Antileukemic Activity of the HSP70 Inhibitor Pifithrin-μ

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3306-3306
Author(s):  
Martin Kaiser ◽  
Andrea Kuehnl ◽  
Jutta Ortiz-Tanchez ◽  
Ouidad Benlasfer ◽  
Cornelia Schlee ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Combination Treatment ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Active Form ◽  
Combination Therapies ◽  
Acute Leukemias ◽  
Genetic Characteristics ◽  
B Lineage

Abstract Abstract 3306 Introduction: Heat shock protein (HSP) 70 is aberrantly expressed in acute leukemias and other hematologic and solid malignancies, promoting tumor cell survival and therapy resistance. Recently, the small molecule pifithrin-μ (2-phenylethynesulfonamide) has been identified as a direct inhibitor of inducible HSP70, showing antiproliferative activity in different cell lines of solid tumors. Here, we analysed the in vitro antileukemic effect of pifithrin-μ in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cell lines, as well as in primary AML blasts. In addition, incubations of pifithrin-μ with cytarabine, the histone deacetylase inhibitor SAHA, the HSP90 inhibitor 17-AAG, and the multikinase inhibitor sorafenib were performed to evaluate the potential use of combination therapies with pifithrin-μ in acute leukemias. Methods: Leukemic cell lines KG-1a (AML), K562 (CML in blast crisis), K562r (cytarabine-resistant K562), NALM-6 (B-lineage ALL), TOM-1 (B-lineage ALL, BCR-ABL pos.), Jurkat (T-lineage ALL), BE-13 (T-lineage ALL) and 9 bone marrow cell samples from newly diagnosed or relapsed AML patients were exposed to pifithrin-μ. Cell viability of all cell lines listed above was quantified by WST-1 assay. Subsequent functional analyses were performed on KG-1a and NALM-6 cells. Apoptosis was determined by annexin-V/7-AAD staining and subsequent flow cytometric analysis. Activated caspase-3 was detected by flow cytometry. Levels of the cell signaling kinase Akt were measured by intracellular staining and FACS analysis. Coincubations of pifithrin-μ with cytarabine, SAHA, 17-AAG or sorafenib were performed in KG-1a, NALM-6 and TOM-1, using WST-1 assays to analyse cytotoxic effects of combination therapies. Results: Pifithrin-μ at low micromolar concentrantions significantly inhibited viability of all acute leukemia cell lines tested, with IC50 values ranging from 2.5 to 12.7 μM independent of the differentiation lineage. Importantly, viability of both cytarabine-sensitive and -resistant K562 cells was effectively inhibited by pifithrin-μ. The median IC50 of primary AML blasts was 8.8 μM, ranging from 5.7 to 11.8 μM with no obvious differences regarding patients' clinical or genetic characteristics. Apoptosis was induced in a time- and dose-dependent fashion with a rate of specific apoptosis of 46% at 4 μM pifithrin-μ for NALM-6 and 36% at 40 μM pifithrin-μ for KG1a. In NALM-6, treatment with 3 μM pifithrin-μ for 24 hours resulted in a significant increase in the cleaved, active form of caspase-3, whereas in KG1a no increase in active caspase-3 was detected. Intracellular concentrations of Akt were markedly reduced after 12 hours incubation of NALM-6 with pifithrin-μ. In NALM-6, KG-1a, and TOM-1 combination treatment of pifithrin-μ at concentrations below the IC50 with either SAHA, 17-AAG or sorafenib resulted in a significant decrease of cell viability compared to corresponding monotherapy. Thus in NALM-6 combination of 2 μM pifithrin-μ with 0.6 μM SAHA inhibited viability by 73%, compared to 22% and 0% inhibition for either drug alone (p<0.05). Combination of 2 μM pifithrin-μ with 2 μM 17-AAG led to 58% inhibition, in contrast the monotherapy inhibited cell viability only in 31% for either drug alone. In NALM-6 and TOM-1, the combination of pifithrin-μ with cytarabine decreased viability significantly (47% and 55%, respectively), whereas the single agents were less effective (22% for 2 μM pifithrin-μ, 24% for 9 nM cytarabine in NALM-6; 26% for 3.5 μM pifithrin-μ and 41% for 40 nM cytarabine in TOM-1). Conclusion: This is, to our knowledge, the first report of the antileukemic effects of the HSP70 inhibitor pifithrin-μ. The inhibitor is highly active against all AML and ALL cell lines tested, including cytarabine resistant cell lines as well as primary leukemic cells. Effectivity of pifithrin-μ could even be increased in combination treatment with other antileukemic agents. Targeting HSP70 might be a promising new therapeutic approach for the treatment of acute leukemias to overcome drug resistance. Thus, our data might build a framework for future clinical trials. Disclosures: No relevant conflicts of interest to declare.

