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1.
Nat Commun ; 15(1): 963, 2024 Feb 01.
Article in English | MEDLINE | ID: mdl-38302473

ABSTRACT

The MYC oncogene is often dysregulated in human cancer, including hepatocellular carcinoma (HCC). MYC is considered undruggable to date. Here, we comprehensively identify genes essential for survival of MYChigh but not MYClow cells by a CRISPR/Cas9 genome-wide screen in a MYC-conditional HCC model. Our screen uncovers novel MYC synthetic lethal (MYC-SL) interactions and identifies most MYC-SL genes described previously. In particular, the screen reveals nucleocytoplasmic transport to be a MYC-SL interaction. We show that the majority of MYC-SL nucleocytoplasmic transport genes are upregulated in MYChigh murine HCC and are associated with poor survival in HCC patients. Inhibiting Exportin-1 (XPO1) in vivo induces marked tumor regression in an autochthonous MYC-transgenic HCC model and inhibits tumor growth in HCC patient-derived xenografts. XPO1 expression is associated with poor prognosis only in HCC patients with high MYC activity. We infer that MYC may generally regulate and require altered expression of nucleocytoplasmic transport genes for tumorigenesis.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Mice , Animals , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Genes, myc , Cell Transformation, Neoplastic/genetics , Carcinogenesis/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic
2.
Cancer Metab ; 9(1): 31, 2021 Aug 16.
Article in English | MEDLINE | ID: mdl-34399819

ABSTRACT

BACKGROUND: Metabolic reprogramming is a central feature in many cancer subtypes and a hallmark of cancer. Many therapeutic strategies attempt to exploit this feature, often having unintended side effects on normal metabolic programs and limited efficacy due to integrative nature of metabolic substrate sourcing. Although the initiating oncogenic lesion may vary, tumor cells in lymphoid malignancies often share similar environments and potentially similar metabolic profiles. We examined cells from mouse models of MYC-, RAS-, and BCR-ABL-driven lymphoid malignancies and find a convergence on de novo lipogenesis. We explore the potential role of MYC in mediating lipogenesis by 13C glucose tracing and untargeted metabolic profiling. Inhibition of lipogenesis leads to cell death both in vitro and in vivo and does not induce cell death of normal splenocytes. METHODS: We analyzed RNA-seq data sets for common metabolic convergence in lymphoma and leukemia. Using in vitro cell lines derived in from conditional MYC, RAS, and BCR-ABL transgenic murine models and oncogene-driven human cell lines, we determined gene regulation, metabolic profiles, and sensitivity to inhibition of lipogenesis in lymphoid malignancies. We utilize preclinical murine models and transgenic primary model of T-ALL to determine the effect of lipogenesis blockade across BCR-ABL-, RAS-, and c-MYC-driven lymphoid malignancies. Statistical significance was calculated using unpaired t-tests and one-way ANOVA. RESULTS: This study illustrates that de novo lipid biogenesis is a shared feature of several lymphoma subtypes. Using cell lines derived from conditional MYC, RAS, and BCR-ABL transgenic murine models, we demonstrate shared responses to inhibition of lipogenesis by the acetyl-coA carboxylase inhibitor 5-(tetradecloxy)-2-furic acid (TOFA), and other lipogenesis inhibitors. We performed metabolic tracing studies to confirm the influence of c-MYC and TOFA on lipogenesis. We identify specific cell death responses to TOFA in vitro and in vivo and demonstrate delayed engraftment and progression in vivo in transplanted lymphoma cell lines. We also observe delayed progression of T-ALL in a primary transgenic mouse model upon TOFA administration. In a panel of human cell lines, we demonstrate sensitivity to TOFA treatment as a metabolic liability due to the general convergence on de novo lipogenesis in lymphoid malignancies driven by MYC, RAS, or BCR-ABL. Importantly, cell death was not significantly observed in non-malignant cells in vivo. CONCLUSIONS: These studies suggest that de novo lipogenesis may be a common survival strategy for many lymphoid malignancies and may be a clinically exploitable metabolic liability. TRIAL REGISTRATION: This study does not include any clinical interventions on human subjects.

