REVIEW Interleukin-3 Receptor in Acute Leukemia

REVIEW

Interleukin-3 receptor in acute leukemia U Testa1, R Riccioni1, D Diverio2, A Rossini1, F Lo Coco3 and C Peschle1 1Department of Hematology and Oncology, Istituto Superiore di Sanita`, Viale Regina Elena 299, Rome, Italy; 2Dipartimento di Biotecnologie Cellulari, Universita` ‘‘La Sapienza’’, Rome, Italy; and 3Cattedra e Divisione di Ematologia, Universita` Tor Vergata, Ospedale S Eugenio, Rome, Italy

Recent studies indicate that abnormalities of the interleukin-3 Several lines of evidence have indicated that AML1/ETO is receptor (IL-3R) are frequently observed in acute myeloid insufficient by itself to induce leukemia, but instead alters the leukemias (AMLs) and may contribute to the proliferative advantage of leukemic blasts. This review analyzes the self-renewal capacities of hematopoietic stem cells, resulting in evidences indicating that the IL-3R represents one of the target the formation of a preleukemic population that lacks a great 5 molecules involved in the stimulation of proliferation of AMLs, growth advantage. For the development of an overt leukemic and the overexpression of the IL-3Ra chain may represent one condition, AML1/ETO-induced effects must be associated with of the mechanisms contributing to the development of a highly the effects of secondary mutations that cooperate with the malignant leukemic phenotype. Furthermore, there is evidence primary mutation and ultimately result in a growth advantage that the IL-3Ra is a marker of leukemic stem cells, at variance 6,7 with normal stem cells that are IL-3RaÀ. Finally, the IL-3R may and in a block of cell differentiation. In line with these represent an important target for the development of new findings, a high frequency of NRAS and c-kit mutations have þ antileukemic drugs. been observed in AML patients with AML1/ETO leukemias. Leukemia (2004) 18, 219–226. doi:10.1038/sj.leu.2403224 Therefore, it was proposed that fusion proteins encoding Published online 4 December 2003 hybrid transcription factors require genetic complementation Keywords: leukemia; growth factors; interleukins; proliferation; with mutant or activated kinases. Activated kinases could differentiation interfere with the cell cycle and complement the differentiation-blocking activity of chimeric transcription factors. This model is supported also by additional important observations: (a) fusion proteins, such as BCR/ABL or TEL/AML1 or AML1/ The current model on the pathogenesis of acute myeloid ETO, are found in normal subjects at a frequency about 100 leukemias (AMLs) 8,9 times the corresponding leukemia rates, thus indicating that their presence in few hemopoietic cells is per se not sufficient Several lines of experimental evidence suggest that multiple for the development of a leukemic condition; (b) the analysis of mechanisms are involved in the genesis of human tumors and, the clonal relationship of concordant leukemia in identical particularly, of AMLs.1,2 Among these different pathogenetic mozygotic twins strongly argues in favor of a parental in utero mechanisms, the occurrence of chromosomic translocations 10 origin of pediatric leukemias. with consequent generation of fusion genes and corresponding In the recent years several genes have been identified, fusion proteins play a major role in the genesis of these diseases. mutated or abnormally expressed in AML, that confer a Stem cells are the main target for these key genetic events in proliferative survival advantage to leukemic blasts. These genes acute leukemias. The major contribution of fusion proteins, such are characterized by their involvement in the control of cell as PML/RARa, AML1/ETO, AML1/Evi1 or CBFb/SMMHC, con- proliferation. Thus, the hemopoietic receptor tyrosine kinase sists in the capacity to block the differentiation process at a Flt3 is constitutively activated in 30–35% of cases of AML, as a specific stage (reviewed in Tenen).3 Studies in transgenic mice consequence of internal tandem-repeat mutations in the harboring the fusion gene in their genome clearly indicate that juxtamembrane domain, or activating loop mutations (reviewed the simple expression of this gene in hemopoietic cells greatly 11 in Gilliland and Griffin). Similarly, Flt3 is frequently over- contributes to the differentiation block and, in some cases, also 12 expressed in secondary, therapy-related AMLs and in lympho- in part to the proliferative stimulation, but per se is not sufficient 13 4 blastic leukemia with MLL rearrangements and with for the development of a leukemic condition. This conclusion 14 hyperdiplody. Finally 5–10% of patients with myelodysplasia is directly supported by several experimental models, such as have mutations of the Flt3 gene. The occurrence of a significant transgenic animals expressing the PML/RARa fusion gene: these association between the presence of Flt3 mutations in AML and animals, in spite a high expression of the transgene, need 1–2 the number of blast cells strongly indicates that the mutated years before the development of leukemia. This time is required receptor induces a proliferative biologic effect in leukemic for the acquisition in hemopoietic cells of additional mutations, blasts. Activating loop mutations have been identified in the mainly occurring at the level of genes involved in the control of hematopoietic receptor tyrosine kinase c-kit in 5% of AML cell proliferation, whose alterations are required for tumor 15 cases. Furthermore, activating loop mutations in RAS family development. Interestingly, similar observations have been also members, mainly NRAS and KRAS, have been identified in 15– made in M2 leukemias associated with the t(8;21) translocation 16 25% of cases. It was estimated that mutations of some and the consequent formation of the AML1/ETO fusion protein. components of the receptor tyrosine kinase pathway (including Flt3, c-kit, VEGFRs, c-fms and RAS) occur in 450% of AML Correspondence: Dr U Testa, Department of Hematology and patients. Oncology, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy; Fax: þ 39 064 938 7087; E-mail: [email protected] Recent studies indicate that abnormalities of the interleukin-3 Received 22 July 2003; accepted 15 October 2003; Published online receptor (IL-3R) are frequently observed in AMLs and may 4 December 2003 contribute to the proliferative advantage of leukemic blasts.17 IL-3R in acute leukemia U Testa et al 220 This review will analyze the evidences indicating that the IL-3R GM-CSFR acquired biologic activity on cell differentiation and may represent one of the target molecules involved in the losted its activity on cell proliferation of FDPC-mix cells.23 stimulation of proliferation in AMLs and particularly, that its According to these observations, it was suggested that IL-3 overexpression may represent one of the mechanisms involved exerts its biologic activities mostly at the level of the control of in the pathogenesis of a highly malignant leukemic phenotype. proliferation of hemopoietic progenitors, while the predominant This review will not cover the basic physiology of the IL-3R, as effect of GM-CSF is on the differentiation of these cells. well as the signal transduction pathways induced by the The analysis of the signal transduction pathways triggered by activation of this receptor since this topic was covered by an IL-3R activation is