Polo-Like Kinase 1 (PLK1) Inhibition Reduces Cell Proliferation and Induces Apoptosis in Childhood Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3529-3529 ◽  
Author(s):  
Stefanie A. Hartsink-Segers ◽  
Carla Exalto ◽  
Steven C. Clifford ◽  
Huib N. Caron ◽  
Rob Pieters ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Response To Therapy ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Pediatric All ◽  
Plk1 Expression ◽  
Low Expression

Abstract Abstract 3529 Polo-like Kinase 1 (PLK1) is an essential regulator of mitosis. It is often overexpressed and a predictor of poor prognosis in different types of solid tumours and adult hematopoietic malignancies, making PLK1 an interesting therapeutic target in these cancer types. No PLK1 inhibitor has entered clinical trials in pediatric malignancies yet. This study therefore aimed to determine the potential of PLK1 as a new target in the treatment of childhood acute lymphoblastic leukemia (ALL), the most common type of childhood cancer. Reverse phase protein arrays were performed to analyze levels of PLK1 protein expression and phosphorylation at T210, the major activating phosphorylation site, in ALL patient samples (n=174) and normal bone marrow mononuclear cells (nBM) (n=11). Both PLK1 expression and T210 phosphorylation were elevated (2.6-fold and 1.8-fold, respectively) in patients compared to nBM (p<0.0001). Furthermore, PLK1 protein expression levels were 1.3-fold higher in ALL cells carrying an E2A-PBX1 translocation than in other ALL genetic subtypes (p<0.0001). Knockdown of PLK1 expression by lentivirally delivered short hairpins led to inhibition of cell proliferation and induction of apoptosis in three precursor B-ALL cell lines and one T-ALL cell line, as indicated by decreased cell numbers and increased cleavage of PARP. This suggests an essential role for PLK1 in maintenance of the leukemic cell. An MTS cell viability assay was used to test the efficacy of the PLK1-specific inhibitor NMS-P937 (NMS-1286937) in primary ALL samples (n=15). When exposed to 120nM of NMS-P937 for 96 hours, there was a significant reverse correlation between PLK1 protein expression and leukemic cell survival (rs=−0.58; p=0.024): patient cells with a high expression were more sensitive than cells with a low expression, whereas nBM cells were resistant up to concentrations in the micromolar range. Cumulative incidence of relapse and non-response to therapy (CIR) did not significantly differ between ALL patients with high and low levels of PLK1 protein (CIR=25.9% vs 15.7%; p=0.215). Patients with a high PLK1 T210 phosphorylation level, however, showed a trend towards a higher CIR than those with a low expression (CIR=27.5% vs 14.0%; p=0.078). Finally, Sanger sequencing of PLK1 mutational hot-spots led to the identification of a new non-synonymous mutation in 1 out of 38 patients. This mutation was located in exon 5 of the PLK1 gene, resulting in Ser335Arg, and did not lead to unusual levels of PLK1 protein or T210 phosphorylation, nor to NMS-P937 sensitivity. Taken together, these results show that PLK1 is overexpressed in pediatric ALL and plays a pivotal role in the proliferation and survival of pediatric ALL cells. Moreover, they underline the potency of PLK1-inhibiting drugs as a valuable addition to current ALL treatment strategies, especially for cases expressing high levels of PLK1 protein. Disclosures: No relevant conflicts of interest to declare.

Systems Modeling of Proliferation Mechanisms in Childhood Acute Lymphoblastic Leukemia

2013 ◽  
pp. 227-256
Author(s):  
George I. Lambrou ◽  
Apostolos Zaravinos ◽  
Maria Adamaki ◽  
Spiros Vlahopoulos
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Systems Approach ◽  
Current Knowledge ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Systems Modeling ◽  
Global Patterns ◽  
Diseased State

Acute Lymphoblastic Leukemia (ALL) is the most common neoplasm in children, but the mechanisms underlying leukemogenesis are poorly understood, despite the existence of several theories regarding the mechanics of leukemic cell proliferation. However, with the advent of new biological principles, it appears that a systems approach could be used in an effective search of global patterns in biological systems, so as to be able to model the phenomenon of proliferation and gain a better understanding of how cells may progress from a healthy to a diseased state. This chapter reviews the current knowledge on proliferation dynamics, along with a discussion of the several existing theories on leukemogenesis and their comparison with the theories governing general oncogenesis. Furthermore, the authors present some “in-house” experimental data that support the view that it is possible to model leukemic cell proliferation and explain how this has been performed in in vitro experiments.

