Evaluation of MAPK and PI3K/AKT Signaling Pathways In Adult Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3240-3240 ◽  
Author(s):  
Gustavo Loureiro ◽  
Daniella Bahia Kerbauy ◽  
Maria de Lourdes ◽  
L. F. Chauffaille ◽  
Maria Lucia M. Lee ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Acute Lymphoblastic Leukemia ◽  
T Lymphocytes ◽  
Cell Line ◽  
Jurkat Cell ◽  
Lymphoblastic Leukemia ◽  
Jurkat Cell Line ◽  
Adult Acute Lymphoblastic Leukemia ◽  
Wbc Count

Abstract Abstract 3240 Introduction: Treatment of adult acute lymphoblastic leukemia (ALL) has shown only modest improvements over the last 2 decades, with overall survival of 15% to 40%. The mitogen-activated protein kinase (MAPK) signaling cascade and the phosphoinosytol-3 phosphate/AKT (PI3K/AKT) pathways are involved in proliferation and differentiation of hematopoietic cells. It has been reported that those pathways are frequently activated in solid tumors and acute myeloid leukemia. However, their role in adult ALL is still uncertain. Better understanding of such pathways is necessary for development of novel therapeutic strategies. Aims: To evaluate the phopho-ERK and phospho-AKT protein expression in ALL at diagnosis and to correlate with biological and clinical parameters. Material and Methods: Twenty eight patients (median age 33y, 14–69y) with ALL at diagnosis were studied. Bone marrow and/or peripheral blood mononuclear cells (PBMC) (10 fresh and 18 cryopreserved cells at diagnosis) were stained using phospho-ERK and phospho-AKT/alexafluor 488 monoclonal antibodies (Cell Signaling Technology, Beverly, MA) and their expression was evaluated by flow cytometry (FACScalibur cytometer and CELL Quest program - BDB, San Jose, CA). The monoclonal antibodies CD19, CD10, CD3, CD45, IgM, CD34, CD7, CD2 were used for leukemic cells characterization by four-colour staining. Healthy donor PBMC and Jurkat cell line were used as controls: normal T lymphocytes are negative for p-ERK and Jurkat cell line express p-ERK and p-AKT in low levels. Samples were analyzed for constitutive expression of p-ERK and p-AKT and also after cell activation by phorbol-myristate-acetate (PMA). The expression of these proteins was evaluated by Kolmogorov-Smirnov test using fluorescence ratio between control isotype and phospho-protein (D). p-ERK and p-AKT expression was also evaluated in fresh and frozen samples of the same patients (2 cases) and similar results were obtained. In addition, patients were evaluated for multidrug resistance (MDR) through p-glycoprotein (PGP) expression and Rhodamine (Rh) efflux test and minimal residual disease (MRD) detection at the end of induction by flow cytometry. Results: Twenty cases were B-ALL (EGIL B-I 3, B-II 9, B-III 8, B-IV 5) and 3 T-ALL. Median WBC count was 25.3×109/L (2.3-373×109/L). The expression of p-ERK and p-AKT varied and the median value of p-ERK expression was D = 0.16 (0.01-0.80) and p-AKT median D = 0.08 (0.00-0.63). Considering these values as cutoff there was no difference regarding the patients` age and WBC count at the diagnosis between the positive and negative groups. In regards to EGIL subtypes, p-ERK expression was higher in T-ALL [median 0.50 (0.18-0.54)] than in B-precursor ALL [median 0.14 (0.01-0.80)] (p=0.03). Conversely p-AKT expression was similar in all cases, although high levels were observed among BIII cases. The frequency of Rh efflux was 88% in pERK negative cases and 66% in positive group but there was no difference on PGP expression. On the contrary, PGP expression and Rh efflux were more frequently seen in p-AKT positive cases (88% and 100%, respectively) than p-AKT negative ones (63% and 60%). MRD analysis was performed in seven patients. Two cases presented detectable MRD (>0.01%) and both were p-ERK positive and p-AKT negative. Interestingly all five MRD (-) cases were p-ERK negative and p-AKT positive. In addition, PMA test showed p-ERK activation of normal T lymphocytes and the expression was increased in 52% of ALL cases upon treatment. Conclusion: MAPK and PI3/AKT activation varied among ALL patients. MAPK pathway showed to be more activated in T-ALL than in precursor-B ALL. The functional analysis of these pathways can address the role in ALL pathogenesis. Both pathways may be potential therapeutic targets for novel therapies. (Support: FAPESP proc.09/51002-8). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Comparative Analysis for Differential Drug Response between Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) and T-Cell Acute Lymphoblastic Leukemia (T-ALL) Patients Using the Cellworks Omics Biology Model (CBM): Mycare-021-03

