Molecular Biology of Polycythemias Josef T

REVIEW ARTICLE

Molecular Biology of Polycythemias Josef T. Prchal

Overview EPO, O2-sensing, and hypoxia-inducible factor Under normal conditions, EPOproduction is mediated ei- Polycythemia is literally translated as "many cells in the ther by reduced red blood cell mass (anemia) or decreased O2 blood". Only erythrocytosis (an alternative term for these saturation of red cell hemoglobin (hypoxemia). Hypoxic stimu- disorders) produces polycythemia since leukocytes and lation results in increased production of hypoxia inducible factor platelets are present in blood in far smaller proportions. (HIF-1), which is the major factor for transcriptional activa- Polycythemia maybe due to increased proliferation or de- tion of the EPOgene (4). HIF-1 is also found in cells that do creased apoptosis of erythroid progenitors, or to delayed not express EPO,suggesting that HIF-1 is part of a widespread erythroid differentiation with an increased number of pro- O2-sensing mechanism providing transcriptional regulation of genitor cell divisions. Prolonged red cell survival, another vascular endothelial growth factor, glycolytic enzymes, and theoretical cause of polycythemia, has not yet been de- other genes (5-8). The identity of the O2 sensor and the mecha- scribed and with intact regulatory mechanismsis unlikely nism by which it regulates HIF-1 are unknownat the present to occur. Primary polycythemiasresult fromabnormali- time. Overall, HIF-1 is a physiologic regulator of genes that ties expressed in hematopoietic progenitors. In contrast, promote cell survival under ischemia and are expressed in re- circulating factors cause secondary polycythemia (1). There sponse to decreased cellular O2 tension (9). HIF-1 which regu- are acquired and congenital causes of both primary and lates vasculogenesis, is required for proper embryonic devel- secondary polycythemia (1). opment, and also plays an important role in carcinogenesis ( 10- (Internal Medicine 40: 681-687, 2001) 1 3). Thus, polycythemia maybe only one phenotypic manifes- tation of a congenital defect of the HIF-1 pathway (13). HIF-1 is composed of two subunits, HIF-1a and HIF-p (an Key words: erythrocytosis, hypoxia inducible factor (HIF), ARTN-like homologue) that form a heterodimer ( 14); only HIF- erythropoietin, hematopoiesis la is regulated by hypoxia. HIF-1a mRNAand protein levels are induced by hypoxia and decay rapidly with return to normoxia. Normoxia-induced ubiquitin-mediated degradation of the HIF-loc protein is the major regulator of HIF-la levels Regulation of Erythropoiesis: Relevance to (8, 15). Von Hippel Lindau (VHL) syndrome is a genetic ab- Polycythemic Disorders normality of this post-translational control. The mutated VHL protein targets HIF for oxygen-dependent proteolysis ( 16, 17), Erythropoiesis is the physiological process of the produc- mediated by a complexmechanismthat involves an interaction and renewal of the red blood cell mass. This process is tion of p subunit ofVHLprotein with other proteins (18, 19). influenced by a numberof hormones, receptors and transcrip- VonHippel Lindau syndrome is characterized by a high pro- tion factors (2). The principal hormone that regulates erythro- pensity for development of renal tumors; polycythemia occurs poiesis is erythropoietin (EPO). In adults, the kidney is the main but is not common.However, hemangioblastomas of the cen- source of EPO. After erythroid commitment, erythroid progeni- tral nervous system have long been associated with secondary tors express their ownEPO(3). In vitro studies have shown polycythemia and these tumors and elevated EPOare some- that variable levels of EPOare required at various stages of times seen with VHLdisease. erythroid maturation (2). Instead of EPO, pluripotent stem cells and primitive erythroid progenitors, i.e. early burst-forming Erythropoietin receptor units-erythroid (BFU-E), require stem cell factor, granulocyte- The interaction of EPO with the EPO receptor (EPOR) leads macrophage colony-stimulating factor (GM-CSF), and/or to homodimerizationand signal transduction that results in a) interleukin 3 (IL-3), and thrombopoietin for growth (2). stimulation of mitogenicity of erythroid progenitor cells, b) erythroid differentiation by induction of erythroid-specific ex-

From the Division of Hematology/Oncology, Baylor College of Medicine, Houston, Texas Reprint requests should be addressed to Dr. Josef T. Prchal, Division of Hematology/Oncology Baylor College of Medicine, 1 Baylor Plaza 802E, Houston, Texas, 77030,USA

Internal Medicine Vol. 40, No. 8 (August 2001) 681 Prchal

