Inhibition of Src family kinases enhances retinoic acid–induced gene expression and myeloid differentiation Michelle B. Miranda, 1 Robert L. Redner, 1 and Daniel E. Johnson 1,2 Departments of 1 Medicine and 2 Pharmacology, University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania Abstract Treatment of acute promyelocytic leukemia with retinoic acid (RA) results in differentiation of the leukemic cells and clinical remission. However, the cellular factors that regulate RA-induced myeloid differentiation are largely unknown, and other forms of acute myelogenous leuke- mia (AML) do not respond to this differentiation therapy. A greater understanding of the molecules that positively or negatively regulate RA-induced differentiation should facilitate the development of more effective differentia- tiontherapies.Inthisstudy,weinvestigatedthepotential role of Src family kinases (SFK) in the regulation of RA- induced gene expression and myeloid differentiation. We report that inhibition of SFKs markedly enhanced RA- induced differentiation in myeloid cell lines and primary AML cells, as assessed by flow-cytometric analysis of cell surface markers, morphologic analysis, and nitroblue tetrazolium reduction. In addition, inhibition of SFKs enhanced expression from retinoic acid receptor (RAR) target genes encoding CCAAT/enhancer binding protein E (C/EBPE), PU.1, intercellular adhesion molecule-1 (ICAM- 1),andcathepsinD.Moreover,aconstitutivelyactiveSrc inhibited RAR-dependent transcription, whereas a kinase- dead Src exerted little effect. These studies provide the first demonstration that SFKs act to negatively regulate RA-induced gene expression and myeloid differentiation and suggest that the combination of SFK inhibition and RA treatment may be therapeutically beneficial in AML. [Mol Cancer Ther 2007;6(12):3081–90] Introduction Acute myelogenous leukemias (AML) are characterized by defective differentiation and excessive accumulation of proliferative progenitor cells. A variety of molecular abnormalities have been shown to be associated with differentiation blockades in AML and include aberrant expression of (a ) fusion oncoproteins such as AML1/ETO (1, 2), PML/RARa (3–5),andCBFh/MYH11 (6); (b ) mutant myeloid transcription factors such as mutant PU.1 (7) and CCAAT/enhancer binding protein a (C/EBPa; ref. 8); and (c ) mutant cytokine receptors such as Flt3-ITD (9) and truncated granulocyte colony-stimulating factor (G-CSF) receptor (10). At the same time, the molecular bases for defective differentiation in many cases of AML remain unknown. A promising strategy for treating AML involves the use of differentiation therapy. Indeed, in the case of acute promyelocytic leukemia (APL), characterized by expression of fusion oncoproteins incorporating the reti- noic acid receptor (RAR; ref. 11), treatment with retinoic acid (RA) results in the induction of myeloid differentiation and clinical remission (12–14). However, despite the success of RA in the treatment of APL, other subtypes of AML fail to respond to this agent, and effective differen- tiation strategies for these malignancies do not exist. Moreover, the cellular factors that regulate RA-induced myeloid differentiation and may contribute to responsive- ness to RA are poorly understood. Emerging evidence has implicated Src family kinases (SFK) as regulators of proliferation and survival of myeloid lineage cells and regulators of cytokine-induced myelopoi- esis (15). The SFK family comprises nine different members, with the primary SFKs expressed in myeloid cells being Hck, Lyn, and Fgr. Early studies revealed that expression of v-Src or constitutively active Hck abrogated cytokine dependence and inhibited granulocyte colony-stimulating factor (G-CSF)–induced granulocytic differentiation in the murine myeloid progenitor cell line 32Dcl3 (16, 17). Sub- sequent studies have shown that Lyn plays an important role in G-CSF–induced production of reactive oxygen species and myeloid cell growth (18, 19). Experiments involving knock-out mice have provided evidence for the regulation of cytokine-stimulated myelopoiesis by SFKs. Granulocytic precursor cells derived from mice deficient in Hck exhibit enhanced proliferation in response to G-CSF (20). Mice lacking Lyn manifest an increase in myeloid progenitors, an enhancement of G-CSF–stimulated gran- ulopoiesis, and development of a myeloproliferative disor- der leading to monocyte/macrophage tumors (20, 21). Similar findings were reported in triple knock-out mice lackingLyn,Fgr,andHck(20).Together,thesestudiesimpli- cate myeloid SFKs as negative regulators of myelopoiesis. Received 8/6/07; revised 9/17/07; accepted 10/12/07. Grant support: NIH grant CA108904 to D.E. Johnson. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Daniel E. Johnson, Division of Hematology/ Oncology, University of Pittsburgh, Room 2.18c, Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213-1863. Phone: 412-623-3245; Fax: 412-623-7768. E-mail: johnsond@pitt.edu Copyright C 2007 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-07-0514 3081 Mol Cancer Ther 2007;6(12). December 2007 on June 15, 2017. © 2007 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst December 7, 2007; DOI: 10.1158/1535-7163.MCT-07-0514