A Sensitized Screen Uncovers a Novel Platelet Phenotype in a Loss-of-Function Jak2 Allele.

Abstract 2516

Poster Board II-493

Our group has undertaken a concerted effort to develop novel heritable mouse strains via ethylnitrosourea (ENU)-based random mutagenesis. We have complemented this strategy to perform a dominant sensitized screen to identify mouse mutants with altered recovery following treatment with 5-fluoruracil (5-FU). We isolated a heritable line with elevated platelets as well as an overshoot in platelet numbers when exposed to 5-FU. Affected mice also displayed megakaryocytosis and centrilobular hepatic lipidosis.

The mutation was mapped to a 8.7Mb region on mouse Chromosome 19, which contained the candidate gene Jak2. The entire coding region of Jak2 was sequenced and a mutation in exon 19 was found. The causative mutation was located at nucleotide 2740 and leads to an A to T transversion in exon 19 of JAK2 and causes a substitution of a Lys to a premature stop codon at amino acid 914. The resulting protein product terminates within the JH1 kinase domain of JAK2.

JAK2⁹¹⁴ is distinct from the JAK2 null allele characterized by the Ihle and Pfeffer labs. For example, JAK2^−/− embryos die at E12.5, whereas JAK2^914/914 embryos survive until E13.5, suggesting that the truncated protein product may serve as a scaffold. Differences were also observed in clonogenic potential of both strains. For example, JAK2^+/− mice had increased CFU-C whereas JAK2^914/+ were normal. Splenic BFU-E and CFU-C were increased in JAK2+/− mice while both cell types were decreased in JAK2^914/+. In contrast, bone marrow CFU-E were increased in both strains of mice and splenic CFU-E were normal in JAK2^+/− and JAK2^914/+. In addition, both mouse strains display elevated platelets when animals are housed under resting conditions.

The hepatic phenotype is phenocopied by Leptin Receptor null mice that are hyperphagic, poorly cold-adapted, sterile and have enhanced fat conversion resulting in lipidosis.

In conclusion, a sensitized screen has shown that JAK2 regulates the response to the chemotherapeutic drug, 5-fluorouracil. These findings have significance to potential myelosuppressive effects of the clinical use of 5-FU in colorectal and pancreatic cancer.