Normal Stem Cell Function Research Articles

Pathobiology of cancer metastasis: a short account

Cancer-initiating cells display aberrant functional and phenotypic characteristics of normal stem cells from which they evolved by accumulation of multiple cytogenetic and/or epigenetic alterations. Signal transduction pathways which are essential for normal stem cell function are abnormally expressed by cancer cells, with a cancer cell phenotype playing an essential role in cancerization and metastasis.Local tumour progression, metastasis and metastatic tumour growth are mediated by direct cell-to-cell and paracrine reciprocal interactions between cancer cells and various stromal cells including fibroblasts, macrophages, bone marrow derived stem cells and progenitor cells. These interactions mediate breakdown of basement membrane barriers and angiogenesis both locally at the invasive front of the primary tumour and at the distant metastatic site; attract primary tumour cells to the candidate metastatic site; and promote proliferation, survival and growth of primary tumour cells and of metastatic cells at their distant site.It is the purpose of this article to highlight the analogies between some of the genetic programs of normal stem cells, and of cancer cells participating in the process of metastasis.

PD08-05: Stat3 Signaling in Human Breast Cancer Stem Cells.

Abstract Recent data suggest the existence of a unique subset of breast cancer cells capable of initiating tumor growth and giving rise to all other cells characteristic of a given tumor. These cells have been termed “cancer stem cells” or “tumor-initiating cells”. Breast cancer stem cells appear to be resistant to chemo- and radiotherapies and may be responsible for recurrence and metastasis in breast cancer patients. Identifying the cellular signaling pathways responsible for breast cancer stem cell maintenance and self-renewal represents a critical hurdle for developing effective therapeutics. The Stat3 pathway is a critical regulator of the function of normal stem cells, and shows altered expression in human breast cancers [4]. Moreover, IL-6, the Stat3 signaling agonist, is required for breast cancer stem cell function in human breast cancer cell lines. All these suggest an important role of Stat3 signaling in breast cancer stem cells. However, due to lack of method for pathway-activity-based live cell separation, whether Stat3 functions in the cancer stem cells themselves or whether it may function in surrounding niche cells remain unknown. We have constructed a lentiviral fluorescent reporter for Stat3 signaling which contains four copies of M67 Stat3 binding sites upstream of enhanced Green Fluorescent Protein. This reporter system enables FACS-sorting of cells with active Stat3 signaling and in vivo/in situ localization of Stat3 responsive cells. In addition, C188-9, the first Stat3 specific competitive inhibitor, has been developed in our collaborator's lab, which provides us an advanced tool in studying Stat3 function. We hypothesize that Stat3 signaling is preferentially active in stemlike subpopulation and depend on non-stem cancer cells to maintain its activation. Three aims to test this hypothesis include: 1. To test whether cancer cells with activated Stat3 signaling are enriched for breast cancer stem cells or whether they may serve as niche cells. 2. To test whether small molecule antagonists of Stat3 signaling can inhibit cancer stem cell function. 3. To identify novel targets of Stat3 signaling that may serve as indicators of responsiveness to Stat3 antagonist or predict treatment response. So far, our GFP reporters for Stat3 signaling effectively report Stat3 activity in both patient xenografts and human breast cancer cell lines in vivo and in vitro, which enables effective separation of Stat3+/Stat3- cells for functional Study. MDA231 and SUM159 tumors with reporters have been established. Tumor cells will be sorted according to Stat3 activity and perform limiting dilution and MSFE assays. Tumors with reporters will also be treated by C188-9, Chemo, or in combination to study synergistic effect of Stat3 inhibitors on tumor growth. Preliminary data based on inhibitor and limiting dilution studies suggested that Stat3 signaling is required for stem cell function. However, neither Stat3+ nor Stat3- cells are self-sufficient in performing these functions, indicating communications between subpopulations. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-05.

Socs2 Is Dispensable for BCR/ABL1-Induced Chronic Myeloid Leukemia-Like Disease in Mice and for Normal Hematopoietic Stem Cell Function,

Abstract 3743 Background:The P210 BCR/ABL1 fusion protein initiates signaling through several down stream pathways such as STAT5, PI3K, AKT, RAS, and WNT. However, only few down stream mediators have so far been thoroughly studied in vivo in the context of BCR/ABL1-mediated induction of CML-like disorder in mice deficient for the specific genes. Suppressor of cytokine signaling 2 (SOCS2) is known as a feedback inhibitor of cytokine signaling, a negative regulator of the STAT5 pathway, and is markedly upregulated in primary bone marrow cells from patients with chronic myeloid leukemia (CML). However, it has not been clear whether SOCS2 is involved in BCR/ABL1 induced cell transformation or whether it is important for normal hematopoietic stem cell (HSC) function. Methods:To evaluate the stem cell function of Socs2, HSCs from wild type and Socs2 deficient mice were competitively transplanted into lethally irradiated recipients. Blood chimerism and lineage distribution were analyzed by flow cytometry at 4 and 18 weeks after transplantation. To investigate the potential role of Socs2 in CML, c-kit enriched Socs2(−/−) and wild type cells were transduced with a BCR/ABL1 expressing retroviral vector and transplanted into lethally irradiated recipients. The in vivo development of malignant disease was analyzed by peripheral blood cell counts and, upon euthanization, by flow cytometry and histopathology. Socs family gene expression was assessed by Q-RT/PCR and Stat5 phosphorylation by Western blotting on BCR-ABL1 expressing cells. Results:Although Socs2 was previously found to be upregulated in long-term repopulating HSCs by gene expression arrays, Socs2 deficient HSCs were indistinguishable from wild type HSCs when challenged in competitive bone marrow transplantation. Furthermore, when expressing BCR/ABL1, both Socs2 deficient and wild type cells induced a CML-like disease with an average survival of three weeks after transplantation. The leukemic mice suffered from elevated white blood cell counts and splenomegaly. To investigate if other Socs family members were upregulated to compensate for the Socs2 deficiency, we compared the expression of the Socs gene family members in Socs2(−/−) with wild type, leukemic cells. The expression levels of all other Socs genes were similar between Socs2(−/−) and Socs2 wt cells, suggesting that compensatory mechanisms of other Socs genes do not account for the lack of Socs2 function. In addition, the finding that the phosphorylation of Stat5 was unaffected in Socs2(−/−) cells indicates that BCR/ABL1-induced Stat5-phosphorylation is insensitive to Socs2 levels. Conclusions:Collectively, our results clarify that Socs2 is dispensable for normal HSC function and for BCR/ABL1-induced CML-like disease. Disclosures:No relevant conflicts of interest to declare.

