Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish S-Adenosylmethionine Synthesis

Ramy R Attia,Lidia A Gardner,Engy Mahrous,Debra J Taxman,Leighton Legros,Sarah Rowe,Jenny P.-Y Ting,Arthur Geller,Malak Kotb

doi:10.1074/jbc.m804159200

Abstract

We exploited the fact that leukemic cells utilize significantly higher levels of S-adenosylmethionine (SAMe) than normal lymphocytes and developed tools that selectively diminished their survival under physiologic conditions. Using RNA interference gene silencing technology, we modulated the kinetics of methionine adenosyltransferase-II (MAT-II), which catalyzes SAMe synthesis from ATP and l-Met. Specifically, we silenced the expression of the regulatory MAT-IIbeta subunit in Jurkat cells and accordingly shifted the K(m L-Met) of the enzyme 10-15-fold above the physiologic levels of l-Met, thereby reducing enzyme activity and SAMe pools, inducing excessive apoptosis and diminishing leukemic cell growth in vitro and in vivo. These effects were reversed at unphysiologically high l-Met (>50 microm), indicating that diminished leukemic cell growth at physiologic l-Met levels was a direct result of the increase in MAT-II K(m L-Met) due to MAT-IIbeta ablation and the consequent reduction in SAMe synthesis. In our NOD/Scid IL-2Rgamma(null) humanized mouse model of leukemia, control shRNA-transduced Jurkat cells exhibited heightened engraftment, whereas cells lacking MAT-IIbeta failed to engraft for up to 5 weeks post-transplant. These stark differences in malignant cell survival, effected by MAT-IIbeta ablation, suggest that it may be possible to use this approach to disadvantage leukemic cell survival in vivo with little to no harm to normal cells.

Highlights

Leukemia are among the deadliest and most common cancers
Expression of methionine adenosyltransferase (MAT)-II␣2 was insignificantly elevated, perhaps reflecting an attempt to increase enzyme synthesis to compensate for the lack of ␤ expression and increase SAMe synthesis
We showed that by silencing the expression of the regulatory ␤ subunit of methionine adenosyltransferase-II (MAT-II), we exerted significant growth disadvantage upon human Jurkat leukemic T cells under physiologic L-Met levels in vitro and in vivo

Summary

Targeting SAMe Metabolism in Leukemia

It is believed that this differential oligomerization of MAT-␣1 is an important adaptation to cope with special metabolic requirements in the liver, where SAMe levels need to be maintained at a certain range inasmuch as a deficiency or excess of SAMe has been associated with serious pathology [23,24,25]. MAT-II activity, SAMe utilization rate, and SAMe pool size are, respectively, 20-, 60-, and 60 –100-fold higher in lymphocytic leukemia, than in normal lymphocytes [4] Based on these previous studies [4, 21, 26, 29], we predicted that if we ablated MAT-II␤ expression, we would shift MAT-II Km L-Met by at least 10-fold above physiologic L-Met levels, and that this would reduce SAMe pool size and selectively diminish the growth of leukemic cells in physiological fluids and extrahepatic tissues. We report that MAT-II␤ subunit specific shRNA successfully silenced the expression of the MAT-II␤ regulatory subunit in the Jurkat leukemic T cell line and increased the enzyme Km L-Met by 10 –15-fold, depleting SAMe pools, inducing excessive apoptosis, and diminishing the growth of these leukemic cells in physiologic L-Met concentrations, both in vitro and in vivo in a humanized NOD/Scid IL-2R␥null mouse model of leukemia

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION