The BCR-ABL1-Regulated hnRNP A1, hnRNP E2, and hnRNP K Are Differentially Expressed Between CD34+ and CD34+/CD38- Ph+ Cells, and After Blastic Transformation of CML.

Abstract 1222

The molecular mechanism leading to disease progression of chronic myelogenous leukemia (CML) still remains to be identified although enhanced BCR/ABL expression and activity seems to play an important role in controlling genomic stability, differentiation, and self renewal of the leukemic cell clone undergoing blastic tranformation by affecting expression and function of RNA binding proteins (RBPs) like hnRNP A1, hnRNP E2, and hnRNP K (Perrotti D. et al. J. Clin Invest. 2010).

We previously reported (Harb J. et al., ASH 2009) that a BCR-ABL1 dosage-dependence and hierarchical organization exists for the expression of hnRNP A1, hnRNP E2, and hnRNP K in cell line models of CML. In fact, as BCR/ABL levels increase, upregulated expression of hnRNP A1 is observed, followed by increased expression of hnRNP E2 and hnRNP K. HnRNP A1 and hnRNP K were also temporally expressed within Lineage^- Sca-1⁺ c-kit⁺ (LSK), common myeloid progenitors (CMP), and granulocyte macrophage progenitors (GMP) in a mouse model (SCLtTA TRE-BCR/ABL) of chronic myeloid leukemia. Interestingly, hnRNP A1 and hnRNP K levels in BCR/ABL⁺ mouse progenitors correlated with disease severity as mice with higher levels of these RBPs presented a more progressed phenotype characterized by increased mixed lineage B220⁺/Mac-1⁺ progenitors in bone marrow and spleen when compared with mice that developed a CML-CP-like phenotype.

Here we show that hnRNP A1, hnRNP E2, and hnRNP K expression levels, as well as BCR/ABL activity are different in HSC (CD34⁺/CD38^-), CMP (CD34⁺/CD38⁺/CD45⁺/IL-3Ra^-), and GMP (CD34⁺/CD38⁺/CD45⁺/IL-3Ra⁺) isolated from peripheral blood of patients in chronic phase (CML-CP) at diagnosis, untreated accelerated phase (CML-AP), and blast crisis (CML-BC). Interestingly, in CML-CP, the highest expression of hnRNP A1 was found in the CD34⁺/CD38^- stem cell fraction and it gradually decreased in the more mature CMP and GMP progenitors (55% and 65% lower, respectively). By contrast, consistent with the role of hnRNP A1 as regulator of progenitor cell proliferation and survival, hnRNP A1 expression progressively increased in the HSC, CMP and GMP fractions isolated from patients in CML-AP and CML-BC. Unlike hnRNP A1, hnRNP E2 and hnRNP K were barely detected in the CD34⁺/CD38^- from CML-CP patients but their expression was markedly pronounced in the HSC fraction of progressed CML patients. In agreement with our cell line data, expression of hnRNP A1 and hnRNP E2 in advanced CML (CML-AP and CML-BC) increases when CD34⁺/CD38^- stem cells undergo maturation toward CMP. Conversely, it appears that hnRNP K expression is the last to increase in CML-BC, suggesting a hierarchical regulation of RBP expression during differentiation and lineage commitment. Expectedly, levels of hnRNP A1 in CMPs increase during disease progression (CP<AP<BC). Because of the highest expression of hnRNP A1 in the CD34⁺/CD38^- fraction in CML-CP, and its previously described role in regulating survival and proliferation of Ph+ CD34+ cells (Iervolino et al., Mol Cell Biol 2002; Neviani et al. Cancer Cell 2005), we lentivirally transduced a hnRNP A1 shRNA into the CD34⁺/CD38^- and CD34⁺/CD38⁺ cell fractions in CML-CP and assessed the role played by this RBP in survival of primitive and committed CML-CP progenitors. Western blot analysis demonstrated that hnRNP A1 was efficiently knockdown (≥ 80% reduction) in both compartments. Due to its ability to regulate BCR-ABL1 expression/function through the SET-PP2A axis, downregulation of hnRNP A1 led to reduced BCR/ABL activity in both stem and progenitor cells. However, cell survival and cytokine-dependent proliferation were severely compromised in committed progenitors transduced with the hnRNP A1 shRNA but not in the shRNA hnRNP A1-expressing CD34⁺/CD38^- cells, consistent with the notion that progenitors but not stem cells are BCR-ABL1 oncogene addicted. Colony forming assays performed with empty vector- and hnRNP A1 shRNA-transduced CD34⁺/CD38^- cells showed a 50% reduction in the number of CFCs upon hnRNP A1 downregulation. Accordingly, levels of the hnRNP A1-regulated anti-apoptotic Bcl-x_L were substantially reduced in CD34⁺/CD38^- cells.

Taken together, these data further implicate RBPs hnRNP A1, hnRNP E2, and hnRNP K in CML disease progression and suggest their possible role in the control of survival of stem and primitive CML-CP progenitors.