Publication Date

2017-07-07

Availability

Open access

Embargo Period

2017-07-07

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2017-06-28

First Committee Member

Julio C. Barredo

Second Committee Member

Ronan T. Swords

Third Committee Member

Theodore J. Lampidis

Fourth Committee Member

Anthony J. Capobianco

Fifth Committee Member

Xiang-Xi (Mike) Xu

Abstract

Development of resistance and relapse remains a major challenge for acute lymphoblastic leukemia (ALL) treatment. Results from our lab and others showed that ALL cells are specifically vulnerable towards disturbance of endoplasmic reticulum (ER) protein homeostasis, which will cause proteotoxic ER stress/unfolded protein response (UPR). The typical ER stress inducer bortezomib (VELCADE) has been approved as a front-line treatment for multiple myeloma. Clinical trials of bortezomib in combination with other therapeutic agents demonstrated its potent anti-leukemic efficacy in relapsed/refractory pediatric ALL patients. Bortezomib functions by inhibiting the proteasome in the ubiquitin-proteasome system (UPS) to impede protein turnover in cancer cells, rendering accumulation of abnormal and/or misfolded proteins to trigger proteotoxic ER stress. However, the ubiquitous existence of proteasome in normal tissue cells compromised the selectivity of this therapeutic strategy as severe side effects have been reported in clinical trials with bortezomib treatment. Nonetheless, the great success of bortezomib in the clinic inspired the development of novel pharmacological strategies focused on targeting upstream components of the UPS. Currently, more than 1000 E3 ligases have been identified and the largest family among them is the cullin-RING ligases (CRLs) which are responsible for degradation of approximately 20% of cellular proteins degraded via UPS. Most of these proteins are critically involved in cell cycle progression, signaling pathway transduction and apoptosis induction. Activation of CRLs requires conjugation of an ubiquitin-like protein NEDD8 (neural precursor cell-expressed developmentally downregulated 8) to near the C-terminal of the scaffold cullin proteins in the CRLs. Consequently, NEDD8 conjugation serves as an upstream regulatory mechanism that can switch "on" and "off" CRL activity by NEDDylation and deNEDDylatin of cullins, respectively. Conjugation of NEDD8 to the cullins happens in three enzymatic steps involving NEDD8 activating enzyme (NAE; E1), UBC12 and UBE2F (E2s) and E3s that will carry out the final step in conjugating NEDD8 to cullins. A novel therapeutic agent called pevonedistat (pevo, MLN4924) was recently developed to specifically inhibit the attachment of NEDD8 to NAE, leading to blockage of NEDD8 conjugation to the cullins and the concomitant accumulation of CRL substrates. Based on the promising in vitro data proving the efficacy of this novel targeting strategy, multiple clinical trials are undergoing to evaluate the safety and efficacy of pevo in melanoma, acute myeloid leukemia and solid tumors. To evaluate the potential therapeutic gain of NEDDylation inhibition for ALL management, our study focused on analyzing the in vitro and in vivo efficacy of pevo in ALL, investigated potential mechanisms of action and studied the potential resistance ALL cells may develop with pevo treatment. Potent in vitro anti-leukemic activities of pevo were observed with IC50 (growth inhibition) and EC50 (cell death) around 200 nM and 400 nM, respectively, for the ALL cell lines studied (T-ALL: Jurkat, CCRF-CEM; Pre-B ALL: NALM6, REH, SUPB15). Mechanistic studies on the cytotoxicity of pevo identified concomitant activation of the mTOR/p70S6K cascade and eIF2a de-phosphorylation which will promote nascent protein synthesis to trigger proteotoxic ER stress/UPR as manifested by accumulation of GRP78 (Ig heavy chain-binding protein (BiP)) and CHOP (CCAAT/enhancer binding protein homologous protein) in pevo-treated ALL cells. Inhibition of nascent protein synthesis using the mTOR inhibitor rapamycin or the general protein synthesis inhibitor cycloheximide rescued pevo-induced proteotoxicity and ALL cell death, indicating pevo-induced ER stress/UPR as one major cytotoxic mechanism of NEDDylation inhibition in ALL cells. We identified CReP as a target of CRL that will accumulate with pevo-mediated NEDDylation inhibition and promotes eIF2a de-phosphorylation. Pevo induced eIF2a de-phosphorylation will compromise the ability of ALL cells to stop protein synthesis and thus, dysregulate the activation of cytoprotective integrated stress response (ISR), leading to sensitization of ALL cells towards chemotherapeutic agents like dexamethasone and others. We also found consistent activation of the MEK/ERK pathway in pevo-treated ALL cells. Blockage of the MEK/ERK pathway significantly potentiated the ALL-killing effect of NEDDylation inhibition in vitro and in vivo. We further identified Ca2+ influx via the Ca2+ release-activated Ca2+ (CRAC) channels activated protein kinase C fl (PKC-fl) which will promote MEK/ERK signaling cascade activation. The pro-survival role of MEK/ERK signaling in ALL cells were attributed to ERK1/2 mediated phosphorylation/neutralization of the pro-apoptotic Bcl-2 family protein BIM. Studies on the effects of NEDDylation blockage on the two essential components of the CRACs, Orai1 and stromal interaction molecule 1 (STIM1) showed that pevo treatment significantly decreased the STIM1 level while keeping Orai1 levels stable. These changes will optimize the Orai1:STIM1 ratio to activate CRAC, leading to Ca2+ influx in pevo-treated ALL cells. Further studies identified a unique upstream open reading ORF (uORF) in the 5'-untranslated region (5'-UTR) of STIM1 mRNA which can regulate STIM1 mRNA translation with phosphorylation and de-phosphorylation of eIF2a. Based on these data, the efficacy, cytotoxic mechanisms and drug-drug interactions with other chemotherapeutic agents of pevo-mediated NEDDylation inhibition in our pre-clinical ALL cell line models and NSG leukemia mouse model were identified, supporting NEDDylation blockage as a novel therapeutic strategy for ALL treatment.

Keywords

NEDDylation; Pevonedistat; Acute Lymphoblastic Leukemia; ER stress; Store Operated Calcium Entry; MEK/ERK signaling

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