out the scope of the present review, and the excellent review, recently published on this journal.18 reader is referred to an excellent review recently published on this journal.18 Here, only a brief summary of this process is briefly oultined. After ligand binding, the IL-3R/GM-CSFR bc Basic background becomes phosphorylated, and through the recruitment of adaptor proteins such as Shc, Grb2, and Sos activates the Ras Normal hematopoiesis is a multistep process in which lineage signaling pathway. The activated Ras pathway in turn stimulates development occurs as a consequence of the initial stochastic Raf followed by the downstream induction of the MAPK differentiation of pluripotent stem cells with the generation of pathway related to ERK1 and ERK2, linked to increased multipotent hemopoietic progenitors and the subsequent differ- expression of the transcription factors c-fos and c-jun. The entiation along the different hemopoietic lineages of these cells activation of the MAPK pathway is not restricted only to ERK1 under the ordered effects of a number of growth factors. These and ERK2, but involves also p38, and JNK/SAPK. Another very growth factors may be classified according to the level of their important transduction pathway induced during the activation biological action as early-acting hemopoietic growth factors of the IL-3R is represented by the phosphatidylinositol 3-kinase (HGFs), multilineage HGFs and late-acting HGFs. The prototype (PI-3K)/AKT pathway: the p85 subunit of PI3 K associates with of multilineage HGF is represented by IL-3. the bc of the activated IL-3R at the level of phosphorylated Ser- The main biologic activity of IL-3 is exerted at the level of the 585,24 recruits PKB/AKT by phosphorylation of its serine progenitor compartment, where this cytokine stimulates the residues and transmits several survival signals. Ultimately, these survival and proliferation of multipotent cells; particularly, IL-3 various transcription pathways influence in a specific way gene stimulates the development of multilineage colonies from transcription activating or repressing a set of transcription normal bone marrow, but displayes also effects at the level of factors, cause cell to progress through cell cycle check points the compartment of hemopoietic precursors, restricted to the via the activation of specific protein kinases and inhibit granulocytic and monocytic lineages.19,20 apoptosis basically through the phosphorylation of specific IL-3, as well as GM-CSF, exerts its biologic activity through proteins involved in the apoptotic cascade. the interaction with cell surface receptors that consist of two As above mentioned, one of the main effects exerted by both subunits, the a subunit specific to each and the b common chain IL-3R and GM-CSFR consists in inducing an inhibitory effect on (bc). The a chain (IL-3Ra, GM-CSFRa) binds IL-3 and GM-CSF, cell apoptosis. In this process, a major role is played by the respectively, with high specificity, but with low affinity.19,20 The activation of the p38 MAP kinase and PI3K/AKT pathways. A key interaction of an a chain with a b chain leads to the formation of target of both these transduction pathways activated by IL-3 is a high-affinity receptor complex able to bind the respective represented by the Mcl-1 gene, a member of the Bcl-2 family of ligand in the range of its physiologic concentrations and to proteins. Mcl-1 is an immediate early gene activated by the IL-3 transduce proliferative, antiapoptotic and differentiative sig- signaling pathways and representes one major component of the nals.19,20 The bc expressed alone, in the absence of a specific a viability response elicited by this cytokine.25 chain, confers little binding affinity for either IL-3 or GM-CSF.21 One additional important target for the PI3K/AKT pathway is Although the bc of the IL-3/IL-5/GM-CSF receptors initiates represented by NFkB: this transcription factor is held in an signal transduction events specific for this receptor family, it inactive, latent state in the cytoplasm by the inhibitor protein apparently does not distinguish between IL-3, IL-5 and GM-CSF. IkB; AKT phosphorylates IkB kinase that in turn phosphorylates It is obvious that the cytokine-specific a-chains are candidates to IkB with its consequent degradation by the proteasome, confer specificity in signaling between IL-3, GM-CSF and IL-5. allowing NFkB to translocate to the nucleus and regulate the The IL-3Ra and GM-CSFRa intracellular domains are relatively expression of a broad of genes such as bcl-2, bcl-XL and A1, all short (about 30 amino acids), but seem to play a critical role in involved in cell survival.26 This pathway is activated by IL-3 via signaling transduction, in that their deletion leads to the phosposerine-585 bc phosphorylation.26 abrogation of receptor signaling. The intracytoplasmic regions According to all these observations it was proposed that the of all three IL-3Ra, GM-CSFRa and IL-5Ra contain a highly antiapoptotic activity of IL-3 could be related to the induction of 27 conserved membrane-proximal proline-rich region, involved in the Bcl-2 family genes, bcl-2, bcl-XL, A1 and mcl-1. receptor signaling. Near this region, a tripeptide whose The screening by microarray gene expression profiling of sequence is different in IL-3Ra compared to GM-CSFRa was genes downmodulated by IL-3 deprivation in an IL-3-dependent recently identified. This tripeptide seems to confer to IL-3R a cell line following IL-3 starvation provided evidence that pim-2 differential biologic activity as compared to GM-CSFR: in fact, is the gene most modulated by IL-3.28 Pim-2, a serine threonine in a multipotential murine hemopoietic precursor cell line kinase, emerged from these studies as a candidate for a key (FDCP-mix cells), the transfection of IL-3R resulted in a mediator of survival signaling by IL-3: in fact, pim-2 knockout or stimulation of cell proliferation, while the transduction of GM- overexpression of a pim-2 dominant-negative mutant, prevented CSFR resulted in a stimulation of cell differentiation;22 the effctive survival signaling by IL-3.28 Pim-2 seems to act as an transduction of a mutatnt GM-CSF receptor containing a activator of several antiapoptotic pathways.29 tripeptide sequence mutated from its own sequence to the IL- Finally, SHP-2, a tyrosine phosphatase, highly expressed in 3R sequence, induced a shift of the biologic activity of GM-CSF hematopoietic cells seems to play multiple and important roles from induction of differentiation to induction of proliferation;23 in IL-3 signal transduction. In fact, the IL-3-induced proliferative similarly, the mutated IL-3R with the tripeptide sequence of the response is markedly dimished in SHP-2 (À/À) cells.30

