Identification of the novel KP fusion in a glioblastoma tumor. (A) A complex amplicon on chromosome 4 is profiled by high-resolution aCGH (median probe interval, 53 bp). Relative amplification of the 3′ portion of PDGFRA and the 5′ portion of the KDR locus was identified, suggesting possible gene fusion. (B) Schematic diagrams of KDR and PDGFRA partial gene structures as well as the formation of the KP fusion transcript, and of the predicted KP, KDR, PDGFRA, and PDGFRA^{Δ8, 9} mutant proteins. Because the KDR gene is on the minus strand of chromosome 4, the creation of this fusion appears to have involved an intrachromosomal inversion. (Ig-like domain) Immunoglobulin-like domain; (S) signal sequence; (TM) transmembrane domain; (JM) juxtamembrane domain; (C) cryptic exon from KDR intron 13. (C) RT–PCR detection of the KP fusion. The presence of the KP fusion was revealed by RT–PCR using different combinations of KDR forward primers and PDGFRA reverse primers. (M) Molecular marker öX174/HaeIII (Invitrogen); (K9 and K13) forward primers in KDR exons 9 and 13, respectively; (P7, P9, and P12) reverse primers in PDGFRA exons 7, 9, and 12. (D) Partial sequence of the KP fusion transcript. Sequencing of RT–PCR products of K13/P12 and K9/P12 identified the sequence around the fusion point of the KP transcript. The transcript is an in-frame fusion of KDR exon 13 (red) to PDGFRA exon 10 (yellow) with an intervening cryptic exon from KDR intron 13 (blue). The green area indicates 8 bases of overlap sequence between intron 13 of KDR and exon 10 of PDGFRA. The translated amino acid sequence is shown below the transcript sequence.

Histopathological features of glioblastomas with the PDGFRA mutant. Representative images of H&E, PDGFRA, GFAP, and Olig2 staining are shown at 20× or 40× magnification. Tumors with the PDGFRA mutation demonstrate histological features of oligodendroglioma, as shown by round nuclei, perinuclear halos, a chicken wire-like capillary network, and minigemistocytes with prominent Olig2 expression. GBM (MSK352x) has a PDGFRA transcript with PDGFRA amplification. Bars, 50 μm.

The KP fusion gene is a transforming oncogene. (A) Cells expressing the KP fusion are morphologically transformed. Cell morphology of NIH3T3 cells expressing mock (panels a,e), the PDGFRA^{Δ8, 9} mutant (panels b,f), the KP fusion (panels c,g), and the kinase-inactive mutant (K831R) of the KP fusion (panels d,h) were photographed at 100× (top panels) or 200× (bottom panels) magnification (bars, 10 μm). (B) Analysis of anchorage-independent growth. NIH3T3 cell lines described in A were maintained on soft agar with 5% calf serum; colony formation in soft agar is shown with 1× (top, panels a–d) or 100× (bottom, panels e–h) magnification after 4 wk (bars, 10 μm). (C) Tumor formation in nude mice. Cell lines as described in A were inoculated subcutaneously into the flanks of nude mice. Each tumor was measured weekly and was photographed after 6 wk of inoculation. (D) The growth curve of tumors as described in C. The KP fusion (n = 6) was compared with mock (n = 6) or K831R (n = 4) at the final point, respectively. (***) P < 0.001. Tumor formation of cells expressing the KP fusion was reproduced four times in an independent experiment. Representative results are shown in C and D.

The PDGFRA^{Δ8, 9} mutant is a recurrent gene rearrangement. (A) RT–PCR detection of the PDGFRA^{Δ8, 9} mutant in glioma tumors. Identification of the PDGFRA^{Δ8, 9} mutant was revealed by RT–PCR analysis with a PDGFRA-S3 and PDGFRA-AS2 primer pair that covers an in-frame junction. The putative PCR products of the wild type and the PDGFRA^{Δ8, 9} mutant are 885 and 642 bp, respectively. Molecular weight (M) is shown in the figure. Sequencing of RT–PCR products from the lower bands of MSK538, MSK543, MSK572, MSK573, KB6, KTS647, and KT648 revealed an in-frame deletion of 243 bp. PMXIG-PDGFRA and PDGFRA^{Δ8, 9} plasmids were used as controls for wild-type PDGFRA (WT) and the PDGFRA^{Δ8, 9} mutant (DM), respectively. KTS647 and KTS648 are samples from the same tumor taken from different regions during resection. Samples with the mutation are marked with an asterisk. (B) PDGFRA gene quantitation in GBM tumors. PDGFRA gene amplification was identified using q-PCR analysis in tumors showing high expression of the PDGFRA transcript (Supplemental Fig. S2). The Y-axis indicates average PDGFRA gene quantities relative to the reference sample and 18S gene. Average DNA quantities greater than four were defined as gene amplification (dotted line). (White bars) Normal lymphocytes; (blue bars) GBMs; (red bars) GBMs with the PDGFRA^{Δ8, 9} mutant; (yellow bars) grade 3 gliomas, including anaplastic astrocytoma, oligodendroglioma, and ependymoma; (green bars) grade 2 gliomas, including diffuse astrocytoma, oligodendroglioma, mixed oligoastrocytoma, and pilomyxoid astrocytoma; (magenta bars) grade 1 gliomas, including pilocytic astrocytoma. (C) Summary of PDGFRA^{Δ8, 9} mutant screening. (TMs) Tumors.

