The Src-Family Kinase Inhibitor PP2 Rescues Inducible Differentiation Events in Emergent Retinoic Acid- Resistant Myeloblastic Leukemia Cells Holly A. Jensen 1 , Lauren E. Styskal 2¤a , Ryan Tasseff 1¤b , Rodica P. Bunaciu 3 , Johanna Congleton 3¤c , Jeffrey D. Varner 1 , Andrew Yen 3 * 1 School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, United States of America, 2 Department of Biological Engineering, Cornell University, Ithaca, New York, United States of America, 3 Department of Biomedical Sciences, Cornell University, Ithaca, New York, United States of America Abstract Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA- inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47 phox expression. However, RA-treated RA- resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid’s biological effects in WT HL60 cells. Citation: Jensen HA, Styskal LE, Tasseff R, Bunaciu RP, Congleton J, et al. (2013) The Src-Family Kinase Inhibitor PP2 Rescues Inducible Differentiation Events in Emergent Retinoic Acid-Resistant Myeloblastic Leukemia Cells. PLoS ONE 8(3): e58621. doi:10.1371/journal.pone.0058621 Editor: Jo ¨ rn Lausen, Georg Speyer Haus, Germany Received November 7, 2012; Accepted February 5, 2013; Published March 15, 2013 Copyright: ß 2013 Jensen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institutes of Health (NIH) (R01 # CA033505 and CA152870) and a NYSTEM New York State Department of Health grant to Andrew Yen. The authors acknowledge the gracious financial support of the National Science Foundation CAREER (NSF #CBET- 0846876) to Jeffrey Varner for support of H.A.J. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Lauren E. Styskal is employed by Quanterix Corporation. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: ay13@cornell.edu ¤a Current address: Currently at Quanterix Corporation, Lexington, Massachusetts, United States of America ¤b Current address: Institute for Systems Biology, Seattle, Washington, United States of America ¤c Current address: Environmental Working Group, Washington DC, Untied States of America Introduction Retinoids, the family of vitamin A derivatives, have long been known to control differentiation processes and have similar mechanisms to those of steroid and thyroid hormones [1]. Retinoic acid (RA) has pro-differentiative and anti-proliferative effects, and is associated with embryonic development, mainte- nance of epithelial linings and prevention of epithelial tumorigen- esis [1]. RA is the current treatment for acute promyelocytic leukemia (APL) [2], and retinoids serve preventative and therapeutic roles in other cancers and diseases [3,4]. However, RA-treated myeloid leukemia cells, and RA-treated patients, may develop RA resistance after continual treatment. Many RA- upregulated proteins may continue to be expressed in RA-resistant lines, indicating that during RA resistance certain signaling pathways remain responsive while others do not. For example, RA-dependent upregulation of the surface marker CD38 is observed in both wild-type and RA-resistant HL60 (this study) and NB4 cells [5]. The HL60 cell line is an attractive, comprehensive model for understanding how RA-induced differentiation and proliferation mechanisms operate. These myeloblastic (FAB M2) leukemia cells have been a durable experimental system since the late 1970s [6]. HL60 cells are bipotent [7] myelomonocytic precursors, capable of being induced to differentiate into monocytes or granulocytes [8,9]. Treatment with all-trans retinoic acid (RA) induces differentiation of HL60 along the granulocytic lineage into neutrophil-like cells [8,9]. In HL60, inducer treatment results in G1/G0 cell cycle arrest and the cells become committed to terminal differentiation. With a doubling time of 20–24 h, HL60 undergo two rounds of cell division after RA treatment and are committed to granulopoiesis by 48 h [10]. RA-induced HL60 cells characteristically upregulate various surface proteins, including CD38 and CD11b. CD11b is an integrin component expressed in neutrophils [11]. CD38, an PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e58621