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-19
Author(s):  
Scott Howard ◽  
Ansu Kumar ◽  
Anusha Pampana ◽  
Yashaswini S Ullal ◽  
Anuj Tyagi ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Jurkat Cell ◽  
Lymphoblastic Leukemia ◽  
Patient Specific ◽  
Therapy Options ◽  
Jurkat Cell Line

Background:Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), an orphan disease, is a sub-type of T-Cell Acute Lymphoblastic Leukemia (T-ALL) with very poor prognosis and limited therapy options. ETP-ALL is a heterogeneous disease with many distinct genomic profiles, often with more myeloid than lymphoid characteristics. However, standard of care (SOC) drugs for acute myeloid leukemia (AML) have shown limited efficacy for ETP-ALL (PMID: 32733662, 25435716). The genomic profiles of ETP-ALL patients have more complex cytogenetics and larger numbers of genomic aberrations when compared to non-ETP-ALL (T-ALL) profiles (PMID: 22237106, 30641417). We present an alternative multi-gene analysis approach using the Cellworks Omics Biology Model (CBM) workflow to identify unique, intersecting protein pathways in patient-specific disease profiles. The CBM predictive workflow was used to design novel personalized therapy options for an ETP-ALL representative PEER human lymphoid cell line in comparison to a T-ALL JURKAT cell line. The predicted combination therapies were then validated in a lab model. Methods:A PEER cell line was selected to represent ETP-ALL and a JURKAT cell line was selected as a representative for non-ETP T-ALL. Next Generation Sequencing (NGS) was performed for the PEER cell line. For the JURKAT cell line, publicly available NGS whole exome sequencing from cBioPortal and Sanger, along with array CGH from Agilent, were used. The genomic data for the PEER and JURKAT cell lines were used as inputs to the CBM to generate dynamic patient-specific disease protein network maps. Biomarkers and pathway characteristics unique to the PEER and JURKAT cell lines were identified. A digital drug library of targeted FDA-approved agents was simulated on the disease models using both single drug agents and drug combinations at varying doses. The treatment impact was assessed by quantitatively measuring drug effect on a cell growth score, which is a composite of the quantified values of cell proliferation, survival and apoptosis along with impact on the patient-specific disease biomarker score. Comparative dose response studies were run to assess IC50 differences for both cell lines. Cellworks VenturaTM predicted novel therapy combinations for the ETP-ALL representative PEER cell line, which were then prospectively validated by in vitro experiments. The same therapy options were predicted to be less effective in the T-ALL representative JURKAT cell line, which was also confirmed by in vitro studies. Results:The CBM predicted three novel combination therapies for the ETP-ALL representative PEER cell line: nilotinib + cytarabine, bortezomib + cytarabine and bortezomib + idarubicin. All three therapies were predicted to be less effective in JURKAT cells. In vitro, PEER cells were sensitive to all 3 combinations, as predicted by the CBM; whereas, JURKAT cell lines were not sensitive to the first 2 combinations (as predicted), but were sensitive to bortezomib + idarubicin. The CBM analysis is supported by scientific rationales for these combinations based on the genomics-driven disease characteristics of the cell-line. The reasons for drug sensitivity and resistance were determined. These combinations were then prospectively validated in vitro on both cell lines and the experimental responses matched the predicted outcomes. Conclusion:The Cellworks Omics Biology Model integrates the multiple genomic abnormalities in a patient to identify disease network characteristics unlike other NGS analytic tools that attempt to interpret the impact of each genomic alteration in isolation. CBM identified 3 novel therapy options for ETP-ALL that were validated in vitro, similar to anecdotal experience in vivo. This predictive technology can improve clinical decision-making and identify novel treatment options. Disclosures Howard: Cellworks:Consultancy;Servier:Consultancy, Other: Speaker;EUSA Pharma:Consultancy;Sanofi:Consultancy, Other: Speaker;Boston Scientific:Consultancy.Kumar:Cellworks Research India Private Limited:Current Employment.Pampana:Cellworks Research India Private Limited:Current Employment.Ullal:Cellworks Research India Private Limited:Current Employment.Tyagi:Cellworks Research India Private Limited:Current Employment.Lala:Cellworks Research India Private Limited:Current Employment.Kumari:Cellworks Research India Private Limited:Current Employment.Joseph:Cellworks Research India Private Limited:Current Employment.Raju:Cellworks Research India Private Limited:Current Employment.Balakrishnan:Cellworks Research India Private Limited:Current Employment.Mundkur:Cellworks Group Inc.:Current Employment.Macpherson:Cellworks Group Inc.:Current Employment.Nair:Cellworks Research India Private Limited:Current Employment.Kapoor:Cellworks Research India Private Limited:Current Employment.

