In Vitro Effects of PI3Kδ Inhibitor GS-1101 (Cal-101) in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3534-3534 ◽  
Author(s):  
Nathalie Y Rosin ◽  
Stefan Koehrer ◽  
Ekaterina Kim ◽  
Susan O'Brien ◽  
William G. Wierda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
P Value ◽  
B Cell Malignancies ◽  
Growth Experiments

Abstract Abstract 3534 Acute lymphoblastic leukemia (ALL) is a highly heterogeneous disease. B-cell acute lymphoblastic leukemia (B-ALL) is characterized by uncontrolled proliferation of immature lymphoid blasts with suppression of normal hematopoiesis. Phosphoinositide 3-kinases (PI3K) transmit activation signals from diverse transmembrane receptors, leading to generation of phosphatidylinositol- 3,4,5-trisphosphate (PIP3) which promotes proliferation, differentiation, migration, and survival in lymphocytes and various other cell types. A knockout mouse model of the PI3K isoform p110δ demonstrates a unique role of p110δ (PI3Kδ) in B cell receptor (BCR) signaling. This is corroborated by clinical efficacy of the PI3Kδ inhibitor GS-1101 in mature B cell malignancies, especially in chronic lymphocytic leukemia (CLL). In contrast to mature B cell malignancies, expression and function of PI3Kδ in B-ALL has not been well characterized. We therefore analyzed PI3Kδ expression and effects of the PI3Kδ inhibitor GS-1101 in B-ALL. To screen efficacy of GS-1101 in B-ALL subsets, we performed viability and proliferation assays, using a panel of B-ALL cell lines, derived from different B-cell development stages (Pro-B: REH, RS4;11, Nalm-20, Nalm-21, TOM-1; Pre-B: Nalm-6, Kasumi-2, KOPN-8, SMS-SB, RCH-ACV, 697; Mature: Tanoue, Ball-1 unknown: CCRF-SB). A key downstream effector of PI3K is the serine/threonine kinase Akt, whose phosphorylation is used as a common readout of PI3K activation status. Western Blot analysis of the 15 cell lines showed almost identical levels of phospho-Akt (Ser473) in all tested cell lines, suggesting constitutive PI3K activity. To investigate the ability of GS-1101 to inhibit B-ALL cell proliferation, we performed cell growth experiments. Among the pre-B cell lines 4 of 6 showed a marked decrease in proliferation, 2 other pre-B cell lines showed a minor decrease. In contrast, none of the pro-B or mature B-ALL cell lines were affected by GS-1101. To explore the effects of GS-1101 on cell cycle of B-ALL cells, cell lines were treated with GS-1101 at concentrations ranging from 0.5μM to 5μM. In accordance with the cell growth experiments, G1 phase arrest and reduced numbers of S phase cells were detected in pre-B cell lines after GS-1101 treatment, but not in the pro-B or mature B cell lines. Next, we examined GS-1101 effects on metabolism of B-ALL cells via XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) staining. Cell lines were treated with GS-1101 concentrations between 0.1μM and 5μM for 3 days prior to XTT measurement. Pre-B cells showed a significant (p-value <0.0001) decrease in normalized absorbance compared to the control (without treatment) indicating a decrease in cellular viability. Finally, preliminary co-culture experiments of primary B-ALL samples and KUSA-H1 bone marrow stromal cells revealed significantly reduced B-ALL cell viability after GS-1101 treatment, signifying that GS-1101 can overcome microenviromental-mediated B-ALL cell protection; this is similar to that in other B cell malignances. In summary, these experiments demonstrate that GS-1101 inhibits growth, cell cycle progression and metabolic activity of pre-B ALL cells. Validation of these data with primary patient samples is ongoing. Disclosures: Lannutti: Gilead Sciences Inc: Employment.

