OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015)

BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors. Follow this format for each person. DO NOT EXCEED FIVE PAGES. NAME: Shannon Mychel Buckley eRA COMMONS USER NAME (credential, e.g., agency login): Buckls02 POSITION TITLE: Assistant Professor

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) DEGREE Completion (if Date FIELD OF STUDY INSTITUTION AND LOCATION applicable) MM/YYYY

University of St. Thomas, St. Paul, MN B.S. 09/96 Biology

University of Minnesota, Mpls, MN Ph.D. 01/09 Molecular and Cellular Biology

NYU School of Medicine, New York, NY Postdoctoral 04/2015 Cancer and Stem Cell Biology

Please refer to the Biographical Sketch sample in order to complete sections A, B, C, and D of the Biographical Sketch.

A. Personal Statement Over the years my graduate and postdoctoral research has built the foundation for my future research interests. My goal is to utilize genomic and proteomic approaches in adult, and leukemic stem cells as well as mouse models to study molecular mechanisms regulating cell fate decisions. Specifically, studying posttranslational modifications by the ubiquitin proteasome system (UPS) in self-renewal, differentiation, and transformation. These goals combine my knowledge gained during my graduate work directly with the experience and expertise of my postdoctoral fellowship. My PhD was completed in January 2009 where I studied the role of the microenvironment in regulating hematopoietic stem cell (HSC) self-renewal and maintenance with my graduate advisor Dr. Catherine Verfailllie at both the University of Minnesota and the Katholieke Universiteit Leuven in Belgium. My work identified novel secreted factors expressed during the development of hematopoiesis that enhance migration, homing, and maintenance of HSC, thus providing further understanding of the stem cell niche. I did my postdoctoral work in the Department of Pathology at New York University School of Medicine working under Dr. Iannis Aifantis. For my postdoctoral research I turned my interest to intrinsic mechanisms regulating cell fate decisions. Employing both pluripotent (embryonic stem cells and induced pluripotent stem cells) and adult stem cells (HSC), we were able to identify novel members of the UPS that play key roles in pluripotency, differentiation and leukemogenesis. Building from these findings I aim in my future research to further explore the role of UPS in regulating self-renewal, differentiation, cellular reprogramming, and transformation by utilizing both proteomic and genomic approaches in stem cell populations. a. Reavie, LR.*, Buckley, SM.*, Loizou, E, Takeishi, S., Abdel-Wahab, O., Aranda-Orgilles, B., Ndiaye-Lobry, D., Ibrahim, S., Nakayama, KI., and Aifantis, I. Regulation of c-Myc ubiquitination controls chronic

myelogenous leukemia initiation and progression. Cancer Cell. 2013; 23(3):362-375. PMID: 23518350 (*contributed equally to the manuscript) b. Buckley, SM., Aranda-Orgilles, B., Strikoudis, A., Apostolou, E., Loizou, E., Moran-Crusio, K., Farnsworth, CL., Koller, AA., Dasgupta, R., Silva, JC., Stadtfeld, M., Hochedlinger, K., Chen, EI., and Aifantis, I. Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system. Cell Stem Cell. 2012; 11(6):783-798. PMID: 23103054

Comment in: Cell Stem Cell. 2013; 11(6):728-730. UPS Delivers Pluripotency. Okita, Y. and Nakayama, K. c. Buckley, SM., Ulloa-Montoya, F., Abts, D., Oostendorp, R., Dzierzak, E., Ekker, SC. and Verfaillie, CM. Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line. Experimental Hematology. 2011; 39(1):114-123. PMID: 20933051

B. Positions and Honors

Positions and Employment 2001-2002 Junior Scientist, Stem Cell Institute, University of Minnesota 2002-2003 Assistant Scientist, Stem Cell Institute, University of Minnesota 2003-2009 Research Assistant/Graduate Student, Stem Institute, University of Minnesota 2006-2009 Research Assistant/Graduate Student, Katholieke Universiteit Leuven, Belgium 2009-2015 Postdoctoral Fellow, NYU School of Medicine 2015-Present Assistant Professor, Department of Genetics, Cell Biology, and Anatomy, University of Nebraska Medical Center

Honors 2005 Keystone Symposia, Molecular Regulation of Stem Cells, Travel Scholarship 2009-2010 Postdoctoral Fellowship, Hematology NIH Research Training Grant 2011-2012 Postdoctoral Fellowship, Hematology NIH Research Training Grant 2011 International Society for Stem Cell Research, travel grant 2012- 2014 Postdoctoral Fellowship, NYU Stem Cell NIH Research Training Grant 2013 Keystone Symposia, Stem Cell Regulation in Homeostasis and Disease, Invited Conference Assistant 2013-2014 NYU Kimmel Stem Cell Center Senior Postdoctoral Fellow

C. Contribution to Science 1. In vivo, HSC receive signals from the local microenvironment in which they reside that regulate self-renewal vs. differentiation cell fate decisions. Utilizing transcriptional profiling of the hematopoietic microenvironment during development, we identified novel secreted that enhance migration, homing, and maintenance of HSC, thus providing further understanding of the stem cell niche, as well as, potential treatments to enhance homing of HSC following HSC transplantation in patients. a. Khurana, S., Margamulijana, L., Joseph C., Schouteden, S., Buckley, SM., and Verfaillie CM. Glypican-3 mediated inhibition of CD26 by TFPI: a novel mechanism in hematopoietic stem cell homing and maintenance. Blood. 2013; 121(14):2587-95. PMID: 23327927 b. Khurana, S., Buckley, S., Schouteden, S., Ekker, S., Petryk, A., Delforge, M., Zwijsen, A., and Verfaillie CM. A novel role of BMP4 in adult hematopoietic stem and progenitor cell homing via Smad independent regulation of Integrin-α4 expression. Blood. 2013; 121(5):781-790. PMID: 23243277 c. Buckley, SM., Ulloa-Montoya, F., Abts, D., Oostendorp, R., Dzierzak, E., Ekker, SC. and Verfaillie, CM. Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line. Experimental Hematology. 2011; 39(1):114-123. PMID: 20933051 d. Buckley, S. and Verfaillie, C. Regulation of Hematopoiesis. In: Blood and Bone Marrow Pathology, 2nd ed. Elsevier, 63-76, 2011.

