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[Cancer Research 65, 7151-7158, August 15, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

OLIG2 (BHLHB1), a bHLH Transcription Factor, Contributes to Leukemogenesis in Concert with LMO1

Ying-Wei Lin1, Ramona Deveney1, Mary Barbara2, Norman N. Iscove2, Stephen D. Nimer3, Christopher Slape1 and Peter D. Aplan1

1 Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland; 2 Department of Medical Biophysics, University of Toronto, Ontario, Canada; and 3 Department of Medicine, Hematology Service, Division of Hematologic Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York

Requests for reprints: Peter D. Aplan, Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, 8901 Wisconsin Avenue, Bethesda, MD 20889-5105. Phone: 301-435-5005; Fax: 301-496-0047; E-mail: aplanp{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
OLIG2 (originally designated BHLHB1) encodes a transcription factor that contains the basic helix-loop-helix motif. Although expression of OLIG2 is normally restricted to neural tissues, overexpression of OLIG2 has been shown in patients with precursor T-cell lymphoblastic lymphoma/leukemia (pre-T LBL). In the current study, we found that overexpression of OLIG2 was not only found in oligodendroglioma samples and normal neural tissue but also in a wide spectrum of malignant cell lines including leukemia, non–small cell lung carcinoma, melanoma, and breast cancer cell lines. To investigate whether enforced expression of OLIG2 is oncogenic, we generated transgenic mice that overexpressed OLIG2 in the thymus. Ectopic OLIG2 expression in the thymus was only weakly oncogenic as only 2 of 85 mice developed pre-T LBL. However, almost 60% of transgenic mice that overexpressed both OLIG2 and LMO1 developed pre-T LBL with large thymic tumor masses. Gene expression profiling of thymic tumors that developed in OLIG2/LMO1 mice revealed up-regulation of Notch1 as well as Deltex1 (Dtx1) and pre T-cell antigen receptor {alpha} (Ptcra), two genes that are considered to be downstream of Notch1. Of note, we found mutations in the Notch1 heterodimerization or proline-, glutamic acid-, serine-, and threonine-rich domain in three of six primary thymic tumors. In addition, growth of leukemic cell lines established from OLIG2/LMO1 transgenic mice was suppressed by a {gamma}-secretase inhibitor, suggesting that Notch1 up-regulation is important for the proliferation of OLIG2-LMO1 leukemic cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The OLIG2 gene, originally designated BHLHB1 (for basic helix-loop-helix class B, 1), is located at 21q22 and encodes a transcription factor that contains the conserved bHLH motif (1–3). Several studies have suggested that OLIG2 expression is normally restricted to neural tissues, especially those of the mature oligodendrocyte lineage as well as embryonic oligodendrocyte precursors (4). Targeted deletion ("knockout") and overexpression studies have shown that OLIG2 and the closely related OLIG1 normally play an important role in oligodendrocyte differentiation and specification (5–9). More recently, it has been shown that human OLIG genes, including OLIG1 (BHLHB2) and OLIG2 (BHLHB1), are overexpressed in oligodendroglioma, in contrast to the low level or absent expression seen in astrocytoma samples (10, 11). This is an important clinical finding because there has been a lack of molecular markers that histologically clearly distinguish oligodendroglioma from astrocytoma.