Hijacking the Niche: The Role of Stromal Osteopontin in the Induction of Leukemic Dormancy within the Bone Marrow Microenvironment

Abstract 754 Introduction:Acute lymphoblastic leukemia (ALL) is a treatable malignancy where initial induction chemotherapy achieves clinical remission in the majority of patients. Relapsed disease, however, occurs in many patients and is significantly more difficult to treat. The majority of relapsed cases are a result of minimal residual disease (MRD) that persists within the bone marrow (BM) after initial chemotherapy. Our evolving knowledge of the importance of the host microenvironment in tumor progression suggests that the stromal microenvironment can protect leukemic cells from chemotherapeutic assault, and that inhibiting the supportive relationship between leukemic blasts and the bone marrow microenvironment (BMM) will provide novel therapeutic opportunities. We therefore aimed to identify and characterize novel stromal signaling mechanisms that retain and support blasts within the malignant BMM. Our preliminary data suggest that osteopontin (OPN), normally secreted by osteoblasts within the marrow, is one such signaling chemokine that is highly upregulated in the leukemic niche. OPN has well-defined roles in both solid tumor metastasis and normal hematopoietic stem cell function within the BMM. Specifically, OPN expression at the endosteal bone surface functions to recruit hematopoietic progenitors to bone where they are induced to become quiescent and maintain long term repopulating potential. We hypothesized that a similar relationship exists between leukemia and OPN resulting in a quiescent population of chemoresistant leukemic blasts at the BM endosteum. Here, we demonstrate that stromal OPN negatively regulates leukemia cell proliferation in the BMM. Methods:A GFP expressing clone of the pre-B ALL cell line Nalm-6 (10 × 106 cells) was engrafted into SCID hosts (6-8 weeks old) via tail vein injection. In vivo imaging was accomplished in live anesthetized mice by reflecting the scalp and imaging the calvarial marrow compartment using real-time multi-photon confocal microscopy. OPN expression in the malignant marrow was imaged by injecting engrafted mice with fluorescently conjugated anti-OPN antibodies 18hrs prior to imaging. For OPN neutralizing experiments, engrafted mice were injected with a cocktail of anti-mouse and anti-human OPN antibodies at a dose of 3 mg/kg. Results:Using PCR, Western blot and ELISA assays, we show that the ALL cell line Nalm-6 expresses OPN and secretes large quantities of OPN into conditioned media. Flow cytometric analysis demonstrates that Nalm-6 also express the cell surface OPN receptors VLA-4 and VLA-5. Furthermore, Nalm-6 cells specifically adhere to OPN in vitro via specific engagement of these integrin receptors. In vivo imaging demonstrates that OPN expression in the BM increases as leukemia progresses and that OPN is highly expressed adjacent to areas of high tumor burden. Specifically, a significant amount of OPN is detected in bony tunnels surrounding the vasculature at the invading tumor front. Using Q-PCR and western analysis, we demonstrate that both host-derived and leukemia-derived OPN are upregulated in malignant BM. In vivo inhibition of the OPN signaling axis in the Nalm-6 xenograft model using neutralizing antibodies directed at both human and murine OPN increased overall tumor burden two-fold as measured by flow cytometry and in vivo imaging (p=0.02, N=7) while simultaneously increasing the Ki-67 positive proliferative tumor population (p=0.029, N=4). Furthermore, IHC analysis of a panel of diagnostic bone marrow biopsies from a diverse cohort of ALL patients demonstrated high OPN expression in the marrow of these patients. Conclusion:Leukemic blasts that have hijacked normal stromal interactions to become quiescent may represent a major source of MRD and patient relapse in ALL. Our data demonstrate that the interaction of leukemic blasts with OPN in the stromal microenvironment reduces the number of cycling blasts and constrains tumor proliferation within the marrow. Current investigations are aimed at combining OPN neutralization with an in vivo model of MRD to determine whether OPN neutralization induces cycling of quiescent blasts, ultimately rendering them sensitive to chemotherapy. The ultimate goal of this work is the development of clinically relevant therapies designed to render leukemic cells more susceptible to chemotherapy by disengaging them from protective interactions with the BM microenvironment. Disclosures:No relevant conflicts of interest to declare.

FLVCR Deletion Does Not Impact Normal Hematopoietic Stem Cell Function but Is Necessary for Erythroid and T-Cell Maturation and Enhances Platelet Formation

