Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>Abstract 3956</jats:p>
<jats:p>Hematopoietic progenitor kinase 1 (HPK1) is a differentially regulated MAP4 kinase (MAP4K) in B lymphocytes (Geisberger et al., 2002; Königsberger et al., 2010). In germinal centres (GCs), B-cell differentiation, selection and Ig-class-switch are modulated by TNF-receptor family members, subsequently activating GC kinases like HPK1 by corresponding adaptor molecules (TRADD and TRAF), or by activated RTKs linked to SH2/SH3 adaptors. Initiating signaling events in the molecular pathogenesis of multiple myeloma (MM) are currently under investigation. A first proteome wide analysis implicated a potential contribution of HPK1 to the pathogenesis of MM demonstrating that selective knock down of HPK1 (MAP4K1) caused a lethal phenotype in MM cells (Tiedemann et al. 2010). Here, we studied expression and regulation of HPK1 in MM and effects on MM phenotype by modulation of HPK1 activity in vitro and in vivo.</jats:p>
<jats:p>HPK1 mRNA and protein expression was examined in a series of human MM cell lines and murine MPC-11 MM cells compared to human B-lymphocytes, B-CLL cells and other non-hematological tissues. Subsequently, HPK1 was overexpressed in MPC-11 cells in a kinase-active (HPK1high) and kinase-deficient (K46E) variant. In addition, HPK1 was knocked down by RNAi in MPC-11 cells (HPK1low). Subsequently, we performed analyses of the transcriptome and proteome, in particular with attention to the kinome, in order to further characterize HPK1-modulated MM cells. In addition, transformation of MM phenotype was assayed by thymidine uptake and induced apoptosis in vitro as well as by variances of subcutaneous tumor growth in murine model with regard to the different HPK1 activities in the respective MM cells (HPK1wt, HPK1high, HPK1K46E, HPK1low and corresponding controls).</jats:p>
<jats:p>HPK1 expression varied in MM cell lines tested (high: MPC-11, RPMI-8226; mid: U-266, NCI-H929, OPM-2, JJN-3, KMS-11, ANBL-6, MM1.S, OCI-MY5, MM1.R; low: L363, KMS-18, UTMC-2). HPK-1 mRNA and protein expression was found to be significantly (2-5fold) lower in purified CD138+/CD38high cells from bone marrow aspirates of MM patients as in CD19+ peripheral blood B-lymphocytes or CD19+/CD20+/CD5+ B-CLL cells. In cells derived from non-hematological tissues HPK1 was found below the detection limit. For further analyses, MPC-11 cells were generated overexpressing HPK1. Of note, kinase-active HPK1high cells showed decreased sensitivity to induced apoptosis by glucocorticoid receptor inhibition compared to HPK1wt and HPK1K46E cells. In addition, we found a consecutive increased phosphorylation of downstream signaling proteins in HPK1high cells (MEKK1, ASK1 or p38, JNK, TAK1, MEK1/2, p70S6K alpha, MAPKAPK2). Pro-apoptotic effects by hyperactivation of JNK and p38 were blocked by HPK1-induced consecutive overexpression of cell-cycle-associated genes (GADD45 gamma, Cip1, MCL-1). This observation may contribute to inhibition of caspase-dependent pathways and protect myeloma cells from dexamethasone-induced apoptosis. These in vitro findings were correlated with an accelerated tumor growth and higher tumor burden in vivo (194% vs. 100% HPK1high vs. HPK1wt and respective K46E control, day 7). In contrast, HPK1 knock down by siRNA resulted in a decline of phosphorylation of cAMP response element binding protein-kinase (CREB) and TGF beta-activated kinase-1 (TAK1), whereas pJNK1/2, pERK1, p70S6K alpha und pMAPKAPK2 were not modulated compared to HPK1wt cells. Also no significant alteration was observed in HPK1low cells regarding proliferation and apoptosis in vitro.</jats:p>
<jats:p>In conclusion, HPK1 is constitutively expressed in MM cells and shows regulatory activity concerning intracellular survival signaling in MM cells. HPK1 may contribute to the malignant phenotype so that a HPK1-targeted approach will be promising. To confirm this hypothesis, a HPK1 knock down approach in vivo is recommended.</jats:p>
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<jats:title>Disclosures:</jats:title>
<jats:p>No relevant conflicts of interest to declare.</jats:p>
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