Colony-forming Capacity Research Articles

Abstract 736: HOXA5 induction by norepinephrine in precursor cells of ovarian cancer

Abstract Chronic stress induced by repetitive activation of the sympathetic nervous system leads to rapid release of catecholamines like norepinephrine (NE). Several studies have shown that increased levels of NE can contribute to tumor progression by promoting tumor growth, migration and invasion of ovarian cancer cells. However, the role of NE in tumor initiation remains mostly unknown. The purpose of this study was to explore the acute and chronic effects of NE on immortalized normal fallopian tube and ovarian cell lines presumed to be the origin of epithelial ovarian cancers. Normal immortalized ovarian (iOSE11) and fallopian tube (iFTE283) surface epithelial cells were treated with 10µM NE for 15 min, 1h and 4h followed by whole-transcriptome RNA-Seq. 234 and 313 differentially expressed genes were identified at 1h and 4h time points in iFTE283 cells respectively; while 53 and 34 differentially expressed genes were identified at 1h and 4h time points in iOSE11 cells. Gene ontology analysis using Panther Statistical Overrepresentation Test was performed on these differentially expressed genes. Panther Protein Class analysis revealed ‘transcription factor' to be the most significantly overrepresented at both 1h and 4h time-points. Promoter enrichment analysis using oPOSSUM database identified transcription factor HOXA5 to be consistently enriched at both time points in both cell lines. Time course studies performed by qPCR and luciferase promoter assays for HOXA5 transcription factor in normal cells (iOSE11, iFTE283), partially transformed cells (iOSE11-p53R175H, iFTE283-p53R175H, iFTE282-p53R175H and iOSE4-cMyc) and cancer cells (OvCAR8, SkoV3) revealed that HOXA5 induction by NE occurred only in normal immortalized cells and partially transformed p53R175H overexpressing cells, but not in cancer cells. In contrast, chronic exposure to NE for 4 months followed by HOXA5 time course study by qPCR revealed attenuation of HOXA5 induction in normal as well as p53R175H overexpressing cells. Additionally, long term NE treatment lead to increased proliferation and colony forming capacity in iFTE283, iFTE283-p53R175H and iFTE282-p53R175H cells. p53R175H overexpressing iFTE283 cells also showed slight increase in chromosomal instability, as measured by karyotype, after 4 months of treatment with NE. These observations suggest that HOXA5 plays a central role in regulating the early response to NE in normal as well as p53R175H overexpressing ovarian epithelial and fallopian tube cells, but this induction is attenuated in cancer cells as well as in normal and partially transformed cells that are exposed to long term NE treatment. We will further assess the induction of HOXA5 by NE through promoter bashing experiments. Citation Format: Sweta Dash, Ling Cen, Sean Yoder, Tania Mesa, Andrew Smith, Chaomei Zhang, Jamie Teer, Guillermo Armaiz-Pena, Alvaro Monteiro. HOXA5 induction by norepinephrine in precursor cells of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 736.

Abstract 1039: Andrographis enhances 5-fluorouracil-induced anti-tumor effect in colorectal cancer through inhibition of DKK1

Abstract Background: Colorectal cancer (CRC) ranks as the third leading cause of cancer-related deaths in the US. In this malignancy, 5-fluorouracil (5FU)-based treatment remains the mainstay as first-line chemotherapy. Unfortunately, ~50-60% of patients treated with 5FU eventually develop resistance, leading to increased mortality. Hence, a better understanding of the molecular mechanisms underlying 5FU-resistance will lead to development of more effective therapeutic strategies. Our previous studies have shown that, andrographis, a botanical with diverse biological activities, synergistically enhanced 5FU-induced pro-apoptotic effects by mediating Wnt/β-catenin pathways. However, a more comprehensive knowledge of andrographis-mediated synergism with 5-FU remains largely unclear. Methods: We first established 5FU resistant isogenic cell lines of HCT116 and SW480 CRC cells (HCT116 5FUR and SW480 5FUR). To determine the synergistic anti-tumor effects of andrographis and 5FU, we performed cell viability, proliferation and colony formation assays by treating parental and 5FUR cells with andrographis, 5FU and their combination. To explore the molecular mechanisms responsible for the synergistic anti-tumor efficacy, we next performed gene expression microarrays to identify candidate pathways and genes that mediated anti-tumor efficacy of andrographis in 5FUR cells. We analyzed an expression profiling dataset from CRC patients (GSE52735) who either responded or did not respond to 5FU. We also studied the andrographis-mediated synergism with 5FU in a xenograft animal model, and in 3D patient-derived tumor organoids. Results: Combined treatment with andrographis and 5FU exhibited significantly superior effects on cell viability (P<0.01), proliferation (P<0.001) and colony formation capacity (P<0.001), compared to treatment with individual treatments. In line with our in vitro findings, the combined treatment also significantly reduced tumor growth in mice xenografts (P<0.0001) as well as patient-derived tumoroids (P<0.001). By analyzing gene expression profiling data from cell lines and CRC patients, we identified that overexpression of DKK1 gene was a critical event for 5FU resistance in CRC. Moreover, qRT-PCR and western blotting data revealed that andrographis resulted in downregulation of 5FU-induced DKK1 overexpression accompanied with the synergistic anti-tumor effects in animal xenografts (P<0.001) and patient-derived tumor organoids (P<0.001), which were reversed following siRNA-mediated silencing of DKK1 (P<0.01). Conclusions: Our data provide novel evidence for andrographis-mediated anti-tumor synergism with 5-FU through DKK1, highlighting that in addition to its preventive efficacy, andrographis may serve as a potential adjunctive treatment to conventional chemotherapeutic drugs in patients with CRC. Citation Format: Yinghui Zhao, Chuanxin Wang, Ajay Goel. Andrographis enhances 5-fluorouracil-induced anti-tumor effect in colorectal cancer through inhibition of DKK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1039.

Hypoxia induces inflammatory microenvironment by priming specific macrophage polarization and modifies LSC behaviour via VEGF-HIF1α signalling.