The human OLIG2 gene was originally identified by virtue of its activation by chromosomal translocation in a precursor T-cell lymphoblastic lymphoma/leukemia (pre-T LBL) patient with a t(14; 21)(q11.2; q22) chromosomal translocation present in the leukemic cells (3). In this case, OLIG2 was overexpressed due to relocation of the TCRA C{alpha} enhancer upstream of the OLIG2 locus on the translocated chromosome. Subsequently, overexpression of OLIG2 has been detected in additional pre-T LBL patients without known t(14;21) translocations (12, 13). Because there are no reports investigating a direct role for overexpression of OLIG2 in malignant transformation, we began a series of studies designed to determine whether overexpression of OLIG2 contributes to oncogenesis, especially leukemogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Northern blot and reverse transcription-PCR analysis. Expression of OLIG2 in human malignant cell lines and human normal tissues was determined by both Northern blot analysis and reverse transcription-PCR (RT-PCR). Total RNA and poly(A) RNA were isolated from cell pellets using Trizol reagent (Invitrogen, Carlsbad, CA) or Poly(A) Pure RNA extraction kit (Ambion, Austin, TX). Ten micrograms of total RNA or one microgram of poly(A) RNA was size fractionated on a 1% agarose/formaldehyde gel, transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and hybridized to a 32P-labeled human OLIG2 probe. For RT-PCR experiments, after contaminating genomic DNA was removed from total RNA, 1 µg of total RNA was reverse transcribed with Superscript II reverse transcriptase and an oligo (dT) primer (Invitrogen). Parallel reactions were done without the addition of Superscript II reverse transcriptase as controls. First strand cDNAs were amplified with human OLIG2 (form A, see text) primers (21PCR01: 5'-CCCTGAGGCTTTTCGGAGCG-3' and 21PCR02: 5'-GCGGCTGTTGATCTTAGACGC-3'), human OLIG2 form B primers (BB312249F: 5'-GTGGGGACTTTGTGCCTGGGCATCG-3' and 21PCR01R: 5'-CGCTCCGAAAAGCCTCAGGG-3'), or human ß-actin primers (hActin-F1: 5'-AGGCCGGCTTCGCGGGCGAC-3' and hActin-R1: 5'-CTCGGGAGCCACACGCAGCTC-3') in a volume of 20 µL. After a "hot start" at 94°C for 3 minutes, 35 cycles of 94°C for 1 minutes, 64°C for 45 seconds, and 72°C for 1 minutes were used, followed by a terminal 10-minute extension at 72°C. To amplify LMO1, first strand cDNAs were amplified with human LMO1 primers (hLMO1Fw: 5'-GCGAAGCAGTCGAGGTGATA-3' and hLMO1Rv: 5'-AAGTGTGCGTGCTGTGACTG-3') in A volume of 20 µL. After a hot start at 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds were used, followed by a terminal 10-minute extension at 72°C. PCR products were analyzed by agarose gel electrophoresis.

Expression of OLIG2 in normal murine hematopoietic cells and tissues. Expression of OLIG2 in normal murine hematopoietic precursors was determined by dot blot analysis. Thirty-seven globally amplified cDNA samples from a wide spectrum of normal murine hematopoietic colonies (14, 15) and normal murine tissues (16) were deposited, 100 ng/spot, on Hybond N+ membranes (17). The sample set comprised #1: pentapotent erythroid/megakaryocytic/macrophage/neutrophil/mast cell; #2: tetrapotent erythroid/megakaryocytic/macrophage/neutrophil; #3: erythroid/megakaryocytic/macrophage; #4: erythroid/megakaryocytic; #5: macrophage/neutrophil; #6: blast-forming unit-erythroid; #7: colony-forming unit-erythroid; #8: precursor macrophage; #9: precursor neutrophil; #10: precursor megakaryocytic; #11: erythroid; #12: neutrophil; #13: mast cell; #14: megakaryocytic; #15: B-cell; #16: macrophage; #17: T-cell; #18-21: fibroblasts; #22: thymus; #23: aorta; #24: kidney; #25: testis; #26: muscle; #27: lung; #28: brain; #29: skin; #30: lymph nodes; #31: bone marrow; #32: heart; #33: ovary; #34: spleen; and #35-37: purified hematopoietic stem cells (18). The membrane was hybridized with 32P-labeled mouse OLIG2 and OLIG1 probes both containing sequence within 300 bases of the 3' transcript termini. The mouse OLIG2 probe was a 0.3 kb PCR fragment that was amplified using mouse OLIG2 primers (mBB1-F1: 5'-GGTGATAGATACACTCTGCAGG-3' and mBB1-R1: 5'-GTTGCTGTGAATATAGTTTGAGC-3'). The mouse OLIG1 probe was a 0.6 kb fragment isolated from an EST clone (AW494459) with NotI and EcoRI.

Generation of OLIG2 transgenic mice and OLIG2-LMO1 double transgenic mice. An OLIG2 cDNA containing the A isoform (see text) was digested with BamHI and cloned into the BamHI site of the pLIT2 vector that has the lck upstream enhancer, immunoglobulin H enhancer, and TCRVß promoter (19). All cloning junctions were sequenced to verify the construct. The construct was microinjected into zygotes obtained from C57Bl6 mice. Lines were maintained by mating with wild-type C57Bl6. OLIG2 transgenic mice were subsequently crossed with LMO1 transgenic mice that were obtained from Dr. Korsmeyer (20). The LMO1 transgenic mice were originally generated on F1 of a cross between C57Bl6 and C3H mice, and were backcrossed with C57Bl6 for five to six generations. The genotype of both the OLIG2 transgene and the LMO1 transgene was determined by PCR.