Leukemia IL-3R in acute leukemia U Testa et al 221 Aberrant activation of both the Ras/MEK/ERK and of the PI3K/ Other studies were focused to evaluate the effect of IL-3Rb AKT pathways is commonly observed in AMLs: the targeting of chain deficiency both in mice and in humans. Mice lacking IL- some key molecules of these pathways may represent an 3Rb chain show a pulmonary alveolar proteinosis-like disease.43 important tool for the development of new therapeutical Interestingly, these animals showed normal hematologic para- approaches.30–34 meters, with the exception of a reduced eosinophil number.43 A similar situation was observed in children with human pulmonary alveolar proteinosis, where in a high percentage of Oncogenic role of IL-3/IL-3R in experimental models cases a deficit of the IL-3Rb chain was observed.44 This syndrome is also observed in an adult form: in this case it is The role of IL-3Ra and b chain genes in oncogenesis has been due to the development of an anti-GM-CSF autoimmunity.45 explored in several animal models. A first series of experiments Interestingly, in a part of the pediatric patients, the development showed that certain mutations of IL-3Rb may determine growth of pulmonary proteinosis is associated to AML.46 In these factor independency and tumorigenicity. A mutant form of IL- patients, the expression of bc chain was markedly reduced, 3Rb chain was isolated from growth factor-independent cells, while IL-3Ra expression was normal.47 After antileukemic which arose spontaneously after infection of a murine factor- treatment, the pulmonary symptoms resolved and the bc dependent hematopoietic cell line (FDC-P1) with a retroviral IL- expression was normal.47 3Rb expression construct.35 Analysis of this mutant shows a small (37 amino acid) duplication of extracellular sequence that includes two conserved sequence motifs: this mutation is IL-3R expression in leukemias sufficient to confer growth factor-independent growth of these cells and induces a tumorigenic phenotype.35 Furthermore, the Initial studies based on the analysis of the biological responses IL-3Rb chains are truncated in spontaneous mutants of a murine elicited in vitro by IL-3 addition and on the study of the binding promyelocytic cell line, determining growth factor indepen- of -IL-3 to membrane receptors showed that most human AML dency, due to constitutive IL-3R activation.36 The expression of blasts proliferate in response to IL-3 and express IL-3Rs.47–50 this truncated IL-3Rb subunit in factor-dependent myeloid cells Particularly, in more than 85% of AML cases, IL-3 elicited a (FDC-P1) did not immediately render the cells autonomous, but clear stimulation of thymidine incorporation and, as a single increased the spontaneous frequency to factor-independent stimulatory agent, was significantly more active than other growth.36 hemopoietic growth factors.47,48 Similarly, the analysis of IL-3 A second mutation of the IL-3Rb chain, V449E, results in binding to AML blasts showed that 480% of AMLs exhibit high- substitution of a valine residue in the transmembrane domain affinity IL-3 receptors.49,50 with glutammic acid.37 Interestingly, when expressed in the IL- More recently, the availability of antibodies specific for the a 3-dependent murine pro-B-cell line, BAF-B03, this mutant, but and b chain of the IL-3R allowed the evaluation in detail of the not the extracellular mutant, could confer growth factor expression of these two receptors chains in leukemias. independency.37 The finding implies that the extracellular and Particularly, antibodies interacting with the N-terminal of the transmembrane mutants signal via different mechanisms, and IL-3Ra chain and blocking the binding of IL-3 to its receptor, as that the extracellular mutant may require other signaling well as antibodies interacting with other regions of the molecule molecules to deliver a mitogenic signal. In line with this that fail to inhibit IL-3 binding or function, have been hypothesis, studies on primary hemopoietic progenitors showed developed.51 In parallel, antibodies specifically interacting with that retroviral expression of the extracellular mutant confers the common IL-3R/GM-CSFR/IL-5R b-chain have been devel- factor-independent proliferation on cells of the neutrophilic and oped, thus allowing to explore the expression of both IL-3R monocytic lineages only, while expression of the transmmeb- chains. The study of normal IL-3Ra during normal hemopoietic rane mutant exerts this effect on cell myeloid lineages, as well as differentiation provided evidence that this receptor chain was in erythroid cells.38 clearly expressed in CD34 þ HPCs and its expression was Mutations of the IL-3Ra gene have been described in A/J maintained during all stages of granulocytic and monocytic mouse and related mice strains. Particularly, bone marrow cells differentiation, while it was lost after the initial stages of of A/J mice are nonresponsive to IL-3 and this property depends erythroid and megakaryocytic differentiation.52 The IL-3R/GM- on an aberrant splicing of IL-3Ra mRNA and impaired CSFR/IL-5R bc was expressed at low levels in CD34 þ cells and expression of the IL-3Ra chain, in that the mutant protein is its expression increased during initial stages of granulocytic and localized inside the cells and not on the cell surface.39 The A/J monocytic differentiation and therefore maintained up to the mice have a high incidence of lung adenomas and leukemias.40 late stages of maturation, while it was transiently expressed in In addition to these models, where the mutation of either the the erythroid and megakaryocytic differentiation only during the IL-3Ra or IL-3Rb chains may contribute to the development of a early stages of differentiation and then disappeared.53 leukemic condition, other studies have suggested a possible The screening of IL-3Ra expression in hemolymphopoietic oncogenetic role related to the level of expression of these malignancies provided evidence that this receptor chain is receptors. Therefore, in v-abl derivatives of the IL-3-dependent expressed in the large majority of B-ALL and AML, is usually not FL5.12 cell line, the induction of an autocrine mechanism of IL- expressed in T-ALL as well as in the majority of lymphomas, is 3 production was associated with an absent downmodulation of highly expressed in hairy cell leukemia.54 These findings have IL-3Rb chain expression.41 In parental cells, as well as in other been confirmed on a large series of acute leukemias, clearly normal cells, IL-3Rb is downmodulated following exposure of indicating that an elevated expression of IL-3Ra is mainly the cells to IL-3. In another study the effect of IL-3Ra and b observed in AML and B-ALL.13 We observed also that IL-3Ra overexpression in FDC-P1 cells was directly evaluated. The chain is overexpressed (ie, it is expressed at levels significantly overexpression of both these receptor chains elicited two major higher than those observed in normal CD34 þ cells, the normal effects: (a) it increased the sensitivity to IL-3 (ie, the cells cells expressing the highest levels of IL-3Ra chain) (Figure 1a), in proliferated in the presence of suboptimal IL-3 concentrations); 40% of patients with B-ALL and in 45% of patients with AML.17 (b) it favored the development of IL-3-independent clones.42 IL-3R-overexpressing AMLs have been observed along all the

Leukemia IL-3R in acute leukemia U Testa et al 222

Figure 1 (a) Flow cytometry analysis of IL-3Ra in six AMLs (from AML1 to AML6). For each of these six AML patients, the single labeling with an anti-IL-3Ra and the double labeling with anti-CD34 and anti-CD71 are shown. In the panel on the left, the reactivity of puriﬁed CD34 þ normal cells is shown for comparison. (b) Left panel: blasts from 10 AMLs have been separated according to the level of IL-3Ra expression into two cell populations: IL-3Ra dim and IL-3Ra bright cells. Right panel: cell cycle analysis shows that cycling cells are observed mainly among IL-3Ra bright cells.

