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New Insights Into Erythropoietin and Epoetin Alfa: Mechanisms of Action, Target Tissues, and Clinical Applications MITCHELL J. WEISS The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA Key Words. Erythropoietin · Signal transduction · Epoetin alfa · Anemia · Cancer · Central nervous system · Apoptosis

L EARNING O BJECTIVES After completing this course, the reader will be able to: 1. Discuss the mechanism of action of endogenous erythropoietin and the therapeutic use of epoetin alfa to stimulate red blood cell production and improve the quality of life in patients with cancer. 2. Explain how epoetin alfa is being investigated in alternate dosing regimens and for anemia prevention in patients with cancer. 3. Describe how functional endogenous erythropoietin receptor signaling pathways have been demonstrated in numerous nonerythropoietic tissues, including in the central nervous system, and relate evidence for the roles of erythropoietin and epoetin alfa beyond erythropoiesis, including the therapeutic implications of these nonerythroid functions. CME

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A BSTRACT Recombinant human erythropoietin (epoetin alfa) has proven beneficial for the treatment of various anemias. The mechanism of action of endogenous erythropoietin and the therapeutic use of epoetin alfa to stimulate red blood cell production and improve the quality of life in cancer patients are reviewed here. Epoetin alfa may also attenuate the cognitive dysfunction associated with cancer therapy. Interestingly, functional endogenous erythropoietin receptor signaling pathways have been demonstrated in numerous nonerythropoietic tissues. Of particular importance, epoetin alfa confers neurotrophic and neuroprotective

effects in cultured neurons and in several animal models for neurologic disease. In one clinical trial, epoetin alfa appeared to limit functional and histologic damage in patients with stroke. Therefore, in cancer patients receiving chemotherapy, the beneficial effects of epoetin alfa could be mediated not only through enhanced erythrocyte production but also via direct effects on the nervous system. Further investigation into the nonerythropoietic effects of epoetin alfa could broaden its clinical utility for patients with cancer and also provide new therapies for various neurologic disorders. The Oncologist 2003;8(suppl 3):18-29

INTRODUCTION Erythropoietin (EPO) is an endogenous cytokine that is essential for erythrocyte development [1]. In adults, the kidneys produce and release EPO in response to hypoxia [2]. EPO

acts by binding to its receptor (EPO-R) and subsequently activating intracellular signal transduction pathways [1]. EPO is essential for life: mice with deletions of the EPO gene or the EPO-R die of anemia in utero [3, 4]. Recombinant human

Correspondence: Mitchell J. Weiss, M.D., The Children’s Hospital of Philadelphia, 316B Abramson Research Building, Philadelphia, Pennsylvania 19104, USA. Telephone: 215-590-0565; Fax: 215-590-4834; e-mail: [email protected] Received August 29, 2003; accepted for publication October 3, 2003. ©AlphaMed Press 1083-7159/2003/$12.00/0

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EPO (epoetin alfa) is used in clinical practice to reduce transfusion requirements during surgery [5] and to treat anemia of various etiologies, including anemia of chronic kidney disease [6], cancer-related or cancer treatment-related anemia [7], anemia related to zidovudine therapy in HIVinfected patients [8], and anemia related to ribavirin therapy for hepatitis C virus infection [9]. The mechanism of action of EPO in erythropoiesis and the effects of epoetin alfa in cancer patients are reviewed here. Subsequently, evidence for the roles of EPO and epoetin alfa outside erythropoiesis and potential therapeutic uses of epoetin alfa beyond anemia treatment are explored. This review is based on an educational session presented at the American Society of Hematology (ASH) meeting held in December 2002, updated and expanded as necessary. Accordingly, many abstracts on EPO biology that were presented at the meeting are discussed herein. While these studies have not yet been published in full in peer-reviewed journals, they reflect the current research in this therapeutic area.

to the nucleus to activate numerous target genes [1], including the apoptosis inhibitor Bcl-x [14]. The inhibition of apoptosis by the EPO-activated JAK2/STAT5/Bcl-x pathway (Fig. 1) is important for erythroid differentiation. JAK2 deficiency causes embryonic death due to the absence of definitive erythropoiesis [15]. Furthermore, mice deficient in STAT5a/5b have anemia that correlates with the decreased expression of Bcl-x and increased apoptosis in early erythroblasts [16]. Finally, full Bcl-x knockout mice died in embryogenesis with extensive apoptosis of immature hematopoietic cells [17], and conditional hematopoietic-specific Bcl-x knockout mice had severe anemia [18]. In both models, Bcl-x was required for the survival of erythroid cells during terminal maturation [18, 19]. A recent study also demonstrated that enforced Bcl-x expression can rescue maturation of EPO-deprived erythroid progenitors in vitro, suggesting that the major erythropoietic function of EPO is to prevent apoptosis and that Bcl-x is a critical effector gene [20]. However, it is important to note that EPO and EPO-R null mice exhibit a more severe erythropoietic defect than that seen in Bcl-x null animals, indicating that EPO fosters erythropoiesis through additional effectors. In addition, the erythroid defect in the absence of STAT5a/5b is milder than that produced by loss of Bcl-x, indicating that STAT5-independent mechanisms for Bcl-x induction exist. In addition to STAT5, EPO induces JAK2-mediated tyrosine phosphorylation and activation of several other intracellular proteins [1]. Examples include: Shc [21], which, in turn, may activate the signaling pathway involved in erythroid cell proliferation [22]; phosphatidylinositol 3-kinase (PI3K) [23],