2. My postdoctoral research in the Aifantis lab focused on the role of the UPS in stem cell fate decisions. Utilizing both pluripotent and adult stem cells, we have shown that ubiquitin E3 ligases regulate mechanisms of

self-renewal and differentiation. The primary focus was on identifying ubiquitin E3 ligases that regulate pluripotency and differentiation, as well as determining substrates, utilizing a combination of genomic and proteomic approaches. We: a) defined “ubiquitin signatures” of ESC (embryonic stem cells), and iPSC (induced pluripotent stem cells) using mass spectrometry-based approaches and, b) identified ubiquitin ligases, and deubiquitinases (DUBs) as essential for ESC pluripotency and differentiation using RNAi-based screens. The second part of my postdoctoral work focused on the role of E3 ligase Fbxw7 in regulating the leukemic stem cells (LSC) in chronic myeloid leukemia (CML). The LSC population in CML is contained to the hematopoietic stem and progenitor population (HSPC) suggesting molecular mechanisms regulating HSC may also regulate initiation and progression of the disease. Utilizing both mouse models of disease and primary patient samples we have identified the role of , Fbxw7, and its substrate cMyc in disease progression, maintenance and initiation. We found that CML requires specific levels of cMyc expression for maintenance of disease and that silencing of Fbxw7 leads to cell death of the cancer stem cell population suggesting Fbxw7 as a potential therapeutic target in CML.

a. Reavie, LR.*, Buckley, SM.*, Loizou, E, Takeishi, S., Abdel-Wahab, O., Aranda-Orgilles, B., Ndiaye-Lobry, D., Ibrahim, S., Nakayama, KI., and Aifantis, I. Regulation of c-Myc ubiquitination controls chronic myelogenous leukemia initiation and progression. Cancer Cell. 2013; 23(3):362-375. PMID: 23518350 (*contributed equally to the manuscript) b. Buckley, SM., Aranda-Orgilles, B., Strikoudis, A., Apostolou, E., Loizou, E., Moran-Crusio, K., Farnsworth, CL., Koller, AA., Dasgupta, R., Silva, JC., Stadtfeld, M., Hochedlinger, K., Chen, EI., and Aifantis, I. Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system. Cell Stem Cell. 2012; 11(6):783-798. PMID: 23103054

Comment in: Cell Stem Cell. 2013; 11(6):728-730. UPS Delivers Pluripotency. Okita, Y. and Nakayama, K. c. Reavie, L., Della Gatta, G., Crusio, K., Aranda-Orgilles, B., Buckley, SM., Thompson, B., Lee, E., Gao, J., Bredemeyer, AL., Helmink, BA., Zavadil, J., Sleckman, BP., Palomero, T., Ferrando, A., and Aifantis, I. Regulation of Hematopoietic Stem Cell Differentiation by a Single Ubiquitin Ligase-Substrate Complex. Nature Immunology. 2010; 11(3):207-15. PMID: 20081848

Complete List of Published Work in MyBibliography: http://www.ncbi.nlm.nih.gov/sites/myncbi/shannon.buckley.1/bibliograpahy/47629632/public/?sort=dat e&direction=ascending

D. Research Support

Current none

Completed Research Support

3T32CA160002-03S1 Lehman, Ruth (PI) 12/16/2013-12/15/2014 NIH Training Program in Stem Cell and Cancer Biology

5T32CA160002-02 Lehman, Ruth (PI) 09/1/2012-08/31/2013 NIH Training Program in Stem Cell and Cancer Biology

5T32HL007151-33 Gardner, Lawrence (PI) 07/1/2011-06/30/2012 NIH Research in the Pathogenesis of Hematologic Disorders

2T32HL007151-31A1 Gardner, Lawrence (PI) 07/1/2009-06/30/2010 NIH Research in the Pathogenesis of Hematologic Disorders Publication List * * 1. Gao, J. , Buckley, SM. , Cang, Y., Goff, S. and Aifantis, I. Reg. The Cul4-Ddb1 ubiquitin ligase complex controls adult and embryonic stem cell differentiation and homeostasis. (eLife, under Revision, *contributed equally to the manuscript)

* * 2. Reavie, LR. , Buckley, SM. , Loizou, E, Takeishi, S., Abdel-Wahab, O., Aranda-Orgilles, B., Ndiaye-Lobry, D., Ibrahim, S., Nakayama, KI., and Aifantis, I. Regulation of c-Myc ubiquitination controls chronic myelogenous leukemia initiation and progression. Cancer Cell. 2013; 23(3):362-375. (*contributed equally to the manuscript)

3. Khurana, S., Margamulijana, L., Joseph C., Schouteden, S., Buckley, SM., and Verfaillie CM. Glypican-3 mediated inhibition of CD26 by TFPI: a novel mechanism in hematopoietic stem cell homing and maintenance. Blood. 2013; 121(14):2587-95.

4. Khurana, S., Buckley, S., Schouteden, S., Ekker, S., Petryk, A., Delforge, M., Zwijsen, A., and Verfaillie CM. A novel role of BMP4 in adult hematopoietic stem and progenitor cell homing via Smad independent regulation of Integrin-〈4 expression. Blood. 2013; 121(5):781-790.