Immunohistochemistry and immunophenotyping. H&E, periodic acid Schiff, CD3 (DAKO, Carpinteria, CA), B220 (CD45R, PharMingen, San Diego, CA), anti-myeloperoxidase (DAKO), and F4/80 (CALTAG, Burlingame, CA) stainings of sections from tissues such as thymus, lymph nodes, spleen, liver, kidney, lung, and tibia were evaluated using conventional staining techniques. Bone marrow cells were harvested from both femur and tibia by flushing with Iscove's modified Dulbecco's medium (IMDM), and assessed by May-Grünwald-Giemsa stained cytospin. Two-color flow cytometry was used to determine the immunophenotype of single-cell suspension prepared from thymus, spleen, and bone marrow. The cells were stained with FITC-conjugated anti-mouse CD8 and phycoerythrin-conjugated anti-mouse CD4 (PharMingen). Diseases were classified according to the Bethesda proposals (21, 22).

Southern blot analysis for TCRB gene rearrangements. Genomic DNA was isolated as previously described (23) and digested with either HindIII or SstI. Digested DNA was size fractionated on a 0.8% agarose gel, denatured, neutralized, and transferred to a nitrocellulose membrane. The nitrocellulose membrane was hybridized to a 32P-labeled 0.4 kb TCRB probe that detects the constant region of the mouse TCRB gene.

Establishment of leukemic cell lines. Bone marrow cells were cultured in IMDM supplemented with 10% fetal bovine serum (FBS). Half of the media was replaced with fresh media once a week until adherent cells covered entire bottom of culture flasks. After that time, 70% of the media was replaced once a week until suspension cells appeared. Suspension cells were then transferred to RPMI 1640 with 10% FBS.

Gene profiling of tumors. RNA was isolated from thymic tumor and compared with pooled RNA obtained from the thymi of 21 healthy nontransgenic littermates. RNAs were isolated using the Trizol reagent and purified using the Qiagen (Valencia, CA) RNeasy mini kit. First strand cDNA was synthesized and dye coupled using a FairPlay Microarray labeling kit (Stratagene, La Jolla, CA). The experimental cDNA probe (thymic tumor) was labeled with Cy3 and the reference cDNA probe (normal thymus) was labeled with Cy5. Purification of the dye-coupled cDNA was done using a Qiagen Mini Elute PCR purification kit. The Cy3-labeled experimental probe was combined with the Cy5-labeled reference probe and the mixture was hybridized to a National Cancer Institute (NCI) production oligo DNA microarray containing 22,272 long oligo (70-mer) features (Compugen, San Jose, CA). The microarray was scanned using an Axon (Union City, CA) GenePix scanner. The fluorescence ratio was quantified for each transcript and reflected the relative abundance of the transcript in experimental mRNA sample compared with the reference mRNA.

Cell growth assay. Cell lines #1928 and #1931 were established from bone marrow of OLIG2-LMO1 double transgenic mice with pre-T LBL and maintained in RPMI 1640 supplemented with 10% FBS. Cell line #1901 was established from bone marrow of a NUP98-HOXD13 (NHD13) transgenic mouse with pre-T LBL and maintained in IMDM media supplemented with 10% FBS (24). F4-6 is a Friend virus–induced erythroleukemia cell line (25) cultured in IMDM media with 10% FBS. Cell lines seeded at a concentration of 2 x 105 cells/mL were treated with 10 µmol/L of Z-IL-CHO ({gamma}-secretase inhibitor XII, Calbiochem, La Jolla, CA) for 48 hours (26). Viable cell counts were done by trypan blue exclusion. Expression of Notch1 was determined by RT-PCR, as previously described (27). To determine whether G1 arrest and apoptosis were induced by Z-IL-CHO, the cells were stained with propidium iodide (Molecular Probes, Eugene, OR) and DNA content was determined by flow cytometry.

