The Reciprocal t(9;22) p96^ABL/BCR fusion Protein Eenhances the Transformation Potential of BCR/ABL and Increases Therapy Resistance in Models of Ph+ Acute Lymphatic Leukemia

Abstract 3539

The t(9;22)(q34;q11) is a balanced translocation. The cytogenetic correlate of der22 is the so-called Philadelphia chromosome (Ph). Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210^BCR/ABL and the m-bcr, encoding for the 185^BCR/ABL fusion proteins, respectively. BCR/ABL is a constitutively activated kinase which induces the leukemic phenotype by the aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The BCR/ABL kinase activity is efficiently targeted by tyrosin-Kinase inhibitors such Imatinib, Nilotinib, or Dasatinib. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40^ABL/BCR, present in 65% of patients suffering from chronic myeloid leukemia (CML) and the p96^ABL/BCR, detectable in 100% of patients with Ph+ acute lymphatic leukemia (ALL). In our previous studies we have shown the leukemogenic potential of the ABL/BCR fusion proteins.

To further disclose the role of ABL/BCR proteins, mainly p96^ABL/BCR, in the transformation process induced by BCR/ABL and the leukemogenesis of Ph+ ALL, we co-expressed p96^ABL/BCR and p185^BCR/ABL retrovirally in the IL-3 dependent murine Ba/F3 pro-lymphocytic cell line. p96^ABL/BCR and p185^BCR/ABL were expressed from P2A peptide-linked multicistronic retroviral vectors, which allow the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The effect of p96^ABL/BCR on the kinase activity of p185^BCR/ABL, was assessed by the rate of autophosphorylation at Y245 and Y412, the BCR/ABL-dependent substrate phosphorylation (CrkL, Bcr) and by the activation of down-stream signaling pathway (Stat, Erk,) determined by Western blotting. Proliferation of the cells was assessed by growth curve and XTT assays upon withdrawal of IL-3. As classical transformation assays we performed focus formation assays (loss of contact inhibition) and colony formation in semi-solid medium (support independent growth) in untransformed Rat-1 fibroblasts. The p96^ABL/BCR expression in primary Ph+ ALL patient derived long term cultures (PDLTCs) was targeted by retrovirally transduced shRNA. The efficient targeting of p96^ABL/BCR was confirmed by western blotting.

Here we report that p96ABL/BCR i.) p96^ABL/BCR enhanced not only the autophosphorylation of p185^BCR/ABL at Y245, but also the activation of all the downstream signaling pathways; ii.) p96^ABL/BCR by itself did not transform Rat-1 cells but impressively increased the number of colonies and foci induced by p185^BCR/ABL in Rat-1 cells; iii.) p96^ABL/BCR increased the proliferation of p185^BCR/ABL-positive Ba/F3 cells; iv.)p96^ABL/BCR reduced the responsiveness to TKI in p185^BCR/ABL positive Ba/F3 cells; v.) targeting the p96^ABL/BCR by shRNA decreased the proliferation of Ph+PDLTCs by the induction of apoptosis and increased their sensitivity towards kinase inhibitors (Imatinib, Nilotinib) and the allosteric inhibition by GNF-2 directed against p185^BCR/ABL.

Taken together these data suggest that p96^ABL/BCR plays an important role in the determination of the leukemic phenotype and the therapy resistance of Ph+ ALL.