Abstract 2363Hematopoietic stem cells (HSCs) are mainly in a quiescent state, but activate and proliferate in response to stress. To maintain quiescence, as well as respond to activation signals, HSCs must coordinately regulate cell cycle status versus metabolic demand. Mismatch in these pathways can lead to HSC exhaustion, failure or malignant transformation. One metabolic pathway that is essential to all cells is heme biosynthesis. Heme can act directly as a signaling molecule by modulating the function of transcriptional (Bach1) or translational (eIFa2 kinase) regulators. In addition, heme can act indirectly through the action of hemoproteins (hemoglobin, oxidases, peroxidases, cytochromes). However, excess free intracellular heme is toxic, therefore organisms have developed “safety valve” pathways to regulate intracellular heme concentrations. The most well studied mechanism is free heme degradation by the heme-oxygenase pathway. We have recently described a second pathway to prevent intracellular heme excess, namely heme export by the cytoplasmic transporter FLVCR. Previously we have shown that FLVCR is essential for erythropoiesis, as FLVCR−/− mice die of anemia (Keel et. al., Science 319: 825, 2008). FLVCR is expressed in many other tissues and blood cells, including HSCs (Lucas et. al., Blood 106:51, 2005). This suggests that FLVCR may be important in HSC function or the development of other lineages, besides red cells. We used a competitive transplantation model to determine the effect of FLVCR deletion on HSC function and lineage maturation. FLVCR−/− or wild type (WT) donor cells and equal numbers of competitor cells were transplanted into lethally irradiated, syngeneic recipients. FLVCR−/− marrow cells reconstitute the granulocyte compartment equally as well as WT cells in serial transplantation (up to tertiary transplants). Equal contributions also remain after myelotoxic stress (radiation or arsenic trioxide). Therefore, FLVCR may be dispensable for normal HSC function under steady-state and stress conditions. As anticipated, FLVCR is required for proerythroblast survival and maturation. Eight weeks after competitive transplantation with FLVCR−/− donor cells, 100% of red cells are derived from competitor cells. This is also seen when the ratio of FLVCR−/−:competitor cells is increased to 95:5 from 50:50. FLVCR−/− cells fail to mature past the proerythroblast stage (CD71pos lin-Ter119neg or lo) and undergo significantly more apoptosis than WT donor cells (88% vs. 36%, p<0.001). FLVCR deletion also blocks T-cell development. Due to biological variability in our experimental model, T-cell numbers were normalized to granulocytes in each determination, in each mouse and expressed as a percentage of the expected value. At 5 weeks post-transplant, the percentage of FLVCR−/− derived T-cells was 65.8% of the percentage of FLVCR−/− derived granulocytes. The percentage of WT derived T-cells was 93.4% of the percentage of WT derived granulocytes (p<0.02). Over time, FLVCR−/− derived T-cells steadily decreased, dropping to 22% of expected values by week 24, and remaining low, whereas T-cells derived from control, WT donor cells remained stable (74–98% of expected values). FLVCR−/− T-cells are arrested at the double positive (CD4+CD8+) stage (Philip et. al., Blood (ASH Abstracts) 114:913, 2009). Surprisingly, loss of FLVCR improves platelet production. FLVCR−/− donor cells make up ∼60% of all platelets (avg. 1.71 x expected, when normalized to granulocytes). In the separate control cohort competitively transplanted with WT donor cells, ∼40% of the platelet population was derived from WT cells (avg. 0.91 x expected, when normalized to granulocytes). Our studies suggest that the increased platelet production likely results from increased megakaryocyte ploidy (e.g. avg. 14.2N in FLVCR−/− vs 5.2N in control) and not from increased megakaryocyte progenitors. Of note, FLVCR−/− deleted mice have thrombocytosis (2221/μL vs 1008/μL, p<0.001 in littermate controls), a finding we initially dismissed as a stress response to severe anemia. Together, these findings reveal stage specific roles for FLVCR in discrete hematopoietic lineages and support the broader hypothesis that heme excess might impair cell division, leading to decreased red and T-cell production, but improved endomitosis and increased platelet production. Disclosures:No relevant conflicts of interest to declare.

FLT3-ITD Knock-in Impairs Hematopoietic Stem Cell Quiescence/Homeostasis, Leading to a Myeloproliferative Neoplasm

Abstract 46In murine hematopoiesis, long-term hematopoietic stem cells (LT-HSCs) are classically defined as Fms-like tyrosine kinase (Flt3) negative with Flt3 positivity often serving as a marker for differentiation and loss of self- renewal capacity. Flt3 has previously been shown to be dispensable for HSC maintenance and myeloid development. Flt3 and Flt3-ligand knockout mice have minor defects in overall hematopoiesis. However, these studies fail to take into account the effects of overlapping or compensatory pathways that may mask important roles Flt3 may play in HSC homeostasis. To date, few studies have addressed the effects of constitutive Flt3 activity on normal hematopoiesis or stem cell function. These mutations in FLT3 are of particular importance as they comprise one of the most common molecular alterations found in human acute myeloid leukemias (AML). Internal tandem duplication (ITD) mutations in FLT3 render the receptor tyrosine kinase constitutively active and portend a poor prognosis for those patients carrying the mutation. Many of these patients respond only transiently to treatment, possibly due to a reservoir of stem-like cells that are able to escape therapy. To study the effects of expression of constitutive activation mutations of FLT3 in primitive HSC compartments, our lab has developed a murine knock-in model in which this mutation is expressed under control of the endogenous murine Flt3 promoter. Flt3-ITD mice develop a myeloproliferative neoplasm (MPN), display imbalances in hematopoietic stem and progenitor cell population distributions (of note, 2–5 fold reductions in primitive LT-HSCs in SLAM (LIN-CD150+CD48−) and KSL-SLAM (Kit+Sca+Lin-CD150+CD48−) populations) and HSC defects (as measured by significantly reduced engraftment potential). Here, we show these perturbations to be the result of a Flt3-ITD-driven, cell-autonomous, overproliferation within a compartment of normally quiescent LT-HSCs, as evidenced by increased BrdU incorporation, cell cycle entry and upregulation of genes that promote cell cycle (Pim1, Ccnd1). This rapid HSC exhaustion and depletion of the LT-HSC compartment is coupled to the activation of downstream Flt3 signaling, most prominently through Stat5 with observed increases in expression of Stat5 targets (e.g. Fos, JunB, Mkp1, Socs3). Furthermore, for the first time, we show that Flt3 is expressed at detectable (albeit low) levels in this MPN-initiating, LT-HSC cell population and capable of exerting significant functional effects in this compartment. Surprisingly, the depletion and defects in HSC function are completely reversible upon treatment with the small molecule inhibitor, sorafenib, while simultaneously leading to near complete ablation of disease. Taken together, our data implicate previously unrecognized roles for Flt3 in the extremely dynamic process of stem cell homeostasis and quiescence, which appears closely coupled to pathogenesis, as well as pointing to Flt3 as an attractive target for targeted therapy. Disclosures:No relevant conflicts of interest to declare.