Enzastaurin Induces Apoptosis and Cell Cycle Arrest in B-Cell Acute Lymphoblastic Leukemia Cell Lines Through AKT Pathway Inhibition and ß-Catenin Accumulation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1350-1350
Author(s):  
Nakhle Saba ◽  
Magdalena Angelova ◽  
Patricia Lobelle-Rich ◽  
Laura S Levy
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Growth Inhibition ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Time Dependent ◽  
Cell Growth Inhibition ◽  
Downstream Target ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 1350 Precursor B-Cell acute lymphoblastic leukemia (B-ALL) is the most common leukemia in children and accounts for 20% of acute leukemia in adults. The intensive induction–consolidation–maintenance therapeutic regimens used currently have improved the 5-year disease free survival to around 80% in children and to 25%-40% in adults. The poorer response in adults is due to the inability to tolerate the intensive chemotherapy, and to the biology of adult disease which is associated with poor-risk prognostic factors. In the present era of target-specific therapy, protein kinase C beta (PKCß) targeting arose as a new, promising, and well-tolerated treatment strategy for a variety of neoplasms, especially in B-cell malignancies. The most frequently examined drug candidate to date is enzastaurin (LY317615.HCl) (ENZ), an acyclic bisindolylmaleimide that is orally administered and selectively inhibits PKCß. PKCß plays a major role in B-cell receptor signaling, but studies describing the role of PKCß in B-ALL are primitive. In the present study, we investigate the effect of ENZ on a variety of B-ALL cells representing the wide spectrum of the disease. Seven B-ALL cell lines were studied: RS4;11 and SEM-K2 [both Pro-B ALL with t(4;11)(q21;q23)], TOM-1 and SUP-B15 [both Ph-positive Pro-B ALL with t(9;22)(q34;q11)], HB-1119 [Pre-B ALL with t(11;19)(q23;p13)], NALM-6 [Pre-B ALL with t(5;12)(q33;p13)], and Reh [Pre-B ALL with t(12;21)(p13;q22)]. Cells were tested against serial dilutions of ENZ (final concentrations: 0.5–20μM) for 24, 48, and 72 hours in flat bottom 96-well plates. MTS assay was performed to quantify cell viability. ENZ induced a dose and time-dependent cell growth inhibition in B-ALL cell lines. RS4;11, SEM-K2, and HB-1119 (all with translocations involving the MLL gene) showed the greatest sensitivity to ENZ, with statistically significant cell growth inhibition starting at 1 μM, a concentration easily achieved in-vivo. TOM-1 and SUP-B15, both Ph-positive ALL, showed the lowest sensitivity to ENZ. The mechanism of ENZ cell growth inhibition was shown by flow cytometric TUNEL assay to involve apoptotic induction and cell cycle inhibition. Because of its relatively high sensitivity to inhibition among B-ALL cells, RS4;11 was selected for further analysis of the effect of ENZ on phosphorylation of AKT and its downstream target GSK3ß. RS4;11 cells were treated with the corresponding IC50 of ENZ for 0.5, 1, 2, 4, 24, and 48 hours. Treatment resulted in a time-dependent loss of AKT phosphorylation, at both ser473 and thr308, and a decrease in GSK3ß phosphorylation starting after 30 minutes and continuing to 48 hours. No effect on total AKT and GSK3ß was observed. By activating GSK3ß, its downstream target ß-catenin was expected to be diminished secondary to phosphorylation and proteasomal degradation. Surprisingly, ENZ induced a rapid and sustained ß-catenin accumulation, in both its nuclear and cytoplasmic forms. This was explained by a transient loss of ß-catenin phosphorylation at ser33-37; no effect on the proteasome activity was observed. Similar effect on total and phosphorylated ß-catenin was observed in all other cell lines. ß-catenin represents a central component of Wnt/ß-catenin canonical pathway which is found to be implicated in ALL pathogenesis. To investigate the effect of ENZ on Wnt/ß-catenin pathway, total RNA (1 μg) from RS4;11 treated for 24 hours with ENZ was profiled on RT2 Profiler™ PCR Array Human WNT Signaling Pathway (SABiosciences) and compared to untreated control. There were 8 genes whose expression changed >3-fold, most prominently c-Myc, c-Jun, and several genes encoding Wnt proteins. This was confirmed by western blot analysis showing that treatment with ENZ resulted in decreased c-Myc and increased c-Jun proteins expression. The latter showed a preliminary effect on p73, a p53 homologue, and is a subject for further investigation. These results indicate that PKCß plays an important role in the malignant process in B-cell ALL, and suggest that ENZ should be considered as a potential treatment, whether in combination or as a single agent monotherapy. Ongoing studies in our lab will detail the mechanism of PKCß inhibition, explain the contribution of ß-catenin accumulation to the cytotoxic effect of ENZ, and possible relationships between PKCß signaling and 11q23 translocation. Disclosures: No relevant conflicts of interest to declare.

