabolizing enzymes to levels comparable to human hepatocytes. Next, using qRT-PCR, we confirmed that CYP3A4, a major drug metabolizing enzyme, is expressed in primary BM stroma but not in multiple myeloma (MM) or acute myeloid leukemia (AML) cells. We have found that BM stroma protects against Bortezomib mediated kill of MM cells [H929 (p<0.01)] and VP16 mediated kill of AML cells [KG1 (p0.01)] (Table). Since CYP3A4 plays a major role in the metabolism of aforementioned drugs, we studied if stromal CYP3A4 contributes to this protection. We found that ketoconazole, a potent CYP3A4 antagonist, overcomes bone marrow stroma-mediated resistance to Bortezomib (p=0.01), and VP16 (p=0.03). Ketoconazole had no effects in stroma free cultures. In addition, shRNA-mediated knockdown of stromal CYP3A4 renders these cells unable to protect malignant cells from chemotherapy (p=0.01). These studies open the possibilities that the stem cell niche serves as a drug-free sanctuary, where malignant stem cells can survive conventional chemotherapy. Targeting stromal P450 enzymes hold promise of improved efficiency of stem cell directed therapies and decrease rate of relapse in hematological malignancies. Table.Effects of VP16 and Bortezomib on cellular and clonogenic recovery of KG1 and H929 cells, respectively. “Stroma-CYP3A4” represents experiments performed on CYP3A4 knockdown stroma. Control shRNA infected stroma showed no differences compared to untransduced cells. Data are presented as mean±SEM from three independent experiments.VP16: KG1 cellular recoveryBortezomib: H929 clonogenic (CFU-C) recoveryLiquidStromaStroma+ketoconazoleStroma- CYP3A4LiquidStromaStroma +ketoconazoleStroma-CYP3A48.10 ± 1.5529.38 ± 0.515.7 ± 2.719.35 ± 0.8315.77 ± 3.289.5 ± 2.549.33 ± 860.35 ± 4.2 Citation Format: Salvador Alonso, Meng Su, Richard Jones, Gabriel Ghiaur. The stem cell niche detoxifies chemotherapy and protects malignant hematopoietic cells via expression of cytochrome P450 enzymes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4842. doi:10.1158/1538-7445.AM2014-4842

Bone marrow stroma‐mediated resistance to FLT3 inhibitors in FLT3‐ITD AML is mediated by persistent activation of extracellular regulated kinase

A consistent pattern of response has been observed when FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3- internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3-ITD AML blasts co-cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti-apoptotic effect is mediated through a combination of direct cell-cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine-activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal-mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3-ITD AML.

Human B cell development in a novel xenograft model that provides selective TSLP and IL-7 stimulation (P4390)

Abstract IL-7 is critical for mouse B cell development and available data suggest that TSLP has overlapping functions. Our previous in vitro studies show that IL-7 expands human B cell progenitors by ~60 fold (JI 2009 82:4255). Our recent experiments provide evidence that TSLP similarly expands human B cell progenitors in vitro and that primary human bone marrow stroma provides an in vivo source of both cytokines. To study interplay between IL-7 and TSLP in early stages of in vivo human B cell lymphopoiesis, we developed a novel xenograft model system that selectively provides IL-7 and/or TSLP stimulation. While mouse IL-7 can act on human cells, TSLP is species-specific. To overcome this obstacle we coupled a xenograft model system engineered to provide human TSLP (hTSLP) with IL-7 neutralizing antibodies. Preliminary data obtained using this model system transplanted with cord blood CD34+ cells shows that the production of B cell precursors is reduce by ~90% in mice that lack both IL-7 and hTSLP stimulation as compared to mice where both are present. The absence of hTSLP stimulation, alone, reduced B cell production by about 50%, while the effects due IL-7 neutralization, alone, were about half this. These data suggest that IL-7 and TSLP play an essential and at least partially overlapping role in human B cell development. Current studies are aimed at defining the differential stage-specific effects of TSLP and IL-7 on survival and proliferation of human B cell progenitors in vivo.

Arachidonic acid drives amoeboid transendothelial invasion through ligand independent activation of EphA2.

