The applicant's previous studies used flow cytometry based methodologies for assessing methotrexate (MTX) resistance/sensitivity markers in small numbers of leukemic blasts. He obtained evidence for a role for inherent MTX resistance in newly diagnosed childhood ALL involving increased levels of dihydrofolate reductase(DHFR), the target enzyme for MTX, and to a minor extent decreased MTX cellular uptake by the reduced folate carrier (RFC). More recently, he found that MTX resistance markers could be lineage specific (the frequent of DHFR overexpression was far greater in T-cell than B-precursor ALL) and also could be acquired as a result of chemotherapy with MTX (their levels increased in relapsed samples). In this application the applicant proposes (in Aim 1) to develop new and more sensitive methods for assaying DHFR levels and MTX membrane transport in children with ALL, including immunofluorescence assay RT-PCR, and direct assays of (3H)MTX uptake. He will correlate these parameters in T-cell and B-precursor ALL blasts at diagnosis with event-free survival, with leukemic relapse in paired diagnosis/relapse specimens and with patient and wild disease related parameters. He will use these methods (in Aim 1) to explore the relationships between the expression of elevated DHFR, or changes in RFC levels or structure, and molecular alterations in key cell cycle controls (p53, p16INK4A, and MDM2) implicated in ALL leukemogenesis. The latter will be assayed by standard molecular approaches including genomic PRT-PCR, and SSCP-PCR. The relationship between p16INK4A expression and MT sensitivity and resistance profiles, and resistance mechanisms will be further studied in p16INK4A negative T-ALL cells transfected with p16INK4A cDNA expression constructs. Finally, (in Aim 3) he will directly examine the possibility that good prognoses of patients with B-precursor ALL whose blast exhibit chromosome 21-specific alterations (hyperdiploidy, t(l2;21)) are partly due to increased expression of RFC and elevated MTX transport activity. Hyperdiploid patients will be identified by standard cytogenetics whereas t(l2;21) patients will be identified by RT-PCR assay of the TEL/AML1 transcript; RFC expression and function will be measured by the methods in Aim 1. In vitro experiments with TEL/AML1-expressing cell lines will be used to verify the results in patient blasts and will also be used to study the molecular basis for increased RFC expression. The results of the proposed studies will provide a framework for further optimizing modern ALL, therapies including MTX and facilitate the development of approaches for circumventing resistance when it does arise.
