expression changes associated with progression and response in chronic myeloid leukemia

Jerald P. Radich*†‡, Hongyue Dai§, Mao Mao§, Vivian Oehler*, Jan Schelter§, Brian Druker†¶, Charles Sawyersʈ, Neil Shahʈ, Wendy Stock**, Cheryl L. Willman†,††, Stephen Friend§, and Peter S. Linsley§

*Divisions of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; §Rosetta Inpharmatics, Seattle, WA 98109; ¶Oregon Health & Science University, University Center, Portland, OR 97239; ʈUniversity of California, Los Angeles, CA 90095; **University of Chicago School of Medicine, Chicago, IL 60637; ††University of New Mexico Cancer Research and Treatment Center, Albuquerque, NM 87131; and †Southwest Oncology Group, Ann Arbor, MI 48106

Communicated by E. Donnall Thomas, Fred Hutchinson Cancer Research Center, Seattle, WA, December 13, 2005 (received for review June 24, 2005) Chronic myeloid leukemia (CML) is a hematopoietic stem cell Imatinib Mesylate, which suppresses the Ph to the point where it is disease with distinct biological and clinical features. The biologic undetectable by cytogenetic evaluation (a complete cytogenetic basis of the stereotypical progression from chronic phase through remission or CCR) in Ͼ70% of newly diagnosed patients with accelerated phase to blast crisis is poorly understood. We used DNA chronic-phase CML disease (4). The long-term durability duration microarrays to compare in 91 cases of CML in of such responses is unknown, as is potential for cure with imatinib. chronic (42 cases), accelerated (17 cases), and blast phases (32 Complete molecular response, defined by the absence of the cases). Three thousand were found to be significantly (P < Bcr-Abl fusion mRNA by RT-PCR, is unusual in patients with CCR 10؊10) associated with phase of disease. A comparison of the gene (5), and patients who achieve a molecular response, but have signatures of chronic, accelerated, and blast phases suggest that imatinib discontinued, show a relatively rapid reemergence of the progression of chronic phase CML to advanced phase (accel- disease (6). Resistance to imatinib occurs (especially in advanced erated and blast crisis) CML is a two-step rather than a three-step phase disease) often accompanied by point mutations in the process, with new gene expression changes occurring early in tyrosine kinase domain of the abl gene (7). The natural history of accelerated phase before the accumulation of increased numbers such relapses is unknown, although some appear to progress rapidly of leukemia blast cells. Especially noteworthy and potentially into advanced stage disease (8). significant in the progression program were the deregulation of The genetic events that cause the progression of chronic phase to the WNT͞␤-catenin pathway, the decreased expression of Jun B blast crisis CML are largely unknown. Numerous genetic abnor- and Fos, alternative kinase deregulation, such as Arg (Abl2), and an malities have been demonstrated, including the acquisition of increased expression of PRAME. Studies of CML patients who additional chromosomal abnormalities including such as a dupli- Ј relapsed after initially successful treatment with imatinib demon- cation of the Ph , isochromosome 17(p) resulting in the disruption ͞ strated a gene expression pattern closely related to advanced of TP53, and less commonly, the deletion of the p15 p16 tumor phase disease. These studies point to specific gene pathways that suppressor genes (especially in lymphoid blast crisis) and the might be exploited for both prognostic indicators as well as new RUNX1-EV11 fusion (9, 10). Genetic instability is apparent, as targets for therapy. evidenced by the in the additional chromosomal changes that occur with progression, but standard assays of instability, such as alter- genetics ͉ blast crisis ͉ PRAME ͉ Wnt ations in minisatellite repeats, are detected infrequently (11, 12). Unfortunately, clinical and molecular tests cannot predict where on the ‘‘’’ of disease progression an individual