221 Background: We have previously shown that the omega-6 poly unsaturated fatty acid arachidonic acid is a potent stimulator of prostate epithelial transendothelial migration across the bone marrow endothelium and can completely restore the invasive stimulatory capacity of adipocyte depleted human bone marrow stroma. Arachidonic acid induces of EphA2 ligand independent signaling via pAkt with subsequent downstream activation of FAK and Src. Here we present further characterization of the arachidonic acid induced EphA2 pathway. Methods: Transendothelial migration of PC-3 cells across the bone marrow endothelial cell line BMEC was followed by time lapse microscopy and by SEM with or without amoeboid or mesenchymal inhibitors. Western blot analysis of the PC-3 cells or their lipid raft (LR) components post AA stimulation was conducted and signaling pathways validated by siRNA knockdown. Results: Initial studies demonstrated a requirement of PC-3 cells to undergo a mesenchymal to amoeboid switch for transendothelial migration towards bone marrow stroma. The mesenchymal to amoeboid switch was also induced by direct treatment with arachidonic acid as characterized by induction of the Rho/Rock pathway and phosphorylation of myosin light chain. Knockdown of EphA2 by siRNA blocked the induction of the mesenchymal to amoeboid switch and prevented transendothelial invasion. Conclusions: Malignant prostate epithelial cells undergo a switch from mesenchymal to amoeboid movement in response to arachidonic acid in order to cross the bone marrow endothelial barrier.

Wnt Signaling Is Associated with Anti-Apoptosis in the Interaction Between Acute Myeloid Leukemia Cells and Stromal Cells

Abstract 1366Recent studies have revealed that strength of the Wnt signaling pathway regulates normal hematopoiesis including hematopoietic stem cells, and aberrant activation of the pathway is involved in the development of several types of leukemias. In the bone marrow microenvironment, stromal cells are rich sources of cytokines and some secreted cytokines have been observed to block induction of cell death in myeloid leukemia cells exposed to chemotherapy. Here, we examined the role of the Wnt signaling pathway on cell-adhesion, proliferation and survival of the stroma-dependent human AML cell line, TRL-01 cells, which we previously established (Ninomiya, et al. Cancer Gen Cyto, 2006). TRL-01 cells were maintained in the co-culturing with the hTERT-transduced human bone marrow stroma cell line, HTS cells, and cell death of TRL-01 cells was induced after removal of HTS cells. Treatment with the Wnt-receptor competitor, secreted Frizzled related protein (sFRP)-1, or the Rho kinase inhibitor, Y29632 (previously reported as an inhibitor of the non-canonical Wnt downstream pathway), but not with the specific inhibitor of the canonical Wnt pathway (DKK-1) induced apoptosis in dose-dependent manners in TRL-01 cells co-cultured with HTS cells. These results suggested that the non-canonical pathway of Wnt signaling might regulate survival of TRL-01 on the stromal cells. Next, we comprehensively investigated transcripts of the Wnt pathway components (10 Frizzleds, 2 LRPs, and 18 Wnts) in TRL-01 cells and HTS cells using RT-PCR. Transcripts of Wnt5A and Wnt9A were expressed in TRL-01 cells, but not in HTS cells. Moreover, the cell death of TRL-01 cells after removal of HTS cells was partially prevented by additional treatment with Wnt5A or Wnt9A not by other Wnt molecules such as Wnt5B. On the other hand, treatment with Wnt3A induced activated nuclear beta-catenin using Western-blotting, however, did not contribute to the survival of TRL-01 cells without the stromal co-culturing. Moreover, we are investigating regulation of the anti-apoptotic downstream pathway molecules in TRL-01 cells as well as effects of other inhibitors targeted for the Wnt signaling. We are also examining expressions of Wnt pathway components in primary AML bone marrow cells and other human bone marrow stroma cell lines such as HS-5 cells. These results imply association of Wnt5A and Wnt9A with anti-apoptosis in the interaction between AML cells and stromal cells, and a possible therapeutic target of AML for overcoming the resistance to chemotherapy in the bone marrow microenvironment. Disclosures:Naoe:Chugai Pharma: Research Funding; Novartis Pharma: Research Funding; Kyowa-Hakko Kirin: Research Funding.

