De novo purine synthesis inhibition and antileukemic effects of mercaptopurine alone or in combination with methotrexate in vivo

Abstract

Methotrexate (MTX) and mercaptopurine (MP) are widely used antileukemic agents that inhibit de novo purine synthesis (DNPS) as a mechanism of their antileukemic effects. To elucidate pharmacodynamic differences among children with acute lymphoblastic leukemia (ALL), DNPS was measured in leukemic blasts from newly diagnosed patients before and after therapy with these agents. Patients were randomized to receive lowdose MTX (LDMTX: 6 oral doses of 30 mg/m 2 ) or high-dose MTX (HDMTX: intravenous 1 g/m 2 ) followed by intravenous MP; or intravenous MP alone (1 g/m 2 ), as initial therapy. At diagnosis, the rate of DNPS in bone marrow leukemia cells was 3-fold higher in patients with T-lineage ALL compared with those with B-lineage ALL (769 ؎ 189 vs 250 ؎ 38 fmol/nmol/h; P ‫؍‬ .001). DNPS was not consistently inhibited following MP alone but was markedly inhibited following MTX plus MP (median decrease 3% vs 94%; P < .001). LDMTX plus MP and HDMTX plus MP produced greater antileukemic effects (percentage decrease in circulating leukocyte counts) compared with MP alone (؊50% ؎ 4%, ؊56% ؎ 3%, and ؊ 20% ؎ 4%, respectively; P < .0001). Full DNPS inhibition was associated with greater antileukemic effects compared with partial or no inhibition (؊63% ؎ 4% vs ؊37% ؎ 4%; P < .0001) in patients with nonhyperdiploid B-lineage and T-lineage ALL.HDMTX plus MP yielded 2-fold higher MTX polyglutamate concentrations than LDMTX plus MP (2148 ؎ 298 vs 1075 ؎ 114 pmol/ 10 9 cells; P < .01) and a higher percentage of patients with full DNPS inhibition (78% vs 53%; P < .001). Thus, the extent of DNPS inhibition was related to in vivo antileukemic effects, and a single dose of intravenous MP produced minimal DNPS inhibition and antileukemic effects, whereas MTX plus MP produced greater antileukemic effects and DNPS inhibition, with full inhibition more frequent after

Acute lymphoblastic leukemia with TEL-AML1 fusion has lower expression of genes involved in purine metabolism and lower de novo purine synthesis

“…No statistical difference was evident among ALL with E2A-PBX translocation, hyperdiploid B-lineage, and B-lineage ALL without defined genetic abnormalities. As previously observed, 7 DNPS was higher in patients with T-lineage ALL compared with the other ALL subtypes (B-lineage versus T-lineage ALL, P ϭ .01; Blineage without TEL-AML1 versus T-lineage ALL, P ϭ .02).…”

“…8,9 The method for quantitating DNPS was modified to yield higher overall median values when compared with the method previously used to quantitate DNPS in ALL blasts. 7,9 However, the previously observed lineage differences in DNPS (T-ALL greater than B-lineage ALL) remained evident with the current DNPS assay. The concentrations of unlabeled adenine and guanine were determined from peak areas of UV chromatograms against a linear calibration curve of standards (1 to 75 nmol on column) prepared in phosphate-buffered saline.…”

“…In addition, hypoxanthine exerts feedback inhibition on DNPS, and the lower intracellular hypoxanthine plus inosine concentrations in T-lineage ALL compared with B-lineage ALL suggest that lower purine recycling and salvage activity may be compensated by increased DNPS in T-lineage ALL. 7 To date, there have been no studies to determine whether there are differences in the rate of DNPS among genetic subtypes of ALL. The current work is the first to reveal that leukemia cells with the TEL-AML1 fusion have a significantly lower rate of DNPS when compared with other genetic subtypes of childhood ALL.…”

“…4 Several in vitro and in vivo studies suggest that there is a lineage difference in the rate of DNPS in leukemic cells, with T-lineage ALL having higher DNPS than B-lineage ALL. 7,17,18 Recent studies 19,20 show that the gene encoding methylthioadenosine phosphorylase, an enzyme involved in the purine salvage pathway, is more frequently deleted in T-lineage ALL than in B-lineage ALL because of its close genomic proximity to the tumor suppressors p16 INK4A /p15 INK4B . In addition, hypoxanthine exerts feedback inhibition on DNPS, and the lower intracellular hypoxanthine plus inosine concentrations in T-lineage ALL compared with B-lineage ALL suggest that lower purine recycling and salvage activity may be compensated by increased DNPS in T-lineage ALL.…”

“…Leukemic cells were obtained by density separation over a Ficoll-Hypaque gradient and washed 3 times with a solution of HEPES (N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid), Hanks buffered solution, and heparin, as previously described in detail. 7 The final cell yield was determined by hemocytometer and percent viability by trypan blue exclusion. …”