OLIG2 (BHLHB1), a bHLH transcription factor, contributes to leukemogenesis in concert with LMO1

Abstract

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.

Fig. 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 methods). Dot #1 denotes the position of the first dot on the upper left-hand 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).

Fig. 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. The upper panel shows hybridization of OLIG2 and the lower panel shows 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).

Fig. 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) Peripheral blood and (B10) bone marrow 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 demonstrate clonal TCRB gene rearrangements in OLIG2-LMO1 double transgenic (#259 and #1939) and OLIG2 only transgenic (#556) mice. Rearranged bands are indicated with arrows.

Fig. 4. Growth suppression of OLIG2/LMO1 cell lines over-expressing Notch1 by γ-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 μM Z-IL-CHO (γ-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 approximately 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 (AL732541 of GenBank). HD: heterodimerization domain, Nm: no mutation