pression of proteins such as globins, glycophorins, spectrin and ing signaling molecules resulting in "receptor cross-talk". This ankyrin, and c) prevention of apoptosis of erythroid progeni- complexinteraction of serum factors, receptors, and post-re- tors (20, 21). The cytoplasmic portion of EPORcontains a posi- ceptor signaling molecules capable of fine control may not be tive growth-regulatory domain that interacts with Janus 2 ty- emulated by in vitro experiments using cell lines, artificial con- rosine kinase (JAK-2) (22). Immediately after EPO binding, ditions, or non-humantissues. JAK-2 phosphorylates itself, the EPOR, and other proteins such The renin-angiotensin system regulates blood pressure, re- as STAT5, thus initiating a cascade of erythroid-specific signal hemodynamics, and fluid and electrolyte homeostasis (41). naling (23, 24). This JAK2/STAT5 signaling plays a nonredun- The primary function of angiotensin during development is the dant, essential role in EPO/EPOR-mediated regulation of eryth- modulation of tissue growth and differentiation (42). The ropoiesis (24-27). growth effects of angiotensin II are dependent on the differen- The C-terminal cytoplasmic portion of EPORalso possesses tiation state of the cells at the time of exposure to angiotensin a negative growth-regulatory domain. Hematopoietic cell phos- II (43). Angiotensin II is a ligand for two distinct receptors, phatase (HCP, also known as SHP1) interacts with this portion type 1 and type 2. The angiotensin type 1 receptor (AT^, which of the EPORand down-modulates signal transduction (23-26). is found in liver, lung, adrenal gland, placenta, pituitary gland, Recruited by EPORY429, HCPattaches to the cytoplasmic aorta, heart, skeletal muscle, lymphocytes, monocytes and plate- EPORdomain and dephosphorylates JAK2.Inactivation of the lets, appears to have a major role in the modulation of cell HCPbinding site was shown to lead to prolonged phosphory- proliferation (44). The therapeutic effects of angiotensin-con- lation of JAK2/STAT5 (26, 27). Another negative regulator of verting enzyme (ACE) inhibitors and losartan, a specific an- erythropoiesis CIS-3 (also known as SOCS) also binds to the tagonist of ATj (45, 46), on post-transplant erythrocytosis or cytoplasmic portion of the EPO. CIS-3, binds to EPORY401 polycythemia in renal transplant patients indirectly link angio- and generates a erythroid specific inhibitory signal (28, 29). tensin II with the regulation of erythropoiesis. Wehave reported Thus, deletion of the C-terminal cytoplasmic portion of EPOR the presence of AT, on erythroid progenitors and found that its in a truncated EPORabolishes negative regulatory elements ligand, angiotensin II, augments EPOstimulation of erythro- and results in increased proliferation of erythroid progenitor poiesis (47). The involvement of JAK-2 kinase in angiotensin cells. II-mediated intracellular events suggests that the signal trans- Attempts to link a number of disorders characterized by duction pathways mediated by EPOand angiotensin II may dysregulation of erythropoiesis with mutations in EPOand overlap (48). EPORgenes have demonstrated that only a small proportion of cases of primary familial and congenital polycythemia Polycythemias Due to Defined (PFCP) are caused by EPORmutations (30, 31). EPORmuta- Molecular Lesions tions are only rarely found in erythroleukemia (32). Poly- cythemia vera (PV) and Diamond-Blackfan anemia are not Secondary Polycythemias: High A ffinity Hemoglobin Mutan ts, caused by mutations in EPO/EPOR(3 1). Methemoglobinemias, and 2, 3 Bisphosphoglyceromutase (Bpgm) Deficiency Insulin-like growth factor-l (IGF-I), angiotensin II and angio- Though first defined as a cause of congenital polycythemia, tensin II type I receptor and erythropoiesis high oxygenaffinity hemoglobinmutants are an uncommon Although in vitro studies of erythropoiesis have provided cause of congenital secondary polycythemia. More than 50 crucial information about the hierarchy of regulation of eryth- variants of both a and p globin genes have been described in ropoiesis, manyof these experiments were performed in the autosomal dominant polycythemia, and are characterized by presence of serum and serum-componentproteins capable of an increased oxygen affinity of hemoglobin (49). The hemo- stimulating as well as inhibiting erythropoietic activity (33- globin tetramer oscillates between the R (relaxed; fully oxy- 38). Using rigorous serum-free conditions, it has been shown genated hemoglobin) and T (tense; fully deoxygenated hemo- that IGF-1 can substitute for EPOfor normal erythroid pro- globin) states of the quaternary protein conformation requir- genitors and even more effectively for PVerythroid progeni- ing the cooperative interaction of globin subunits. Mutations tors (34, 39). Furthermore, it