5. Buckley, SM., Aranda-Orgilles, B., Strikoudis, A., Apostolou, E., Loizou, E., Moran-Crusio, K., Farnsworth, CL., Koller, AA., Dasgupta, R., Silva, JC., Stadtfeld, M., Hochedlinger, K., Chen, EI., and Aifantis, I. Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system. Cell Stem Cell. 2012; 11(6):783-798. Comment in: Cell Stem Cell. 2013; 11(6):728-730. UPS Delivers Pluripotency. Okita, Y. and Nakayama, K. nd 6. Buckley, S. and Verfaillie, C. Regulation of Hematopoiesis. In: Blood and Bone Marrow Pathology, 2 ed. Elsevier, 63-76, 2011. 7. Buckley, SM., Ulloa-Montoya, F., Abts, D., Oostendorp, R., Dzierzak, E., Ekker, SC. and Verfaillie, CM. Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line. Experimental Hematology. 2011; 39(1):114-123.

8. Reavie, L., Della Gatta, G., Crusio, K., Aranda-Orgilles, B., Buckley, SM., Thompson, B., Lee, E., Gao, J., Bredemeyer, AL., Helmink, BA., Zavadil, J., Sleckman, BP., Palomero, T., Ferrando, A., and Aifantis, I. Regulation of Hematopoietic Stem Cell Differentiation by a Single Ubiquitin Ligase-Substrate Complex. Nature Immunology. 2010; 11(3):207-15.

9. Sahin, B., Schwartz, RE., Buckley, SM., Heremans, Y., Hu, W., Verfaillie, CM. Isolation and Characterization of Liver Derived Stem Cells from Unmanipulated Rat Liver. Liver Transplantation. 2008; 14(3):333-45.

10. Serafini, M., Dylla, S., Oki, M., Heremans, Y., Jiang, Y., Buckley, SM., Pelacho, B., Burns, T., Frommer, S., Rossi, D., Bryder, D. Panoskaltsis-Mortari, A., O’Shaughnessy, M., Nelson-Holte, M., Weissman, IL., Blazar, BR., Verfaillie, CM. Complete Lymphohematopoietic Reconstitution by Multipotent Adult Progenitor Cells. Journal of Experimental Medicine. 2007; 204(1):129-39.

11. Lakshmipathy, U., Buckley, S., Verfaillie C. transfer via nucleofection into adult and embryonic stem cells. Stem Cell Assays, Methods Molecular Biology. 2007; 407:115-126.

12. Liu B, Buckley SM, Lewis ID, Goldman AI, Wagner JE, van der Loo JC. Homing defects of cultured human hematopoietic cells in the NOD/SCID mouse is mediated by Fas/CD95. Experimental Hematology. 2003; 31(9):824-32. 13. Van der Loo JC, Liu BL, Goldman AI, Buckley SM, Chrudimsky KS. Optimization of Gene Transfer into Primitive Human Hematopoietic Cells of Granulocyte-Colony Stimulating Factor-Mobilized Peripheral Blood Using Low-Dose Cytokines and Comparison of a Gibbon Ape Leukemia Virus Versus an RD114- Pseudotyped Retroviral Vector. Human Gene Therapy. 2002; 13(11):1317-30 Buckley, SM Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! PROJECT TITLE: Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

Scientific Abstract

Mantle cell lymphoma (MCL) is a rare and aggressive non-Hodgkin’s lymphoma. The majority of MCL patients have mutations leading to overexpression of CyclinD1 in the pre-B cell population subsequently preceding extensive proliferation and blocks in differentiation originating in the mantle zone of the lymph node. Recently next generation sequencing has identified a number of new novel mutations in MCL patients including the ubiquitin E3 ligase UBR5. E3 ubiquitin ligases serve as the substrate recognizing component for degradation by the ubiquitin proteasome system. Approximately 18% of MCL patients were found to have mutations within the HECT domain of UBR5, which can accept and transfer ubiquitin molecules to the substrate. Interestingly in hematopoietic lineages the B-lymphoid populations including the pre-B cell highly express UBR5 compared to the myeloid and T-lymphoid compartments. These findings suggest that understanding the role of UBR5 in hematopoiesis will provide insights to mantle cell lymphoma transformation, progression and possible future therapeutics treatment, in addition to, basic understanding of hematopoietic cell specification.

Lay Abstract

Mantle cell lymphoma (MCL) is a raw and aggressive form of non-Hodgkin’s lymphoma. Currently MCL patients have poor outcomes and often undergo bone marrow transplantation as part of their treatment. Recently new novel mutations have been identified in patients promising new avenues of research to understand MCL. Here we aim to study the role of the protein UBR5 which has been found to be mutated in ~18% of MCL patients to gain new knowledge and understanding of disease initiation, progression, and future therapies.

DESCRIPTION OF RESEARCH PROPOSED:

A. Specific Aims

The hematopoietic system is maintained and replenished throughout life by hematopoietic stem cells (HSC) that are capable of differentiation to all hematopoietic lineages. Differentiation pathways to specific hematopoietic lineages have been well studied over the years. However, it has been suggested that disruption of differentiation pathways leads to blocks in differentiation and initiation of hematopoietic malignancies. Research has focused on a number of transcription factors networks that regulate cell fate decisions in hematopoiesis however accumulating evidence suggests that protein degradation by the ubiquitin proteasome system regulates decisions of self-renewal, differentiation, and survival. We recently identified a number of ubiquitin E3 ligases that regulate cell fate decisions utilizing genomic and proteomic approaches in embryonic stem cells (ESC), including UBR5, a HECT domain E3 ligase. Interestingly, UBR5 is disrupted in a number of cancers and more recently been found to be mutated in ~18% of patients with mantle cell lymphoma (MCL). These mutations are found within the HECT domain, which can accept and transfer ubiquitin molecules to the substrate. Ubr5 null mice die in utero due to defects in yolk sac vascularization and although Ubr5 is expressed in hematopoietic cell lineages, the role in hematopoietic development and maintenance is unknown. Determining the role of UBR5 in hematopoiesis will provide insights to mantle cell lymphoma transformation, progression and treatment in addition to basic understanding of hematopoietic cell specification.

