Implications of retroviral and oncogene activity in chronic myelogenous leukemia

Implications of Retroviral and Oncogene Activity in Chronic Myelogenous Leukemia Isadore Brodsky, Howard R. Hubbell, David R. Strayer, and David H. Gillespie

ABSTRACT: Chronic myelogenoas leukemia (CML) is a stem cell disease which, on a clinical level, progresses from the release from growth control of normally differentiated cells ( a preleukemic state) to an acute leukemia. On a molecular level, the evolution of CML to acute leukemia is a multistep process. We propose that an early step, at the stem cell level, is acquisition of the ability for gene movement, which allows subsequent submicroscopic and chromosomal rearrangements that cause changes in the growth characteristics and regulation of the stem cell. A specific platelet DNA polymerase (PDP - reverse transcriptase) may play a role in gene movement. The characteristic reciprocal translocation of chromosomes #9 and #22, causing the activation of the c-abl oncogene, appears to be responsible for the uncontrolled cellular growth. Yet, other growth factors (e.g., platelet derived growth factor) and activated oncogenes (e.g., c-sis) must be responsible for the stimulation, progression, and variability seen during the course of the disease. Because CML is a progressive disease with clinically definable stages, CML appears to be a model system for the study of the molecular basis of the progression of preleukemia to leukemia specifically, and preneoplasia to aggressive neaplasia in general.

INTRODUCTION Chronic myelogenous leukemia (CML) is a clonal stem cell hematologic neoplasm characterized by a balanced reciprocal translocation b et w een chromosomes # 9 and #22 [t(9;22)] in 9 0 % - 9 5 % of all cases [1]. The translocation specifically involves bands 9q34.1 and 22q11.21 [2,3]. In 5 % - 1 0 % of patients, variant translocations can occur but these also usually involve chromosomes # 9 and #22 along with a third chromosome. Translocations that involve c h r o m o s o m e #22 and a c h r o m o s o m e other than # 9 occur in less than 2% of all cases [4]. Translocation of genetic information from c h r o m o s o m e # 9 to #22 is a consistent abnormality, as demonstrated in studies that have shown by in situ hybridization variable recipient chromosomes for the translocated fragment of c h r o m o s o m e #22 but a constant translocation of the fragment of c h r o m o s o m e # 9 to c h r o m o s o m e # 2 2 [5]. Advances in molecular biology suggest that the evolution of CML into acute leu-

From the Barry Ashbee Leukemia Research Laboratories, Department of Hematology/Oncology,Hahnemann University,Philadelphia, PA. Address requests for reprints to Dr. Isadore Brodsky, The Barry Ashbee Leukemia Research Laborataries, Department of Hematology/Oncology, Hahnemann University, Broad and Vine Streets, Philadelphia, PA 19102. Received June 29, 1986 accepted July 9, 1986.

15 © 1987 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York. NY 10017

Cancer Genet Cytogenet 26:15-23 (1987) 0165-4608/87/$03.50

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kemia is a multistep process [6, 7] that must take into consideration a) gene movement [8-11] in which increased levels of platelet DNA polymerase (PDP - reverse transcription) are implicated [12, 13]; b) activity of the sis oncogene, which is located on chromosome #22 [11, 14, 15] and the growth changes caused by plateletderived growth factor (PDGF - the protein product of the sis oncogene, derived from the gene or genes encoding a platelet-derived growth factor) [16, 171; c) altered transcription of c-abl oncogene, w h i c h is located on chromosome # 9 [18 22]; and d) clonal evolution of chromosomes [23, 24], which might result in the activation of other oncogenes. These events lead inevitably to blast crisis. Epidemiologic results involving i n d i v i d u a l s who received high doses of radiation from the detonation of nuclear weapons and subsequently d e v e l o p e d CML show clearly that the time between the inciting event (e.g., radiation) and the development of CML (Ph) is about 5-14 years. Therefore, the appearance of disease is a late event in the pathophysiology of CML [26]. Polycythemia vera (PV), a relatively benign myeloproliferative disorder, recently has been d o c u m e n t e d among participants of nuclear weapons tests with exposure to low level radiation [27]. The latent period between radiation exposure and disease was 14.5 years (mean); 14 years (median), a latent period similar to that noted for CML [26]. In the above schema, activation of the c-abl oncogene is closely correlated with the d e v e l o p m e n t of Ph chromosome and, therefore, is a relatively late event in the pathophysiology of CML. In contrast increased PDP levels are likely to be an early event in the pathogenesis of CML perhaps coinciding with the "first hit" initiating the disease process [12,251. GENE MOVEMENT