has also been observed that affecting the equilibrium between R and T states result in a anephric patients with no detectable EPOand normal hemat- change of oxygen affinity. Manyhigh oxygen affinity mutants ocrits have elevated IGF- 1 levels (38). Most of the circulating are located in the al/(32 interface of the hemoglobin tetramer. IGF-1 is bound to six known high affinity IGF-1 binding pro- Somemutations interfere with the binding of 2, 3-bisphos- teins (IGFBP l -IGFBP6).Compared to normal subjects, IGFBP- phoglycerate (2, 3-BPG) to hemoglobin; others have an amino 1 is increased four-fold in patients with PV. IGFBP-1is also a acid substitution at the C-terminus of one of the globin sub- powerful stimulator of normal and PVerythropoiesis in vitro units that interferes with binding of heme. The functional con- (39, 40). However, while the studies using a single cytokine in sequence of increased hemoglobin oxygen affinity is decreased a serum-free media improved our knowledge of the physiologi- delivery of oxygen into the peripheral tissues and compensa- cal processes in health and disease, they mayalso result in tory polycythemia. This assures normal oxygen tissue deliv- misleading conclusions. After all, cells in vivo are exposed to ery, thus patients inheriting these mutations are generally as- multiple cytokines that activate different receptors, someshar- ymptomatic. Since manyof these mutants are electrophoreti-

682 Internal Medicine Vol. 40, No. 8 (August 2001) Molecular Biology of Polycythemias cally silent, the determination of hemoglobin oxygen dissocia- (58) as the affected father, uncle and grandfather) did not have tion kinetics and P50 is the best initial screening for suspected polycythemia. However, this child had the typical in vitro eryth- congenital secondary polycythemia. If a co-oximeter is not roid progenitor EPO hypersensitivity seen in PFCP, indicating available, P50 can be mathematically estimated from a venous the presence of unidentified epigenetic factors that masked the blood gas measurement, providing that both the venous pO2 full phenotypic expression of the EPORmutation (59). and hemoglobin oxygensaturation are measured(50). De- In some families linkage of PFCPwith EPORmutations creased P50 indicates either mutant hemoglobin or 2, 3 bisphos- has been conclusively excluded, further indicating a dominant phoglyceromutase (BPGM)deficiency, a rare cause of increased lesion in an unidentified gene(s) either at the level of the EPOR hemoglobin oxygen affinity (5 1). Methemoglobinemias. Poly- signaling pathwayor another erythropoiesis regulating path- cythemia due to congenital methemoglobinemiais always as- way (60, 61). To further examine the role of EPORmutations sociated with easily discernable coexistent cyanosis and as such in the pathogenesis of PFCP, we examined 50 unrelated PFCP does not represent a diagnostic dilemma. Methemoglobin is a subjects for EPORand found only five (10%) of the patients derivative of hemoglobin in which the ferrous ions (Fe2+) are had EPORmutations (62). These findings strongly suggest that oxidized to the ferric (Fe3+) state. Oxygen binds reversibly to mutations in EPORare not the major genetic defect in the patho- Fe2+ in deoxyhemoglobin but not to methemoglobin. Further- genesis of PFCPand the gene(s) responsible for the majority more, the presence of Fe3+ hemes in some subunits of hemo- of PFCP cases remain to be defined. globin tetramer increases the oxygen affinity of the accompa- nying Fe2+ hemes. A compensatory polycythemia develops, Polycythemias with Incompletely Defined ensuring normal oxygen delivery to tissue. There are three types Molecular Lesions of hereditary methemoglobinemia: hemoglobin M(Hb M) disease, a dominantly inherited due to a mutation of one of the Chuvash Polycythemia globin genes; NADH-cytochromeb5 reductase deficiency; and The only endemic congenital polycythemia is the recessively cytochrome b5 deficiency (49); the latter two are recessively inherited Chuvash polycythemia (CP) found in the Chuvash inherited. people (of Asian ethnic origin) in Central Russia (63-66). Thrombotic and hemorrhagic vascular complications lead to Primary Familial and Congenital Polycythemia (PFCP) early mortality, usually before the age of 30 years, in these PFCP(also called benign erythrocytosis, and familial eryth- individuals (67). The serum EPOconcentrations in the poly- rocytosis) is an uncommoncause of polycythemia, but based cythemic patients are significantly higher compared to healthy on our experience, it is a far more prevalent cause of congeni- first degree family members. The elevated EPOlevel suggests tal polycythemias than the high oxygen affinity hemoglobin a secondary polycythemia