Effects of Rituximab on JAK-STAT and NF-κB signaling pathways in acute lymphoblastic leukemia and chronic lymphocytic leukemia

2019 ◽  
Vol 44 (4) ◽  
pp. 499-509 ◽  
Author(s):  
Ayşegül Dalmızrak ◽  
Nur Selvi Günel ◽  
Burçin Tezcanlı Kaymaz ◽  
Fahri Şahin ◽  
Güray Saydam ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Signaling Pathways ◽  
Cell Lines ◽  
B Lymphocyte ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Protein Expressions ◽  
Apoptotic Effect

AbstractObjectivesRituximab is a monoclonal antibody that targets the B-lymphocyte surface antigen CD20. It is used in the treatment of some diseases including B-cell chronic lymphocytic leukemia (B-CLL). There are a lot of data regarding effect of Rituximab on lymphoma cells. But, there is no satisfactory information about the effect of Rituximab on the signaling pathways in leukemia cells. In this study, it was aimed to understand the effect of Rituximab on JAK-STAT and NF-κB signaling pathways in B-cell acute lymphoblastic leukemia (B-ALL) and B-CLL.Material and methodsApoptotic effect of Rituximab in the TANOUE (B-ALL) and EHEB (B-CLL) cell lines were evaluated by using the Annexin V method. mRNA expression levels of STAT3 and RelA were analysed by quantitative RT-PCR (Q-PCR). Alterations in STAT3 and RelA protein expressions were detected by using a chromogenic alkaline phosphatase assay after Western Blotting.ResultsRituximab had no apoptotic effect on both cell lines. Complement-mediated cytotoxicity was only detected in EHEB cells. mRNA and protein expressions of STAT3 and RelA genes were decreased following Rituximab treatment.ConclusionOur preliminary results suggest that the use of Rituximab might be effective in B-ALL though both signaling pathways.

scholarly journals FTY720 demonstrates promising preclinical activity for chronic lymphocytic leukemia and lymphoblastic leukemia/lymphoma

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 275-284 ◽  
Author(s):  
Qing Liu ◽  
Xiaobin Zhao ◽  
Frank Frissora ◽  
Yihui Ma ◽  
Ramasamy Santhanam ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Raji Cell ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Pp2a Activation

FTY720 is an immunosuppressant developed to prevent organ transplant rejection. Recent studies indicate an additional role for FTY720 in inducing cell apoptosis. We demonstrate here that FTY720 mediates toxic effects in cell lines representing different B-cell malignancies and primary B cells from patients with chronic lymphocytic leukemia (CLL). In contrast to previous reports in T-cell lines, FTY720-induced toxicity in the Raji cell line and primary CLL B cells is independent of activation of caspases or poly(ADP-ribose) polymerase processing. Further, pancaspase inhibitor Z-VAD-fmk failed to rescue these cells from apoptosis mediated by FTY720. FTY720 induced down-regulation of Mcl-1 but not Bcl-2 in CLL B cells. Overexpression of Bcl-2 failed to protect transformed B cells from FTY720-induced apoptosis, suggesting a Bcl-2–independent mechanism. Interestingly, FTY720 induced protein phosphatase 2a (PP2a) activation and downstream dephosphorylation of ERK1/2, whereas okadaic acid at concentrations that inhibited the FTY720-induced PP2a activation also resulted in inhibition of FTY720-mediated apoptosis and restoration of baseline ERK1/2 phosphorylation in primary CLL cells, indicating a role for PP2a activation in FTY720-induced cytotoxicity. Further, FTY720 treatment resulted in significant prolonged survival in a xenograft severe combined immunodeficiency (SCID) mouse model of disseminated B-cell lymphoma/leukemia. These results provide the first evidence for the potential use of FTY720 as a therapeutic agent in a variety of B-cell malignancies, including CLL.