3.
Nat Commun ; 11(1): 2860, 2020 06 05.
Article in English | MEDLINE | ID: mdl-32503978

ABSTRACT

The MYC oncogene drives T- and B- lymphoid malignancies, including Burkitt's lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). Here, we demonstrate a systemic reduction in natural killer (NK) cell numbers in SRα-tTA/Tet-O-MYCON mice bearing MYC-driven T-lymphomas. Residual mNK cells in spleens of MYCON T-lymphoma-bearing mice exhibit perturbations in the terminal NK effector differentiation pathway. Lymphoma-intrinsic MYC arrests NK maturation by transcriptionally repressing STAT1/2 and secretion of Type I Interferons (IFNs). Treating T-lymphoma-bearing mice with Type I IFN improves survival by rescuing NK cell maturation. Adoptive transfer of mature NK cells is sufficient to delay both T-lymphoma growth and recurrence post MYC inactivation. In MYC-driven BL patients, low expression of both STAT1 and STAT2 correlates significantly with the absence of activated NK cells and predicts unfavorable clinical outcomes. Our studies thus provide a rationale for developing NK cell-based therapies to effectively treat MYC-driven lymphomas in the future.


Subject(s)
Burkitt Lymphoma/immunology , Killer Cells, Natural/immunology , Lymphoma, T-Cell/immunology , Proto-Oncogene Proteins c-myc/metabolism , Adoptive Transfer , Animals , Burkitt Lymphoma/mortality , Cell Line, Tumor/transplantation , Disease Models, Animal , Gene Expression Regulation, Neoplastic/immunology , Humans , Immunologic Surveillance/genetics , Interferon Type I/pharmacology , Interferon Type I/therapeutic use , Killer Cells, Natural/drug effects , Killer Cells, Natural/transplantation , Lymphoma, T-Cell/drug therapy , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/pathology , Male , Mice , Primary Cell Culture , Proto-Oncogene Proteins c-myc/genetics , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology
4.
Cell Metab ; 30(3): 556-572.e5, 2019 09 03.
Article in English | MEDLINE | ID: mdl-31447321

ABSTRACT

Lipid metabolism is frequently perturbed in cancers, but the underlying mechanism is unclear. We present comprehensive evidence that oncogene MYC, in collaboration with transcription factor sterol-regulated element-binding protein (SREBP1), regulates lipogenesis to promote tumorigenesis. We used human and mouse tumor-derived cell lines, tumor xenografts, and four conditional transgenic mouse models of MYC-induced tumors to show that MYC regulates lipogenesis genes, enzymes, and metabolites. We found that MYC induces SREBP1, and they collaborate to activate fatty acid (FA) synthesis and drive FA chain elongation from glucose and glutamine. Further, by employing desorption electrospray ionization mass spectrometry imaging (DESI-MSI), we observed in vivo lipidomic changes upon MYC induction across different cancers, for example, a global increase in glycerophosphoglycerols. After inhibition of FA synthesis, tumorigenesis was blocked, and tumors regressed in both xenograft and primary transgenic mouse models, revealing the vulnerability of MYC-induced tumors to the inhibition of lipogenesis.


Subject(s)
Carcinogenesis/genetics , Lipogenesis/genetics , Proto-Oncogene Proteins c-myc/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism , Animals , Cell Line, Tumor , Fatty Acids/biosynthesis , Female , Gene Expression Regulation, Neoplastic , Heterografts , Humans , Male , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-myc/genetics
5.
J Immunother Cancer ; 6(1): 125, 2018 11 20.
Article in English | MEDLINE | ID: mdl-30458889

ABSTRACT

Interleukin-12 (IL-12) is a promising candidate for cancer immunotherapy because of its ability to activate a number of host immune subsets that recognize and destroy cancer cells. We found that human hepatocellular carcinoma (HCC) patients with higher than median levels of IL-12 have significantly favorable clinical outcomes. Here, we report that a messenger RNA (mRNA) lipid nanoparticle delivering IL-12 (IL-12-LNP) slows down the progression of MYC oncogene-driven HCC. IL-12-LNP was well distributed within the HCC tumor and was not associated with significant animal toxicity. Treatment with IL-12-LNP significantly reduced liver tumor burden measured by dynamic magnetic resonance imaging (MRI), and increased survival of MYC-induced HCC transgenic mice in comparison to control mice. Importantly, IL-12-LNP exhibited no effect on transgenic MYC levels confirming that its therapeutic efficacy was not related to the downregulation of a driver oncogene. IL-12-LNP elicited marked infiltration of activated CD44+ CD3+ CD4+ T helper cells into the tumor, and increased the production of Interferon γ (IFNγ). Collectively, our findings suggest that IL-12-LNP administration may be an effective immunotherapy against HCC.


