Abstract A20: Development and validation of a novel acute myeloid leukemia xenograft model that is dependent on the JAK2V617F mutation for growth in vivo
Authors: Lee Leslie , Hanan Emily , Zhou Aihe , Rawson Thomas , Li Ji , Kholi Pawan Bir , Barrett Kathy , Chang Christine , Chen Jacob , Ubhayakar Savita , Siu Mike , Kenny Jane , Blair Wade , Ghilardi Nico , Sampath Deepak 
Published: June 2012

Background: The Janus-associated kinase (JAK) proteins are a family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that play an important role in cellular survival, proliferation, and differentiation. A gain of function mutation (V617F) in the pseudo-kinase domain of JAK2 is detected at high frequency in Philadelphia-negative myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. To further elucidate the role of JAK2V617F on cell growth in vivo we established and validated a novel xenograft model using the acute myeloid leukemia cell line, SET2, which is heterozygous for the mutation.

Materials and Methods: SET2 cells containing stable doxycycline-inducible JAK2V617F shRNA clones were generated by lentiviral transduction. Cell proliferation and viability were assessed by incorporation of 3H-Thymidine or by ATP quantitation. Cell-cycle and apoptosis markers were evaluated by Western blotting using antibodies against cyclin D1, PIM1, cleaved caspase 3 and 7 and PARP. Phospho-STAT5 and total STAT5 were measured using both western blotting and ELISA. A SET2 shRNA clone was selected for in vivo growth in SCID beige female mice and treated with doxycycline or a JAK2 inhibitor (JAK2i). Plasma and tumor drug levels were measured by LC/MS.

Results: Genetic knockdown using a doxycycline-inducible JAK2V617F shRNA confirmed SET2 cells are dependent on this gain of function mutation for growth based on inhibition of cell proliferation in the presence of doxycycline. The latter was corroborated with a selective JAK2i (Ki= 4nM) that potently inhibited SET2 cell viability (EC50 = 355nM). The JAK2i rapidly suppressed phosphorylation of STAT5 (EC50 = 313nM) and induced cycle arrest at the G0–G1 phase based on downregulation of cyclin D1 expression. The JAK2i also effectively inhibited expression of PIM-1 kinase, a STAT5 target gene, and activated cleavage of procaspase 3 and 7 and DNA-repair enzyme PARP to induce apoptosis in a time- and dose-dependent manner. A SET2 xenograft model was established by in vivo selection of a variant of the doxycycline- JAK2V617F shRNA parental cell line. Its dependence on JAK2V617F was confirmed by treatment with doxcycline and oral administration of a JAK2i, which resulted in significant tumor growth inhibition in vivo. Investigation of the PK/PD relationship underlying the activity of JAK2i provided direct evidence of phospho-STAT5 suppression and induction of apoptosis for its antitumor effect in vivo. Plasma drug levels correlated with the duration and magnitude of suppression.

Conclusion: A JAK2V617F dependent SET2 xenograft model has been established and validated by shRNA gene knockdown and pharmacologically with a JAK2i in vivo. JAK2V617F inhibition results in suppression of STAT5 phosphorylation, cell-cycle arrest and induction of apoptosis in vitro and in vivo.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A20.