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BCR-FGFR1 regulated by dimerization and chaperonin Hsp90
Ganetespib (Figure 7A). The BCR-FGFR1 derivatives were more sensitive to the Ganetespib than FGFR1-K656E, pos- sibly due to the lack of HSP90 association with FGFR1- K656E (Figure 6).
Furthermore, in order to determine if the effects of Ganetespib were synergistic with FGFR inhibition, a com- binatorial experiment was performed on NIH3T3 cells expressing either BCR-FGFR1 or FGFR1 derivatives, which were simultaneously treated with Ganetespib and a TKI. These cells were treated with a constant 15 nM Ganetespib and were dosed with increasing concentra- tions of BGJ398, a potent FGFR inhibitor (Figure 7B). A potentially synergistic effect is observed between Ganetespib and BGJ398, as foci production among cells expressing either BCR-FGFR1 or FGFR1 derivatives dra- matically decreases. These data suggest that the use of Hsp90 inhibition combined with TKI treatment may be beneficial for BCR-FGFR1 expressing cells.
The effect of Ganetespib was also examined using 32D IL-3-dependent cells. The IL-3-independent proliferation of the BCR-FGFR1 expressing 32D cells was reduced by treatment with Ganetespib as measured by MTT assay (Figure 7C). The Ganetespib did not affect the normal dependence of the cells on IL-3, as shown in Figure 7D. Therefore, these data suggest that BCR-FGFR1 is depend- ent on the molecular chaperone Hsp90 for cellular trans- formation.
Discussion
Considerable advances have been made in our under- standing of the molecular basis of hematological cancers. Since the identification of BCR-ABL almost 60 years ago,29 over 500 oncogenic translocations have been identified in hematopoietic cancers alone, which emphasizes the importance of identifying and characterizing these onco- genic drivers.30 With the emergence of personalized medi- cine, the characterization of activators of SCLL, such as BCR-FGFR1, is critical in determining additional therapeu- tic targets. Although the use of TKI to treat SCLL is becoming more commonplace, TKI treatment often results in drug resistance in patients, highlighting the need for additional therapies for SCLL.31
Biological and biochemical characterization of BCR-FGFR1
Through the data presented, we were able to extensive- ly characterize the fusion protein BCR-FGFR1. We demonstrate that the N-terminal fusion of BCR results in constitutive activation of FGFR1. Through our cell signal- ing studies, we demonstrate that BCR-FGFR1 activates ERK/MAPK and JAK/STAT pathways, and possesses transforming activity in NIH3T3 cells (Figure 1-2). However, BCR-FGFR1-K514A was unable to activate either pathway, or transform NIH3T3 cells, indicating that BCR-FGFR1 relies on the kinase activity of FGFR1 for acti- vation. Furthermore, 32D cells expressing BCR-FGFR1 or BCR-FGFR1-K656E were able to proliferate in the absence of IL-3, indicating the oncogenic potential of this fusion protein (Figure 3).
LC-MS/MS data additionally demonstrate that BCR- FGFR1 relies on the kinase activity of FGFR1 for onco- genic activity. Phosphorylation on key tyrosine residues in the FGFR1 kinase domain, including Y653 and Y654 in the
activation loop, is observed in BCR-FGFR1 and BCR- FGFR1-K656E, which is absent in kinase-dead BCR- FGFR1-K514A (Figure 4). Although novel phosphorylation sites were detected on BCR, these phosphoacceptor sites do not appear to be critical for the cell transformation or oncogenic ability of BCR-FGFR1 (Table 1).
Novel therapeutic targets for SCLL induced by BCR-FGFR1
Currently, hematopoietic stem cell transplantation is the primary curative option for patients who have SCLL. Here, we have described two novel potential therapeutic approaches: disruption of ionic bonding that stabilizes BCR oligomerization, and inhibition of the chaperonin Hsp90 complex.
The coiled-coil oligomerization domain of BCR has pre- viously been demonstrated to be essential for the trans- forming ability of BCR-ABL.21 However, the requirement of electrostatic interactions within the coiled-coil domain for oligomerization has not been investigated. Here, we have described a novel inhibition of BCR-FGFR1 mediated cell transformation through abolishing three salt bridge interactions in the coiled-coil domain of BCR. This abla- tion of cell transformation is seen through focus forming assays, as the disruption of these salt bridges in the BCR-FGFR1 mutant E34R/E46R/E52R (mutant 11) almost completely abolishes focus formation (Figure 5). Furthermore, the intrahelical Salt Bridge #3, formed by residue Glu52 with Arg55 in BCR, plays a crucial role in providing stability for the coiled-coil domain in BCR. The apparent ability of Arg55 to form complex salt bridges with Glu34 and Glu52 highlights the potential importance of cooperativity exhibited by complex salt bridges and their contribution to protein stability.32 Taken together, these data suggest that these three salt bridges provide a critical role in the activation of BCR-FGFR1. The potential loss of oligomerization and near absence in focus forma- tion suggests that the coiled-coil domain of BCR is an attractive therapeutic target for SCLL.
Additionally, we have shown that BCR-FGFR1 is a client of the Hsp90 chaperone complex, and potentially uses this complex to avoid proteasomal degradation in the cell. Previous work has shown that the FGFR1OP2-FGFR1 fusion is also a client of the Hsp90 complex, and that tar- geting the Hsp90 complex resulted in reduced activity of this fusion protein.33 The interaction and dependence of BCR-FGFR1 on the Hsp90 complex is established through cell transformation assays, and analysis of downstream cell signaling (Figure 6-7). A decrease in overall expression of BCR-FGFR1 is detected with the addition of Ganetespib, a potent Hsp90 inhibitor. Furthermore, BCR- FGFR1 expressing cells treated with Ganetespib displayed a reduction in STAT and MAPK activation, and no FGFR1 phosphorylation. Additionally, the transformation ability of cells expressing BCR-FGFR1 decreases when treated with increasing concentrations of Ganetespib (Figure 7A), indicating that this fusion protein relies on the Hsp90 complex for cellular stability, and is sensitive to Hsp90 inhibition. We also examined the combined effects of Ganetespib with the FGFR inhibitor BGJ398 in NIH3T3 cell transformation assays (Figure 7B), and in 32D cell pro- liferation assays in the absence and presence of IL-3 (Figure 7C-D). Taken together, these data show that BCR- FGFR1 may rely on the Hsp90 molecular chaperone com- plex to avoid proteasomal degradation.34
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