BackgroundLeukemia stem cells (LSCs) play pivotal roles in leukemogenesis, and are closely implicated in leukemia relapse and chemoresistance. LSCs are tightly modulated by hypoxic exposure and macrophage-conditioned microenvironment. Nevertheless, the impacts on the biology of LSCs imposed by the interaction of hypoxia and macrophage polarization remain elusive.MethodsIn the study, LSCs characterized by CD34+CD38− immunophenotype sorted from KG1α and primary AML cells were employed as in vitro and ex vivo cell models. mRNA and protein expressions of cytokine/chemokine of cells under normoxic and hypoxic conditions were determined by RT-PCR and western blot. Macrophage polarization, cell cycle and apoptosis were determined by flowcytometry. Cell viability was assayed by CCK-8.ResultsMacrophages preferentially presented with M2 polarization phenotype characterized by upregulated VEGF and CCL17 cytokine/chemokine profile, when stimulated by specific set of cytokines under hypoxic exposure, and induced an anti-inflammatory microenvironment. LSCs exhibited significantly increased cell viability, colony-forming capacity and chemoresistance when co-incubated in hypoxic conditioned medium (H-CM) primed by polarized M1 macrophages. VEGF expression was upregulated in LSCs which in turn activated survivin expression. VEGF-mediated upregulation of survivin could be abolished by inhibition of VEGF receptor, but not blocked by survivin-targeting siRNA. In addition, survivin upregulation exerted antiapoptotic effects and was associated with increased chemoresistance. Finally, VEGF mediated transcriptional induction of HIF-1α of LSCs coincubated in H-CM, and HIF-1α induction in turn enhanced chemoresistance and reduced cell apoptosis.ConclusionsTo our best knowledge, this is the first study that focus to explore molecule players and interacting signal pathways regulating LSC biology under hypoxic exposure. It reveals that hypoxia preferentially skew macrophage M2 polarization with specific cytokine profile and proinflammatory microenvironment, which impacts malignant behavior of LSCs. VEGF-HIF-1α interaction is closely implicated in sustaining LSCs survival under hypoxic exposure and might be of potential target of novel therapy.

Human Primary Airway Basal Cells Display a Continuum of Molecular Phases from Health to Disease in Chronic Obstructive Pulmonary Disease.

Airway basal cells are crucial for regeneration of the human lung airway epithelium and are believed to be important contributors to chronic obstructive pulmonary disease (COPD) and other lung disorders. To reveal how basal cells contribute to disease and to discover novel therapeutic targets, these basal cells need to be further characterized. In this study, we optimized a flow cytometry-based cell sorting protocol for primary human airway basal cells dependent on cell size and NGFR (nerve-growth factor receptor) expression. The basal cell population was found to be molecularly and functionally heterogeneous, in contrast to cultured basal cells. In addition, significant differences were found, such as KRT14 expression exclusively existing in cultured cells. Also, colony-forming capacity was significantly increased in cultured cells showing a clonal enrichment in vitro. Next, by single-cell RNA sequencing on primary basal cells from healthy donors and patients with Global Initiative for Chronic Obstructive Lung Disease stage IV COPD, the gene expression revealed a continuum ranging from healthy basal cell signatures to diseased basal cell phenotypes. We identified several upregulated genes that may indicate COPD, such as stress response-related genes GADD45B and AHSA1, together with with genes involved in the response to hypoxia, such as CITED2 and SOD1. Taken together, the presence of healthy basal cells in stage IV COPD demonstrates the potential for regeneration through the discovery of novel therapeutic targets. In addition, we show the importance of studying primary basal cells when investigating disease mechanisms as well as for developing future cell-based therapies in the human lung.

Abstract LB131: Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells

Abstract Aim: We investigated the mechanisms driving the concomitant overexpression of ABCG2 and CD133 in cancer stem cells (CSCs) and examined the effect of modulating the underlying machinery on anticancer drug response. Background: Multidrug resistance is a major obstacle to chemotherapy. It is usually associated with increased efflux of anticancer drugs by ATP-binding cassette transporters including ABCG2. ABCG2 is also a key survival factor for CSCs, which lead to recurrence. CSCs have been identified in patient tumor specimens and in cancer cell lines. The CSC hypothesis suggests that anticancer therapies have to target and destroy all residual CSCs in order to produce a durable response. CD133 is a well-characterized stem cell biomarker and it plays crucial role in the self-renewal and tumorigenesis of CSCs. Importantly, ABCG2 and CD133 are co-expressed in CSCs, yet the mechanism(s) driving their concomitant high expression remain elusive. mRNA polyadenylation is a key step regulating the maturation of eukaryotic mRNAs by adding a poly A tail to their 3' ends. Alternative polyadenylation, commonly observed in cancer cells, is a phenomenon responsible for the production of multiple mRNA isoforms bearing different length of 3'-untranslated region (3'UTR). Method: Sub-population of cancer cells from three ABCG2-overexpressing multidrug resistant colon cancer cell lines were sorted by Hoechst dye exclusion or CSC markers (DCLK1+LGR5+BMI1+), respectively, as side population (SP) and putative CSC cells. Expression of ABCG2, CD133, and a few master mRNA polyadenylation trans-acting regulators was compared in SP (or CSC) versus non-SP (or non-CSC) cells. Altered 3'UTR isoforms of ABCG2 and CD133 were examined by 3'RACE assay and 3'UTR isoform specific PCR. The anticancer activity of common chemotherapeutic drugs was examined after genetic modulation of the key mRNA polyadenylation regulators identified. Result: The mRNA stability and expression of both ABCG2 and CD133 were found to be remarkably higher in SP cells (or CSC cells) than in non-SP cells (or non-CSC cells). By 3'RACE assay, shortening of ABCG2 and CD133 3'UTR was evidenced in SP (and CSC) cells but not in non-SP (and non-CSC) cells, thereby increasing mRNA stability and/or translation by escaping the repression from regulatory elements found on long mRNA 3'UTR. Expression of two master mRNA polyadenylation regulators (PABPN1 and CSTF2) was found to be remarkably lower and higher, respectively, in SP (and CSC) than in non-SP (and non-CSC) cells, presumably allowing the preferential utilization of proximal 3'UTR to mediate high expression of ABCG2 and CD133. Genetic silencing of CSTF2 was shown to enhance the anticancer effect of SN-38 and reduce the colony formation capacity of CSC cells. Conclusion: A novel mRNA 3'UTR shortening machinery was shown to mediate the simultaneous overexpression of ABCG2 and CD133 in SP and CSC cells. It may represent useful drug target for circumvention of resistance and eradication of CSCs. Citation Format: Kenneth K.W. To. Shortening of mRNA 3′ untranslated region mediates simultaneous overexpression of ABCG2 and CD133 in putative cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB131.

Cellular and Molecular Comparison of Glioblastoma Multiform Cell Lines.