Synergistic Antileukemic Interactions Between Valproic Acid and Cytarabine: A Potential Means to Improve the Treatment of Pediatric Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2084-2084
Author(s):  
Chengzhi Xie ◽  
Holly Edwards ◽  
Lisa Polin ◽  
Hui Zhou ◽  
Xuanlin Wang ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Clinical Trials ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Active Form ◽  
Acute Leukemias ◽  
Induction Of Apoptosis ◽  
Acute Myeloid

Abstract Abstract 2084 Poster Board II-61 Acute myeloid leukemia (AML) accounts for one fourth of acute leukemias in children, but it is responsible for more than half of the leukemia deaths in this patient population. In contrast to the tremendous success in the treatment of acute lymphoblastic leukemia over the last three decades, resulting in a >80% cure rate, improvements in AML therapy have been limited. Resistance to cytarabine (ara-C), the most active drug in the treatment of AML, is a major cause of treatment failure in this disease. Therefore, new therapies for children with AML need to be developed to overcome drug resistance, decrease relapse rate, and reduce short- and long-term adverse effects of treatment. Histone deacetylase inhibitors (HDACIs) possess antitumor activity and are currently being tested in clinical trials for the treatment of a variety of different cancers. Valproic acid (VPA), an FDA-licensed drug for treating both children and adults with epilepsy, also acts as an HDACI and can induce apoptosis in leukemic cells but not normal cells. In this study, we hypothesized that VPA synergizes with ara-C in antileukemic activity by inducing apoptosis in AML cells. To model this concept and to provide the basis for future clinical studies, we examined the effects of VPA on sensitivities to ara-C in 8 AML cell lines derived from patients (4 were children) with different subtypes of AML and in AML blasts collected at the time of diagnosis from 10 children with de novo AML treated at Children's Hospital of Michigan. We demonstrated synergistic antileukemic interactions between ara-C and VPA in all of the AML cell lines and additive to synergistic antileukemic interactions between the two drugs in the patient samples by standard isobolograms and calculation of combination indexes. It is interesting to note that MV4-11 [which harbors t(4;11)] and Kasumi-1 [which harbors t(8;21)] cells were substantially more sensitive to VPA than the other AML cell lines. Analogous to the Kasumi-1 cells, diagnostic blasts from t(8;21) AML cases (n=4) were significantly more sensitive to VPA than blasts from non-t(8;21) AML cases (n=6) (mean VPA IC50 0.51 mM vs 1.95 mM, p=0.0095) and showed median 53.9-fold increased ara-C sensitivities when combined with VPA at concentrations of 0.5 mM or lower. By contrast, non-t(8;21) AML blasts only showed median 2.1-fold increased ara-C sensitivities when combined with 0.5 mM VPA (p=0.048). In a pilot experiment, treatment of SCID mice with K562 xenograft tumors with combined Palmo-ara-C and VPA resulted in a 31% T/C and a 0.8 gross log cell kill compared to treatments with Palmo-ara-C (67% T/C) or VPA alone (100% T/C), establishing unambiguous in vivo synergy. Real-time RT-PCR analyses revealed changes in transcript levels for hENT1 and cytidine deaminase in Kasumi-1 cells post VPA and ara-C treatment alone or in combination. However, these changes would antagonize ara-C sensitivity in Kasumi-1 cells, suggesting that the effects of VPA or ara-C alone or in combination on expression of genes related to ara-C transport and metabolism do not contribute to the observed synergistic effects in AML cells. Interestingly, ara-C and VPA co-treatment resulted in synergistic induction of apoptosis and S-phase arrest in Kasumi-1 cells determined by flow cytometry analysis with annexin V and PI staining. The synergy between ara-C and VPA in induction of apoptosis in Kasumi-1 cells was accompanied by synergistic activation of caspase-3, induction of both total and acetylated p53 (ac-p53), and release of the active form of Bax determined by caspase-3 assays, co-immunoprecipitation, and Western blotting. Collectively, these results suggest that VPA enhances ara-C sensitivity in Kasumi-1 cells most likely by modulating levels of total and ac-p53 proteins and then release of the active form of Bax to trigger apoptosis. Based on our laboratory results, VPA has been incorporated into a treatment arm for high risk AML patients enrolled in the St. Jude Children's Research Hospital (SJCRH) clinical trial AML08: “A Randomized Trial of Clofarabine Plus Cytarabine Versus Conventional Induction Therapy and of Natural Killer Cell Transplantation Versus Conventional Consolidation Therapy in Patients with Newly Diagnosed Acute Myeloid Leukemia”. The results of our study provide compelling evidence to support the use of VPA in combination with ara-C in clinical trials for treating different risk groups of pediatric AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Direct Effect of Septic Plasma in Human Cell Lines Viability