This leads to the hypothesis that the ubiquitin proteasome system (UPS), specifically E3 ligase UBR5 can control differentiation and lymphoma transformation. We test this hypothesis in the following Aims:

! 1! Buckley, SM Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Aim1: Understanding the role of Ubr5 in B-lymphoid lineage specifications and mantle cell lymphoma. In Aim 1, we propose both in vitro experiments in normal and malignant cells to understand the role of Ubr5 in Mantle Cell Lymphoma and B-lymphoid lineage specification. In vitro we will use bone marrow derived B-cell progenitors to determine if Ubr5 alters lymphoid differentiation, survival, proliferation, and/or DNA damage.

Aim 2: Mimicking UBR5 mutations to determine role in disease initiation and progression. In Aim 2, we will use a murine disease model to determine if Ubr5 plays a role in disease initiation and progression. Finally, we propose to target Ubr5 to mimic mutations previously identified in MCL patients to study lymphoma initiation and progression.

B. Background and Significance

We believe that this seed grant award can further the understanding of ubiquitin ligase, UBR5, function in hematopoietic differentiation and lymphomagenesis, as well as it may provide a future therapeutic target. There have been limited studies that address the role of the protein degradation by the ubiquitin proteasome system (UPS) during hematopoietic lineage specification. The studies proposed can significantly affect both basic and translational research as they could identify novel ubiquitin-regulated substrates important for hematopoiesis, immune regeneration, and leukemia/lymphoma maintenance. Most importantly, the UPS is amenable to small molecule targeting opening the way for possible future therapeutics. Proteasome inhibitors such as bortezomib have shown success for the treatment of blood cancers, including mantle cell lymphoma. Further supporting the translational importance of the UPS and suggests that targeting of specific elements of the UPS could lead to future breakthroughs in both basic research and cancer therapy.

Background: Hematopoietic maintenance and development. During gestational life, lymphopoiesis occurs in the thymus and spleen, and subsequently in the secondary lymphoid organs such as lymph nodes. At E11- 12, at which time T-lymphoid progenitors are also detected in the aorta-gonad-mesonephros (AGM) region, the first lymphoid progenitors can be detected in the thymus1-3. These progenitors then undergo maturation where they progressively become restricted to the T-cell lineage3. At the same time the population expands significantly. By contrast B-cell development occurs largely in the fetal liver, where from E14 B-cell progenitors expand peaking at the perinatal stage4. At E11, cells cluster at the site of the spleen in the dorsal mesogastrium, but the spleen is not identifiable until around E135. By embryonic day 13 cells derived from the fetal liver start to migrate to the spleen. By E14-16, hematopoietic progenitors such as CFU-E and CFU-GEMM can be found in the spleen, a phenomenon that persist in mice till after birth, even though the frequency of colony forming cells decreases around 3 weeks postnatally6. Around E17, coinciding with initiation of lymphopoiesis, cells from the thymus immigrate in the spleen6. Migration of HSC from the fetal liver to the marrow commences around E17, and peaks in the immediate pre-natal period at which time the bone marrow (BM) becomes the chief hematopoietic organ.

Hematopoietic differentiation is a highly ordered process that depends on extrinsic and intrinsic signaling pathways. Adult hematopoiesis is maintained throughout life by the hematopoietic stem cells (HSCs), which are self-renewing capable of generating all hematopoietic lineages 7-10. The hematopoietic stem and progenitor populations is marked by cell surface profile of Lineage-Sca1+c-kit+ (LSK) subset 11. The LSK fraction can be further divided into subsets of quiescent or Long Term (LT)-HSC (CD34-48-CD150+LSK), short-term or “actively self-renewing” (ST)-HSC (CD34+48-CD150-LSK) and multipotential progenitors (MPP, CD34+48+CD150-CD135-/+) cells 12. Homeostasis and differentiation of stem cells depends on interactions with their microenvironment and transcription factor signaling that determine hematopoietic lineage specification.

Mantle Cell Lymphoma. Mantle Cell Lymphoma (MCL) is a non-Hodgkin lymphoma (NHL) that represents ~6% of al NHL. Malignant transformation occurs in the mantle zone of the lymph node where the transformed pregerminal center B-lymphocyte population accumulates due to uncontrolled proliferation 13. The majority of MCL cases are marked by a translocation between 11 and 14 t(11;14), resulting in overexpression of cyclinD1. Recently additional mutations have been identified in MCL patients including ATM, ! 2! Buckley, SM Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! TP53, NOTCH, CCDN1, and UBR5 14,15. Although MCL initiates within the lymph nodes, infiltration can occur in the bone marrow (BM), liver, and lungs. Limited treatments are available for patients with MCL resulting in poor survival rates and outcomes. The proteasome inhibitor, Bortezomib has recently been successful in patients with progressed disease16, however many patients undergo autologous bone marrow transplants.