Correlation of karyotype and immunophenotype in childhood acute lymphoblastic leukemia.

1988 ◽  
Vol 6 (1) ◽  
pp. 56-61 ◽  
Author(s):  
C H Pui ◽  
D L Williams ◽  
P K Roberson ◽  
S C Raimondi ◽  
F G Behm ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Chromosomal Change ◽  
Cytogenetic Feature

To correlate leukemic cell karyotype with immunophenotype, we studied 364 children with acute lymphoblastic leukemia (ALL). A prognostically favorable cytogenetic feature, hyperdiploidy greater than 50 chromosomes, was found in 33% of cases classified as common ALL antigen positive (CALLA+) early pre-B (common) ALL, in contrast to 18% of pre-B cases (P = .012), 5% of T cell cases (P less than .001), and none of the B cell cases (P less than .001) or cases of CALLA negative (CALLA-) early pre-B ALL (P = .002). The frequency of translocations, an adverse cytogenetic feature, was significantly lower in CALLA+ early pre-B ALL cases (35%) than in B cell (100%; P less than .0001), pre-B (59%; P less than .001), or CALLA- early pre-B (62%; P = .016) cases. Thus, patterns of chromosomal change differ widely among the major immunophenotypic groups of ALL and may account for reported differences in responsiveness to treatment.

High CD45 (PTPRC) Expression Is Associated with An Overall Poor Outcome in Childhood Acute Lymphoblastic Leukemia Treated on the ALL-BFM 2000 Protocol and Exerts An Especially Pronounced Effect in Intermediate Risk Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 742-742
Author(s):  
Gunnar Cario ◽  
Rita Mitlohner ◽  
Martin Zimmermann ◽  
Renja Romey ◽  
Peter Rhein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Protein Expression ◽  
Lymphoblastic Leukemia ◽  
Treatment Resistance ◽  
Intermediate Risk ◽  
Prognostic Relevance ◽  
Risk Patients ◽  
Low Expression