FAB AML subtypes. However, the analysis of the membrane Given the peculiar properties of leukemic blasts expressing immunophenotype of AMLs expressing high levels of IL-3Ra high levels of IL-3Ra, we then explored the possible association chain showed several interesting findings: usually, these between elevated IL-3Ra expression and the response to leukemias show high expression of CD34 (Figure 1a), CD33 therapy. In this context, we observed that: (i) there is a good and CD13 antigens, constantly associated with a low CD15 and correlation between IL-3Ra levels and the number of leukemia CD11b expression; interestingly, the large majority of IL- blasts at diagnosis, suggesting a role for IL-3Ra in leukemic blast Ra þþþ AMLs dislpay a clearly detectable expression of both proliferation/survival; (ii) patients with elevated levels of IL-3Ra c-kit and flt3, with a high proportion of leukemic blasts being c- on their leukemic blasts exhibited a reduced number of kit þ and flt3 þ . complete remissions, higher relapse rate and a lower survival The biological consequences of this overexpression pattern than patients with normal/low IL-3Ra levels.17 According to the were investigated in AMLs, showing that: (i) in all patients, the ensemble of these findings it was suggested that in AML blasts expressing elevated IL-3Ra levels exhibit higher cycling deregulated expression of IL-3Ra may contribute to the activity (Figure 1b) and increased resistance to apoptosis proliferative advantage of the leukemic blasts and, hence, to a triggered by growth factor deprivation; (ii) spontaneous Stat5 poor prognosis.17 phosphorylation was frequently observed only in AML patients In line with these findings, studies on cell lines have clearly exhibiting high levels of IL-3Ra expression; (iii) following IL-3 shown that IL-3R overexpression induces oncogenic effects: treatment, Stat5 was activated at significantly higher levels in particularly, IL-3Ra overexpression did not completely relieve blasts with elevated IL-3Ra expression than in blasts with IL-3Ra factor dependency, but allowed the cells to proliferate in the levels comprised within the normal range.17 presence of suboptimal concentrations of IL-3.41 On the basis of

Leukemia IL-3R in acute leukemia U Testa et al 223 these observations, it was concluded that a deregulation of IL- progress to evaluate whether there is an overlapping between 3Ra expression may provide a proliferative advantage to Flt3 mutations and IL-3Ra overexpression. hemopoietic cells growing under conditions in which the From a more general point of view it of interest to note that cytokine is limiting and allows the development of a leuke- Flt3 mutations are considered in recent views on the pathogen- mia.41 esis of acute leukemias as the prototype of class I mutations, that In addition to these findings, another study extended the is, of those mutations that induce a proliferative advantage to analysis of IL-3Ra to subpopulations of AML blasts and, leukemic cells.3 In addition to the Flt3 mutations, also the IL-3Ra particularly, to leukemic stem cells (ie, the subpopulation of overexpression should be considered, although it is not a leukemic blasts, corresponding to cells that maintain the mutation, another type of abnormality of leukemic cells leukemic process and that seemingly represent the main target contributing, like Flt3 mutations, to confer to leukemic blasts a of the initial leukemic transformation).55 The analysis of an AML proliferative advantage. This conclusion is directly supported by subpopulation enriched in leukemic stem cells (ie, 34 þ /38À) the analysis of animal models of development of acute showed that these cells express high levels of IL-3Ra, while in the leukemia, where IL-3R stimulation, as well as Flt3 mutations, normal counterpart (ie, in 34 þ /38À cells isolated from normal cooperated with chimeric transcription factors in inducing a controls) low level of IL-3Ra have been observed, thus suggesting leukemic condition.69 that IL-3Ra represents an unique marker for AML stem cells.55 Within the family of receptors that use the common bc, the According to these findings, it was shown that the isolation of overexpression of IL-3Ra in AMLs is specific in that both the 34 þ /38À/IL-3Ra þþ cells represent an efficient system for the GM-CSFRa and the IL-5Ra chains are not overexpressed. purification of leukemic stem cells in AML patients.56 Particularly, the GM-CSFRa is preferentially expressed in It is clear that the mechanism through which an over- myeloid compared to lymphoid leukemias and its level of expressed IL-3Ra may induce a growth advantage in AML blasts expression is in the majority of cases comparable to those is related to the induction of a constitutive Stat5 activity. The observed in normal hemopoietic cells at a comparable degree of constitutive Stat5 activation is a pathway involved in many differentiation.17 hematologic malignancies. Translocations resulting in TEL- In addition to the IL-3Ra, few studies have explored the JAK257 or TET-PDGFbetaR58 constructs lead to the constitutive expression of bc in acute leukemias.The expression of bc was Stat5 activation and to IL-3 independency. Similarly, in chronic preferentially observed in AMLs, at levels of expression myeloid leukemia (CML), Stat5 is constitutively activated comparable to those observed in the normal counterpart. through a mechanism involving the interaction between Bcr/ Interestingly, in many leukemic patients, a markedly increased 59 70 Abl and the hemopoietic cell kinase (HCK). The inhibition of level of a truncated form (bIT) of the bc was observed. This bIT the Bcr/Abl kinase activity by specific inhibitors blocks the has a tail of 46 amino acids, instead of 432 for bc, with 23 constitutive Stat5 activation and growth of CML cells.60 amino acids in common with bc and a new sequence of 23 70 Interestingly, recent studies provided evidence that the Flt3 amino acids. The proportion of bIT increased to 90% of total mutations are also able to activate the Stat5 pathway. The Flt3 bc content in blast cells of a significant proportion of AML 62 receptor, a tyrosine kinase, is constitutively activated either by patients. The bIT was unable to transduce a proliferative signal internal tandem duplication or by point mutations in 430% of and it was proposed that it could act as a dominant negative AML patients.11 It was clearly shown in large series