Glioblastoma multiform (GBM) is one of the most severe tumor types. It is highly invasive and characterized as a grade IV neoplastic cancer. Its resistance to chemotherapy-temozolomide (TMZ treatment)-in combination with tumor treating fields (TTFields), limits the cure of GBM. Therefore researchers are searching for new treatment options to increase the length of recurrence time and improve overall survival for GBM patients. Several cell lines have been established and are in use to understand the molecular basis of GBM and to test the developed drugs. On one hand, it is highly advantageous to utilize multiple cell lines with different genetic backgrounds to gain more insight into the characterization and treatment of the disease. However, on the other hand, characteristics of these cell lines such as proliferation rate, invasion, and colony formation capacity differ greatly among these cells. Hence, a detailed comparison concerning molecular and cellular features of commonly used cell lines is essential. In this study, cell proliferation and apoptosis rate, cell migration capacity, and gene expression profile of U87, Ln229, and SvGp12 cells have been investigated and compared.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 1 (IGF2BP1) Is a Prognostic Biomarker and Associated with Chemotherapy Responsiveness in Colorectal Cancer.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an RNA-binding protein and serves as a post-transcriptional fine-tuner regulating the expression of mRNA targets. However, the clinicopathological roles of IGF2BP1 in colorectal cancer (CRC) remains limited. Thus, we aimed to elucidate the clinical significance and biomarker potentials of IGF2BP1 in CRC. A total of 266 specimens from two sets of CRC patients were collected. IGF2BP1 expression was studied by immunohistochemical (IHC) staining. The Kaplan-Meier survival plot and a log-rank test were used for survival analysis. The Cox proportional hazards model was applied to determine the survival impact of IGF2BP1. Public datasets sets from The Cancer Genome Atlas (TCGA) and Human Cancer Metastasis Database (HCMDB), receiver operating characteristic (ROC) plotter, and two CRC cell lines, HCT-116 and DLD-1, were used for validating our findings. We showed that IGF2BP1 was overexpressed in tumor specimens compared to 13 paired normal parts by examining the immunoreactivity of IGF2BP1 (p = 0.045). The increased expression of IGF2BP1 in primary tumor parts was observed regardless of metastatic status (p < 0.001) in HCMDB analysis. IGF2BP1 expression was significantly associated with young age (59.6% vs. 46.7%, p-value = 0.043) and advanced stage (61.3% vs. 40.0%, p-value = 0.001). After controlling for confounding factors, IGF2BP1 remained an independent prognostic factor (HR = 1.705, p-value = 0.005). TCGA datasets analysis indicated that high IGF2BP1 expression showed a lower 5-year survival rate (58% vs. 65%) in CRC patients. The increased expression of IGF2BP1 in chemotherapy non-responder rectal cancer patients was observed using a ROC plotter. Overexpression of IGF2BP1 promoted the colony-forming capacity and 5-fluorouracil and etoposide resistance in CRC cells. Here, IGF2BP1 was an independent poor prognostic marker in CRC patients and contributed to aggressive phenotypes in CRC cell lines.

A Metabolic Interplay Coordinated By Hlx Balances Hematopoietic Stem Cell Maintenance and Differentiation

Upregulation of the non-clustered homeobox transcription factor HLX (H2.0-like Homeobox) is frequently observed in patients with acute myeloid leukemia, but its mechanism of action remains obscure. Since developmental pathways are often reactivated in cancer, we asked whether hlx1 plays a role during hematopoietic development in zebrafish. Endothelial specific overexpression of human HLX led to aberrant production of hematopoietic stem and progenitor cells (HSPCs) and increased numbers of immature myeloid cells at 48 hpf (hours post fertilization), thereby mimicking a preleukemic phenotype. On the other hand, hlx1 morphants had diminished numbers of HSPCs, a defect that could be rescued by overexpressing human HLX under control of either an endothelial or HSPC specific promoter. To delve into the molecular mechanism of HLX function, we performed expression analysis of sorted endothelial cells from zebrafish that overexpress HLX and hlx1 morphants. Overexpression of HLX led to downregulation of mitochondrial electron chain enzymes (NDUFS4, SDHD, CYTB, COX7A2) and upregulation of the peroxisome proliferator activated receptor family member δ (PPARδ). Downregulation of hlx1 led to diminished expression of PPARδ. Analysis of open chromatin by ATAC-seq revealed altered chromatin accesibility around the vicinity of these genes in hlx1 morphants. To validate functionally these results we measured the mitochondrial membrane potential by Tetramethylrhodamine, Methyl Ester, Percholate (TMRM). TMRM was decreased upon HLX overexpression and increased in hlx1 morphants. These results suggested that HLX regulates the fate of hematopoietic cells by fine tuning mitochondrial membrane potential and PPARδ. Indeed PPARδ agonists could rescue HSPC generation in zebrafish morphants, while PPARδ antagonists relieved the differentiation block of myeloid cells. Direct regulation of mitochondrial genes and PPARδ by HLX was established by chromatin immunoprecipitation in human cell lines. To test whether there is a conserved, intrinsic role of HLX in primary hematopoietic progenitor cells, we used human progenitor CD34+ cells. In accordance with our results in zebrafish, overexpression of HLX led to increased numbers of colonies in colony forming unit assays and a myeloid differentiation block, whereas downregulation greatly compromised the ability of CD34+ cells to form colonies. HLX overexpression resulted in diminished oxidative phosphorylation and mitochondrial membrane potential whereas downregulation of HLX coincided with increased mitochondrial membrane potential. PPARδ agonists and antagonists could rescue TMRM staining and the colony forming capacity in HLX knockdown CD34+ cells and alleviated the myeloid differentiation block upon HLX overexpression, respectively. In summary, our findings show that HLX balances hematopoietic stem and progenitor cell maintenance and differentiation by modulating their metabolic status. DisclosuresNo relevant conflicts of interest to declare.

Ca2+ Oscillations and NFATC1 -Activation Enable Oncogenic Signaling in Pre-B Cell Transformation and Leukemogenesis

Background: Nuclear factor of activated T cells (NFATC1) regulates activation and differentiation of B and T cells. Activation of NFATC1 requires sustained Ca2+ influx by store-operated Ca2+ entry (SOCE). Our group previously showed that oncogenic signaling from the BCR-ABL1 kinase in Ph+ ALL results in autonomous oscillatory Ca2+ signaling (Klein et al., J Exp Med 2004; Feldhahn et al., J Exp Med 2005). However, the significance of Ca2+ oscillations remained unclear. Here we examined the function of Orai and Stim1/2, the central mediators of SOCE, and the role of Ca2+ oscillations in the survival of BCR-ABL1 -driven pre-B ALL cells.Results: To investigate the consequence of inducible deletion of SOCE-effector genes, Orai1, Stim1, and Stim2, we have generated BCR-ABL1 - and CreERT2-transduced murine pre-B Orai1f/f and Stim1/2f/f cells. Both Orai1 - and Stim1/2 -deletion impaired Ca2+ influx from the extracellular space when Ca2+ uptake from cytosol to endoplasmic reticulum was pharmacologically inhibited. Consistently, both Orai1 - and Stim1/2 -deletion significantly attenuated the amplitude and frequency of autonomous Ca2+ oscillations, compared with that of empty vector controls. To determine whether the impaired Ca2+ signaling associated with cell survival and proliferation, we next analyzed their growth and colony-forming capacity. As expected, both Orai1 - and Stim1/2 -deleted pre-B ALL cells exhibited growth disadvantage and reduced colony-forming capacity, compared with empty vector controls. Flow cytometry studies of Annexin V revealed that both Orai1 - and Stim1/2 -deletion induced apoptosis in the BCR-ABL1 -transformed cells. These results suggest that autonomous Ca2+ oscillations are needed to circumvent apoptosis, and enhance cell growth and self-renewal in BCR-ABL-driven ALL cells. Interestingly, even in normal pre-B cells and in the absence of autonomous Ca2+ oscillations, both Orai1 - and Stim1/2 -deletion caused growth disadvantage suggesting the existence of similar cellular function to Ca2+ oscillations in normal pre-B cells. Subsequently, we studied NFATc1 activity by Western blot, as reflected by nuclear localization, in pre-B ALL cells before and after Orai1 - and Stim1/2 -deletion. NFATc1 is known as one of a central NFAT family member that is activated in lymphocytes upon BCR and TCR stimulation. While Orai1fl/fl and Stim1/2fl/fl B-lineage ALL cells transduced with empty vector controls displayed marked nuclear accumulation of NFATc1, Cre-mediated deletion of Orai1 and Stim1/2 strongly reduced nuclear accumulation of NFATc1, with slight increases in the cytoplasmic fraction. To examine the consequences of NFATc1 inhibition, we generated BCR-ABL1 - and CreERT2-transduced murine pre-B Nfatc1f/f and observed its effect on cell growth. As expected, inducible ablation of Nfatc1 resulted in rapid depletion of pre-B ALL cells from the cell culture compared to empty vector controls. Importantly, a NFAT-calcineurin association inhibitor, INCA-6, displayed growth inhibitory effects in all six patient-derived xenograft of B cell ALL tested, including samples from patients who relapsed after initially successful chemotherapy. These results suggest that the Ca2+-NFAT signal may suppress apoptosis, and promote drug-resistance and relapse in human pre-ALL cells.Conclusions: Taken together, our findings showed that SOCE has a critical role in NFAT activation and survival of BCR-ABL1 -driven pre-B ALL cells. Orai1- and Stim1/2-mediated autonomous Ca2+ oscillatory signal is required for BCR-ABL1-dependent proliferation. This signal may bypass the NFAT activation which suppress apoptosis and enhance proliferation in BCR-ABL-driven ALL cells. DisclosuresNo relevant conflicts of interest to declare.

