A Novel Cell Line, Tnet-2, Derived from "Triple-Negative" Essential Thrombocythemia

【Introduction】 Myeloproliferative neoplasm (MPN) is a heterogeneous group of disorders, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). It is known that mutations of JAK2, CALR, or MPL gene are causative alternations of MPNs. However, 10-15% of MPNs have no mutations in any of these three genes, so-called "triple negative" (TN) MPN. It remains unclear that what genetic alternations cause TN-MPN. Cell lines derived from clonal hematopoietic disorders are useful tools to characterize diseases as well as to identify effective reagents for such diseases. To our knowledge, there is no cell line derived from TN-MPN although there are several lines with JAK2 V617F or CALR mutation. We have encountered an aggressive ET case with TN phenotype. Three years after initial diagnosis, blastic transformation occurred in this patient. Both conventional chemotherapy and allogeneic transplantation failed to cure the patient. After relapse, various therapies including ruxolitinib (JAK inhibitor) failed to control the disease. We have successfully established a cell line from the leukemic cells of this patient. We have also characterized the cell line by whole genome sequencing (WGS), RNA sequencing (RNA-Seq), as well as in vitro assays.

【Material and Methods】 We have established a novel cell line, TNET-2, from the leukemic cells. WGS and RNA-Seq were performed at Beijing Genome Institute (BGI) Japan. Proliferation assays were performed in the presence or absence of ruxolitinib, AC220 (FLT3 inhibitor), giltertinib (FLT3/AXL inhibitor), etoposide, cytarabine or hydroxycarbamide.

【Results】 The patient's leukemic cells have been cultured in RPMI1640 culture media with 10% fetal bovine serum and antibiotics. The cells were exponentially growing more than one year and the cell line, TNET-2, has been established. WGS has found 116 missense mutations in 110 genes encoding proteins. Also, five nonsense mutations causing early termination codons were found. InDel mutations causing frame-shifts were found in 28 genes. In-frame deletions were found in seven genes and in-frame insertions were found in nine genes. No mutations were found in JAK2, MPL, or CALR gene. A nonsense mutation was found in ASXL1 gene. Multiple genes encoding proteins associated with myeloid proliferation/differentiation, including CBL, A-RAF, HOXB9 and BCL11B, were mutated. Genes involved in DNA repair, for example ERCC6, were also mutated. Genes involved in apoptosis including CASP7, TRAK1 and SOCS3 were mutated. RNA-Seq revealed high expression of IRAK1 mRNA as well as low expression of TET2. Cell proliferation assays showed TNET-2 cells were resistant to ruxolitinib and conventional chemotherapeutic reagents including etoposide, cytarabine and hydroxycarbamide. However, both AC220 and giltertinib induced cell death.

【Discussion】 It has been reported that alternations of ASXL1 and/or TET2 genes are common in TN-MPNs. Consistent with the reports, a nonsense mutation of ASXL1 gene and low expression of TET2 were detected in TNET-2 cells. Besides these well-known genetic changes, we have found more than 150 mutated genes. Some of them seem "actionable" / "targetable" mutations. There are no optimal treatments targeting an altered signaling pathway for TN-MPNs. Our novel cell line is a useful tool to characterize this subtype of MPN and find novel treatment options for this rare type of disease. In summary, we have, for the first time, established a cell line derived from TN-MPN and found novel altered signaling pathways by WGS, RNA-Seq and in vitro assays.