scholarly journals Inhibition of telomerase activity and induction of apoptosis byRhodospirillum rubrumL-asparaginase in cancer Jurkat cell line and normal human CD4+ T lymphocytes

2017 ◽  
Vol 6 (11) ◽  
pp. 2697-2712 ◽  
Author(s):  
Dmitry D. Zhdanov ◽  
Vadim S. Pokrovsky ◽  
Marina V. Pokrovskaya ◽  
Svetlana S. Alexandrova ◽  
Mikhail A. Eldarov ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cell Line ◽  
Jurkat Cell ◽  
Telomerase Activity ◽  
Jurkat Cell Line ◽  
Normal Human ◽  
Induction Of Apoptosis

scholarly journals Measurable Residual Disease Assessed by Flow-Cytometry Is a Stable Prognostic Factor for Pediatric T-Cell Acute Lymphoblastic Leukemia in Consecutive SEHOP Protocols Whereas the Impact of Oncogenetics Depends on Treatment

2021 ◽  
Vol 8 ◽  
Author(s):  
Nerea Vega-García ◽  
Sara Perez-Jaume ◽  
Elena Esperanza-Cebollada ◽  
Clara Vicente-Garcés ◽  
Montserrat Torrebadell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Late Time ◽  
Time Points ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Wbc Count ◽  
The Impact ◽  
Measurable Residual Disease

Robust and applicable risk-stratifying genetic factors at diagnosis in pediatric T-cell acute lymphoblastic leukemia (T-ALL) are still lacking, and most protocols rely on measurable residual disease (MRD) assessment. In our study, we aimed to analyze the impact of NOTCH1, FBXW7, PTEN, and RAS mutations, the measurable residual disease (MRD) levels assessed by flow cytometry (FCM-MRD) and other reported risk factors in a Spanish cohort of pediatric T-ALL patients. We included 199 patients treated with SEHOP and PETHEMA consecutive protocols from 1998 to 2019. We observed a better outcome of patients included in the newest SEHOP-PETHEMA-2013 protocol compared to the previous SHOP-2005 cohort. FCM-MRD significantly predicted outcome in both protocols, but the impact at early and late time points differed between protocols. The impact of FCM-MRD at late time points was more evident in SEHOP-PETHEMA 2013, whereas in SHOP-2005 FCM-MRD was predictive of outcome at early time points. Genetics impact was different in SHOP-2005 and SEHOP-PETHEMA-2013 cohorts: NOTCH1 mutations impacted on overall survival only in the SEHOP-PETHEMA-2013 cohort, whereas homozygous deletions of CDKN2A/B had a significantly higher CIR in SHOP-2005 patients. We applied the clinical classification combining oncogenetics, WBC count and MRD levels at the end of induction as previously reported by the FRALLE group. Using this score, we identified different subgroups of patients with statistically different outcome in both Spanish cohorts. In SHOP-2005, the FRALLE classifier identified a subgroup of high-risk patients with poorer survival. In the newest protocol SEHOP-PETHEMA-2013, a very low-risk group of patients with excellent outcome and no relapses was detected, with borderline significance. Overall, FCM-MRD, WBC count and oncogenetics may refine the risk-stratification, helping to design tailored approaches for pediatric T-ALL patients.

