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Targeted inhibition of FLT3 overcomes the block to myeloid differentiation in 32Dcl3 cells caused by expression of FLT3/ITD mutations

Rui Zheng, Alan D. Friedman and Donald Small

Article Figures & Data

Figures

  • Fig. 1.

    Ligand-independent phosphorylation of FLT3/ITD in 32D cells.

    Total cellular protein extracts derived from 1 × 107parental 32D cells and 32D subclones transduced with the pBabePuro vector, pBabePuro-FLT3, and pBabePuro-FLT3/ITD were immunoprecipitated with anti-FLT3 antibody. Cells were incubated without (lanes 1, 3, 5, 7) and with (lanes 2, 4, 6, 8) FL (100 ng/mL) for 5 minutes before extracts were made. The immunoprecipitates were resolved by 8% SDS-PAGE and subjected to immunoblot analysis with antiphosphotyrosine antibody 4G10 (top panel). The same membrane was stripped and reprobed with anti-FLT3 antibody (bottom panel).

  • Fig. 2.

    FLT3/ITD blocks 32D cell morphologic and functional differentiation to granulocytes.

    32D/pBabe, 32D/FLT3, and 32D/FLT3/ITD cells were washed and transferred from medium containing IL-3 (1 ng/mL) to medium containing G-CSF (20 ng/mL) for 9 days. Every other day, fresh G-CSF–containing medium was added to cells to replace the old medium. (A) Cytospins were prepared on days 0 and 9. The cytospins were subjected to Wright-Giemsa staining followed by light microscopy. Original magnification, × 100. (B) Total cellular RNA from 32D/pBabe (lanes 1-6), 32D/FLT3 (lanes 7-12), and 32D/FLT3/ITD (lanes 13-18) cells was prepared from 1 × 107cells after 0, 1, 3, 5, 7, and 9 days in G-CSF. RNA (15 μg) from each time point was then subjected to Northern blotting with MPO, lysozyme, and actin cDNA probes.

  • Fig. 3.

    FL stimulation slows the differentiation of 32D cells expressing wild-type FLT3.

    32D/FLT3 cells were washed and transferred from medium containing IL-3 to medium containing G-CSF (20 ng/mL) with and without FL (100 ng/mL) and cultured for 12 days. Fresh medium with G-CSF with and without FL was used to replace the old medium every 48 hours. (A) Cytospins were prepared on days 0, 9, and 12. The morphologic features of the cells were visualized by means of Wright-Giemsa staining followed by light microscopy. Original magnification, × 100. (B) Total cellular RNA was prepared from 1 × 107 32D/FLT3 cells after 0, 1, 3, 5, 7, and 9 days in medium containing G-CSF without FL (lanes 1-6) or with FL (100 ng/mL; lanes 7-12). RNA (15 μg) was then subjected to Northern blotting with MPO, lysozyme, and actin cDNA probes.

  • Fig. 4.

    CEP-701 inhibits the kinase activity of FLT3/ITD and the proliferation of 32D/FLT3/ITD cells.

    (A) 32D/FLT3/ITD cells were treated with CEP-701 at increasing concentrations for 1 hour at 37°C. Cell lysates were immunoprecipitated with anti-FLT3 antibody and immunoblotted with antiphosphotyrosine antibody (top panel). The same membrane was then stripped and probed with anti-FLT3 antibody (bottom panel). (B) Effects of CEP-701 on the growth of 32D/FLT3/ITD cells and 32D/pBabe cells were evaluated by counting the number of viable cells for 11 days. 32D/FLT3/ITD cells were seeded at an initial density of 2 × 105/mL in 10% FCS/RPMI medium supplemented with various concentrations of CEP-701 with and without G-CSF (20 ng/mL). 32D/pBabe cells were seeded at the same density in medium containing IL-3 (1 ng/mL) either with or without CEP-701 (5 nM), or G-CSF. The medium was replenished every other day, and the cell densities were adjusted to 2 × 105/mL. Viable cells were counted on the basis of trypan blue exclusion. Results shown are the means from triplicate assays.

  • Fig. 5.

    CEP-701 overcomes the FLT3/ITD-mediated block of differentiation in 32D/FLT3/ITD cells.

    32D/FLT3/ITD cells were treated with or without CEP-701 (5 nM) in the presence of G-CSF (20 ng/mL) for 11 days. Every other day, fresh medium containing G-CSF with or without CEP-701 was added to cells to replace the old medium. (A) Cytospins were prepared on days 0 and 11. The morphologic features of the cells were visualized by means of Wright-Giemsa staining followed by light microscopy. Original magnification, × 100. (B) Total cellular RNA was prepared from 1 × 107 cells on days 0, 1, 3, 5, 7, 9, and 11 of culture without (lanes 1-7) or with (lanes 8-14) CEP-701. RNA (15 μg from each sample) was then subjected to Northern blotting with MPO, lysozyme, and actin cDNA probes.

  • Fig. 6.

    Regulation of C/EBPε, c-myc, and c-myb in 32D/FLT3 and 32D/FLT3/ITD cells.

