scholarly journals The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome Positive Acute Lymphatic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2402-2402
Author(s):  
Afsar Mian ◽  
Anahita Rafiei ◽  
Claudia Oancea ◽  
Oliver G. Ottmann ◽  
Martin Ruthardt
Keyword(s):  
Stem Cell ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Acute Lymphatic Leukemia ◽  
Abl Kinase ◽  
Lymphatic Leukemia ◽  
Syngeneic Mouse Model ◽  
Philadelphia Chromosome Positive

Abstract The successful targeting of BCR/ABL by selective ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib alone is unable to eradicate the leukemic clone in Philadelphia chromosome positive (Ph+ ) leukemia. The t(9;22)(q34;q11) is a balanced translocation. Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. The constitutively activated BCR/ABL kinase is responsible for the leukemic transformation through an aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients with chronic myeloid leukemia (CML) and the p96ABL/BCR, detectable in 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 p96ABL/BCR for the pathogenesis of Ph+ ALL. We co-expressed p96ABL/BCRand p185BCR/ ABL from a p2A peptide-linked multi-cistronic retroviral vector, which allows the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The co- expression of p96ABL/BCR enhanced the kinase activity and, as a consequence, the transformation potential of p185BCR/ABL in factor dependent progenitor cells and untransformed fibroblasts. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and primary Ph+ ALL patient-derived long-term cultures (PD-LTCs). Furthermore p96ABL/BCR negatively influenced the response to AKI in these models as shown by an increased response to AKI when p96ABL/BCR was down-regulated. Our in vitro and in vivo stem cell studies on murine fetal liver cells and adult HSCs revealed a functional hierarchy between p96ABL/BCR and p185BCR/ABL. In fact, p96ABL/BCR strongly increased stem cell capacity in replating efficiency and colony forming unit-spleen day 12 (CFU-S12) assays, whereas p185BCR/ABL showed no effect. In contrast co-expression of p96ABL/BCR and p185BCR/ABL increased significantly both serial replating potential and CFU-S12 colony formation as compared to p96ABL/BCR alone. In a syngeneic mouse model co-expression of p96ABL/BCR abolished the capacity of p185BCR/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 p96ABL/BCR for the pathogenesis of Ph + ALL. Disclosures No relevant conflicts of interest to declare.

The Reciprocal t(9;22) p96ABL/BCR fusion Protein Eenhances the Transformation Potential of BCR/ABL and Increases Therapy Resistance in Models of Ph+ Acute Lymphatic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3539-3539
Author(s):  
Anahita Rafiei ◽  
Afsar Mian ◽  
Anna Metodieva ◽  
Claudia Oancea ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Western Blotting ◽  
Fusion Proteins ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Therapy Resistance ◽  
Acute Lymphatic Leukemia ◽  
Lymphatic Leukemia

Abstract 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 p210BCR/ABL and the m-bcr, encoding for the 185BCR/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 p40ABL/BCR, present in 65% of patients suffering from chronic myeloid leukemia (CML) and the p96ABL/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 p96ABL/BCR, in the transformation process induced by BCR/ABL and the leukemogenesis of Ph+ ALL, we co-expressed p96ABL/BCR and p185BCR/ABL retrovirally in the IL-3 dependent murine Ba/F3 pro-lymphocytic cell line. p96ABL/BCR and p185BCR/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 p96ABL/BCR on the kinase activity of p185BCR/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 p96ABL/BCR expression in primary Ph+ ALL patient derived long term cultures (PDLTCs) was targeted by retrovirally transduced shRNA. The efficient targeting of p96ABL/BCR was confirmed by western blotting. Here we report that p96ABL/BCR i.) p96ABL/BCR enhanced not only the autophosphorylation of p185BCR/ABL at Y245, but also the activation of all the downstream signaling pathways; ii.) p96ABL/BCR by itself did not transform Rat-1 cells but impressively increased the number of colonies and foci induced by p185BCR/ABL in Rat-1 cells; iii.) p96ABL/BCR increased the proliferation of p185BCR/ABL-positive Ba/F3 cells; iv.)p96ABL/BCR reduced the responsiveness to TKI in p185BCR/ABL positive Ba/F3 cells; v.) targeting the p96ABL/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 p185BCR/ABL. Taken together these data suggest that p96ABL/BCR plays an important role in the determination of the leukemic phenotype and the therapy resistance of Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.