lies at the time hronic myeloid leukemia (CML) is a hematopoietic stem cell of the diagnosis, and this makes it impossible to adapt therapy to the Cdisease with distinct biological and clinical features. CML degree of risk that faces an individual CML patient. We also cannot usually presents in chronic phase, in which the clonal expansion of yet identify the subset of patients who are most likely to benefit from mature myeloid cells leads to an elevated white blood cell count. a specific treatment option. This study is aimed at determining Without curative intervention, chronic-phase CML will invariably changes in gene expression that occur in the evolution of the chronic transform through a phase of ‘‘acceleration,’’ often heralded by the phase to blast crisis, with the hope of identifying genes and pathways appearance of increased immature myeloid cells in the bone that may be useful as prognostic markers or targets for therapeutics. marrow and peripheral blood, as well as new cytogenetic changes in addition to the Philadelphia (Ph). Progression then Results proceeds to blast crisis, with immature blast cells overwhelming the Phase-Specific Gene Expression in CML. We first examined the genes production of normal hematopoetic elements. Blast crisis is highly associated with the phase of disease. We used as a reference a pool resistant to treatment, with death generally occurring from infec- of 200 chronic phase bone marrows, and studied 91 individual cases tion and bleeding complications secondary to the absence of of CML in chronic phase (n ϭ 42), accelerated phase by blast count normal granulocytes and platelets. The median time from diagnosis criteria (n ϭ 9) or by the occurrence of additional clonal cytogenetic of chronic phase CML to progression to accelerated phase is Ϸ3–4 changes (n ϭ 8), blast crisis (n ϭ 28), and four cases of blast crisis years, but the range of timing is quite broad, encompassing from 0.5 in remission after chemotherapy. An ANOVA analysis revealed to 15 years (1). Ϸ3,500 genes (from a total of Ϸ24,000 genes) differentially ex- There are several treatment options for CML. All treatments are pressed across the different phases of disease by using a minimum more successful when administered during the chronic phase disease than in accelerated or blast phase. The only known curative therapy for CML is stem cell transplantation, a complex and Conflict of interest statement: No conflicts declared. potentially toxic modality that carries a high potential for morbidity Freely available online through the PNAS open access option. and mortality (2). Nontransplant therapy includes IFN-␣, which Abbreviations: CML, chronic myeloid leukemia; CCR, complete cytogenetic remission. produces a major reduction in the proportion of Ph-positive cells Data deposition: The sequence reported in this paper has been deposited in the Gene and extends the natural history of the disease in Ϸ10–20% of Ontology (GO) database (accession no. GSE4170). patients cases, with some alive and in remission for Ͼ10 years (3). ‡To whom correspondence should be addressed. E-mail: [email protected]. IFN-␣ has largely been replaced by the tyrosine kinase inhibitor, © 2006 by The National Academy of Sciences of the USA

2794–2799 ͉ PNAS ͉ February 21, 2006 ͉ vol. 103 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510423103 Downloaded by guest on September 30, 2021 Fig. 1. Genes associated with CML progression. Sam- ples from patients with of CML cases in chronic phase, accelerated by cytogenetic criteria only, accelerated phase, blast crisis, and blast crisis ‘‘in remission’’ were compared to a pool of chronic phase RNA (See Mate- rials and Methods for details). Approximately 3,500 genes were significantly associated with progressive disease at a significance level of P Ͻ 10Ϫ11. Each row represents one sample, and each column represents one gene. Red color indicates overexpression relative to the control pool, and green color indicates low expression.

