Compensatory Signaling From ROR1 and the Pre-B Cell Receptor Promote Survival of t(1;19) Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2466-2466
Author(s):  
Vincent T Bicocca ◽  
Bill H Chang ◽  
Markus Muschen ◽  
Brian J. Druker ◽  
Jeffrey W Tyner
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Immunoblot Analysis ◽  
Cell Receptor ◽  
Cell Surface Receptor

Abstract Abstract 2466 BACKGROUND: Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the t(1;19) ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against t(1;19) ALL cells due to its capacity to inhibit tyrosine kinases necessary for transduction of pre-B cell receptor (preBCR) signaling. Finally, we show that ROR1 and the preBCR activate mutually compensatory signaling pathways, suggesting that optimal therapeutic regimens would include agents targeting both pathways. METHODS: To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients with siRNAs and determined cell viability using an MTS assay. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cells lines and primary ALL cells by treating samples with a library of small-molecule inhibitors and cell viability was assessed by MTS. Signaling pathways disrupted by inhibitor treatment or ROR1 knockdown were interrogated by phospho-protein arrays and confirmed by immunoblot analysis. RESULTS: The RAPID assay identified a unique sensitivity to the cell surface receptor ROR1 in a subject identified with t(1;19) pediatric ALL. Similar sensitivity was not observed in ALL patients of alternative cytogenetic subtypes. Examination of additional ALL patient samples revealed conserved overexpression of the ROR1 transcript in t(1;19)-positive specimens with absence of ROR1 expression in t(1;19)-negative samples. Cell lines and early passage xenograft cells confirmed overexpression and functional dependence of t(1;19)-positive cells on ROR1. A subsequent kinase inhibitor screen of t(1;19) ALL cell lines and patient samples revealed universal sensitivity to the FDA-approved drug dasatinib. Further examination revealed the dasatinib targets, BTK and LYN, which signal downstream of the preBCR as the viability dependent targets of dasatinib in t(1;19) ALL. Inhibition of the preBCR results in transient loss of AKT activity and, surprisingly, upregulation of ROR1. Analysis of signaling pathways after silencing of ROR1 or dasatinib treatment revealed compensatory signaling pathways emanating from ROR1 and the preBCR that both serve to regulate AKT activity. Consequently, combination of ROR1 knockdown and dasatinib treatment resulted in additive impairment of cell viability compared with either treatment alone. CONCLUSION: The cell surface receptor ROR1 is consistently overexpressed in t(1;19) ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Similarly, t(1;19) cells are sensitive to the kinase inhibitor dasatinib due to activity against the preBCR. Combined targeting of ROR1 and the preBCR with dasatinib yields an additive viability effect due to compensatory signaling pathways aimed at regulating AKT. These results demonstrate a novel mechanism of AKT regulation in t(1;19) ALL as well as a therapeutic strategy for treatment of t(1;19) ALL. Disclosures: Druker: MolecularMD: Equity Ownership, OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t; Ariad Pharmaceuticals: Consultancy; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; Bristol-Myers Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Cylene: Consultancy.

ROR1 as a Therapeutic Target In E2A-PBX1-Positive Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 539-539
Author(s):  
Vincent Bicocca ◽  
Bill H Chang ◽  
Markus Muschen ◽  
Brian J Druker ◽  
Jeffrey W Tyner
Keyword(s):  
Tyrosine Kinase ◽  
Cell Viability ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Mononuclear Cells ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Fusion Product ◽  
Rapid Assay ◽  
Pediatric All