Prominin-1/CD133 Is Involved in the Lineage Decision As Well As a in the Sensitivity to Molecular Therapy in Ph+ Acute Lymphatic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2579-2579
Author(s):  
Anna T. Metodieva ◽  
Heike Pfeifer ◽  
Afsar Mian ◽  
Anja Vogel ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Point Mutations ◽  
Myelogenous Leukemia ◽  
Molecular Therapy ◽  
Cross Resistance ◽  
Acute Lymphatic Leukemia ◽  
Hematopoietic Stem ◽  
Abl Kinase ◽  
Lymphatic Leukemia

Abstract Abstract 2579 Chronic myelogenous leukemia (CML) and Philadephia chromosome-positive acute lymphatic leukemia (Ph+ ALL) is caused by the t(9;22) which fuses the BCR to ABL resulting in a deregulated tyrosine kinase activity. ABL Kinase inhibitiors (AKI) such as imatinib, are effective in the early stage CML, but in advanced stages patients relapse as a result of point mutations within the BCR/ABL. However a certain group of resistant patients do not have point mutations, which can explain the resistance. The mechanisms of resistance remain often unknown. CD133 (PROMININ-1 or PROM-1; in the mouse: Prom-1) is a novel hematopoietic stem/progenitor cell (HSPC) marker, but its biological function is nearly unknown. Its expression is found on various leukemic cells. It has been shown that PROM-1-negative colon cancer-initiating cells were more aggressive than PROM-1-positive cells, suggesting that cancer-initiating cells are in fact PROM-1-negative. We examined the role of PROM-1 in the normal and BCR/ABL-induced malignant haematopoiesis in Prom-1−/− in comparison to Prom+/+ murine HSPCs. Our results suggest that PROM-1 plays an important role in the induction of the BCR/ABL related leukemic phenotype. BCR/ABL induced a significantly higher colony number in Prom−/− as compared to Prom+/+ HSPCs in factor independent CFU-Assays, which did not respond to 1μM Imatinib. In fact in comparison to Prom +/+ HSPCs Prom −/− HSPCs exhibited in presence as well as in absence of BCR/ABL a different response to Imatinib characterized by a significantly increase of immature c-Kit and Sca-1-positive cells. Furthermore in a transduction/transplantation model, BCR/ABL induced a significantly higher rate of ALLs (50%) in the Prom−/− than in Prom+/+ background, where only CML-like diseases were seen. Based on these results we studied the role of PROM-1 for the non-mutational resistance in Ph+ ALL. We investigated the expression of PROM-1 in different non mutational resistance models of Ph+ ALL among the patient derived cell lines (SupB15 and Tom-1) as well as 7 primary long term cultures derived from patient with Ph+ ALL (PDLTCs). These PDLTCs exhibit several grades of resistance against established ABL-kinase inhibitors (AKI). Furthermore there is a cross resistance against other molecular therapy approaches, such as allosteric inhibition by GNF-2 or oligomerization inhibition by competitive peptides. In these cells there was a direct relationship between PROM-1 expression and response to AKIs. In fact sensitivity to AKIs increased with the expression of PROM-1. In these PDLTCs targeting PROM-1 by siRNA reduced and the over expression of PROM-1 increased the response to AKIs. Interestingly, also the only PDLTC harboring the BCR/ABL-T315I was PROM-1 negative. Furthermore the induction of resistance by increasing concentrations of either Imatinib or Nilotinib in the Ph+ ALL line SupB15 was accompanied by a progressive reduction of PROM-1 expression on the surface (CD133). In summary, our data show for the first time the importance of PROM-1/CD133 for the determination of the leukemic phenotype and as a potential marker for resistance to AKIs in Ph+ ALL, which is actually under examination in cohorts of Ph+ ALL patients with non mutational resistance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome-Positive Acute Lymphatic Leukemia

PLoS Genetics ◽  
2015 ◽  
Vol 11 (4) ◽  
pp. e1005144 ◽  
Author(s):  
Anahita Rafiei ◽  
Afsar Ali Mian ◽  
Claudia Döring ◽  
Anna Metodieva ◽  
Claudia Oancea ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Philadelphia Chromosome ◽  
Acute Lymphatic Leukemia ◽  
Lymphatic Leukemia ◽  
Philadelphia Chromosome Positive

scholarly journals The role of stem cell transplantation in the management of Philadelphia chromosome-positive acute lymphoblastic leukemia

2018 ◽  
Vol 9 (12) ◽  
pp. 357-368 ◽  
Author(s):  
Jose-Maria Ribera ◽  
Jordi Ribera ◽  
Eulalia Genescà
Keyword(s):  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Molecular Response ◽  
Hematopoietic Stem ◽  
Philadelphia Chromosome Positive

The concurrent administration of tyrosine kinase inhibitors (TKIs) with standard chemotherapy together with allogeneic hematopoietic stem cell transplantation (alloHSCT) has improved the outcome of patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Although to date, no study has shown alloHSCT to be inferior to chemotherapy plus TKIs in any subgroup of adult Ph+ ALL, there is some evidence suggesting no additional benefit of alloHSCT in patients with deep molecular responses to intensive chemotherapy with a second-generation, and especially, third-generation TKI. As none of these positive and negative studies are controlled, randomized trials are needed to fully define the role of alloHSCT in Ph+ ALL, especially in those with deep molecular response. However, if studies combining TKIs with new approaches such as immunotherapy lead to durable responses, alloHSCT in the first complete remission could be avoided in the near future in the majority of patients with Ph+ ALL.