Abstract Abstract 742 Further improvement of outcome in childhood acute lymphoblastic leukemia (ALL) could be achieved by identifying additional high-risk (HR) patients who then may benefit from an intensified treatment. In trial ALL-BFM 2000, the HR group was defined by inadequate initial response to induction treatment [poor prednisone response on treatment day eight (PPR), non remission on treatment day 33, and/or a high load of minimal residual disease (MRD, ≥10E-3) after 12 weeks of treatment (TP2)] and/or by positive cytogenetics for a t(4;11) or t(9;22). No MRD already on treatment day 33 defined standard risk (SR) patients, a measurable MRD at a low level characterized the intermediate risk (IR) group. Of importance, the majority of relapses occurred within this heterogeneous group of patients. In order to identify potential new stratification markers we earlier compared gene expression profiles of MRD resistance (HR) and sensitive (SR) ALL in a case-control setting (Cario et al, Blood 2005). Subsequently, we aimed at confirming the potential prognostic relevance of genes identified and their respective proteins in representative study populations. CD45 (also PTPRC, protein-tyrosine phosphatase, receptor-type, C) was one of these candidate genes. In order to assess its prognostic relevance, CD45 gene expression was first analyzed by quantifiable RT-PCR in a set of 555 precursor B-ALL (pB-ALL); its protein expression subsequently in 422 pB-ALL patients by flow cytometry. About one third of patients were included in both study sets. Normalization of protein expression was done by assessing the density of surface expression relative to its density on normal lymphocytes. The 90th percentile was used as a cut-off to distinguish a CD45-high from a CD45-low expression group in both analyses. In gene expression analysis we observed a significant association of a high CD45 expression with a high white blood cell count at diagnosis (WBC) (P = 0.0004), NCI-HR (P = 0.03) as well as presence of the MLL-AF4 rearrangement (P < 0.0001). Moreover, a high CD45 expression was associated with in-vivo treatment resistance as defined by MRD (P = 0.0025). Analyzing CD45 protein expression confirmed the association of a high expression with a high WBC (P < 0.0001), NCI-HR (P = 0.0002) as well as presence of the MLL-AF4 rearrangement (P < 0.0001). Moreover, although the association to treatment resistance was lower (P = 0.055), patients with a high CD45 expression had a significantly worse 5-years EFS probability of 62±8% compared to 82±2% for those in the low-expression group (P=0.002). Focussing on the IR group, patients with a high CD45 expression had a very poor outcome (EFS 45±15%) as compared to those with a low expression (EFS 86±3%, P < 0.0001). This effect was mainly related to a higher cumulative relapse incidence (55±16% vs. 13±3%, P < 0.0001). Of interest, no significant differences in EFS were seen in HR patients. Based on our results, consideration of CD45 protein expression may serve as additional stratification tool in BFM-based protocols to further refine true non-high-risk patients with a low risk of relapse by identifying additional patients at high relapse risk. Of importance, in view of the fact that CD45 expression was not prognostic in the high-risk group, patients with a high CD45 expression currently treated on non high risk arms, may potentially benefit from an intensified treatment in the HR arm. Disclosures: No relevant conflicts of interest to declare.

FLT3 inhibition selectively kills childhood acute lymphoblastic leukemia cells with high levels of FLT3 expression

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 812-820 ◽  
Author(s):  
Patrick Brown ◽  
Mark Levis ◽  
Sheila Shurtleff ◽  
Dario Campana ◽  
James Downing ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Gene Rearrangements ◽  
Childhood All ◽  
Activating Mutations ◽  
Mll Gene ◽  
Flt3 Inhibitor ◽  
Targeted Agent

AbstractFMS-like tyrosine kinase 3 (FLT3) is almost universally expressed in B-precursor childhood acute lymphoblastic leukemia (ALL). Cases of ALL with MLL gene rearrangements and those with high hyperdiploidy (&gt; 50 chromosomes) express the highest levels of FLT3, and activating mutations of FLT3 occur in 18% of MLL-rearranged and 28% of hyperdiploid ALL cases. We determined the antileukemic activity of CEP-701, a potent and selective FLT3 inhibitor, in 8 ALL cell lines and 39 bone marrow samples obtained at diagnosis from infants and children with various subtypes of ALL. CEP-701 induced pronounced apoptotic responses in a higher percentage of samples that expressed high levels of FLT3 (74%, n = 23) compared with samples with low levels of expression (8%, n = 13; P = .0003). Sensitivity to FLT3 inhibition was particularly high in samples with MLL gene rearrangements (82%, n = 11; P = .0005), high hyperdiploidy (100%, n = 5; P = .0007), and/or FLT3 mutations (100%, n = 4; P = .0021). Seven of 7 sensitive samples examined by immunoblotting demonstrated constitutively phosphorylated FLT3 that was potently inhibited by CEP-701, whereas 0 of 6 resistant samples expressed constitutively phosphorylated FLT3. We conclude that the FLT3 inhibitor CEP-701 effectively suppresses FLT3-driven leukemic cell survival. Clinical testing of CEP-701 as a novel molecularly targeted agent for the treatment of childhood ALL is warranted.