Abstract Abstract 539 Background: Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. These lesions can result in aberrant tyrosine kinase expression and activity required for leukemogenesis. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. The role of E2A-PBX1 in the development of acute leukemia remains unclear. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the E2A-PBX1 ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against E2A-PBX1-positive ALL cells. Methods: To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients by electroporating siRNAs individually targeting each member of the tyrosine kinase gene family. Four days later, we determined the cell viability and tabulated sensitivity of the cells to any individual tyrosine kinase. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cell lines and primary ALL cells by treating samples with a library of small-molecule inhibitors consisting of 90 cell-permeable inhibitor compounds. Inhibitors are plated at four serial dilutions to allow IC50 calculations. The effect of each drug on cell viability is determined at day three by an MTS cell viability assay. Results: The RAPID assay identified a unique sensitivity to the receptor tyrosine kinase-like orphan receptor 1 (ROR1) in a subject identified with E2A-PBX1-positive B cell precursor pediatric ALL. Similar sensitivity was not observed in patients of other leukemic backgrounds or ALL patients of alternative cytogenetic subtypes. Examination of mononuclear cells from this patient by reverse-transcriptase-PCR revealed overexpression of the ROR1 transcript compared with ALL patients lacking the E2A-PBX1 fusion product. Examination of 12 additional E2A-PBX1-positive ALL patient samples revealed universal overexpression of ROR1 within the E2A-PBX1 background. Analysis of E2A-PBX1-positive cell lines and early passage xenograft cells showed both overexpression of ROR1 and sensitivity to siRNA-mediated ROR1 silencing, confirming the ROR1 dependent survival observed in primary cells with the RAPID assay. Finally, since ROR1 is defined as a tyrosine kinase, we performed a kinase inhibitor screen and identified universal sensitivity of E2A-PBX1-positive cell lines and patient samples to the FDA-approved drug dasatinib. Hence, dasatinib is suggested as a potential therapeutic for E2A-PBX1-positive ALL patients. Conclusion: The cell surface receptor ROR1 is consistently overexpressed in E2A-PBX1-positive ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Finally, the kinase inhibitor dasatinib is suggested as a novel therapeutic tool for treatment of E2A-PBX1-positive ALL based on universal sensitivity of E2A-PBX1 samples to this kinase inhibitor. Disclosures: Druker: Molecular MD: Equity Ownership.

Pre-BCR Signaling Activity Predicts Sensitivity To Syk Kinase Inhibition In B Cell Acute Lymphoblastic Leukemia (B-ALL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 614-614
Author(s):  
Stefan Koehrer ◽  
Richard E. Davis ◽  
Greg Coffey ◽  
Ekaterina Kim ◽  
Nathalie Y. Rosin ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Lymphocyte ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Lymphocyte Development ◽  
Bcr Signaling ◽  
Associated Proteins ◽  
Cell Acute Lymphoblastic Leukemia ◽  
B Lymphocyte Development

