High PDGFRA Expression Does Not Serve As Effective Therapeutic Target in ERG-Deleted B-Cell Precursor Acute Lymphoblastic Leukemia

Abstract

Background and aimsSeveral oncogenic aberrations of receptor tyrosine kinases occur in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we describe a subgroup of pediatric patients lacking common chromosomal aberrations but with high gene expression of platelet-derived growth factor receptor alpha (PDGFRA). Oncogenic fusions involving the tyrosine kinase domain of PDGFRA are recurrent in eosinophilic leukemia and are responsive to kinase inhibitors (Cools et al. N Engl J Med 2003). One case with a FIP1L1-PDGFRA fusion was reported in adult BCP-ALL (Roberts et al. J Clin Oncol 2016). Therefore, we tested for genomic aberrations underlying the overexpression of PDGFRA and evaluated the inhibition of PDGFRα as treatment option in pediatric BCP-ALL.MethodsExpression profiling was performed using Affymetrix U133 plus 2.0 arrays. BCR-ABL1 -like cases were identified using the 110 probe set expression signature (Den Boer et al. Lancet Oncol 2009). High PDGFRA was defined as log2 expression of probe set 203131_at >5.9. FISH was performed using Cytocell FIP1L1/CHIC2/PDGFRA probes. Targeted locus amplification (de Vree et al. Nat Biotechnol 2014) was performed using custom-designed primers. Cells were treated for 10 minutes with 10 ng/mL PDGF-BB with or without 10 µM imatinib or 1 µM CP673451 and analyzed by western blot. Cell viability after 4 day exposure to imatinib (0.2µM - 1.6µM - 12.5µM) or CP673451 (0.02µM - 0.16µM - 1.25µM) was assessed by flow cytometry.ResultsGene expression analysis of 654 pediatric ALL cases identified high expression of PDGFRA in 26 of 574 BCP-ALL cases (4.5%). High PDGFRA expression was exclusively found in cases belonging to the B-other (n=22) or BCR-ABL1 -like (n=4) subtypes, and not present in ETV6-RUNX1, high hyperdiploid, TCF3-PBX1, BCR-ABL1 and KMT2A -rearranged BCP-ALL subtypes or T-ALL. PDGFRA expression was confirmed by RT-qPCR, and also found in the BCP-ALL cell line Nalm6. Using RT-PCR we did not detect any of the six reported PDGFRA fusions, which involve FIP1L1, STRN, BCR, CDK5RAP2, ETV6, and KIF5B, nor did we detect novel structural aberrations affecting PDGFRA by FISH. We studied the PDGFRA locus at high resolution by targeted locus amplification. Following DNA cross-linking, digestion, and proximity-based re-ligation, targeted amplification was performed from viewpoints in intron 2 and exon 15 of PDGFRA. In the eosinophilic cell line EOL1 we confirmed an interstitial deletion on chromosome 4 which rearranges FIP1L1 intron 12 to PDGFRA exon 12. The results obtained from Nalm6 and primary BCP-ALL samples with high PDGFRA expression did not indicate genomic aberrations of PDGFRA.ERG deletions were present in 10 of 16 PDGFRA -high cases (63%), compared with 0% in PDGFRA -low BCP-ALL cases (n=71; p<0.001). Vice versa, all ERG-deleted cases expressed high levels of PDGFRA . Recently, a novel BCP-ALL subtype was described with rearrangements of the transcription factor DUX4 and deletion of ERG (Lilljebjörn et al. Nat Commun 2016; Yasuda et al. Nat Genet 2016; Zhang et al. Nat Genet 2016). Furthermore, the high PDGFRA-expressing cell line Nalm6 carries a DUX4 rearrangement (Yasuda et al. Nat Genet 2016) and PDGFRA is highly expressed in DUX4 BCP-ALL and its transcription start site directly bound by DUX4 (Zhang et al. Nat Genet 2016). Therefore, we regard it as highly likely that the PDGFRA-high cases represent this novel DUX4- rearranged subtype.Because transcription factors are difficult to target with small molecule inhibitors, PDGFRα inhibition could represent a treatment option. Exposure to the PDGFR inhibitors imatinib and CP673451 reduced phosphorylation of PDGFRα in Nalm6 and primary BCP-ALL cells. However, ex vivo drug exposure revealed no cytotoxic effect of the PDGFR inhibitors on PDGFRA-high BCP-ALL cells, either in the presence or absence of the ligand PDGF-BB. Interestingly, we found a marked ex vivo sensitivity towards prednisolone in PDGFRA-high cases, in line with the good prognosis reported for ERG -deleted/DUX4-rearranged BCP-ALL.ConclusionsWe found high PDGFRA expression, in the absence of genomic aberrations of the PDGFRA locus, in a subgroup of BCP-ALL patients with frequent ERG deletions. PDGFR inhibition caused abrogation of PDGFRα phosphorylation but was not cytotoxic in PDGFRA-high expressing BCP-ALL cells. Therefore, high PDGFRA expression does not serve as effective therapeutic target in ERG -deleted BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.