Immunodepletion of MDSC By AMV564, a Novel Tetravalent Bispecific CD33/CD3 T Cell Engager Restores Immune Homeostasis in MDS in Vitro

Myelodysplastic syndromes (MDS) are age-dependent and genetically diverse hematopoietic neoplasms associated with inflammation, cytopenias, myeloid dysplasia and risk for acute myeloid leukemia (AML) progression. Mounting evidence indicates that emergence of somatic gene mutations arising in hematopoietic stem and progenitor cells (HSPC) with age plays an important role in MDS pathogenesis. Our investigations indicate that sustained activation of innate immune response directs inflammation in the bone marrow (BM) microenvironment that contributes to hematopoietic and immune impairment through local generation of inflammatory cytokines, expansion of regulatory T- cells, and up-regulation and activation of pattern recognition receptors. Critical cellular effectors are the inflammation-associated immature myeloid cells termed myeloid-derived suppressor cells (MDSCs). Human MDSCs, distinguished phenotypically by CD33high and HLA-DR−Lin−, expand and are activated in cancers. We previously reported that MDSCs are profoundly expanded in the local BM microenvironment of MDS where they contribute to MDS hematopoietic BM failure. MDSCs suppress T-cell responses and impair CD4+/CD8+ function, suggesting that this may be a key mechanism fostering clonal escape from anti-tumor immune response. Indeed, the MDSC population size limits the benefit of immune checkpoint antagonists in the treatment of solid tumors and effective strategies to deplete MDSCs are lacking. Therefore, we hypothesized the high CD33 surface density in MDSCs can be exploited for selective immune-depletion by the T-cell engager AMV564, a novel CD33/CD3 tetravalent bispecific antibody that recognizes both CD33 and CD3, currently in clinical development for AML (NCT03144245). Depletion of MDSCs may restore immune function, hematopoiesis and improve sensitivity to checkpoint inhibitors. Primary BM mononuclear cells (BMMNC) from 15 MDS patients were treated with AMV564 or isotype IgG control for 5-7 days in vitro . AMV564 treatment of MDS BMMNC eliminated CD33+ MDSCs in a dose-dependent manner and expanded CD4+ and CD8+ T-cells compared to controls (P ≤0.001). Proliferation of CD4+ and CD8+ T-cells, measured by Brdu incorporation, more than doubled with AMV564 treatment (P ≤0.001). IFNγ, a T-cell activation biomarker, dramatically increased in AMV564 treated cells indicating T-cells are fully activated by AMV564 at concentrations as low as 0.7 ng/ml (P ≤0.01). Moreover, depletion of MDSCs and immunological restoration was accompanied by significant improvement in colony-forming capacity after AMV564 treatment (P ≤0.01). Checkpoint inhibition alone with 10 µg/ml of anti-PD1 antibody did not result in reduction of MDSCs and T-cell activation in MDS BMMNCs, demonstrating that anti-PD1 alone is insufficient to appropriately activate T-cells or modulate MDSC cell number. Interestingly, combined treatment of MDS BMMNC with anti-PD1 and AMV564 yielded more than additive T-cell activation and increased colony forming capacity (P ≤0.01), indicating the potential to augment MDS-specific immune responses. Our findings demonstrate that AMV564 treatment selectively depletes MDSCs to reactivate T lymphocytes by improving both quantity and quality of immune responses leading to the improvement of hematopoiesis. These preclinical data provide a strong rationale for clinical investigation of this innovative approach in patients with MDS. DisclosuresGuenot:Amphivena Therapeutics, Inc.: Employment. Fox:Amphivena Therapeutics, Inc.: Consultancy.

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.

Blocking CD70/CD27 Signaling in Combination with Hypomethylating Agents Eradicates Human CD34+ AML Stem and Progenitor Cells in Vitro and In Vivo

Leukemia stem cells (LSCs) are the origin of acute myeloid leukemia (AML) and are resistant to standard therapeutic regimens resulting in frequent relapse of the disease and poor prognosis especially for AML patients not fit for intensive chemotherapy. Consequently, LSCs represent a major obstacle for AML therapy. We recently identified the interaction of the TNF ligand CD70 and its receptor CD27 on LSCs as a promising therapeutic strategy to target LSCs.In this study, we demonstrate for the first time that treatment with hypomethylating agents (HMA, azacitidine or decitabine) significantly up-regulates CD70 mRNA and protein expression on primary CD34+ stem/progenitor cells from newly diagnosed AML patients independent of the cytogenetic/molecular risk category in vitro (mRNA: 4.3±1.6 fold increase, FACS: 4.0±0.9 fold increase vs. vehicle treatment, n=15). Similarly, CD34+ stem/progenitor cells from AML patients after 5 days decitabine administration (20mg/kg by continuous intravenous infusion over 1 hour repeated daily) exhibited a significant increase in CD70 expression as analyzed by flow-cytometry (6.8±1.1 fold increase vs. diagnostic sample, n=6). In contrast, CD70 expression of CD34+ stem/progenitor cells from healthy donor bone marrow (n=3) was not affected by the treatment.Consequently, co-treatment of CD34+ stem/progenitor cells from AML patients of each risk group (n=3) with the HMA decitabine and a blocking αCD70 monoclonal antibody strongly reduced colony-forming and re-plating capacity in vitro compared to single agent treatment. To confirm and extend our findings towards disease-initiating CD34+ AML LSCs in vivo, we performed patient-derived AML xenografts (PDX) from one patient with intermediate and one patient with adverse cytogenetic risk profile. Combining decitabine treatment with CD70 blockade significantly reduced AML engraftment in blood, spleen and BM compared to controls. Moreover, co-treatment significantly reduced frequency and absolute numbers of CD34+CD38- and CD34+CD38-CD45RA- AML LSCs in the BM as analyzed phenotypically by FACS and eliminated human LSCs as analyzed in limiting dilution PDX experiments compared to single treatments. Importantly, CD34+ hematopoietic stem/progenitor cells from healthy human bone marrow (n=3) were only marginally affected by the co-treatment.In summary, combining HMAs with blocking CD70/CD27 signaling could represent a novel strategy to eradicate human LSCs. DisclosuresVan Rompaey:argenX: Employment. Leupin:argenX: Employment. De Haard:argenX: Employment.