MECHANISM OF ACTION OF EPO IN ERYTHROPOIESIS In adult kidneys, hypoxia gives rise to increased EPO expression stimulated by the DNA binding protein, hypoxiainduced factor-1 (HIF-1) [2, 10]. EPO is secreted into the plasma and, upon arrival in the bone marrow, binds to EPO-Rs on the surface of erythroid progenitor cells [11]. This association triggers a conformational change that brings EPO-Rassociated Janus family tyrosine protein kinase 2 (JAK2) molecules into close proximity, stimulating their activation by transphosphorylation [1, 12, 13]. Subsequently, JAK2 molecules phosphorylate eight tyrosine residues in the cytoplasmic domain of the EPO-R, which then serve as docking sites for various Epo Src homology 2-domain-containing P SHC EpoR Grb2 intracellular signaling proteins [1]. RAS P Jak2 GAB1 SOS These proteins, in turn, are tyrosine P Vav P P P SHP1 phosphorylated and activated. One P GAP P of these proteins is a signal transP MAPKKK RAF-1 SHP2 P P ducer and activator of transcription IRS-2 MAPK (STAT5) that, on phosphorylation P by JAK2, dissociates from the EPOP STAT5 PI 3-K R, dimerizes, and then translocates P P

Figure 1. JAK2/STAT5/Bcl-x signal transduction pathway of the EPO-R. Adapted with permission from Cheung and Miller [1]. Molecular mechanisms of erythropoietin signaling. Nephron 2001;87:215-222. Adapted with permission of S. Karger AG, Medical and Scientific Publishers, Basel, Switzerland.

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which may promote survival of erythroid cells [22], and phospholipase C-γ1, which may play a role in erythroid cell proliferation [24]. In this regard, loss of the p85α subunit of PI3K impairs fetal liver erythropoiesis, although this mutation appears to inhibit signaling through the c-Kit receptor to a greater extent than does mutation of the EPO-R [25]. ERYTHROPOIETIC EFFECTS OF EPOETIN ALFA IN PATIENTS WITH CANCER Rationale for Treatment of Cancer-Related Anemia Up to 75% of patients with cancer experience anemia during the course of their disease, either due to the cancer itself (tumor infiltration of the bone marrow and/or anemia of chronic disease) or to the myelosuppressive effects of chemotherapy or radiation therapy [7, 26]. Several lines of evidence indicate that aggressive treatment of anemia is an important aspect of cancer therapy. First, a quantitative review of published data from patients with cancer suggests that anemia may be an independent prognostic factor for survival, with a 65% overall estimated greater relative risk of death [27]. In a retrospective analysis of patients undergoing chemotherapy and thoracic radiotherapy for stage IIIA/IIIB non-small cell lung cancer, declining hemoglobin (Hb) levels were highly predictive of survival [28]. The 2-year survival rates were 51% for patients whose Hb level decreased 30% [28]. While these observations might simply reflect impaired erythrocyte production in patients with more aggressive malignancies or greater systemic illness, there are some indications that anemia can interfere with cancer therapy. For example, anoxic tumor cells are two to three times more resistant to radiation therapy than are normally oxygenated cells [29, 30]. In addition, low Hb levels have been associated with tumor hypoxia, lower rates of local failures, and lower survival rates during radiation therapy for multiple tumor types [30-36]. In addition to the possible negative effects of anemia on cancer treatment and clinical outcomes, chronic anemia and the resulting fatigue impair the quality of life (QOL) of patients with cancer, and the use of epoetin alfa to boost erythrocyte production in those patients with cancer-related anemia has resulted in clinically important and statistically significant improvements in QOL [37]. Although red blood cell transfusion is also a treatment option for anemia, its associated problems include the potential for transmission of infections, limited availability of adequate blood supplies, and short-term resolution of symptoms. Epoetin alfa is more convenient to administer, reduces infectious risks, and maintains more constant steady-state Hb levels. Epoetin alfa also may offer potential benefits beyond stimulating erythropoiesis (described later).

EPO: Functions Beyond Erythropoiesis Clinical Practice Guidelines for the Use of Epoetin Alfa in Cancer-Related Anemia Evidence-based clinical practice guidelines for the use of epoetin alfa in patients with cancer have been developed by the American Society of Clinical Oncology (ASCO) in conjunction with ASH [38] and by the National Comprehensive Cancer Network (NCCN) [39]. The ASCO/ASH guidelines provide a thorough overview of the use of epoetin alfa therapy in patients with various forms of cancer based on data included in an evidence report assembled by the Blue Cross Blue Shield Association Technology Evaluation Center [38]. The panel reviewed and summarized many clinical studies involving patients with solid tumors or hematologic malignancies and with chemotherapy- or cancer-related anemia. Briefly, the ASCO/ASH guidelines recommend the use of epoetin alfa in patients with chemotherapy-associated anemia when Hb levels decline to ≤10 g/dl, with the decision of whether to treat less severe anemia (i.e., Hb of >10 g/dl to