Scientific Resources

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1,also known as CD71),which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fcγ receptors and the complement component C1q,suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition,in 2 disseminated multiple myeloma xenograft mouse models,we show that a single dose of ch128.1Av results in significant antitumor activity,including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally,we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches,including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies. View Publication

Pim kinases are Ser/Thr kinases with multiple substrates that affect survival pathways. These proteins are overexpressed in acute myeloid leukemia (AML) blasts and we hypothesized that Pim kinase inhibition would affect AML cell survival. Imidazo[1,2-b]pyridazine compound,SGI-1776 inhibits Pim-1,Pim-2 and Pim-3,and was evaluated in AML-cell line,-xenograft model,and -primary blasts. Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis and we investigated its effect on Pim kinase functions. Phosphorylation of traditional Pim kinase targets,c-Myc(Ser62) and 4E-BP1 (Thr36/Thr47),were both decreased in actively cycling AML cell lines MV-4-11,MOLM-13 and OCI-AML-3. Levels of antiapoptotic proteins Bcl-2,Bcl-x(L),XIAP,and proapoptotic Bak and Bax were unchanged; however,a significant reduction in Mcl-1 was observed. This was correlated with inhibition of global RNA and protein synthesis and MCL-1 transcript decline after SGI-1776 treatment. These data suggest that SGI-1776 mechanism in AML involves Mcl-1 protein reduction. Consistent with cell line data,xenograft model studies with mice bearing MV-4-11 tumors showed efficacy with SGI-1776. Importantly,SGI-1776 was also cytotoxic in AML primary cells,irrespective of FLT3 mutation status and resulted in Mcl-1 protein decline. Pim kinase inhibition may be a new strategy for AML treatment. View Publication

In human B-acute lymphoblastic leukemia (B-ALL),RAG1-induced genomic alterations are important for disease progression. However,given that biallelic loss of the RAG1 locus is observed in a subset of cases,RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf(-/-)Rag1(-/-) mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model,we identified a new,Rag1-independent leukemia-initiating mechanism originating from a Sca1(+)CD19(+) precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM,a similar CD34(+)CD19(+) population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans. View Publication

Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs,substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγc(null) (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples,39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However,33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients,but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction,and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function,and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies. View Publication

Melanoma,a potentially lethal skin cancer,is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses,limited knowledge exists on the role of mature B cells. We describe an approach,including a cell-based ELISA,to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (Ptextless0.0001). Interestingly,we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (Ptextless0.0001). Overall,28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly,a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients,which is reduced with disease progression,adding to previous reports of tumor-reactive antibodies in patient sera,and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. View Publication

Recent evidence suggests that enhanced aldehyde dehydrogenase (ALDH) activity is a hallmark of cancer stem cells (CSC) measurable by the aldefluor assay. ALDH1A1,one of 19 ALDH isoforms expressed in humans,was generally believed to be responsible for the ALDH activity of CSCs. More recently,experiments with murine hematopoietic stem cells,murine progenitor pancreatic cells,and human breast CSCs indicate that other ALDH isoforms,particularly ALDH1A3,significantly contribute to aldefluor positivity,which may be tissue and cancer specific. Therefore,potential prognostic application involving the use of CSC prevalence in tumor tissue to predict patient outcome requires the identification and quantification of specific ALDH isoforms. Herein we review the suggested roles of ALDH in CSC biology and the immunohistological studies testing the potential application of ALDH isoforms as novel cancer prognostic indicators. View Publication

4-Hydroxy-5-methoxy-2,3-dihydro-1H-[1,3]benzodioxolo[5,6-c]pyrrolo[1,2-f]-phenanthridium chloride (NK314) is a benzo[c] phenanthridine alkaloid that inhibits topoisomerase IIα,leading to the generation of DNA double-strand breaks (DSBs) and activating the G(2) checkpoint pathway. The purpose of the present studies was to investigate the DNA intercalating properties of NK314,to evaluate the DNA repair mechanisms activated in cells that may lead to resistance to NK314,and to develop mechanism-based combination strategies to maximize the antitumor effect of the compound. A DNA unwinding assay indicated that NK314 intercalates in DNA,a property that likely cooperates with its ability to trap topoisomerase IIα in its cleavage complex form. The consequence of this is the formation of DNA DSBs,as demonstrated by pulsed-field gel electrophoresis and H2AX phosphorylation. Clonogenic assays demonstrated a significant sensitization in NK314-treated cells deficient in DNA-dependent protein kinase (DNA-PK) catalytic subunit,Ku80,ataxia telangiectasia mutated (ATM),BRCA2,or XRCC3 compared with wild-type cells,indicating that both nonhomologous end-joining and homologous recombination DNA repair pathways contribute to cell survival. Furthermore,both the DNA-PK inhibitor 8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (NU7441) and the ATM inhibitor 2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933) significantly sensitized cells to NK314. We conclude that DNA-PK and ATM contribute to cell survival in response to NK314 and could be potential targets for abrogating resistance and maximizing the antitumor effect of NK314. View Publication

