Sabine L. A. Plasschaert

Expression of Multidrug Resistance–Associated Proteins Predicts Prognosis in Childhood and Adult Acute Lymphoblastic Leukemia

Purpose: Patients with acute lymphoblastic leukemia (ALL) are treated with a variety of chemotherapeutic drugs, which can be transported by six multidrug resistance–associated proteins (MRP). These MRPs have strongly overlapping functional activities. The aim of this study was to investigate the expression levels of MRP1 to MRP6 and study their effect on prognosis. Experimental Design: The mRNA expression levels of MRP1 to MRP6 were analyzed by quantitative real-time PCR in leukemic blasts of 105 de novo ALL patients (adults, n = 49; children, n = 56) including 70% B-lineage and 30% T-lineage ALL patients. Results: Adults showed a higher expressions of MRP1 (P = 0.008), MRP2 (P = 0.026), and MRP3 (P = 0.039) than children. Interestingly, this difference disappeared when patients were categorized based on clinical outcome. Relapsed patients showed a higher expression of all MRP genes, except MRP4. For the total group of ALL patients, the expressions of MRP1, MRP2, MRP3, MRP5, and MRP6 predicted relapse. Moreover, high expression of all MRP genes, except MRP4, was associated with a reduced relapse-free survival in children and adults (MRP1, P = 0.005; MRP2, P = 0.008; MRP3, P = 0.001; MRP5, P = 0.016; MRP6, P = 0.037). Conclusions: The present study shows that a subset of ALL patients with high MRP expression has an unfavorable prognosis independently of age.

Influence of functional polymorphisms of the MDR1 gene on vincristine pharmacokinetics in childhood acute lymphoblastic Leukemia

Our objective was to investigate the effect of single nucleotide polymorphisms (SNPs) in the P‐glycoprotein MDR1 gene on vincristine pharmacokinetics and side effects in childhood acute lymphoblastic leukemia.

Prognosis in childhood and adult acute lymphoblastic leukaemia: a question of maturation?

Acute lymphoblastic leukaemia (ALL) is a disease diagnosed in children as well as adults. Progress in the treatment of ALL has led to better survival rates, however, children have benefited more from improved treatment modalities than adults. Recent evidence has underscored that the difference in characteristics and biology of adult versus childhood ALL might be the result of a different origin. According to the two-hit paradigm of Knudson, to develop cancer two genetic events are necessary. It has been suggested, that in childhood ALL the first genetic event happens in the more mature lymphoid committed progenitor cells, whereas in adult ALL the first hit occurs in multipotent stem cells. This review compares patient characteristics, the extent of the disease, leukaemic cell characteristics and treatment between childhood and adult ALL. This is discussed in relation to the hypothesis that the maturation stage of the cells, from which the leukaemia arises, is responsible for the differential behaviour of adult and childhood ALL.

TUMOUR REVIEWPrognosis in childhood and adult acute lymphoblastic leukaemia: a question of maturation?☆

Acute lymphoblastic leukaemia (ALL) is a disease diagnosed in children as well as adults. Progress in the treatment of ALL has led to better survival rates, however, children have benefited more from improved treatment modalities than adults. Recent evidence has underscored that the difference in characteristics and biology of adult versus childhood ALL might be the result of a different origin. According to the two-hit paradigm of Knudson, to develop cancer two genetic events are necessary. It has been suggested, that in childhood ALL the first genetic event happens in the more mature lymphoid committed progenitor cells, whereas in adult ALL the first hit occurs in multipotent stem cells. This review compares patient characteristics, the extent of the disease, leukaemic cell characteristics and treatment between childhood and adult ALL. This is discussed in relation to the hypothesis that the maturation stage of the cells, from which the leukaemia arises, is responsible for the differential behaviour of adult and childhood ALL.

High functional P-glycoprotein activity is more often present in T-cell acute lymphoblastic leukaemic cells in adults than in children

There is a distinct difference in prognosis between childhood versus adult acute lymphoblastic leukaemia (ALL). To define whether multidrug resistance (MDR) genes might contribute to this distinction, the expression and functional activity of P-glycoprotein (P-gp) and MDR associated proteins (MRP) were determined with RT-PCR (MDR-1, MRP1, MRP2, MRP3) and flow cytometry (P-gp and MRP). Patient samples were obtained from 36 children and 35 adults with de novo ALL. Of these patients, 38 showed a T-lineage and 33 showed a B-lineage immunophenotype. In the samples, large variability in P-gp activity (0.8-4.9) and MRP activity (1.1-13.9) was observed. Most T-ALL patients with high P-gp activity were adults (89%). The mRNA expression of MDR-1 correlated weakly with P-gp activity. In contrast, MRP activity did not correlate with the mRNA expression of MRP1, MRP2 and MRP3. In T-ALL, a worse overall survival and event-free survival was observed with increasing P-gp activity. P-gp activity had no prognostic impact in B-lineage ALL. In addition, high MRP activity did not influence treatment outcome in either T- or B-lineage ALL. Multivariate Cox regression analysis, showed P-gp activity to be the only unfavourable prognostic factor for overall survival in T-ALL. In conclusion, this study demonstrates the prognostic relevance of P-gp activity in T-ALL. Since the majority of the patients with high P-gp activity were adults, P-gp might contribute to the poor prognosis of adult T-ALL.