statistical significance cutoff of P Ͻ 10Ϫ11 (Fig. 1 and Table 4, which drial genes, RNA-binding genes, and biosynthesis genes, is published as supporting information on the PNAS web site). This reflecting the increased proliferation and metabolism of progres- set of genes identified in the progression from chronic to blast phase sive disease. Advanced-phase CML, compared to chronic phase, is referred to here as the ‘‘phase reporter’’ gene set. exhibited a decreased expression of genes involved in structural We examined the proposed biologic function of the genes integrity and adhesion, as well as decreases in expression of genes associated with CML phases by applying a biological annotation involved in inflammatory and immune response. In addition, program based on the (GO) and KEGG annotations. several protooncogenes and tumor suppressor genes are differen- The major functional groups of genes in the phase reporter gene set tially expressed in advanced phase CML, including N- and H-ras, are shown in Table 5, which is published as supporting information FLT3, yes, AF1q, CBFB, WT1, ORALOV1, Bcl-2, and PTPN11. on the PNAS web site. The functional groups most highly correlated with disease phase (accelerated͞blast phase relative to chronic CML Appears to Be a ‘‘Two-Step’’ Disease. We examined whether the phase) included increased expression of nuclear genes, mitochon- progression of CML best fit a three-step model as often described MEDICAL SCIENCES

Fig. 2. Comparison of CML blasts to normal CD34ϩ cells and correction for the pattern of gene expression. (A) Phase genes were corrected for normal CD34ϩ gene expression (ANOVA, P Ͻ 1 ϫ 10Ϫ8). The gene expression of normal CD34ϩ cells was subtracted from each disease sample. The resulting pattern reflects genes associated with progression independent of normal blast biology. (B) Relative gene expression of the ‘‘top ten’’ genes found to be up-regulated in advanced phase disease compared to chronic phase.

Radich et al. PNAS ͉ February 21, 2006 ͉ vol. 103 ͉ no. 8 ͉ 2795 Downloaded by guest on September 30, 2021 in the literature (chronic phase 3 accelerated phase 3 blast crisis), Table 1. Functional annotation of ‘‘progression’’ genes or whether the expression data were best explained by a two-step Keyword Number (%)* Examples P value model (chronic phase 3 advanced phase; i.e., accelerated or blast phase). We compared the gene expression of accelerated phase Ribosome 18 (21) ROK13A 9 ϫ 10Ϫ11 Ϫ cases versus blast crisis cases (Fig. 4, which is published as support- Wnt signaling 16 (11) Cadherin, MDI1, 2 ϫ 10 5 ing information on the PNAS web site). The correlation of expres- Prickle 1, FZD2 22 (22) BSZ1A, HIST1H2AE 3 ϫ 10Ϫ11 sion level between accelerated and blast phase gene expression was ϫ Ϫ9 ϭ Sugar 45 (26) RP1A, ALDOC, 4 10 strong (r 0.81). In addition, the correlation of gene expression metabolism G6PD between accelerated phase and cases of accelerated phase defined Myeloid 27 (14) CEBPA, CEBPE, 3 ϫ 10Ϫ8 by new cytogenetic abnormalities alone (i.e., chronic phase mor- differentiation FOXO3A phology but additional chromosomal changes besides the Ph) was Apoptosis 42 (10) GADD45G, BCL2, 2 ϫ 10Ϫ7 also high (r ϭ 0.61). These observations suggest that the difference FOXO3A, MCL1 of gene expression between accelerated and blast phase is a DNA damage 36 (10) GASDD45G, 2 ϫ 10Ϫ7 quantitative change in expression rather a qualitative change. response FANCG, XRN2 Bold functions are up-regulated in progression; italic functions are down- Identification of ‘‘Progression-Specific’’ Gene Expression. We next regulated. investigated how the phase reporter gene set was influenced by the *The number of genes present on the array with the specific keyword; in gene expression signature of leukemia blasts and how these CML parentheses are the percentage of significant genes found differentially blasts compared to normal immature CD34ϩ cells. First, we made expressed among the keyword class. a direct comparison of the gene expression signature of six samples of normal CD34ϩ cells with CML blast samples containing Ͼ70% ␦ blasts (Fig. 5, which is published as supporting information on the MZF- and EF1 -Controlled Genes Are Aberrantly Expressed in Progres- PNAS web site). We found that the gene expression pattern of CML sion. Gene expression networks defined by specific promoter reg- blast cells was very similar to normal CD34ϩ cells. To uncover gene ulation were analyzed in the progression and phase reporter gene expression unique to CML progression (that is, associated with sets (Table 2). The most significantly network of genes showing phase of disease, but not normal CD34ϩ expression), we used two aberrant control