Rapid molecular response during early induction chemotherapy predicts a good outcome in childhood acute lymphoblastic leukemia

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 790-794 ◽  
Author(s):  
E. Renate Panzer-Grümayer ◽  
Monika Schneider ◽  
Simon Panzer ◽  
Karin Fasching ◽  
Helmut Gadner
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Risk Stratification ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Early Response ◽  
Response To Therapy ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Induction Treatment ◽  
Molecular Response ◽  
Significant Difference

Early response to therapy is an independent prognostic factor in childhood acute lymphoblastic leukemia. Although most patients have rapid early responses, as detected by morphology, 15% to 20% of patients have relapses. The authors evaluated residual disease by molecular methods on day 15 of minimal residual disease (MRD) therapy and compared these data with their recently established MRD-based risk stratification, defined by MRD levels 5 weeks after induction treatment and before consolidation. All 68 children treated according to current Berlin-Frankfurt-Münster (BFM) protocols went into morphologically complete remission after induction. There was a significant difference in outcome between children with rapid disease clearance and those with high levels of day-15 MRD (P = .035). Among patients with high levels of day-15 MRD, only the MRD-based risk stratification was predictive of the outcome. All patients with negative or low day-15 MRD had excellent prognoses and were in the MRD-based low-risk group. Thus, after only 2 weeks of treatment, the authors were able to identify a patient population of 20% who may benefit from the least intensive treatment.

scholarly journals Response: Genes associated with response to therapy in childhood acute lymphoblastic leukemia

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2487-2488
Author(s):  
Christian Flotho ◽  
Elaine Coustan-Smith ◽  
Deqing Pei ◽  
Cheng Cheng ◽  
Guangchun Song ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Response To Therapy ◽  
Childhood Acute Lymphoblastic Leukemia

scholarly journals Targeting High Dynamin2 Expression By Restoring Ikaros Function in Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2713-2713
Author(s):  
Zheng Ge ◽  
Jianyong Li ◽  
Baoan Chen ◽  
Sinisa Dovat ◽  
Chunhua Song
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Primary Cells ◽  
Proliferation Assay ◽  
High Relapse Rate ◽  
Leukemic Cell Lines ◽  
Ck2 Inhibitor

Abstract Background: Dynamin-2 (DNM2) is a GTPase essential for intracellular vesicle formation and trafficking, cytokinesis and receptor endocytosis. Mutations in DNM2 are common in early T-cell precursor acute lymphoblastic leukemia (ALL). However, DNM2 expression in other types of ALL is not reported. Ikaros, encoded by IKZF1, is a transcriptor factor functioned as a tumor suppress gene, and its dysfunction is associated with poor survival and high relapse rate in ALL. Casein Kinase II (CK2) inhibition could restore Ikaros function in high-risk leukemia and CK2 inhibitor-CX4945 showed the therapeutic efficacy on high-risk leukemia with human-derived xenograft mouse model. It is still undetermined if Ikaros regulates DNM2 expression in the leukemic cells. Methods: The 151 patients' and 30 volunteers' BM samples were collected between June 2008 and June 2014 at the First Affiliated Hospital of Nanjing Medical University. The ALL diagnosis was made according to the morphologic, Immunophenotypic, cytogenetic, and molecular criteria of WHO Diagnosis and Classification of ALL (2008).Cytogenetic and molecular analyses as previously reported. The DNM2 expression was determined by qPCR in the patients. All the patients were divided into high or low DNM2 expression groups (Q4 vs Q1-3) and the cutoff was determined by SPSS 17.0. For quantitative parameters, overall differences between the cohorts were evaluated using a Mann - Whitney U -test. For qualitative parameters, overall group differences were analyzed using a χ2 test. All statistical analyses were performed using the SPSS 17.0 and P<0.05 was considered statistically significant. The effect of Ikaros on DNM2 gene expression was observed by qPCR in the leukemic cells expressed Ikaros or Ikaros ShRNA. Ikaros binding with promoter of DNM2 was evaluated by chromatin immunoprecipitation assay following quantitative real-time PCR in leukemic cells. The effect of DNM2 inhibitor on cell proliferation was performed by WST-1 cell proliferation assay, and the synergy of Casein Kinase inhibitor which restores Ikaros function with DNM2 inhibitor on cell proliferation of leukemic cells was analyzed by CalcuSyn. Results: We studied DNM2 mRNA level in adults with B- and T-cell ALL, and found DNM2 is more highly expressed compared with normals in both forms of ALL. High DNM2 expression is significantly associated with poor overall survival (OS), high relapse rate, and leukaemia cell proliferation markers particularly in B-ALL. DNM2 expression is significantly higher in the patients with IKZF1 deletion compared to that of without deletion. Ikaros directly binds the DNM2 promoter in Nalm6 (B-ALL) and CEM (T-ALL) leukemic cells. Ikaros suppresses the transcription of DNM2 with luciferase reporter assay. Retroviral transduction of Ikaros results in the down-regulation of DNM2 in the leukemic cells. CK2 inhibitor, TBB increases Ikaros binding to promoter of DNM2 and suppresses DNM2 expression in an Ikaros-dependent manner in both leukemic cell lines and primary cells. TBB induced-increase of H3K9me3 binding on the promoter of DNM2 was also observed in leukemic cell lines and primary cells. Finally, DNM2 inhibitor-MiTMAB significantly suppresses the cell proliferation of Nalm6 and CEM cells with the WST-1 cell proliferation assay and has significantly synergistic effect with Ck2 inhibitor, CX-4945 in the cells. Conclusion: High DNM2 expression is associated with Ikarosdys-regulation, revealing their potential roles on the development of ALL. DNM2 inhibitor MiTMAB inhibits cell proliferation and has synergistic effect with CK2 inhibitor CX4945 in leukemic cells. Disclosures No relevant conflicts of interest to declare.

