Gefitinib potentiates myeloid cell differentiation by ATRA

Acute myeloid leukemias (AMLs) are characterized by a variety of genetic defects that contribute to the failure of these cells to undergo differentiation to mature granulocytes or monocytes/macrophages. A primary consequence of this failure to differentiate is the accumulation of proliferative blasts in the bone marrow and peripheral blood. In the case of one subtype of AML, acute promyelocytic leukemia (APL; FAB M3), differentiation can be induced by all-trans retinoic acid (ATRA), a treatment that is curative for many acute promyelocytic leukemia patients.^1,2 However, ATRA has not proven to be effective in the treatment of other subtypes of AML, underscoring the need to identify new compounds that can either induce differentiation as mono-therapies or enhance the efficacy of ATRA therapy. Here we report that the tyrosine kinase inhibitor gefitinib markedly enhances ATRA-induced differentiation in myeloid cell lines.

We and others have shown that ATRA-induced monocytic and granulocytic differentiation is dependent on activation of serine/threonine kinases in the mitogen-activated protein kinase kinase/extracellular-regulated kinase (MEK/ERK) signaling pathway.³ In addition, we have reported that pharmacologic inhibition of Src family kinases results in enhancement of ATRA-induced myeloid differentiation.⁴ These results indicate that tyrosine kinases in the Src family kinase family act as negative regulators of ATRA-induced differentiation. Recently, Stegmaier et al.⁵ have shown that the pharmacologic inhibitor gefitinib stimulates differentiation of the myeloid cell lines HL-60, Kasumi-1 and U937. Gefitinib is a small molecule inhibitor of the epidermal growth factor receptor tyrosine kinase and has been approved by the Food and Drug Administration for treatment of non-small cell lung cancer.⁶ Interestingly, the epidermal growth factor receptor does not appear to be expressed in AML cells, indicating that the differentiation-inducing effects of gefitinib are likely due to inhibition of, as yet unidentified, cellular tyrosine kinases.⁵ The inhibition of a variety of cellular kinases by low micromolar concentrations of gefitinib has been reported.⁷ Despite an unclear mechanism of action, we sought to determine whether gefinitib might act in synergy with low-dose ATRA to stimulate myeloid differentiation.

As shown in Figure 1, gefitinib (10µM; kindly provided by AstraZeneca, London, UK) dramatically enhanced ATRA-induced differentiation of HL-60 cells. In Figure 1a, induction of differentiation was assessed using flow cytometric analysis of the myeloid marker protein CD11b. Treatment of HL-60 cells for 3 days with 2 µm of ATRA alone resulted in 19.2% CD11b-positive cells, while only 1.9% of cells treated with gefitinib (10µm) alone were CD11b-positive. By contrast, cells treated simultaneously with ATRA and gefitinib scored as 65.2% CD11b-positive. Similar results were obtained in three independent experiments. The ability of gefitinib to enhance ATRA-induced HL-60 cell differentiation was confirmed using assays that scored differentiation on the basis of nitro-blue tetrazolium staining (Figure 1b) or cellular morphology (Figure 1c). Strikingly, enhancement of ATRA-induced differentiation was observed even when gefitinib was added as late as 24 h after initiation of ATRA treatment (data not shown), suggesting that inhibition of later or prolonged kinase signaling is important.

Figure 1.

Gefitinib ententes ATRA-induced differentiation of HL-60 cells, HL-60 cells were left untreated or were treated with ATRA alone (2 µm), gefitinib alone (10 µm) or ATRA plus gefitinib. After 72 h, cells were harvested and induction of myeloid differentiation was assessed. (a) Flow cytometric analysis of CD11b expression. Numbers indicate the percent of CD11b-postive cells. (b) Nitroblue tetrazolium reduction assays of differentiation. For each data point, a total of 300 cells were counted from three different fields. (c) Morphological analysis of differentiation, Granulocytic differentiation was assessed using Hema 3-stained cytospin preparations. For each data point, 300 cells were counted from three different fields. Similar results were seen in three independent experiments. ATRA, all-trans retinoic acid.

The ability of gefitinib to enhance ATRA-induced differentiation was not restricted to HL-60 cells. Treatment of NB-4 cells with the suboptimal dose of 0.01 µm ATRA resulted in 6.5% CD11b-positive cells after 3 days, while treatment with gefitinib alone (10µm) generated only 2.8% CD11b-positive cells (Figure 2a). The combination of ATRA and gefitinib, however, yielded 41.7% CD11b positivity. Similarly, the combination of ATRA and gefitinib resulted in enhanced induction of U937 cell differentiation, as assessed by CD11b expression analysis (Figure 2b).

Figure 2.

Gefitinib potentiates ATRA-induced differentiation of NB-4 and U937 cells, (a) NB-4 cells were left untreated or were treated for 72 h with the indicated concentrations of ATRA alone, gefitinib alone or ATRA plus gefitinib. Flow cytometric analysis of CD11b expression was used to assess differentiation. (b) U937 cells were left untreated or were treated for 72 h with the indicated concentrations of ATRA, gefitinib or ATRA plus gefitinib. Similar results were seen in three independent experiments. ATRA, all-trans retinoic acid.

In prior studies, activation of the MEK/ERK pathway has been shown to be important for ATRA-induced myeloid differentiation. To determine whether activation of this pathway is required for the enhanced differentiation observed with the combination of ATRA and gefitinib, HL-60 cells treated with the ATRA/gefitinib combination were simultaneously incubated with the MEK-1/-2 inhibitor U0126 (10µm). Previous experiments have shown that 10µm U0126 results in nearly complete inhibition of the MEK/ERK pathway in myeloid lineage cells.⁸ As shown in Figure 3, inhibition of the MEK/ERK pathway using U0126 resulted in abrogation of HL-60 cell differentiation by the ATRA/gefitinib combination. This suggests that the MEK/ERK pathway lies downstream of the signaling components impacted by either ATRA or gefitinib.

Figure 3.

The MEK/ERK pathway is essential for gefitinib enhancement of ATRA-induced differentiation. HL-60 cells were left untreated or were treated for 72 h with ATRA alone (2 µm), U0126 alone (10 µm), ATRA plus gefitinib or ATRA plus gefitinib plus U0126. Numbers indicate the percent of cells staining positive for CD11b. ATRA, all-trans relinoic acid.

In conclusion, we have demonstrated that the tyrosine kinase inhibitor gefitinib markedly enhances ATRA-induced differentiation of myeloid cell lines. It remains to be determined whether gefitinib enhancement of ATRA-induced differentiation will be observed in vivo at clinically achievable doses. In addition, it remains unclear which cellular kinases are being targeted by gefitinib to elicit enhancement of ATRA action, although their elucidation may provide novel targets for therapeutic intervention in AML. In the meantime, application of Food and Drug Administration-approved gefitinib, or related inhibitors, should be considered as a means of enhancing ATRA sensitivity in AML.