The ubiquitin-proteasome system (UPS). Temporally coordinated destruction of key cell regulatory proteins by the UPS represents an important control mechanism to ensure that specific protein functions are turned off at the right time, in the right compartment, and in a unidirectional fashion. Proteins are covalently tagged with chains of the small protein ubiquitin, which allows recruitment to the proteasome for proteolysis. The polyubiquitin chain is assembled on the substrate protein via an enzymatic cascade, in which ubiquitin is activated by covalent linkage to an E1 ubiquitin activating enzyme and transferred to an E2 ubiquitin conjugating enzyme, before an E3 ubiquitin ligase mediates transfer to a lysine residue in the substrate or a lysine in the growing polyubiquitin chain. Notably, the ultimate regulation of the reaction is dictated by the E3, which determines substrate specificity. Accordingly, the encodes two E1s, 39 E2s, and over 500 E3s. They are divided into groups based on their recognition sequences and interaction with its target substrate. The majority of E3 ligases are classified into two classes including HECT E3, and RING E3 ligases 23. Moreover, apart from the function of distinct ligase complexes, the UPS is also regulated by a class of enzymes responsible for removing ubiquitin conjugates from substrates. These deubiquitinating enzymes (DUBs) may serve a number of purposes: to recycle ubiquitin, to process ubiquitin precursors, or to edit previously ubiquitinated proteins among other functions 24.

The ubiquitin E3 ligase, Ubr5. Ubr5 is a large ~300kDa protein that belongs to the HECT domain class of E3 ligases and has a conserved carboxyl terminal HECT domain. Unlike RING E3 ligases that interact with E2 ubiquitin conjugating enzymes the conserved cysteine within the HECT domain can forms bond with ubiquitin before transferring the ubiquitin molecule to the target substrate17. Ubr5 has been shown to either poly- and mono-ubiquitinate substrates including β-catenin, RNF168, and CDK918,19,20. Ubr5 also contains two protein- protein interacting domains, a UBA domain and RING like zinc finger domain suggested being required for substrate interaction21. The Ubr5 null mice die during embryogenesis due to defects in yolk sac vascularization22. Ubr5 mutations have been identified in a number of solid tumors, however mutations are found throughout the gene. Interestingly, recently mutations have also been found in patients with MCL14. Approximately 60% of mutations in MCL are located in the HECT domain, and lead to loss of the conserved cysteine residue suggesting that mutations inhibit ubiquitin conjugation of Ubr5 substrates.

C. Preliminary Studies.

Identification of UPS regulators of pluripotency and differentiation. To study in detail the role of the UPS in embryonic stem cell (ESC) differentiation and pluripotency, we employed a combination of genomic and proteomic approaches. First, using mass spectrometry approaches we defined the ubiquitin Figure 1; Silencing of UBR5 leads to loss of landscape in mouse ESC, differentiated ESC, induced pluripotency and increased differentiation. A) z& pluripotent stem cells (iPSC), and mouse embryonic score!of!siRNA!screen!for!UPS!!regulating!! pluripotency..! B)! Nanog&GFP! expression! fibroblasts (MEF). This approach identified a large following! silencing! by! siRNA! C)! heat! map! of! number of pluripotent cell-specific ubiquitinated gene! expression! profile! for! pluripotency! genes! proteins. Second, to start identifying the enzymes and! early! differentiation! genes! in! identified! regulators! in! D)! morphology! of! two! responsible for these modifications, we used RNAi- representative!colonies!following!transfection!of! based screens, targeting the majority of known siRNA!at!day!6.!E)!AP+!ES&like!colony!at!Day!14! members of the UPS under both conditions of self- following!siRNA!knockdown!of!UBR5!relative!to! ! non&target!control. 3! Buckley, SM Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! renewal and differentiation. These functional screens identified a large number of putative novel regulators and suggested that proteasome function is essential for both ESC differentiation and pluripotency. These studies revealed that ubiquitin E3 ligase Ubr5 silencing leads to loss of maintenance and induction of pluripotency. To evaluate the role of the UPS in pluripotency vs. lineage differentiation cell fate decisions, we used siRNA libraries against E1 activating enzymes, E2 conjugating enzymes, E3 ligases, and deubiquitinating enzymes, as well as, predicted E3 ligases, which consisted of approximately 640 predicted and known UPS genes. The initial screen revealed 22 genes, including Ubr5 that led to significant negative effects on ESC self-renewal (z- score >1) (Figure 1A&B). Interestingly, following silencing of Ubr5 whole transcriptome profiling revealed up- regulation of early ectoderm specific genes suggesting Ubr5 plays a key role in lineage specification (Figure 1C). Additionally, when Ubr5 was silenced during cellular reprogramming morphology changes consistent with cellular reprogramming were not observed and the efficiency of generation of induced pluripotent stem cells was significantly reduced (Figure 1D&E). These combined studies suggest that Ubr5 plays a key role in cell fate decisions and lineage commitment.

Ubr5 in hematopoiesis. Little is known about the role of Ubr5 in hematopoiesis due to the Ubr5 null mouse dies during embryogenesis prior to the onset of definitive hematopoiesis at E10.522. However the recent identification of mutations specifically in the ubiquitin conjugation domain, HECT, of Ubr5 suggests a role in lymphomagenesis Ubr5. Utilizing transcriptional profiling in mouse hematopoietic cell lineages, we found that Ubr5 is expressed in all hematopoietic populations, however the highest expression is in the IgM+ B- and pre-B lymphocytes (Figure 2A). To determine if silencing would affect hematopoietic differentiation, we designed shRNAs targeting Ubr5 (Figure 2B). Silencing of Ubr5 in hematopoietic stem and progenitor cells led to significant decrease in hematopoietic progenitor colony forming potential with a greater impact on the more primitive GEMM colonies (Figure 2C&D). However no increase in cell death, apoptosis, or cell cycle was observed (Data Not Shown). Interestingly, the colonies following knockdown of Ubr5 were more mature lineage Figure 2; UBR5 is expressed in hematopoietic lineages and silencing leads to loss of progenitor cell activity. A) expression profiling in hematopoietic populations B) and analysis of cells within the colonies relative knockdown of UBR5 shRNAs. C&D) Colony forming units per 1,000 shRNA+ depicted definitive myeloid cells suggesting that Lin- bone marrow cells C) total colonies. D) of GEMM colonies. loss of Ubr5 led to hematopoietic differentiation similar to what was seen in ESC differntiation.

