Abstract
FLT3 expression level in leukemia cells is associated with specific histone modifications that are associated with presence or absence of MLL fusion genes
FLT3 is a class III receptor tyrosine kinase that is normally expressed in hematopoietic stem/progenitor cells. FLT3 expression is lost as hematopoietic cells differentiate. Based on murine knockout studies, FLT3 signaling is known to be important in the development of myeloid progenitors, B lymphoid progenitors, NK cells and dendritic cells. The FLT3 gene is mutated in about one third of acute myeloid leukemia. Apart from the activating mutations, it is also over-expressed in wide range of pre-B and myeloid leukemias. It is particularly highly expressed in leukemias harboring rearrangements of MLL at 11q23. The quantitative level of FLT3 expression in these leukemias is orders of magnitude higher than in germline (wild type) MLL leukemias of similar lineage. The MLL protein is known to have histone methyltransferase activity which resides in the C-terminal SET domain, which predominantly mediates H3K4 methylation. The MLL fusion proteins that result from MLL rearrangements lack the SET domain, but form complexes that interact with DOT1L, a H3K79 methyltransferase. These histone modifying properties of germline and rearranged MLL are central to the function of these proteins as master regulators of target gene expression. The mechanism(s) regulating the level of expression of FLT3 in hematopoietic cells have not been described. Given the association of high FLT3 expression levels with MLL rearrangements, and the recent elucidation of the role of the MLL gene and its fusion proteins in histone modification, we hypothesized that histone modifications may play an important role in the regulation of FLT3 expression. On histone H3, acetylation on lysines 9 and 14 and methylation on lysines 4, 36, and 79 are linked to active transcription, whereas tri-methylation on lysines 9 and 27 is linked to transcriptional repression. We analyzed the modification of histone H3 at lysine 9 (acetylation and tri-methylation) and 14 (acetylation) at the FLT3 promoter in different cell lines including pre-B ALL, monocytic AML, T-cell ALL and adenocarcinoma, with a range of quantitative FLT3 expression. For each of these cell lines, we performed ChIP using H3K9/14 acetyl and H3K9 tri-methyl antibodies followed by Real Time PCR with FLT3 promoter specific primers. The results are summarized in Table 1.
Table 1
Cell line Lineage/origin Cytogenetics FLT3 expression mutational status ddC1 FLT3 promoter Acetyl/Methyl Ratio Acetyl H3K9 Methyl H3K9 MV4-11 monocylic AML MLL-AF4 High/TTD 11.1 5.55 2.00 Kopn 8 pre-BALL MLL-ENL High/wt 10.4 5.75 1.81 SEM pre-BALL MLL-AF4 High/wt 13.5 7.9 1.71 Nalm 6 pre-BALL ;(5;12) Low/wt 9 10.1 0.89 Jurkat TALL hypotetraploid Negative/wt 5.4 84 0.64 HeLa cervical CA aneuploid Negative/wt 4.05 9.5 0.43
We found that cell lines with robust FLT3 expression have higher acetylation at H3K9/14 than those with no or low FLT3 expression. Conversely, cell lines with no/low FLT3 expression have higher tri-methylation at H3K9 than those with high FLT3 expression. For individual cell lines, the ratio of acetyl H3K9/14 to tri-methyl H3K9 correlated with FLT3 expression. Furthermore, comparison of similar lineage cell lines with and without MLL rearrangements supports the hypothesis that the MLL mutational status (rearranged vs. germline) may dictate these differences. Kopn 8 (MLL rearranged, high FLT3) and Nalm6 (MLL germline, low FLT3) are both pre-B ALL leukemia cell lines that show converse H3K9/14 acetylation to H3K9 tri-methylation ratios. Together, these findings suggest that FLT3 expression may be controlled in part by histone modifications at its promoter, and that the mutational status of the MLL gene at 11q23 may be an important determinant of these modifications. In ongoing studies that will be reported at the meeting, we are expanding these studies to include additional H3 lysines (such as H3K4 and H3K79), additional cell lines, and primary patient leukemia samples. We are also performing ChIP assays on various sorted fractions of normal human bone marrow to ascertain whether histone modifications play a role in FLT3 expression during normal hematopoiesis.
Ash2 acts as an ecdysone receptor coactivator by stabilizing the histone methyltransferase Trr
