Requirement of the E2F3 Transcription Factor for BCR/ABL Leukemogenesis.

Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34⁺ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3’UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BC^CD34+ compared to CML-CP^CD34+ progenitors from paired patient samples and to normal CD34⁺ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL⁺ lineage negative (Lin⁻) progenitors was markedly impaired in BCR/ABL⁺ E2F3^−/− compared to BCR/ABL-transduced E2F3^+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P<0.001). Furthermore, subcutaneous injection of shE2F3-expressing BCR/ABL⁺ cells into SCID mice markedly impaired in vivo tumorigenesis (>80% reduction in tumor burden, P<0.01). Accordingly, BCR/ABL leukemogenesis was strongly inhibited in SCID mice intravenously injected with E2F3 shRNA-expressing 32D-BCR/ABL cells and in mice transplanted with BCR/ABL-transduced Lin⁻ bone marrow cells from E2F3^−/− mice. Specifically, we demonstrate that reduced or absent levels of E2F3 resulted in dramatically decreased numbers of circulating BCR/ABL⁺ cells as determined by nested RT-PCR at 4 weeks post-injection (P=0.0001), normal splenic architecture and bone marrow cellularity and the absence of infiltrating myeloid blasts into non-hematopoietic compartments (i.e. liver). By contrast, SCID mice transplanted with vector-transduced 32D-BCR/ABL cells or BCR/ABL⁺ E2F3^+/+ Lin⁻ BM progenitors showed signs of an overt acute leukemia-like process with blast infiltration of hematopoietic and non-hematopoietic organs. Altogether, these data outline the importance of E2F3 expression for BCR/ABL leukemogenesis and characterize a new potential therapeutic target for the treatment of patients with advanced phase CML.