Sequence analysis of Notch1 heterodimerization and proline-, glutamic acid-, serine-, and threonine-rich domains. The heterodimerization (HD) and proline-, glutamic acid-, serine-, and threonine-rich (PEST) domains of Notch1 were sequenced to determine whether there is a mutation in these domains. The primers used to amplify exons 26 and 27 of the HD domain were i25F1 (5'-GGCTGAGTTTCTTTAGAGTC-3') and i26R1 (5'-CCTCCCCTGAGGTTACACCT-3'), and i26F1 (5'-GAGTGTCCCATTGCGGGGCT-3') and i27R1 (5'-TGCAGAGGTCAGAAAGTGTT-3'), respectively. To amplify the PEST domain, the primers PEST1 (5'-TACCAGGGCCTGCCCAACAC-3') and PEST2 (5'-GCCTCTGGAATGTGGGTGAT-3') were used. The PCR was done using the same protocol as the RT-PCR (27). Subsequently, the PCR products were isolated from agarose gels, and the sequence was compared with that of the wild-type murine Notch1 genomic sequence (GenBank accession no. AL732541).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expressions of OLIG genes in normal hematopoietic tissues, leukemic cell lines, and other malignant cell lines. Although expression of OLIG2 has been thought to be restricted to neural tissues, we previously found overexpression of OLIG2 in a patient with pre-T LBL (1–3). To determine whether other leukemic cells expressed OLIG2, we examined OLIG2 expression in five leukemic cell lines (HEL, HSB-2, CEM, Jurkat, and HL60) and two glioblastoma cell lines (A172 and U87) by Northern blot analysis. Strikingly, two of those leukemic cell lines (HEL and HL60) expressed higher level of OLIG2 mRNA than the glioblastoma cell lines (A172 and U87) used as positive controls. Furthermore, HL60 also expressed OLIG1, which was not detected in glioblastoma cell lines (Fig. 1A).



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  		Figure 1. OLIG1 and OLIG2 expression in leukemic cell lines and normal hematopoietic cells. A, Northern blot analysis of OLIG1 and OLIG2 expression in leukemic cell lines and glioblastoma cell lines (A172 and U87). B, dot blot analysis of OLIG1 and OLIG2 expression in normal murine hematopoietic precursors. The membrane contains cDNA from 37 distinct normal hematopoietic colonies and tissues (see Materials and Methods). Dot #1 denotes the position of the first dot on the top left corner. There was only a single hybridization signal detectable in each membrane, which was cDNA from whole brain at position #28. Hybridization intensities on the hematopoietic cell samples (#1-17) did not exceed the background intensity on the negative control spot at position #18 (PCR primer concatamers amplified in the absence of cellular RNA template).

 
We considered the possibility that OLIG2 is normally expressed in a subset of normal hematopoietic cells. To investigate this possibility, we determined expression of OLIG1 and OLIG2 using a dot blot analysis of amplified cDNA from a wide spectrum of mouse hematopoietic cells (14, 15), as well as other tissues including thymus, aorta, kidney, testis, muscle, lung, brain, skin, lymph nodes, bone marrow, heart, ovary, and spleen (16). Significant hybridization of the OLIG1 and OLIG2 probes was detected on brain cDNA but not on any of the normal hematopoietic cell samples (Fig. 1B). Because OLIG2 was not expressed in normal hematopoietic cells, and previously was found to be overexpressed in the leukemic cells from patients with pre-T LBL, we considered the possibility that OLIG2 might be expressed only in malignant hematopoietic cells. We expanded the survey to 41 leukemic cell lines using RT-PCR. The survey revealed that OLIG2 was strongly expressed in HL60 (promyelocytic), KBM5, THP-1 (erythroleukemic), HI-MEG (megakaryocytic), and MEG-01 (megakaryocytic), and weakly expressed in MO-91 (undifferentiated), HEL, LAMA84, KMOE-1, TF-1 (erythroleukemic), and CHRF-288 (megakaryocytic; Table 1; Supplementary Fig. S1). To determine whether ectopic OLIG2 expression was restricted to hematopoietic malignancies, we determined OLIG2 expression in the "NCI-60" panel of malignant cell lines (28, 29). Thirteen of the 60 cell lines in this panel expressed OLIG2: three central nervous system (CNS) tumor cell lines (SF-295, SNB-19, and U251) and 10 non-CNS malignant cell lines. These 10 cell lines included five melanoma cell lines (UACC-257, SK-MEL2, SK-MEL28, M14, and SK-MEL5), three non–small cell lung carcinoma cell lines (NCI-H522, HOP-62, and NCI-H23), one breast cancer cell line (MDA-MB-435), and the HL60 leukemic cell line. Thus, OLIG2 expression was detected not only in normal and malignant neural tissues but also in malignant cell lines derived from skin, lung, breast, and hematopoietic tissues (Table 1; Supplementary Fig. S2).