in progression with those that contained a MZF ␦ Ͻ Ϫ15 Ͻ Ϫ11 approaches. First, we found genes differentially expressed between promoter or a EF1 promoter sequence (P 10 and 10 , CML blast crisis and normal immature CD34ϩ cells (Fig. 6, which respectively; Figs. 8 and 9, which are published as supporting is published as supporting information on the PNAS web site, 368 information on the PNAS web site). Also significantly associated genes with ANOVA P Ͻ 0.1%). Secondly, we mathematically with progression were genes bearing SPI-B, Yin Yang, and AHR- Ϫ subtracted the normal CD34ϩ signature from each of the CML ARNT promoter sequences (all with P values Ͻ10 9). sample and examined the resulting effect on the phase reporter signals (Fig. 2A, 386 genes with P Ͻ 10Ϫ8, and Table 6, which is Gene Expression Profiles of Cases Treated with Imatinib. We studied published as supporting information on the PNAS web site). These the gene expression of CML patients for whom imatinib therapy two approaches identified 103 overlapping genes found in both gene was ineffective. We examined 15 cases of CML treated with sets (Table 7, which is published as supporting information on the imatinib, including nine patients who initially achieved a CCR on PNAS web site, hypergeometric P value: 1.3 ϫ 10Ϫ99). Table 8, imatinib therapy, but then relapsed back into an apparent chronic which is published as supporting information on the PNAS web site phase by morphologic examination; three cases who had achieved (identified by the second approach), shows the ‘‘top ten’’ genes up- a complete hematologic but no cytogenetic response; and three late and down-regulated that are associated with progression, indepen- chronic phase patients. All but one of these the chronic phase dent of normal CD34ϩ expression, based on log 10 ratio of patients who relapsed after a CCR had a point mutation in abl1, expression compared to the chronic phase pool. These genes presumably abrogating imatinib activity (Table 3). In addition, only suggest a deregulation of genes of transcriptional regulation (GLI2, one of the relapsing cases had additional cytogenetic lesions. WT1, FOS, FOSB), signal transduction (SOCS2, Rras2, IL8), and Fig. 3 shows the expression pattern of these 15 CML cases. We apoptosis (GAS2). Also significantly associated with progression found that the cases that appeared in chronic phase after relapsing was PRAME (Preferentially Expressed Antigen of Melanoma), a after an achievement of CCR had expression patterns similar to gene with unknown function, but associated with myeloid malig- advanced disease. This can be demonstrated by segregating all nancy (13, 14). The expression levels of these genes are shown in CML cases by the correlation of gene expression signature between Fig. 2B. the boundaries of ‘‘most chronic’’ cases (bottom of the heat map) Gene functions of the progression gene set were organized by and ‘‘most advanced’’ gene expression (top of the heat map) for all GO and KEGG functional annotation (Table 1; for the full list, see 3,000 genes in the phase reporter gene set. The majority of the poor Tables 9–11, which are published as supporting information on the response patients have gene expression profiles more consistent PNAS web site). Of special relevance was aberrance in genes with advanced disease rather than chronic phase. Both cases with ͞␤ involved in the WNT -catenin signaling pathway, alterations in T315I mutations, which have been shown to have especially poor nucleosome genes, and genes involved in differentiation and apo- prognosis (8, 15), have expression signatures more similar to ␤ ptosis. In all, 16 genes associated with the -catenin pathway were advanced disease than chronic phase (red arrows). significantly deregulated in progression (e.g., cadherin, protocad- hedrin, PRICKLE1, CSNK1E, PLCB1, FZD2, LRP6, SMAD3, MDF1, etc.; see Fig. 7, which is published as supporting information Table 2. Promoter sequences differentially represented on the PNAS web site). in progression Progression appears to be associated with expression changes of P value in progression P value in several cytoskeletal and adhesion molecules. Thus, there is an Promoters genes phase genes increase in CD47, Crebl1, and ITGA5 expression, and a decrease in proto-cadhedrin, cadhedrin, actin, and ␤-actin. In addition, the MZF-1 Ͻ10Ϫ15 Ͻ10Ϫ15 abl family member abl2 (ARG) was significantly up-regulated in EF1␦ 9 ϫ 10Ϫ11 1 ϫ 10Ϫ9 Ϫ Ϫ disease progression; by contrast, abl1, which is both expressed from SPI-B 1 ϫ 10 10 2 ϫ 10 11 ϫ Ϫ9 ϫ Ϫ11 the normal chromosome 9 and the t(9, 22) translocation, was not Yin Yang 1 10 7 10 AHR-ARNT 2 ϫ 10Ϫ9 5 ϫ 10Ϫ10 differentially expressed with progression.