Characterisation of Naive Immunological Profile of Umbilical Cord Blood: The Role of Mucosal-Associated Invariant T Cells and Functional Defects of T Cells and Natural Killer Cells

Patients undergoing allogeneic haematopoietic stem cell (HSC) transplantation are susceptible to infection and reactivation of viruses, such as cytomegalovirus, due to impairment of the T cell compartment following intensive immunosuppressive therapy. There is a growing interest in evaluating cell-mediated immunity to improve the management of these infections. Over the last two decades, umbilical cord blood (UCB) has emerged as an alternative source of cellular products for the treatment of a range of haematological disorders. UCB HSCs have a number of distinguishing characteristics which differentiate them from adult HSCs, including increased proliferation and increased in vitro colony forming capacity. The use of UCB as HSC grafts for allogeneic stem cell transplant has a number of advantages, including immediate availability and reduced risk of graft-versus-host disease despite donor-recipient HLA disparity. However, the limited number of HSCs present in a single UCB collection and incomplete immune reconstitution because of delayed HSC engraftment has restricted the use of cord blood in stem cell transplantation. In addition, UCB transplantation is associated with lack of transferred adoptive immunity, resulting in transplant related morbidity and mortality due to infectious complications. While the human neonatal immune system is considered to be immature compared with its adult counterpart, the role of the innate immune cells in the immunological naïvety is not fully understood.Here, we describe the collection and characterisation of UCB units collected from a series of Irish mothers who delivered term infants by elective caesarean at a major Irish maternity hospital. Pre analytical variables included maternal factors such as gestation at delivery, blood group, age, medical history and BMI, and neonatal factors such as birth weight and gender. Mononuclear cells (MNC) were isolated and the immunological profile was analyzed by flow cytometry for the surface markers CD3, CD4, CD5, CD8, CD19, CD34, CD45, CD56, CD161 and the Vα24Jα17, Vα7.2, Vδ1, Vδ2 and Vδ3 T cell receptors. Progenitor cell colony forming ability was assessed by CFU-GM and BFU-E assays. Stimulation of T cells, by cross-linking of CD3 and CD28 using tetrameric antibodies, and Natural Killer (NK) cells, with IL12/IL18 and target cells, was assessed by measurement of surface expression of CD107a and intracellular production of IFNγ, IL4 and IL17a.We showed no difference in the overall numbers of B cells, T cells (CD4+ and CD8+) and γδ T cells between UCB (n=23) and adult blood (n=4); however, NK cells (p<0.001) and mucosal-associated invariant T (MAIT) cells (p<0.0001) were significantly depleted in UCB. Stimulated UCB T cells and NK cells (n=17) showed significantly reduced IFNγ production compared to adult blood counterparts (n=4) (p<0.01) Following thaw of cryopreserved UCB MNC, we observed no significant change in CFU-GM, CD34+ cell concentration or immunological profile/function compared to fresh MNC (n=7).Our study suggests that reduced numbers of circulating innate immune cell populations, as well as reduced cytokine responses following stimulation, may contribute to the naïve immunological phenotype and hyporesponsiveness of UCB. Further investigation and manipulation of these innate immune cell populations could improve UCB transferred adoptive immunity potential and assist in the management of post allogeneic stem cell transplant viral infections. DisclosuresNo relevant conflicts of interest to declare.

Acquired Gene Mutation of MAP4K2 Identified from Myelodysplastic Syndromes Patients Confers Azacitidine Resistance

Myelodysplastic syndromes (MDS) is clinically characterized by ineffective hematopoiesis and frequent development of acute myeloid leukemia (AML). Accumulating literature has addressed a variety of abnormalities in MDS hematopoietic stem cells, including chromosomal abnormalities, genetic mutations and epigenetic changes. At present, however, these recent discoveries have hardly led to improvement in patient outcomes. Except for allogeneic stem cell transplantation as a potentially curative treatment, few treatment options are available. Among them, DNA methyltransferases inhibitor azacitidine has been widely used against MDS and AML with proven efficacy in overall survival improvement, but subsequent recurrence is inevitable even after marked response. In this study, we searched genomic mutations associated with azacitidine resistance in high risk MDS patients by next generation sequencing, using longitudinally collected samples from patients undergoing azacitidine treatment in our institution. Mutational analysis was performed by whole exome sequencing in 4 consecutive cases, with patients' BM samples taken at the point of diagnosis, during treatment, after relapse. Control sequencing was performed using T cell fraction from during-treatment samples. We verified variant allele frequency of the detected mutations by targeted deep sequencing, to identify gene mutations strongly related to clinical azacitidine resistance.We identified an acquired gene mutation of MAP4K2, which is a member of the serine/threonine protein kinase family and activated by TNF-α/TRAF2 signal. MAP4K2 is required for the efficient activation of JNKs by stress-induced pathway. The mutation was exclusively found in the region encoding the protein-binding domain, which is required for signal transduction of JNK and p38 pathway by binding MAP3Ks such as MEKK1. We introduced the mutated form of MAP4K2 retrovirally into the human AML cell line Kasumi-1, and assessed its impact on azacitidine resistance. We found that the MAP4K2 mutation recaptured azacitidine resistance without affecting proliferation in the absence of azacitidine. In addition, another AML cell line HL60 with MAP4K2 mutation demonstrated higher growth ratio compared with control vector or MAP4K2 wild type after azacitidine treatment. Moreover, Kasumi-1 cells harboring MAP4K2 mutation showed low apoptosis rate and increased colony forming capacity after azacitidine treatment. We knocked in the mutations into MAP4K2 alleles Kasumi-1 cells using CRISPR/Cas9 system and found that Kasumi-1 cells with heterozygous MAP4K2 mutation similarly showed azacitidine resistance compared with control cells.We examined whether associated with stress-induced cytokines are associated with MAP4K2 mutation-induced azacitidine resistance. We revealed that MAP4K2 mutation increased TNF-α expression after azacitidine treatment. In addition, the mutant-form of MAP4K2 exhibited enhanced stability compared with the wild type. These results imply that MAP4K2 mutation we detected in this study is gain of function mutation that confers altered cytokine signaling and growth advantage on MDS cells by stabilizing MAP4K2 protein.In summary, we found that MAP4K2 mutation in MDS patients who have lost azacitidine responsiveness. Mutant MAP4K2 played a causative role in azacitidine resistance. Targeting MAP4K2 has become an attractive therapeutic strategy to conquer azacitidine resistance in MDS. DisclosuresTsukamoto:Pfizer Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Research Funding; Astellas Pharma Inc.: Research Funding. Masamoto:Pfizer Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Astellas Pharma Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Kurokawa:Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Celgene Corporation: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Pfizer Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau.

