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Transforming activities of the NUP98-KMT2A fusion
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Figure 5. iNUP98-KMT2A acute myeloid leukemia cells express low levels of Hox genes and are resistant to small molecule menin and Brd4 inhibitors. (A) Expression of HoxA, -B, and -C genes was quantified by quantitative polymerase chain reaction (qPCR) analysis in total bone marrow from wildtype (WT) control (CTRL) cells, leukemic cells from iNUP98-KMT2A mice as well as from mice transplanted with retrovirally-transduced KMT2A-ENL (rKMT2A-ENL) and rKMT2A-AF9 cells. Expression relative to that of WT cells is shown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way analysis of variance (ANOVA) relative to control, n=3. (B) rKMT2A-AF9 and iNUP98-KMT2A leukemic blasts were treated with the menin inhibitor, MI-2-2, for 48 h at the indicated concentrations and the cell cycle phase was analyzed by flow cytometry. The figure shows the percentage of cells in each phase of the cell cycle. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two- way ANOVA relative to cells treated with dimethylsulfoxide (DMSO), n=2. (C) rKMT2A-AF9 and iNUP98-KMT2A leukemic blasts were treated with the BRD4 inhibitor, JQ1, for 48 h at the indicated concentrations. Cell cycle phase was analyzed by flow cytometry. The figure shows the percentage of cells in each phase of the cell cycle. *P<0.05, two-way ANOVA relative to DMSO-treated cells, n=2.
accompanied by spontaneous mutations in Nras, Kras and Cbl; however, no such mutations were found in three iNUP98-KMT2A mice that developed AML31 (data not shown). Several studies also demonstrated leuke- mogenic cooperation of NUP98-HOXD13 with overex- pression of Meis1, MN1, or loss of p53 or p15INK4B.32-35 Intercrossing of transgenic NUP98-HOXD13 mice with Flt3-internal tandem duplication (ITD) knock-in mice resulted in acceleration to a fully-penetrant AML pheno- type.36 In contrast to NUP98-HOXD13, we observed that transplantation of iNUP98-KMT2A BM cells retrovirally overexpressing FLT3-ITD did not accelerate the disease (data not shown).36
In the presence or absence of FLT3-ITD, pre-leukemic NUP98-HOXD13 cells expressed significantly increased levels of HoxA7, HoxA9, HoxB4, HoxB6, HoxB7, HoxC4 and HoxC6 mRNA.30,36 In sharp contrast, BM cells from diseased iNUP98-KMT2A mice expressed significantly reduced lev- els of the HoxA-B-C gene cluster compared to normal BM cells, recapitulating what was shown in primary NUP98- KMT2A+ AML cells.19 Expression levels of HoxA-B-C genes below those observed in normal HSPC suggest that the iNUP98-KMT2A fusion may affect the function of normal KMT2A in a dominant-negative manner.
Previous work showed that KMT2A plays a critical role in cell cycle progression.37 Interestingly, under base- line conditions, we found a higher proportion of iNUP98-KMT2A Lin- cells in S-phase when compared to WT cells supporting previous studies indicating that ablation of normal KMT2A function results in defective S-phase cell cycle entry. The percentage of both WT and iNUP98-KMT2A cells in S-phase decreased following exposure to ionizing radiation (data not shown) as has been demonstrated previously.38 Interestingly, compared to control cells, in vitro doxycycline treatment of both iNUP98-KMT2A BM-derived HSPC and MEF led to the accumulation of cells in the G1-phase, mirroring what was seen in LSK cells taken from iNUP98-KMT2A mice that had been on doxycycline food for several months. Further experiments with MEF showed that expression of iNUP98-KMT2A abrogated cellular senescence: com- pared to WT cells, iNUP98-KMT2A MEF on doxycycline never showed any signs of crisis and could easily be propagated for >45 passages. In contrast, iNUP98- KMT2A cells off doxycycline showed signs of a crisis at passages 10-12 but were able to escape and could also be propagated for over 40 passages (data not shown), suggest- ing that very low expression of iNUP98-KMT2A is suffi-
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