Together these studies define a role for the UPS in cell fate decisions, and more specifically suggest a role of Ubr5 in hematopoietic differentiation.

D. ProgressReport (forRenewalApplicationsonly) N/A

E. Research Design and Methods

Aim1: Understanding the role of Ubr5 in B-lymphoid lineage specifications and mantle cell lymphoma. Ubr5 null mice die in utero due to defects in yolk sac vascularization and although Ubr5 is expressed in hematopoiesis, the role in hematopoietic maintenance and development is unknown. Interestingly, Ubr5 is disrupted in a number of cancers and more recently been found to be mutated in 18% of patients with mantle ! 4! Buckley, SM Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! cell lymphoma (MCL)14. These mutations are found within the HECT domain, which can accept ubiquitin molecules that are then transferred to the substrate. Our findings of Ubr5 having a role in ESC differentiation along with the identification of Ubr5 mutations in lymphoma allude to a role of Ubr5 in hematopoiesis may provide insights on mantle cell lymphoma transformation and hematopoietic development. To test this hypothesis we will;

Aim 1A: Determine role of Ubr5 in B-lymphoid differentiation in vitro. We have previously showed that silencing of Ubr5 leads to a reduction in myeloid progenitor populations and rapid differentiation, however the effect on B-lymphoid differentiation is unknown. We will isolate hematopoietic progenitor cells (Lin-) from the bone marrow and transduce the cells with retroviral expressing shRNAs against Ubr5. Cells will be plated in pre-B cell methylcellulose based colony forming cultures supplemented with IL-7 and analyzed for their potential to give rise to colonies as well as their ability to subsequently replate to determine their proliferative potential, an indicator of tumor transformation. Cell surface markers (B220, CD45, IgM, CD19) will also be followed by flowcytometry during in vitro differentiation to follow lineage specification towards the B-Lymphoid lineage. Since MCL is marked by overexpression of cyclinD1, in parallel cells will also be co-transduced with retrovirus expressing cyclinD1 cDNA. These studies will determine if silencing of Ubr5 alone or in conjunction with cyclinD1 leads to a block in differentiation and enhanced proliferative potential of B-lymphoid progenitors.

Aim 1B: Impact of loss of UBR5 in MCL cell lines. In order to further understand the role of Ubr5 in MCL we will perform proliferation and survival experiments. We will transduce MCL cell lines, JEKO-1 and Mino, and a normal human transformed B-lymphoblast cell line with shRNAs against UBR5 and monitor for growth, survival and death. It is important to note that we have sequenced these MCL cell lines and they do not possess mutations in the UBR5 gene. Proliferation and survival will be monitored by flowcytometry using BrdU for cell cycle and AnnexinV for apoptosis/death. We expect if UBR5 plays a role in tumor initiation and progression increased proliferation and survival will be observed.

Aim 1C: Determine if loss of UBR5 in MCL cells leads to increased DNA damage. UBR5 has been linked to regulating DNA damage pathways. In order to determine if loss of UBR5 in MCL cell lines leads to increased DNA damage, we will transduce cells MCL cell lines, JEKO1 and Mino along with bone marrow derived mouse hematopoietic progenitor cells with shRNAs against a non-target control or Ubr5. DNA damage will be evaluated following treatment with double stranded break agents (doxorubicin, ionizing radiation) and analyzed for markers of DNA damage repair including gamma H2AX and 53BP1 staining.

Aim 2: Mimicking UBR5 mutations to determine role in disease initiation and progression. Currently there is only one mouse model of mantle cell lymphoma, which takes up to 20 months for disease initiation. Here we propose to generate a conditional knockout mouse model of Ubr5 that mimics the truncated protein formed by MCL patient mutations. This model will help determine if Ubr5 plays a role in disease initiation or progression. We also propose bone marrow transplantations utilizing the Eµ-CyclinD1T268A mouse bone marrow to generate MCL and further elucidate the role of Ubr5 in disease.

Aim 2A: Determine role of Ubr5 knockdown on MCL disease initiation and progression. As previously mentioned MCL is characterized by a translocation t(11;14) leading to overexpression of cyclinD1. CyclinD1 is targeted for ubiquitination following phosphorylation by GSK3β 23. Gladden et. al. generated a mouse model of MCL by expressing phosphorylation mutant CyclinD1 under the control of the immunoglobin enhancer (Eµ- CyclinD1T268A)24. The mutation leads to constitutive nuclear expression of cyclinD1 and mice develop B-cell lymphoma. Malignancy is in the spleen and lymph nodes with infiltration of tumor cells in the lung and intestine. Although the mice develop B-cell lymphoma they have late latency of the disease (survival ~13-21 months of age) suggesting that corroborating lesions my drive disease initiation. It is important to note that Eµ-CyclinD1 expression alone is not enough to generate malignancy. To determine if Ubr5 silencing accelerates disease and corroborate, we will isolate BM from Eµ-CyclinD1T268A mice and retroviral infect with shRNA against Ubr5. Infected cells will be transplanted intravenously into lethally irradiated C57Bl6 mice. Mice will be monitored for signs of disease and enlarged lymph nodes. Disease initiation and progression will be followed by both B- ! 5! Buckley, SM Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! lymphocyte development by flowcytometry and histology of lymphoid organs. Eµ-CyclinD1T268A mice will be kindly provided by Alan Diehl (Medical University of South Carolina).

