Our data support a two-step model by which FGFR3 activates RSK2 and mediates transformation signals in hematopoietic cells. The first step involves tyrosine phosphorylation at Y529 of RSK2 by FGFR3, which facilitates binding of the inactive form of ERK to RSK2 in the initial step of ERK-dependent RSK2 activation (, Step 1). This binding, which is required for phosphorylation and activation of RSK2 by ERK, in turn promotes the second step where ERK is activated via the Ras/Raf/ MEK/MAPK pathway downstream of FGFR3, leading to ERK-mediated phosphorylation and activation of RSK2 (, Step 2). Thus, FGFR3 plays a dual role in the activation of RSK2 by both assisting inactive ERK binding to RSK2 and activating ERK to phosphorylate and activate RSK2. Moreover, inhibition of RSK2 by specific siRNA or small molecule inhibitor fmk effectively induces apoptosis in human t(4;14)-positive myeloma cells that express FGFR3, which demonstrates the importance of the RSK2 pathway in FGFR3-related myeloma. These studies therefore demonstrate that RSK2 is a critical signaling effector of FGFR3 and may represent a potential therapeutic target in hematologic malignancies associated with dysregulated FGFR3.
Proposed Two-Step Model that FGFR3 Activates RSK2 to Mediate Hematopoietic Transformation Involving Tyrosine Phosphorylation at Y529 in RSK2
Tyrosine phosphorylation at Y529 may provide an additional docking site to promote the binding of inactive ERK to the C terminus of RSK2. Future detailed structural studies would illuminate this process. FGFR3 might not be the only upstream tyrosine kinase that phosphorylates RSK2 at Y529 as we have observed that upon treatment of EGF, RSK2 is tyrosine phosphorylated at Y529 and activated in 293T cells that do not express FGFR3 (S.K., S.D., T.-L.G., A.G., S.L., H.J.K., R.D.P., and J.C., unpublished data). This suggests that phosphorylation at Y529 might be a general requirement for RSK2 activation through the ERK/MAPK pathway. Further studies to identify the alternative upstream tyrosine kinase(s) of RSK2 as well as the role of phospho-tyrosine residues besides Y529 in the activation and function of RSK2 are warranted.
Although Y529 is highly homologous in both RSK1 and RSK2, RSK1 was not detected to be tyrosine phosphorylated in our proteomics studies (data not shown). This is in consonance with our observations that FGFR3 specifically activates RSK2 but fails to significantly activate RSK1 in Ba/F3 cells (data not shown) as well as the lack of apoptosis induced following siRNA knockdown of RSK1 in FGFR3-expressing human myeloma cells (). Thus, FGFR3 may specifically signal through RSK2 to mediate transformation signaling.
Fmk as a highly specific RSK inhibitor induces significant apoptosis in primary CD138-positive myeloma cells from FGFR3-expressing, t(4;14)-positive multiple myeloma patient with minimal nonspecific cytotoxicity (). Interestingly, the sensitivity to fmk is different among t(4;14)-positive HMCLs. Fmk induces significant apoptosis in KMS11, OPM1, and LP1 cells, whereas KMS18 cells are relatively resistant ( and ). This difference suggests that there may be other oncogenic abnormalities that are not responsive to fmk treatment in KMS18 myeloma cells in addition to FGFR3. For example, in addition to activation of FGFR3
, the t(4;14)(p16;q32) also results in creation of a chimeric fusion transcript between IGH
domain) (Chesi et al., 1998b
). Larger numbers of cell lines will need to be evaluated to determine efficacy of fmk in HMCLs that overexpress FGFR3.
Fmk as a first generation RSK inhibitor shows promising but so far limited effectiveness in treatment of FGFR3-expressing myeloma cells, compared to the FGFR3 inhibitor PKC412 (). Fmk was designed to specifically target the CTD auto-kinase domain of RSK1, 2, and 4; however, it cannot completely abrogate the phosphorylation of Ser386 of RSK2 (;Cohen et al., 2007
;Cohen et al., 2005
). Cohen et al. recently reported that a fmk derivative, fmk-pa, inhibits RSK Ser386 phosphorylation at saturating concentrations following stimulation of phorbol ester, but has no effect on RSK activation by lipopolysaccharide (Cohen et al., 2007
). These findings together suggest that RSK CTD-dependent autophosphorylation at Ser386 is context dependent, and alternative kinases may exist and bypass the CTD requirement and phosphorylate Ser386 in the RSK2 hydrophobic motif, which therefore limits the therapeutic effects of fmk. Indeed, PDK1 can phosphorylate RSK at Ser386 in vitro, and Ser386 is also within the identified consensus phosphorylation motif of RSK NTD domain (Richards et al., 2001
). On the other hand, the RSK2 NTD transkinase domain is responsible for phosphorylation of RSK2 substrates such as histone H3 and BAD. Thus, targeting RSK CTD and NTD should have different physiological effects, which warrants future studies to test other potent RSK inhibitors that inhibit RSK NTD, such as BI-D1870 (Sapkota et al., 2007
), or compounds target both kinase domains of RSK2. Such inhibitors may have enhanced therapeutic efficacy to inhibit FGFR3-mediated transformation signaling.
Fmk is also able to induce significant apoptotic cell death in the t(4;14)-negative human myeloma cell line RPMI8226 that harbors an active RAS
K12 mutation (), suggesting a wider therapeutic implication of targeting RSK in treatment of both FGFR3-positive and -negative multiple myeloma. Activating mutations of FGFR3 have been identified in human bladder and cervical carcinomas (Cappellen et al., 1999
). Thus, our findings may also have therapeutic implications with regard to solid tumors associated with dysregulation of FGFR3.