http://repub.eur.nl/res/aut/7803/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/7773/ 2006-06-02T00:00:00Z Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) is characterized by a high incidence (~80%) of rearrangements of the MLL gene, resistance to several important chemotherapeutic drugs, and a poor treatment outcome. With overall survival rates for infant ALL not exceeding 50%, current chemotherapeutic regimens clearly are not sufficient to adequately treat ALL in infants. Therefore, infant ALL urgently requires innovative therapeutic strategies in order to improve prognosis. For this it is of utmost importance to understand this malignancy by accurately studying its unique molecular and biological properties. Chapter 2 comprises a review describing important aspects of infant ALL, including MLL rearrangements, the cell of origin, the prenatal origin of this leukemia, the etiology and risk factors, prognostic factors, cellular drug resistance, and putative therapeutic approaches to improve prognosis. Chapter 3 describes a study investigating the mechanism underlying the remarkable sensitivity of infant ALL cells to Ara-C. This study demonstrates that elevated expression of the gene encoding the human equilibrative nucleoside transporter 1 (ENT1), on which Ara-C is mainly dependent to permeate the cell membrane, provides a plausible explanation for this phenomenon. Since Ara-C is a drug that is typically used to treat acute myeloid leukemia (AML), and MLL rearranged infant ALL cells often display myeloid characteristics, a reasonable hypothesis would be that Ara-C sensitivity (as a result of increased ENT1 expression) is associated with the presence of MLL rearrangements. In chapter 4 we address this hypothesis, and show that there is no direct association of the presence of MLL rearrangements and sensitivity to Ara-C. In chapter 5 we investigated whether the mechanism underlying Ara-C sensitivity in childhood AML is similar to the mechanism in infant ALL (i.e. increased ! ENT1 expression). This study revealed that elevated expression of ENT1 in childhood AML samples not only predicts sensitivity to Ara-C, but also appeared to be associated with sensitivity towards other nucleoside analogue drugs such as cladribine, decitabine, and gemcitabine. Since infant ALL cells in vitro are resistant to multiple chemotherapeutic drugs, infant ALL patients may legitimately be classified as multidrug resistant. Therefore, a plausible explanation for the chemo-resistant character of infant ALL cells could be the involvement multidrug resistance proteins that function as specialized membrane pumps capable of trafficking chemotherapeutic drugs out of the cell. In chapter 6 we investigated whether drug resistance in infant ALL is a consequence of increased drug efflux mediated by multidrug resistance pumps. Given the poor response of infant ALL patients to current chemotherapeutic regimes, it is of utmost importance to explore innovative therapeutic strategies. For this, we set out to search for molecular targets suitable to direct therapy against. In collaboration with Dr. Scott Armstrong (Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA) we observed that the gene encoding Fms-like tyrosine kinase receptor 3 (FLT3) is highly expressed in MLL rearranged ALL samples. As a consequence of high-level expression we demonstrated that FLT3 is constitutively activated in an MLL rearranged ALL cell line, promoting leukemic cell proliferation and survival. In chapter 7 we studied whether high-level expression of FLT3 is associated with constitutive FLT3 signaling in primary MLL rearranged infant ALL cells. Moreover, we investigated whether inhibition of FLT3 using a small molecule FLT3 inhibitor may represent a novel therapeutic approach against this type of leukemia. Constitutively activated FLT3 also frequently occurs in primary AML cells, predominantly caused by specific mutations within the FLT3 gene. In chapter 8 we studied whether these activating mutations also occur in MLL rearranged infant ALL, and if so, how frequently these genetic abnormalities occur. For this we screened the entire FLT3 gene for the presence of known and novel mutations, and demonstrate that the main etiology for constitutive FLT3 signaling