zd-9331 and Leukemia

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate deple

Topics: Biological Transport; Drug Resistance, Multiple; Drug Screening Assays, Antitumor; Folic Acid; Folic Acid Antagonists; gamma-Glutamyl Hydrolase; Glutamates; Guanine; Homeostasis; Humans; Leukemia; Methotrexate; Ornithine; Pemetrexed; Peptide Synthases; Polyglutamic Acid; Pterins; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

Cellular methotrexate (MTX) resistance may cause treatment failure in childhood common/preB-acute lymphoblastic leukemia (c/preB-ALL), T-lineage ALL (T-ALL), and acute myeloid leukemia (AML). The ex vivo potency of several antifolates (MTX, trimetrexate [TMQ], GW1843U89, multitargeted antifolate [MTA], Raltitrexed, and ZD9331) was studied via in situ inhibition of thymidylate synthase (TS). After short-term exposure, relapsed c/preB-ALL (rALL, n = 21), T-ALL (n = 22), and AML (n = 22) were 3-fold, 10-fold, and 6-fold less sensitive to MTX (P

Topics: Antimetabolites, Antineoplastic; Child; Child, Preschool; Drug Resistance, Neoplasm; Folic Acid Antagonists; Humans; Leukemia; Methotrexate; Quinazolines; Thiophenes; Treatment Outcome; Trimetrexate

ZD9331 is a non-polyglutamatable, potent quinazoline antifolate inhibitor of thymidylate synthase (TS). In an effort to clarify the exact mechanism of resistance to this novel TS inhibitor, we examined the molecular alterations in its target enzyme TS, the transport protein (reduced folate carrier, RFC), and folylpolyglutamate synthetase (FPGS) in a human acute lymphoblastic leukemia cell line, MOLT-3, made resistant to ZD9331. A 310-fold resistant subline was established after 6 months exposure to the drug at concentrations up to 7 microM, and was designated MOLT-3/ ZD9331. MOLT-3/ZD9331 showed crossresistance to CB3717 (4.8-fold), raltitrexed (63-fold) and methotrexate (MTX) (120-fold), but retained sensitivity to trimetrexate (0.88-fold). The resistant cells demonstrated impaired initial cellular uptake and low accumulation of [3H]MTX in accordance with a decreased expression of RFC1, suggesting the downregulation of RFC. However, Southern blot analysis demonstrated no change in gene copy number nor gross rearrangement of RFC1 in the resistant cells. In addition, MOLT-3/ZD9331 showed amplification of the TS gene with a concomitantly increased level in the gene expression. In contrast, the expression of FPGS did not alter. These results demonstrate that continuous exposure of the cells to ZD9331 leads not only to a decreased expression of RFC1 but also to TS gene amplification and overexpression. The resistant mechanisms are likely to be regulated both at a genetic and a transcriptional level for different resistance phenotypes in the ZD9331-resistant MOLT-3 cells.

Topics: Acute Disease; Antimetabolites, Antineoplastic; Antineoplastic Agents; Carrier Proteins; Drug Resistance, Neoplasm; Humans; Leukemia; Membrane Proteins; Membrane Transport Proteins; Methotrexate; Peptide Synthases; Quinazolines; Thymidylate Synthase; Tumor Cells, Cultured