In addition to gross chromosomal abnormalities, recent work implicates the phenomenon of submicroscopic gene m o v e m e n t and consequential regulatory changes in h u m a n leukemia and cancer [8-11]. Retroviral gene movement and translocations resulting in onc gene deregulation are special cases presently receiving intense experimental attention [7, 28]. Our laboratories continue to explore the possibility that a h u m a n reverse transcriptase might mediate gene movements leading to leukemia and cancer. PDP is an enzyme extracted from retrovirus-like particles of h u m a n platelets, w h i c h is capable of supporting DNA synthesis from RNA [12, 13, 29]. PDP is unlike any k n o w n cellular DNA polymerase but it has several similarities to reverse transcriptase. It is as yet indistinguishable from the DNA polymerase discovered in and purified from retroviral-like particles in the cytoplasm of h u m a n cancer cells [30, 31]. Most strikingly, both PDP and h u m a n cancer cell polymerase are arranged in their respective particles with RNA primers and templates, so that a short c o m p l e m e n t of DNA can be synthesized in vitro [12, 13]. The cancer cell polymerase activity was detected in virtually all cancer cases, including those during remission of acute leukemia [30-32]. Present at low levels in platelets of normal persons, PDP activity was elevated in platelets of nearly all i n d i v i d u a l s with PV and CML. The highest levels of PDP are found in CML and are elevated very early in the course of CML [12, 13] (Fig. 1). Because PDP provides a m e c h a n i s m for gene movement, PDP elevation probably precedes the onset of s y m p t o m s and clinical findings. In PV, successful c h e m o t h e r a p y with busulfan is associated with a fall in PDP levels to normal range prior to any hematologic change, such as lowering the platelet count [25, 29] (Fig. 2). In contrast, CML patients seldom show a PDP drop (Fig. 3) even after clinical remission is achieved. Clinically longlasting remissions with busulfan are the rule in PV, whereas, remissions in CML are shortlived. The available results concerning PDP suggest that elevated PDP levels connote increased cancer risk. We have suggested that increased risk arises because PDP sometimes regulates the expression of or amplifies oncogenes. In this model, PDP

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elevation is an early event, particularly in regard to CML, whereas oncogene changes occur m u c h later, virtually coincidental with the demonstration of the Ph chromosome, clonal expansion, and the ultimate blast crisis [2, 25]. We have measured PDP and oncogene expression in chronic phase CML patients receiving c,-interferon [33] (Figs. 4 and 5). Interferon induces clinical remission in a p p r o x i m a t e l y 90% of CML patients in chronic phase [34]. While the expression of several oncogenes r e m a i n e d unchanged during therapy, abl oncogene expression decreased significantly w i t h i n a few days (Fig. 6). Later, hematologic remission was achieved. The relation between the CML p h e n o t y p e and PDP levels is not straightforward. Patients who achieve hematologic remission with busulfan do not show reduction in PDP levels to the normal range or loss of the Ph chromosome. Of two patients who received treatment with interferon, one clearly showed persistence of elevated PDP whereas the other d i d not. Decreases in the Ph chromosome have been obtained with prolonged treatment with interferon (J. Gutterman and M. Talpaz, personal communication). Preliminary experiments suggest that PDP levels in such patients normalize (unpublished results with M. Talpaz). These observations suggest that examination of PDP and oncogene expression longitudinally during therapy of CML will add substantially to our u n d e r s t a n d i n g of CML at the molecular level [33]. abl AND sis ONCOGENES, PDGF, AND THE MEGAKARYOCYTE Gale and Canaani [22] reported an abnormal 8 Kb transcript of the c-abl oncogene in the cells of chronic and blast crisis phases of CML patients. Collins et al. [19, 20] confirmed that CML cells contained a novel 8.2 kb abl-related RNA and that the levels of abl-related message were even eight times higher in CML cell lines from patients in blast crisis, c o m p a r e d with CML cells obtained during chronic phase CML or non-CML cells. In K562 leukemia cells, Konopka et al. [35] demonstrated that the translocation of c-abl to c h r o m o s o m e #22 unmasks associated tyrosine kinase activity of c-abl. They further stress that CML is a multistep process [6, 7, 35] and that the generation of the Ph appears to be a step that follows initial prolif-

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eration of preleukemic cells but may not be sufficient to cause CML. Our own studies confirm that the c-abl oncogene shows variable overexpression in the chronic phase of CML. Generally, reports of higher c-abl oncogene expression during chronic phase appear to be associated with more extensive purification of mononuclear cells [33]. The results are consistent with the idea that only some blood cells possess elevated levels of c-abl mRNA during the chronic phase and these cells accumulate in the consequent evolving leukemia. Our results with interferon therapy suggest a farily simple relationship between c-abl oncogene expression and persistence of the CML phenotype: namely, the maintenance of leukocytosis requires c o n t i n u e d c-abl oncogene expression in some or all blood cells. On the one hand, r e d u c e d expression may lead to loss of CML phenotype, whereas, additional changes in c-abl oncogene or other oncogenes may lead to transformation, clonal evolution, and termination in a blast crisis. In this context, however, the term blast crisis requires better definition, especially if oncogenes are to be invoked as causative factors of this phenomenon. Following the chronic phase of CML there is a period of transformation that may be characterized by anemia, progressive leukocytosis, thrombocytopenia, thrombocytosis, and/or progressive splenomegaly. This period may last several months to 1 year and ultimately terminate in blast crises that may be myeloblastic, promyelocytic, monocytic, lymphoblastic, or megakaryoblastic. In the acute n o n l y m p h o c y t i c blast crises, clonal evolution, as monitored by chromosomal changes, occurs but in the lymphoblastic crises choromosomal clonal evolution is not evident [23, 24]. The presence of the B-cell marker HLA.DR+ in chronic phase CM~L w o u l d suggest that blast crisis, w h e n it occurs, w o u l d be l y m p h o i d rather than m y e l o i d [36]. Can one invoke activation of c-abl to explain all these permutations and combinations plus progres-