2018 ◽  
Vol 47 (1-3) ◽  
pp. 270-276
Author(s):  
Grazia Maria Virzì ◽  
Chiara Borga ◽  
Chiara Pasqualin ◽  
Silvia Pastori ◽  
Alessandra Brocca ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cell Viability ◽  
Cell Lines ◽  
Caspase 3 ◽  
Test Group ◽  
Organ Injury ◽  
Enzyme Linked Immunosorbent Assay ◽  
Caspase 9 ◽  
Renal Tubular Cells

Background: Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients’ plasma. Methods: We enrolled 26 septic patients (“test” group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit. Results: We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937). Conclusion: Humoral mediators in septic patients’ plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages.

scholarly journals 2193

2017 ◽  
Vol 1 (S1) ◽  
pp. 58-59
Author(s):  
Houda Alachkar ◽  
Martin Mutonga ◽  
Amanda de Albuquerque ◽  
Rucha Deo ◽  
Gregory Malnassy ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Leucine Zipper ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Hematologic Malignancies ◽  
Cytotoxic Activities ◽  
Knock Down ◽  
Protein Levels

OBJECTIVES/SPECIFIC AIMS: Unlike the high cure rates (90%) of children with acute lymphoblastic leukemia (ALL), that of adults is still lagging behind and better therapies are needed. Maternal embryonic leucine-zipper kinase (MELK) is aberrantly upregulated in cancer, and implicated in cancer stem cell survival. A recent study has identified FOXM1, a MELK substrate, as a therapeutic target in B cell ALL (B-ALL). Thus, we hypothesized that MELK may act as a therapeutic target in ALL via targeting FOXM1 activity. METHODS/STUDY POPULATION: Western blot and qPCR were used to assess MELK expression in 14 ALL cell lines. Knock-down and kinase inhibition approaches targeting MELK expression and function, followed by CCK-8 and Annexin V (flow cytometry) assays to measure cell viability and apoptosis, respectively. RESULTS/ANTICIPATED RESULTS: MELK was significantly upregulated in patients with ALL (oncomine data analysis). MELK was also significantly higher in B-ALL and T-ALL cell lines compared with that in blood cells of healthy donors. MELK knock-down significantly decreased cell viability (40%–70%, p<0.05, Fig. 1) in ALL cells, and induced apoptosis (~40%). OTS167, a potent MELK inhibitor exhibited cytotoxic activities in both B and T-ALL cells. The IC50 of OTS167 ranged from 20 to 60 nM; we also found a significant increase in apoptosis (p<0.05). Mechanistically, MELK inhibition resulted in decrease of FOXM1 protein levels 3 hours post-treatment. DISCUSSION/SIGNIFICANCE OF IMPACT: MELK is highly expressed in ALL and represents a novel therapeutic target likely via modulating FOXM1 activity. Functional and mechanistic studies will complement and ensure the success of the undergoing Phase I/II clinical trial of OTS167 in patients with refractory or relapsed AML, ALL, and other advanced hematologic malignancies.

Adoptive T-Cell Therapy for B-Cell Acute Lymphoblastic Leukemia: Preclinical Studies

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3531-3540 ◽  
Author(s):  
Angelo A. Cardoso ◽  
J. Pedro Veiga ◽  
Paolo Ghia ◽  
Hernani M. Afonso ◽  
W. Nicholas Haining ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Adoptive T Cell Therapy ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
T Cell Lines

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.

