MLL-rearranged B lymphoblastic leukemias selectively express the immunoregulatory carbohydrate-binding protein galectin-1

Abstract

Purpose: Patients with mixed lineage leukemia (MLL)-rearranged B-lymphoblastic leukemias (B-ALL) have an unfavorable prognosis and require intensified treatment. Multiple MLL fusion partners have been identified, complicating the diagnostic evaluation of MLL rearrangements. We analyzed molecular markers of MLL rearrangement for use in rapid diagnostic assays and found the immunomodulatory protein, Galectin-1 (Gal-1), to be selectively expressed in MLL-rearranged B-ALL.

Experimental design: Transcriptional profiling of ALL subtypes revealed selective overexpression of Gal-1 in MLL-rearranged ALLs. For this reason, we analyzed Gal-1 protein expression in MLL-germline and MLL-rearranged adult and infant pediatric B-ALLs and cell lines by immunoblotting, immunohistochemistry, and intracellular flow cytometry of viable tumor cell suspensions. Because deregulated gene expression in MLL-rearranged leukemias may be related to the altered histone methyltransferase activity of the MLL fusion protein complex, we also analyzed histone H3 lysine 79 (H3K79) dimethylation in the LGALS1 promoter region using chromatin immunoprecipitation.

Results: Gal-1 transcripts were significantly more abundant in MLL-rearranged B-ALLs. All 32 primary MLL-rearranged B-ALLs exhibited abundant Gal-1 immunostaining, regardless of the translocation partner, whereas only 2 of 81 germline-MLL B-ALLs expressed Gal-1. In addition, Gal-1 was selectively detected in newly diagnosed MLL-rearranged B-ALLs by intracellular flow cytometry. The LGALS1 promoter H3K79 was significantly hypermethylated in MLL-rearranged B-ALLs compared with MLL-germline B-ALLs and normal pre-B cells.

Conclusion: In B-ALL, Gal-1 is a highly sensitive and specific biomarker of MLL rearrangement that is likely induced by a MLL-dependent epigenetic modification.

Fig. 1

Gal-1 is overexpressed in MLL-rearranged B-ALL cell lines and primary tumors. A, Gal-1 protein expression in B-ALL cell lines (left) and primary tumors (right) with or without MLL-rearrangements [MLL-r, MLL-g (MLL-germline), respectively]. B and C, immunohistochemical analyses of Gal-1 in representative primary B-ALLs with known MLL status from two independent series. B, series 1 bone marrow biopsies were analyzed; C, series 2 bone marrow aspirates were assessed. B and C, representative primary B-ALLs with specific MLL translocations t(4;11), t(11;19), t(9;11), or t(10;11) or germline MLL are shown.

Fig. 2

Gal-1 is detected by intracellular flow cytometry in MLL-rearranged B-ALL cell lines and primary tumors. Intracellular flow cytometry was performed on B-ALL cell lines (A) and viable primary tumor specimens from four B-ALL patients with known MLL translocation status (B). B, mean fluorescence intensity for control and anti–Gal-1 immunostaining was as follows: P1, 21.5 versus 108; P2, 24.2 versus 117; P3, 20.3 versus 35.5; and P4, 26.6 versus 63.3.

Fig. 3

LGALS1 promoter exhibits enrichment of H3K79 dimethylation in MLL-rearranged B-ALL. A, LGALS1 H3K79diMe in B-ALL cell lines with known MLL status. B, H3K79diMe ChiP-chip analysis of primary MLL-rearranged and MLL-germline B-ALLs and normal CD34/CD19⁺ cells. C, quantitative analysis of Gal-1 promoter H3K79 dimethylation in normal pre-B cells, MLL-germline (MLL-g), and MLL-rearranged (MLL-r) ALLs.