NEDD8-activating Enzyme Inhibitor Pevonedistat Research Articles

Phase 1 study of NEDD8 activating enzyme inhibitor pevonedistat in combination with chemotherapy in pediatric patients with recurrent or refractory solid tumors (ADVL1615)

PurposeThe objective of this study was to determine the recommended Phase 2 dose (RP2D) of pevonedistat, a first in class inhibitor of NEDD8 activating enzyme, in combination with irinotecan (IRN) and temozolomide (TMZ) in children with cancer. MethodsThis Phase 1 study used a rolling 6 design to evaluate escalating doses of pevonedistat in combination with standard doses of IRN and TMZ in pediatric patients with recurrent/refractory solid or CNS tumors. During cycle 1, pevonedistat was administered intravenously on days 1, 8, 10, and 12, with IRN (IV, 50 mg/m2) and TMZ (orally, 100 mg/m2), on days 8–12 of a 28-day cycle. In subsequent cycles, pevonedistat was administered on days 1, 3, and 5, with IRN/TMZ on days 1–5 of a 21-day cycle. ResultsThirty patients enrolled; all were eligible and evaluable for toxicity. Six patients each enrolled on pevonedistat dose levels (DL) 1 (15 mg/m2), 2 (20 mg/m2), 3 (25 mg/m2) and 4 (35 mg/m2) as well as an expanded pharmacokinetic (PK) cohort at DL4. The maximum tolerated dose (MTD) was not exceeded. 2/12 (17 %) patients treated at the RP2D (35 mg/m2) experienced a cycle 1 dose limiting toxicity (DLT). IRN is unlikely to affect the pharmacokinetics of pevonedistat. Two patients had a partial response and 6 patients had prolonged stable disease (> 6 cycles). ConclusionsPevonedistat in combination with IRN/TMZ is well tolerated in children with solid or CNS tumors. The RP2D of pevonedistat is 35 mg/m2 on days 1, 3, 5 in combination with IRN/TMZ.

Phase 1b/2 Study of Escalating Doses of the NEDD8 Activating Enzyme Inhibitor Pevonedistat Administered in Combination with Standard Induction Therapy (Cytarabine and Idarubicin) in Newly Diagnosed High Risk Acute Myeloid Leukemia

Introduction: Pevonedistat is a potent and selective small-molecule inhibitor of the NEDD8-activating enzyme that has demonstrated clinical activity in acute myeloid leukemia (AML). Preclinical studies suggest that pevonedistat synergistically enhances the activity of cytarabine in AML. We investigated the safety and efficacy of pevonedistat in combination with 7+3 cytarabine and idarubicin induction chemotherapy in adults with high-risk AML. Methods: This open-label, multicenter phase 1b/2 study (NCT03330821) enrolled patients aged ≥18 years with newly diagnosed high-risk AML, defined as therapy-related AML, AML with antecedent myelodysplastic syndrome or AML with myelodysplasia-related changes, the presence of adverse genetic features per ELN 2017 guidelines, or being age ≥55. Eligible patients had an ECOG status of 0-2. Patients were administered pevonedistat on days 1, 3, and 5 of each 28-day cycle in combination with cytarabine 100 mg/m 2 on days 1-7 and idarubicin 12 mg/m 2 on days 1-3. In the phase 1b dose-escalation part of the study, a standard 3+3 design was used to evaluate 2 dose levels of pevonedistat: 15 and 20 mg/m 2. Consolidation consisted of up to 4 cycles of cytarabine (1-3 g/m 2 q12h) on days 1, 3, and 5 or transplant. For pharmacodynamic (PD) assessment, RT-PCR assays were performed on peripheral blood samples to measure expression levels of 7 NRF-2 target genes (ATF3, GCLM, GSR, NQ01, SLC7A11, SRXN1, and TXNRD1). The primary objective for the phase 1b study was to determine the RP2D of pevonedistat in combination with cytarabine and idarubicin. The primary objective for the phase 2 study was to determine the composite complete response rate (CRc; CR+CRi). Secondary objectives included relapse-free survival (RFS), overall survival (OS), and safety and tolerability. Results: A total of 28 patients were enrolled at 5 sites in the US between August 2018 and August 2021. Six patients were enrolled in the phase 1b study and 22 patients in phase 2. Baseline patient characteristics are shown in Table 1. The median age was 61 years (range 47-74). Per the 2017 ELN risk classification, 8 of 28 patients (29%) had favorable risk, 8 (29%) had intermediate risk, and 12 (43%) had adverse risk. All 28 patients were included for safety and response assessments. In phase 1b, none of the 6 patients experienced dose-limiting toxicity at 15 or 20 mg/m 2. Thus, the RP2D of pevonedistat was determined to be 20 mg/m 2. The CRc rate was 64% (95% CI 44-81) with 17 patients achieving CR and 1 patient achieving CRi. Of the 18 patients who achieved CR or CRi, 16 (89%) achieved MRD negativity by multiparameter flow cytometry, and 7 (39%) proceeded to HSCT. The CRc rates for patients in the ELN 2017 favorable, intermediate, and adverse risk groups were 100% (95% CI 63-100), 63% (95% CI 29-89), and 42% (95% CI 18-71), respectively. Eight patients were included in the interim PD analysis. Among patients who achieved CR or CRi (n=6), 4 (ATF3, GSR, SLC7A11, and SRXN1) of the 7 NRF-2 target genes measured were significantly upregulated at 5 hours post-pevonedistat induction compared to baseline. There was no significant increase in NRF-2-regulated gene transcript levels among patients with refractory disease (n=2). The median follow-up was 31.1 months (range 19.1-55.9). The median RFS was 24.4 months (95% CI 7-54.3+) and median OS was 25 months (95% CI 11.9-55.9+). The estimated 1-year and 2-year RFS were 72% (95% CI 52-93) and 55% (95% CI 23-78), respectively. The estimated 1-year and 2-year OS were 68% (95% CI 51-85) and 53% (95% CI 34-72), respectively. The most common grade ≥3 treatment-related adverse events were anemia (61%), febrile neutropenia (25%), and hypophosphatemia (18%). No treatment-related deaths were observed. Conclusion: Pevonedistat in combination with cytarabine and idarubicin was well tolerated among adults with newly diagnosed, high-risk AML and induced deep remissions with promising RFS and OS.

