Comprehensive Quantitative Proteomic Profiling of the Pharmacodynamic Changes Induced By MLN4924 in Acute Myeloid Leukemia Cells Reveals Rational Targets for Combination Therapy
Authors: Visconte Valeria , Nawrocki Steffan T. , Kelly Kevin R. , Han Yingchun , Possemato Anthony , Beausoleil Sean , Nazha Aziz , Maciejewski Jaroslaw P. , Carraway Hetty E. , Advani Anjali S. , Sekeres Mikkael A. , Carew Jennifer S. 
Published: October 2019

Abstract
NEDDylation controls the ubiquitination and proteasomal degradation of proteins that are critical for cell survival, oncogenic transformation, and therapeutic sensitivity. MLN4924 (4924, Pevonedistat) is a first-in-class inhibitor of NEDDylation that has been evaluated in multiple phase I trials. Despite its preliminary efficacy in patients with relapsed/refractory acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (MDS), the specific pharmacodynamic (PD) effects that mediate the anti-leukemic activity of 4924 have not been completely defined. We conducted comprehensive proteome profiling of MV4-11 FLT3 ITD+ cells to determine the global impact of inhibiting NEDDylation with 4924 on the AML proteome. MV4-11 cells were treated with 4924 (1 μM) for 24 hours and processed for high-throughput proteome quantification. Using a 2-fold PD change cut-off, 47 of 3,812 unique detected proteins were significantly upregulated by 4924 treatment (P < 0.05). The effects of 4924 on the levels of selected proteins were confirmed by immunoblotting. 4924 triggered increased levels of many established NEDD8 substrates including CDT1, p27, KEAP1, NUSAP1, and MLX. Other notable factors elevated by 4924 treatment included RRM2, BRD2, NQO1, regulators of cellular redox status (GCLM, TXNRD1, HMOX1), the DNA helicase DNA2, and the DNA replication factor ESCO2. Reactome network analysis demonstrated that the significantly affected proteins primarily clustered in the cell cycle, mitosis, and stress response pathways. A comparison of our proteomic data with a comprehensive SILAC analysis performed in A375 melanoma cells similarly treated with MLN4924 revealed that 34% (16/47) of the pharmacodynamically increased proteins were identical between the two analyses. These findings suggest that the repertoire of proteins that are modulated by 4924 may be tumor-type dependent.
Notably, several of the proteins modulated by 4924 in our study could represent biomarkers for patient stratification. For example, the chromodomain helicase DNA binding protein CHD3 (fold change: 2.23) was reported to be elevated in patients with MYST3-CREBBP AML or AML with a monocytic phenotype and high FLT3 expression that experienced short complete remissions following conventional therapy. It would be worthwhile to investigate whether patients with high basal CHD3 levels derive less benefit from treatment with 4924. We also detected drug-induced changes in 27 members of the RNA helicase family including DDX41, in which mutations were recently reported in AML and MDS. Out of these 27 helicases, DDX24 and DDX54 were most increased (1.74- and 1.51-fold, respectively). Although the impact of drug treatment on individual helicases fell below our set threshold of significance, the collective data suggest that 4924 may have a previously undefined class effect on RNA helicase function. Further investigation is required to assess whether NEDD8 plays a novel role in the regulation of RNA helicases and to determine how this may impact 4924 efficacy.
Additionally, several of the targets that were elevated following treatment with 4924 are directly actionable with existing approved and investigational drugs. For example, a proof of concept FLT3-ITD+AML xenograft study confirmed that known effects of azacitidine (AZA) on RRM2 antagonized MLN4924-mediated upregulation of RRM2 and synergistically increased efficacy (P <0.01), implying a potential relationship between RRM2 expression and clinical response that could be explored in the ongoing trial of 4924 plus AZA in elderly patients with AML (NCT0181426). The ability of 4924 to increase BRD2 levels may also heighten the sensitivity of AML cells to BET inhibitors, which represents another clinical implication that could be seamlessly translated into the clinic and investigated in trials of existing BET inhibitor combinations. We are currently assessing this possibility in preclinical models of AML. In summary, our study demonstrates that high-throughput proteomic technology is a powerful tool with potential applications in patient refinement and the identification of rational actionable targets for precision combination therapeutic strategies. These findings support the implementation of high-throughput proteomics as a synergistic complement to genomics in novel anticancer drug development.

Disclosures
Sekeres: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.