The functional interplay between the t(9;22)-associated fusion proteins BCR/ABL and ABL/BCR in Philadelphia chromosome-positive acute lymphatic leukemia

Abstract

The hallmark of Philadelphia chromosome positive (Ph(+)) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph(+) leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96(ABL/BCR) for the pathogenesis of Ph(+) ALL. The co-expression of p96(ABL/BCR) enhanced the kinase activity and as a consequence, the transformation potential of p185(BCR/ABL). Targeting p96(ABL/BCR) by RNAi inhibited growth of Ph(+) ALL cell lines and Ph(+) ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96(ABL/BCR) and p185(BCR/AB)L in hematopoietic stem cells. Co-expression of p96(ABL/BCR) abolished the capacity of p185(BCR/ABL) to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96(ABL/BCR) for the pathogenesis of Ph(+) ALL.

Fig 1. Effect of p96^ABL/BCR on the proliferation and transformation capacity of p185^BCR/ABL-positive cells.

(A) Schematic representation of proviruses encoding the transgenes used in this experiment; (B) IL-3 independent growth of Ba/F3 cells expressing the indicated fusion proteins. Proliferation was measured using XTT-assay and growth by dye exclusion using trypan blue. The results are given as means of 3 independent experiments ± SD; (C) Detection of the expression of transgenes in Ba/F3 cells and activation/phosphorylation status of Crkl, Stat5 and Erk1/2 by immunoblotting using the indicated antibodies. The bar graphs represent the relative quantification of phosphorylated with respect to total protein of p185^BCR/ABL (p185) and p96^ABL/BCR-p185^BCR/ABL co-expressing cells (p96 + p185); (D) Transformation assays in Rat-1 cells. For focus formation retrovirally transduced Rat-1 cells were incubated for 15 days in 24-well plates. All experiments were performed at least three times with similar results. For colony formation assay seeded at 5x10³ cells/well in soft-agar in 6-well-plates. After 15 days, the colonies were counted. The mean of three independent experiments each performed in triplicates ± SD is given; (E) Effect of co-expression of p96^ABL/BCR and p185^BCR/ABL on the proliferation of human HSCs. Proliferation of lentivirally transduced CD34⁺/CD38^- HSCs was measured using XTT-assay. The mean of three independent experiments is given ± SD.

Fig 2. Targeting p96^ABL/BCR in Ph⁺ ALL cells.

(A) SupB15 and K562 cells were lentivirally transduced with shRNA against p96^ABL/BCR (siR961 and siR962) and a control shRNA (NTC). The expression of p96^ABL/BCR and/or BCR was detected by immunoblotting using anti-BCR antibody. Tubulin was used as loading control. Proliferation was measured using XTT-assay after 3 days. One representative experiment in triplicates ± SD of at least three yielding similar results is given; (B) The effect of targeting p96^ABL/BCR by shRNA in SupB15 on STAT5 and ERK1/2 pathway was detected using the indicated antibodies; (C) Down-regulation of p96^ABL/BCR in Ph⁺ ALL PD-LTCs by shRNA. Ph⁺ ALL PD-LTCs—PH: fully TKI-responsive; BV: TKI-resistant; as controls were used: HP (Ph^- ALL patient) and VG: t(12;9)-TEL/ABL-positive ALL. The effect of shRNAs on the expression of p96^ABL/BCR was tested by immunoblotting using the indicated antibodies and by q-RT-PCR for PH and BV. The Ct values were normalized to that of GAPDH and results are represented as 2^-ΔΔCt. Proliferation was measured by XTT-assay. The mean of at least experiments is given ± SD.

Fig 3. Role of p96^ABL/BCR for response to selective TKI.

PH and BV cells were transduced lentivirally with shRNAs against p96^ABL/BCR; (A) Treatment with different concentrations of imatinib (0.1, 0.5, 1 and 2 μM). All experiments were performed in triplicates for a total of three experiments, which gave similar results. One representative experiment is given ± SD; (B) Treatment with different concentrations of GNF-2 (0.1, 0.5, 1 and 2 μM). All experiments were performed in triplicates for a total of three, all yielding similar results. One representative experiment is given ± SD.

Fig 4. Effect of the t(9;22) fusion proteins the serial replating potential of murine fetal liver HSCs.

(A) Schematic representation of the experimental procedure. Sca1⁺/lin^- cells were immunomagnetically isolated from murine fetal liver and the cells were transduced with the indicated retroviruses and plated in semi-solid medium supplemented with growth factors for determination of the serial replating potential; (B) Colony numbers were counted on day 10, cells were harvested and serially replated (I-VI-plating rounds).

Fig 5. Stem cell colonogenic potential of t(9;22) fusion proteins.

(A) Experimental strategy for studying the influence of t(9;22) fusion proteins on the biology of murine HSCs. Sca1⁺/lin^- bone marrow (BM) cells were infected with the indicated retroviruses and maintained for 9 days in liquid culture supplemented with the indicated growth factors. 1 x 10⁴ cells were inoculated into lethally irradiated recipients that were sacrificed at day 12 after transplantation; (B) Number of colonies in the spleens (n = 3); the experiment was performed a total of three times with similar results. One representative experiment is given; (C) Gene expression profile induced by t(9;22) fusion proteins in spleen from the CFU-S12. Clustering was done by selecting genes with the highest SD and sorted according to the similarity in expression level; (D) Seven representative cellular pathways known to be influenced by BCR/ABL related to cell cycle regulation, proliferation and apoptosis are presented here. The numbers indicate the number of differentially expressed genes between p185 ^BCR/ABL and p96 ^ABL/BCR + p185^BCR/ABL (p96+p185)-positive cells from the total number of the genes (related to each of the pathways); (E) Total RNA was isolated from spleens from the CFU-S12. The expression levels of Tp53, Gadd45α, and Cdkn1a were analyzed using q-RT-PCR. The Ct values were normalized to that of Gapdh and results are represented as 2^-ΔΔCt. The mean of three independent experiments each done in triplicates is given ± SD; (F) PH and BV were lentivirally transduced with shRNA against p96^ABL/BCR (siR961 and siR962) and a control shRNA (NTC). The expression of GADD45α was detected by q-RT-PCR. The Ct values were normalized to that of GAPDH and results are represented as 2^-ΔΔCt.

Fig 6. The leukemogenic potential of t(9;22) fusion proteins.

(A) Schematic representation of the experimental procedure. Sca1⁺ bone marrow cells were infected with the indicated retroviruses and inoculated into sub-lethally irradiated mice. Empty vector-transduced cells were used as control; (B) Kaplan Maier curves present the probability of survival upon primary induction of leukemia and re-transplantation of leukemic cells in order to induce secondary (II) leukemia; (C) May-Grünwald-Giemsa staining of cytospins from BM and spleens of p185^BCR/ABL and p96+p185-positive leukemia of one representative mouse in each group; (D) Expression of differentiation specific surface markers (Mac1: monocytes- macrophage, Gr1: granulocytes and B220: mature B-cells) of one representative mouse in p185^BCR/ABL and p96 ^ABL/BCR + p185^BCR/ABL (p96+p185) groups. (E) Co-expression of differentiation specific surface markers (Mac1/Gr-1—myeloid leukemia; B220/CD19: B-cell leukemia) of one representative mouse in p185^BCR/ABL and p96 ^ABL/BCR + p185^BCR/ABL (p96+p185) groups and secondary transplanted sup.