of patients interfering with the signal-transducing properties of the bc, and that the presence of a mutated Flt3 was associated to a poor could play a role in the differentiation blockade of leukemic prognosis and to the development of a leukemia with an cells.70 aggressive phenotype (high blast cell counts at diagnosis), In addition to abnormalities of IL-3R expression, in some findings similar to those observed in the group of AML patients leukemias, an autocrine production of IL-3 by leukemic cells overexpressing the IL-3Ra chain (reviewed in Gilliland and was found. This condition was studied in detail in CML. A first Griffin11 and Stirewalt and Radich61). Recent studies clearly series of studies showed that the expression of BCR/ABL confers indicate that the mechanism through which the mutated Flt3 autonomous growth properties on a variety of cell lines confer to leukemic blasts a growth advantage is mainly related dependent on IL-3 for their growth.71 Acquisition of growth to the capacity to constitutively activate the Stat5 transduction factor independence was associated with the induction of IL-3 pathway. In fact, it was shown that the majority of AML patients production.71 This mechanism of autocrine IL-3 production was with Flt3 mutations and particularly with ITD mutations exhibit shown to be activated in primitive hemopoietic progenitor a constitutive Stat5 activation.62 Similarly, Sta5 is activated in (CD34 þ ) cells of CML patients: this IL-3 production activates an lymphoid leukemic cells with D835/I836 mutatation.63 Inter- autocrine stmulatory loop that leads to constitutive Stat5 estingly, the wt Flt3 receptor does not signal through the Stat5 activation.71 Interestingly, this autocrine IL-3 production me- pathway, but via AKT and MAPK pathways.64,65 Importantly, the chanism declines when leukemic cells differentiate. There is incubation of these leukemic blasts with inhibitors of the Flt3 evidence that IL-3 production by CML cells is directly and kinase activity elicited an inhibition of Stat5 activity and of the rapidly induced by BCR/ABL in early hemopoietic progenitor 72 expression of Bcl-XL (a target gene of Stat5), thus indicating that cells. Importantly, the shift of early CML progenitors from a the constitutive Flt3 receptor signaling is responsible for Stat5 growth factor-dependent growth to autonomous growth is activation.65,66 In line with these observations, microarray associated with acquisition to synthesize and release IL-3.73 analysis showed that the target genes of mutationally activated Similarly, there is evidence that a significant proportion of Flt3 resembled more closely those of the IL-3R than those of AMLs secrete IL-3 or GM-CSF.74,75 In a study carried out on 53 ligand-activated wt Flt3.67 The role of Flt3 mutations in the AML patients, clearly detectable levels of IL-3 have been genesis of highly, alignant forms of leukemia renders FLT3 an detected in about 80% of the leukemic blast cell culture obvious target of therapy. Several small-molecule FlT3 inhibitors supernatants.76 The highest IL-3 secreting capacity was ob- are under development and are under evaluation in phase I/II served in M1/M2/M3 AMLs.76 It is of interest to note that in this studies.68 study was reported the capacity of IL-3 to stimulate the Since a constitutive Stat activation was also observed in AML production of secondary cytokines, such as IL-6, TNF-a and patients exhibiting IL-3Ra overexpression, experiments are in IL-1b by leukemic cells.76 Interestingly, IL-3 or GM-CSF

Leukemia IL-3R in acute leukemia U Testa et al 224 production by leukemic blasts was observed also at the level of However, the DT-GM-CSF produced dose-limiting toxicity due early progenitor leukemic cells, that is, at the level of the to liver injury related to the binding of GM-CSF receptors leukemic blasts that initiate and maintain the leukemic present on the surface of liver macrophages.84 Since IL-3Rs were process.77 not present on liver macrophages, it is expected that DT-IL3 may It is of interest to note that an autocrine Flt3 activating loop have a better toxicity profile and a wider therapeutic index than was recently described in AML cells.78 In fact, it was observed DT-GM-CSF. that the majority of AMLs not only express the Flt3 receptor but possess also the capacity to synthesize and secrete its ligand.78 This autocrine Flt3 ligand production leads to an autocrine stimulatory loop with consequent constitutive Flt3 autopho- sphorylation and activation.78 Interestingly, this phenomenon References occurs both in patients with Flt3 mutataions and with wild-type Flt3. The autocrine Flt3 ligand production was also found at the 1 Hahn WC, Weinberg RA. Rules for making human tumor cells. N level of very early leukemic progenitors, thus suggesting that the Engl J Med 2002; 347: 1593–1603. 2 Hahn WC, Weinberg RA. Modelling the molecular circuity of Flt3 activation may play a role in the stimulation of early cancer. Nat Rev Cancer 2002; 2: 331–341. 77 leukemic progenitors. 3 Tenen DG. Disruption of differentiation in human cancer: AML shows the way. Nat Rev Cancer 2003; 3: 89–101. 4 Kelly LM, Kutok JL, Williams IR, Boulton CI, Amaral SM, Curley J Development of therapeutical approaches tailored to the et al. PML/RARalpha and FLT3/ITD induce an APL-like disease in IL-3R mouse model. Proc Natl Acad Sci USA 2002; 99: 8283–8288. 5 Dowining JR. The core-binding factor leukemias: lessons learned from murine models. Curr Opin Genet Dev 2003; 13: 48–54. Taking advantage on the development of the studies on IL-3 and 6 Yuan Y, Zhou L, Miyamoto T, Iwasaki H, Zhang DE. AML1-ETO IL-3R expression in AMLs, several investigators have explored expression is directly involved in the development of acute the efficacy of drugs based on a diphteria toxin (DT) human IL-3 myeloid leukemia in the presence of additional mutations. Proc fusion protein. The basic idea is that the fusion protein interacts Natl Acad Sci USA 2001; 98: 10398–11403. with the leukemic blasts expressing high levels of IL-3R, is 7 Higuchi M, O’Brien D, Kumuravelu P, Lenny N, Yeoh EJ, Downing JR. Expression of a conditional AML1-ETO oncogene bypasses internalized and exerts its highly cytotoxic effects. These studies embryonic lethality and establishes a murine model of human have been initially carried out in murine models showing that t(8;21) acute myeloid leukemia. Cancer Cell 2002; 1: 63–74. DT-murine IL-3 