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In the blast crisis phase of chronic myelogenous leukemia (CML), Bcr-Abl+ myeloblasts fail to undergo terminal maturation. The extracellular signal–regulated kinase (Erk) mitogen-activated protein (MAP) kinase has been shown to mediate terminal differentiation of myeloid cells. Interestingly, Bcr-Abl+ CML cell lines established from blast crisis were found to have low Erk MAP kinase activity. In this study, we analyzed the role of the Gab2 docking protein in regulation of the Erk MAP kinase in Bcr-Abl+K562 human CML cells. Overexpression of Gab2 in K562 cells resulted in transcriptional activation of the c-fos serum response element (SRE) promoter, whereas overexpression of SHP2, Grb2, and CrkL had no effect. Activation of the c-fos SRE transcriptional activity by Gab2 required tyrosine 604, which is a SHP2 docking site on Gab2, and the SHP2 tyrosine phosphatase activity. Elk1, c-Jun, and CHOPtrans-reporting assays indicated that overexpression of Gab2 selectively activated the Erk2-Elk1 signaling pathway. To determine cellular consequences of elevating the Gab2 level in K562 cells, stable cell lines for doxycycline-inducible expression of the wild-type Gab2 (Gab2WT) and an SHP2-binding defective Gab2 (Gab2Tyr604Phe) were established. Analysis of these cell lines indicated that induction of Gab2WT expression, but not Gab2Tyr604Phe expression, led to Erk activation, growth arrest, cell spreading, and enlargement; expression of megakaryocyte/platelet lineage–specific integrins αIIb/β3 (CD41/CD61); and upregulation of RNA for megakaryocyte/platelet proteins. All of these changes are characteristics of megakaryocytic differentiation. Together, these results reveal Gab2 as a limiting signaling component for Erk MAP kinase activation and terminal differentiation of K562 CML cells.

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma, graft-versus-host disease, and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway, within a single day, without added cytokines, as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized, as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature, common to ECP-processed monocytes from normal subjects, and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway, this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances, ECP may potentially offer a general source of therapeutic DCs.

Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34+ cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood, and reduced ploidy in bone marrow megakaryocytes. In conclusion, MKL1 promotes physiologic maturation of human and murine megakaryocytes.