The Reliability and Specificity of c-kit for the Diagnosis of Acute Myeloid Leukemias and Undifferentiated Leukemias

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 596-599 ◽  
Author(s):  
M.C. Bene ◽  
M. Bernier ◽  
R.O. Casasnovas ◽  
G. Castoldi ◽  
W. Knapp ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Cell Lineage ◽  
Specific Marker ◽  
Acute Leukemias ◽  
Routine Basis ◽  
Immunological Classification ◽  
B Lineage ◽  
Acute Myeloid

Abstract We document findings on c-kit (CD117) expression in 1,937 pediatric and adult de novo acute leukemia cases, diagnosed in five single European centers. All cases were well characterized as to the morphologic, cytochemical, and immunologic features, according to the European Group for the Immunological Classification of Leukemias (EGIL). The cases included 1,103 acute myeloid leukemia (AML), 819 acute lymphoblastic leukemia (ALL), 11 biphenotypic acute leukemia (BAL), and 4 undifferentiated (AUL). c-kit was expressed in 741 (67%) AML cases, regardless of the French-American-British (FAB) subtype, one third of BAL, all four AUL, but only in 34 (4%) of ALL cases. The minority of c-kit+ ALL cases were classified as: T-cell lineage (two thirds), mainly pro-T–ALL or T-I, and B lineage (one third); cells from 62% of these ALL cases coexpressed other myeloid markers (CD13, CD33, or both). There were no differences in the frequency of c-kit+ AML or ALL cases according to age being similar in the adult and pediatric groups. Our findings demonstrate that c-kit is a reliable and specific marker to detect leukemia cells committed to the myeloid lineage, and therefore should be included in a routine basis for the diagnosis of acute leukemias to demonstrate myeloid commitment of the blasts. c-kit expression should score higher, at least one point, in the system currently applied to the diagnosis of BAL, as its myeloid specificity is greater than CD13 and CD33. Findings in ALL and AUL suggest that c-kit identifies a subgroup of cases, which may correspond to leukemias either arising from early prothymocytes and/or early hematopoietic cells, both able to differentiate to the lymphoid and myeloid pathways.

Valproic Acid as a Promising Co-Treatment With Paclitaxel and Doxorubicin in Different Ovarian Carcinoma Cell Lines

2016 ◽  
Vol 26 (9) ◽  
pp. 1546-1556 ◽  
Author(s):  
Patrycja Kwiecińska ◽  
Erik Taubøll ◽  
Edyta Grzyb ◽  
Elżbieta Fiedor ◽  
Anna Ptak ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Valproic Acid ◽  
Cell Viability ◽  
Dna Fragmentation ◽  
Cell Lines ◽  
Cancer Cell ◽  
Caspase 3 ◽  
Ovarian Cancer Cell ◽  
Tubulin Acetylation ◽  
Ovarian Cancer Cell Lines

ObjectiveThe current preferred treatment of ovarian cancer is combination chemotherapy, usually a platinum-based drug coupled with paclitaxel (PTX). Here, we investigated whether co-treatment with valproic acid (VPA) could increase the efficiency of various ovarian cancer drugs—PTX, doxorubicin (DOX), carboplatin (CBP), and cyclophosphamide (CP)—in different ovarian cancer cell lines.MethodsThree different ovarian cancer cell lines (OVCAR-3, TOV-21G, and TOV-112D) were treated with chemotherapeutic drugs, alone or in combination with VPA. Cell viability (XTT assay), caspase-3 activity, and the expression of cell cycle– and apoptosis-related genes and proteins were assessed. Furthermore, the effects of these drugs on α-tubulin acetylation and DNA fragmentation were investigated.ResultsPaclitaxel and DOX decreased cell viability and increased caspase-3 activity, and co-treatment with VPA enhanced this effect. Carboplatin and CP had no effect. Responses to treatment with PAX and DOX together with VPA on gene expression profile were highly variable and depended on the cell line investigated. However, a common feature in all cell lines was an increased expression ofCDKN1A,CCNE1,PARP1, andPARP3. Co-treatment with VPA enhanced the effect of DOX and PAX on most protein expressions investigated in TOV-21G and TOV-112D cell lines, whereas in OVCAR-3, the most effect was seen with DOX with VPA. Valproic acid did not increase PTX-induced α-tubulin acetylation. An additive effect of DOX with VPA on DNA fragmentation was observed in TOV-21G and TOV-112D cell lines but not in the OVCAR-3.ConclusionsOur results indicate that VPA could be a promising agent in combined anticancer therapy for ovarian cancer, with the combination of VPA and DOX being the most effective. Certainly, additional in vivo and ex vivo experiments are necessary to investigate the molecular mechanisms of action underlying the cellular effects reported here and to study possible clinically relevant effects in ovarian cancer explants.

