Don M. Wojchowski, Ph.D.
Maine Medical Center Research Institute
81 Research Dr
Scarborough ME, 04074
Phone: (207) 396-8258
Fax: (207) 396-8179
Don Wojchowski is a Colby College graduate who developed research skills in endocrinology at the Boston Children's Hospital, performed doctoral studies in Cell Biology at the University of Massachusetts Amherst, and pursued postdoctoral investigations in Hematology/Oncology at Harvard Medical School.
Subsequently, Dr Wojchowski served as tenured Professor and Immunobiology Program Director at The Pennsylvania State University, with a research focus on hematopoiesis, hematopoietic growth factors and erythropoiesis.
Since returning to Maine and joining MMCRI in 2003, Dr Wojchowski has continued to advance nationally funded investigations in hematology. He also serves as Director of the MMCRI NIH-sponsored Center of Biomedical Research Excellence in Stem and Progenitor Cell Biology. Dr. Wojchowski is the recipient of several NIH career development awards, has served as full member on Hematology and Cell & Molecular Hematology NIH study sections, and is an Editorial Board Member for Blood (Journal of the American Society of Hematology).
Investigations in the Wojchowski laboratory aim to gain new insight into cell and molecular mechanisms that support hematopoietic cell growth, survival and development.
One present focus is on the biological effects and action mechanisms of hematopoietic growth factors (HGFs). In particular, the Erythropoietin (EPO) and EPO receptor (EPOR) system provides a highly scientifically informative and clinically important model. In response to hypoxia EPO is expressed by, and secreted from, a rare subset of renal interstitial fibroblasts. EPO then acts as an essential HGF predominantly within bone marrow to support the formation of erythroid cells. At steady-state, this involves the remarkable production of ~200 billion red cells per day. Clinically, rhEPO is used to treat the anemia of chronic kidney disease, cancer chemotherapy, AIDS (Zidovudine therapy) and lower-risk myelodysplastic syndrome. Additionally, EPO may exert meaningful cytoprotective effects in injured non-hematopoietic tissues. However, rhEPO frequently induces hypertension, is associated with thrombolytic events, may worsen the course of certain cancers, and is a high cost to national health care systems. Further impetus for understanding EPO's action modes relates to the recent development of new EPOR agonists as erythropoietic stimulating agents whose action mechanisms (and side-effects) likewise now become important to understand.
With regards to our laboratory's ongoing investigations of EPO/EPOR action, one set of goals (specific aims) is outlined below that summarizes certain proteomic-based studies. These studies uncover exciting new EPO/EPOR targets and signal transducers.
In addition, as spring-boarded by our transcriptome-based discoveries of novel EPO/EPOR target genes, a parallel set of ongoing investigations (specific aims) is outlined that seeks first to define functional roles for Trib3 (and Trib2) as pseudokinases engaged during anemia and stress erythropoiesis. And second to better understand a novel EPO/EPOR pathway to the cytoprotection of erythroblasts against leached lysosomal B and L cathepsins.
Strong funding is in place, and new opportunities exist for talented postdoctoral fellows and/or research staff to assume lead roles in these and related investigations.
PROTEOMIC BASED IN-ROADS TO THE DISCOVERY AND FUNCTIONAL CHARACTERIZATION OF NOVEL EPO/EPOR/JAK2 TARGETS AND SIGNAL TRANSDUCERS:
Following the cloning of EPO and its receptor, early investigations contributed in basic ways to our understanding of EPO and HGF action. However, model systems, approaches, reagents, and tools plus databases were limited as compared to contemporary means. Subsequent studies have underscored that certain central EPO action models are likely incorrect. BclxL, for example, is an important cytoprotectant for maturing erythroblasts, but does not so much appear to be strongly induced via EPO/EPOR/ JAK2/Stat5 signals, and EPO efficiently cytoprotects Bcl-xL deficient EPCs. Additionally, it's becoming clear that EPO/EPOR/JAK2 complexes target important new transducers. Illustrating examples include an EPO induced Spi2A serpin that cytoprotects EPCs against leached lysosomal cathepsins, and Erythroferrone as a TNF cytokine that's induced by EPO in erythroblasts, and acts to inhibit hepcidin, stabilize ferroportin, and boost iron efflux.