In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

Abstract 758Primary leukemia stem cells (LSCs) reside in an in vivo microenvironment that supports the growth and survival of malignant cells. Despite the increasing understanding of the importance of niche interactions and primary cell biology in leukemia, many studies continue to focus on cell autonomous processes in artificial model systems. The majority of strategies to-date that attempt to define therapeutic targets in leukemia have relied on screening cell lines in culture; new strategies should incorporate the use of primary disease within a physiologic niche. Using a primary murine MLL-AF9 acute myeloid leukemia (AML) model highly enriched for LSCs, we performed an in vivo short hairpin RNA (shRNA) screen to identify novel genes that are essential for leukemia growth and survival. LSCs infected with pools of shRNA lentivirus were transplanted and grown in recipient mice for 2 weeks, after which bone marrow and spleen cells were isolated. Massively parallel sequencing of infected LSCs isolated before and after transplant was used to quantify the changes in shRNA representation over time. Our in vivo screens were highly sensitive, robust, and reproducible and identified a number of positive controls including genes required for MLL-AF9 transformation (Ctnnb1, Mef2c, Ccna1), genes universally required for cell survival (Ube2j2, Utp18), and genes required in other AML models (Myb, Pbx1, Hmgb3).In our primary and validation screens, multiple shRNAs targeting Integrin Beta 3 (Itgb3) were consistently depleted by more than 20-fold over two weeks in vivo. Follow up studies using RNA interference (RNAi) and Itgb3−/− mice identified Itgb3 as essential for murine leukemia cells growth and transformation in vivo, and loss of Itgb3 conferred a statistically significant survival advantage to recipient mice. Importantly, neither Itgb3 knockdown or genetic loss impaired normal hematopoietic stem and progenitor cell (HSPC) function in 16 week multilineage reconstitution assays. We further identified Itgav as the heterodimeric partner of Itgb3 in our model, and found that knockdown of Itgav inhibited leukemia cell growth in vivo. Consistent the therapeutic aims or our study, flow cytometry on primary human AML samples revealed ITGAV/ITGB3 heterodimer expression. To functionally assess the importance of gene expression in a human system, we performed another RNAi screen on M9 leukemia cells, primary human cord blood CD34+ cells transduced with MLL-ENL that are capable of growing in vitro or in a xenotransplant model in vivo. We found that ITGB3 loss inhibited M9 cell growth in vivo, but not in vitro, consistent with the importance of ITGB3 in a physiologic microenvironment.We explored the signaling pathways downstream of Itgb3 using an additional in vivo, unbiased shRNA screen and identified Syk as a critical mediator of Itgb3 activity in leukemia. Syk knockdown by RNAi inhibited leukemia cell growth in vivo; downregulation of Itgb3 expression resulted in decreased levels of Syk phosphorylation; and expression of an activated form of Syk, TEL-SYK, rescued the effects of Itgb3 knockdown on leukemia cell growth in vivo. To understand cellular processes controlled by Itgb3, we performed gene expression studies and found that, in leukemia cells, Itgb3 knockdown induced differentiation and inhibited multiple previously published LSC transcriptional programs. We confirmed these results using primary leukemia cell histology and a model system of leukemia differentiation. Finally, addition of a small molecule Syk inhibitor, R406, to primary cells co-cultured with bone marrow stroma caused a dose-dependent decrease in leukemia cell growth. Our results establish the significance of the Itgb3 signaling pathway, including Syk, as a potential therapeutic target in AML, and demonstrate the utility of in vivo RNA interference screens. Disclosures:Armstrong:Epizyme: Consultancy.

A Small Molecule Inhibitor Targeting CREB and CBP Inhibits Proliferation of AML Cells In Vitro and In Vivo,

Abstract 3607The cAMP Response Element Binding Protein, CREB, is a nuclear transcription factor that is a downstream target of signaling pathways regulating memory, glucose homeostasis, cell proliferation, differentiation, and survival. We previously demonstrated that CREB is overexpressed at both the protein and mRNA levels in leukemia blasts and in leukemia stem cells. AML patients who overexpress CREB in their bone marrow have an increased risk of relapse and decreased event-free survival. To determine whether CREB is sufficient for leukemogenesis, we created a transgenic mouse in which CREB is expressed under the control of a myeloid specific hMRP8 promoter. Bone marrow progenitors from CREB transgenic mice had higher proliferative potential and replating ability. These mice developed myeloproliferative disease after one year but not acute leukemia, suggesting that CREB is not sufficient to induce myeloid transformation. To determine whether CREB is necessary for leukemia cell proliferation, we transduced AML cells with lentiviral CREB shRNA. We observed that downregulation of CREB led to decreased AML cell proliferation and survival in vitro. Furthermore, our results demonstrated that CREB knockdown inhibits the growth of AML cells in vivo without affecting normal hematopoietic stem cell function. Together, these results strongly suggest that CREB acts as a proto-oncogene and is a potential target for AML therapy.CREB is activated through phosphorylation, leading to the recruitment of the histone acetyltransferase, CREB binding protein (CBP) and subsequent target gene expression. XX-650-23 (MW: 288.3) is a small molecule that was identified through in silico screening methods to inhibit the interaction between CREB and CBP. We tested the effects of the drug on various AML cell lines using MTT assays and trypan blue exclusion. The IC50 ranged from 700 nM to 2 μM after 72 hours of drug treatment. However, treatment of normal human bone marrow progenitors cultured in methylcellulose containing XX-650-23 at a concentration of 10 μM had no effect on colony numbers. We also tested the in vivo effects of XX-650-23 using xenograft NOD-SCID IL-2Rgamma null (NSG) mouse models. To assess toxicity, mice were treated with the drug at various concentrations, ranging from 10 to 20 mg/kg, by intraperitoneal injection (IP) once daily for 28 days. We did not observe any weight loss or hematologic, renal, hepatic, or cardiac toxicity in the mice. We also performed pharmacokinetic analysis to determine the half-life and stability of the drug. After 1 hour of treatment, the serum drug concentration was 33 nM. The estimated drug mean residence time was 7.5 hours. Plasma clearance divided by IP absorption fraction was 9.6 L/min/kg. The mice were treated daily with drug (17.5mg/kg IP) or vehicle control once MV4-11 cells reached a tumor size of 300mm3 or at the time cells were injected. Our results demonstrated significant inhibition of tumor growth with treatment of the drug started on the day of injection of cells compared to waiting until the tumor reached 300mm3. Together, these data suggest that a small molecule inhibitor targeting CREB and CBP interaction is a potential avenue for drug development. We are currently studying the mechanisms by which XX-650-23 inhibits AML cell proliferation and analyzing the effects of combining the drug with chemotherapy. Disclosures:No relevant conflicts of interest to declare.