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  		Table 1. OLIG2 expression in leukemic/lymphoma and NCI-60 cell line panel

 
OLIG2 transgenic mice. To investigate the oncogenic potential of misexpressed OLIG2 in vivo, we generated transgenic mice that overexpressed OLIG2 in the thymus using an lck promoter (19). Because the original description of human OLIG2, as well as the mouse and rat homologues, did not identify the OLIG2 initiation codon (1–3), we used 5' rapid amplification of cDNA ends to identify the initiation codon. We identified two alternative upstream exons, designated 1B and 1A. We then used RT-PCR to determine which 5' exon was most commonly used. Although we detected exon 1A spliced to exon 2 (Form A) in a wide spectrum of samples, including normal brain and the aforementioned malignant cell lines (see Fig. 2 and Supplementary Figs. S1 and S2), we detected exon 1B spliced to the 3' portion of exon 1A and subsequently exon 2 (form B) only in the HEL cell line. Therefore, we used an OLIG2 form A cDNA to generate the transgenic mice.



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  		Figure 2. Generation of OLIG2 transgenic mice. A, OLIG2 splice forms A and B are generated by alternate splicing as indicated. B, splice form A of the OLIG2 cDNA was cloned into the BamHI site of the pLIT2 vector (19). C, expression of the transgene was determined by Northern blot analysis. Top, hybridization of OLIG2; bottom, EtBr staining of the gel as a loading control. A clear OLIG2 signal is seen in thymus and a faint signal in bone marrow (BM).

 
Transgenic mice were generated on a C57Bl6 background by pronuclear injection and three independent founders (B1, D5, and K4), all male, were identified. High levels of OLIG2 mRNA were detected in the thymus, and lower levels in the bone marrow (Fig. 2C) in RNA from the offspring of mouse D5; similar levels were detected in F1 mice from the other two founders (data not shown). We followed a cohort of 69 transgenic and 76 nontransgenic control littermates from these three lines for 24 months; none of the transgenic mice developed a malignant tumor.

OLIG2-LMO1 double transgenic mice. We considered the possibility that OLIG2 might not be sufficient for leukemia and might require collaborative events to induce leukemia. For instance, the SCL (Tal1) gene, which encodes a transcription factor bearing the bHLH motif, was not sufficient to induce leukemia in mice when SCL was misexpressed under control of the SIL promoter (30), but was able to induce leukemia in concert with overexpression of LMO1. SCL-LMO1 double transgenic mice developed aggressive pre-T LBL within 6 months with nearly 100% penetrance (23, 30). Because OLIG2, like SCL, encodes a class B bHLH transcription factor, we crossed the OLIG2 transgenic mice to LMO1 transgenic mice to investigate whether OLIG2 overexpression might collaborate with LMO1 overexpression to induce leukemia. lck-OLIG2 (line D5) mice were mated to lck-LMO1 mice, and the offspring were observed for signs of leukemia. Fifty-seven percent (8 of 14) of OLIG2-LMO1 double transgenic mice died by the age of 14 months, whereas all of the wild-type control littermates survived. In addition, 8% (1 of 13) of LMO1 only transgenic mice and 12% (2 of 16) of OLIG2 only transgenic mice developed signs of pre-T LBL during the 14-month study period (Fig. 3A). Necropsy findings from these mice revealed an aggressive pre-T LBL. All OLIG2-LMO1 double transgenic mice with pre-T LBL had markedly enlarged thymic tumors and pleural effusion (Fig. 3B1). Additional gross findings included hepatosplenomegaly and localized (inguinal) or generalized lymphadenopathy (Fig. 3B2). Histologic examination revealed a perivascular infiltration of CD3-positive lymphoblasts in the lung, as well as interstitial and perivascular infiltration of CD3-positive lymphoblasts in the kidney and liver, respectively (Fig. 3B3-8). Examination of peripheral blood and bone marrow revealed lymphoblasts characterized by a high nuclear-cytoplasmic ratio and variably condensed chromatin (Fig. 3B9-10). The malignant blasts were positive for CD3, CD4, and CD8 (Fig. 3C1). Clonal rearrangements of the TCRB gene were identified by Southern blot analysis of tumors from OLIG2-LMO1 double transgenic mice (Fig. 3C2). These findings were consistent with a diagnosis of pre-T LBL. Moreover, pre-T LBL cell lines were established from the bone marrow of three independent OLIG2-LMO1 double transgenic mice and have been maintained in RPMI 1640 supplemented with 10% FBS for longer than 14 months. These cell lines continue to express OLIG2 as well as LMO1, similar to findings seen with the primary thymic tumors (Fig. 4A).