RNAi Identifies Survivin as a Potential Target for Therapy In Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5108-5108
Author(s):  
Bill H Chang ◽  
Jeffrey W Tyner ◽  
Abdusebur Jemal ◽  
Mathew Thayer ◽  
Brian J Druker
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Transplant ◽  
Pediatric Acute Lymphoblastic Leukemia ◽  
Potential Target ◽  
Functional Screen ◽  
Pediatric All

Abstract Abstract 5108 Background: Pediatric Acute Lymphoblastic Leukemia (ALL) remains the most common pediatric malignancy. Despite advances in treatment and outcomes, there continue to be subsets of patients that are refractory to standard intensive chemotherapy and hematopoietic stem cell transplant. Therefore, novel gene targets for therapy are needed to further advance treatment for this disease. Survivin, a member of the chromosome passenger complex and inhibitor of apoptosis has been shown to be over-expressed in malignant cells and in relapsed disease. Therefore, survivin may be a potential target for therapy in pediatric ALL. Further, RNAi technology can be used as a rapid functional screen to identify target genes crucial for viability in ALL cell lines and in primary patient samples. Design/methods: Pediatric lymphoblastic cell lines and fresh primary mononuclear cells from newly diagnosed patients with ALL were used for all studies. Survivin was silenced by transfecting cells (via electroporation or transductin (IDT)) with siRNA SMARTpools obtained from Dharmacon. Protein expression was identified 48 hours after treatment with siRNA. Viability was measured using a standard methane-thiosulfonate viability assay. Activation of apoptosis was identified using the Guava nexin Annexin V binding assay. Result: Survivin is highly expressed in multiple ALL cell lines and in many primary ALL samples. Silencing of survivin expression with siRNA decreases viability and increases apoptosis in ALL cell lines and in primary ALL samples. Decreased protein expression of approximately fifty percent was sufficient to cause an increase in cell death. Finally, inhibition of P53 expression abrogates this phenotype suggesting that the mechanism of cell death induced following survivin silencing involves activation of the P53 pathway. Conclusion: Gene silencing with siRNA can be used as a rapid functional screen to identify potential targets for therapy. Implementing this technique identified survivin as a potential target for therapy in pediatric ALL. The results of these experiments will be used as a foundation to develop a comprehensive understanding of the mechanisms of survivin dependence in pediatric ALL. Future studies will also be designed to develop a model system for targeting of survivin in a therapeutically-relevant manner such that these findings can be translated into improved treatments for patients with ALL. Disclosures: No relevant conflicts of interest to declare.

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