Abstract B lymphocyte development proceeds in a stepwise fashion and is tightly linked to the generation of a functional B cell receptor (BCR). At the preB cell stage B lymphocyte progenitors express the precursor B cell receptor (pre-BCR), an immature form of the BCR consisting of two µ heavy chains (µHC) and two surrogate light chains (SLC). Pre-BCR expression marks the proB to preB transition and induces a burst in preB lymphocyte proliferation. In 20% of the cases B cell acute lymphoblastic leukemia (B-ALL) arises from lymphocytes arrested at the pre-BCR positive stage of lymphocyte development (preB-ALL). Due to the essential role of the pre-BCR for preB cell proliferation we hypothesized that pre-BCR signaling also is involved in the maintenance of preB-ALL. Consequently, pharmacological inhibition of Spleen tyrosine kinase (Syk), the main transducer of pre-BCR signaling, may serve as effective treatment for this subtype of B-ALL. We analyzed a panel of six ALL cell lines (SMS-SB, RCH-ACV, Nalm-6, Kasumi-2, 697, KOPN-8) arrested at the pre-BCR+ stage of B lymphocyte development (cytoIgµ+, sIgM-). Assessment of the baseline phosphorylation levels of the pre-BCR associated kinases Lyn, Syk and Btk by immunoblotting and subsequent densitometric analysis allowed us to assign B-ALL cells into groups with either high levels of Lyn, Syk and Btk phosphorylation or with low or absent phosphorylation of these kinases, respectively. Moreover cell lines with highly phosphorylated Lyn, Syk and Btk also exhibited lower surface pre-BCR expression than cell lines with low phosphorylation levels. As pre-BCR activation is followed by its rapid internalization the concomitant presence of low pre-BCR expression and high phosphorylation of pre-BCR associated proteins suggests increased pre-BCR pathway activity. When we investigated the impact of pharmacological inhibition of the pre-BCR associated kinase Syk through the highly specific inhibitor PRT060318, preB-ALL cell lines with highly phosphorylated pre-BCR associated molecules turned out to be more sensitive to Syk inhibition (IC50 < 1.6µM) than preB-ALL cell lines with less phosphorylation (IC50 > 3.9µM). In proliferation assays PRT060318 inhibited preB-ALL proliferation in a dose dependent manner, whereas PRT060318 did not induce apoptosis in concentrations as high as 5µM. This supports the notion that pre-BCR signaling activity may be more relevant for preB-ALL proliferation than for preB-ALL viability. In line with these results the pre-BCR- proB-ALL cell lines REH and RS4;11 were highly resistant to Syk inhibition in all functional assays (IC50 > 10µM), suggesting that pre-BCR expression is a prerequisite for sensitivity to Syk inhibition. To examine the molecular changes following pre-BCR inhibition, ALL cells were treated with increasing concentrations of PRT060318 (100nM-5µM) for two hours and then subjected to immunoblotting. Syk inhibition led to a dose dependent decrease in AKT phosphorylation in all preB-ALL cell lines and subsequently reduced phosphorylation of FOXO transcription factors. In the resistant proB-ALL cell line REH, AKT and FOXO phosphorylation were not affected. Gene expression analysis of the preB-ALL cell lines RCH-ACV and Nalm-6 further suggested that PRT060318 interferes with pre-BCR signaling. Treatment with 1µM PRT060318 for 72h reduced the expression of genes associated with pre-BCR signaling (e.g. BCL6, CD22, PTPN6) and Ingenuity Pathway Analysis identified pre-BCR signaling as the main target of PRT060318 in both cell lines (p<0.05). We are currently validating the GEP analysis by quantitative PCR and immunoblotting. In conclusion, we provide evidence for the efficacy of Syk inhibition in pre-BCR+ ALL. Moreover we were able to correlate the baseline phosphorylation status of pre-BCR associated proteins and pre-BCR expression levels with the sensitivity of preB-ALL to the Syk inhibitor PRT060318. These findings provide a first rationale for the clinical testing of Syk inhibitors in preB-ALL, and suggest that activation status of pre-BCR associated molecules can help in selecting preB-ALL cases that are particularly sensitive to Syk inhibition. Disclosures: Coffey: Portola Pharmaceuticals: Employment.