MicroRNA Regulation of Cancer Stem Cells

Cancer stem cells (CSC), or cancer cells with stem cell properties, have been reported in many human tumors and are thought to be responsible for tumor initiation, therapy resistance, progression, relapse, and metastasis. Despite their potential clinical importance, how CSCs are regulated at the molecular level is not well understood. MicroRNAs (miRNA), small noncoding RNAs that play critical roles in normal stem cell functions during development, have emerged as important regulators of CSCs as well. In this review, we summarize the current major findings of miRNA regulation of various CSCs and discuss our recent findings that miR-34a suppresses prostate CSCs and metastasis by directly repressing CD44. This recent progress has important implications for understanding how CSCs are intricately regulated by networks of miRNAs and for developing novel mechanism-based miRNA therapeutics that specifically target CSCs.

MSI2 protein expression predicts unfavorable outcome in acute myeloid leukemia

AbstractMSI2 is highly expressed in human myeloid leukemia (AML) cell lines, and high expression of MSI2 mRNA is associated with decreased survival in AML, suggesting its use as a new prognostic marker. To test this, we measured MSI2 protein level by immunohistochemistry in 120 AML patients. Most cases (70%) showed some nuclear or cytoplasmic positivity, but the percentage of positive cells was low in most cases. Despite this, MSI2 protein expression was negatively associated with outcome, particularly for patients with good cytogenetic subgroup. For practical diagnostic purposes, the strongest significance of association was seen in cases with > 1% of cells showing strong MSI2 staining, these having a very poor outcome (P < .0001). Multivariate analysis with cytogenetic category, age, white cell count, and French-American-British subtype demonstrated that nuclear MSI2 levels were independently predictive of outcome (P = .0497). These results confirm the association of MSI2 expression with outcome in AML at the protein level and demonstrate the utility of MSI2 protein as a clinical prognostic biomarker. In addition, although positive at some level in most cases, its prognostic power derived from few positive cells, supporting its role in control of normal hematopoietic stem cell function and highlighting its role in disease progression.

Glioma Stem Cell Proliferation and Tumor Growth Are Promoted by Nitric Oxide Synthase-2

Malignant gliomas are aggressive brain tumors with limited therapeutic options, and improvements in treatment require a deeper molecular understanding of this disease. As in other cancers, recent studies have identified highly tumorigenic subpopulations within malignant gliomas, known generally as cancer stem cells. Here, we demonstrate that glioma stem cells (GSCs) produce nitric oxide via elevated nitric oxide synthase-2 (NOS2) expression. GSCs depend on NOS2 activity for growth and tumorigenicity, distinguishing them from non-GSCs and normal neural progenitors. Gene expression profiling identified many NOS2-regulated genes, including the cell-cycle inhibitor cell division autoantigen-1 (CDA1). Further, high NOS2 expression correlates with decreased survival in human glioma patients, and NOS2 inhibition slows glioma growth in a murine intracranial model. These data provide insight into how GSCs are mechanistically distinct from their less tumorigenic counterparts and suggest that NOS2 inhibition may be an efficacious approach to treating this devastating disease.

Head neck squamous cell carcinoma c‐Met+ cells display cancer stem cell properties and are responsible for cisplatin‐resistance and metastasis

c-Met, the tyrosine kinase receptor for hepatocyte growth factor, is overexpressed in a variety of tumors in which it plays a central role in malignant transformation. Although c-Met has also been determined to be a critical signaling molecule in normal stem cell function, the potential role of c-Met as a single marker for cancer stem cells (CSCs) has not been previously examined. In our study, we reported that human head neck squamous cell carcinoma (HNSCC) cells expressing c-Met were capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumors, and isolation of HNSCC cells using a second marker CD44 could further enhance upon the in-vivo tumorigenicity. We also reported that c-Met(+) HNSCC cells could readily make spherical colonies in nonadherent culture conditions, in contrast, c-Met(-) population did not; these spherical colonies could be passaged multiple times without loss of colony-forming capability. Furthermore, we showed that c-Met(+) HNSCC cells have increased expression of self-renewal pathways are spared by cisplatin treatment and are responsible for mediating metastasis. These results indicated that c-Met could serve as a novel marker for CSCs at least in HNSCC, and the highly chemoresistant and metastatic capabilities of c-Met(+) HNSCC population make them an important cell type to better define and understand their function.

