ALBERT

Description: (Abelson helper integration site-1) is a novel oncogene that was initially identified by provirus insertional mutagenesis in v-abl-induced murine pre-B cell lymphoma as a candidate cooperate oncogene. The Ahi-1 protein has a SH3 domain, multiple SH3 binding sites and WD-repeat domains, suggesting novel signaling activities. We have recently demonstrated that AHI-1 is highly deregulated in human leukemic cells, particularly in BCR-ABL+ leukemic stem cells from patients with chronic myeloid leukemia (CML). Overexpression of Ahi-1 in primitive hematopoietic cells confers a growth advantage in vitro and induces leukemia in vivo; these effects can be enhanced by BCR-ABL, a fusion oncogene that plays a major role in the genesis of CML. Conversely, RNAi-mediated suppression of AHI-1 in BCR-ABL-transduced lin−CD34+ human cord blood cells and leukemic stem/progenitor cells from CML patients reduces their growth autonomy in vitro. Interestingly, a direct physical interaction between AHI-1 and BCR-ABL at endogenous levels has been identified in CML cells and this interaction complex further mediates tyrosine kinase inhibitor response/resistance of CML stem/progenitor cells. To further investigate regulatory roles of Ahi-1 in mediating BCR-ABL transforming activities and altered signaling, we have now evaluated co-operative effects of Ahi-1 in a BCR-ABL inducible BaF3 cell line model in which the level of expression of p210BCR-ABL can be variably down-regulated by exposure to doxycycline (Dox). These experiments showed that reduction in BCR-ABL protein expression in the presence of Dox resulted in a corresponding decrease in growth factor independence both in liquid suspension cultures and in semi-solid cultures and an increase in Annexin V+ apoptotic cells in vitro. Interestingly, stable co-expression of Ahi-1 in BCR-ABL inducible BaF3 cells under these stringent conditions enabled them to grow continuously in liquid suspension culture, with fewer Annexin V+ apoptotic cells, and to produce more factor independent CFCs than cells transduced with BCR-ABL alone (10–30 fold). Strikingly, Ahi-1 co-transduced cells also displayed greater resistance to imatinib and, in the presence of IL-3, produced as many CFCs as were produced by the same cells without treatment of imatinib. In contrast, BCR-ABL-transduced cells alone showed a significant reduction of CFC output in response to imatinib. Western blot analysis further demonstrated that co-expression of Ahi-1 in BCR-ABL inducible cells resulted in sustained phosphorylation of BCR-ABL and enhanced activation of JAK2/STAT5 compared to BCR-ABL inducible cells alone when BCR-ABL expression was downregulated in the presence of Dox. Moreover, physical interaction between Ahi-1 and BCR-ABL was demonstrated by co-IP studies in Ahi-1 co-expressed BCR-ABL inducible cells. Taken together, these results provide direct evidence of the regulatory role of Ahi-1 in BCR-ABL-mediated transformation and imatinib response that is associated with altered BCR-ABL phosphorylation and JAK2/STAT5 activation.

Description: It is currently established that the immune system plays a critical role in the control of chronic myelogenous leukemia (CML). Several cell populations including T cells, dendritic cells (DC) and NK cells have been shown to exhibit potential anti-leukemic activities. The possible role of plasmacytoid dendritic cells (pDC) in this anti-leukemic response has not been explored so far. As pDC are the major source of interferon(IFN)-α in vivo, the frequency and the function of circulating pDC were compared in CML patients and in healthy subjects. Three groups of CML patients were studied: chronic phase patients (CP) (i.e. at diagnosis or unresponsive to treatment); patients in complete/major cytogenetic remission induced by IFN-α and patients in complete/major cytogenetic remission induced by imatinib mesylate (IM). Flow cytometric analysis of pDC (assessed by the strong co-expression of CD123 and CD303, formerly BDCA2) showed a dramatic depletion of pDC compartment in CP patients (n = 11) as compared to healthy donors (n = 9) (0.99 versus 5.71 pDC/μL of blood, respectively). Moreover, their functional capacity to produce IFN-α after peripheral blood mononuclear cells (PBMC) stimulation with influenza virus was abolished (883 versus 8201 pg/ml of supernatant, respectively). Patients treated with IFN-α exhibited a complete and long-termed restoration of pDC compartment (4.88 pDC/μL of blood) and of IFN-α production (6932 pg/ml), which was sustained even after the treatment was stopped. In contrast, although IM therapy led to the complete restoration of IFN-α production (6662 pg/ml), its effect on circulating pDC number remained solely partial (2.82 pDC/μL of blood). This last observation and the absence of any linear relationship between pDC number and IFN-α production in patients in complete remission suggested that treatments, and more particularly IM, were able to induce “pDC-independent” pathways of in vivo IFN-α production. Consistently with this hypothesis, we found that pDC-depleted PBMC from patients in cytogenetic remission were able to produce IFN-α, whereas those from healthy donors were not. As a conclusion, our results show for the first time that IFN-α and IM treatments reverse the quantitative and functional impairment of pDC compartment in CP CML patients. They also suggest that these treatments are able to induce differentiation mechanisms leading to the generation of new IFN-α producing cell populations.

