New insights into MLL gene rearranged acute leukemias using gene expression profiling: shared pathways, lineage commitment, and partner genes A Kohlmann 1 , C Schoch 1 , M Dugas 2 , S Schnittger 1 , W Hiddemann 1 , W Kern 1 and T Haferlach 1 1 Laboratory for Leukemia Diagnostics, Department of Internal Medicine III, Ludwig-Maximilians University, Munich, Germany; and 2 Department of Medical Informatics, Biometrics and Epidemiology, Ludwig-Maximilians University, Munich, Germany Rearrangements of the MLL gene occur in both acute lymphoblastic and acute myeloid leukemias (ALL, AML). This study addressed the global gene expression pattern of these two leukemia subtypes with respect to common deregulated pathways and lineage-associated differences. We analyzed 73 t(11q23)/MLL leukemias in comparison to 290 other acute leukemias and demonstrate that 11q23 leukemias combined are characterized by a common specific gene expression signature. Additionally, in unsupervised and supervised data analysis algorithms, ALL and AML cases with t(11q23) segre- gate according to the lineage they are derived from, that is, myeloid or lymphoid, respectively. This segregation can be explained by a highly differing transcriptional program. Through the use of novel biological network analyses, essential regulators of early B cell development, PAX5 and EBF, were shown to be associated with a clear B-lineage commitment in lymphoblastic t(11q23)/MLL leukemias. Also, the influence of the different MLL translocation partners on the transcriptional program was directly assessed. Interestingly, gene expression profiling did not reveal a clear distinct pattern associated with one of the analyzed partner genes. Taken together, the identified molecular expression pattern of MLL fusion gene samples and biological networks revealed new insights into the aberrant transcriptional program in 11q23/MLL leukemias. Leukemia (2005) 19, 953–964. doi:10.1038/sj.leu.2403746 Published online 7 April 2005 Keywords: acute leukemia; gene expression; microarray; MLL; t(11q23) Introduction The MLL gene (also termed ALL-1, HRX, and TRX1) located at chromosome band 11q23 is a recurrent target of chromosomal translocations in acute leukemias, particularly prevalent in infant leukemias and treatment-related secondary leukemias, and associated with dismal prognosis. 1–3 Reciprocal transloca- tions associated with the MLL gene result in in-frame fusion transcripts with various partner genes from at least 50 distinct gene loci. 4 In addition, a partial tandem duplication of the MLL gene has been reported. 5 The class of oncogenic MLL fusion proteins consists of the N-terminal portion of the MLL protein fused to C-terminal portions of a fusion partner. Experimental systems in which MLL fusion proteins were generated to induce leukemia in mice demonstrated that this fusion to a C-terminal partner is necessary for immortalization. Two critical regions within MLL were identified: a region with three AT hook DNA-binding motifs and the DNA methyltransferase homology region. 6 The MLL fusion partners act via dominant gain of function and seem to play a role in two main functional categories, namely signaling molecules that normally localize to the cytoplasm/cell junctions or nuclear factors implicated in regulatory processes of transcription. 7 With respect to the oncogenic activation of MLL in leukemia So and Cleary proposed two mechanisms. One subset of fusion partners already displays the required transcriptional activation potential required for leukemogenesis. The other subset acts via their homodimerization or oligodimerization domains and therefore can lead in a dimerization-dependent pathway to deregulated transcription. 8 Interestingly, distinct MLL fusion partners suggest a possible role in the tropism of the leukemia. Certain partner proteins not only convert MLL to an oncogenic fusion protein but also direct the lineage susceptibility for transformation. MLL-AF4 expres- sing leukemias are mainly diagnosed as pro B ALL (acute lymphoblastic leukemia), whereas, for example, fusion partners AF9, AF6, or AF10 are common in myelomonocytic or monoblastic AML (acute myeloid leukemia) subtypes. 9 Microarrays simultaneously assess the abundance of thou- sands of mRNA transcripts. 10 During the past few years powerful algorithms have been developed and adapted to mine micro- array data. 11 More recently also applications to interprete gene expression signatures in terms of pathways and networks have evolved. In this study, from a series of 363 acute leukemia patient samples hybridized to a set of high-density microarrays representing a near complete human genome, analyses were performed to (i) identify t(11q23)/MLL gene signatures compared to numerous specific subtypes of acute leukemias, (ii) discrimi- nate t(11q23)/MLL-positive AML from t(11q23)/MLL ALL sam- ples, (iii) investigate signatures correlated with MLL-AF9 and other MLL partner genes, and (iv) decipher common biological networks. Specifically we addressed the question how the differing MLL partner genes influence the global gene expression signature and whether pathways could be identified to explain the molecular determination of MLL leukemias occurring in both the myeloid and lymphoid lineages. Materials and methods Patient samples This study included bone marrow samples from 363 adult acute leukemia patients at diagnosis representing distinct precursor B-ALL subtypes t(11q23)/MLL, t(8;14), t(9;22) and precursor T-ALL as well as AML subtypes with t(11q23)/MLL, t(8;21), t(15;17), inv(16), or complex aberrant karyotype (Supplementary Table 1). In AML with t(11q23)/MLL, 15 out of 48 cases were therapy-related. Seven out of these 15 cases followed exposure to topoisomerase II inhibitors. All samples were sent between December 1998 and February 2004 for reference diagnostics to our laboratory and were registered in our leukemia database. 12 Received 25 October 2004; accepted 26 January 2005; Published online 7 April 2005 Correspondence: A Kohlmann, Laboratory for Leukemia Diagnostics, Department of Internal Medicine III, Ludwig-Maximilians University, Marchioninistr. 15, 81377 Munich, Germany; Fax: þ 49 89 7095 4971; E-mail: alexander.kohlmann@med.uni-muenchen.de Leukemia (2005) 19, 953–964 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu