Complex Oncogene Dependence in Microrna-125A– Induced Myeloproliferative Neoplasms
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Complex oncogene dependence in microRNA-125a– induced myeloproliferative neoplasms Shangqin Guoa,b, Haitao Baib,c, Cynthia M. Megyolaa,b, Stephanie Halenea,d,e, Diane S. Krausea,e,f, David T. Scaddeng, and Jun Lua,b,e,1 bDepartment of Genetics, dDepartment of Medicine, fDepartment of Laboratory Medicine, aYale Stem Cell Center, and eYale Cancer Center, Yale University School of Medicine, New Haven, CT 06520; gCenter for Regenerative Medicine, Massachusetts General Hospital, Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Boston, MA 02114; and cDepartment of Hematology, Shanghai First People’s Hospital Shanghai 200080, China Edited by Sherman M. Weissman, Yale University, New Haven, CT, and approved August 30, 2012 (received for review August 2, 2012) Deregulation of microRNA (miRNA) expression can lead to cancer neoplasms, MPNs) are a collection of hematopoietic diseases initiation and progression. However, limited information exists on the with common features (reviewed in refs. 13 and 14), such as function of miRNAs in cancer maintenance. We examined these issues overproduction of one or more myeloid/erythroid/platelet line- in the case of myeloproliferative diseases and neoplasms (MPN), ages and splenomegaly. In some patients, the MPN will progress a collection of hematopoietic neoplasms regarded as preleukemic, to acute myeloid leukemia, leading to the notion that MPN is thereby representing early neoplastic states. We report here that one of the preleukemic states of a full-blown myeloid leukemia. microRNA-125a (miR-125a)–induced MPN display a complex manner In this study, we used genomics and genetics to explore the role of oncogene dependence. Following a gain-of-function genomics of miRNAs in MPN and the dependence of the early neoplastic screen, we overexpressed candidate miR-125a in vivo, which led MPN state on an oncogenic miRNA. We report a barcode genetic to phenotypes consistent with an atypical MPN characterized by screen with the use of a unique miRNA expression library, which led to the identification of miR-125 miRNAs in inducing hyper- leukocytosis, monocytosis, splenomegaly, and progressive anemia. sensitivity toward cytokine signaling in both cell line and primary The diseased MPN state could be recapitulated in a doxycycline-in- bone marrow cells. Using a unique inducible model for miRNA ducible mouse model. Upon doxycycline withdrawal, the primary expression, we show that miR-125a–induced MPN display a com- MPN phenotypes rapidly resolved after the discontinuation of miR- plex manner of dependence on the continued expression of miR- 125a overexpression. However, reinduction of miR-125a led to com- 125a. Although the hematopoietic phenotypes of the initial miR- plex phenotypes, with some animals rapidly developing lethal ane- 125a–induced MPN appear reversible, re-exposure to the same mia with extensive damages in the spleen. Forced expression of oncogenic miRNA causes significantly altered disease manifesta- miR-125a resulted in elevated cellular tyrosine phosphorylation tion, with damages in multiple tissues indicating irreversible sys- and hypersensitivity toward hematopoietic cytokines. Furthermore, temic changes. Molecularly, we identify several protein phos- we demonstrate that miR-125a targets multiple protein phospha- phatases as previously unrecognized targets of miR-125a and the tases. Our data demonstrate that miR-125a–induced MPN is ad- associated elevation in cellular tyrosine phosphorylation. Our dicted to its sustained overexpression, and highlight the complex work demonstrates that oncogenic miRNA dependence is not nature of oncogenic miRNA dependence in an early neoplastic state. limited to advanced disease states. Our work also implies that profound systemic changes could occur during an early neoplastic oncogene addiction | PTPN18 | spleen fibrosis disease state and its remission, which then modifies the nature of the lesion upon re-encountering the same oncogene. he expression of microRNAs (miRNAs) is frequently deregu- Results Tlated in human cancers (1, 2). Alteration in miRNA gene expression or function can promote cancer initiation and pro- Functional Genomics Screen Identified miR-125 Family miRNAs as gression in vivo, leading to the recognition that this class of small Conferring Reduced Cytokine Dependence. Hematopoietic cells noncoding RNAs can function as both oncogenes and tumor from MPN patients often display hypersensitivity toward cytokines suppressors (3–5). Much less is known, however, regarding the (15–17). To search for miRNAs that regulate cytokine sensitivity, role of miRNAs in cancer maintenance. A special type of cancer we designed a barcode-based functional genomics screen in BaF3 maintenance is related to oncogene dependence/addiction, in cells, which strictly depends on IL-3 for survival and proliferation which the maintenance of cancer phenotype is dependent on the (18) (Fig. 1A). We constructed a retroviral miRNA expression li- sustained