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  		Figure 3. Pre-T LBL in transgenic mice. A, survival curve for offspring of OLIG2 and LMO1 transgenic mice. Survival differences from wild-type control littermates are shown using Student's t test. B1, typical large thymic tumor (arrow) from an OLIG2/LMO1 transgenic mouse (mouse #1939). B2, hepatomegaly (arrow), splenomegaly (arrow with dashed line), and lymphadenopathy (arrowhead) were also common features (mouse #1939). B3-4, lung infiltration with CD3-positive blasts (mouse #1928). B5-6, kidney infiltration with CD3-positive blasts (mouse #259). B7-8, liver infiltration with CD3-positive blasts (mouse #1939). B9-10, peripheral blood (B9) and bone marrow (B10) infiltrated with lymphoblasts characterized by high-nuclear cytoplasmic ratio and variably condensed chromatin (mouse #1939). B11, pre-T LBL in OLIG2 only transgenic mice characterized by massively enlarged thymus (arrowhead; mouse #556). C1, immunophenotype of leukemic blasts from bone marrow displaying immature CD4+CD8+ (mouse #1928 and #1931). C2, Southern blot analysis of DNA from thymic tumors show clonal TCRB gene rearrangements in OLIG2-LMO1 double transgenic (#259 and #1939) and OLIG2 only transgenic (#556) mice. Arrows, rearranged bands.

 


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  		Figure 4. Growth suppression of OLIG2/LMO1 cell lines overexpressing Notch1 by {gamma}-secretase inhibitor. A, expression of the OLIG2 and LMO1 transgenes was determined by RT-PCR. Primary thymic tumors and pre-T LBL cell lines established from bone marrow (BMCL) continued to express the expected transgenes; mice #259, #1928, and #1931 were double transgenic, #556 was OLIG2 only, and #255 was an LMO1 only transgenic mouse. B, the indicated cell lines were cultured in the presence of 10 µmol/L Z-IL-CHO ({gamma}-secretase inhibitor) for 48 hours. Viable cells were identified by trypan blue exclusion. Expression of Notch1, OLIG2, and LMO1 was determined by RT-PCR. Growth of the OLIG2-LMO1 double transgenic cells (#1928 and #1931) was suppressed ~100-fold, whereas growth of the control cell lines (#1901 and F4-6) was not. C, mutation analysis in primary thymic tumors from double transgenic mice. The nucleotide numbers refer to mouse Notch1 genomic sequence (GenBank accession no. AL732541). HD, heterodimerization domain; Nm, no mutation.

 
The pre-T LBL that developed in OLIG2 or LMO1 only transgenic mice was similar to that seen in the double transgenic mice. Bone marrow and peripheral blood were heavily invaded by malignant lymphoblasts, and the mice had markedly enlarged thymi (Fig. 3B11), hepatosplenomegaly, and generalized lymphadenopathy. The lymphoblasts were positive for CD3 and the malignant blasts showed clonal TCRB gene rearrangements (Fig. 3C2).

Expression profile of OLIG2-LMO1 tumors. To identify genes that might collaborate with OLIG2 and LMO1 during leukemic transformation, or identify genes of which expression might be affected by OLIG2 and/or LMO1, we compared the gene expression profiles of four thymic tumors with those of normal thymus from nontransgenic littermates by means of a two-color microarray assay. Table 2 lists genes that were up-regulated more than 3-fold in the thymic tumors compared with normal thymus. Several genes that are considered to provide growth and/or survival signals were up-regulated. These genes included Saa3 (serum amyloid A), Aldh1b1 (aldehyde dehydrogenase 1 family, member B1), Tfrc (transferrin receptor), Eif3s9 (eukaryotic translation initiation factor), and Eef2 (eukaryotic translation elongation factor). Interestingly, we also found that Notch1 was up-regulated an average of 3.5-fold in OLIG2-LMO1 thymic tumors, and two genes that are thought to be downstream targets of Notch1 [Dtx1 (Deltex1) and Ptcra (pre T-cell antigen receptor {alpha})] were up-regulated 10.2-fold and 3.1-fold, respectively.