and therefore one wouldexpect nor- mutants or 2, 3-BPGdeficiency. Typically inherited as an au- mal responsiveness of erythroid progenitors to EPO. However, tosomal dominant disorder, PFCPis characterized by an el- the erythrocyte progenitors of persons with Chuvash poly- evated erythrocyte mass and hemoglobin concentration, hy- cythemia are EPOhypersensitive (67, 68), consistent with a persensitivity of erythroid progenitors to EPOin serum con- primary polycythemia. Thus, Chuvash polycythemia blurs the taining clonogenic cultures, low serum EPOlevel, normal he- distinction between primary and secondary polycythemia. moglobin oxygen dissociation, and absence of progression to Based on clinical and laboratory data, we hypothesized that leukemia (1 , 52-54). All PFCP-causing mutations described CP may be due to dysregulation in the oxygen-sensing path- thus far are due to truncation of the cytoplasmic EPORdomain way, and that hypoxia inducible factor 1 alpha (HIF-loe) may (1 , 55). Studies with myeloid cell lines retrovirally transfected play a role in the pathogenesis of this disease. To test this hy- with murine tuuncated EPORsuggested that the truncated pothesis, Western blot analysis using EBVtransformed B-lym- EPORmay, in fact, impede erythropoiesis due to a deletion of phocyte cell lines from CPindividuals revealed higher normoxic a PI-3-kinase anti-apoptotic binding domain (56). However, HIF- loc protein and mRNAlevels. These data indicate that CP the recent creation of mice bearing normal humanand mutant is an inherited disorder of hypoxia sensing. However,muta- disease-causing human EPORsfacilitated our understanding tions ofHIF-loc, EPO,and EPORgenes as a cause of CPwere of the structure/function relationship of EPORand should prove ruled out (67, 68) and the involved gene(s) remain to be iden- useful for studies of augmented EPOsignaling in erythroid tified. and non-erythroid tissues. A polycythemic phenotype, found in both homozygousand heterozygous mice for mutant EPOR Polycythemia Vera (PV) (57), confirms the central roles of JAK2and HCPin the regu- PV, is the most commonof the primary polycythemias. As lation of erythropoiesis. with other myeloproliferative syndromes, PV is caused by an The effect of a truncated EPORin the host milieu is not acquired mutation in a single hematopoietic progenitor that always predictable. Whenthe molecular basis of polycythemia leads to increased erythrocyte mass and variably increased plate- in the index family that led to the original description of the lets and myeloid cells. While the identity of gene(s) and the PFCP disease entity (52) was examined, it was initially con- nature of the disease causing the mutation responsible for PV cluded that EPORwas not linked with PFCPsince a child in remains elusive, somecellular biological characteristics of PV the third generation (who inherited the same EPORhaplotype have been established and these findings should facilitate the Internal Medicine Vol. 40, No. 8 (August 2001) 683 Prchal eventual identification of the PVresponsible gene and the elu- rified early hematopoietic precursors (87, 88). Other studies cidation of its molecular pathology (69). have shownthat PVerythroid progenitors exhibit up to three- Clonality. Since PV arises from a single hematopoietic pro- fold higher protein tyrosine phosphatase activity comparedto genitor, all or the vast majority of the circulating myeloid cells normal cells (89). are clonal (70). The mutation leading to clonal myeloid expan- Dysregulation of the expression of a numberof other genes, sion in PVaffects the pluripotent stem cell as a variable pro- including the thrombopoietin receptor and Bcl-xL, has been portion of B lymphocytes are a part of PVclone; however, the observed in PV subjects (90, 91). Several other abnormalities majority of the T and NKcells are polyclonal (71). in PV include platelet dysfunction (92, 93), differences in oxi- In vitro responses of PVprogenitors. PVbone marrowor dative responses of PVneutrophils and monocytes (94), an in- peripheral blood progenitor cells form erythroid colonies in creased platelet-derived growth factor mRNAlevel in mega- the absence of exogenous EPO(1, 72-75), a phenomenonnot karyocytes of MPD(95) and increased tyrosine phosphatase in observed in normal bone marrowsamples (76). The erythroid PV progenitors (96, 97); however, to our knowledge none of colony assay is a useful tool to distinguish PVfrom secondary these abnormalities is specific for PVAll of these abnormali- polycythemias (1, 76), ET (77) and PFCP (52, 76). Anti-EPO ties suggest that the PVdefect alters a numberof cellular func- and anti-EPOR neutralizing antibodies completely suppress or tions and is not restricted