Gain-of-Function of ASXL1 Truncating Protein in the Pathogenesis of Myeloid Malignancies

Introduction: Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We and others have reported that loss of ASXL1 impairs hematopoietic stem/progenitor cell functions (HSC/HPCs) and leads to myeloid dysplastic syndrome (MDS) in mice. It remains controversial whether truncating mutations in ASXL1 result in gain- or loss-of-function, or whether they confer dominant-negative activity in vivo . Answering this question is critical for determining how to target these mutations therapeutically.Methods: We generated a transgenic mouse model using HS321/45-vav vector that expresses the Asxl1 Y588X cDNA under the control of the Vav1 promoter. The transgene contains the entire coding region of mouse Asxl1 (full-length) with a stop codon mutation at the Y588 site (Asxl1 Y588X), leading to a production of truncating protein ASXL1aa1-587 in the hematopoietic system only. Utilizing the transgenic model, we performed a serial hematopoietic analyses, including peripheral blood (PB) counts, histological characterization on blood smear, tissue sections of femur and spleen, and flow cytometric analysis. Bone marrow(BM) transplantation was performed to identify the effects of truncating ASXL1aa1-587 on HSC/HPCs functions. RNA-seq and ATAC-seq analyses were performed to survey the differentially expressed genes in HSC/HPCs of WT and Asxl1 Y588X Tg mice. Liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation (IP) experiments were performed to identify the specific protein binding partner of ASXL1aa1-587. An epigenetic drug screening was used to identify specific targets to block the gain-of-function protein, ASXL1aa1-587, in the pathogenesis of myeloid malignancies in vivo.Results: The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patientswith ASXL1 mutations. Competitive transplantation of WT and Asxl1Y588XTg BM cells revealed that expression of A SXL 1 aa587 was associated with a higher percentage of CD45.2+ cells in the PB of the recipients, indicating that A SXL11-587aa expression increases HSC self-renewal capacity in vitro and in vivo. ATAC- and RNA-Seq analyses revealed that the ASXL11-587aa truncating protein expression results in more open chromatin in cKit+ cells compared to wildtype cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. LC-MS) and co-IP experiments showed that ASXL11-587aa acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hyper-sensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors.Conclusion: This study demonstrates that ASXL11-587aa plays a gain-of-function role in promoting myeloid malignancies and that BET bromodomain inhibitors significantly reduced the colony forming capacity of ASXL1Y588XTgHSPCs. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Lgr5 Functions As Negative Regulator of Wnt Signaling in B Cells and Is Critical for Self-Renewal of Normal and Transformed B Cells

Background and significance: The identification of leukemia-initiating cells (LIC) in myeloid leukemias allowed for focused therapeutic approaches to prevent drug-resistance and relapse. In B cell leukemia and lymphoma, LIC occur at a high frequency (Rehe 2013), are phenotypically diverse (Aoki 2015) and can arise from any stage of B cell development (Le Viseur 2008). While self-renewal of HSCs and other organ-stem cells is regulated by a developmental hierarchy, self-renewal in the B cell lineage is induced by positive selection events, i.e. induced by antigen-receptor (BCR) signaling when B cells bind to their cognate antigen.Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) is a Wnt target gene and through binding to its ligand R-spondin, Lgr5 modulates Wnt signaling strength. Lgr5 is widely used as stem cell marker in multiple organs, however its expression levels are very low in hematopoietic cells.Results: Unexpectedly, B cells upregulate Lgr5-expression by >20-fold, (i) when they express the pre-BCR for the first time and (ii) when they encounter antigen for the first time in germinal centers. Engagement of BCR signaling on B cell lymphomas strongly increased LGR5 expression, which was sensitive to inhibition of SYK and BTK kinases in the BCR pathway.In addition, in patients with pediatric or adult ALL, or B cell chronic lymphocytic leukemia (CLL), higher than median mRNA levels of LGR5 at the time of diagnosis were associated with poor clinical outcome and higher likelihood of drug-resistance and relapse. For these reasons, we studied the function of Lgr5 during normal B cell development and malignant transformation. Conditional ablation of Lgr5fl/fl alleles during earliest stages of B cell development (Mb1-Cre) resulted in a >100-fold reduction of absolute B cell numbers. In addition, inducible deletion of Lgr5 in cultured pre-B and mature B cells caused rapid cell death, demonstrating that Lgr5 function is an essential requirement for B cell development. Most studies in epithelial cells suggest a role of Lgr5 as potentiator of WNT-signaling. However, Cre-mediated deletion of Lgr5 in B cells caused cell death in parallel with massive accumulation of nuclear b-catenin. Our genetic experiments in leukemia models demonstrated that LGR5 is critical for leukemia-initiation and survival. Cre-mediated deletion of Lgr5 and b-catenin accumulation abolished colony forming capacity and the ability of leukemia cells to initiate fatal disease in transplant recipients. Likewise, inducible activation of a gain-of-function mutant of b-catenin resulted in rapid cell death in B cells.Conclusion: Lgr5 is widely established as a marker of organ stem cells with self-renewal capacity. In the absence of the developmental stem cell hierarchy in B cells, we conclude that Lgr5 expression is a biomarker of positive selection in B cells. Lgr5-expression and positive selection is induced by BCR-engagement (e.g. by antigen) or oncogenic mimics of BCR signaling in B cell malignancies (e.g. transforming oncogenes that engage the BCR pathway). Unlike in epithelial cells, LGR5 expression in B cells restricts the levels of nuclear b-catenin and enables B cell survival through negative regulation of Wnt-signaling. DisclosuresNo relevant conflicts of interest to declare.

