A study published yesterday in the Journal of Clinical Investigation reports the whole-genome sequencing of a mouse acute promyelocytic leukemia (APL) genome. This is a subtype of AML, characterized by the presence of a t(15;17) translocation that creates the PML-RARA fusion oncoprotein. In mice, you can induce expression of PML-RARA transgenically, and they’ll develop APL after a latency period that’s often a year or more. This suggests that the oncogene alone is not sufficient to cause disease, but requires additional progression mutations. The identity of these mutations, and the means by which they contribute to APL development, are largely unknown.
Generating A Mouse APL Cohort
Wartman et al performed have a mouse model of APL in which a human PML-RARA cDNA is knocked in to the 5′ UTR of the cathespin G gene on chromosome 14 of the 129/SVJ strain. The fusion protein is expressed in myeloid progenitor cells, and the mice develop myoproliferative disease that evolves into “acute leukemia with promyelocytic features” after a latency of 7-18 months. In the current study, they back-cr0ssed transgenic mice onto the C57BL/6 background for 10 generations. The goal here was to reduce “passenger” variation inherent to the 129/SVJ genome. A large cohort was established for sample banking; some 60% of the resulting mice went on to develop APL.
Whole-genome Sequencing and Mutation Detection
To identify disease-contributing mutations, the authors performed whole-genome sequencing on the diploid tumor genome of a mouse that developed APL after 335 days. Three runs on the Illumina GAIIx platform yielded 59.64 Gbp (billion base pairs) of sequence, roughly 16x haploid coverage of the mouse genome. But the subsequent SNP calling revealed a bit of a surprise: over 100,000 variants. Unfortunately, a matched germline sample for the donor mouse hadn’t been preserved; it was thought unnecessary because of the back-crossing.
So the authors did the next best thing: sequenced the 129/SVJ genome, using pooled samples from 6 young wild-type males. At roughly 30x coverage, some 4.95 million SNPs between 129/SVJ and C57BL/6 were identified. These served to eliminate 79,339 of the tumor SNVs. Another 17,179 occurred in “contiguous blocks” of 4+ SNPs within 40 kbp, which were presumed passenger mutations arising from genetic drift. That left 15,628 potential somatic SNVs, of which 31 were heterozygous “tier 1″ (coding) variants.
Pursuit of the Coding Mutations
PCR and 3730 sequencing confirmed 8 of 31 coding SNVs as valid mutations. Two were synonymous (silent) mutations and not given further consideration. The remaining 6 were screened in a collection of 89 mouse APL tumors. Three were recurrent among litter-mates, presumably de novo germline mutations in a common ancestor. Two weren’t recurrent at all. Only one remained: a missense mutation (V657F) in the pseudokinase domain of Jak1. Intriguingly, this mutation is homologous to a recently described activating mutation (Jak1 V658F) found in human APL and ALL tumors. The orthologous position in human Jak2 is commonly mutated (V617F) in myoproliferative neoplasms, or MPN.
JAK Mutations in Human Cancers
In the commentary article, Rampal and Levine note that more than 30 mutations in JAKs have been reported in various human cancers including AML, ALL, breast cancer, lung cancer, and others. Janus kinases (JAKs) are protein tyrosine kinases involved in the transduction of cytokine receptor signaling. Since these are predominantly gain-of-function mutations, JAKs are an appealing target for new cancer drugs. Indeed, some JAK inhibitors have already entered late-stage clinical trials for treatment of MPN.
In the last part of the paper, the authors demonstrate that a pan-JAK inhibitor reduced APL colony formation with similar efficacy to all-trans retinoic acid, the standard therapeutic agent for APL. Thus, their work not only serves to elucidate the pathogenesis of leukemia, but also points the way to new strategies for treating this type of cancer.
Wartman, L., Larson, D., Xiang, Z., Ding, L., Chen, K., Lin, L., Cahan, P., Klco, J., Welch, J., Li, C., Payton, J., Uy, G., Varghese, N., Ries, R., Hoock, M., Koboldt, D., McLellan, M., Schmidt, H., Fulton, R., Abbott, R., Cook, L., McGrath, S., Fan, X., Dukes, A., Vickery, T., Kalicki, J., Lamprecht, T., Graubert, T., Tomasson, M., Mardis, E., Wilson, R., & Ley, T. (2011). Sequencing a mouse acute promyelocytic leukemia genome reveals genetic events relevant for disease progression Journal of Clinical Investigation DOI: 10.1172/JCI45284
Rampal, R., & Levine, R. (2011). Finding a needle in a haystack: whole genome sequencing and mutation discovery in murine models Journal of Clinical Investigation DOI: 10.1172/JCI57200