With the overall goal of better understanding EPO's actions, we've undertaken aggressive phosphoproteomic based interrogations. Via a focus initially on EPO/EPOR- regulated phosphotyrosine (pY) targets, this LC-MS/MS approach is meeting with exciting success. "RHEX" provides one supporting example as a novel human (pro)erythroblast plasma membrane protein that regulates EPO-dependent erythroid development.
Thus, new functional insight into how EPO exerts its essential effects on human EPC's is being provided via the following Aims and Approaches:
#1: PTM motif proteomic approaches are being applied to discover important new EPO/EPOR targets and transducers. Novel EPO targets in human erythroid precursors are defined by LC-MS/MS of immune-adsorbed tryptic and/or GluC peptides modified (due to EPO) at select signaling node motifs as pY, ubiquitin-KeGG, T*PP/T*P , PXS*P, and RXXS/T sites. Proteomic data on new EPO/EPOR targets and PTMs will next be mined, and assembled into integrated public databases.
#2:For Thioredoxin Interacting Protein, our studies reveal that EPO modulates TXNIP at a novel stabilizing TPP motif, and that TXNIP's knockdown sharply limits EPC cell growth and development. GOF, LOF and rescue approaches are being used to determine specifically how TXNIP acts as an important new mediator of EPO-dependent erythropoiesis.
#3: We also are working to determine the functional roles, and action mechanisms, of two additional new mediators of EPO/EPOR dependent human EPC formation, PTPN18 and RHEX: For Protein-Tyrosine Phosphatase N18, we demonstrate PTPN18 to be a positive regulator of EPC growth and of JAK2's activation, and are now seeking to define PTPN18's bio and molecular effects on hEPCs, and on EPOR/JAK2/RHEX signaling. RHEX's specific effects on hEPC development also are being defined using primary EPC systems, plus LOF and GOF approaches. Signaling routes affected by RHEX (and EPO- regulated RHEX pY132 & pY141 motifs) will be determined using pathway-specific proteomic and gene array analyses, and mutant rescue approaches.
NOVEL PRO-ERYTHROPOIETIC AGENTS THAT SUPPORT ERYTHROID PRECURSOR FORMATION DURING ANEMIA/STRESS:
Erythropoiesis is a profound balancing-act, and limited slippage (especially chronic) can compromise tissue oxygenation, and health. Stress to the erythron is heightened in CKD, chemo- and radiation cancer therapies, MDS, inflammation, iron dysregulation and hemoglobinopathies. EPO is used to treat the anemia of CKD, MDS and cancer chemotherapy. A significant percent of CKD and MDS patients, however, are EPO- unresponsive. For thalassemia and SCD, therapies involve transfusions, daily hydroxyurea dosing (SCD) and BMT. Exciting progress nonetheless is being made in the discovery of new stress erythropoiesis regulators. Examples include soluble activin receptor lessening of the anemia of multiple myeloma; roles for a ZFP36L2 RNA binding protein in mediating glucocorticoid effects on BFUe expansion; and anemia- modulating effects of a SMAD-targeting TMPRSS6 protease in β-thalassemia.
The above examples illustrate that to gain insight towards new anemia therapies, it's essential to uncover and understand the specifics of stress erythropoietic pathways. Via EPO action studies in primary erythroid progenitors, we have discovered two new stress- specific erythropoietic factors, and are working to understand the pathways they control to regulate erythroid cell formation. One involves an EPO/EPOR/Stat5 Spi2A serpin pathway; reveals erythroblast lysosomes to become hyper-sensitive to damage during stress; and points to cathepsin inhibitors as cytoprotective agents. A second involves actions of Trib3 pseudokinase as a novel EPO/EPOR mediator that governs both early, and late stages of stress erythropoiesis.
