Abstract
Introduction
Hodgkin Lymphoma (HL) accounts for 11% of all lymphomas and despite being one of the most curable lymphomas, 20% of HL patients still ultimately die of their disease. Similarly, a proportion of cases of primary mediastinal B cell lymphoma (PMBCL) have refractory disease or early relapse and frequently fail second-line therapy. Development of more targeted therapeutic approaches is impeded by the lack of knowledge about the mutational landscape in the cancer genomes of these lymphomas. PTPN1 is a protein tyrosine phosphatase gene that encodes the protein, PTP1B. PTP1B dephosphorylates tyrosine residues on many activated kinases to maintain cellular homeostasis. As overactive receptor kinases are critical oncogenic events in cancer, we hypothesized that constitutively active Janus kinase-Signal transducer and activation of transcription (JAK-STAT) observed in HL and PMBCL are in part due to a mutated PTPN1 gene with an impaired functional ability to dephosphorylate this constitutive signaling pathway.
Methods and samples
Biopsies at the time of primary diagnosis were obtained for 49 PMBCL and 30 HL patients from the British Columbia Cancer Agency, Arizona Lymphoma Repository and the Hôpital Henri Mondor Pathology Department. DNA from PMBCL samples, microdissected Hodgkin Reed Sternberg (HRS) cells and 12 lymphoma-derived cell lines were extracted for PTPN1 exonic PCR amplification (nested PCR was used for HRS cell DNA) and Sanger sequencing. PTPN1 was silenced in a HL cell line (KMH2) by lentiviral transduction of a vector expressing shRNA and confirmed by quantitative real time (qRT) PCR. Wild type and mutant PTPN1 cDNA were cloned into the mammalian expression vector pcDNA 3.1 and expressed in HEK-293 cells. Protein expression of clinical samples, silenced and expressed cells were analyzed by immunohistochemistry and western blotting. Comparisons between groups were performed using two-sample student t tests.
Results
After exclusion of reported single nucleotide polymorphisms (SNPs) and silent mutations, 16 PTPN1 coding sequence mutations were found in our PMBCL cohort, corresponding to 14 mutations (29%) in clinical samples and 2 in PMBCL-dervied cell lines. Twelve additional mutations were discovered in our HL cohort, corresponding to 6 mutations (20%) in HRS cell samples and another 6 in HL-derived cell lines. In total, 14 (54%) missense, 4 (15%) frameshift, 3 (12%) single amino acid deletions, 4 (15%) nonsense mutations, and 1 (4%) promoter mutation were observed. Eight of these mutations were confirmed as somatic by sequencing of matched constitutional DNA. Silencing of PTPN1 resulted in hyperphosphorylation of JAK1, JAK2, STAT3, STAT5, STAT6 and up-regulation of the oncogenes, MYC and BCL6. Ectopic expression of nonsense and missense PTPN1 mutants in HEK-293 cells led to sustained phosphorylation of STAT6 in comparison to the empty vector control (densitometric values Q9* 0.5 vs. 1.0, R156* 0.7 vs. 1.0, M74L 0.4 vs. 1.0 and M282L 0.8 vs. 1.0). Furthermore, no phosphatase activity was observed for the nonsense mutants and moderate phosphatase activity for the missense mutants using a tyrosine phosphatase-specific substrate (fold change Q9* 2.0, R156* 1.9, M74L 46.7, M282L 46.0 and WT 58.3, compared to empty vector control). Immunohistochemical analysis showed that PTPN1 mutations correspond to decreased protein expression in PMBCL (p=0.03).
Discussion
PTPN1 is recurrently mutated in PMBCL and HL contributing to constitutive JAK-STAT signaling and oncogene dysregulation. These data suggest PTPN1 mutations as novel driver alterations in these lymphomas and might provide a novel, rational therapeutic target for treating HL and PMBCL patients.
Disclosures:
Savage: Eli-Lilly: Consultancy. Connors:F Hoffmann-La Roche: Research Funding; Roche Canada: Research Funding.
Reporter gene HEK 293 cells and WNT/Frizzled fusion proteins as tools to study WNT signaling pathways