Inhibitor of the CREB Proto-Oncogene Suppresses AML Cell Proliferation and Induces Apoptosis In Vitro and In Vivo

AbstractAbstract 3270The cAMP Response Element Binding Protein, CREB, is a nuclear transcription factor that is critical for cell proliferation, differentiation, and survival in a variety of cell types. We previously demonstrated that CREB is overexpressed in leukemia blasts from greater than 60% of patients with AML. CREB overexpression is also associated with an increased risk of relapse and decreased event free survival in a cohort of AML patients. To understand the role of CREB in leukemogenesis, a transgenic mouse was created by overexpressing CREB under the control of the hMRP8 promoter in Gr-1/Mac-1 + myeloid cells. Bone marrow progenitors from these mice have increased proliferative potential and replating ability. CREB transgenic mice developed myeloproliferative disease after one year, but not acute myeloid leukemia suggesting that CREB is not sufficient to induce complete transformation. Given these results, we hypothesized that CREB could be a potential target for AML therapy. To test this possibility, we knocked down CREB using lentiviral shRNAs in primary mouse hematopoietic stem cells in vitro and in vivo. Downregulation of CREB resulted in significant decrease in myeloid colony numbers in methylcellulose colony assays. However, there was no effect of CREB knockdown in hematopoietic stem cell numbers or long-term engraftment by stem cell transduction and transplantation experiments. In contrast, inhibition of CREB by shRNAs in human myeloid leukemia cells and Ba/F3 cells transduced with T315I mutant BCR-ABL resulted in suppression of growth of leukemia cells in vitro and in NOD-SCID mice. Taken together, these data suggest that CREB preferentially inhibits the growth of leukemia cells without significantly affecting normal hematopoietic stem cell proliferation or function. To target CREB in AML cells, a small molecule, XX-650-23 (MW: 288.3), was developed to inhibit the interaction between CREB and its binding partner, the histone acetyltransferase CBP (CREB Binding Protein). We tested the effects of the drug on AML cell lines Molm-13 and MV-411 in vitro using MTT assays and trypan blue exclusion. The IC50 for both cell lines was between 1–2 micromolar concentration after 72 hours of treatment. Our results also demonstrated that treatment of MV-411 cells with XX-650-23 not only inhibited cell proliferation but also resulted in apoptosis. Treatment of normal human bone marrow progenitors with XX-650-23 had no effect on colony numbers in methylcellulose containing IL-3, IL-6, and Stem Cell Factor up to 10 micromolar concentration. NOD-SCID IL-2Rgamma null (NSG) mice treated with the drug at 10, 15, 17.5, and 20 mg/kg IP once daily for 28 days had no evidence of weight loss or toxicity to organs as measured by peripheral blood counts and chemistries to assess kidney and liver function. We next tested the drug in xenograft models of AML with MV-411 cells (2×106) injected subcutaneously into the flanks of NSG mice. Three groups of 5 mice were treated with either XX-650-23 or vehicle control at 17.5 mg/kg IP daily at the time of injection of MV-411 cells or after the tumors reached a size of 300 mm3. Serum drug concentration 1 hour after first injection was 33 nM. Estimated drug mean residence time was 7.5 hours, and plasma clearance was 9.6 ml/min/kg, assuming complete IP absorption. Our results demonstrated a significant inhibition of tumor growth with treatment of the drug and MV-411 cells at the same time on Day 1. There was also an effect of the drug alone when treated after the tumor reached a size of 300 mm3, although less dramatic. These data suggest that development of a drug that targets CREB is a novel approach to treat AML. We are currently optimizing the treatment of AML cells with XX-650-23 in mice and investigating the downstream signaling pathways to understand the mechanism by which XX-650-23 suppresses proliferation and induces apoptosis of AML cells. Disclosures:Sakamoto:Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.

The miR-17-92 microRNA Polycistron Regulates MLL Leukemia Stem Cell Potential by Modulating p21 Expression

Despite advances in defining the critical molecular determinants for leukemia stem cell (LSC) generation and maintenance, little is known about the roles of microRNAs in LSC biology. Here, we identify microRNAs that are differentially expressed in LSC-enriched cell fractions (c-kit(+)) in a mouse model of MLL leukemia. Members of the miR-17 family were notably more abundant in LSCs compared with their normal counterpart granulocyte-macrophage progenitors and myeloblast precursors. Expression of miR-17 family microRNAs was substantially reduced concomitant with leukemia cell differentiation and loss of self-renewal, whereas forced expression of a polycistron construct encoding miR-17-19b miRNAs significantly shortened the latency for MLL leukemia development. Leukemias expressing increased levels of the miR-17-19b construct displayed a higher frequency of LSCs, more stringent block of differentiation, and enhanced proliferation associated with reduced expression of p21, a cyclin-dependent kinase inhibitor previously implicated as a direct target of miR-17 microRNAs. Knockdown of p21 in MLL-transformed cells phenocopied the overexpression of the miR-17 polycistron, including a significant decrease in leukemia latency, validating p21 as a biologically relevant and direct in vivo target of the miR-17 polycistron in MLL leukemia. Expression of c-myc, a crucial upstream regulator of the miR-17 polycistron, correlated with miR-17-92 levels, enhanced self-renewal, and LSC potential. Thus, microRNAs quantitatively regulate LSC self-renewal in MLL-associated leukemia in part by modulating the expression of p21, a known regulator of normal stem cell function.

Overlapping Genes May Control Reprogramming of Mouse Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) and Breast Cancer Stem Cells

Recent findings suggest the possibility that tumors originate from cancer cells with stem cell properties. The cancer stem cell (CSC) hypothesis provides an explanation for why existing cancer therapies often fail in eradicating highly malignant tumors and end with tumor recurrence. Although normal stem cells and CSCs both share the capacity for self-renewal and multi-lineage differentiation, suggesting that CSC may be derived from normal SCs, the cellular origin of transformation of CSCs is debatable. Research suggests that the tightly controlled balance of self-renewal and differentiation that characterizes normal stem cell function is dis-regulated in cancer. Additionally, recent evidence has linked an embryonic stem cell (ESC)-like gene signature with poorly differentiated high-grade tumors, suggesting that regulatory pathways controlling pluripotency may in part contribute to the somatic CSC phenotype. Here, we introduce expression profile bioinformatic analyses of mouse breast cells with CSC properties, mouse embryonic stem (mES) and induced pluripotent stem (iPS) cells with an emphasis on how study of pluripotent stem cells may contribute to the identification of genes and pathways that facilitate events associated with oncogenesis. Global gene expression analysis from CSCs and induced pluripotent stem cell lines represent an ideal model to study cancer initiation and progression and provide insight into the origin cancer stem cells. Additionally, insight into the genetic and epigenomic mechanisms regulating the balance between self-renewal and differentiation of somatic stem cells and cancer may help to determine whether different strategies used to generate iPSCs are potentially safe for therapeutic use.