scholarly journals Targeted Therapy for ETV6-RUNX1-Driven B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 62-62
Author(s):  
Roel Polak ◽  
Marc B. Bierings ◽  
Cindy S. van der Leije ◽  
Rosanna E.S. van den Dungen ◽  
Mathijs A. Sanders ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Fusion Protein ◽  
Cell Viability ◽  
B Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Growth Arrest ◽  
Leukemic Cells ◽  
Cell Precursor

Abstract Background: Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the favorable prognostic parameters of this B-ALL subgroup, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. The molecular mechanisms underlying ETV6-RUNX1-driven leukemia are largely unknown. Increased knowledge of these mechanisms is essential to develop novel therapeutic strategies to selectively target ETV6-RUNX1-positive leukemia. Objectives: This study aims to identify and target the molecular drivers behind ETV6-RUNX1-positive BCP-ALL. Results: Gene expression profiling of leukemic blasts of 654 ALL patients revealed that the class III PI3-kinase Vps34, an important regulator of autophagy, was exclusively up-regulated in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (2.7-fold; p ≤ 10-30). In addition, ectopic expression of ETV6-RUNX1 in cord blood-derived hematopoietic progenitor cells (CB-HPCs) significantly induced expression of Vps34 1.3-fold already 40 hours after transduction (p ≤ 0.05). This suggests that the Vps34-autophagy pathway is activated by ETV6-RUNX1, which may mechanistically explain the leukemogenic and pro-survival properties ascribed to ETV6-RUNX1. In correspondence, Ingenuity Pathway Analysis (IPA) predicted a pro-survival and pro-proliferative phenotype in ETV6-RUNX1 transduced CB-HPCs and highlighted a network of up-regulated transcription factors, including HEY1, EGR1, GATA1 and GATA2 (2 – 25-fold up-regulation; p ≤ 0.05). Luciferase reporter assays revealed that not only the ETV6-RUNX1 fusion protein, but also the ETV6-RUNX1-induced target genes HEY1, EGR1 and GATA1 positively regulate Vps34 promoter activity (5 – 13-fold up-regulation; p ≤ 0.01).Lentiviral knockdown experiments were performed to elucidate the importance of Vps34 expression in ETV6-RUNX1-positive BCP-ALL cells. Knockdown of all Vps34 transcript variants, with two independent constructs, led to complete growth arrest of the ETV6-RUNX1-positive cell lines REH and AT2, while this only led to a decrease in proliferation of the ETV6-RUNX1-negative cell line NALM6. This growth arrest was caused by a significant induction of apoptosis (more than 4-fold 7 days after transduction; p ≤ 0.001) and a significantly reduced percentage of cycling cells (1.3-fold 7 days after transduction; p ≤ 0.05). Analysis of p62 protein expression by western blot and reverse phase protein arrays revealed that the levels of autophagy were significantly higher in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (p ≤ 0.001). In addition, knockdown of ETV6-RUNX1 and Vps34 significantly reduced autophagy, quantified with confocal microscopy, in ETV6-RUNX1-positive cells with 50% and 84%, respectively (p ≤ 0.01). Furthermore, pharmacological inhibition of autophagy with hydroxychloroquine (HCQ) significantly reduced cell viability of BCP-ALL cell lines and primary patient-derived BCP-ALL cells (p ≤ 0.001). Treatment of the ETV6-RUNX1-positive BCP-ALL cell lines REH and AT2 with 20 µg/mL HCQ resulted in a 82% and 95% reduced cell viability, while the viability of ETV6-RUNX1-negative BCP-ALL cell lines and T-ALL cell lines were reduced to a lesser extent (NALM6: 43%; TOM-1: 50%; Loucy: 40%; Jurkat: 0%). Importantly, HCQ selectively sensitized ETV6-RUNX1-positive leukemic cells to L-asparaginase treatment in clinically relevant concentrations. Treatment of primary ETV6-RUNX1-positive patient cells with 10 µg/mL HCQ resulted in a 70% reduction in cell survival during L-asparaginase exposure (p ≤ 0.01). This sensitization was not observed in ETV6-RUNX1-negative BCP-ALL cells. Conclusion: The ETV6-RUNX1 fusion protein activates autophagy via Vps34, which is essential for survival and proliferation of ETV6-RUNX1-positive cells. Inhibition of autophagy in primary ETV6-RUNX1-positive leukemic cells inhibited cell survival and sensitized these cells to L-asparaginase treatment. These results indicate that autophagy inhibition may provide a novel means to sensitize L-asparaginase-resistant ETV6-RUNX1-positive BCP-ALL patients. 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.

Flow Cytometric Diagnosis of the Cell Lineage and Developmental Stage of Acute Lymphoblastic Leukemia by Novel Monoclonal Antibodies Specific to Human Pre–B-Cell Receptor

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4317-4324 ◽  
Author(s):  
Keiko Tsuganezawa ◽  
Nobutaka Kiyokawa ◽  
Yoshinobu Matsuo ◽  
Fujiko Kitamura ◽  
Noriko Toyama-Sorimachi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Lineage ◽  
Cell Receptor ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Cytoplasmic Staining ◽  
Flow Cytometric

Abstract Three novel monoclonal antibodies (MoAbs) have been established that recognize distinct epitopes of a human pre–B-cell receptor (pre-BCR) composed of a μ heavy (μH) chain and a λ5/VpreB surrogate light (SL) chain. HSL11 reacts with λ5 whereas HSL96 reacts with VpreB. Intriguingly, HSL2 does not bind to each component of the pre-BCR but does bind to the completely assembled pre-BCR complex. Flow cytometric analyses with cytoplasmic staining of a panel of human cell lines showed that HSL11 and HSL96 specifically stained cell lines derived from the pro–B and pre–B-cell stages of B-cell development. In contrast, HSL2 stained exclusively cell lines derived from the pre–B-cell stage. These results prompted us to explore the possibility of clinical application of these MoAbs for the determination of the cell lineage and developmental stage of acute lymphoblastic leukemia (ALL). Whereas none of mature B-lineage ALLs (B-ALLs), T-lineage ALLs (T-ALLs), and acute myeloid leukemias analyzed were stained in the cytoplasm with these three MoAbs, the vast majority of non–B- and non–T-ALLs (53 out of 56 cases) were found positive for either λ5, Vpre-B, or both in their cytoplasm. Among these 53 cytoplasmic SL chain-positive ALLs, 19 cases were also positive for cytoplasmic μH chain, indicative of pre–B-cell origin. Interestingly, 6 out of these 19 pre–B-ALL cases were found negative for cytoplasmic staining with HSL2. From these results, we propose a novel classification of B-ALL in which five subtypes are defined on the basis of the differential expression of SL chain, μH chain, pre-BCR, and light chain along the B-cell development.