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  		Table 2. Genes up-regulated more than 3-fold in thymic tumors

 
Growth of OLIG2/LMO1 tumor cell lines is inhibited by {gamma}-secretase inhibitors. Because it has been shown that aberrant expression of Notch1 is associated with the pathogenesis of pre-T LBL (31–33), we investigated whether Notch1 up-regulation was important for the continued growth of OLIG2-LMO1 thymic tumors. Notch1 is a transmembrane receptor of which signaling normally requires {gamma}-secretase–mediated proteolytic processing. On cleavage by {gamma}-secretase, the intracellular domain of Notch1 (ICN1) gains access to the nucleus where it functions as a regulator of transcription. To determine if the up-regulated Notch1 was functionally important for the growth of OLIG2-LMO1 cells, pre-T LBL cell lines (#1928 and #1931) established from OLIG2-LMO1 double transgenic mice were cultured in the presence of the {gamma}-secretase inhibitor Z-IL-CHO. Growth of OLIG2-LMO1 double transgenic cells was markedly suppressed by Z-IL-CHO, whereas the growth of control cell lines that expressed little (#1901, a pre-T LBL cell line established from a NHD13 transgenic mouse) or no (the erythroleukemia cell line F4-6) Notch1 was not suppressed (Fig. 4B). The OLIG2-LMO1 cell line #1931 showed a decreased number of cells in S-G2-M phase and an increased number of sub-G1 apoptotic cells compared with the F4-6 control cell line following Z-IL-CHO treatment (Supplementary Fig. 3).

Mutations of Notch1 in OLIG2-LMO1 double transgenic thymic tumors. Because it has been shown that 50% of human pre-T LBL had activating mutations that involved the HD and PEST domains of NOTCH1, we investigated whether primary OLIG2-LMO1 double transgenic thymic tumors had Notch1 mutations. We found that three of six primary tumors had mutations in either HD or PEST domain (Fig. 4C). Two of these three tumors showed single base substitutions in one allele of the HD domain that subsequently altered the proteins [mouse #259: nucleotide 58,374 T>C (W>R); mouse #262: nucleotide 58,462 T>A (L>Q)]. Another tumor had a frame-shift mutation in one allele of the PEST domain that resulted in an introduction of a premature stop codon [mouse #1939: nucleotides 66,508-66,510 AGG>AGGG].


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Overexpression of OLIG2 (BHLHB1) has been shown in oligodendroglioma samples and has been proposed to be a specific diagnostic marker for oligodendroglioma (10, 11, 34). In this study, we confirmed that three glioblastoma cell lines overexpressed OLIG2, whereas three astrocytoma cell lines did not express OLIG2. Interestingly, we found OLIG2 expression in non–small cell lung carcinoma, melanoma, breast cancer, and a wide spectrum of leukemic cell lines. In contrast, none of the colon (n = 7), prostate (n = 2), renal (n = 6), or ovarian (n = 4) cancer cell lines expressed OLIG2. Eleven of the 41 leukemic cell lines that we investigated expressed OLIG2, and we and others have detected OLIG2 overexpression in patients with pre-T LBL (3, 12, 13). In summary, overexpression of OLIG2 was found in a wide spectrum of malignant cells leading to the speculation that overexpression of OLIG2 might be oncogenic.

In the current study, we focused on a leukemogenic effect of OLIG2. We initially found that none of 69 OLIG2 transgenic mice developed malignancy. However, when we crossed OLIG2 mice to LMO1 mice, 2 of 16 mice transgenic for OLIG2 only developed pre-T LBL at the age of 9 and 12 months. Although the cause for the discrepancy between these two cohorts is unknown, the low penetrance of disease suggested that overexpression of OLIG2 was a relatively weak oncogenic trigger and required additional cooperative events to induce leukemia. This hypothesis is supported by the observation that OLIG2-LMO1 double transgenic mice developed pre-T LBL. The penetrance of pre-T LBL in the double transgenic mice was remarkably elevated (57%) compared with that of OLIG2 only transgenic (12%), LMO1 only transgenic (8%), and nontransgenic control (0%) mice. Whereas the OLIG2 only transgenic mice developed pre-T LBL at a relatively advanced age (9 and 12 months) and a single LMO1 only transgenic mouse developed the disease at the age of 13 months, OLIG2-LMO1 double transgenic mice developed pre-T LBL as early as 4 months of age. These findings (increased penetrance and early onset of disease) indicated that OLIG2 and LMO1 synergistically collaborated during leukemic transformation.

We investigated the events that might be downstream of or collaborate with OLIG2 and LMO1 using two-color microarray expression profiling of OLIG2-LMO1 tumors. A large number of genes that contribute to cell growth and/or survival were found up-regulated in OLIG2-LMO1 thymic tumors. We noted with interest that Notch1 was one of the most highly and consistently up-regulated genes. Expression of Notch1 in thymic tumors from OLIG2-LMO1 double transgenic mice was more than 3-fold up-regulated compared with that of normal control thymus. In addition, two genes that are considered to be downstream of Notch1 (Dtx1 and Ptcra) were also up-regulated.