Parallel Molecular and Immunological Monitoring of Minimal Residual Disease in Adult Acute Lymphoblastic Leukemia (ALL) Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2294-2294
Author(s):  
E.N. Parovichnikova ◽  
J.R. Davidyan ◽  
I.V. Galtzeva ◽  
V.L. Surin ◽  
I.A. Vorobiev ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Detection Sensitivity ◽  
Induction Phase ◽  
Adult Acute Lymphoblastic Leukemia ◽  
Tumor Clearance ◽  
Minimal Residual

Abstract Though the initial response to induction therapy, indicating rapid tumor clearance, is currently considered to be one of the most important prognostic factors, detection of submicroscopic levels of minimal residual disease (MRD), by means of PCR or immunophenotyping after induction and during different stages of treatment, has also become important in predicting outcome in ALL patients. We initiated a study using parallel PCR and flow cytometry to monitor MRD in adult acute lymphoblastic leukemia (ALL) patients treated by standard approach. Bone marrow samples were analyzed at diagnosis, after pre-phase (one week of steroid therapy to determine steroid responsiveness), at 8-weeks post induction, at consolidation, before maintenance (+7–8 mo), and during maintenance for the detection of clonal IgH, TCR-gamma rearrangements by PCR (The level of detection sensitivity is 10−3 to 10−4). Specific markers were identified for 88% of the patients (30/34); prolonged monitoring was carried out in 22 patients with follow up from 2 to 24 months. Fifteen of those patients were analyzed by three-color flow cytometry analysis with a panel of 20 monoclonal antibodies for patient-specific aberrant leukemia-associated phenotype (LAP) at diagnosis and before maintenance (10−4). Contrary to PCR analysis, LAPs were detected in all patients. The most common LAPs were CD19/34/10, CD 19/34/TdT, and CD19/34/IgM. Clinical CR was achieved in 85% of patients. Prednisone resistance was detected in 71% of patients. MRD was detected by PCR in all patients after pre-phase, in 21/22 (95%) after 1st induction phase, in 11/15 (73%) after 2nd induction phase, in 10/15 (67%) after consolidation, and in 8/14 (57%) before maintenance. Flow cytometry data coincides with molecular parameters: only 4 of 10 patients examined before maintenance (+7–8 mo) had no cells with LAP. Comparing our parallel measurements we have noted discrepancies in 3 of 10 cases. Two immunologically positive patients were PCR-negative, and one PCR-positive patient was immunologically negative. All patients (n=3) whose PCR probes were positive more than twice, relapsed at different time points; no relapses were noted in once positive patients (n=3), and 1of 8 PCR-negative patients relapsed. Our data indicate that both methods are effective and relevant in detection of MRD. They show very slow tumor clearance in ALL patients. MRD positivity reflects the higher relapse probability. More patients can be followed for MRD with flow-cytometry, due to higher detection rate of LAP.

Characterization of binding sites for neuropeptide FF on T lymphocytes of the Jurkat cell line

Peptides ◽  
1995 ◽  
Vol 16 (1) ◽  
pp. 105-111 ◽  
Author(s):  
M. Minault ◽  
J.C. Lecron ◽  
S. Labrouche ◽  
G. Simonnet ◽  
J. Gombert
Keyword(s):  
T Lymphocytes ◽  
Cell Line ◽  
Binding Sites ◽  
Jurkat Cell ◽  
Jurkat Cell Line ◽  
Neuropeptide Ff

scholarly journals Clinical significance of occult central nervous system disease in adult acute lymphoblastic leukemia. A multicenter report from the Campus ALL Network

Haematologica ◽  
2019 ◽  
Vol 106 (1) ◽  
pp. 39-45
Author(s):  
Maria Ilaria Del Principe ◽  
Elisa Buzzatti ◽  
Alfonso Piciocchi ◽  
Fabio Forghieri ◽  
Massimiliano Bonifacio ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Flow Cytometry ◽  
Nervous System ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Central Nervous System Disease ◽  
Nervous System Disease ◽  
System Disease ◽  
Adult Acute Lymphoblastic Leukemia ◽  
The Status

In acute lymphoblastic leukemia, flow cytometry detects more accurately leukemic cells in patients' cerebrospinal fluid compared to conventional cytology. However, the clinical significance of flow cytometry positivity with a negative cytology - occult central nervous system disease - is not clear. In the framework of the national Campus ALL program, we retrospectively evaluated the incidence of occult central nervous system disease and its impact on outcome in 240 adult patients with newly diagnosed acute lymphoblastic leukemia. All cerebrospinal fluid samples were investigated by conventional cytology and flow cytometry. The presence of ≥10 phenotypically abnormal events, forming a cluster, was considered as flow cytometry positivity. No central nervous system involvement was documented in 179 patients, while 18 were positive by conventional morphology and 43 were occult central nervous system disease positive. The relapse rate was significantly lower in central nervous system disease negative patients and the disease-free and overall survival were significantly longer in central nervous system disease negative patients than in those with manifest or occult central nervous system disease positive. In multivariate analysis, the status of manifest and occult central nervous system disease positivity was independently associated with a worse overall survival. In conclusion, we demonstrate that in adult acute lymphoblastic leukemia patients at diagnosis flow cytometry can detect occult central nervous system disease at high sensitivity and that the status of occult central nervous system disease positivity is associated with an adverse outcome. (Clinicaltrials.gov NCT03803670)

scholarly journals The JAK Inhibitor Ruxolitinib Is Effective in Treating T Cell Acute Lymphoblastic Leukemia with Gain of Function Mutations in IL-7R Alpha

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1330-1330 ◽  
Author(s):  
Emilee Senkevitch ◽  
Julie Hixon ◽  
Caroline Andrews ◽  
Joao T Barata ◽  
Wenqing Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
Jak Inhibitors ◽  
Nsg Mice ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Acute lymphoblastic leukemia (ALL) results from transformation of immature B or T cells, and is the most common pediatric cancer. Though the current cure rate of ALL is 80-90%, it is important to understand the underlying biology of ALL in order to develop refined therapies for patients who fail to respond to conventional chemotherapy as well as to reduce its toxicity. The IL-7 receptor (IL-7R) signaling pathway is necessary for the proliferation and survival of T cells. Together with collaborators, we have shown that 9% of patients with T cell acute lymphoblastic leukemia (T-ALL) have gain of function mutations in IL-7R alpha. These mutations promote homodimerization of IL-7R alpha subunits, resulting in constitutive activation of this pathway via Janus Kinase 1 (JAK1). As the JAK-STAT pathway is downstream of the IL-7 receptor, we hypothesized that JAK inhibitors could be used to treat T-ALL patients with IL-7R mutations. To demonstrate this, we first established a cell line model of T-ALL driven by constitutive IL-7R signaling. The model cells were established by transforming the D1 thymocyte cell line with a mutated IL-7R alpha derived from a patient sequence. These cells termed "D1_hIL7R_P1" are also GFP+, which allows us to monitor the proliferation of the cells in vivo.I have showed that D1_hIL7R_P1 cells delivered intravenously result in an aggressive leukemia with morbidity within 18-21 days. Ruxolitinib, a JAK1 inhibitor, inhibits survival, proliferation, and STAT5 activation of D1_hIL7R_P1 cells in vitro. To treat this leukemia in vivo, I administered ruxolitinib for 5 days at a dose of 150 mg/kg to mice starting 8 days after D1_hIL7R_P1 engraftment. Tissues were then harvested for analysis of GFP+ cells as a measure of leukemic burden via flow cytometry. Ruxolitinib reduced leukemic cells from 15% in the blood (as demonstrated in untreated mice) to 5%, from 30% to 10% in the spleen, and 40% to 20% in the lungs. With these promising results, I have acquired T-ALL patient samples that can be xenografted into NSG mice. TALL#5 cell line successfully engrafts in 30 days and can be detected in the bone marrow, spleen, and peripheral blood by flow cytometry. Additionally, TALL#5 expresses human IL-7R alpha and is sensitive to ruxolitinib treatment in vitro. I have also identified two T-ALL cell lines, DND41 and KOPTK1, which express high levels of IL-7R alpha. These cell lines are also potential candidates for studying the effects of ruxolitinib on T-ALL in NSG mice. TALL#5, DND41, and KOPTK1 will be engrafted into mice, and after leukemia is established, mice will be treated with ruxolitinib. If successful, this will give us insight into the effectiveness of JAK inhibitors in treating ALL, and which patients can be recruited for future clinical trials. Disclosures Off Label Use: Ruxolitinib is a JAK1/2 inhibitor that is FDA approved for myelofibrosis. My experiments involve pre-clinical studies looking at the efficacy of ruxolitinib in leukemic mice..

