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ative option for SCLL patients is hematopoietic stem cell transplantation. Although both Ponatinib and Pemigatinib (INCB054828) have been used to treat patients with mixed results, few other alternative treatment plans exist for patients who are either awaiting or are unable to receive transplantation.4,5 This work focuses on the t(8;22)(p11;q11) chromosomal translocation resulting in the BCR-FGFR1 fusion protein with exon 4 of the break- point cluster region (BCR) fused to exon 9 of FGFR1. Although BCR was first identified fused to Abelson murine leukemia viral oncogene homolog-1 (ABL), also known as the Philadelphia chromosome, BCR has since then been identified fused to ret proto-oncognene (RET), Janus kinase 2 (JAK2), and platelet derived growth factor receptor alpha (PDGFRA).6-9 Although a common fusion partner, the endogenous function of the BCR gene remains obscure. The fusion protein BCR-FGFR1 retains the coiled-coil dimerization/oligomerization domain, putative serine/threonine kinase domain, and partial RhoGEF domain from BCR.10
The BCR-FGFR1 fusion is not well characterized, and this work seeks to elucidate the underlying mechanisms behind BCR-FGFR1 mediated SCLL. Although tyrosine kinase inhibitor therapies (TKI) are traditionally used to treat certain hematological cancers, the use of TKI often results in drug resistance in patients. Thus, it is crucial to determine additional therapeutic strategies in treating hematopoietic cancers. Here we suggest disruption of the BCR coiled-coil dimerization domain and Hsp90 inhibi- tion as novel therapeutic targets for BCR-FGFR1 driven SCLL. Data presented here may also allow for additional approaches in treating BCR-ABL mediated CML, due to the similarity between BCR-ABL and BCR-FGFR1 fusion proteins.
Methods
DNA Constructs
The BCR gene (pSG65-Bcr) was purchased from Addgene (Watertown, MA, USA) and was subcloned into pcDNA3. FGFR1 and FGFR1-K656E were previously described.11 To construct BCR- FGFR1, a BamHI site was introduced by PCR-based site-directed mutagenesis after amino acid L584 in BCR and before amino acid V429 in FGFR1. This unique internal BamHI site was used togeth- er with an upstream site of EcoRI to subclone the 5’ region of BCR into the FGFR1 pCDNA3 plasmid, creating a fusion breakpoint of BCR exon 4 fused to FGFR1 exon 9. The BamHI site adds 6 bases which code for a GS linker between the 5’ BCR and the 3’ FGFR1. FGFR1-K514A12 and all other mutations described were intro- duced by PCR-based site-directed mutagenesis. FGFR1 or BCR- FGFR1 clones were subcloned into the pLXSN expression plasmid for use in NIH3T3 or 32D cells. Details of plasmid DNA used are in the Online Supplementary Material and Methods.
starvation at 10% CO2, 37°C. Transfected NIH3T3 cells were maintained with 0, 10, 20, 23, 25, or 30 nM Ganetespib for 14 days, and fixed and scored as described. For combination drug treatment, 15 nM Ganetespib was used with the FGFR inhibitor BGJ398 at 0, 2.5, or 10 nM. All cell assays were performed a min- imum of three times. More detailed information is provided in the Online Supplementary Materials and Methods.
Interleukin-3 (IL-3) independent growth in 32D cells
A total of 1x106 32D cells were electroporated (1,500V, 10 ms, 3 pulses) by Neon Transfection system using 30 mg of FGFR1 or BCR-FGFR1 derivatives in pLXSN in triplicate. 48 h after transfec- tion, cells were selected with 1.5 mg/mL Geneticin (G418) for 10 days to generate stable cell lines before starting IL-3 independent growth assays. Triplicate flasks were seeded with the cell lines at 4x104 cell/mL in the presence or absence of mouse IL-3. In addi- tion, 1 nmol/L of FGF and 30 ug/mL of heparin was added to a set of flasks in the absence of IL-3. On days 1, 3, 5, 7, and 9 samples were counted and measured for MTT metabolic activity as described.16 For Ganetespib treatment, cells were seeded with 0, 2.5, or 5.0 nM Ganetespib –/+ IL-3. MTT metabolic activity was measured on days 3, 5, and 7. A concentration of 10 nM or higher of Ganetespib was found to be toxic to 32D cells in the presence of IL-3.
Mass spectrometry sample preparation
Liquid chromatography/mass spectroscopy (LC-MS/MS) for phosphopeptide analysis was as described previously.16 Complete information is available in the Online Supplementary Material and Methods.
Results
Signaling cascade activation by BCR-FGFR1
The role of BCR in this fusion protein has remained unclear, as BCR-FGFR1 retains the coiled-coil oligomeriza- tion domain, putative serine/threonine kinase domain, and partial RhoGEF domain from BCR. In order to eluci- date if BCR-FGFR1 relies on the tyrosine kinase activity of FGFR1, a K514A kinase dead mutation,12 or a K656E kinase activating mutation,11,15 were introduced in the FGFR1 tyrosine kinase domain in both FGFR1 and BCR- FGFR1 backgrounds (Figure 1A). These studies were per- formed in HEK293T cells, as they have previously been used in FGFR signal transduction and protein phosphory- lation studies.16 HEK293T cells expressing either full- length FGFR1 or BCR-FGFR1 variants were analyzed for MAPK, STAT3 and STAT5 activation, and FGFR1 receptor phosphorylation. Expression of FGFR1 shows slight acti- vation of the MAPK pathway, while expression of the activated FGFR1-K656E shows elevated phosphorylation levels of MAPK (Figure 1B, panel 7). Expression of BCR- FGFR1 or BCR-FGFR1-K656E also resulted in MAPK phos- phorylation. Additionally, STAT3 and STAT5 phosphory- lation were strongly elevated by BCR-FGFR1 and BCR- FGFR1-K656E, in comparison to FGFR1 or FGFR1-K656E, indicating that BCR-FGFR1 induces both MAPK and STAT pathway activation (Figure 1B, panel 3 and 5).
To examine the phosphorylation of each fusion con- struct compared with FGFR1, FGFR1-K656E and FGFR1- K514A were expressed in HEK293T cells, collected and immunoprecipitated with a C-terminal FGFR1 antibody, and probed for tyrosine phosphorylation. An increase in tyrosine phosphorylation was observed in both BCR-
Cell transfection, immunoprecipitation, immunoblot analysis
HEK293T cells were transfected with 3 mg of the pcDNA3 plas- mid constructs using calcium phosphate transfection as described.13 Immunoblotting was performed as described.14 NIH3T3 focus assays were performed as described.15 Number of foci were, normalized by transfection efficiency, and quantitated relative to a positive control +/-standard error of the mean (SEM).
The Hsp90 inhibitor, Ganetespib was added to HEK293T trans- fected cells at a concentration of 200 nM for 4 hours (h) during cell
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BCR-FGFR1 regulated by dimerization and chaperonin Hsp90
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