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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA076641-02
Application #
2856494
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Wu, Roy S
Project Start
1998-01-01
Project End
2002-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Wayne State University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Medyouf, Hind; Gao, Xiuhua; Armstrong, Florence et al. (2010) Acute T-cell leukemias remain dependent on Notch signaling despite PTEN and INK4A/ARF loss. Blood 115:1175-84
Larson Gedman, A; Chen, Q; Kugel Desmoulin, S et al. (2009) The impact of NOTCH1, FBW7 and PTEN mutations on prognosis and downstream signaling in pediatric T-cell acute lymphoblastic leukemia: a report from the Children's Oncology Group. Leukemia 23:1417-25
LaFiura, K M; Edwards, H; Taub, J W et al. (2008) Identification and characterization of novel AML1-ETO fusion transcripts in pediatric t(8;21) acute myeloid leukemia: a report from the Children's Oncology Group. Oncogene 27:4933-42
Ge, Yubin; Haska, Christina L; LaFiura, Katherine et al. (2007) Prognostic role of the reduced folate carrier, the major membrane transporter for methotrexate, in childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group. Clin Cancer Res 13:451-7
Matherly, Larry H; Hou, Zhanjun; Deng, Yijun (2007) Human reduced folate carrier: translation of basic biology to cancer etiology and therapy. Cancer Metastasis Rev 26:111-28
Yang, L V; Wan, J; Ge, Y et al. (2006) The GATA site-dependent hemogen promoter is transcriptionally regulated by GATA1 in hematopoietic and leukemia cells. Leukemia 20:417-25
Liu, Mingjun; Ge, Yubin; Payton, Scott G et al. (2006) Transcriptional regulation of the human reduced folate carrier in childhood acute lymphoblastic leukemia cells. Clin Cancer Res 12:608-16
Flatley, Robin M; Payton, Scott G; Taub, Jeffrey W et al. (2004) Primary acute lymphoblastic leukemia cells use a novel promoter and 5'noncoding exon for the human reduced folate carrier that encodes a modified carrier translated from an upstream translational start. Clin Cancer Res 10:5111-22
Matherly, Larry H; Goldman, David I (2003) Membrane transport of folates. Vitam Horm 66:403-56
Zhang, L; Taub, J W; Williamson, M et al. (1998) Reduced folate carrier gene expression in childhood acute lymphoblastic leukemia: relationship to immunophenotype and ploidy. Clin Cancer Res 4:2169-77