Mechanisms of AXL overexpression and function in Imatinib-resistant chronic myeloid leukemia cells

AXL is a receptor tyrosine kinase of the TAM family, the function of which is poorly understood. We previously identified AXL overexpression in Imatinib (IM)-resistant CML cell lines and patients. The present study was conducted to investigate the role of AXL and the mechanisms underlying AXL overexpression in Tyrosine Kinase Inhibitor (TKI)-resistant CML cells. We present evidence that high AXL expression level is a feature of TKI-resistant CML cells and knockdown of AXL sensitized TKI-resistant cells to IM. In addition, expression of wild-type AXL but not a dominant negative form of AXL confers IM-sensitive CML cells the capacity to resist IM effect. AXL overexpression required PKCα and β and constitutive activation of ERK1/2. Accordingly, GF109203X a PKC inhibitor, U0126 a MEK1 inhibitor and PKCα/β knockdown restore sensitivity to IM while PKCα or PKCβ overexpression in CML cells promotes protection against IM-induced cell death. Finally, using luciferase promoter activity assays we established that AXL is regulated transcriptionally through the AP1 transcription factor. Our findings reveal an unexpected role of AXL in resistance to TKI in CML cells, identify the molecular mechanisms involved in its overexpression and support the notion that AXL is a new marker of resistance to TKI in CML

BCL2L10 is a predictive factor for resistance to Azacitidine in MDS and AML patients

Azacitidine is the leading compound to treat patients suffering myelodysplastic syndrome (MDS) or AML with less than 30% of blasts, but a majority of patients is primary refractory or rapidly relapses under treatment. These patients have a drastically reduced life expectancy as compared to sensitive patients. Therefore identifying predictive factors for AZA resistance is of great interest to propose alternative therapeutic strategies for non-responsive patients. We generated AZA-resistant myeloid cell line (SKM1-R) that exhibited increased expression of BCL2L10 an anti-apoptotic Bcl-2 family member. Importantly, BCL2L10 knockdown sensitized SKM1-R cells to AZA effect suggesting that increased BCL2L10 expression is linked to AZA resistance in SKM1-R. We next established in 77 MDS patients that resistance to AZA is significantly correlated with the percentage of MDS or AML cells expressing BCL2L10. In addition, we showed that the proportion of BCL2L10 positive bone marrow cells can predict overall survival in MDS or AML patients. We propose a convenient assay in which the percentage of BCL2L10 expressing cells as assessed by flow cytometry is predictive of whether or not a patient will become resistant to AZA. Therefore, systematic determination of BCL2L10 expression could be of great interest in newly diagnosed and AZA-treated MDS patients

Long-term outcomes after reduced-intensity conditioning allogeneic stem cell transplantation for low-grade lymphoma: a survey by the French Society of Bone Marrow Graft Transplantation and Cellular Therapy (SFGM-TC).

International audienceBACKGROUND AND OBJECTIVES: High-dose chemotherapy with allogeneic stem cell transplantation (SCT) has proven to be a successful treatment for low-grade lymphoma (LGL), but is associated with considerable transplant-related mortality (TRM). In an effort to reduce toxic mortality while maintaining the graft-versus-leukemia effect, allogeneic SCT has been combined with a reduced-intensity conditioning (RIC) regimen. The aim of this study was to determine the outcome of patients with LGL treated with RIC allogeneic SCT. DESIGN AND METHODS: This retrospective multicenter study included 73 patients with relapsed or refractory LGL allografted after a RIC regimen between 1998 and 2005 whose data were recorded in a French registry. RESULTS: Patients received a median of three lines of therapy prior to RIC allogeneic SCT. The most widely used conditioning regimens were fludarabine + busulfan + antithymocyte globulin (n=43) and fludarabine + total body irradiation (n=21). Prior to allografting, patients were in complete response (CR; n=21), partial response (PR; n=33) or had chemoresistant disease (n=19). The median follow-up was 37 months (range, 16 to 77 months). In patients in CR, PR and chemoresistant disease, the 3-year overall survival rates were 66%, 64% and 32%, respectively, while the 3-year event-free survival rates were 66%, 52% and 32%, respectively. The 3-year cumulative incidences of TRM were 32%, 28% and 63%, respectively. The incidence of relapse was 9.6%. INTERPRETATION AND CONCLUSIONS: Although associated with significant TRM, RIC allogeneic SCT in advanced chemosensitive disease leads to long-term survival

Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients

Imatinib sensitizes T-cell lymphocytes from chronic myeloid leukemia patients to FasL-induced cell death: a brief communication.