(A) The KP fusion (KP) and PDGFRA^{Δ8, 9} mutant (DM) are both constitutively active TKs. (Top panel) After 24 h of serum-free conditions, cells expressing mock (lane 1), the PDGFRA^{Δ8, 9} mutant (lane 2), and the KP fusion (lane 3) were immunoprecipitated (IP) with anti-c-myc agarose, and the immunoblots (IB) were probed with anti-phosphotyrosine antibody. Whole-cell lysates were assayed with the indicated antibodies for input of immunoprecipitation or detection of phosphotyrosine (Y720) and downstream signaling. (B) In vitro kinase assay of NIH3T3 cells expressing mock (lane 1), the KP fusion (lane 2), and the kinase-inactive mutant (K831R) of the KP fusion (lane 3). (Top panel) Whole-cell lysates from each cell line were immunoprecipitated with anti-c-myc agarose after 24 h of serum-free conditions, and then subjected to an in vitro kinase reaction with [γ-³²P] ATP. (Bottom panel) Whole-cell lysates were also subjected to immunoblot analysis with anti-c-myc antibody for input of immunoprecipitation. Autophosphorylation of the KP fusion was detected by autoradiography. (C) The KP fusion behaves as a constitutively active PDGFRA. Cells expressing mock (lane 1), the KP fusion (lane 2), and the kinase-inactive mutant (K831R) of the KP fusion (lane 3) were harvested after 24 h of serum-free conditions. As a control, the cells expressing WT-PDGFRA (lane 4) and WT-KDR (lane 5) were stimulated with PDGF-BB (PB) or VEGF165 for 5 min after 24 h of serum-free conditions, respectively. The cells were then subjected to immunoblot analysis with the indicated antibodies. (D) The KP fusion behaves as an active kinase in various cells. (Left panel) Cos 7 cells. (Middle panel) T98G glioma cells. (Right panel) primary glial cells. DNA plasmids indicated were introduced with transient transfection (Cos 7 cells) or with retroviral infection (T98G and primary glial cells). Cos 7 cells were incubated for 12 h in serum-free DMEM after 60 h of transfection, and were immunoprecipitated with anti-HA agarose. Whole-cell lysates were then subjected to immunoblot analysis with the indicated antibodies. T98G and primary glial cells were immunoprecipitated with anti-myc agarose after 12 or 24 h of serum-free conditions, and were then subjected to immunoblot analysis with the indicated antibodies as well. Cells expressing H-RasV12 were used as a control of the experiment. (E) Immunoprecipitation analysis of KP fusion homodimerization in NIH3T3 cells. (F) Immunoprecipitation analysis of KP fusion homodimerization and heterodimerization in 293 cells. Representative blots from reproduced experiments are shown.

PDGFRA mutants are potential therapeutic targets of the RTK inhibitors PTK787 and Gleevec. (A) PTK787 and Gleevec inhibit tyrosine phosphorylation with active downstream signals of PDGFRA mutants in a concentration-dependent manner. Cells expressing the PDGFRA^{Δ8, 9} mutant or KP fusion were treated with 0.1% DMSO (D) and PTK787 (P1: 1 μM PTK787; P10: 10 μM PTK787) or Gleevec (G1: 1 μM Gleevec; G10: 10 μM Gleevec) at the indicated drug concentrations. After 2 h of treatment, the cells were harvested and whole-cell lysates were subjected to immunoblot analysis with the indicated antibodies. A representative blot is shown. (B) The effects of PTK787 and Gleevec on the transformed phenotype of NIH3T3 cells expressing PDGFRA mutants. Cells expressing the KP fusion or PDGFRA^{Δ8, 9} mutant were treated with 0.1% DMSO, PTK787, or Gleevec at the indicated drug concentrations. Cell morphology was observed and documented at 100× magnification after 72 h of individual treatment (bars, 10 μm). (C) The effects of PTK787 and Gleevec on anchorage-independent growth of PDGFRA mutants. Cells expressing the KP fusion or PDGFRA^{Δ8, 9} mutant were maintained on soft agar with 5% calf serum containing 0.1% DMSO, PTK787, or Gleevec at the indicated drug concentrations. Fresh medium containing 5% calf serum with DMSO, PTK787, or Gleevec was added onto the upper agar every 4 d. The colonies were photographed at 100× magnification after 3 wk. DMSO control was compared with 1 μM PTK787 treatment against the KP fusion or PDGFRA^{Δ8, 9} mutant cells, respectively. (***) P < 0.0001). Bars, 10 μm. (D) The effect of treatment with various inhibitors on the transformed phenotype induced by PDGFRA mutants. The NIH3T3 cells expressing the KP fusion or PDGFRA^{Δ8, 9} mutant were treated with 0.1% DMSO, 10 μM U0126, 10 μM LY294002, 1 nM rapamycin, and the indicated combinations of inhibitors. Cell morphology was photographed at 100× magnification after 72 h of individual treatment (bars, 10 μm).