Effect of Protein Kinase C β Specific Inhibition On Acute Lymphoblastic Leukemia Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4817-4817
Author(s):  
Nakhle S Saba ◽  
Hana F Safah ◽  
Laura S Levy
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Growth Inhibition ◽  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Chromosomal Abnormality ◽  
Lymphoblastic Leukemia ◽  
Specific Inhibitor ◽  
B Cell Lymphoma ◽  
Specific Inhibition

Abstract Abstract 4817 Acute lymphoblastic leukemia (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 basically 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, PKCβ targeting arose as a new, promising, and well tolerated treatment strategy in a variety of neoplasms, especially in B-cell malignancies. It showed encouraging results in preclinical and clinical studies involving chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma. PKCβ plays a major role in B-cell receptor signaling, but studies describing the role of PKCβ in B-cell ALL are lacking. In the present study, we measured the sensitivity of a variety of B-cell ALL cell lines to PKCβ specific inhibition. Three cell lines were studied: RS4;11 (characterized by the t(4;11) chromosomal abnormality), TOM-1 (characterized by the t(9;22) chromosomal abnormality), and REH (characterized by the t(12;21) chromosomal abnormality). Cells were tested for PKCβ1 and PKCβ2 expression by immunoblot. Cell viability was measured when PKCβ-specific inhibitor at concentrations of 1, 2.5, 5, 10, 20 and 30 μM was added for 48 hours in the presence of 10% fetal bovine serum (FBS). MTS assay was performed to quantify cell viability. Results showed that all three cell lines express PKCβ1 and PKCβ2. Treatment with PKCβ-specific inhibitor resulted in a dose-dependent inhibition of cell proliferation; Sensitivity was evident at 1 μM for RS4;11 cell line, and at 2.5 μM for TOM-1 and REH cell lines, with 10% cell growth inhibition; Growth inhibition increased to 90% for all cell lines at an inhibitor concentration of 30 μM. These results indicate that PKCβ plays an important role in the malignant process in B-cell ALL, and suggest that PKCβ targeting should be considered as a potential treatment, whether in combination with the current regimens used or as a single agent monotherapy. Ongoing studies in our lab will detail the mechanism of PKCβ and adverse cytogenetics like t(4;11) and t(9;22). Disclosures: No relevant conflicts of interest to declare.

Development of a Small-Molecule Inhibitor Screen to Rapidly Identify Key Signaling Pathways in Leukemogenesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 708-708
Author(s):  
Jeffrey W Tyner ◽  
Luke Fletcher ◽  
Wayne Yang ◽  
Stephen T Oh ◽  
Jason R. Gotlib ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Viability ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Small Molecule ◽  
Tyrosine Kinases ◽  
Leukemic Cell ◽  
Small Molecule Inhibitor ◽  
Kinase Activation ◽  
Molecule Inhibitor

Abstract Abstract 708 Aberrantly activated tyrosine kinases and their associated signaling pathways are critical to leukemogenesis and primary acute myeloid leukemia (AML) cell viability. While aberrant kinase activation has been confirmed in a significant percentage of AML, constitutive phosphorylation of STAT5, a marker of tyrosine kinase activation, is present in the majority of AML samples indicating that as yet unidentified tyrosine kinases can be aberrantly activated and contribute to leukemogenesis. Efforts to identify activating tyrosine kinase mutations using high-throughput sequencing have identified low frequency mutations of uncertain functional significance. Because these studies failed to detect additional high-frequency kinase mutations, the identity and mechanism of tyrosine kinase activation may be unique in many AMLs. To avoid the imitations of high-throughput sequencing, we have developed a functional assay that can rapidly and simultaneously identify therapeutic targets while providing therapeutic options. Methods: To rapidly identify activated kinase pathways in individual, primary AML samples, we have developed a small-molecule inhibitor array which includes 90 small-molecule, cell-permeable inhibitor compounds including a core of 36 tyrosine kinase inhibitors that covers the majority of the tyrosine kinome. Many of the inhibitors are available for clinical use or are in clinical development. In this assay, inhibitors were placed in 96-well plates at four serial dilutions to allow IC50 calculations. Three days after adding primary AML cells to each well, we performed an MTS cell viability assay to evaluate the effect of each inhibitor on cell viability. Because most inhibitors affect multiple kinases, we compared target specificities of compounds that decrease primary AML cell viability with those that have no effect to identify potential targets. Results: In preliminary proof-of-principal experiments, we tested leukemia cell lines with known activating tyrosine kinase mutations and Ba/F3 cell lines expressing activated tyrosine kinases. Appropriate inhibitor sensitivity profiles were obtained in CMK cells which depend on a JAK3 A572V mutation for viability, MKPL-1 cells with an activating CSF1R translocation, and in a Ba/F3 line expressing JAK2 V617F. In addition to the primary target, downstream targets were frequently identified; MKPL-1 cells also showed sensitivity to phosphoinositol 3-kinase and NFKB inhibitors. Thus, not only primary targets but the downstream signaling pathways critical to leukemic cell viability can be highlighted using this assay. To date, we have analyzed approximately 150 primary leukemia and lymphoma samples. In some cases, targets could be identified by comparison of overlapping kinase specificities for compounds that decreased leukemic cell viability and subtraction of possible kinase targets inhibited by compounds that had no effect on viability. However, many cases exhibited complex, often unique, inhibitor sensitivity profiles that complicated target identification. Comparison with sensitivity profiles for known aberrantly activated kinases was useful when available. Accordingly, additional leukemia cell lines and Ba/F3 lines that depend on a single aberrantly activated tyrosine kinase for viability are being evaluated. Automated scripts that correlate the leukemic cell inhibitor sensitivity with the inhibitor target specificity are also in preparation. Conclusions: These preliminary data demonstrate that the small-molecule inhibitor functional assays can rapidly identify disease causing genes, provide insights into their mechanism of action, and suggest therapeutic options. The distinct patterns of tyrosine kinase sensitivity in these samples support the hypothesis that tyrosine kinases and related pathways contributing to leukemogenesis in each patient may be different and that targeted therapy will be most effective when administered on an individualized basis. Disclosures: Druker: OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding.

Quantitative Proteomic Analysis Reveals Maturation As a Mechanism Underlying Glucocorticoid Resistance in Childhood Acute Lymphoblastic Leukemia and PAX5 As a Re-Sensitising Therapeutic Target

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1411-1411
Author(s):  
Lindsay Nicholson ◽  
Caroline Evans ◽  
Elizabeth C Matheson ◽  
Lynne Minto ◽  
Christopher Keilty ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Proteomic Analysis ◽  
Lymphoblastic Leukemia ◽  
Childhood All ◽  
Resistant Phenotype

Abstract Abstract 1411 Glucocorticoids (GC), such as prednisolone and dexamethasone, are an integral component of the multi-agent treatment of childhood acute lymphoblastic leukemia (ALL). GC-resistance is a significant prognostic indicator of a poor treatment outcome and remains a clinical problem, with the underlying mechanisms still unclear. Mutation or loss of the primary mediator of GC-action, the glucocorticoid receptor (GR), underlies the GC-resistant phenotype in several commonly used leukemic cell lines. However, these events are rare in primary leukemic cells, with relatively few examples in vivo. This suggests that it may be possible to reverse the GC-resistant phenotype pharmacologically. We have used an iTRAQ proteomics approach for hypothesis generation of potential mechanisms for GC-resistance in childhood ALL. To achieve this, we compared a well-characterized GC-sensitive cell line, PreB 697, and a GC-resistant sub-clone (R3F9), both bearing wildtype GR, in a comparative proteomic experiment using 4-channel isobaric tagging for relative and absolute quantification (iTRAQ). A comparison of protein profiles before and after dexamethasone exposure of the two cell lines identified two transcription factors involved in B-cell differentiation, PAX5 and IRF4, to be differentially upregulated in the PreB 697 compared to the R3F9 cell line in response to GC. Experimentally, there was approximately 50% reduction in PAX5 basal protein expression in R3F9 compared to its GC-sensitive parent, a finding which was also evident in four other resistant sub-lines. This was accompanied by a decreased expression of CD19 and CD10, indicative of an increased B-cell maturation state. The reduced PAX5 level in the GC-resistant cell lines was not due to mono-allelic loss or mutation and mRNA levels were not significantly altered, suggestive of a post-transcriptional mechanism for PAX5 protein reduction. Paradoxically, knockdown of PAX5 reversed the GC-resistant phenotype of the R3F9 cell line such that the apoptotic response to dexamethasone was similar to that of the GC-sensitive parent line as measured by Annexin V staining (R3F9: mean 52.22%, SD 12.54%, n=3; PreB 697: mean 67.23%, SD 9.96%, n=3) and cell viability assays. This chemosensitization after PAX5 knockdown was specific to GC, with no difference in cell viability observed in either cell line after exposure to daunorubicin, vincristine or L-asparaginase when compared to negative siRNA or mock controls. This increase in GC-sensitivity was coupled with a significant upregulation of GR and its transcriptional target, GILZ. We also showed an enhanced GC response after PAX5 knockdown in two out of eight primary, diagnostic pre-B lineage ALL patient samples. Thus, in this ALL cell line model, quantitative proteomic analysis revealed increased maturation as a recurrent mechanism underlying GC-resistance and identifies PAX5 as a possible therapeutic target to fully re-sensitise GC-response in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Flow Cytometric Diagnosis of the Cell Lineage and Developmental Stage of Acute Lymphoblastic Leukemia by Novel Monoclonal Antibodies Specific to Human Pre–B-Cell Receptor