High-Throughput Screens Identify NEDD8 Inhibition As a Strategy to Augment Natural Killer Cell Cytotoxicity Against Blood Cancers

Natural killer (NK) cell-based therapies are amongst the emerging immunotherapeutic approaches that aim to target malignant cells refractory to standard therapies. Currently used strategies to augment the NK cell anti-cancer function of NK cells include cytokine treatments and the use of feeder expansion protocols for culturing NK cells. Recent studies have indicated that NK cell cytotoxicity and function may also be enhanced by certain anti-cancer drugs. However, the effects of the hundreds of available oncology drugs on NK cell function have not been systematically evaluated. Here, we perform large-scale high-throughput testing of NK cell-drug combinations using co-culture models with diverse blood cancer cells, identifying the NEDD8-activating enzyme inhibitor pevonedistat as a promising candidate for augmenting NK cell anti-cancer responses. To identify oncology drugs synergizing with NK cell-based immunotherapy, we established a high-throughput drug sensitivity and resistance testing (DSRT) platform to investigate effects of 527 approved drugs and investigational compounds on NK cell cytotoxicity against blood cancer cell lines. Overall, 10 cell lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and multiple myeloma (MM), were screened in combination with primary expanded NK cells. Target cells were treated for 24 hours with drugs at five different concentrations both with and without NK cells. In NK cell co-culture, an effector-target (ET) cell ratio at which half of the target cells were killed was selected for experiments. Most effective compounds were then selected for further testing using multiplexed single-cell RNA sequencing (scRNA-seq) and analysis of secreted cytokines (Figure 1). Overall, the results from our DSRT platform demonstrated varying effects of different drug classes on NK cell cytotoxicity against blood cancer cell lines. In general, 5-10% of drugs had NK cell cytotoxicity-enhancing effects, 30-40% inhibited NK function and 35-50% of drugs had no effect. Similarities in results between cell lines highlighted the dependence of NK cell-drug synergistic effects on disease pathobiology. Across multiple disease models, pevonedistat (MLN4924), a selective NEDD8 activating enzyme inhibitor that has been studied as treatment for relapsed/refractory AML, showed strong synergistic effects with NK cells. For example, in OCI-AML-3 (AML), pevonedistat alone showed no effect on target cell viability, but in combination with NK cells at a 1:4 ET ratio, it resulted in 98% target cell killing at 100 nM. Similar effects were also seen in MM1.S (MM), although a higher concentration (10000 nM) of pevonedistat was required (Figure 2). Results from scRNA-seq after combination treatment with NK cells and pevonedistat revealed distinct transcriptomic changes in both NK cells and target cells when compared to controls. In both NK cells and target cells, pevonedistat caused upregulation of TXN, TALDO1, TXNRD1, NQO1, SRXN1 and SLC7A11,collectively indicating effects on the NRF2 signaling pathway. The upregulation of these genes correlated with the tested concentration of pevonedistat, with notably higher expression when treated with 1000 nM pevonedistat compared to 100 nM. Changes were also observed in the secretion of cytokines detected from the cell culture media, where concentrations of IL-1β, IL-2, IL-6, IL-8, IL-10, IFN-γ and TNF-α all increased up to 300% compared to controls. In HEL (AML) and LAMA-84 (CML), where pevonedistat had only minor effects on NK cell cytotoxicity, the expression TNF and TNFRSF1B at baseline and after pevonedistat treatment were notably lower, when compared to cell lines where the combination therapy led to stronger responses. This suggests a possible mechanism for pevonedistat-NK cell synergy, given that previous reports have demonstrated a synergy between pevonedistat and TNF-α to activate apoptosis. In conclusion, we discovered a cell context-specific synergy between pevonedistat and NK cells, which increased NK cell cytotoxicity against malignant cells in vitro. In addition, our results demonstrate the potential of using a high-throughput platform for studying NK cell-drug interactions against malignant cells, while aiming to improve treatment for hematological malignancies through combination immunotherapies.