scholarly journals The NSD2 p.E1099K Mutation in Relapse Pediatric Acute Lymphoblastic Leukemia Is Linked to Mercaptopurine Resistance

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3962-3962
Author(s):  
Jason Saliba ◽  
Joanna Pierro ◽  
Nikki Ann Evensen ◽  
Anita Qualls ◽  
Natasha Belsky ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Counting ◽  
Type I ◽  
Type Ii ◽  
Set Domain

Abstract Introduction: While the outcome for children with acute lymphoblastic leukemia (ALL) has improved dramatically, the prognosis for those who relapse remains poor. One of the most common alterations found at relapse is the p.E1099K missense change within the SET domain of NSD2, a histone methyltransferase that di-methylates histone 3 lysine 36 (H3K36). NSD2 has 3 isoforms, two of which, Type II (canonical) and REIIBP (C-terminal), contain the SET domain, and another, Type I (N-terminal), that does not. The p.E1099K mutation leads to increased enzymatic activity, but pathways leading to a clonal advantage are unknown in ALL. Methods: We used short hairpin RNAs (shRNAs) to target knockdown of two combinations of NSD2 isoforms: shI/II targets Types I and II, shII/RE targets Type II and REIIBP. Three different B-cell lines (Reh, 697, and KOPN-8) with 2 wildtype (WT) copies of NSD2 were stably transduced with shII/RE. Two B-Cell lines, RS4;11 and RCH-ACV, heterozygous for the NSD2 p.E1099K mutation, were transduced with shI/II and shII/RE. As a control, each B-cell line was stably transduced with a scrambled non-targeting (NT) shRNA. NSD2 knockdown was confirmed by Western Blots. Cell lines were treated for 5 days with chemotherapy agents commonly used in pediatric ALL treatments (mercaptopurine (MP), cytarabine, methotrexate, prednisone, and doxorubicin). Cytotoxicity was assessed by CellTiter- Glo® and significance between IC50s was determined by ANOVA and post hoc Tukey test. Cell proliferation was measured by cell counting with trypan blue. Cell cycle progression in RS4;11 lines was monitored with Edu staining and flow cytometry with and without exposure to MP. Results: Similar to previously reported results, knockdown of NSD2 in the 3 WT B-cell lines had no effect on cell proliferation. However, shI/II reduced growth by 40% in RS4;11 and 20% in RCH-ACV, while shII/RE decreased proliferation by 45% in RS4;11 and 55% in RCH-ACV when compared to their NT control. In RS4;11, both shI/II and shII/RE led to a similar 10% decrease in cells progressing through S phase compared to NT, which could be due to either a slower progression through cell cycle or less cells entering the cell cycle. Knockdown of NSD2 resulted in sensitivity to 6MP compared to NT in both RS4;11 and RCH-ACV lines. RS4;11 shII/RE had an IC50 3.2-fold more sensitive ( p<.01) and the RS4;11 shI/II IC50 was 1.25-fold more sensitive (NS) versus the NT control. Similarly, RCH-ACV shII/RE had an IC50 3.4-fold more sensitive (p<.01) and the RCH-ACV shI/II IC50 was 2.6-fold more sensitive (p<.01) compared to the NT control. No significant changes in drug sensitivity were noted for the 3 WT NSD2 knockdown B-cell lines compared to their NT controls. During a 120 hour exposure to MP, 34% more RS4;11 shII/RE cells were arrested in the G phase than NT controls, while 26% more RS4;11 shI/II cells were arrested in G phase relative to NT controls. This result indicates MP exposure leads to a reduced percentage of knockdown cells able to progress through the cell cycle. Overall, simultaneously reduced expression of Type II and REIIBP had a greater effect of on cell proliferation and MP response compared to the co-reduction of Types I and II NSD2 in the p.E1099K heterozygous cell lines. Conclusion: The p.E1099K mutation confers a growth advantage and resistance to MP, a cornerstone of ALL therapy. Concurrent reduction of Type II and REIIBP expression by shII/RE resulted in the largest impact on proliferation and MP sensitivity. Both of these isoforms include the SET domain containing the p.E1099K mutation, which indicates one or both isoforms could be responsible for changes in the chromatin state and other possible alterations that lead to a clonal advantage. Based on our findings, determining the mechanism of resistance to MP imparted by NSD2 p.E1099K is now a top priority. Disclosures No relevant conflicts of interest to declare.

