2458 Despite dramatic improvements in treatment over the past 40 years, acute lymphoblastic leukemia (ALL) remains one of the most common causes of death from disease in childhood. Glucocorticoids are among the most effective agents used in the treatment of lymphoid malignancies, and patient response to treatment is an important determinant of long-term outcome in childhood ALL. In spite of its clinical significance, the molecular basis of glucocorticoid resistance is still poorly understood. The aim of this study was to develop an experimental model system to define clinically relevant mechanisms of glucocorticoid resistance in childhood ALL. An in vivo model of childhood ALL has been developed in our laboratory, using patient biopsies established as xenografts in immune-deficient nonobese diabetic severe-combined immunodeficient (NOD/SCID) mice. This model is highly representative of the human disease (Lock et al., Blood, 99: 4100-4108, 2002). The in vivo responses of these xenografts to the glucocorticoid dexamethasone (DEX) correlated significantly with patient outcome (p 1 μM) in xenografts from six patients, five of whom died of their disease. In contrast, four DEX-sensitive xenografts (IC50 values 2-fold in sensitive xenografts within 8 hours of treatment. In contrast, Bim induction was dramatically attenuated in DEX-resistant xenografts. These results have identified a clinically significant and novel mechanism of glucocorticoid resistance in childhood ALL, which occurs downstream of receptor-ligand interactions, but upstream of the signalling pathway resulting in Bim induction and apoptosis.

Novel mechanisms of glucocorticoid resistance in childhood acute lymphoblastic leukemia.

Histone Deacetylase Inhibitor Depsipeptide (FK228) Induces Apoptosis in Leukemic Cells by Facilitating Mitochondrial Translocation of Bax, Which Is Enhanced by Proteasome Inhibitor Bortezomib.

In chapter 1 we introduce various anti-cancer drugs and focus on histone deacetylase inhibitors (HDACIs). Histone deacetylases are enzymes which catalyze the removal of acetyl groups from histones and other proteins, thereby regulating gene activity and protein function and activity. Several HDACI are currently tested in clinical trials, but their mechanisms of action are poorly understood. Chapter 2 describes a functional genetic screen which was conducted to gain insight into the mechanisms of action of HDACI. We introduced a cDNA library into transformed murine cells and selected the cells for resistance to the HDACI PXD101. Overexpression of the genes encoding for retinoic acid receptor alpha (RARa) and PRAME made the cells resistant to treatment with PXD101 and other HDACI. RARa is a nuclear hormone receptor which activates gene transcription upon binding of its ligand retinoic acid (RA). Treatment of cells with HDACIs activated RA signaling, but ectopic RARa and PRAME repressed RA signaling. Conversely, cells without RARs and cells with RNA interference (RNAi) to PRAME were hypersensitive to HDACIs. Melanoma tumors with knockdown of PRAME were sensitized to treatment with RA, HDACI, or both. Treatment of cells with HDACI and RA together synergistically activated RA signaling. These results indicate a role for RA signaling in HDACI-induced cell cycle arrest and apoptosis. Chapter 3 contains a detailed analysis of the human tumor antigen PReferentially expressed Antigen of MElanoma (PRAME). PRAME bound to RARa and repressed RA signaling, resulting in inhibition of physiological responses triggered by RA, such as growth arrest, differentiation and apoptosis. PRAME itself acted as a transcriptional repressor. PRAME bound the polycomb group protein EZH2, which is known as a silencer of gene expression by histone methylation. Melanoma cells contain high levels of PRAME protein and are resistant to RA, but could be rendered responsive to RA by knockdown of PRAME using RNAi. Similarly, melanoma tumors with PRAME knockdown were sensitive to RA treatment. In chapter 4, a third gene is described that was identified in the genetic screen from chapter 2. The protein encoded by this gene, UNC45A (SMAP-1), inhibited RA signaling induced by HDACI and by RA and acted as a transcriptional repressor. UNC45A also attenuated ligand-induced transactivation by progesterone receptor and PPAR receptors. UNC45A bound to RARa and RXRa and inhibited growth arrest and differentiation by RA. Chapter 5 describes a functional genetic screen with the HDACI SAHA using an RNAi library. Knockdown of HR23B rendered cells resistant to apoptosis induced by SAHA. HR23B is known to shuttle ubiquitylated proteins to the proteasome for degradation. SAHA was found to inhibit proteasome activity. SAHA and proteasome inhibitors induced HR23B protein levels and triggered apoptosis, which was prevented by knockdown of HR23B. SAHA has been approved for the treatment of advanced cutaneous T cell lymphoma (CTCL) and CTCL biopsies were found to contain high levels of HR23B protein. In Chapter 6, we have investigated the PRAME expression levels in breast cancer. PRAME expression was a prognostic marker for poor clinical outcome, as it was associated with shortened metastasis-free and overall survival. Chapter 7 reviews the current literature on PRAME expression and function in cancer. PRAME is expressed in a variety of human cancers, including melanomas, breast cancers, lung cancers, brain tumors and leukemias and in a limited number of normal tissues. It is not known if PRAME plays the same role in all cancers in which it is expressed. Finally, chapter 8 is a general discussion of the new findings presented in this thesis. We also describe data on bexarotene, an RXR-selective drug that is approved for treatment of CTCL. Cotreatment of cells with bexarotene and HDACI induced RA signaling in a synergistic manner and induced CTCL cell death in an additive manner. HDACI induced a downregulation of RXRa protein levels in CTCL cells, which was associated with apoptosis. These observations are consistent with the findings in our genetic screen from chapter 2. Together, we have shown that functional genetic screening can be used as an approach to gain new insights in the pathways that are targeted by clinically relevant compounds.

Functional genetic screens as tools to discover signaling pathways targeted by cancer drugs

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Functional genetic screens as tools to discover signaling pathways targeted by cancer drugs