fusion protein is toxic for murine IL-3-bearing 8 Biernaux C, Loos M, Sels A, Huez G, Stryckmans P. Detection of leukemic cell lines, but spares the majority of normal mouse major bcr-abl gene expression at very low level in blood cells of bone marrow-repopulating stem cells.79,80 Two subsequent some healthy individuals. Blood 1995; 86: 3118–3122. studies have evaluated the effect of a DT human IL-3 fusion 9 Mori H, Colman SM, Xias Z, Ford AM, Healy LE, Donaldson et al. Chromosome translocations and covert leukemic clones are protein on AML blasts. In an initial study, it provided generated during normal fetal development. Proc Natl Acad Sci preliminary evidence that a DT human IL-3 fusion protein was USA 2002; 99: 8242–8247. more cytotoxic for AML blasts than for normal bone marrow 10 Greaves MF, Wiemels J. Origins of chromosome translocations in hemopoietic progenitors.81 In a second study, the issue was childhood leukemia. Nat Rev Cancer 2003; 3: 1–11. studied in more detail showing that: (i) a high percentage of 11 Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and AML progenitors or AML LTC-IC (‘long-term culture-initiating leukemia. Blood 2002; 100: 1532–1542. 12 Quian Z, Fernald AA, Gadley LA, Larson RA, LeBeau M. cells’) were killed by the IL-3 fusion protein, with most of the Expression profiling of CD34+ hematopoietic stem/progenitor cells surviving progenitors being cytogenetically normal; (ii) a reveal distinct subtypes of therapy-related acute myeloid leukemia. significant proportion of normal hemopoietic progenitors was Proc Natl Acad Sci USA 2002; 99: 14925–14930. killed by, while LTC-ICs were not significantly killed by DT/IL-3; 13 Armstrong SA, Kung AL, Mobon AL, Griffin JD, Korsmeyer SJ. (iii) a significant proportion of leukemic, but not of normal Inhibition of Flt3 in MLL: validation and therapeutic target repopulating stem cells, as evaluated by the NOD/SCID assay, identified by gene expression based classification. Cancer Cell 2003; 3: 173–183. was killed by the DT/IL-3; (iv) the in vivo administration of DT/ 14 Yeoh EJ, Rosse ME, Shurtleff SA. Classification, subtype discovery, IL-3 markedly reduces the engraftment of AML cells in NOD/ and prediction of outcome in pediatric acute lymphoblastic SCID mice.82 On the basis of these observations, the same leukemia by gene expression profiling. Cancer Cell 2002; 1: investigators have evaluated the antileukemic activity of DT- 133–143. human IL-3 in the treatment of immunocompromised mice 15 Beghini A, Peterlongo P, Rigamonti CB, Larizza L, Cairoli R, Morra engrafted with human IL-3R-positive AML blasts. The results of E et al. c-Kit mutations in core binding factor leukemias. Blood 2000; 95: 726–727. these studies showed a substantial killing of malignant 16 Kyoi H, Nasc T, Niakano Y, Jinnai I, Shimazaki C. Prognostic progenitors in vivo by DT-IL3, with a potency inferior to that implication of FLT3 and N-RAS mutations in acute myeloid of a standard antileukemic cytotoxic drug, such as cytarabine, leukemia. Blood 1999; 93: 3074–3080. but with a potency comparable to that displayed by other agents 17 Testa U, Riccioni R, Militi S, Coccia E, Stellacci E, Samoggia P et al. for target-specific therapy (such as DT-GM-CSF or anti-CD33- Elevated expression of IL-3Ra in acute myelogenous leukemia is calicheamicin).83 associated with enhanced blast proliferation, increased cellularity, and poor prognosis. Blood 2002; 100: 2980–2988. These observations warrant the further preclinical develop- 18 Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, ment of DT-IL-3. This drug could be useful in the treatment of Wang XY, Algate PA et al. Signal transduction, cell-cycle patients refractory to standard chemotherapy or in association rgeulatory, and anti-apoptotic pathways regulated by IL-3 in with chemotherapy. Furthermore, DT-IL3 could have a toxicity hematopoietic cells: possible sites for intervention with anti- profile more favorable than DT-GM-CSF. In fact, the fusion neoplastic drugs. Leukemia 1999; 13: 1109–1166. protein between GM-CSF and diphteria toxin has been 19 Begley CJ, Woodcock JM, Stovoski FC, Lopez AF. The structural and functional basis of cytokine receptor activation: lessons from developed as a clinical grade reagent and was used for the the common b subunit of the granulocyte–macrophage colony treatment of AML patients resistant to standard chemotherapy: stimulating factor, interleukin-3 and IL-5 receptors. Blood 1997; on 31 treated patients, three remissions have been observed.84 89: 1471–1482.

Leukemia IL-3R in acute leukemia U Testa et al 225 20 Miyajima A, Mui AL, Ogorochi T, Sakamaki K. Receptors for 38 Ichihara M, Hara T, Takagi M, Cho LC, Gorman DM, Miyajima A. granulocyte–macrophage colony-stimulating factor, interleukin-3, Impaired interleukin-3 (IL-3) response on the A/J mouse is caused and interleukin-5. Blood 1993; 82: 1960–1974. by a branch point deletion of the IL-3 receptor a subunit gene. 21 Kitamura T, Sato N, Arai K, Miyajima A. Expression cloning of the EMBO J 1995; 14: 939–950. human IL-3 receptor cDNA reveals shared beta subunit for the 39 Hara T, Ichihara M, Takagi M, Miyajima A. Interleukin-3 (IL-3) human IL-3 and GM-CSF receptors. Cell 1991; 66: 1165–1174. poor-responsive inbred mouse strains carry the identical deletion 22 Evans C, Pierce A, Winter SA, Spooncer E, Heyworth C, Whetton of a branch point in the IL-3 receptor a subunit gene. Blood 1995; AD. Activation of granulocyte–macrophage colony-stimulating 85: 2331–2336. factor and interleukin-3 receptor subunits in a multipotential 40 Algate PA, Steelman LS, Mayo MW, Miyajima A, McCubrey JA. hematopoietic progenitor cell line leads to differential effects on Regulation of the interleukin-3 (IL-3) receptor by IL-3 in the fetal development. Blood 1999; 94: 1504–1514. liver-derived FL5.12 cell line. Blood 1994; 83: 2459–2468. 23 Evans C, Ariffin S, Pierce A, Whetton AD. Identification of primary 41 Steelman LS, Algate PA, Blalock WL, Wang XY, Prevost KD, Hoyle structural features that define the differential actions of IL-3 and PE et al. Oncogenic effects of overexpression of the interleukin-3 GM-CSF receptors. Blood 2002; 100: 3164–3174. receptor on hematopoietic cells. Leukemia 1996; 10: 528–542. 24 Guthridge MA, Stomski F, Bany EF, Winnel W, Woodcock JM, 42 Nishinakamura R, Nakayama N, Hirabayashi Y, Inoue T, Aud D, McClure BJ et al. Site-specific serine phosphorylation of the IL-3 Mori T et al. Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor is required for hemopoietic cell survival. Mol Cell 2000; receptor exhibit lung pathology and impaired immune response, 6: 99–108. with beta II receptor-deficient mice are normal. Immunity 1995; 2: 25 Wang JM, Lai Mz, Yang-Yen HF. Interleukin-3 stimulation of mcl-1 211–222. gene transcription involves activation of the PU.1 transcription 43 Robb L, Drinkwater CC, Metcalf D, Li R, Kontgen F, Nicola NA factor through a p38 mitogen-activated protein kinase-dependent et al. Hematopoietic and lung abnormalities in mice with a null pathway. Mol Cell Biol 2003; 23: 1896–1909. mutation of the common b subunit of the receptors for 26 Guthridge MA, Barry EF, Felquer FA, McClure BJ, Stomski FC, granulocyte–macrophage colony-stimulating factor and interleu- Ramshaw H et al. The phosphosrine-585-dependent pathway of kins 3 and 5. Proc Natl Acad Sci USA 1995; 92: 9565–9569. the GM-CSF/IL-3/IL-5 receptors mediates hemopoietic cell survival 44 Dirksen U, Nishinakamura R, Groneck P, Hattenhorst U, Nogee L, through activation of NF-kB and induction of bcl-2. Blood 2003, in Murray R et al. Human pulmonary alvoeolar proteinosis associated press. with a deficit in GM-CSF/IL-3/IL-55 receptor common beta chain 27 Karlsson R, Engstrom M, Jonsson M, Karlberg P, Pronk CJ, Richter expression. J Clin Invest 1997; 100: 2211–2217. A et al. Phosphatidylinositol 3-kinase is essential for Kit ligand- 45 Bonfield TL, Raychaudhuri B, Malur A, Abraham S, Trapnell BC, mediated survival, whereas interleukin-3 and Flt3 ligand induce Kavuru MS et al. PU.1 regulation of human alveolar macrophage expression of antiapoptotic Bcl-2 family genes. J Leukoc Biol 2003; differentiation requires granulocyte macrophage colony stimulat- 74: 923–931. ing factor (GM-CSF). Am J Physiol Lung Cell Mol Physiol 2003; 28 Fox CJ, Hammerman PS, Cinalli RM, Master SR, Chodosh LA, 285: L1132–L1136. Thompson CB. The serine/threonine kinase Pim-2 is a transcrip- 46 Dirksen U, Hattenhorst U, Schneider P, Schrotewn H, Gobel U, tionally regulated apoptotic inhibitor. Genes Dev 2003; 17: Bocking A et al. Defective expression of granulocyte–macrophage 1841–1854. colony-stimulating factor/interleukin-3/interleukin-5 receptor 29 White E. The pims and outs of survival signaling: role for the Pim-2 common b chain in children with acute myeloid leukemia protein kinase in the suppression of apoptosis by cytokines. Genes associated with respiratory failure. Blood 1998; 92: 1097–1103. Dev 2003; 17: 1813–1816. 47 Delwel R, Salem M, Pellens C, Dorssers L, Wagemaker G, Clarks S 30 Yu WM, Hawley TS, Hawlet RG, Qu CK. Catalytic-dependent and et al. Growth regulation of human acute myeloid leukemia: effects -independemt roles of SHP-2 tyrosin phosphatase in interleukin-3 of five recombinant hematopoietic factors in a serum-free cluture signaling. Oncogene 2003; 22: 5995–6004. system. Blood 1988; 72: 1944–1949. 31 Lee JT, McCubrey JA. The Raf/MEK/ERK signal transduction 48 Vellenga E, Ostapovicz D, O’Route B, Griffin JD. Effects of cascade as a target for chemotherapeutic intervention. Leukemia recombinant IL-3, GM-CSF, and G. CSF on proliferation of 2002; 16: 486–507. leukemic clonogenic cells in short-term and long-term cultures. 32 Chang F, Lee JT, Navolanic PM, Blalock WL, Franklin RA, Leukemia 1987; 8: 584–589. McCubrey JA. Involvment of PI3K/AKT pathway in cell cycle 49 Budel LM, Touw IP, Delwel R, Clark SC, Lowenberg B. Interleukin- progression, apoptosis and neoplastic transformation: a target for 3 and granulocyte–macrophage colony-stimulating factor recep- cancer chemotherapy. Leukemia 2003; 17: 590–603. tors on human acute myelocytic leukemia cells and relationship to 33 Chang F, Steelman LS, Lee JT, Shelton JG, Navolanic PM, Franklin the proliferative response. Blood 1989; 74: 565–571. RA et al. Signal transduction mediated by Ras/Raf/MEK/ERK 50 Budel LM, Elber O, Hoogerbrugger H, Delwel R, Mahmoud LA, pathway from cytokine receptors to transcription factors: potential Lowenberg B et al. Common binding structure for granulocyte– targeting for therapeutic intervention. Leukemia 2003; 17: 1263– macrophage colony stimulating factor and interleukin-3 on human 1293. acute myeloid leukemia cells and monocytes. Blood 1990; 75: 34 Shelton JG, Steelman LS, Lee JT, Knapp SL, Blalock WL, Moye PW 1439–1445. et al. Effects of the RAF/MEK/ERK and PI3K/Akt signal transduction 51 Sun Q, Woodcock JM, Rapoport A, Stumski FC, Korpelainen EI, Ba pathways on the abrogation of cytokine-dependence and preven- CJ et al. Monoclonal antibody 7G3 recognizes the N-terminal tion of apoptosis in hematopoietic cells. Oncogene 2003; 22: domain of the human interleukin-3 (IL-3) receptor alpha-chain and 2478–2492. functions as a specific IL-3 receptor antagonist. Blood 1996; 87: 35 D’Andrea R, Rayner J, Moretti P, LopezA, Goodall GJ, Gonda TJ 83–92. et al. A mutataion of the common receptor subunit for interleukin- 52 Testa U, Fossati C, Samoggia P, Masciulli R, Mariani G, Hassan HJ 3 (IL-3), granulocyte–macrophage colony-stimulating factor, and et al. Expression of growth factor receptors in unilineage IL-5 that leads to ligand independence and tumorigenicity. Blood differentiation culture of purified hematopoietic progenitors. Blood 1994; 83: 2802–2808. 1996; 88: 3391–3406. 36 Hannemann J, Hara T, Kawai M, Miyajima A, Ostertag W, 53 Militi S, Riccioni R, Parolini I, Sposi NM, Samoggia P, Pelosi E et al. Stocking C. Sequential mutations in the interleukin-3 (IL3)/ Expression of interleukin 3 and granulocyte–macrophage colony granulocyte macrophage colony-stimulating factor/IL5 receptor stimulating factor receptor common chain betaC, betaIT in normal b-subunit genes are necessary for the complete conversion to hematopoiesis: lineage specificity and proliferation-independent growth autonomy mediated by a trucated bc subunit. Mol Cell Biol induction. Br J Haematol 2000; 111: 441–451. 1995; 15: 2402–2412. 54 Munoz L, Nomdedeu JF, Lopez O, Carnicer MJ, Bellido M, Aventin 37 Mc Cormack MP, Gonda TJ. Expression of activated mutants of the A et al. Interleukin-3 receptor alpha chain (CD123) is widely human interleukin-3/interleukin-5/granulocyte–macrophage col- expressed in hematologic malignancies. Haematologica 2001; 86: ony-stimulating factor receptor common b subunit in primary 1261–1269. hematopoietic cells induces factor-independent proliferation and 55 Jordan CJ, Upchrch D, Szilvassy SJ, Guzman ML, Howard DS, differentiation. Blood 1997; 90: 1471–1481. Pettigrew AL et al. The interleukin-3 receptor alpha is a unique