2796 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510423103 Radich et al. Downloaded by guest on September 30, 2021 Table 3. Characteristics of cases treated with imatinib Phase of disease at Phase of disease Code initiation of imatinib at time of sample Cytogenetics Mutation(s)

2 Chronic Relapsed chronic 100% Phϩ; NEW:t(1;12) in all cells M244V 4 Chronic Relapsed chronic 100% Phϩ T315I 5 Chronic Relapsed chronic 80% Phϩ F359V 6 Chronic Relapsed chronic 100% Phϩ M351T, F317L 8 Chronic Relapsed chronic 100% Phϩ M351T 10 Chronic Relapsed chronic 100% Phϩ E255K 11 Chronic post-allo BMT Relapsed chronic 100% Phϩ T315I 12 Chronic Relapsed chronic 100% Phϩ L248V 14 Chronic Relapsed chronic 100% Phϩ M351T, H396R

BMT, bone marrow transplantation.

However, there were gene expression features that were unique progression persists in a subpopulation of CML cells during to the imatinib relapsers. Examples of genes unique to imatinib therapy. resistance failure are those of serine threonine kinases (CTRL, In our analysis, we first used unsorted CML samples to develop MAP21K14, CLK3), mitogen-activated protein kinase (MKNK2), gene signatures associated with the phase of disease. This data set the tyrosine kinase oncogene FYN, and the drug exporter ABCC3. gives a more global picture of the transformation of disease, and is Fig. 10, which is published as supporting information on the PNAS important because any diagnostic assay for progression would web site, compares relapsed imatinib cases against blast crisis cases, optimally be performed on whole blood or marrow, rather than and several areas (boxed) indicate genes that are expressed in selected cell subpopulations. To control for any gene signatures that reverse direction in these two states (Tables 12 and 13, which are might be common to an immature blast phenotype, we subtracted published as supporting information on the PNAS web site, lists a normal CD34ϩ signature from the CML samples. This left us with genes in the first box. See Table 12 for the rest of the imatinib a progression gene set that should be highly enriched in genes resistance genes). associated with CML progression, but not reflecting normal CD34ϩ cell gene expression. This gene set may be more indicative of the Discussion biology of CML progression, and give more insight to pathways that The biology and treatment of CML is dictated by the phase of might be exploited by new therapeutic agents. Moreover, the disease, as the efficacy of all therapies (transplantation, IFN, or progression gene set may contain genes able to distinguish early imatinib) are most successful when used during the chronic evidence of progression in immunophenotypically similar immature phase, in comparison to accelerated phase and͞or blast crisis. cells. Understanding the biology of progression may provide clinical The demonstration that the gene expression pattern between diagnostic markers of progression and offer insights into new accelerated and blast phases are very similar suggests that the strategies for treatment. The data presented here suggest that crucial steps in progression are at the transition of chronic to the progression of chronic phase CML to advanced phase CML accelerated phase, before obvious morphologic, cytogenetic, or is a two-step process, with progression associated a block of clinical evidence of progression. This has clinical implications, differentiation and apoptosis, a shift toward turning on expres- because these patients might benefit from more aggressive therapy. sion of genes involved in the nucleosome, with alterations in cell In addition, the observation that gene signatures of blast crisis can adhesion, and activation of alternative signaling pathways. In be seen in accelerated phase patients by cytogenetic criteria only addition, it appears that relapse after initial successful treatment and blast crisis cases in remission (both of which have similarly low with imatinib may be associated with gene expression patterns blast counts as patients with chronic phase disease), demonstrates similar to advanced phase CML, suggesting that the process of two important points. First, it points out the difficulty of correlating MEDICAL SCIENCES Fig. 3. Gene expression in patients with resistance to Imatinib failure cases. Cases were ranked and sorted by the correlation of summed gene expression, with cases representing the most ‘‘chronic phase-like’’ and most ‘‘blast crisis-like’’ forming the boundaries of gene ex- pression patterns. Cases that had a poor response to imatinib are designated by a blue dot in the ‘‘IM-cases’’ box to the right of the heat map. The red arrows point to the two cases that had the T315I Abl mutation.