Aim 2B: Generation of HECT domain mutations in mice, modeling mutations found in MCL patients. In order to mimic human MCL mutations we have designed a targeting vector to generate a mouse model recapitulating human mutation. Approximately 60% of the mutations found in UBR5 in MCL were located in the HECT leading to a truncating protein lacking a cysteine important for ubiquitin conjugation14. In collaboration with Ingenious Targeting Laboratory we designed a conditional knockout strategy utilizing the cre-lox system. Here we flanked exon 58 with lox p sites that when crossed to a cre expressing mouse model will delete exon 58 leading to a truncated protein lacking the ubiquitin binding cysteine (Figure 3). ESC were targeted by electroporation and targeting was confirmed by both PCR (Figure 3B). Tetraploid complementation will be utilized to provide ESC derived mice in ~18 days to provide rapid generation of targeted mice25. We will utilize the Cre-Lox system to delete Ubr5 in the adult hematopoietic system (Mx-Cre), and specifically in the B- lymphoid (CD19-Cre) compartment to determine if loss of Ubr5 leads to hematopoiesis defects and/or generation of hematopoietic malignancies. Both Cre mouse strains are commercially available through The Jackson Laboratory. Hematopoiesis will be monitored utilizing flowcytometry for lineage specific cell surface markers. Hematopoietic organs, bone marrow, spleen, and thymus will be analyzed for cell composition and histology.

Pitfalls/Limitations: An alternative to look at disease initiation and progression, is to cross Eµ-CyclinD1T268A mice with the conditional HECT domain mutant mouse in Aim 2B to follow disease progression and initiation. Also, in both aims we utilize shRNAs to silence Ubr5, however in the patients mutants are thought to lose the C-terminal HECT domain, as an alternative to more recapitulate the mutations in patients we could utilize the CRISPR/Cas9 system and design gRNAs near the HECT domain eliminating the ability to transfer ubiquitin to its substrate.

Conclusions: This funding opportunity Figure 3; Targeting Strategy for HECT mutant. A) endogenous locus, targeting vector would provide the preliminary data and and targeted locus B) Strategy to screen ESC. more importantly the mouse model that mimics the UBR5 mutations in MCL patients to apply for future external funding opportunities. Aim 1 can be accomplished, and experiments in Aim2 can be initiated in the timeline of the grant. Aim 2 proposes mouse models of disease and a conditional knockout model that take more than a year to generate data, but will be used in future studies to further unravel the role of UBR5 in hematopoiesis and mantle cell lymphoma. The combination of my PhD and postdoctoral research have provided me a foundation in normal and malignant hematopoiesis, which can now be used to further elucidate the molecular mechanisms regulating hematopoietic lineage specification and pathways disrupted in cancer. The proposed experiments will further unravel the ubiquitin-related molecular mechanisms that regulate induction of lineage specification and transformation.

F. Statement of Cancer Relevance

We believe that post-translational regulation of hematopoietic differentiation by the UPS, specifically the substrate recognizing ubiquitin E3 ligases and “proteomics” centered approaches could indeed be one of the ! 6! Buckley, SM Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! important future directions in the identification of novel therapeutic targets in leukemia and lymphoma. Within the E3 ligase family, Ubr5 is an intriguing ligase for further study for a number of reasons: 1) there are limited known UBR5 ubiquitin substrates; 2) Loss of Ubr5 plays a role in ESC differentiation; 3) UBR5 is highly expressed in hematopoietic tissue, specially in the IgM+ B-cell and pre-B populations; 4) mutations in mantle cell lymphoma suggests a role in hematopoietic differentiation and lymphomagenesis This proposal advances the understanding of the general mechanisms underlying hematopoietic lineage commitment. Also, a number of cancers rely on aberrant mechanisms of differentiation, providing further evidence that role of the UPS could have a broad impact not only in hematopoietic differentiation but also more broadly in other forms of cancer. Moreover the UPS provides candidates for future advances in cancer therapeutics with the goal of providing targets for drug discovery.

! 7! Buckley, SM Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! G. References