International audienceThere is now substantial evidence that imatinib may affect immune responses, especially those mediated by T lymphocytes. Fas (CD95/Apo-1), a cell death receptor, is a key regulator of the immune system. We have explored the consequences of treatment on the Fas system in chronic myeloid leukemia patients treated with imatinib. In comparison with healthy controls, we found not only a mild blood lymphopenia but also impairment of phytohemagglutinin activation in CD4Fas and CD8Fas lymphocytes of imatinib-treated patients. Moreover, these lymphocyte populations were more sensitive to FasL-induced cell death in relation to an increase in Fas expression at the cell surface. Taken together, these results reveal the role of Fas receptor in the lymphopenia observed in patients treated with imatinib, with potential deleterious consequences on antileukemic responses against this immunogenic hematological malignancy

Viremia Copy-Years as a Predictive Marker of All-Cause Mortality in HIV-1-Infected Patients Initiating a Protease Inhibitor-Containing Antiretroviral Treatment.

Equipe CHU UB (EA) Pôle MERS CT3 Hors EnjeuInternational audienceViremia copy-years (VCY) has been reported as a short-term predictor of mortality. We evaluated the association of this parameter with 10-year outcome within the APROCO-COPILOTE cohort. Prospective data from 1281 HIV-1-infected patients who started a first protease inhibitor-containing regimen in 1997-1999 were analyzed. Patients with baseline plasma viral load (pVL) > 500 copies per milliliter and at least 2 pVL measures from the eighth month of follow-up were selected. VCY was calculated individually over the follow-up as the area under the pVL curve. Multivariate Cox models analyzed the relation between all-cause mortality and the following variables: age, sex, geographical origin, transmission group, HIV infection duration, ART-naive, pVL at baseline, time-dependent CD4 count, and VCY. Nine hundred seventy-nine patients were followed up for a median of 10 years (interquartile range: 5-11.5). At baseline, median (interquartile range) values for duration of HIV infection, pVL, and CD4 cell count were 43 (4-95) months, 4.6 (3.9-5.2) log10 copies per milliliter, and 278 (125-416) cells per cubic millimeter, respectively. At censoring date, 77 patients (8%) had died. VCY >1.4 log10 copies × yrs/mL was an independent predictor of death (hazard ratio: 2.0; 95% confidence interval: 1.2 to 3.5), which was no longer the case after adjustment for the latest pVL value [risk ratio (RR): 1.2 for 1 additional log10 copies per milliliter; 95% confidence interval: 1.1 to 1.4]. VCY was associated with mortality in HIV-infected patients under combined antiretroviral therapy but did not overweigh the predictive value of the latest pVL. VCY might be more useful as a marker of persistent viral replication than for routine clinical care

All tyrosine kinase inhibitor-resistant chronic myelogenous cells are highly sensitive to Ponatinib.

International audienceThe advent of tyrosine kinase inhibitor (TKI) therapy has considerably improved the survival of patients suffering chronic myelogenous leukemia (CML). Indeed, inhibition of BCR-ABL by imatinib, dasatinib or nilotinib triggers durable responses in most patients suffering from this disease. Moreover, resistance to imatinib due to kinase domain mutations can be generally circumvented using dasatinib or nilotinib, but the multi-resistant T315I mutation that is insensitive to these TKIs, remains to date a major clinical problem. In this line, ponatinib (AP24534) has emerged as a promising therapeutic option in patients with all kinds of BCR-ABL mutations, especially the T315I one. However and surprisingly, the effect of ponatinib has not been extensively studied on imatinib-resistant CML cell lines. Therefore, in the present study, we used several CML cell lines with different mechanisms of resistance to TKI to evaluate the effect of ponatinib on cell viability, apoptosis and signaling. Our results show that ponatinib is highly effective on both sensitive and resistant CML cell lines, whatever the mode of resistance and also on BaF3 murine B cells carrying native BCR-ABL or T315I mutation. We conclude that ponatinib could be effectively used for all types of TKI-resistant patients

Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors.

International audienceImatinib is used to treat chronic myelogenous leukemia (CML), but resistance develops in all phases of this disease. The purpose of the present study was to identify the mode of resistance of newly derived imatinib-resistant (IM-R) and PD166326-resistant (PD-R) CML cells. IM-R and PD-R clones exhibited an increase in viability and a decrease in caspase activation in response to various doses of imatinib and PD166326, respectively, as compared with parental K562 cells. Resistance involved neither mutations in BCR-ABL nor increased BCR-ABL, MDR1 or Lyn expression, all known modes of resistance. To gain insight into the resistance mechanisms, we used pangenomic microarrays and identified 281 genes modulated in parental versus IM-R and PD-R cells. The gene signature was similar for IM-R and PD-R cells, accordingly with the cross-sensitivity observed for both inhibitors. These genes were functionally associated with pathways linked to development, cell adhesion, cell growth, and the JAK-STAT cascade. Especially relevant were the increased expression of the tyrosine kinases AXL and Fyn as well as CD44 and HMGA2. Small interfering RNA experiments and pharmacologic approaches identified FYN as a candidate for resistance to imatinib. Our findings provide a comprehensive picture of the transcriptional events associated with imatinib and PD166326 resistance and identify Fyn as a new potential target for therapeutic intervention in CML