Description: Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence of the pathognomonic Philadelphia chromosome and the chimeric BCR-ABL oncoprotein with deregulated tyrosine kinase activity. It has been shown previously that T cell immunity contributes to the control of CML, and several arguments suggest an implication of NKT cells in this anti-tumoral immunity. We thus compared frequency, phenotype and functions of blood NKT cells (defined by the CD1d tetramer+ Vα24+ staining) in healthy subjects and patients with CML. Three groups of patients were studied, including Patients in chronic phase (CP) (either at diagnosis or unresponsive to treatment) patients in major/complete cytogenetic remission induced by interferon-alpha (IFN-α) or patients in major/complete cytogenetic remission induced by imatinib mesylate (IM, a specific inhibitor of the BCR-ABL tyrosine kinase). Our results showed that blood NKT cells frequency was not significantly different between healthy donors (n = 17), CP patients (n = 14) and IM-treated patients (n = 16) (0.062 % versus 0.079 % versus 0.041 % respectively). On the other hand, this frequency defined as above was found to be dramatically decreased in patients in complete remission after IFN-α therapy ( 0.01 %, n = 15 patients). We have then analyzed from the phenotypic point of view NKT cells from these three groups. This ex vivo phenotypic study showed that NKT phenotype (expression of CCR7 and CD161) was clearly modified in the IFN-treated group as compared to IM-treated or CP patients and healthy donors, with a clear enrichment in CD161-CCR7+ NKT cells (49% versus 26%, 22% and18% respectively). This CD161-CCR7+ phenotype has been described as the central memory T cell phenotype, with increased lymph-node homing and antigen-presenting cell-stimulating capacities. We have then performed functional studies of NKT cells measuring their proliferative response to α-galactosylceramide (αGC) as a specific triggering antigen. NKT proliferative response to α-GC was abolished in CP patients (2-fold expansion versus 83-fold in healthy donors). This functional impairment was found to be restored in patients treated with IM and in patients treated with IFN-α (106-fold and 20-fold expansion respectively), although this latter group had a strongly depleted NKT compartment. More interestingly, the incubation of CP CML cells in the presence of IM (0.5 and 1 micromolar, n = 5) led to the partial restoration of the NKT cell reactivity to α-GC (29-fold expansion versus α-GC alone). Thus, our results suggest that IFN-α therapy leads to the generation of "central memory-like phenotype" NKT cells, which could play an important role in the long-term remissions observed in these patients. Moreover, our results strongly suggest that IM is able to partially restore the antigenic-response of CML NKT cells in vitro and in vivo, suggesting a role of BCR-ABL in the anergic state of these cells as this was observed at diagnosis. The IM-induced restoration of NKT cell proliferation defect in CP patients suggest that the antileukemic effect of IM could also be partially due to this action in vivo. Cellular mechanisms involved in this phenomenon are currently under study.