Notch1 is required to maintain definitive hematopoiesis (35) and induces T-cell differentiation, at least in part, by inhibiting B-cell differentiation of common lymphoid progenitors (36). It has been shown that activated Notch1 induces pre-T LBL in a murine bone marrow transduction model with complete penetrance (33). More recently, it was reported that over 50% of pre-T LBL patients lacking Notch1-related chromosomal translocations showed activating mutations of Notch1 (37). Thus, deregulated Notch1 can contribute to the pathogenesis of pre-T LBL. The Notch1 protein is a transmembrane receptor that binds multiple ligands including {Delta} and Jagged. On ligand binding, the intracellular domain of Notch1 (ICN1) is proteolytically cleaved by {gamma}-secretase from the transmembrane portion and transferred into the nucleus, where it activates downstream signaling pathways (38). It has previously been shown that {gamma}-secretase inhibitors suppressed the growth of Notch1-transformed pre-T LBL cells (39). We investigated whether up-regulation of Notch1 in the OLIG2-LMO1 tumors was important for the transformed phenotype using pre-T LBL cell lines established from OLIG2-LMO1 double transgenic bone marrow. These pre-T LBL cell lines showed both cell death and growth inhibition in the presence of a {gamma}-secretase inhibitor (Fig. 4 and Supplementary Fig. S3), whereas two control cell lines that did not overexpress Notch1 were unaffected by the {gamma}-secretase inhibitor. Thus, Notch1 expression is important for cell survival in malignant cells that overexpress OLIG2 and LMO1. We also investigated whether the activation of Notch1 was induced by activating mutation in the HD and/or PEST domain as reported for pre-T LBL patients (37). Similar to the result that has been shown in human pre-T LBL, 50% (3 of 6) of OLIG2-LMO1 double transgenic tumors had activating mutations in either the HD or PEST domain.

SCL/Tal-1, a bHLH transcription factor like OLIG2, has been shown to bind directly to E2A (40, 41); this inhibition of E2A by SCL was shown to be enhanced by overexpression of LMO1 (23). The inhibition of E2A function by overexpression of SCL and LMO1 is likely to be important for leukemogenesis given that E2A null mice develop pre-T LBL (42, 43). We have previously shown that E2A function was suppressed by OLIG2 overexpression in a transient transfection assay (3). Given that coexpression of LMO1 and OLIG2 was more strongly oncogenic than expression of either protein alone, it seems reasonable to suggest that LMO1 might potentiate the OLIG2-mediated inhibition of E2A function, and exert its oncogenic effect through inhibition of E2A.

We noted that PDGFR{alpha} was not up-regulated (average 1.0 ± 0.2-fold) in thymic tumors compared with that of normal thymus. PDGFR{alpha} overexpression previously was detected in several oligodendroglioma samples, leading to the suggestion that PDGFR{alpha} is a downstream target of OLIG2 that might contribute to the pathogenesis of oligodendroglioma (2, 44–47). However, given that PDGFR{alpha} was not up-regulated in pre-T LBL tumors from OLIG2 and OLIG2/LMO1 mice, it seems plausible that the downstream targets of OLIG2 might be cell type specific.

In summary, we found that OLIG2 overexpression was not restricted to malignant oligodendrocytes but was also seen in malignant cells of skin, lung, breast, and hematopoietic origin. We showed that OLIG2 overexpression was only weakly oncogenic in thymocytes and required collaborative events to induce a highly penetrant leukemia. Putative collaborative events include up-regulation of LMO1, Notch1, as well as other cell proliferation signals.


   Acknowledgments
 
Grant support: Specialized Center of Research grant from the Leukemia and Lymphoma Society (S.D. Nimer).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank R. Keith Humphries of University of British Columbia for his gift of the pLIT2 vector, Stan Korsmeyer of Howard Hughes Medical Institute for the lck-LMO1 mice, Lionel Feigenbaum of the NCI transgenic core facility for microinjection and animal husbandry, Trang Hoang of the University of Montreal for helpful discussions, Donal MacGrogan of the Memorial Sloan-Kettering Cancer Center for preparation of RNA from human leukemic cell lines, and Junji Tsurutani of the NCI for valuable discussion in cell cycle analysis.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/25/05. Revised 6/12/05. Accepted 6/15/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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