Breast cancer resistance protein (BCRP) in acute leukemia

Multidrug resistance, cross-resistance to structurally and functionally unrelated drugs, is an important cause of treatment failure in acute leukemia. Multidrug resistance can result from the overexpression of ATP-dependent efflux pumps, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family. Recently a novel transporter has been identified, which is called breast cancer resistance protein (BCRP), ABCG2 or mitoxantrone resistance protein. BCRP confers resistance to chemotherapeutic agents, such as mitoxantrone, doxorubicin and daunorubicin. This review describes BCRP detection techniques and the normal physiology of BCRP. The role of BCRP in the physiology of hematopoietic stem cells is addressed as well as the involvement of BCRP in multidrug resistance in acute leukemia. In AML and ALL, several studies showed that BCRP is expressed and functionally active at low, but variable levels. However, further studies are warranted to investigate its effect on clinical outcome, and explore whether patients could benefit from the combination of BCRP inhibitors and chemotherapy.

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma.

Abstract 5826: Essential role for cyclic-AMP responsive element binding protein 1 (CREB1) phosphorylation in the survival of medulloblastoma patients

Prognosis for recurrent medulloblastoma patients is extremely poor, with a median survival of 26.8 months. Treatment options for recurrent medulloblastoma patients are limited and ineffective due to resistance to chemotherapeutic agents and radiotherapy. Therefore, more research is required to address the high mortality of medulloblastoma patients that have therapy-resistant medulloblastoma. In a previous, study, we showed that cyclic-AMP responsive element binding protein (CREB1) was highly phosphorylated in primary medulloblastoma specimens. CREB is an important transcription factor involved in cerebellar cell proliferation and survival. In various cancers types, the expression of CREB is associated with prognosis. Untill now, CREB expression and its relation with overall survival has not been studied in medulloblastoma patients. In this study, we determined CREB phosphorylation levels and mRNA levels of CREB and its transcriptional co-activators CREBBP and EP300 using peptide phosphorylation and gene expression arrays in a cohort of 50 pediatric medulloblastoma patients. Furthermore, we performed shRNA-mediated knockdown of CREB, CREBBP and EP300 mRNA levels in medulloblastoma cell line DAOY. Knockdown was confirmed by means of qRT-PCR and western blotting. Next, cell viability assays were used to investigate the effect of chemotherapeutic reagents (etoposide and/or cisplatin) on cell viability in CREB, CREBBP or EP300 knockdown cells or in combination with a small molecule CREB inhibitor (KG-501).Low CREB phosphorylation levels in combination with low CREBBP and EP300 mRNA levels was significantly associated with poor overall survival (p=0.0001) with a median survival of 11.7 months in medulloblastoma patients. High CREB phosphorylation in combination with high CREBBP and EP300 mRNA levels was associated with a favourable prognosis (5 year-overall survival: 91%). Supervised clustering of gene expression data based upon low versus high CREB phosphorylation and CREBBP/EP300 mRNA levels and subsequent gene ontology analysis showed significant enrichment of genes associated with cerebellar and neuronal differentiation and development. Furthermore, knockdown with shCREB.3, shCBP.1-2 or shEP300A completely rescued DAOY cells from the etoposide-induced decrease in cell viability that was observed in the scrambled control. Similarly, addition of the CREB inhibitor KG-501 (2μM) completely rescued DAOY cells from the etoposide-induced decrease in cell viability and partially rescued DAOY cells from the cisplatin-induced decrease in cell viability.Together, these findings support an important role for CREB in neuronal differentiation, chemosensitivity and in the overall survival of pediatric medulloblastoma patients. Citation Format: Walderik W. Zomerman, Sabine L. Plasschaert, Frank J. Scherpen, Harm Jan J. Lourens, Geesina C. Huizinga, Eelco W. Hoving, Wilfred F. den Dunnen, Sophia Bruggeman, Eveline S.J.M. de Bont. Essential role for cyclic-AMP responsive element binding protein 1 (CREB1) phosphorylation in the survival of medulloblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5826. doi:10.1158/1538-7445.AM2017-5826

Exogenous HGF Bypasses the Effects of ErbB Inhibition on Tumor Cell Viability in Medulloblastoma Cell Lines

Recent clinical trials investigating receptor tyrosine kinase (RTK) inhibitors showed a limited clinical response in medulloblastoma. The present study investigated the role of micro-environmental growth factors expressed in the brain, such as HGF and EGF, in relation to the effects of hepatocyte growth factor receptor (MET) and epidermal growth factor receptor family (ErbB1-4) inhibition in medulloblastoma cell lines. Medulloblastoma cell lines were treated with tyrosine kinase inhibitors crizotinib or canertinib, targeting MET and ErbB1-4, respectively. Upon treatment, cells were stimulated with VEGF-A, PDGF-AB, HGF, FGF-2 or EGF. Subsequently, we measured cell viability and expression levels of growth factors and downstream signaling proteins. Addition of HGF or EGF phosphorylated MET or EGFR, respectively, and demonstrated phosphorylation of Akt and ERK1/2 as well as increased tumor cell viability. Crizotinib and canertinib both inhibited cell viability and phosphorylation of Akt and ERK1/2. Specifically targeting MET using shRNA’s resulted in decreased cell viability. Interestingly, addition of HGF to canertinib significantly enhanced cell viability as well as phosphorylation of Akt and ERK1/2. The HGF-induced bypass of canertinib was reversed by addition of crizotinib. HGF protein was hardly released by medulloblastoma cells itself. Addition of canertinib did not affect RTK cell surface or growth factor expression levels. This manuscript points to the bypassing capacity of exogenous HGF in medulloblastoma cell lines. It might be of great interest to anticipate on these results in developing novel clinical trials with a combination of MET and EGFR inhibitors in medulloblastoma.

Exogenous HGF bypasses ErbB inhibition on tumor cell viability in medulloblastoma cell lines

Recent clinical trials investigating receptor tyrosine kinase (RTK) inhibitors showed limited clinical responses in medulloblastoma. An emerging concept is the role of the tumor microenvironment in bypassing targeted therapies by producing ligands that can compensate for the drug-inhibited RTK via alternative routes of pathway activation. The aim of this study is to investigate the role of well-known CNS expressed growth factors in relation to the effects of hepatocyte growth factor receptor (MET) and epidermal growth factor receptor family (EGFR, ErbB2-4) inhibition on tumor cell viability and downstream signaling in medulloblastoma cell lines. Five different medulloblastoma cell lines (DAOY, RES256, UW402, UW426, and UW473) were treated with crizotinib or canertinib, targeting MET or EGFR, ErbB2-4, respectively. Upon treatment, cells were stimulated with the CNS expressed growth factor VEGF-A PDGF-AB, HGF, FGF-2 or EGF basic. Subsequently, a cell viability assay was used to measure cell viability upon growth factor stimulation, compared to non-growth factor stimulated cells. Expression levels of respective receptor tyrosine kinases (RTK’s) and growth factors were analyzed using flow cytometry and human growth factor antibody arrays. Phosphorylation status of RTK9s and downstream signaling effectors was visualized using western blot analysis and human phospho-kinase antibody arrays. We observed high MET and EGFR cell surface expression levels. Addition of HGF or EGF phosphorylated MET or EGFR, respectively, and resulted in downstream phosphorylation of Akt and ERK1/2 as well as increased tumor cell viability. Whereas crizotinib and canertinib inhibited cell viability and phosphorylation of Akt and ERK1/2, addition of HGF to canertinib significantly enhanced cell viability and phosphorylation of Akt and ERK1/2. The HGF induced bypass of canertinib was reversed by the addition of crizotinib. HGF protein was hardly released by medulloblastoma cell lines. This suggests that MET activation is mainly dependent on paracrine HGF contribution. In conclusion, these data point to the bypassing capacity of well-known CNS expressed paracrine growth factors (e.g. HGF) in medulloblastoma cell lines and provides a potential explanation for the limited clinical response of single RTK inhibitors in medulloblastoma clinical trials. It might be of great interest to anticipate on these results in developing novel clinical trials with a combination of tyrosine kinase inhibitors, targeting for example MET and EGFR in medulloblastoma. Citation Format: Walderik W. Zomerman, Sabine LA Plasschaert, Sander H. Diks, Harm-Jan Lourens, Tiny Meeuwsen-de Boer, Eelco W. Hoving, Wilfred FA den Dunnen, Eveline SJM de Bont. Exogenous HGF bypasses ErbB inhibition on tumor cell viability in medulloblastoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4025. doi:10.1158/1538-7445.AM2015-4025