Subject(s)
Carcinoma, Hepatocellular/genetics , Genes, myc/genetics , Interleukin-12/metabolism , Liver Neoplasms/genetics , RNA, Messenger/metabolism , Animals , Carcinogenesis , Carcinoma, Hepatocellular/pathology , Female , Humans , Interleukin-12/genetics , Lipids , Liver Neoplasms/pathology , Male , Mice , Nanoparticles
6.
Oncotarget ; 9(5): 5517-5528, 2018 Jan 19.
Article in English | MEDLINE | ID: mdl-29464015

ABSTRACT

Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options. Genomic amplification and/or overexpression of the MYC oncogene is a common molecular event in HCC, thus making it an attractive target for drug therapy. Unfortunately, currently there are no direct drug therapies against MYC. As an alternative strategy, microRNAs regulated by MYC may be downstream targets for therapeutic blockade. MiR-17 family is a microRNA family transcriptionally regulated by MYC and it is commonly overexpressed in human HCCs. In this study, we performed systemic delivery of a novel lipid nanoparticle (LNP) encapsulating an anti-miR-17 oligonucleotide in a conditional transgenic mouse model of MYC driven HCC. Treatment with anti-miR-17 in vivo, but not with a control anti-miRNA, resulted in significant de-repression of direct targets of miR-17, robust apoptosis, decreased proliferation and led to delayed tumorigenesis in MYC-driven HCCs. Global gene expression profiling revealed engagement of miR-17 target genes and inhibition of key transcriptional programs of MYC, including cell cycle progression and proliferation. Hence, anti-miR-17 is an effective therapy for MYC-driven HCC.

7.
Cancer Immunol Immunother ; 67(1): 47-60, 2018 01.
Article in English | MEDLINE | ID: mdl-28905118

ABSTRACT

The delivery of immunomodulators directly into the tumor potentially harnesses the existing antigen, tumor-specific infiltrating lymphocytes, and antigen presenting cells. This can confer specificity and generate a potent systemic anti-tumor immune response with lower doses and less toxicity compared to systemic administration, in effect an in situ vaccine. Here, we test this concept using the novel combination of immunomodulators anti-CTLA4, -CD137, and -OX40. The triple combination administered intratumorally at low doses to one tumor of a dual tumor mouse model had dramatic local and systemic anti-tumor efficacy in lymphoma (A20) and solid tumor (MC38) models, consistent with an abscopal effect. The minimal effective dose was 10 µg each. The effect was dependent on CD8 T-cells. Intratumoral administration resulted in superior local and distant tumor control compared to systemic routes, supporting the in situ vaccine concept. In a single tumor A20 model, injection close to the tDLN resulted in similar efficacy as intratumoral and significantly better than targeting a non-tDLN, supporting the role of the tDLN as a viable immunotherapy target in addition to the tumor itself. Distribution studies confirmed expected concentration of antibodies in tumor and tDLN, in keeping with the anti-tumor results. Overall intratumoral or peri-tDLN administration of the novel combination of anti-CTLA4, anti-CD137, and anti-OX40, all agents in the clinic or clinical trials, demonstrates potent systemic anti-tumor effects. This immunotherapeutic combination is promising for future clinical development via both these safe and highly efficacious routes of administration.


Subject(s)
Antibodies, Monoclonal/therapeutic use , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Colonic Neoplasms/therapy , Immunotherapy/methods , Lymphoma/therapy , Sentinel Lymph Node/pathology , Animals , CTLA-4 Antigen/immunology , Colonic Neoplasms/immunology , Disease Models, Animal , Drug Therapy, Combination , Female , Humans , Lymphoma/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, OX40/immunology , Remission Induction , Sentinel Lymph Node/drug effects , Tumor Burden/drug effects , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunology
8.
Stem Cell Reports ; 2(4): 457-72, 2014 Apr 08.
Article in English | MEDLINE | ID: mdl-24749071

ABSTRACT

Hematopoiesis in the embryo proceeds in a series of waves, with primitive erythroid-biased waves succeeded by definitive waves, within which the properties of hematopoietic stem cells (multilineage potential, self-renewal, and engraftability) gradually arise. Whereas self-renewal and engraftability have previously been examined in the embryo, multipotency has not been thoroughly addressed, especially at the single-cell level or within well-defined populations. To identify when and where clonal multilineage potential arises during embryogenesis, we developed a single-cell multipotency assay. We find that, during the initiation of definitive hematopoiesis in the embryo, a defined population of multipotent, engraftable progenitors emerges that is much more abundant within the yolk sac (YS) than the aorta-gonad-mesonephros (AGM) or fetal liver. These experiments indicate that multipotent cells appear in concert within both the YS and AGM and strongly implicate YS-derived progenitors as contributors to definitive hematopoiesis.