Enhancement of stress-induced apoptosis in B-lineage cells by caspase-9 inhibitor

Blood ◽  
2004 ◽  
Vol 104 (9) ◽  
pp. 2873-2878 ◽  
Author(s):  
Nisha Shah ◽  
Rebecca J. Asch ◽  
Alana S. Lysholm ◽  
Tucker W. LeBien
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Adherent Cell ◽  
Cell Contact ◽  
Caspase 9 ◽  
B Lineage ◽  
B Lineage Cells

Abstract We have established human B-lineage (BLIN) acute lymphoblastic leukemia cell lines that retain a dependency on fibroblast monolayers for survival and proliferation. Eight hours following removal from adherent cell contact BLIN cells undergo a decrease in mitochondrial transmembrane potential and an increase in annexin V binding. Unexpectedly, the caspase-9 inhibitor (C9i) benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone enhanced the appearance of apoptotic cells within 8 hours following removal of BLIN cells from fibroblast monolayers. C9i enhancement of apoptosis was dose dependent and did not occur with irreversible inhibitors of caspases-2, -3, -6, and -8. C9i also enhanced apoptosis in cord blood-derived CD19+ B-lineage cells (but not myeloid cells) removed from murine stromal cells. Longer exposure (&gt; 18 hours) to C9i culminated in apoptosis in a panel of B-lineage acute lymphoblastic leukemia (ALL) cell lines in the presence or absence of fibroblast monolayers, as well as in 2 proliferating leukemic cell lines (RAMOS and CEM). BLIN-4L cells made deficient in caspase-9 by RNA interference exhibited no resistance to apoptotic signals and actually showed increased apoptotic sensitivity to staurosporine. These collective results suggest that a 4-amino acid caspase inhibitor of caspase-9 can promote apoptosis and that at least some types of apoptotic pathways in B-lineage ALL do not require caspase-9.

IPI-504, a Novel, Orally Active HSP90 Inhibitor, Prolongs Survival of Mice with BCR-ABL T315I CML and B-ALL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2183-2183 ◽  
Author(s):  
Cong Peng ◽  
Julia Brain ◽  
Yiguo Hu ◽  
Linghong Kong ◽  
David Grayzel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Median Survival ◽  
Combination Treatment ◽  
Lymphoblastic Leukemia ◽  
Hsp90 Inhibitor ◽  
Leukemic Cells ◽  
Prolonged Survival ◽  
Hsp90 Inhibition ◽  
T315i Mutation

