Role Of BCR Research Articles

Role of BCR and FNBP1 Proteins in Phagocytosis as a Model of Membrane Rearrangements with Chronic Myelogenous Leukemia

Role of BCR and FNBP1 Proteins in Phagocytosis as a Model of Membrane Rearrangements with Chronic Myelogenous Leukemia

BCR: a new target in resistance mediated by BCR/ABL-315I?

Targeting BCR/ABL with Tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias but the "gatekeeper" mutation T315I confers resistance against all approved TKIs, with the only exception of ponatinib, a multi-targeted kinase inhibitor. Besides resistance to TKIs, T315I also confers additional features to the leukemogenic potential of BCR/ABL, involving endogenous BCR. Therefore we studied the role of BCR on BCR/ABL mutants lacking functional domains indispensable for the oncogenic activity of BCR/ABL. We used the factor independent growth of murine myeloid progenitor 32D cells and the transformation of Rat-1 fibroblasts both mediated by BCR/ABL. Here we report that T315I restores the capacity to mediate factor-independent growth and transformation potential of loss-of-function mutants of BCR/ABL. Targeting endogenous Bcr abrogated the capacity of oligomerization deficient mutant of BCR/ABL-T315I to mediate factor independent growth of 32D cells and strongly reduced their transformation potential in Rat-1 cells, as well as led to the up-regulation of mitogen activated protein kinase (MAPK) pathway. Our data show that the T315I restores the capacity of loss-of-function mutants to transform cells which is dependent on the transphosphorylation of endogenous Bcr, which becomes a putative therapeutic target to overcome resistance by T315I.

Association between gene and miRNA expression profiles and stereotyped subset #4 B-cell receptor in chronic lymphocytic leukemia

In this study we investigated specific biological and clinical features associated with chronic lymphocytic leukemia (CLL) patients carrying stereotyped BCR subset #4 (IGHV4–34) among a prospective cohort of 462 CLL/MBL patients in early stage (Binet A). All subset #4 patients (n = 16) were characterized by the IGHV mutated gene configuration, and absence of unfavorable cytogenetic lesions, NOTCH1 or SF3B1 mutations. Gene and miRNA expression profiling evidenced that the leukemic cells of subset #4 cases showed significant downregulation of WDFY4, MF2A and upregulation of PDGFA, FGFR1 and TFEC gene transcripts, as well as the upregulation of miR-497 and miR-29c. The transfection of miR-497 mimic in primary leukemic CLL cells induced a downregulation of BCL2, a known validated target of this miRNA. Our data identify biological characteristics associated with subset #4 patients, providing further evidence for the putative role of BCR in shaping the features of the tumor cells in CLL.

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

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. DisclosuresNo relevant conflicts of interest to declare.

Role of BCR in Down-Modulation of BCR-ABL Oncogenicity in CML

Bcr-Abl acquires its transforming ability through its up-regulated Abl tyrosine kinase activity. Bcr is a phosphoprotein with a novel serine/threonine kinase activity encoded by its first exon. Over-expression of BCR in K562 cells produces a phosphoserine form of Bcr and interferes with the oncogenic effects of BCR-ABL in mice (Lin et al., Oncogene 2001). We have recently shown the inhibitory effects of Bcr on Bcr-Abl, in a nude mouse solid tumor model. Expression of BCR/GFP in TonB210 cells used for injection delayed tumor formation and tumors were 50% smaller compared to the TonB210/GFP control. In contrast two point mutants in the BCR kinase domain (Y360F and S354A), not only blocked Bcr's inhibitory effects but enhanced the oncogenic effects of BCRABL (Perazzona et.al. Oncogene, 2008). Similar Bcr effects were observed in a mouse leukemia model. We are investigating the mechanism of the interaction between Bcr and Bcr-Abl proteins. Using TonB210 cells, in which BCR-ABL expression is controlled by a tetracycline-inducible promoter and Bcr is stably transduced by lentivirus infection, we observed that increasing levels of Bcr-Abl expression increased the levels of the Bcr protein. Treatment of TonB210 cells with imatinib mesylate decreased the levels of Bcr- Abl and surprisingly the Bcr protein as well, indicating that the tyrosine kinase function of Bcr-Abl is required to up-regulate Bcr protein expression. In addition, withdrawal of doxycycline also reduced Bcr-Abl and Bcr protein levels, confirming that Bcr-Abl is required for increased expression of the Bcr protein. In order to examine the levels of Bcr in cells lacking Bcr-Abl, we transduced BCR/GFP with lentivirus infection into BaF3 and 32D cells. Surprisingly, these cell lines expressed extremely low levels of Bcr, despite 90% expression of the GFP marker. Expression of Bcr was restored by overnight treatment with the proteasome inhibitor calpain inhibitor I. Forced expression of Bcr-Abl in BCR- transduced cells restored high expression of Bcr protein, confirming that Bcr- Abl is required for preventing degradation of the Bcr protein. Together these findings indicate that Bcr-Abl up-regulated Bcr expression by interfering with proteasomemediated degradation of the Bcr protein. Additional studies indicated that Bcr increases expression of the myeloid membrane surface marker Mac-1 in Bcr-Abl TonB210 cells, which originated from the mouse pro-B cell BaF3. We propose that Bcr may play a role in generating the myeloid phenotype caused by Bcr-Abl in CML patients and may be an important player in the chronic phase of CML by down-modulating Bcr-Abl.

Quality assurance in the field of biomedical analyses: The role and the contribution of the BCR program of the European communities

It is now widely recognized that the Western World has reached a high standard of living through the improvement of technologies and their application to environmental protection, safety, and health. The quality of life results in particular from improvement in health care comprising all measures of prevention, early detection, and treatment of diseases. To this end a large number of biomedical analyses are carried out, and their reliability must be guaranteed. For this purpose in most of the EC Member States a system of quality assurance (QA) for these analyses has been established. From this standpoint QA plays a key role in the improvement of analyses carried out in European clinical laboratories. The involvement of the Community Bureau of Reference (BCR) program in relation to QA is outlined. Emphasis is also placed on the role of BCR in fostering collaboration between organizers of QA schemes as well as on the concept of and means of securing traceability of results in this field.