We analyzed the TS-2 acute lymphoblastic leukemia (ALL) cell line that contains a t(1;19)(q23;p13.3) but lacks E2A-PBX1 fusion typically present in leukemias with this translocation. We found that the t(1;19) in TS-2 fuses the 19p13 gene DAZAP1 (Deleted in Azoospermia-Associated Protein 1) to the 1q23 gene MEF2D (Myocyte Enhancer Factor 2D), leading to expression of reciprocal in-frame DAZAP1/MEF2D and MEF2D/DAZAP1 transcripts. MEF2D is a member of the MEF2 family of DNA binding proteins that activate transcription of genes involved in control of muscle cell differentiation, and signaling pathways that mediate response to mitogenic signals and survival of neurons and T-lymphocytes. DAZAP1 is a novel RNA binding protein expressed most abundantly in the testis. We demonstrate that MEF2D/DAZAP1 binds avidly and specifically to DNA in a manner indistinguishable from that of native MEF2D and is a substantially more potent transcriptional activator than MEF2D. We also show that DAZAP1/MEF2D is a sequence-specific RNA-binding protein. MEF2D has been identified as a candidate oncogene in murine retroviral insertional mutagenesis studies. Our data implicate MEF2D in human cancer and suggest that MEF2D/DAZAP1 and/or DAZAP1/MEF2D contribute to leukemogenesis by altering signaling pathways normally regulated by wild-type MEF2D and DAZAP1.

Our study shows that R702 mutations result in especially large platelets and inclusion bodies being faint and mostly invisible on conventionally stained blood smears. We further demonstrated that poly(A)+ RNA content but not NMMHC-IIA accumulation is responsible for the morphological appearance/stainability of inclusion bodies on stained blood smears and the amount of poly(A)+ RNA is decreased in those with R702 mutations.

The binding of all-trans retinoic acid (ATRA) to the ligand-binding region in the E-domain of retinoic acid receptor-α (RARα) modifies the transcriptional activity of RARα protein. ATRA probably induces differentiation of acute promyelocytic leukemia (APL) cells by binding to the E-domain of the RARα portion (RARα/E-domain) of PML/RARα chimeric protein. Therefore, molecular alteration in the RARα/E-domain of the chimeric gene is one mechanism by which patients with APL may acquire resistance to ATRA therapy. In this study using reverse transcription-polymerase chain reaction and single-strand conformation polymorphism, DNA segments amplified from the RARα/E-domain in fresh APL cells of 23 APL patients (8 males and 15 females from 4 to 76 years of age) were screened for mutations. Of those patients, 3 patients (1 with de novo and 2 with relapse) had clinical resistance to ATRA therapy. We found mutations in the RARα/E-domain of PML/RARα chimeric gene exclusively in the 2 patients who exhibited ATRA-resistance at relapse, whereas the mutations were not detected at their initial onset. Interestingly, these patients received a prolonged or intermittent administration of ATRA before relapse with ATRA-resistance. The mutations lead to the change of amino acid in the ligand-binding region of RARα/E-domain, Arg272Gln, or Met297Leu according to the amino acid sequence of RARα, respectively. Further study demonstrated that the in vitro ligand-dependent transcriptional activity of the mutant PML/RARα protein was significantly decreased as compared with that of wild-type PML/RARα. These findings suggest that mutations in the RARα/E-domain of the PML/RARα chimeric gene may confer clinical resistance to ATRA therapy in patients with APL.

No abstract