scholarly journals Therapeutic potential of SGN-CD19B, a PBD-based anti-CD19 drug conjugate, for treatment of B-cell malignancies

Blood ◽  
2017 ◽  
Vol 130 (18) ◽  
pp. 2018-2026 ◽  
Author(s):  
Maureen C. Ryan ◽  
Maria Corinna Palanca-Wessels ◽  
Brian Schimpf ◽  
Kristine A. Gordon ◽  
Heather Kostner ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Lymphoblastic Leukemia ◽  
Preclinical Models ◽  
B Cell Malignancies ◽  
Key Points

Key Points SGN-CD19B is broadly active in vitro against malignant B-cell lines, including double-hit and triple-hit lymphoma cell lines. SGN-CD19B shows significant antitumor activity in vivo in preclinical models of B-NHL and B-cell–derived acute lymphoblastic leukemia.

Restraining the Proliferation of Acute Lymphoblastic Leukemia Cells by Genistein through Up-regulation of B-cell Translocation Gene-3 at Transcription Level

2019 ◽  
Vol 8 ◽  
Author(s):  
Masoumeh Abedi Nejad ◽  
Mohsen Nikbakht ◽  
Masoomeh Afsa ◽  
Kianoosh Malekzadeh
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cancer Cells ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Suppressor Gene ◽  
Transcription Level ◽  
Hematopoietic Malignancy ◽  
Proliferative Effect ◽  
Cancer Agent

Background: Acute lymphoblastic leukemia (ALL) is a highly prevalent pediatric cancer accounting for approximately 78% of leukemia cases in patients younger than 15 years old. Different studies have demonstrated that B-cell translocation gene 3 (BTG3) plays a suppressive role in the progress of different cancers. Genistein is considered a natural and biocompatible compound and a new anti-cancer agent. In this study, we evaluate the effect of genistein on BTG3 expression and proliferation of ALL cancer cells. Materials and Methods: ALL cell lines (MOLT4, MOLT17, and JURKAT) were cultured in standard conditions. Cytotoxicity of genistein was detected using MTT assay. The cells were treated with different concentrations of genistein (10, 25, 40, and 55μM) for 24, 48, and 72 hours, and then cell viability and growth rate were measured. The quantitative real-time polymerase chain reaction was applied to investigate the effect of genistein on BTG3 expression. Results: The percentage of vital cells treated with genistein significantly decreased compared to the non-treated cells, showed an inverse relationship with an increasing genistein concentration. The present study suggests a dose of 40μM for genistein as a potent anticancer effect. Genistein could elevate BTG3 for 1.7 folds in MOLT4 and JURKAT and 2.7 folds in MOLT17 cell lines at transcription level conveged with 60 to 90% reduction in the proliferation rate of cancer cells. Conclusion: Up-regulation of BTG3 as a tumor suppressor gene can be induced by genistein. It seems that BTG3 reactivation can be introduced as another mechanism of anti-proliferative effect of genistein and could be considered as a retardant agent candidate against hematopoietic malignancy.[GMJ. 2019;inpress:e1229]

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