    32D/FLT3 cells were washed and transferred from medium containing IL-3 to medium containing G-CSF (20 ng/mL) without (lanes 1-6) or with (lanes 7-12) FL (100 ng/mL) and cultured for 9 days. 32D/FLT3/ITD cells were treated without (lanes 13-19) or with (lanes 20-26) CEP-701 (5 nM) in the presence of G-CSF (20 ng/mL) for 11 days. Every other day the medium was replaced. Total cellular RNA was extracted from 1 × 107 32D/FLT3 cells on days 0, 1, 3, 5, 7, and 9. Total cellular RNA was also prepared from 1 × 107 32D/FLT3/ITD cells on days 0, 1, 3, 5, 7, 9, and 11. RNA (15 μg from each sample) was then subjected to Northern blotting with C/EBPε, c-myc, c-myb, and actin cDNA probes.

Tables

  • Table 1.

    Differentiation of 32Dcl3 cells is blocked by expression of FLT3/ITD

    CellsDay 0Day 9
    32D/pBabe32D/FLT332D/FLT3/ITD32D/pBabe32D/FLT332DFLT3/ITD
    Myeloblasts, %81.3 ± 2.585.7 ± 3.882.3 ± 4.514.3 ± 2.511.3 ± 3.285.3 ± 5.0
    Intermediates, %18.7 ± 2.514.3 ± 3.817.7 ± 4.564 ± 3.666.7 ± 4.514.7 ± 5.0
    Granulocytes, %00 0 21.7 ± 4.014.7 ± 5.00
    • Differential counts of 32D/pBabe, 32D/FLT3, and 32D/FLT3/ITD cell cultures induced by G-CSF (20 ng/mL) were performed on days 0 and 9.

    • Values are the mean percentages, ± SD, of cells from 3 independent counts examined by light microscopy after Wright-Giemsa staining of the cytospins. “Intermediates” include promyelocytes, myelocytes, and metamyelocytes.

  • Table 2.

    FL stimulation slows, but does not block, differentiation of 32D/FLT3 cells

    CellsG-CSFG-CSF+FL
    Day 0Day 9Day 12Day 0Day 9Day 12
    Myeloblasts, %82.7 ± 5.013.7 ± 2.514.2 ± 1.283.7 ± 3.251.7 ± 2.517.5 ± 0.5
    Intermediates, %17.3 ± 5.063.7 ± 4.656.8 ± 2.816.3 ± 3.246.3 ± 3.859.7 ± 6.2
    Granulocytes, %0 22.6 ± 2.529.0 ± 2.80 2.0 ± 1.022.8 ± 6.6
    • Differential counts of 32D/FLT3 cell cultures induced by G-CSF (20 ng/mL) with and without FL (100 ng/mL) were performed on days 0, 9, and 12.

    • Values are the mean percentages, ± SD, of cells from 3 independent counts examined by light microscopy after Wright-Giemsa staining of the cytospins. “Intermediates” include promyelocytes, myelocytes, and metamyelocytes.

  • Table 3.

    CEP-701 overcomes the FLT3/ITD-mediated block of differentiation in 32D cells

    CellsG-CSFG-CSF + CEP-701
    Day 0Day 11Day 0day 11
    Myeloblasts, %84.7 ± 3.885.7 ± 5.988.0 ± 3.014.7 ± 4.7
    Intermediates, %15.3 ± 3.814.3 ± 5.912.0 ± 3.066.7 ± 5.5
    Granulocytes, %0 0 0 18.7 ± 3.5
    • Differential counts of 32D/FLT3/ITD cell cultures treated with G-CSF (20 ng/mL) with and without CEP-701 (5 nM) were performed on days 0 and 11.

    • Values are the mean percentages, ± SD, of cells from 3 independent counts examined by light microscopy after Wright-Giemsa staining of the cytospins. “Intermediates” include promyelocytes, myelocytes, and metamyelocytes.

  • Table 4.

    Cell death occurs during the differentiation of 32D/FLT3/ITD and 32D/pBabe cells

    Day 0, %Day 1, %Day 11, %
    32D/FLT3/ITD11.2 ± 2.511.1 ± 1.78.8 ± 0.6
    32D/FLT3/ITD+G-CSF11.2 ± 2.58.6 ± 1.39.9 ± 0.4
    32D/FLT3/ITD+G-CSF+CEP-70111.2 ± 2.518.5 ± 1.136.5 ± 2.9
    32D/pBabe+IL-36.6 ± 1.09.5 ± 0.311.8 ± 0.6
    32D/pBabe+IL-3+CEP-7016.6 ± 1.05.9 ± 0.36.1 ± 0.5
    32D/pBabe+G-CSF6.6 ± 1.016.8 ± 1.642.7 ± 0.3
    • 32D/FLT3/ITD cells were cultured in medium with or without G-CSF (20 ng/mL) in the presence or absence of CEP-701 (5 nM). 32D/pBabe cells were cultured either in medium containing IL-3 (1 ng/mL) with and without CEP-701 (5 nM) or in medium containing G-CSF (20 ng/mL). Early and late apoptotic cells were detected by annexin V/7-AAD staining followed by flow cytometry on days 0, 1, and 11.

    • Values are the mean percentages, ± SD, of cells from triplicate experiments.