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4317-4324 ◽  
Author(s):  
Keiko Tsuganezawa ◽  
Nobutaka Kiyokawa ◽  
Yoshinobu Matsuo ◽  
Fujiko Kitamura ◽  
Noriko Toyama-Sorimachi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Lineage ◽  
Cell Receptor ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Cytoplasmic Staining ◽  
Flow Cytometric

Three novel monoclonal antibodies (MoAbs) have been established that recognize distinct epitopes of a human pre–B-cell receptor (pre-BCR) composed of a μ heavy (μH) chain and a λ5/VpreB surrogate light (SL) chain. HSL11 reacts with λ5 whereas HSL96 reacts with VpreB. Intriguingly, HSL2 does not bind to each component of the pre-BCR but does bind to the completely assembled pre-BCR complex. Flow cytometric analyses with cytoplasmic staining of a panel of human cell lines showed that HSL11 and HSL96 specifically stained cell lines derived from the pro–B and pre–B-cell stages of B-cell development. In contrast, HSL2 stained exclusively cell lines derived from the pre–B-cell stage. These results prompted us to explore the possibility of clinical application of these MoAbs for the determination of the cell lineage and developmental stage of acute lymphoblastic leukemia (ALL). Whereas none of mature B-lineage ALLs (B-ALLs), T-lineage ALLs (T-ALLs), and acute myeloid leukemias analyzed were stained in the cytoplasm with these three MoAbs, the vast majority of non–B- and non–T-ALLs (53 out of 56 cases) were found positive for either λ5, Vpre-B, or both in their cytoplasm. Among these 53 cytoplasmic SL chain-positive ALLs, 19 cases were also positive for cytoplasmic μH chain, indicative of pre–B-cell origin. Interestingly, 6 out of these 19 pre–B-ALL cases were found negative for cytoplasmic staining with HSL2. From these results, we propose a novel classification of B-ALL in which five subtypes are defined on the basis of the differential expression of SL chain, μH chain, pre-BCR, and light chain along the B-cell development.

scholarly journals Evaluation of Idelalisib with B-Cell Receptor or Orthogonal Pathway Inhibitors in Diffuse Large B-Cell Lymphoma Cell Lines in Vitro and In Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1845-1845 ◽  
Author(s):  
Sarah Meadows ◽  
Anella Yahiaoui ◽  
Rick Sorensen ◽  
Zhi-Hua Cui ◽  
Robert Brockett ◽  
...  
Keyword(s):  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Tumor Regression ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Bet Inhibitor ◽  
Large B Cell Lymphoma

