Abstract
De novo and acquired drug resistance and subsequent relapse remain major challenges in acute lymphoblastic leukemia (ALL). We previously identified that pevonedistat (TAK-924, MLN4924), a first-in-class inhibitor of NEDD8 activating enzyme (NAE), elicits ER stress and has potent in vitro and in vivo efficacy against ALL. However, in pevonedistat-treated ALL cell lines, we found consistent activation of the pro-survival MEK/ERK pathway, which has been associated with relapse and poor outcome in ALL. We uncovered that inhibition of the MEK/ERK pathway in vitro and in vivo sensitized ALL cells to pevonedistat. The observed synergistic apoptotic effect appears to be mediated by inhibition of the MEK/ERK pro-survival cascade leading to de-repression of the pro-apoptotic BIM protein. Mechanistically, Ca2+ influx via the Ca2+-release-activated Ca2+ (CRAC) channel induced protein kinase C β2 (PKC-β2) was responsible for activation of the MEK/ERK pathway in pevonedistat-treated ALL cells. Sequestration of Ca2+ using BAPTA-AM or blockage of store-operated Ca2+ entry (SOCE) using BTP-2 both attenuated the compensatory activation of MEK/ERK signaling in pevonedistat-treated ALL cells. Pevonedistat significantly altered the expression of Orai1 and stromal interaction molecule 1 (STIM1), resulting in significantly decreased STIM1 protein levels relative to Orai1. Further, we identified eIF2α as an important post-transcriptional regulator of STIM1, suggesting that pevonedistat-induced eIF2α de-phosphorylation selectively down-regulates translation of STIM1 mRNA. Consequently, our data suggest that pevonedistat potentially activates SOCE and promotes Ca2+ influx leading to activation of the MEK/ERK pathway by altering the stoichiometric Orai1:STIM1 ratio and inducing ER stress in ALL cells.
Highlights
Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children and adolescents and has an overall 5-year event-free survival (EFS) of 80%, whereas adults with ALL have a 5-year EFS rate under50% [1, 2]
Consistent with our hypothesis, we found that the addition of BAPTA-AM significantly inhibited pevonedistat-induced p-ERK1/2 activation in all three ALL cell lines examined (Figure 1C), supporting a mechanistic role of Ca2+ in mediating MEK-ERK pathway activation in pevonedistat-treated ALL cells
This is evidenced by direct measurement of intracellular calcium using the Ca2+ binding Fluo-8 AM dye, and the Ca2+-release-activated Ca2+ (CRAC) inhibitor BTP-2 which clearly demonstrated that pevonedistat www.impactjournals.com/oncotarget increased Ca2+ influx in ALL cells, and that activation of p-ERK1/2 was significantly reduced in presence of the Ca2+ chelator BAPTA-AM or the PKC inhibitor enzastaurin
Summary
Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children and adolescents and has an overall 5-year event-free survival (EFS) of 80%, whereas adults with ALL have a 5-year EFS rate under50% [1, 2]. There is a need to develop novel, more targeted strategies, that increase cure rates and mitigate against immediate and late toxicities. In this context, we identified the NEDD8activating enzyme (NAE) as a novel therapeutic target for ALL therapy, resulting in potent in vitro and in vivo anti-leukemic effects [4]¬. We demonstrated pevonedistat induced apoptosis in ALL cells occurred by dysregulating cellular translation machinery causing induction of proteotoxic endoplasmic reticulum (ER) stress. Activation of both mTOR and UPR/eIF2α pathways [4]¬ were key mediators of this mechanism. We interpreted the induction of MEK/ERK as a compensatory survival mechanisms in response to pevonedistat cytotoxicity, but the mechanisms responsible for MEK/ERK activation remained unclear
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