Description: Imatinib mesylate (IM) is currently the first line treatment for patients with Chronic Myeloid Leukemia (CML) allowing induction of major molecular responses in the majority of patients. Nevertheless, resistance develops in a small proportion of patients principally due to missense mutations in the ABL-kinase domain of the BCR-ABL fusion gene. New drugs, such as Dasatinib or Nilotinib, which have recently been designed to overcome IM-resistant mutants, remain inefficient against the mutation at the gatekeeper position (T315I). Thus, this mutation is likely to become the most critical hurdle in patients who fail to benefit from ABL competitive inhibitors. Consequently, when a T315I mutation is detected, a quantitative monitoring of 315 mutants seems to be crucial to evaluate the response to new therapies. For that purpose, we developed a sensitive quantitative-RT-PCR method using allele specific primers to quantify mRNA harboring the T315I mutation. Each cDNA from blood samples were tested in duplicates for ABL, BCR-ABL and BCR-ABL-315I mRNA expression. Serial dilutions of a p210BCR-ABL-315I plasmid were used to construct standard curves allowing the determination of numbers of ABL, BCR-ABL and BCR-ABL-315I mRNA copies. Results were expressed as the BCR-ABL/ABL ratio and BCR-ABL-315I/ABL. To ensure the quality of the results, several controls were analyzed together with patient samples: distilled water, cDNA from healthy blood donors, cDNA from CML patients at diagnosis and cDNA from IM-resistant CML patients exhibiting a T315I mutation. Using this protocol, we retrospectively studied cDNA samples from 5 patients with an acquired resistance to IM therapy due to the presence of the T315I mutation. The periods of follow-up went from 6 months to 2 years. The BCR-ABL/ABL and BCR-ABL-315I/ABL ratios were determined for all cDNA samples and the kinetics for the two transcripts were carried out for each patient. The Q-RT-PCR method, with the standard curves and the different controls, appears to be efficient enough to allow a reliable investigation. Figure shows the results obtained for patient D and C. In patient D, showing a total resistance to IM, a slow and constant increase in the expression of the BCR-ABL-315I mRNA was observed. Conversely, in patient C, BCR-ABL-315I mRNA increased more rapidly after an initial IM-responsive phase. The differential kinetics observed between the two patients suggest that target stem cells for T315I mutation could have some hierarchical heterogeneity with regard to their emergence. Similar results were obtained for the 3 other patients. In conclusion, the quantitative RT-PCR assay that we developed to estimate the relative amount of BCR-ABL mRNA exhibiting the T315I mutation could be of major interest not only for the evaluation of the mechanism of resistance but also for the follow-up of T315I-bearing CML stem cells in patients undergoing targeted therapy using new non-ATP competitive BCR-ABL inhibitors. Figure Figure

Description: Human ES cell lines represent an invaluable tool of research for the study of normal and pathological development. In this study we asked whether the human ES cell lines currently used for research purposes are derived from single outgrowths using X-linked clonality analysis. Amongst the cell lines tested, the female H9 cell line was informative at the Humara (human androgen receptor) locus by the presence of two CAG repeats allowing distinction of paternal (Xp) and maternal (Xm) X chromosomes. Clonal analysis was performed upon Hpa II digestion of DNA purified from total H9 cells (passages 27–61), single H9 cell clones generated from single cell cloning, single embryoid bodies (EB) (day 4, day 14) as well as from single hematopoietic progenitors. The same analysis was performed after induction of hematopoietic differentiation in liquid cultures in the presence of hematopoietic permissive conditions (co-culture in the presence of OP-9 cell line). Clonality was evaluated by calculating the Relative Corrected Index (RCI) which is the ratio of the intensity of peaks generated by Hpa II-digested and undigested DNA. In several experiments, all passages of H9 cell lines examined ( p27, p28, p31, p61) were found to exhibit a monoclonal pattern with disappearance of the same allele A upon Hpa II digestion (RCI 〉 10). 13 clonal cultures of H9 obtained by single cell cloning exhibited also the same clonal pattern with digestion of the same allele and RCI values between 6–14. Similarly, DNA purified from EB’s at different stages was found to be clonal. In DNA obtained from 75 individual hematopoietic progenitors (CFU-GM, CFU-G), clonal analysis revealed the same clonal pattern in each, with disappearance of the same allele A. However, clonal analysis performed in 10 hematopoietic colonies, revealed unexpectedly, an equal digestion of Humara alleles with RCI 〈 2 (polyclonal pattern). Finally, in two experiments, hematopoietic cells recovered from liquid cultures exhibited an absolute clonal pattern (Total disappearance of the same allele A). Interestingly, quantification of X-Inactivation Specific Transcript (XIST) RNA in H9 and day14 EB’s by RT-PCR did not show any evidence of XIST expression both before and after differentiation. Altogether, our data suggest that XCI has already been initiated in undifferentiated H9 cells and this inactivation is maintained, at least in the Humara locus in the absence of XIST. The monoclonal pattern of the cell line can be due to the fact that H9 ES cell line might have arisen from a limited number of stem cells having undergone XCI at a very early stage, with maintenance of the methylation status at this locus during mesodermal and hematopoietic commitment. Conversely, hematopoietic differentiation might occur from several mesodermal progenitors having inactivated the same X chromosome in H9 at the Humara locus. Despite the fact that hematopoietic progenitors and differentiated hematopoietic cells originating from H9 exhibited also a clonal pattern, a partial reactivation of the inactive X chromosome during hematopoietic differentiation could occur, and may explain the “polyclonal” pattern obtained in 10 CFU-GM analyzed. Overall, X-linked clonality testing could be an important issue before considering the potential clinical applications, in all female hES cell lines.