The Combination of GROβ and AMD3100 Leads to Rapid and Robust Mobilization of Hematopoietic Stem Cells in Nonhuman Primates

▪Introduction: Mobilized peripheral blood grafts are currently the predominant source of hematopoietic stem and progenitor cells (HSPC) for both autologous and allogeneic transplantation. The most common clinical hematopoietic stem cell mobilization protocol is five days of Filgrastim (G-CSF). This regimen requires daily injections, has been associated with bone pain and often results in unpredictably low yields. A rapid mobilization method that ideally only required a single treatment and had robust and predictable kinetics would be a significant improvement over the current standard of care. In mice, a unique CXCR2 agonist, GROβ, induces rapid mobilization of stem and progenitor cells 15 minutes after a single injection. When co-administered with plerixafor (AMD3100), an inhibitor of CXCR4, a synergistic increase in mobilization results, with a graft enriched in highly engraftable hematopoietic stem cells (Hoggatt et al., Blood 2016 128:368). Here, we present data demonstrating that combination treatment with GROβ and AMD3100 synergistically mobilizes CD34+ cells and colony forming units (CFU) in nonhuman primates (NHP).Methods: Cynomolgus macaques were injected with G-CSF (10 µg/kg/day, s.c. for four days), AMD3100 (1 mg/kg, s.c. once) or GROβ (various doses and routes) alone and in combination with AMD3100. Blood was collected at various times post treatment and analyzed by multicolor flow cytometry to quantitate HSPC numbers. Additional aliquots of mobilized blood were plated in methylcellulose and CFU were enumerated seven days later.Results: We tested the effect of GROβ alone or in combination with AMD3100 in Cynomolgus monkeys. As published previously, administration of G-CSF, AMD3100 or G-CSF+AMD3100 induced significant mobilization of hematopoietic stem and progenitor cells. In comparison, a single injection each of GROβ+AMD3100 yielded 4-fold more CD34+ cells compared to four days of G-CSF (p < 0.01). While the combination of G-CSF+AMD3100 mobilized significantly more CD34+ cells compared to GROβ+AMD3100 (p < 0.0001), the numbers of CD34+ CD90+ CD45RA- cells, representing the most primitive of hematopoietic stem and progenitor cells, were equivalent between treatment groups. Similarly, GROβ+AMD3100 treatment elicited 7-fold more CFU compared to animals treated with G-CSF (p < 0.01), 3.5-fold more CFU compared to AMD3100 (p < 0.01) and equivalent CFU compared to G-CSF+AMD3100. Importantly, the frequency of CFU among the CD34+ cells was significantly greater with GROβ+AMD3100 (1 in 4) compared to G-CSF (1 in 10) or G-CSF+AMD3100 (1 in 25). In contrast to the increased CD34+ numbers and CFU, white blood cell (WBC) and neutrophil counts were significantly lower with GROβ+AMD3100 compared to AMD3100 alone or G-CSF+AMD3100.Conclusions: We describe a rapid mobilization method that within four hours of a single treatment results in robust hematopoietic mobilization in nonhuman primates. The GROβ+AMD3100 regimen enriches for primitive CD34+ CD90+ CD45RA- stem and progenitor cells with increased colony forming capacity compared to CD34+ cells mobilized with G-CSF+AMD3100, suggesting a significant graft quality difference with the new regimen. The ability to rapidly mobilize primitive HSC with increased CFU frequency may allow effective single day mobilization of HPSCs for patients requiring a HSCT and be especially useful for HSC based gene therapy and gene editing protocols where HSPC grafts enriched for primitive HSC may reduce manufacturing costs and ensure durability of the gene modified HSC. The ability to achieve increased HSPC numbers without dramatically increasing WBC and neutrophils may be especially advantageous in patient populations where G-CSF mobilization is not well tolerated such as Sickle Cell Disease, or where G-CSF leads to increased disease activity such as in autologous transplant for Multiple Sclerosis. A single treatment that results in robust mobilization that is equivalent to G-CSF may allow for a one-day collection method of donor stem cells, and these findings in nonhuman primates support clinical exploration. DisclosuresFalahee:Magenta Therapeutics: Employment, Equity Ownership. Goncalves:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Morrow:Magenta Therapeutics: Employment, Equity Ownership. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

The Traf2 and Nck-Interacting Kinase Is a Conserved Regulator of Self-Renewal in Hematopoietic and Leukemia Stem Cells

The Traf2 and Nck-interacting kinase (TNIK) is a member of the germinal center kinase (GCK) family. We have recently found that signaling through the tumor necrosis factor superfamily receptor CD27 activates Wnt target genes through Traf2-TNIK-β-catenin signaling, promoting leukemia development and progression in acute and chronic myeloid leukemia (AML and CML).To explore how TNIK signaling regulates hematopoietic and leukemia stem cells (HSCs and LSCs), we generated a constitutive TNIK-/- knockout mouse.Under steady-state conditions, total bone marrow (BM) cellularity of naïve TNIK-/- mice was slightly reduced (mean BM count (x108): TNIK-/- = 1.42; TNIKwt/wt = 2.15, p < 0.0132). In contrast, no differences in absolute number of lineage negative (Lin-) cells, as well as the more primitive subset Lin-; Sca1+; c-kit+ (LSK) cells could be observed. Similarly, proportions of long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs) and multipotent progenitors (MPPs) did not differ between TNIK-/- and TNIKwt/wt mice. Functionally, naïve TNIK-/- and TNIKwt/wt HSCs displayed in vitro a comparable serial colony formation capacity in methylcellulose. Altogether these data suggest that TNIK does not affect the maintenance and self-renewal of HSCs under steady-state conditions.Next the impact of TNIK on hematopoietic recovery after genotoxic stress was analyzed in vivo. TNIK-/- and TNIKwt/wt mice were injected i.p. with 5-Fluorouracil (5-FU) (150mg/g bodyweight) and BM cellularity as well as HSC phenotype and function were assessed 9 days post injection. The total BM cellularity was significantly higher in TNIK-/- mice compared to controls (mean BM count (x106/ml): TNIK-/- =10.25; TNIKwt/wt =4.55, p< 0.0382). Furthermore, LSKs were significantly reduced in frequency in TNIK-/- mice (% LSK of whole BM: TNIK-/- = 2.93; TNIKwt/wt = 7.073, p < 0.0009) but not in absolute numbers. Interestingly, however, the total number LT-HSCs was significantly reduced in the BM of TNIK-/- mice (mean LT-HSC count (x103): TNIK-/- = 5.628; TNIKwt/wt = 16.725, p < 0.0101). These results were functionally confirmed in vivo by competitive BM transplantation (BMTx) of whole BM from 5-FU treated mice into lethally irradiated secondary recipients. Mice receiving BM from 5-FU-treated TNIK-/- mice showed a significantly reduced peripheral engraftment capacity 6 weeks post BMTx compared to controls (mean peripheral blood chimerism: TNIK-/- = 64.6%; TNIKwt/wt = 35.4%, p < 0.0001), suggesting that TNIK signaling mediates HSC maintenance during genotoxic stress conditions in vivo.In parallel the role of TNIK signaling was analyzed in myeloid LSCs. CML-like disease was induced in mice by transplanting pMSCV-BCR-ABL1-IRES-GFP transduced TNIK-/- or TNIKwt/wt LSKs into BL/6 mice. Even though CML developed similarly as indicated by measuring BCR-ABL1-GFP+ granulocytes in the peripheral blood, primary TNIK-/- CML mice survived significantly longer compared to TNIKwt/wt CML mice (Median survival (days): TNIK-/- = 33, TNIKwt/wt = 23.5, p < 0.0216) with 2 out of 7 mice surviving long-term. Next, we determined the effect of TNIK signaling on the functionality of FACS sorted Lin-; GFP+; Sca1+; c-kit+ LSC in vitro in serial colony forming assays and in vivo in secondary transplantations. Serial colony formation capacity of TNIK-/- CML LSCs was significantly reduced after re-plating in vitro (CFUs fold change (TNIK-/- vs TNIKwt/wt): 1st Plating = 0.89, P<0.5149, 2nd Plating = 0.42, 9 < 0.0002). In line with these findings, only 1 out of 9 mice secondarily transplanted with LSCs from primary TNIK-/- CML mice developed CML within 90 days, while all 9 recipients receiving TNIKwt/wt LSCs succumbed to the disease within 23 days after transplantation (p < 0.0001). These results suggest that TNIK signaling also promotes self-renewal of CML LSCs.To summarize, we have described for the first time TNIK signaling as a commonly conserved regulator in stress-induced hematopoiesis and CML, which promotes stem cell self-renewal and maintenance. As TNIK inhibitors are currently developed for the treatment of different kinds of solid tumors, TNIK may be an attractive therapeutic target for LSCs in myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