Abstract Background: Idelalisib, a selective oral inhibitor of PI3Kd, is approved for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with follicular lymphoma who have received at least 2 prior therapies. Despite remarkable clinical efficacy, complete responses are rare, highlighting the need to identify more effective therapies, including combinations of novel agents. GS-4059 (ONO-4059) is an investigational next generation Bruton's tyrosine kinase (BTK) inhibitor with improved selectivity compared with ibrutinib. We report here the results of the combination of a PI3Kd inhibitor and GS-4059 in a diffuse large B-cell lymphoma (DLBCL) xenograft model, demonstrating supportive data for our ongoing combination trial in B-cell malignancies (NCT02457598). Additionally, we investigated preclinical orthogonal combination approaches for DLBCL. Methods: Growth inhibition was assessed using CellTiter-Glo Assay after 96 h incubation with idelalisib and GS-4059. CB17-SCID mice were irradiated, implanted subcutaneously with TMD8, and treated BID PO with the PI3Kd inhibitor GS-649443, GS-4059, or coformulated combination when tumors reached 200 mm3. Lysates from tumors or cell cultures were analyzed by Simple Western (Protein Simple). Synergy for antiproliferative effects was assessed using Chalice software (Horizon Discovery, Inc., Lehar et al., Nature Biotech, 2009). Results: Idelalisib and GS-4059 potently inhibited the ABC subtype DLBCL cell line TMD8, which is a B-cell receptor (BCR)-dependent line that exhibits chronic activated B-cell signaling due to mutations in CD79A/CD79B and MYD88 (Kim Y. et al., Hum Pathol, 2014). When a clinically relevant single concentration of idelalisib or GS-4059 was added in combination to a dose responsive effect of the other, a shift in EC50 on cell viability was seen. GS-4059 (50 nM) shifted the EC50 of idelalisib from 141 nM to 5 nM, a 28-fold shift. Idelalisib (1 µM) shifted the EC50 of GS-4059 from 27 nM to 2 nM, a 14-fold shift. Evaluation of downstream signaling pathways implicated in malignant B-cell survival and proliferation showed enhanced inhibition of pAkt S437, pBTK Y223, pErk1/2 T202/Y204, and MYC with a combination of idelalisib and GS-4059, more than either single agent alone. When TMD8 xenografts were treated with a PI3Kd tool compound, GS-649443, GS-4059 or a combination of the 2 inhibitors, a statistically significant decrease in tumor volume was seen as well as tumor regression, when compared with single agent effects (Figure 1A). Evaluation of TMD8 tumor lysates showed strong suppression of pAkt S437, pBTK Y223, pS6RP S235/236, and MYC in tumors treated with both GS-649443 and GS-4059 (Figure 1B). pS6RP S235/236 and MYC, in formalin-fixed paraffin-embedded (FFPE) TMD8 tumors, were profoundly inhibited in tumors treated with combination therapy compared to the monotherapies (Figure 1C). Since the combination of a PI3Kd inhibitor and GS-4059 led to TMD8 tumor regression, an effect correlated to strong down-modulation of MYC, the combination of idelalisib with a bromodomain and extra-terminal (BET) family inhibitor was explored as a potential new orthogonal combination approach for DLBCL. A panel of DLBCL cell lines was evaluated for inhibition of cell viability by idelalisib in combination with GS-5829, a BET inhibitor currently being evaluated in a phase 1 clinical trial. At clinically relevant concentrations, the combination of idelalisib and GS-5829 showed synergistic effects on cell viability in 2 of 6 ABC subtype, 4 of 5 GCB subtype, and 2 of 2 double-hit DLBCL cell. As compared with combination with other agents that inhibit the BCR pathway (GS-4059) or the Bcl-2 pathway (ABT-199), the broadest activity across cell lines was seen with the combination of idelalisib and GS-5829. Conclusion: Idelalisib and GS-4059 demonstrated synergistic inhibition of the TMD8 xenograft with concomitant inhibition of MYC. Screening of other targeted agent combinations in a panel of DLBCL lines revealed broad preclinical activity for the BET inhibitor GS-5829 in combination with idelalisib. This represents a potential orthogonal approach for a new therapeutic strategy for the treatment of B-cell malignancies. Figure 1A Figure 1A. Figure 1B Figure 1B. Figure 1C Figure 1C. Disclosures Meadows: Gilead Sciences: Employment. Yahiaoui:Gilead Sciences: Employment. Sorensen:Gilead Sciences: Employment. Cui:Gilead Sciences: Employment. Brockett:Gilead Sciences: Employment. Keegan:Gilead Sciences: Employment. Tannheimer:Gilead Sciences: Employment.

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