Description: It is currently established that the immune system plays a critical role in the control of chronic myelogenous leukemia (CML). Several cell populations including T cells, dendritic cells (Dc) and NK cells have been shown to exhibit potential anti-leukemic activities. We have previously shown that plasmacytoid dendritic cells (pDc) differentiation and IFN-a production was impaired along the chronic phase (CP) of CML and that the Tyrosine- Kinase (TK) activity of BCR-ABL was responsible for this impairment. Moreover, patients in complete cytogenetic remission (CCyR) after imatinib mesylate (IM) or IFN-a therapy present a partial restoration of the pDc compartment and IFN-a production. Some recent results (Esashi et al., Immunity, 2008) indicate that, in mice, STAT-5 but not STAT-3, block the expression of IRF-8, a transcription factor required for the differentiation of pDc and that the GM-CSF-induced STAT5 activation interferes with pDc differentiation. As STAT3 and STAT5 proteins are important targets of BCR-ABL TK activity, we postulate that their recruitment could be responsible for the CML pDc impairment and could be involved in the persistent pDc dysfunction in CR patients. We decided to investigate the effect of BCR-ABL or GM-CSF on STAT-3, STAT-5 and IRF8 status ex vivo, on CMN of patients who have achieved sustained CCyR and major molecular response (MMR) after IFN-a therapy and in whom treatment has been discontinued (n=8) compared to CMN of healthy subjects (HS) (n=5). The same investigations were also performed in vitro with the model of pDc differentiation of CD34+ haematopoietic progenitors from CP patients and HSs, using the combination of Stem Cell Factor, Flt3-Ligand and Thrombopoietin,. Flow cytometry analysis showed that the homeostasis of the IFN-a production was altered in CCyR patients : pDc (determined by the coexpression of CD303, CD123 and CD4), represent 34.6 ± 14.9 % of the IFN-a producing cells after stimulation by Influenza virus and intracellular detection of IFN-a, whereas in healthy donors (HD) IFN-a producing cells were principally pDc (72.1 ± 20.5 %of IFN-a+ cells, p

Description: Chronic myeloid leukemia (CML) is a clonal multilineage myeloproliferative disorder arising from the neoplastic transformation of a pluripotent hematopoietic stem cell that acquires a unique BCR-ABL fusion gene. The BCR-ABL oncoprotein displays constitutively elevated tyrosine kinase activity that deregulates cellular proliferation and apoptosis control through effects on several common signal transduction cascades, including the PI3K/AKT, JAK2/STAT5, and NF-kB pathways. The current first line treatment for CML involves administration of the tyrosine kinase inhibitor imatinib mesylate (IM) that has shown promise in treating chronic phase CML patients. However, early relapses and IM-resistant disease have emerged and are frequently associated with mutations in the BCR-ABL kinase domain that affect inhibitor binding. AHI-1 (Abelson helper integration site 1) is a recently discovered oncogene that has been demonstrated to be highly deregulated in a CML cell line (K562) and in primary leukemic stem/progenitor cells from CML patients. AHI-1 contains several unique domains that are indicative of signalling functions, including both an SH3 and a WD40-repeat domain. We have recently demonstrated that overexpression of murine Ahi-1 is able to transform IL-3 dependent Baf3, resulting in cells able to grow in the absence of growth factors. When these transduced cells were injected into sublethally irradiated NOD/SCID immunodeficient mice, the mice developed leukemia, demonstrating the oncogenic properties of Ahi-1. Interestingly, these in vitro and in vivo effects can be enhanced by co-transduction of BCR-ABL in these cells. In addition, a direct interaction between AHI-1 and BCR-ABL at endogenous levels was identified in K562 cells and this protein interaction complex further mediated IM response/resistance in CML stem/progenitor cells. To further investigate AHI-1’s involvement in mediating this cellular resistance to IM, AHI-1 was either stably overexpressed in K562 cells by transduction of EF1a-AHI-1-IRES-YFP lentivirus or was suppressed in K562 cells using a lentiviral-mediated RNA interference approach. Interestingly, overexpression of AHI-1 in K562 cells significantly increased cellular survival in the presence of 1, 5 and 10 uM of IM as measured by a viability assay; survival