The discovery of synergistic clinical activity between all-trans retinoic acid (ATRA) and chemotherapy has achieved 70-90% long-term remissions in APL. Adjustments in scheduling of ATRA/chemotherapy and utilization of alternative agents may eventuate in universal cure without undue long-term adverse effects. Despite this optimism, some patients continue to relapse, and the results of translational research indicate that much can be learned from continued clinical-laboratory correlative studies. One promising prognostic approach is the use of real-time quantitative RT-PCR (RQ-RT-PCR) to assess the relationship of the level PML-RARa, which is paradoxically the primary leukemogenic and the primary target molecule for ATRA response, to disease outcome. This application seeks to determine if previously-published risk-assessments for quantitative cut-off levels of PML-RARa can be confirmed, including validation of blood as a substititute for marrow, and standardized for clinical decision-making. It also seeks to further understand observations of dynamic fluctuations in low PML-RARa levels that were usually consistent with continuing remission but in approximately 20% of cases were associated with relapse. About 30% of relapse cases had mutations in the ligand binding domain of PML-RARa, which regulates the transcription of a genes containing a retinoic acid response element (RARE) in the promoter region. In tracking pre-relapse samples from patients harboring PML-RARa mutations at relapse, we found that mutant APL subclones frequently emerge in the absence of recent ATRA selection pressure or may not be selected from a low-level by continuing ATRA therapy. These observations suggest that, in contrast to other gene-targeted therapeutics, e.g., imatinib in CML, that initial ATRA exposure may impart other molecular changes to the PML-RARa mutation-harboring subclone that predispose it to delayed emergence in the absence of proximate ATRA selection. Based on a recent report that PML-RARa can recruit DNA methyltransferases to ATRA target gene promoters, we hypothesize that aberrant hypermethylation by mutant PML-RARa is a primary molecular alteration in initially-dormant mutant APL subclones that eventually break loose from host control due to secondary mutations that produce a proliferative advantage.
Specific Aim (SA) 1 of this proposal deals with confirming the relationship of PML-RARa mutant subclones to ATRA selection pressure and disease relapse in a large patient cohort, using relapse samples from 1 Phase III and 2 Phase II APL clinical trials. SA2 investigates the relationship of the PML-RARa mutations to ATRA-targeted and global gene promoter methylation in relapse APL cells, as well as in an in vitro model. SA3 proposes the use of Affymetrix TM gene expression analysis of mutant vs non-mutant and pretreatment APL cells to assess whether a signature gene expression profile is associated with putative central hypermethylation events (or an alternative molecular mechanism) in relapse PML-RARa subclones. Accomplishment of these aims may define a novel mechanism of gene-targeted drug resistance and provide greater understanding of clonal evolution in a therapeutic setting leading to relapse.
Showing the most recent 10 out of 27 publications