Radich et al. PNAS ͉ February 21, 2006 ͉ vol. 103 ͉ no. 8 ͉ 2797 Downloaded by guest on September 30, 2021 morphology with the biology of the disease. Secondly, the pene- study of the control of MZF1 and EF1␦ may be particularly fruitful tration of a progression gene expression signature into a ‘‘chronic in understanding the molecular mechanisms of CML progression. phase’’ appearing bone marrow suggests that progression is not Given that efficacy of treatment in CML (be it with IFN, merely an absolute block of differentiation, but that abnormal gene imatinib, or transplantation) is so intimately associated with phase expression signals persist in normal appearing differentiated cells. of disease, one wonders whether those chronic phase patients in This is critically important because it provides the basis for testing whom imatinib therapy is ineffective have genetic features of for progression genes in unsorted bone marrow samples from advanced phase invisible to routine pathological and cytogenetic ‘‘chronic phase’’ patients. examination. Imatinib failures are a reasonable setting to explore Bcr-Abl has a wholesale range of biological activities. Critical in this possibility. Although imatinib can cause cytogenetic remissions the transformation process is the activation of the Ras͞mitogen- in the majority of chronic phase cases, treatment failure, especially activated protein kinase (MAPK) pathways, which has broad secondary to point mutations, is an increasingly important problem. effects on changes in cell adhesion (through Rho), proliferation It has previously been demonstrated that the probability of devel- (MAPK pathway), and apoptosis (through Akt) (16, 17). Imatinib’s oping a point mutation depends largely on the time from diagnosis efficacy results from a blockade of these effects. Imatinib is less to initiation of therapy (8). This finding implies that the genetic successful for advanced-phase disease; this may be because these mechanisms that lead to point mutations are relentless, and there- leukemia have become less dependent on the pathways that ima- fore the treatment of ‘‘late’’ chronic phase patients (i.e., Ͼ 1 year tinib blocks. Thus, we found that the MAPK pathway was relatively from diagnosis) may be undermined by genetic changes that have underexpressed in advanced disease compared to chronic phase, already occurred. Branford et al. (8) demonstrated that patients but other signaling pathways, including those involving cytokines who developed P-loop point mutations had a very poor outcome, (IL3RA, SOCS2), alternative ras pathways (Rras2), and those with approximately half dying within a year of relapse. These involved in cell adhesion (WNT͞␤-catenin) are activated. The observations are in keeping with our demonstration that many activation of these pathways may allow progression even in the face imatinib failures have gene expression changes similar to advanced of therapeutic blockade of Bcr-Abl activated pathways. In addition, disease, despite their benign pathological appearance. The predic- abl2 (Abl related gene, or ARG) is also up-regulated in progression. tive power of genetic markers of response and progression will be As opposed to abl1, ARG is a cytoplasmic protein (18). The important to test prospective in future clinical studies of imatinib signaling targets of ARG are unknown, and although broadly and similar compounds. expressed in tissue, its only known functional role apparent from Several of the genes found in the progression set might serve as knockout mouse models appears to be in the nervous system (19, early markers of progression in diagnostic assays, and may serve as 20). ARG has been associated with myeloid leukemia in the context therapeutic targets, as well. For example, PRAME was originally of TEL͞ARG translocations (21, 22). ARG shares Ͼ90% homol- identified as a tumor antigen recognized by cytotoxic T cells against ogy of its tyrosine kinase domain with abl1, and ARG tyrosine a melanoma surface antigen (13, 39). Like similar antigens MAGE, kinase activity is inhibited by imatinib at similar drug concentra- BAGE, or GAGE, PRAME is expressed in some solid tumors; tions (18, 23). However, given that Bcr-Abl amplification is con- unlike these other antigens, however, it has been found to be sidered to