Abstract Development of mutations within the kinase domain is a major drug-resistance mechanism for tyrosine kinase inhibitors (TKIs) in cancer therapy. In CML (chronic myeloid leukemia), a disease driven by the constitutively active BCR-ABL oncoprotein, no available TKIs have been effective in treating patients with the BCR-ABL T315I mutation. Heat shock protein 90 (Hsp90) is a highly conserved, constitutively expressed molecular chaperone that facilitates folding of client proteins like BCR-ABL, and affects the stability of these proteins. Several labs have shown that Hsp90 inhibition in vitro results in the degradation of BCR-ABL T315I and induces potent killing of these cell lines. However, these results have not been demonstrated in animal models for BCR-ABL-induced CML and B-ALL (B-cell acute lymphoblastic leukemia, a disease that does not respond well to TKIs including imatinib and dasatinib). Thus, IPI-504, an orally administered Hsp90 inhibitor, was evaluated in murine models of CML and B-ALL. Treatment of mice with wild type (WT)- or T315I BCR-ABL-induced CML with IPI-504 resulted in BCR-ABL protein degradation and a decrease in circulating BCR-ABL positive cells. In response to treatment with vehicle the median survival time of WT and T315I CML mice is approximately 20 days. While the T315I CML mice were resistant to imatinib with a median survival of 21 days, IPI-504 (50 and 100 mg/kg, PO TIW) demonstrated dose-dependent prolonged survival of these mice by 30 and 70 days, respectively (p<0.001 for both doses). Both imatinib and IPI-504 similarly prolonged survival of mice with BCR-ABL-WT-induced CML. In the T315I CML mice prolonged survival of the IPI-504 treated cohort was associated with decreased peripheral blood BCR-ABL positive leukemia cells during treatment, less splenomegaly and improved pulmonary histopathlogy at necropsy. In CML mice receiving mixed BCR-ABL-WT- or T315I-transduced donor bone marrow cells, Hsp90 inhibition more potently suppressed T315I-expressing leukemia clones relative to the WT clones, consistent with in vitro studies where T315I BCR-ABL was more sensitive to IPI-504 induced degradation in cell lines than WT BCR-ABL. Combination treatment with IPI-504 and imatinib was more effective than either treatment alone in prolonging survival of mice bearing both WT and T315I leukemic cells. IPI-504 also significantly prolonged survival of B-ALL mice bearing the T315I mutation (p<0.001). These results provide a rationale for use of an Hsp90 inhibitor as a novel approach to overcoming resistance to TKIs as well as the potential for first line combination treatment in CML patients. The potential for IPI-504 to eliminate mutant kinases via Hsp90 inhibition provides a new therapeutic strategy for treating BCR-ABL-induced CML, ALL as well as other cancers resistant to treatment with TKIs.

Activation of the JNK Pathway Mediates Chemoresistance in T-Cell Acute Lymphoblastic Leukemia by Phosphorylation and Proteosomal Degradation of BimEL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2372-2372
Author(s):  
Kam Tong Leung ◽  
Karen Kwai Har Li ◽  
Samuel Sai Ming Sun ◽  
Paul Kay Sheung Chan ◽  
Yum Shing Wong ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cytochrome C ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
Jnk Pathway ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Induced Apoptosis

Abstract Despite progress in the development of effective treatments against T-cell acute lymphoblastic leukemia (T-ALL), about 20% of patients still exhibit poor response to the current chemotherapeutic regimens and the cause of treatment failure in these patients remains largely unknown. In this study, we aimed at finding mechanisms that drive T-ALL cells resistant to chemotherapeutic agents. By screening etoposide sensitivity of a panel of T-ALL cell lines using DNA content and PARP cleavage as apoptosis markers, we identified an apoptosis-resistant cell line, Sup-T1. Western blot analysis and caspase activity assay showed that Sup-T1 cells were deficient in etoposide-induced activation of caspase-3 and caspase-9. In addition, mitochondrial cytochrome c release was not evident in etoposide-treated Sup-T1 cells. However, addition of exogenous cytochrome c in cell-free apoptosis reactions induced prominent caspase-3 activation, indicating that the chemoresistance observed in Sup-T1 cells was due to its insusceptibility to the drug-induced mitochondrial alterations. Analysis of the basal expression of the Bcl-2 family proteins revealed that the levels of Bcl-2 was higher in Sup-T1 cells, while Bax and BimEL levels were lower, when compared to etoposide-sensitive T-ALL cell lines. Gene silencing using antisense oligonucleotide to Bcl-2 and overexpression of Bax did not resensitize cells to etoposide-induced apoptosis. On the contrary, transient transfection of BimEL into Sup-T1 cells significantly restored etoposide sensitivity. Further experiments revealed that the lack of BimEL expression in Sup-T1 cells was due to the rapid degradation of newly-synthesized BimEL by the proteosomal pathway, as treatment of Sup-T1 cells with a proteosome inhibitor significantly restored the protein level of BimEL. Moreover, treatment with proteosome inhibitor resulted in mobility shift of BimEL, which was sensitive to phosphatase digestion. Furthermore, treatment of Sup-T1 cells with JNK inhibitor resulted in accumulation of BimEL, and pretreatment with JNK inhibitor restored sensitivity of Sup-T1 cells to etoposide-induced apoptosis, indicating that constitutive activation of the JNK pathway in Sup-T1 cells was responsible for promoting BimEL phosphorylation, and this may serve as a signal targeting BimEL to the proteosome for degradation. Altogether, our findings provide the first evidence that JNK activation correlates inversely with BimEL level by promoting its phosphorylation and degradation. This, in turn, reduces the sensitivity of T-ALL cells to chemotherapeutic agents.