Leukemia IL-3R in acute leukemia U Testa et al 226 marker for human acute myelogenous leukemia stem cells. kin-3 (IL-3), and IL-5 with increased mRNA expression in some Leukemia 2000; 14: 1777–1784. patients with acute leukemia. Blood 1998; 91: 54–63. 56 Guzman ML, Swiderski CF, Howard DS, Grimes BA, Rossi RM, 71 Jiang XJ, Lopez A, Holyoake T, Eaves A, Eaves C. Autocrine Szilvassy SJ et al. Preferential induction of apoptosis for primary production and action of IL-3 and granulocyte colony-stimulating human leukemic stem cells. Proc Natl Acad Sci USA 2002; 99: factor in chronic myeloid leukemia. Proc Natl Acad Sci USA 1999; 16220–16225. 96: 12804–12809. 57 Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, 72 Jiang XJ, Yip C, Eisterer W, Chaladon Y, Stuible M, Eaves C. Mauchauffe M et al. A TEL-JAK2 fusion protein with constitutive Primitive interleukin 3 null hemopoietic cells transduced with kinase activity in human leukemia. Science 1997; 278: BCR-ABL show accelerated loss after culture of factor indepen- 1309–1312. dence in vitro and leukemogenic activity in vivo. Blood 2002; 100: 58 Wheadon H, Welham MJ. The coupling of TEL/PDGFbetaR to 3731–3740. distinct functional responses is modulated in the presence of 73 Holyoake TL, Jiang X, Jorgensen HG, Graham S, Alcom MJ, Laid C cytokine: involvment of a mitogen-activated protein kinases. Blood et al. Primitive quiescent leukemic cells from patients with chronic 2003; 102: 1480–1489. myeloid leukemia spontaneously initiate factor-independent 59 Klejman A, Schreiner SJ, Nieberowska-Skorska M, Slupianek A, Wi growth in vitro in association with up-regulation of expression of M, Smithgall TE et al. The Src family kinase Hck couples BCR/ABL interleukin-3. Blood 2001; 97: 720–728. to STAT5 activation in myeloid leukemia cells. EMBO J 2002; 21: 74 Young DC, Griffin JD. Autocrine secretion of GM-CSF in acute 5766–5774. myeloblastic leukemia. Blood 1986; 68: 1178–1181. 60 Huang M, Dorsey JF, Epling-Burnette PK, Mimmanapalli R, 75 Ailles LE, Gurhard B, Hogge DE. Detection and characterization of Lamboski TH, Laughran TP et al. Inhibition of Bcr-Abl kinase primitive malignant and normal progenitors in patients with acute activity by PD0970 blocks constitutive activation of Stat5 and myelogenous leukemia using long-term culture with supportive growth of CML cells. Oncogene 2002; 21: 8804–8816. feeder layers and cytokines. Blood 1997; 90: 2555–2564. 61 Stirewalt DL, Radich JP. The role of FLT3 in haematopoietic 76 Nowak R, Oelsahlagel U, Gurth H, Range U, Albrecht S, Krebs U malignancies. Nat Rev Cancer 2003; 3: 650–665. et al. Relations between IL-3-induced proliferation and in vitro 62 Spiekermann K, Bagrintseva K, Scwab R, Scmieja K, Hiddemann cytokine secretion of bone marrow cells from AML patients. W. Overexpression and constitutive activation of FLT3 induces Cytokine 1999; 11: 435–442. STA5 activation in primary acute myeloid leukemia blast cells. 77 Guan Y, Gerhard B, Hogge DE. Detection, isolation, and Clin Cancer Res 2003; 9: 2140–2150. stimulation of quiescent primitive leukemic progenitor cells from 63 Taketani T, Taki T, Sugita K, Furuichi Y, Ishii E, Hanada R et al. patients with acute myeloid leucemia (AML). Blood 2003; 101: FLT3 mutataions in the activation loop of tyrosine kinase domain 3142–3149. are frequently found in infant acute lymphoblastic leukemia (ALL) 78 Zhang R, Levis M, Piloto O, Baldwin BR, Gorin NC, Beran M et al. with MLL rearrangements and pediatric ALL with hyperdiploidy. FLT3 ligand causes autocrine signaling in acute myeloid leukemia Blood 2003, in press. cells. Blood 2003, In press. 64 Murata K, Kumagai H, Kawashima T, Tamitsu K, Irie M, Nakajima 79 Chan CH, Blazar BR, Greenfield L, Kreitman RJ, Vallera DA. H et al. Selective cytotoxic mechanism of GTP-14564, a novel Reactivity of murine cytokine fusion toxin, diphteria toxin390- tyrosine kinase inhibitor in leukemia cells expressing a constitu- murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor tively active FLT3. J Biol Chem 2003; 278: 32892–32898. cells. Blood 1996; 88: 1445–1456. 65 Minami Y, Yamamoto K, Kiyoi H, Ueda R, Saito H, Naoe T. 80 Vallera DA, Seo SY, Panoskaltis-Mortari A, Griffin JD, Blazar BR. Different anti-apoptotic pathways between wild-type and mutated Targeting myeloid leukemia with a DT390-mIL-3 fusion immuno- FLT3: insights into therapeutic targets in leukemia. Blood 2003; toxin: ex vivo and in vivo studies in mice. Prot Eng 1999; 12: 102: 2969–2975. 779–785. 66 Mizuki M, Schwable J, Steur C, Choudhary C, Agrawal S, Sarin B 81 Frankel AE, McCubrey JA, Delatte S, Ramage J, Kiser M, Kucera GL et al. Suppression of myeloid transcription factors and induction of et al. Diphteria toxin fused to human interleukin-3 is toxic to blasts STAT response genes by AML-specific Flt3 mutations. Blood 2003; from patients with myeloid leukemia. Leukemia 2000; 14: 101: 3164–3173. 576–585. 67 Kelly L, Claek J, Gilliland DG. Comprehensive genotypic analysis 82 Feuring_buske M, Frankel AE, Alexander RL, Gerhard B, Hogge of leukemia: clinical and therapeutic implications. Curr Opin DE. A diphteria toxin-interleukin 3 fusion protein is cytotoxic to Oncol 2002; 14: 10–18. primitive acute myeloid leukemia progenitors, but spares normal 68 Levis S, Small D. IT Does matter in leukemia. Leukemia 2003; 17: progenitors. Cancer Res 2002; 62: 1730–1736. 1738–1752. 83 Black JH, McCubrey JA, Willingham MC, Ramage J, Hogge DE, 69 Phan V, Shultz DB, Truong BT, Blake TJ, Brown AL, Gonda TJ et al. Frankel AE. Diphteria toxin-interleukin-3 fusion protein (DT388IL3) Cooperation of cytokine signaling with chimeric transcription prolongs disease-free survival of leukemic immunocompromised factors in leukemogenesis: PML-retinoic acid receptor alpha mice. Leukemia 2003; 17: 155–159. blocks growth factor-mediated differentiation. Mol Cell Biol 84 Franker AE, Powell BL, Hall PD, Case D, Kreitman RJ. Phase I trial 2003; 23: 4573–4585. of a novel diphteria toxin/granulocyte macrophage colony- 70 Gale RE, Freebunn RW, Khwaya A, Chopra R, Linch DC. A stimulating factor fusion protein (DT388GMCSF) for refractory or truncated isoform of the human b chain common to the receptors relapsed acute myeloid leukemia. Clin Cancer Res 2002; 8: for granulocyte–macrophage colony-stimulating factor, interleu- 1004–1013.

Leukemia