ETO2 Regulates Cell-Fate Genes and Controls Relapse in Acute Myeloid Leukemia

The cell-fate of hematopoietic stem cells (HSCs) and their malignant version, leukemia stem cells (LSCs), are controlled by antagonistic functions of two classes of genes. We defined these genes as pro-HSC/LSC and anti-HSC/LSC genes based on their abilities to increase or decrease the steady state numbers of stem cells. Expansion of LSCs is thought to be an early step towards relapse (Lapidot, Sirard et al. 1994, Huntly and Gilliland 2005, Vasanthakumar and Godley 2014), a medical condition with no effective cures. We show here that the expression of both pro- and anti-HSC/LSC genes are coordinately controlled by ETO2, a transcriptional corepressor of E-protein family of transcription factors (Zhang, Kalkum et al. 2004, Gow, Guo et al. 2014). In both HSCs and acute myeloid leukemia (AML) cells, ETO2 occupies active chromatin regions that encompass enhancers and promoters of pro- and anti-HSC/LSC genes. ETO2 orchestrates the binding of epigenetic modifiers and transcription factors to enhance the expression of self-renewal and survival genes while reducing the expression of growth arrest and pro-inflammatory genes. Further supporting a role of ETO2 in maintaining the self-renewable population of HSCs and LSCs, depleting ETO2 compromises self-renewal of HSCs and diminishes in vivo propagation of AML cells.Analysis of multiple patient datasets supports the conclusion that relapse requires a threshold level of ETO2 expression. Attaining this threshold level requires a conserved gene activation mechanism mediated by GCN5 histone acetyltransferase. Inhibiting GCN5 reduces ETO2 and abolishes colony-formation capacity of primary AML cells. We also show that the conserved and diverged functions between ETO2 and AML1-ETO, a leukemia fusion protein expressed in t(8;21) AML, combined with a newly-discovered ability of AML1-ETO to repress ETO2 transcription, defines the unique properties of t(8;21) AML and explains its long-observed favorable prognosis. Importantly, we show that despite an enriched expression of HSC/LSC genes, relapse is paused in t(8;21) AML due to AML1-ETO-mediated downregulation of ETO2 and requires overcoming this repression to proceed. Thus, targeting GCN5/ETO2 axis to achieve a similar effect as that imposed by AML1-ETO is expected to dramatically improve therapeutic efficacy of AML, whose current survival rate is worse than most cancers and leukemias (Supported by NIH grants R01HL093195-01A1 and R21CA178513 to JZ and T32 training grant T32GM008306-26A1 to NS).Gow, C. H., C. Guo, D. Wang, Q. Hu and J. Zhang (2014). “Differential involvement of E2A-corepressor interactions in distinct leukemogenic pathways.” Nucleic Acids Res 42(1): 137-152.Huntly, B. J. and D. G. Gilliland (2005). “Leukaemia stem cells and the evolution of cancer-stem-cell research.” Nat Rev Cancer 5(4): 311-321.Lapidot, T., C. Sirard, J. Vormoor, B. Murdoch, T. Hoang, J. Caceres-Cortes, M. Minden, B. Paterson, M. A. Caligiuri and J. E. Dick (1994). “A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.” Nature 367(6464): 645-648.Vasanthakumar, A. and L. A. Godley (2014). “On the origin of leukemic species.” Cell Stem Cell 14(4): 421-422.Zhang, J., M. Kalkum, S. Yamamura, B. T. Chait and R. G. Roeder (2004). “E protein silencing by the leukemogenic AML1-ETO fusion protein.” Science 305(5688): 1286-1289. DisclosuresNo relevant conflicts of interest to declare.

Iron Overload Results in Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation By Impairing Hematopoiesis

Background:Clinical data suggest that pre-transplant iron overload due to high transfusion requirements may affect graft function after allogeneic hematopoietic stem cell transplantation (allo-HSCT) .OBJECTIVE:To determine the impact of iron overload and its underlying mechanism on poor graft function (PGF) .METHODS:In the setting of allo-HSCT, a BALB/C mice and a C57/BL6 (H-2b) were used as recipient and donor, respectively. Recipient mice were injected intraperitoneally with iron sucrose with the cumulative doses of 50 mg, 100 mg, and 200 mg in three experimental groups, while the control with phosphate-buffered saline. All mice received total body irradiation for HSCT. Plasma iron levels and Iron stores of liver, spleen and bone marrow (BM) were measured. Hematological parameter, CD34+ cells and the hematopoietic colony-forming capacity were monitored to evaluate the hematopoietic recovery. Bone marrow derived mesenchymal stem cells (BM-MSCs) were isolated and assessed for proliferation. Immunohistochemical analysis assessed the BM microenvironment, expression of haematopoietic chemokines and immunological parameters.RESULTS:Iron overload aggravated PGF as observed by the results that peripheral blood cells were significantly lower in number and recovered less rapidly with a dose-dependend manner, compared with that of controls (P<0.05). In the experimental groups, BM was hypocellular, CD34+ cell counts and colony-forming units were significantly decreased (P<0.05). BM-MSCs showed a longer double time than control (P<0.05). Furthermore, the immunohistochemical analysis demonstrated the immunological impairment and the low expression of stem cell factor -1 and vascular endothelial growth factor-1.CONCLUSION:Current evidence shows that iron overload is an important risk factor for PGF by damaging hematopoietic stem cells and the microenvironment, which highlights the need for optimizing iron management to improve the transplantation outcomes. DisclosuresNo relevant conflicts of interest to declare.