High Pro-Apoptotic Effects of 17-Allylamino-17-Demethoxy Geldanamycin (17-AAG) In Philadelphia Acute Lymphoblastic Leukemia (Ph+ ALL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3226-3226
Author(s):  
Emmanuelle Tavernier-Tardy ◽  
Pascale Flandrin ◽  
Francoise Solly ◽  
Jérôme Cornillon ◽  
Karine Augeul-Meunier ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Apoptotic Proteins ◽  
Hsp 90

Abstract Abstract 3226 Heat Shock Protein 90 (HSP 90), a 90 Kda molecular weight protein, is one of the most abundant cytosolic chaperone, acting in protein folding, refolding and degradation and of particular importance in cell survival. HSP 90 is overexpressed in acute leukemia cells and this high expression is related with a poor prognosis. This ATP-dependent chaperone could be an interesting potential drug target. 17-AAG, a Geldanamycin derivates, is a HSP 90 inhibitor with a promising antitumor activity. Very few reports have evaluated the effects of 17-AAG in acute lymphoblastic leukemia (ALL). The aim of this work is to study the cytotoxicity of 17-AAG and to compare Philadelphia positive (Ph+) ALL to common B-cell ALL. Two human leukemia cell lines, Reh (common B-cell ALL) and SUPB15 (Ph+ ALL) were used in this study. We identified 63 consecutive patients treated in our institution for ALL (44 common B-cell ALL and 19 Ph+ ALL) and leukemic samples were collected at diagnosis from bone marrow aspiration after patient's consent. The expression of HSP 90, pro-apoptotic BAX protein, anti-apoptotic bcl-2 and bcl-xl proteins was studied by flow cytometry. Cell lines and ALL patient's cells were cultured in RPMI 1640 and exposed to various concentrations of 17-AAG. Apoptosis was evaluated by an Annexin V and an activated caspase-3 staining by flow cytometry. Results were expressed as percentage of positive cells. Pro-apoptotic effect of 17-AAG in Ph+ ALL cells was superior when compared to common B-cells with a 100% mortality rate after exposure to [10μM] 17-AAG for 24 hours for SUPB15 cell line whereas 24% of cell surviving was noted for Reh cell line. Similar results were observed with patient's leukemic samples (as shown in figure). The susceptibility of Ph+ ALL cells to 17-AAG was confirmed by the assessment of the IC50, estimated at 1.1 μM for SUPB15 cells versus 2.9 μM for ReH cells. As assessed by Annexin V binding and activated caspase-3 staining, 17-AAG induced apoptosis in ALL cells. As regard Ph+ ALL, the median percentage of Annexin-V positive cells and caspase-3 positive cells after exposure to [5μM] 17 AAG for 24 hours was 54% and 57% respectively and increased up to 99% and 99,5% after exposure for 48 hours. Spontaneous apoptosis in control cell culture was measured in 1% at 24 hours and 3% at 48 hours. The pattern of expression of HSP90 and apoptotic proteins was different between common B-cell ALL and Ph+ ALL cells. The percentage of HSP90 positive cells was 62% for Reh cell line whereas it reached 100% for SUP B15 cell line. As shown in the Table, the percentage of HSP90-positive cells and anti-apoptotic bcl-2 and bcl-xl proteins expression were higher for Ph+ ALL samples when compared to common B-cell ALL samples. HSP90 Bcl-2 Bcl-xl Bax Common B-cell ALL samples n=44 32.5% (±19.8) 33% (± 21.4) 28.5% (±19.5) 33% (±17) Ph+ ALL samples n = 19 79% (±7.5) 82% (±8.4) 84% (±7.8) 12% (±4.3) When ALL cells were exposed in culture to various concentrations of 17-AAG, there was no change in HSP90 expression but we observed a down-regulation in bcl-2 and bcl-xl expression and an up-regulation in bax expression. In summary, we showed that HSP90 and anti-apoptotic proteins were expressed at a higher level on Ph+ ALL cells when compared to common B-cell ALL. High percentage of HSP90-positive cells was associated with high sensitivity to 17-AAG. 17-AAG is a new targeted therapy that induces the apoptotic death of leukemic cells via a caspase-3 dependant way. It would be interesting to test its antileukemic activity in combination with chemotherapeutic agents to study additional or synergistic effects. Despite therapeutic improvement with the development of tyrosine kinase inhibitors (TKI) in the treatment of Ph+ ALL, relapse remains a major problem. Considering that Bcr-Abl constitutes HSP 90 substrates and depends on this chaperone for its maturation and conformational maintenance, 17-AAG could be of particular interest for Ph+ ALL disease, in combination with TKI. We can hypothesise that this drug could restore the sensitivity to TKI treatment for patients with Bcr-Abl mutation. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document