1 Itoi, M., Kawamoto, H., Katsura, Y. & Amagai, T. Two distinct steps of immigration of hematopoietic progenitors into the early thymus anlage. International immunology 13, 1203-1211 (2001). 2 Ohmura, K. et al. Immature multipotent hemopoietic progenitors lacking long-term bone marrow- reconstituting activity in the aorta-gonad-mesonephros region of murine day 10 fetuses. J Immunol 166, 3290-3296 (2001). 3 Havran, W. L. & Allison, J. P. Developmentally ordered appearance of thymocytes expressing different T-cell antigen receptors. Nature 335, 443-445 (1988). 4 Cumano, A. & Paige, C. J. Enrichment and characterization of uncommitted B-cell precursors from fetal liver at day 12 of gestation. The EMBO journal 11, 593-601 (1992). 5 Brendolan, A., Rosado, M. M., Carsetti, R., Selleri, L. & Dear, T. N. Development and function of the mammalian spleen. Bioessays 29, 166-177 (2007). 6 Wolber, F. M. et al. Roles of spleen and liver in development of the murine hematopoietic system. Experimental hematology 30, 1010-1019 (2002). 7 Schwarz, B. A. & Bhandoola, A. Circulating hematopoietic progenitors with T lineage potential. Nat Immunol 5, 953-960 (2004). 8 Hirose, J. et al. A developing picture of lymphopoiesis in bone marrow. Immunol Rev 189, 28-40 (2002). 9 Martin, C. H. et al. Efficient thymic immigration of B220+ lymphoid-restricted bone marrow cells with T precursor potential. Nat Immunol 4, 866-873 (2003). 10 Akashi, K., Traver, D., Kondo, M. & Weissman, I. L. Lymphoid development from hematopoietic stem cells. Int J Hematol 69, 217-226. (1999). 11 Weissman, I. L. Normal and neoplastic stem cells. Novartis Found Symp 265, 35-50; discussion 50-34, 92-37 (2005). 12 Wilson, A. et al. Dormant and self-renewing hematopoietic stem cells and their niches. Ann N Y Acad Sci (2007). 13 Royo, C. et al. The complex landscape of genetic alterations in mantle cell lymphoma. Seminars in cancer biology 21, 322-334, doi:10.1016/j.semcancer.2011.09.007 (2011). 14 Meissner, B. et al. The E3 ubiquitin ligase UBR5 is recurrently mutated in mantle cell lymphoma. Blood, doi:10.1182/blood-2013-01-478834 (2013). 15 Kridel, R. et al. Whole transcriptome sequencing reveals recurrent NOTCH1 mutations in mantle cell lymphoma. Blood 119, 1963-1971, doi:10.1182/blood-2011-11-391474 (2012). 16 Herrmann, A. et al. Improvement of overall survival in advanced stage mantle cell lymphoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27, 511-518, doi:10.1200/JCO.2008.16.8435 (2009). 17 Rotin, D. & Kumar, S. Physiological functions of the HECT family of ubiquitin ligases. Nature reviews. Molecular cell biology 10, 398-409, doi:10.1038/nrm2690 (2009). 18 Cojocaru, M. et al. Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B. J Biol Chem 286, 5012-5022, doi:10.1074/jbc.M110.176628 (2011). 19 Hay-Koren, A., Caspi, M., Zilberberg, A. & Rosin-Arbesfeld, R. The EDD E3 ubiquitin ligase ubiquitinates and up-regulates beta-catenin. Molecular biology of the cell 22, 399-411, doi:10.1091/mbc.E10-05-0440 (2011). ! 8! Buckley, SM Role of E3 ligase, UBR5, in hematopoietic differentiation and malignant transformation

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 20 Gudjonsson, T. et al. TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged . Cell 150, 697-709, doi:10.1016/j.cell.2012.06.039 (2012). 21 Wilkinson, C. R. et al. Proteins containing the UBA domain are able to bind to multi-ubiquitin chains. Nat Cell Biol 3, 939-943, doi:10.1038/ncb1001-939 (2001). 22 Saunders, D. N. et al. Edd, the murine hyperplastic disc gene, is essential for yolk sac vascularization and chorioallantoic fusion. Molecular and cellular biology 24, 7225-7234, doi:10.1128/MCB.24.16.7225- 7234.2004 (2004). 23 Diehl, J. A., Cheng, M., Roussel, M. F. & Sherr, C. J. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes & development 12, 3499-3511 (1998). 24 Gladden, A. B., Woolery, R., Aggarwal, P., Wasik, M. A. & Diehl, J. A. Expression of constitutively nuclear cyclin D1 in murine lymphocytes induces B-cell lymphoma. Oncogene 25, 998-1007, doi:10.1038/sj.onc.1209147 (2006). 25 Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. & Roder, J. C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America 90, 8424-8428 (1993). 26 Henderson, M. J. et al. EDD mediates DNA damage-induced activation of CHK2. J Biol Chem 281, 39990-40000, doi:10.1074/jbc.M602818200 (2006). 27 Munoz, M. A. et al. The E3 ubiquitin ligase EDD regulates S-phase and G(2)/M DNA damage checkpoints. Cell cycle 6, 3070-3077 (2007). 28 Duan, S. et al. FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas. Nature 481, 90-93, doi:10.1038/nature10688 (2012). 29 Buckley, S. M. et al. Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system. Cell stem cell 11, 783-798, doi:10.1016/j.stem.2012.09.011 (2012).

! 9! BUDGET

Category Cost Personnel

$12,500 A Salary support or a rauate stuent or 0 calenar monts

Materials and Supplies

Lab Supplies: $12,000 Cores: $11,500 $32,500 Animal Facility: $9,000

Equipment

Travel

Other Direct Costs

Total Cost $45,000 Budget Justification:

Personnel

Graduate Students, 6.00 calendar support requested.

Dr. Buckley is a new faculty member at University of Nebraska Medical Center starting her position August 10th, 2015. This year she is recruiting a graduate student, who will be rotating September 2015 till March 2016 with recruited students expected to join around April 2016. Graduate student stipend at University of Nebraska Medical Center is $25,000/yr.

6.00 months salary ~$12,500

Materials and Supplies

Lab Supplies: $12,000 The proposed project requires a significant amount of lab supplies. These include cell culture Media, serum, cytokines, enzymes, antibodies, transfection reagents, DNA and RNA preparation kits, protein biochemistry supplies etc.

Based on our experience we request $1000/month ($12,000/year).

Cores: $11,500

a. Flow cytometry/FACS sorting Facility: Our project will relay on flow cytometry to analyze hematopoietic surface markers, as well as, cell sorting to purify populations for transplant. We estimate a total cost of approximately $3,000/year.

b. Transgenic Mouse Facility: Our project will be generating a new mouse models and we will use the transgenic facility to inject our targeted ESC into blastocyst and chimera development. We estimate a total cost of approximately $8,500.

Animal Care: $9,000

Animal Facility Costs: We anticipate the use of a number (~35) mouse cages. The mice will be used as hosts for transplantation and crossing transgenic models. The current per diem for our animals is $0.97/day/cage. We will be using an absolute minimum of 35 cages, which include both breeding and experimental cages. The total cost will be (25*0.97*365)~ $9,000/year.