of these cells was similar to that observed in an IM resistant K562 cell line reported to be highly resistant to IM in vitro. Furthermore, suppression of AHI-1 had the opposite effect, with cells displaying heightened sensitivity to IM at concentrations as low as 1 uM. Phosphorylation and protein expression levels of several proteins known to be involved in BCR-ABL signalling, including JAK2, STAT5, AKT and NF-kB (P105, P50, and P65 subunits), were then quantified by Western blot analysis. Interestingly, elevated phosphorylation and protein expression levels of JAK2, and STAT5 and total protein expression levels NF-kB p105/p55 subunits were observed in both the AHI-1 overexpressing K562 cells and IM resistant K562 cells, while reduced phosphorylation and protein expression of these same proteins was observed in AHI-1 suppressed K562 cells. Differential expression of phosphorylated NF-kB p65 subunit at serine 536 was observed, while total protein expression levels did not significantly differ. Phosphorylated AKT expression levels were only affected in AHI-1 suppressed K562 cells, and total AKT protein expression was not affected in AHI-1 overexpressed or suppressed cells. Interestingly, AHI-1 protein expression was highly elevated at endogenous levels in the IM resistant K562 cells relative to a parental K562 cell line. These findings suggest that AHI-1 may play an important role in mediating cellular resistance to IM through activation of several signalling proteins involved in BCR-ABL signalling pathway, including JAK2 and STAT5.

Description: Recent data suggest that there is a major discrepancy between the expression of BCR-ABL in primitive hematopoietic stem cells as compared to their differentiated counterparts where the expression is much lower. This high expression level predisposes this compartment to a major genetic instability leading to a mutator phenotype. The “gatekeeper” mutation T315I is the most problematic of these mutations leading to a resistance to all three clinically available tyrosine kinase inhibitors (TKI). This mutation is suspected to generate a specific signalling distinct from non-mutated BCR-ABL with high transforming potential but less TK activity. In order to study the effects of both mutated and non-mutated BCR-ABL in a primitive stem cell context, we have transduced retrovirally (Bosc 23 packaging cell line) both BCR-ABL and BCR-ABL T315I as well as an empty GFP control retrovirus, into day+5 embryoid bodies (EB’s) derived from D3 murine embryonic stem (ES) cells. In several experiments, the transduction efficiency was found to vary between 10–45% as evaluated by GFP expression at day+ 3 post-transduction. EB’s were then dissociated and put in hematopoietic differentiation conditions using clonogenic assays and in liquid culture in the presence of OP-9 stroma and hematopoietic growth factors including SCF, IL6 and IL3. Cells were collected weekly and analyzed with regard to their hematopoietic commitment, their amplification potential, their phenotype and their morphology as well as expression of BCR-ABL. In clonogenic assays, BCR-ABL and T315I6transduced cellD induced GFP+ growth-factor-independent colonies. In hematopoietic differentiation conditions, empty vector- and BCR-ABL-transduced cells underwent approximately 8-population doublings in vitro. On the other hand, BCR-ABL T315I mutant-transduced cells underwent a major expansion during this time with 16-Population doublings in 4 weeks. The addition of imatinib mesylate (1 and 2 mM) to cell cultures stimulated cell growth in T315I-BCR-ABL- transduced cells as compared to controls. Interestingly, phenotypic analysis demonstrated the appearance of CD45+ CD34+ cells in BCR-ABL-transduced cells as early as week+2–3 whereas this hematopoietic differentiation appeared to be delayed in BCR-ABL-T315I-transduced cells. Thus, our data suggest that in the context of a primitive hematopoietic stem cell context, T315I BCR-ABL exhibits a higher transforming potential. Current experiments underway are testing the long-term repopulating ability of BCR-ABL and BCR-ABLT315I- expressing cells in NOD/SCID reconstitution assays as well as the occurrence of ABL-kinase mutations in the presence and in the absence of TKI. Thus, the murine ES-cell-derived hematopoiesis could be an important experimental tool to recapitulate the early stages of hematopoiesis and the role of T315I mutation in the rare, primitive stem cell populations.