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors,very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel,aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally,array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes,thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis,potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4,a GTPase regulator located in the commonly deleted 7q31 region,was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets,providing further validation of our findings. Finally,DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis,thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region. View Publication

Chromosomal translocations of the mixed lineage leukemia (MLL) gene are a common cause of acute leukemias. The oncogenic function of MLL fusion proteins is,in part,mediated through aberrant activation of Hoxa genes and Meis1,among others. Here we demonstrate using a tamoxifen-inducible Cre-mediated loss of function mouse model that DOT1L,an H3K79 methyltransferase,is required for both initiation and maintenance of MLL-AF9-induced leukemogenesis in vitro and in vivo. Through gene expression and chromatin immunoprecipitation analysis we demonstrate that mistargeting of DOT1L,subsequent H3K79 methylation,and up-regulation of Hoxa and Meis1 genes underlie the molecular mechanism of how DOT1L contributes to MLL-AF9-mediated leukemogenesis. Our study not only provides the first in vivo evidence for the function of DOT1L in leukemia,but also reveals the molecular mechanism for DOT1L in MLL-AF9 mediated leukemia. Thus,DOT1L may serve as a potential therapeutic target for the treatment of leukemia caused by MLL translocations. View Publication

Oncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and,accordingly,ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes,however,inhibit ARF/p53 and only infrequently select for ARF or p53 mutations,suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK,the initiating oncogene of anaplastic large cell lymphomas (ALCLs),induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly,human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a,which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence,and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors. View Publication

PURPOSE: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC),an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. EXPERIMENTAL DESIGN: MG18L,containing a U(S)3 deletion and an inactivating LacZ insertion in U(L)39,was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs,alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002,triciribine,GDC-0941,and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. RESULTS: MG18L was severely neuroattenuated in mice,replicated well in GSCs,and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs,whereas their combination with MG18L synergized in killing GSCs and glioma cell lines,but not human astrocytes,through enhanced induction of apoptosis. Importantly,synergy was independent of inhibitor sensitivity. In vivo,the combination of MG18L and LY294002 significantly prolonged survival of mice,as compared with either agent alone,achieving 50% long-term survival in GBM-bearing mice. CONCLUSIONS: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM,being especially effective when combined with PI3K/Akt pathway-targeted agents. View Publication

Markers that reliably identify cancer stem cells (CSC) in ovarian cancer could assist prognosis and improve strategies for therapy. CD133 is a reported marker of ovarian CSC. Aldehyde dehydrogenase (ALDH) activity is a reported CSC marker in several solid tumors,but it has not been studied in ovarian CSC. Here we report that dual positivity of CD133 and ALDH defines a compelling marker set in ovarian CSC. All human ovarian tumors and cell lines displayed ALDH activity. ALDH(+) cells isolated from ovarian cancer cell lines were chemoresistant and preferentially grew tumors,compared with ALDH(-) cells,validating ALDH as a marker of ovarian CSC in cell lines. Notably,as few as 1,000 ALDH(+) cells isolated directly from CD133(-) human ovarian tumors were sufficient to generate tumors in immunocompromised mice,whereas 50,000 ALDH(-) cells were unable to initiate tumors. Using ALDH in combination with CD133 to analyze ovarian cancer cell lines,we observed even greater growth in the ALDH(+)CD133(+) cells compared with ALDH(+)CD133(-) cells,suggesting a further enrichment of ovarian CSC in ALDH(+)CD133(+) cells. Strikingly,as few as 11 ALDH(+)CD133(+) cells isolated directly from human tumors were sufficient to initiate tumors in mice. Like other CSC,ovarian CSC exhibited increased angiogenic capacity compared with bulk tumor cells. Finally,the presence of ALDH(+)CD133(+) cells in debulked primary tumor specimens correlated with reduced disease-free and overall survival in ovarian cancer patients. Taken together,our findings define ALDH and CD133 as a functionally significant set of markers to identify ovarian CSCs. View Publication