to cytokine receptor signal transduc- abolish the formation of erythroid colonies in normal as well tion only, and is not specific for PV(98). as in PFCP samples, but not in PV samples (78, 79). Twoother intriguing possible mechanismsof PVpathophysi- However, these observations hold true only in the presence ology have been recently reported. The EPORgene can pro- of serum. In serum-free culture systems, whencomparedto duce several mRNAsby alternative splicing (99); one of the normal cells, PVprogenitor cells exhibit hypersensitivity to resulting peptide products that inhibits EPORcontrolled eryth- IGF- 1 but intriguingly, no differences are observed in responses ropoiesis was recently reported to be absent in some PVpa- to EPO(34, 39). In other culture systems, hypersensitivity of tients (100). A second finding of particular interest is the per- PVerythroid progenitors to other cytokines (interleukin-3 , IL- sistence of transcription of PRV-1gene, a novel memberof the 3 ; granulocyte-monocyte colony stimulating factor, GM-CSF; UPARreceptor superfamily (101). The mRNAfor this gene and stem cell factor, SCF) was reported, but to a lesser magni- was detectable in PVgranulocytes but not in normal subjects tude than with IGF-1 (80, 81). These data suggest that the pu- or in those with secondary polycythemia due to pulmonary dis- tative PVdefect affects signaling downstream from the cytokine ease (101); however the peptide levels of the gene were the receptors, resulting in increased proliferative responsiveness same in granulocytes of normal and PVsubjects. This gene of PVcells to cytokine stimulation (82). However,we recently does not appear to be mutated, and its continuous transcription observed similar IGF- 1 hypersensitivity of native PFCPeryth- in the terminally differentiated myeloid cells appears to be a roid progenitors due to truncated EPOR(83), in serum-free secondary phenomenon(Pahl HL, personal communication but not in serum containing media, suggesting that increased September and November 2000). Nevertheless, if this defect is signaling of the IGF- 1 signal transduction pathway is unlikely found to be indeed specific for PV (i.e., not found in PFCP or to be the primary PVdefect. However, serum-free experiments in congenital high EPOstates such as Chuvash polycythemia) should be interpreted with caution; while they provide valu- this finding could potentially lead to the development of a rapid, able insight into the regulation of erythropoiesis, they can not specific PVdiagnostic test. substitute for in vivo complexinteractions of serum factors with In the rare familial cases of PV, the predisposition for PVis cellular receptors orchestrating the fine control of erythropoie- inherited in an autosomal dominant fashion and is typically sis. seen in elderly family members. This suggests that a newmu- Molecular basis of abnormal cytokine responses of PVpro- tation is acquired and the disease mayresult from the loss of genitor cells. The number and ligand affinity of EPORon eryth- heterozygosity. The existence of these families should greatly roid progenitors was found to be identical in normal, PV, and facilitate the search for PV causing gene mutation(s) ( 102, 103). in progenitors from subjects with PFCP(54). Furthermore, no While the molecular target of an acquired mutation leading to mutations were found in the coding sequences of EPORof PV PVremains to be elucidated, our knowledge of the disease- subjects (84). Thus abnormalities of the EPORare not directly specific defects is increasing. The increasing number of recog- involved in PV. nized instances of familial incidence of PV suggests that in The putative lesion associated with PVmay result in in- these families the predisposition for PV is inherited as a domi- creased tyrosine kinase activity or decreased tyrosine phos- nant trait, and that PVis acquired as a newmutation that leads phatase activity associated with cytokine receptors (85). Stud- to a clonal hematopoiesis. The existence of these families pro- ies of mice deficient in hematopoietic cell phosphatase (SHP- vides a unique opportunity for isolation of the mutations in the 1; also knownas HCP, SH-PTP1), a negative regulator of a gene leading to PV. numberof cytokine receptors, suggested that "loss-of-function" mutations in SHP- 1 may result in cytokine independent or hy- References persensitive proliferation of cells (86). No mutations, however, were found in the coding region of SHP-1and the amount of l)Prchal JT, Sokol L. Benign erythrocytosis and other familial and con- SHP-1 protein was normal in PVgranulocytes and highly pu- genital polycythemias. Eur J Haematol 57: 263-268, 1996.

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