play a role in imatinib resistance (24, 25), ARG over- overexpressed in Ͼ25% of leukemia, and has been found to be expression in blast crisis could theoretically contribute to the induced by Bcr-Abl in CML cell lines (14, 40). Indeed, PRAME relative resistance to imatinib found with progressive disease, either overexpression has been described as one of the few features that as independently acting on aberrant signaling, or acting as an characterize the transient myeloproliferative syndrome of Down’s imatinib ‘‘sink.’’ syndrome from the progressive acute megakaryoblastic leukemia Two recent observations on the molecular biology of progression found in that disorder (41). Very recently, insights into the function in CML are relevant to this study. First, activation of the WNT͞ of PRAME make it an attractive target for potential therapy. ␤-catenin pathway was observed in primary cell samples from PRAME is a nuclear protein, which seems to act as a dominant patients with CML (26). Secondly, it was recently observed that repressor of retinoic acid (RAR) signaling (42). In cell line mice deficient in Jun B develop a disease much like CML (27). Our models, the forced overexpression of PRAME blocked RAR- data complement these findings, as we found broad dysregulation mediated differentiation, growth arrest, and apoptosis. Thus, ther- of WNT͞␤-catenin pathway as well as decreased Jun B expression. apeutic maneuvers to block the PRAME͞RAR effect may allow A link between these pathways may be the gene MDFI (I-mfa), an for differentiation and cell death, and be particularly effective in inhibitor of myogenic basic helix–loop–helix transcription factors attacking advanced phase CML. (28). MDFI interacts with axin, which is involved in binding and In comparison to other types of leukemia, there have been few modulating free ␤-catenin, and thus changes in MDFI may influ- papers exploring the use of microarrays on the biology of CML ence ␤-catenin mediated gene activation (29). In addition, MDFI (43–45). It is difficult to make a direct comparison of these studies and axin interaction also influences WNT and Jun signaling (30). and ours, given the different types of samples obtained (some Our analysis suggests that genes controlled by MZF1 and EF1␦ unsorted, some AC133ϩ or CD34ϩ selected), the difference in the may be particularly important in progression. MZF1 is a member array platforms, etc. Nonetheless, in general, the functional changes of the Kruppel family of , and was originally of progression (changes in differentiation, apoptosis, cell adhesion, cloned from a cDNA library from a blast crisis CML patient (31). and inflammatory response) remained as common themes across MZF1 appears to play a critical role in hematopoietic stem cell these studies. Moreover, in keeping with a recent publication that differentiation, including modulation of CD34 and c- expres- found that the high expression of elastase (ELA2) was associated sion (32, 33). MZF1Ϫ/Ϫ knockout mice display an increase in with a long period of indolent disease (45), in our data, ELA2- hematopoietic progenitor proliferation, which continues in long- decreased expression is associated with progression. term culture conditions (32). These data suggest MZF1 deregula- These findings have the potential to influence therapy of tion may disrupt normal differentiation, promoting the progression CML. Patients who present with gene expression patterns sug- to advanced disease. EF1␦ is related to the Smad zinc finger gestive of advanced phase disease might benefit by moving to proteins that play an important role in TGF-␤ gene regulation. more aggressive (i.e., transplantation) or other investigational EF1␦ has been shown to compete with basic helix–loop–helix therapies. Moreover, PCR assays of individual genes (or small activators, and is implicated in modulation of MyoD regulated sets of genes) may be used to monitor patients early in the course pathways (34, 35). It is not known whether EF1␦ directly influences of imatinib therapy for signs of progression to advanced disease. MDFI expression. Of note is that both MZF1 and EF1␦ have been Lastly, new targets to interfere with progression, or reverse shown to influence cadherin expression (36–38). Thus, the further imatinib resistance, may be exploited.