in MLL rearranged infant ALL cells merely is over-expression of wild-type FLT3. In addition to exploring the use of over-expressed genes as therapeutic targets for infant ALL, we also studied genes that appeared to be under-expressed in infant ALL as compared to other ALL subtypes. Chapter 9 describes a study demonstrating that MLL rearranged infant ALL is characterized by the silencing of the tumor suppressor gene FHIT, and how this phenomenon provides a rationale for the use of demethylating drugs as therapeutic intervention for these patients. Finally, chapter 10 summarizes the work presented in this thesis, accommodated with general conclusions and future perspectives. Chapter 11 comprises a concise layman's summary of this thesis in Dutch. http://repub.eur.nl/res/pub/8242/ 2005-01-01T00:00:00Z Acute lymphoblastic leukemia (ALL) in infants is characterized by rearrangements of the mixed lineage leukemia (MLL) gene, drug resistance, and a poor treatment outcome. Therefore, novel therapeutic strategies are needed to improve prognosis. Recently, we showed that FLT3 is highly expressed in MLL rearranged ALL (MLL). Here we demonstrate FLT3 expression in infants with MLL (n = 41) to be significantly higher compared to both infant (n = 8; P < .001) and noninfant patients with ALL (n = 23; P = .001) carrying germline MLL genes. Furthermore, leukemic cells from infants with MLL were significantly more sensitive to the Fms-like tyrosine kinase 3 (FLT3) inhibitor PKC412 (N-benzoyl staurosporine) than noninfant ALL cells, and at least as sensitive as internal tandem duplication-positive (ITD+) AML cells. Surprisingly, activation loop mutations only occurred in about 3% (1 of 36) of the cases and no FLT3/ITDs were observed. However, measuring FLT3 phosphorylation in infants with MLL expressing varying levels of wild-type FLT3 revealed that high-level FLT3 expression is associated with ligand-independent FLT3 activation. This suggests that infant MLL cells displaying activated FLT3 as a result of overexpression can be targeted by FLT3 inhibitors such as PKC412. However, at concentrations of PKC412 minimally required to fully inhibit FLT3 phosphorylation, the cytotoxic effects were only fractional. Thus, PKC412-induced apoptosis in infant MLL cells is unlikely to be a consequence of FLT3 inhibition alone but may involve inhibition of multiple other kinases by this drug. http://repub.eur.nl/res/pub/8186/ 2003-01-01T00:00:00Z Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL) gene rearrangements, a poor outcome, and resistance to chemotherapeutic drugs. One exception is cytosine arabinoside (Ara-C), to which infant ALL cells are highly sensitive. To investigate the mechanism underlying Ara-C sensitivity in infants with ALL, mRNA levels of Ara-C-metabolizing enzymes were measured in infants (n = 18) and older children (noninfants) with ALL (n = 24). In the present study, infant ALL cells were 3.3-fold more sensitive to Ara-C (P =.007) and accumulated 2.3-fold more Ara-CTP (P =.011) upon exposure to Ara-C, compared with older children with ALL. Real-time quantitative reverse trancriptase-polymerase chain reaction (RT-PCR) (TaqMan) revealed that infants express 2-fold less of the Ara-C phosphorylating enzyme deoxycytidine kinase (dCK) mRNA (P =.026) but 2.5-fold more mRNA of the equilibrative nucleoside transporter 1 (hENT1), responsible for Ara-C membrane transport (P =.001). The mRNA expression of pyrimidine nucleotidase I (PN-I), cytidine deaminase (CDA), and deoxycytidylate deaminase (dCMPD) did not differ significantly between both groups. hENT1 mRNA expression inversely correlated with in vitro resistance to Ara-C (r(s) = -0.58, P =.006). The same differences concerning dCK and hENT1 mRNA expression were observed between MLL gene-rearranged (n = 14) and germ line MLL cases (n = 25). An oligonucleotide microarray screen (Affymetrix) comparing patients with MLL gene-rearranged ALL with those with nonrearranged ALL also showed a 1.9-fold lower dCK (P =.001) and a 2.7-fold higher hENT1 (P =.046) mRNA expression in patients with MLL gene-rearranged ALL. We conclude that an elevated expression of hENT1, which transports Ara-C across the cell membrane, contributes to Ara-C sensitivity in MLL gene-rearranged infant ALL.