Minimal residual disease–based role of imatinib as a first-line interim therapy prior to allogeneic stem cell transplantation in Philadelphia chromosome–positive acute lymphoblastic leukemia

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 3068-3070 ◽  
Author(s):  
Seok Lee ◽  
Dong-Wook Kim ◽  
Yoo-Jin Kim ◽  
Nak-Gyun Chung ◽  
Yoo-Li Kim ◽  
...  
Keyword(s):  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
First Line ◽  
Philadelphia Chromosome Positive

Abstract Fourteen adults with Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) were studied to evaluate the role of imatinib prior to allogeneic stem cell transplantation (SCT). Of these, 12 patients were in complete hematologic response (CHR), and 2 were refractory. Imatinib was administered as an interim schedule after each chemotherapy course. After the first imatinib cycle, 11 patients remained in sustained CHR with a decrease in the BCR-ABL/ABL ratios (0.89 logs), and one refractory patient achieved CHR. Meanwhile, 2 patients were resistant to imatinib. Ten patients receiving a second imatinib cycle following consolidation showed sustained CHR, including 2 molecular CR, with a further decrease in the BCR-ABL/ABL ratios (0.19 logs). Twelve patients underwent SCT in a favorable status, and of these, 11 are still alive in a leukemia-free status at 9 to 28+ months after SCT. First-line imatinib interim therapy appears to be a useful strategy to bridge the time to SCT for patients with Ph+ ALL.

scholarly journals Transphosphorylation of Endogenous BCR Mediates the Effect of T315I on the Transformation Potential of BCR/ABL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4523-4523
Author(s):  
Afsar Mian ◽  
Isabella Haberbosch ◽  
Oliver G. Ottmann ◽  
Martin Ruthardt
Keyword(s):  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Point Mutations ◽  
Resistance Mutation ◽  
Loss Of Function ◽  
Resistance Mutations ◽  
Biological Features ◽  
Syngeneic Mouse Model ◽  
Transformation Potential

Abstract Targeting of BCR/ABL by ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib is a proven concept in Philadelphia chromosome positive (Ph+ ) leukemia. In the majority of cases the acquisition of resistance is related to point mutations in BCR/ABL, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). Noteworthy, Ph+ leukemias, both CML and Ph+ ALL, never emerge at diagnosis with a BCR/ABL harboring a resistance mutation even if the clone with the mutation is already existing and detectable by very sensitive methods. This indicates that the mutations confer biological features to the clones unveiled by the treatment. One can hypothesize that the presence of mutations such as the T315I confers a growth disadvantage with respect to native BCR/ABL. We and others have previously shown that the resistance mutations may influence the biology of BCR/ABL regarding its transformation potential. The aim of the study was to determine whether the ‘‘gatekeeper’’ mutation T315I is able not only to induce biochemical modification of BCR/ABL responsible for the to resistance of patients against first and second generation AKI, but also to confer biological features to BCR/ABL influencing its leukemogenic potential. We recently showed that T315I is able to fully restore factor independent growth in Ba/F3 cells of loss of function mutants (LOFM) of BCR/ABL such as that lacking the Y177 (Y177F) or the N-terminal coiled coil oligomerization interface (ΔCC), which was accompanied by a transphosphorylation of endogenous BCR. Based on these findings we systematically investigated the influence of T315I on the biology of BCR/ABL and the role of BCR in this process. As models we used a syngeneic mouse model of BCR/ABL induced CML-like disease, factor dependent 32D and Ba/F3 cells, and Ph+ ALL patient derived long term culture (PD-LTCs). These models allowed the direct comparison of BCR/ABL with BCR/ABL-T315I. Furthermore we took advantage of LOFM such as ΔCC-BCR/ABL and Y177F-BCR/ABL which we studied for their capacity to mediate either transformation potential in Rat-1 fibroblasts (contact inhibition and anchorage-independent growth) and/or factor independent growth in 32D cells in the presence of T315I. The role of the transphosphorylation of BCR was assessed by RNAi against BCR in 32D cells and the Rat-1 cells. Here we show that proliferation of 32D or Ba/F3 cells or the PD-LTCs expressing BCR/ABL-T315I was significantly slower than that of correspondent cells expressing native BCR/ABL. Also the induction of a CML-like disease in syngeneic mice was significantly delayed in the presence of T315I (median: BCR/ABL - 27 days; BCR/ABL-T315I - 61 days). On the other hand T315I was able to restore both transformation potential and factor independent growth of LOFM of BCR/ABL in Rat-1 and 32D cells, respectively. This was accompanied by a transphosphorylation of endogenous BCR at Y177, which led to an activation of Ras/Erk1/2 pathway. This effect of T315I on factor independent growth and transformation mediated by the LOFM and related activation of Ras/Erk1/2 was reverted by targeting the BCR with RNAi. Taken together these data suggest that T315I confer biological features to BCR/ABL which are unveiled only upon treatment or in the presence of LOFM. How these features may influence the destiny of the BCR/ABL-T315I clone in the patient and the role of Ras/Erk1/2 pathway in this process has to be further investigated. Disclosures No relevant conflicts of interest to declare.

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