Using novel KO mouse models, and primary erythroid progenitor cell (EPC) systems, we are advancing the following studies: #1 employs our Spi2A-KO model to define key Spi2A-linked pathways that protect (or compromise) developing erythroblasts and their lysosomes during stress erythropoiesis. In particular, Spi2A LOF effects on β-thalassemia and iron imbalance are being determined as are Spi2A-KO effects on erythroblast autophagy and damage incurred by erythroblast lysosomes due to oxidative stress. #2: For Trib3, we're employing a novel KO model to determine Trib3's specific contributions to stress erythropoiesis. Roles for Trib3 pseudokinase during β-thalassemia and iron imbalance further are being investigated. #3: We're also working to translate our findings on murine erythroblast lysosomal compromise, and on Trib3's proerythropoietic effects, to primary human erythroblasts. This includes defining lysosomal compromise in human CD34(+) derived β-thalassemic erythroblasts, together with effects of pharmacological inhibition of leached lysosomal cathepsins on erythroblast cytoprotection and macro-autophagy. LOF and GOF studies likewise are being used to define pro-erythropoietic roles of Trib3 during human erythroid progenitor cell formation.
[note: For a further listing of publications please click here]
Verma R, Su S, McCrann DJ, Green JM, Leu K, Young PR, Schatz PJ, Silva JC, Stokes MP, Wojchowski DM. RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development. J Exp Med. 2014;211:1715-22. PMCID: PMC4144737
Kuhrt D, Wojchowski DM. Emerging EPO and EPO receptor regulators and signal transducers. Blood. 2015 Apr 17. [Epub ahead of print]
Li L, Byrne SM, Rainville N, Su S, Jachimowicz E, Aucher A, Davis DM, Ashton-Rickardt PG, Wojchowski DM. Serpin Spi2A as a novel modulator of hematopoietic progenitor cell formation. Stem Cells. 2014;32:2550-6. PMCID: PMC4138266
Dev A, Byrne SM, Verma R, Ashton-Rickardt PG, Wojchowski DM. Erythropoietin-directed erythropoiesis depends on serpin inhibition of erythroblast lysosomal cathepsins. J Exp Med. 2013;210:225-32. PMCID: PMC3570101
Singh S, Verma R, Pradeep A, Leu K, Mortensen RB, Young PR, Oyasu M, Schatz PJ, Green JM, Wojchowski DM. Dynamic ligand modulation of EPO receptor pools, and dysregulation by polycythemia-associated EPOR alleles. PLoS One. 2012;7:e29064. PMCID: PMC3257245.
Singh S, Dev A, Verma R, Pradeep A, Sathyanarayana P, Green JM, Narayanan A, Wojchowski DM. Defining an EPOR- regulated transcriptome for primary progenitors, including Tnfr-sf13c as a novel mediator of EPO- dependent erythroblast formation. PLoS One. 2012;7:e38530. PMCID: PMC3396641.
Sathyanarayana P, Dev A, Pradeep A, Ufkin M, Licht JD, Wojchowski DM. Spry1 as a novel regulator of erythropoiesis, EPO/EPOR target, and suppressor of JAK2. Blood. 2012;119:5522-31. PMCID: PMC3369686.
Wojchowski DM, Sathyanarayana P, Dev A. Erythropoietin receptor response circuits. Curr Opin Hematol. 2010;17:169-76. PMCID: PMC2855390.
Sathyanarayana P, Houde E, Marshall D, Volk A, Makropoulos D, Emerson C, Pradeep A, Bugelski PJ, Wojchowski DM. CNTO 530 functions as a potent EPO mimetic via unique sustained effects on bone marrow proerythroblast pools. Blood. 2009;113:4955-62. PMCID: PMC2686145.
Menon M, Karur V, Bogacheva O, Bogachev O, Cuetara B, Wojchowski DM. Signals for stress erythropoiesis are integrated via an erythropoietin receptor phosphotyrosine-343-Stat5 axis J Clin Invest. 2006;116:683-94. PMCID: PMC1386105.