Targeting neddylation sensitizes colorectal cancer to topoisomerase I inhibitors by inactivating the DCAF13-CRL4 ubiquitin ligase complex

Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.

Abstract B022: The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer

Abstract Colorectal cancer (CRC) is a heterogeneous disease with a wide spectrum of clinical outcomes, from indolent resectable disease to aggressive-metastatic cases. Primary and acquired resistance limits the efficacy of available treatments, and the identification of effective drug combinations is needed to further improve patients' outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induces tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a drop-out loss-of-function synthetic lethality screening with a shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at various levels could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. Our results suggested possible therapeutic opportunities in specific CRC clinical settings. We further focused on HER2-amplified CRC and found marked sensitivity to pevonedistat in a CRC cell line carrying HER2 amplification. In these cells, long-term in vitro treatment with a combination of lapatinib and trastuzumab induces the emergence of colonies of long-term persisters (i.e., cells surviving to several weeks of HER2/EGFR blockade). These persisters display extremely slow growth and senescence features, however they fully recover upon removal of the blockade, mimicking disease relapse in patients after treatment suspension. Interestingly, persister colonies maintained sensitivity to pevonedistat, displaying a marked decrease when pevonedistat was added after six weeks of HER2/EGFR blockade, independently of the continuation of the blockade. These results unveil the possibility of employing pevonedistat in HER2-amplified CRC patients subsequently to tumor stabilization or regression by HER-2/EGFR blockade. Citation Format: Federica Invrea, Alessandro Carugo, Consalvo Petti, Giulio Draetta, Enzo Medico. The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B022.

176 (PB056) - The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer

Background: Colorectal cancer (CRC) is a heterogeneous disease with a wide spectrum of clinical outcomes, from indolent resectable disease to aggressive-metastatic cases. Primary and acquired resistance limits the efficacy of available treatments, and the identification of effective drug combinations is needed to further improve patients’ outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies.

Abstract 2611: The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer

Abstract Colorectal cancer (CRC) is a heterogeneous disease with a wide spectrum of clinical outcomes, from indolent resectable disease to aggressive-metastatic cases. Primary and acquired resistance limits the efficacy of available treatments, and the identification of effective drug combinations is needed to further improve patients' outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a "drop-out" loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. Our results suggested possible therapeutic opportunities in specific CRC clinical settings. We further focused on HER2 amplified CRC cells and we observed cooperation between pevonedistat and HER2/EGFR blockade by the trastuzumab + lapatinib combination -the current standard treatment for HER2-amplified CRC. In addition, to confirm cooperation in reducing colony formation in vitro, we explored the effect of pevonedistat on long-term persisters, i.e., cell surviving to several weeks of HER2/EGFR blockade. Interestingly, we observed a marked decrease of cell colonies when pevonedistat was added to persisters survived to three weeks of trastuzumab + lapatinib treatment. These results unveil the possibility of testing in vivo the addition of pevonedistat subsequently to tumor stabilization or reduction by HER2/EGFR blockade, to promote further tumor regression. Citation Format: Federica Invrea, Alessandro Carugo, Sabrina Arena, Alberto Bardelli, Giulio F. Draetta, Enzo Medico. The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2611.