A High Frequency of Blasts with the PNH Phenotype in Patients with ALL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1575-1575
Author(s):  
David Araten ◽  
Katie J Sanders ◽  
Dan Anscher ◽  
Leah Zamechek ◽  
Stephen P Hunger ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Lymphoblastic Leukemia ◽  
Normal Individuals ◽  
Wide Range ◽  
Blast Cells ◽  
B Lineage ◽  
Inactivating Mutation ◽  
Mutant Frequency

Abstract Abstract 1575 Poster Board I-601 It has been proposed that if more than 2 mutations are required for leukemia to arise, then an elevation in the mutation rate would be necessary for these mutations to coincide in the same cell, based on the rarity of spontaneous mutations (Loeb 1991). Hypermutability could occur transiently, for example, due to mutagens, or permanently, due to inactivation of repair pathways. Genome-wide analysis has indicated that in acute lymphoblastic leukemia (ALL), more than 2 somatic mutations are often found (Mullighan et al 2007), suggesting that hypermutability is likely to occur at least in some cases. To investigate this hypothesis, we have used our method for the detection of rare cells that have acquired a spontaneous inactivating mutation in the PIG-A gene. Expanded populations of stem cells with PIG-A mutations in the setting of bone marrow failure are the hallmark of paroxysmal nocturnal hemoglobinuria (PNH), but under most other circumstances, PIG-A mutations are thought to be growth neutral in vitro and in vivo. PIG-A is on the X-chromosome, and therefore, a single inactivating mutation is sufficient to produce the PNH phenotype, characterized by a lack of glycosylphosphatidylinositol (GPI)-linked proteins and an inability to bind to the FLAER reagent. Furthermore, it is known from patients with PNH and from cell lines that a very broad spectrum of mutations can inactivate PIG-A. This combination of features makes PIG-A uniquely suited as a sentinel gene for spontaneous mutagenesis in humans, which is otherwise very difficult to quantitate. Here we have analyzed a panel of blast cells derived from ficolled marrow samples from patients with B-lineage ALL, obtained from the Children's Oncology Group ALL Cell Bank. To determine the frequency of spontaneous PIG-A mutations, samples were stained sequentially with an Alexa-488 conjugate of the FLAER reagent, a mixture of murine anti-CD55 and anti-CD59 antibodies, FITC-conjugated rabbit anti-mouse immunoglobulin secondary antibody, and PE-conjugated anti-CD45 antibody. The blast population was identified based on forward and side scatter, dim expression of CD45, and exclusion of propidium iodide. Control GPI (+) and GPI (-) cells from a patient with PNH served as a control. Among 19 cases of B lineage ALL studied, the frequency of blast cells with the PNH phenotype varied over a very wide range, and the distribution of mutant frequency values was seen to be bimodal. One group, representing about 40% of the cases of ALL, had a median mutant frequency of 6.8 × 10-6, which is very similar to the proportion of granulocytes that acquire the PNH phenotype due to spontaneous PIG-A mutations in normal individuals (Araten et al, 1999). These values are also close to the frequency of mutants reported for the HPRT, GPA, and XK genes in normal individuals. The other 60% of the samples had a median mutant frequency of 465 × 10-6, which is far above the range of this parameter in normal individuals. Among 6 established cell lines derived from T-ALL, the mutant frequency ranged from 4.1 ×10-6 to 265 × 10-6. Here as well, the distribution suggested that in this subtype of ALL, there may be two distinct phenotypes with respect to this parameter. Based on this data, we believe that an increase in inactivating mutations is not essential for the development of ALL, but that it is a common feature of this condition. We cannot rule out the possibility that samples with a low mutant frequency would demonstrate some form of genomic instability that would not be detected by our assay, such as an increase in gene duplications or translocations. Further investigations are needed to determine whether hypermutability as detected by this assay is associated with distinct cytogenetic abnormalities, clinical features at presentation, and outcome. Disclosures No relevant conflicts of interest to declare.

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