The Broad Spectrum Bcl-2 Inhibitor Apogossypol Induces Apoptosis and Differentiation of Blast Crisis Chronic Myeloid Leukemia Stem Cells.

Abstract 3275Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant. Introduction:Leukemia stem cells (LSC) are rare, self-renewing cells capable of proliferation and differentiation into the bulk of cells that make up a leukemia. LSC, like normal tissue-specific stem cells, can be highly quiescent and resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. While traditional chemotherapy may successfully eradicate the bulk of tumor cells, it often fails to kill LSC resulting in their reactivation and eventual relapse of disease.Apoptosis resistance in cancer often involves deregulation of Bcl-2 family proteins. Bcl-2 family expression also seems to have an important role in normal stem cell function as expression changes of Mcl-1, Bcl-2, and Bcl-XL in mouse hematopoietic stem and progenitors characterize the differentiation of hematopoietic cells to different lineages.Several studies have linked expression of Bcl-2 family members to the development of blast crisis chronic myeloid leukemia (BC CML) in vitro, however there has been little data on the role of Bcl-2 expression in apoptosis resistance in human LSC. Also, various data has shown that CML cells become increasingly resistant to BCR-ABL inhibition with progression to blast crisis. In BC CML, LSC are enriched in the progenitor population and can be serially transplanted in immunodeficient mice leading to BC CML in vivo. We hypothesize that human BC CML LSC may resist chemotherapy by overexpression of Bcl-2 family proteins and maintenance of quiescence. We analyzed Bcl-2 family protein expression in BC CML LSC and analyzed whether these cells were sensitive to chemotherapy treatment in vitro. We further analyzed whether BC CML LSC could maintain quiescence in vivo. Finally, we tested the efficacy of the broad spectrum Bcl-2 antagonist apogossypol on BC CML LSC in vitro and in vivo. Methods:Bcl-2 and Mcl-1 protein expression was measured in 1° BC CML LSC by intracellular FACS analysis and compared to expression in normal and chronic phase CML cells. CD34+ cells isolated from serially transplanted BC CML LSC were used for all subsequent studies. To assess whether BC CML cells maintained quiescence in vivo, they were stained with DiR, an infrared fluorescent cell membrane dye, transplanted into neonatal mice, and analyzed for DiR fluorescence 18 weeks later. Dividing cells will distribute DiR to all daughter cells leading to dilution of the dye and a decrease in fluorescent signal per cell. LSC drug resistance was tested in vitro by culturing the cells with etoposide, dasatinib, and apogossypol and by measuring apoptosis by FACS using annexin-V/7-AAD staining. Finally, apogossypol efficacy was tested in vivo in LSC transplanted mice. At 8 weeks post-transplantation, mice were treated for 3 weeks with apogossypol or vehicle and then analyzed for human hematopoietic cell engraftment and apoptosis by FACS. Results:1° BC CML progenitors expressed significantly higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. LSC cultured in vitro were also resistant to etoposide and dasatinib-induced apoptosis. Apogossypol treatment in vitro however led to a dose-dependent increase in cell death and apoptosis and resulted in a significant increase in the frequency of lin+ staining cells along with a significant shift in the frequency of the common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) populations compared to vehicle treated controls.Mice transplanted with BC CML LSC developed diffuse myeloid sarcomas and had high levels of human engraftment in the liver, spleen, and bone marrow. Human cells in tumors and the liver were uniformly DiR- while cells engrafted in the spleen and bone marrow retained DiR fluorescence. In vivo treatment with apogossypol led to a significant reduction in human cell engraftment in mouse bone marrow compared to vehicle controls. Consistent with the results in vitro, there was also a significant increase in the frequency of lin+ staining cells in engrafted mouse spleens as well as a significant decrease in engrafted GMP in the mouse bone marrow. Conclusions:Our results demonstrate that BC CML LSC are highly resistant to conventional chemotherapy but are sensitive to apogossypol in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC by inducing apoptosis as well as by inducing differentiation. Disclosures:Goff:Coronado Biosciences: Research Funding. Tesi:Coronado Biosciences: President and CEO. Jamieson:Coronado Biosciences: Research Funding. Jamieson:Coronado Biosciences: Research Funding.

Role of mTOR in Hematopoiesis and Hematopoietic Stem Cell Regulation.

