Molecular Insights in MLL Rearranged Acute Leukemia

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.

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