Subject(s)
Embryonic Development , Hematopoietic Stem Cells/metabolism , Multipotent Stem Cells/metabolism , Animals , Antigens, Surface/metabolism , CD11a Antigen/genetics , Cell Differentiation , Cell Lineage , Colony-Forming Units Assay , Embryonic Development/genetics , Female , Hematopoietic Stem Cells/cytology , Immunophenotyping , Leukosialin/genetics , Leukosialin/metabolism , Mice , Multipotent Stem Cells/cytology , Phenotype , Proto-Oncogene Proteins c-kit/metabolism , Yolk Sac/embryology
9.
Stem Cells ; 30(10): 2114-27, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22899386

ABSTRACT

Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC.


Subject(s)
Cell Transformation, Neoplastic/pathology , Mammary Neoplasms, Experimental/pathology , Neoplastic Stem Cells/ultrastructure , Animals , Cell Transformation, Neoplastic/metabolism , Epithelium/ultrastructure , Female , Fluorescent Dyes , Genes, Reporter , Green Fluorescent Proteins , Humans , Image Processing, Computer-Assisted , Mammary Neoplasms, Experimental/metabolism , Mammary Tumor Virus, Mouse/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence, Multiphoton , Molecular Imaging , Neoplastic Stem Cells/transplantation , Signal Transduction , Tumor Microenvironment , Wnt1 Protein/metabolism
10.
Nat Biotechnol ; 30(6): 531-42, 2012 May 27.
Article in English | MEDLINE | ID: mdl-22634564

ABSTRACT

To identify early populations of committed progenitors derived from human embryonic stem cells (hESCs), we screened self-renewing, BMP4-treated and retinoic acid-treated cultures with >400 antibodies recognizing cell-surface antigens. Sorting of >30 subpopulations followed by transcriptional analysis of developmental genes identified four distinct candidate progenitor groups. Subsets detected in self-renewing cultures, including CXCR4(+) cells, expressed primitive endoderm genes. Expression of Cxcr4 in primitive endoderm was confirmed in visceral endoderm of mouse embryos. BMP4-induced progenitors exhibited gene signatures of mesoderm, trophoblast and vascular endothelium, suggesting correspondence to gastrulation-stage primitive streak, chorion and allantois precursors, respectively. Functional studies in vitro and in vivo confirmed that ROR2(+) cells produce mesoderm progeny, APA(+) cells generate syncytiotrophoblasts and CD87(+) cells give rise to vasculature. The same progenitor classes emerged during the differentiation of human induced pluripotent stem cells (hiPSCs). These markers and progenitors provide tools for purifying human tissue-regenerating progenitors and for studying the commitment of pluripotent stem cells to lineage progenitors.


Subject(s)
Endoderm/cytology , Endothelium, Vascular/cytology , Mesoderm/cytology , Pluripotent Stem Cells/cytology , Trophoblasts/cytology , Animals , Antigens, Surface/chemistry , Biomarkers/chemistry , Cluster Analysis , Endoderm/chemistry , Endothelium, Vascular/chemistry , Flow Cytometry , Glutamyl Aminopeptidase/genetics , Humans , Mesoderm/chemistry , Mice , Mice, Transgenic , Pluripotent Stem Cells/chemistry , Pluripotent Stem Cells/classification , Receptors, CXCR4/biosynthesis , Receptors, CXCR4/genetics , Trophoblasts/chemistry
11.
Proc Natl Acad Sci U S A ; 109(17): 6662-7, 2012 Apr 24.
Article in English | MEDLINE | ID: mdl-22451913

ABSTRACT

CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.


Subject(s)
Antigens, Differentiation/metabolism , CD47 Antigen/immunology , Neoplasms/immunology , RNA, Messenger/genetics , Receptors, Immunologic/metabolism , Antibodies/immunology , CD47 Antigen/genetics , Cell Division/immunology , Flow Cytometry , Humans , Neoplasms/pathology , Neoplasms/therapy , Phagocytosis/immunology , Prognosis , Survival Analysis
12.
Nat Biotechnol ; 29(9): 829-34, 2011 Aug 14.
Article in English | MEDLINE | ID: mdl-21841799

ABSTRACT

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.


Subject(s)
Antibodies, Monoclonal/metabolism , Pluripotent Stem Cells/cytology , Polysaccharides/metabolism , Stage-Specific Embryonic Antigens/metabolism , Teratoma/pathology , Animals , Biomarkers , Cell Differentiation , Cells, Cultured , Computational Biology , Flow Cytometry , Gene Expression Regulation , Humans , Immunohistochemistry , Mice , Mice, Inbred BALB C , Microarray Analysis , Pluripotent Stem Cells/chemistry , Real-Time Polymerase Chain Reaction , Teratoma/metabolism
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