BCR/ABL Dosage Hierarchically and Temporally Influences hnRNP A1, hnRNP K and hnRNP E2 Expression in Hematopoietic Stem and Progenitor Cells.

Abstract 191

The molecular mechanism leading to the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase (CML-CP) to the rapidly fatal blast crisis (CML-BC) are still unclear although a plausible assumption is that enhanced expression of BCR/ABL, as that observed in most of patients undergoing progression, represents the factor promoting clonal evolution of CML. Given that a) BCR/ABL levels are increased in the CML-BC stem/leukemia-initiating cell population; b) a causal relationship exists between levels and activity of the BCR/ABL oncoprotein and aberrant mRNA processing, nuclear export and/or translation; and c) molecular and/or pharmacologic interference with the expression and/or activity of the BCR/ABL-regulated RNA binding proteins (hnRNP A1, hnRNP K and hnRNP E2) antagonizes both in vitro and in vivo BCR/ABL leukemogenesis by impairing proliferation, inhibiting survival and/or restoring differentiation of BCR/ABL+ hematopoietic progenitors; we hypothesized that BCR/ABL initiates a hierarchical activation of signals leading to a temporally-organized increase in the expression/function of specific RNA binding proteins (RBPs), and that this represents an essential step for disease progression.

To determine whether expression of hnRNP A1, K and E2 is hierarchically regulated by BCR/ABL and at which stage of the CML stem/progenitor cell development it occurs, we first transduced 32Dcl3 cells with the MigR1-BCR/ABL construct and allowed the clones from a 32D-BCR/ABL^low cell population to become BCR/ABL^high/hnRNP E2^high /C/EBPa⁻ within 21 days of culture in the presence of IL-3. Western blots indicate that BCR/ABL-dependent full induction of hnRNP A1 expression precedes that of hnRNP K and E2 which occurs only after BCR/ABL levels and activity increase to levels capable of conferring cytokine-independent growth and differentiation arrest, suggesting that hnRNP A1 may have the role of a “gatekeeper”, as it allows the increase of BCR/ABL expression through inhibition of PP2A. This, in turn, will promote disease progression in part by inducing expression and activity of hnRNP K and E2 that, as we previously reported, regulate survival, proliferation and differentiation of CD34⁺ CML-BC progenitors. Notably, we observed a similar pattern of hnRNP induction in the lymphoid BCR/ABL-inducible TonB210157 cells.

We also have evidence that differences in hnRNP A1, hnRNP K and hnRNP E2 expression exist between the stem and progenitor cell fractions of BCR/ABL+ primary cells and that they are differentially regulated in leukemic and normal cells. In fact, FACS analysis followed by intracellular protein staining performed on bone marrow- and spleen-derived LSK (Lin⁻/Sca-1+/c-kit⁺), common myeloid progenitors (CMPs) (Lin⁻/Sca-1⁻/c-kit⁺/CD34⁺, FCgRII/III^dim) and granulocyte monocyte progenitors (GMPs) (Lin⁻/Sca-1⁻/c-kit⁺/CD34⁺/FCgRII/III^bright) from leukemic SCLtTA-BCR/ABL double-transgenic mice showed that levels of hnRNP A1 were 3 to 5-fold higher in CMP/GMP than LSK cell fractions (LSK<CMP<GMP). By contrast, in non-induced animals, hnRNP A1 expression was overall markedly inhibited (10-fold lower) with respect to leukemic mice and progressively decreased during LSK maturation into GMPs (LSK>CMP>GMP). Likewise, hnRNP K and E2 levels were 2-fold increased in the progenitors compared to the LSK of leukemic animals, whereas an opposite trend in the expression of hnRNP K was observed in the LSK and CMPs/GMPs of non-induced animals. Moreover, hnRNP K levels in the leukemic CMPs/GMPs were 30 to 40-fold higher than those detected in the same cell fractions from non-induced animals. Interestingly, the highest levels of hnRNP A1 and K in the CMP/GMP fractions correlated with the development of a lymphoid blast crisis-like phenotype as determined by the 30% increase in splenic B220⁺/Mac-1⁺ cells.

Altogether these data suggest that hierarchical and temporal changes in the expression of hnRNPs occur upon BCR/ABL transformation in stem and progenitor cells and during disease progression. Furthermore, these results are consistent with the reported role of the BCR/ABL-regulated hnRNP A1, K and E2 as a positive regulator of BCR/ABL stability through the SET-dependent inhibition of PP2A, a direct enhancer of Myc translation, and as an inhibitor of C/EBPa-dependent myeloid maturation of blast crisis CML progenitors, respectively.