Abstract 1490Poster Board I-513The mammalian target of rapamycin (mTOR) integrates nutrients, growth factors, and cellular energy status to control protein synthesis that determines cell growth and metabolism. It is also known that mTOR plays an essential role in cell survival by regulating Akt/PKB signaling. By using the inhibitor rapamycin, mTOR has previously been suggested to regulate proliferation of megakaryocyte progenitors and late stage of megakaryocyte differentiation without a general impact on normal hematopoiesis or hematopoietic stem cell (HSC) function. Due to limitations of rapamycin and the early lethality of conventional mTOR gene targeted mice, the physiological role of mTOR in blood development remains undefined. In this study, we have utilized an inducible conditional mTOR knockout mouse model by crossbreeding mTORflox/flox mice with Mx-Cre mice that allow interferon-induced mTOR deletion in the bone marrow following a transplantation and polyI:C induction protocol, in an effort to determine the genetic role of mTOR in hematopoiesis. Depletion of mTOR drastically affected hematopoiesis in a blood cell autonomous manner in Mx-Cre;mTORflox/flox bone marrow transplant recipients: the mice showed marked reduction in BM cellularity and in the numbers of myeloid and lymphoid lineage cells, erythrocytes, and platelets in peripheral blood, bone marrow, and thymus, leading to bone marrow failure, blood cell exhaustion and lethality. In vitro colony-forming activities by bone marrow or spleen progenitors were completely abolished in the absence of mTOR. Interestingly, the number and frequency of HSCs in bone marrow (Lin−Sca-1+c-Kit+) increased transiently while the number of early progenitors (CMP, GMP, MEP, CLP) detected by cell surface markers remained unchanged or only mildly affected in the mutant mice within 14 days after polyI:C treatment. Concomitantly, mTOR deletion led to a massive egress of HSCs from bone marrow to distal organs including spleen (∼60-fold increase). Transplantation of mTOR−/− bone marrow cells into NOD-SCID mice or competitive transplantation of mTOR−/− bone marrow cells into BoyJ mice further demonstrated that mTOR deficiency caused a complete failure in HSC engraftment and repopulation. Surprisingly, at the cellular level these phenotypes are associated with increased proliferation of HSCs in vivo and in vitro by 60% and 2.5-fold, respectively, as assessed by 5-bromodeoxyuridine incorporation assays whereas the cell survival index appears to be unaffected. Moreover, mTOR−/− HSCs and progenitor cells displayed impaired adhesion to fibronectin CH296 fragment (∼30% decrease) and migration toward SDF-1α gradients (∼30% decrease). At the molecular level, gene chip microarray analysis of mTOR−/− HSCs revealed that the cell cycle regulators myb, wee1, FANCD2, and FANCE were significantly downregulated while Rb and E2F5 were upregulated, the survival/apoptosis regulators MCL1 and BCL2L1 were upregulated, and the actin cytoskeleton and cell extracellular matrix adhesion regulators Arp2/3 complex subunit 5, paxillin, laminin α5, integrin β3, and myosin light chain 6B were upregulated. Further, immunoblotting analysis of isolated Lin− cells showed that SCF-stimulated activation of translational regulators S6K and 4E-BP and survival regulator Akt were abolished upon mTOR deletion. Taken together, these data suggest that mTOR is a critical regulator of HSC quiescence, self-renewal, and engraftment through the regulation of cell cycle, survival and actin cytoskeleton signals, and is essential in multiple stages of hematopoiesis. DisclosuresCancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

The Wnt Receptor, Lrp5, Is Expressed by Mouse Mammary Stem Cells and Is Required to Maintain the Basal Lineage

BackgroundEctopic Wnt signaling induces increased stem/progenitor cell activity in the mouse mammary gland, followed by tumor development. The Wnt signaling receptors, Lrp5/6, are uniquely required for canonical Wnt activity. Previous data has shown that the absence of Lrp5 confers resistance to Wnt1-induced tumor development.Methodology/Principal FindingsHere, we show that all basal mammary cells express Lrp5, and co-express Lrp6 in a similar fashion. Though Wnt dependent transcription of key target genes is relatively unchanged in mammary epithelial cell cultures, the absence of Lrp5 specifically depletes adult regenerative stem cell activity (to less than 1%). Stem cell activity can be enriched by >200 fold (over 80% of activity), based on high Lrp5 expression alone. Though Lrp5 null glands have apparent normal function, the basal lineage is relatively reduced (from 42% basal/total epithelial cells to 22%) and Lrp5−/− mammary epithelial cells show enhanced expression of senescence-associated markers in vitro, as measured by expression of p16Ink4a and TA-p63.Conclusions/SignificanceThis is the first single biomarker that has been demonstrated to be functionally involved in stem cell maintenance. Together, these results demonstrate that Wnt signaling through Lrp5 is an important component of normal mammary stem cell function.

Downregulation of miRNA-200c Links Breast Cancer Stem Cells with Normal Stem Cells

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells.

N-Myc Regulates Expression of Pluripotency Genes in Neuroblastoma Including lif, klf2, klf4, and lin28b

myc genes are best known for causing tumors when overexpressed, but recent studies suggest endogenous myc regulates pluripotency and self-renewal of stem cells. For example, N-myc is associated with a number of tumors including neuroblastoma, but also plays a central role in the function of normal neural stem and precursor cells (NSC). Both c- and N-myc also enhance the production of induced pluripotent stem cells (iPSC) and are linked to neural tumor stem cells. The mechanisms by which myc regulates normal and neoplastic stem-related functions remain largely open questions. Here from a global, unbiased search for N-Myc bound genes using ChIP-chip assays in neuroblastoma, we found lif as a putative N-Myc bound gene with a number of strong N-Myc binding peaks in the promoter region enriched for E-boxes. Amongst putative N-Myc target genes in expression microarray studies in neuroblastoma we also found lif and three additional important embryonic stem cell (ESC)-related factors that are linked to production of iPSC: klf2, klf4, and lin28b. To examine the regulation of these genes by N-Myc, we measured their expression using neuroblastoma cells that contain a Tet-regulatable N-myc transgene (TET21N) as well as NSC with a nestin-cre driven N-myc knockout. N-myc levels closely correlated with the expression of all of these genes in neuroblastoma and all but lif in NSC. Direct ChIP assays also indicate that N-Myc directly binds the lif promoter. N-Myc regulates trimethylation of lysine 4 of histone H3 in the promoter of lif and possibly in the promoters of several other stem-related genes. Together these findings indicate that N-Myc regulates overlapping stem-related gene expression programs in neuroblastoma and NSC, supporting a novel model by which amplification of the N-myc gene may drive formation of neuroblastoma. They also suggest mechanisms by which Myc proteins more generally contribute to maintenance of pluripotency and self-renewal of ESC as well as to iPSC formation.