presence of one of the initial driving genetic lesions, brary, by PCR-amplifying specific miRNA-encoding and flanking even though cancer cells may have acquired additional muta- regions from human genomic DNA and inserting the amplified tions. Indeed, oncogene addiction has been demonstrated for a fragments into a retroviral vector. The resulted library contains 273 short list of protein-coding genes in vivo [e.g., c-myc, BCR/ABL, human miRNA expression constructs (see SI Methods and Dataset Her-2, and p53 (6–7)], many of which are clinically validated S1). The library vector supports overexpression in BaF3 cells (Fig. therapeutic targets (8, 9). In the case of miRNAs, withdrawal of S1C). For the functional screen, we reasoned that BaF3 cells car- ectopic miR-21 is known to cause regression in a mouse model of rying a miRNA that confers reduced IL-3 dependence will have lymphoma (10), indicating that some established lymphomas a growth advantage and be amplified under critically low IL-3 may be dependent on miR-21 overexpression. conditions. Consequently, this miRNA insert should be over- Interestingly, recent studies on p53 demonstrate that the represented in the cell pool after selection with low or no cytokine, phenotype of oncogene addiction can be dependent on the stage of cancer progression (11, 12). In mouse lung cancer models driven by the loss of p53 and mutant k-ras, genetic restoration of Author contributions: S.G., D.S.K., D.T.S., and J.L. designed research; S.G., H.B., C.M.M., p53 leads to significant loss of tumor mass in more advanced and J.L. performed research; S.G., C.M.M., S.H., D.S.K., and J.L. analyzed data; and S.G. and tumor cells but not in the earlier, less aggressive ones. These J.L. wrote the paper. studies suggest that early-stage neoplasms may provide a context The authors declare no conflict of interest. for complex oncogene addition phenomena. However, de- This article is a PNAS Direct Submission. pendence on oncogene behavior in early-stage neoplasms in vivo 1To whom correspondence should be addressed. E-mail: [email protected]. has not been much explored in other types of malignancies. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Myeloproliferative diseases (also known as myeloproliferative 1073/pnas.1213196109/-/DCSupplemental. 16636–16641 | PNAS | October 9, 2012 | vol. 109 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1213196109 Downloaded by guest on September 23, 2021 A Infect library Low IL-3 role of these two miRNAs in inducing cytokine hypersensitivity in BaF3 cells. We used miR-125b-1 in all experiments because BaF3 Cells miR-125b-2 produces exactly the same mature product. In- DNA DNA terestingly, the expression of miR-125a/b is highly enriched in Genomic Genomic – PCR hematopoietic stem cells (20 22). Translocation and overexpres- PCR sion have been reported in acute myeloid leukemia and MPN patients (23–25). Overexpression of miR-125a or miR-125b greatly enhances the hematopoietic stem cell pool and output, B Bead-based bar-code detection and can induce MPN in mice (21, 26). This result demonstrates miRNA- or Control- the effectiveness of the screen to identify relevant MPN-inducing transduced cells miRNAs, and suggest that miR-125 miRNAs may regulate cy- Day 0 Time tokine signaling in primary cells. Mix Constitutive Overexpression of miR-125a Leads to MPN. Although Non-transduced leukemia is induced by overexpression of miR-125b in several compe tor cells Flow Cytometry Analysis models (21, 24, 26–28), myeloproliferation could be the pre- dominant effect of miR-125a overexpression (20, 29, 30). To CD fi 10 50 address oncogene addiction in the context of MPN, we rst Vector o Vector assessed the effects of constitutive miR-125a overexpression in 8 40 o miR-125b-1 miR-125a vivo to gauge the phenotypes of an inducible miR-125a model to 6 30 be described later. Specifically, we transduced donor bone mar- 4 20 row cells with miR-125a or a control vector, each containing 2 10 a GFP marker, and transplanted into lethally irradiated recipient 0 GFP+/GFP- cell ra 0 GFP+/GFP- cell ra animals. Although displaying prominent multilineage engraft- 0 5 10 15 20 Days 0 5Days 10 15 ment, miR-125a–recipient animals over time manifested multiple phenotypes indicative of an atypical MPN, including leukocytosis, Fig. 1. A functional screen identifies miR-125 family miRNAs conferring monocytosis, splenomegaly, liver leukocyte infiltration, and pro- reduced cytokine dependence in BaF3 cells. (A) Schematic of the screen. BaF3 gressive anemia (Fig. 2A and Fig. S2C). Leukocytosis in miR-125a cells were infected with a pooled viral library containing 273 miRNAs. Ge- recipients progressively worsened with time (Fig. S2A), which was nomic DNA was harvested before and after selection in low IL-3, amplified fl MEDICAL SCIENCES fi the result of an increase in myeloid cells. Indeed, ow cytometry with construct-speci c primers and detected on a bead-based platform. fi + + Colored hairpins and lines illustrate hypothetical miRNA integrations and showed a signi cant elevation of the GFP Mac1 population amplicons.