The Novel CXCR4 Antagonist POL5551 Decreases Surface CXCR4 (s-CXCR4) Expression, Inhibits Chemotaxis, and Enhances Chemosensitivity in Acute Lymphoblastic Leukemia (ALL)

Abstract 780 Background:The interaction between the cell surface receptor CXCR4 and the chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow microenvironment. We previously demonstrated that CXCR4 is an important mediator of chemotherapy resistance, as chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) cell lines and primary samples led to increased SDF-1-mediated chemotaxis and increased protection by normal human bone marrow stroma from chemotherapy-induced apoptosis. We also showed that stromal protection and chemotherapy resistance could be reversed by treatment with the FDA-approved CXCR4 inhibitor plerixafor, both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged ALL. Therefore, disruption of the CXCR4/SDF-1 axis is a rational means to target extrinsic survival mechanisms in acute leukemia.The novel Protein Epitope Mimetic (PEM) POL5551 is a selective and potent antagonist of CXCR4. Treatment with POL5551 inhibits vascular accumulation of CXCR4+ smooth muscle cells but its effects on ALL have not been reported. We hypothesized that treatment of ALL cell lines with POL5551 would 1) decrease s-CXCR4 expression, 2) inhibit SDF-1-mediated chemotaxis, and 3) reverse stromal-mediated protection from chemotherapy-induced apoptosis. Methods/Results:Pre-B cell ALL (697, HB11;19, NALM-6, SEMK2) and T cell ALL cell lines (CCRF-CEM-1301, Jurkat, Molt-4) were treated with dose ranges of POL5551. Cells were harvested at multiple time points over 72 hours and s-CXCR4 was measured by FACS. S-CXCR4 was potently and markedly reduced in all cell lines, with IC50 levels of <5 nM at 1 hour and IC50 levels of <20 nM at 48 hours. In comparison, 3- to 30-fold higher doses of plerixafor were needed to achieve similar levels of reduction. Simultaneous measurement of cell proliferation using the WST-1 proliferation assay demonstrated that treatment with POL5551 neither increased nor decreased leukemia cell proliferation in a significant manner.To ascertain the functionality of s-CXCR4 inhibition, we performed chemotaxis assays. Leukemia cells were treated with 10 nM POL5551 or vehicle control and placed into hanging cell culture inserts. Migration through a permeable membrane toward an SDF-1 gradient was then measured after 24 hours. Compared to control-treated cells, POL5551-treated cells had significantly decreased SDF-1-induced chemotaxis (average 38% reduction in chemotaxis in pre-B cell lines, p<0.001; average 41% reduction in T cell lines, p=0.05).We also performed co-culture experiments with normal human bone marrow stroma in the presence and absence of POL5551 to further demonstrate the functional effects of s-CXCR4 inhibition. Specifically, we cultured leukemia cells off stroma (O), on stroma (S), or pretreated with POL5551 for 30 minutes prior to plating on stroma (P+S). Cells from each culture condition were then treated with dose ranges of chemotherapy. Following treatment, we measured apoptosis by staining with Annexin V/7-AAD. IC10 through IC90 values were obtained using Calcusyn. To quantify stromal protection, we calculated a Protective Index (PI), defined as the S IC values divided by the O IC values. Thus, PI >1 signified stromal protection, while PI ≤1 signified no stromal protection. To quantify the ability of POL5551 to reverse stromal protection, we calculated a Reversal Index (RI), defined as the P+S IC values divided by the O IC values. Therefore, PI > RI indicated a decrease in stromal protection, while RI ≤1 indicated a reversal of stromal protection. Overall, stroma protected leukemia cells from chemotherapy-induced apoptosis. Importantly, treatment with POL5551 abrogated stromal-mediated protection and restored chemosensitivity (eg, PI 1.182 vs. RI 0.956 for NALM-6 treated with daunorubicin +/− 20 nM POL5551, p<1×10e-9). Conclusions:The novel CXCR4 antagonist POL5551 is a potent inhibitor of CXCR4 in pre-B and T ALL cell lines with activity at nanomolar concentrations in decreasing s-CXCR4 expression, inhibiting SDF-1-induced chemotaxis, and reversing stromal-mediated protection from chemotherapy in vitro. Therefore, if our findings are confirmed in primary samples and in vivo, interruption of leukemia-microenvironment signaling with POL5551 may prove to be an effective strategy in the treatment of pre-B and T cell ALL. Disclosures:Chevalier:Polyphor Ltd: Employment. Dembowsky:Polyphor Ltd: Employment.