2798 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510423103 Radich et al. Downloaded by guest on September 30, 2021 Materials and Methods or Molecular Function categories (www.ebi.ac.uk͞GOA), or to Patient Samples. All samples were obtained under the auspices of KEGG pathways (www.genome.jp͞kegg͞pathway.html). Gene lists institutional review board approve protocols. Samples came from (input sets) were queried for enrichment of members of specific the Fred Hutchinson Cancer Research Center, the Southwest functional classes or pathways relative to the background frequency. Oncology Group (SWOG) Myeloid Repository, the University of The significance (P value) of enrichment was computed by using the Oregon Health Sciences Center, the University of California, Los hypergeometric probability distribution. Reported in each case are Angeles, or the University of Chicago. RNA extraction was either the numbers of genes in the input set (input gene count), number performed immediately, or in the case of samples stored in a liquid of genes in a particular category or pathway in the input set (overlap nitrogen repository, after thawing. All RNA samples were quality gene count), and number of genes in a particular category among tested by analysis of ribosomal RNA peaks using an ABI Bioalyzer. all genes present on the array (set gene count). The total number The definition of chronic, accelerated and blast crisis was based on of unique genes on the array is 24,132. the criteria of Sokal et al. and the International Bone Marrow Transplant Registry (46, 47). Thus, chronic phase was defined as Methods to Common Promote Site Analysis. The common promoter Ͻ10% blasts, the accelerated phase was defined as 10–30% blasts sites were based on the predictions derived from the database or Ͻ10% blasts with clonal evolution, and blast crisis was defined (oPOSSUM) by Wasserman et al. (www.cisreg.ca). The hypergeo- as Ͼ30% blasts. metric P value for enrichment of a particular binding site was computed by comparing the number of genes with the binding site Amplification, Labeling, and Hybridization. The procedures of RNA from a signature gene set to that from a background set (i.e., all amplification, labeling, and the hybridization to arrays, as well as genes represented on the microarray). the specifics of the array platforms, has been published (48). Controls. We compared gene expression signatures from the sites Analytic Methods and Results. Each individual sample was hybrid- contributing samples to confirm that there were no site-signatures ized to a pool of chronic phase samples. The log10 (Ratio) of confounding the analysis. intensity of individual samples to the pool were used for the Because some samples of blast crisis came from peripheral blood subsequent analysis. Before selecting features by ANOVA, 25,000 rather than bone marrow, we compared three samples in which genes on the array were first screened for evidence of differential simultaneous samples were available from bone marrow and pe- regulation by requiring P value of regulation Ͻ1% in more than ripheral blood. Gene expression was extremely well correlated (r ϭ three experiments, where P value of regulation is based on the 0.97–0.99; Fig. 11, which is published as supporting information on platform error model. Features differentiating progression stages the PNAS web site). were selected by ANOVA test. This work was supported in part by National Institutes of Health Grants Functional Annotation of Gene Lists. Genes represented on the CA-18029 and CA-85053 (to J.P.R.) and the Howard Hughes Medical microarray were annotated by assignment to GO Biological Process Institute (B.D. and C.S.).

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