MOLECULAR PATHOLOGY OF ACUTE PROMYELOCYTIC LEUKEMIA

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Non-random chromosomal translocation t(15;17)(q22;q2l) is a consistent feature of acute promyelocytic leukemia (APL). We and others have shown that the retinoic acid receptor alpha (RARA) gene is involved in the breakpoint. We have cloned and characterized the cDNA of the fusion transcript RARA/myl, myl/RARA and the normal myl cDNA. Our results indicated that both fusion transcripts are able to translate into a fusion protein. Using these DNA sequence information, we are able to analyze the breakpoint sites of eight APL by PCR amplification. We found that the breakpoint sites clustered within two different introns of the myl gene. DNA sequence analysis of these breakpoint sites demonstrated that the fusion transcripts of all APL are able to utilize the correct reading frame. Since RARA is a transcription regulator, the putative fusion proteins RARA/myl and myl/RARA may be potentially oncogenic. Amino acid sequence analysis of myl revealed a cysteine-rich domain similar to those found in a new family of DNA-binding proteins. It is possible that myl may be a novel transcription factor. In this proposal we plan to study the role of t(15;17) translocation breakpoint in the pathogenesis of APL. Two hypotheses are proposed. Hypothesis A: The t(15;17) breakpoint in APL transcribes fusion transcripts RARA/myl and myl/RARA which are oncogenic and is responsible for the pathogenesis of APL. Retroviral mediated gene transfer technique will be used to introduce the recombinant retrovirus constructs pGDRARA/myl and pGDmyl/RARA into the human bone marrow culture. The effect on the expression of these fusion transcripts on clonogenicity, differentiation and tumorigenicity will be studied. We will further study the leukemogenesis of APL by transplantation of the recombinant virus infected bone marrow in an animal model. Hypothesis B: Down regulation of the RARA as a result of t(15;17) translocation, is responsible for the pathogenesis of APL. Two different experiments will be designed to study this hypothesis. (1) Antisense oligodeoxynucleotides against the RARA mRNA will be used to inhibit the translation of RARA in bone marrow culture. The effect on the suppression of RARA translation on clonogenicity and differentiation will be studied. (2) To study the effect of increased expression of RARA and myl genes in the APL cell line, NB4, by gene transfection, on differentiation. We believe that these studies should help understand the molecular mechanism of the pathogenesis of APL.