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sion? Gale and Canaani [22] were unable to demonstrate sis transcription in leukemia cells obtained from patients with CML They stressed that the sis oncogene, in contrast to the c-abl oncogene, shows great variability of the chromosome to which sis is reciprocally translocated. They concluded that the sis oncogene is not important in the pathophysiology of CML. This may be true at a relatively late stage in the development of CML, when the translocation of the c-abl oncogene from chromosome # 9 to #22, the transcription of an abnormal 8-Kb mRNA of c-abl, and the increased expression of the abnormal c-abl oncogene occurs in CML cells [5, 1 8 21]. We propose that it is in the early stages of CML prior to formation of the Ph chromosome that the constitutive turn on of PDP activity, sis oncogene, and PDGF are involved in the pathophysiology of CML. With PDP we have already invoked the concept of gene rearrangements. Recent data clearly indicates that PDGF is involved with oncogene activation [37]. The megakaryocyte produces PDGF and

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marked increase in megakaryocytopoiesis is characteristic of the early stages of PV and CML. Of course, it is quite possible that the sis oncogene and PDGF will play a role in the later stages of CML. The idea that “cancer risk” at a cellular level represents sustained (inappropriate) growth of genetically unstable cells due to excess growth factor or hyperresponsiveness to growth factor is attractive. Proliferation of uncommitted hematopoietic stem cell descendants rendered genetically unstable by radiation might represent a CML risk [26]. It is conceivable that PDGF could be involved in this proliferation, either by acting directly on stem cells or by causing expansion of marrow reticulum that might serve as “feeder” cells. Sis gene alterations have been implicated in genetic predisposition to meningiomas in one family with chromosome #22 alterations [t(14;22)]. In addition, alterations in sis gene expression are common in primary tumors of brain connective tissue, tumors that often are benign

Figure 5

Effect of interferon therapy on PDP (A), platelets (---), and WBC (--) in patient Dacs with CML. High-dose IFN therapy induced a significant decrease in WBC and platelets. There was an initial decrease in PDP at 79 months followed by an increase at 83 months.

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B = before IFN A = after IFN Figure 6 Oncogene expression in two CML patients treated with interferon. Blood samples were obtained before (B) and (A) interferon therapy. Mononuclear blood cells were purified from whole blood by Ficoll gradient centrifugation and divided into two aliquots, one for mRNA immobilization and one for DNA immobilization. Samples taken for mRNA and DNA immobilization were handled as described previously [33, 40]. The immobilized nucleic acids were hybridized to [32p]-labeled v-abl or human c-mos oncogene probes. Both patients showed significant decreases in c-abl and c-mos oncogene transciption after interferon treatment.

[38]. Furthermore, production of PDGF and PDGF receptors are characteristic of unique hematopoietic cell line c o m p o s e d of megakaryocyte precursors [39]. The "paracrine" production of PDGF could generate a self-sustaining automitogenic cell mass. This scheme w o u l d give partial explanation for the p h e n o m e n o n of obtaining a putative primitive megakaryoblast cell line from normal donors [39]. Thus, evidence is emerging implicating the sis gene in cancer risk and in hematopoietic cell proliferation. PERSPECTIVE OF CML

CML appears to be a model system to study the d e v e l o p m e n t of cancer in general from the first preneoplastic states through aggressive neoplasia. CML is a progressive disease with steps of gene movement, growth factor stimulation, oncogene activation, and clonal evolution leading to acute leukemia. Although busulfan can adequately control chronic phase disease for a limited period of time, CML is programmed to develop into acute leukemia with only allogeneic bone marrow transplantation in chronic phase being able to cure the disease. With continued advances in molecular biology with such techniques as in s i t u hybridization of cells and chromosomes, and gene diagnosis, we will be able to d e c i p h e r the steps leading to

22

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c l i n i c a l l y a p p a r e n t CML a n d f u r t h e r p u s h b a c k t h e f r o n t i e r s of early d e t e c t i o n [40]. T h e m a j o r q u e s t i o n r e g a r d i n g p r e v e n t i o n of CML is, C a n w e d i a g n o s e t h e d i s e a s e p r i o r to t h e a p p e a r a n c e of P h ? In o r d e r to u n r a v e l t h e s e r i d d l e s t h a t h o p e f u l l y will lead to p r e v e n t i o n a n d m o r e effective t r e a t m e n t of m y e l o p r o l i f e r a t i v e d i s o r d e r s , close c o l l a b o r a t i o n is e s s e n t i a l a m o n g h e m a t o l o g i s t s , m o l e c u l a r b i o l o g i s t s , g e n e t i cists, a n d e p i d e m i o l o g i s t s . Supported in part by U.S. Public Health Service Grants CA29545 and CA37020 from the National Cancer Institute.

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