Synthetic Lethality Screening Highlights Colorectal Cancer Vulnerability to Concomitant Blockade of NEDD8 and EGFR Pathways.

Simple SummaryIdentification of effective therapies for clinically aggressive, treatment-resistant colorectal cancer (CRC) remains an unmet clinical need. Targeted therapies against the epidermal growth factor receptor (EGFR) signaling axis lead to clinical benefits only in a small fraction of patients due to primary and acquired resistance. We previously showed that the NEDD8 pathway inhibitor pevonedistat induced tumor stabilization in preclinical models of aggressive CRC. Here, through synthetic lethality screenings, we found that pevonedistat could be successfully combined with EGFR pathway-targeted treatments in BRAF-mutant and RAS-RAF wild-type CRCs originally resistant to BRAF and EGFR blockade. We found that combined blockade of NEDD8 and EGFR pathways reverted compensatory feedback loops that reduced the efficacy of single treatments. Our results provide preclinical validation of a promising therapeutic strategy for clinically aggressive CRC resistant to EGFR and BRAF-targeted treatments.Colorectal cancer (CRC) is a heterogeneous disease showing significant variability in clinical aggressiveness. Primary and acquired resistance limits the efficacy of available treatments, and identification of effective drug combinations is needed to further improve patients’ outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a “drop-out” loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. These results unveil possible therapeutic opportunities in specific CRC clinical settings.

Abstract 5211: Targeting Neddylation synergistically augments the activity of cisplatin in head and neck cancer models to yield long-term survival by modulating key DNA damage and redox pathways

Abstract Head and neck cancer is a broad term for neoplasms of the epithelial lining of the anatomical sites of the upper aerodigestive tract. Very few novel therapeutics have been developed for the treatment of advanced head and neck cancer with cisplatin continuing to be a key standard of care agent. Innovative and more effective therapeutic strategies are urgently needed. The inappropriate loss of several NEDD8-cullin-RING ubiquitin ligase (CRL) targets has been linked to cancer progression and drug resistance. Our data show that overexpression of NEDD8 activating enzyme (NAE), a key proximal regulator of the NEDDylation cascade, is correlated with inferior survival in head and neck cancers suggesting that this pathway may be a promising new therapeutic target. We hypothesized that disrupting NEDDylation with the first-in-class NAE inhibitor pevonedistat (MLN4924, PEV) would be an effective new approach for the treatment of head and neck cancers. Initial testing demonstrated that PEV potently impaired the NEDDylation cascade as evidenced by decreased NEDDylated cullins and upregulation of the CRL target cell cycle inhibitors, p21 and p27. In addition, PEV induced a dramatic G2 cell cycle arrest, polyploidy, and activation of the DNA damage response. PEV treatment also potently decreased cell viability and induced apoptosis in a panel of head and neck cancer cell lines with diverse genetic features. Given the potent DNA damage-related aspects of PEV's mechanism of action, we hypothesized that it would be a rational combination partner for standard cisplatin therapy. Consistent with this hypothesis, co-treatment with PEV and cisplatin promoted synergistic levels of DNA damage as measured by comet assay, an accumulation of gamma-H2AX, and quantification of DNA fragmentation. The combination also cooperated to promote redox alterations that were necessary for apoptosis induction and linked to the DNA damaging effects of both drugs. Importantly, synergistic anticancer activity was observed in cell lines from multiple anatomical sites, suggesting that this combination has broad efficacy across many subtypes of head and neck cancer. To further evaluate the therapeutic benefit of the PEV and cisplatin combination, we determined its safety and efficacy in a head and neck cancer xenograft model. Consistent with our in vitro data, the combination significantly decreased tumor proliferation, induced apoptosis, and augmented DNA damage in vivo. While both PEV and cisplatin displayed significant activity as monotherapies, combination treatment resulted in a dramatic decrease in tumor burden and also led to long-term animal survival in every treated animal (10/10 mice remained disease-free at day 100). Importantly, the PEV and cisplatin combination was therapeutically selective and very well tolerated in mice. Our collective data provide a strong rationale for the clinical investigation of NAE inhibition as a novel strategy to augment cisplatin efficacy in patients with head and neck cancer. Citation Format: Trace Jones, Claudia Espitia, Jennifer Carew, Steffan T. Nawrocki. Targeting Neddylation synergistically augments the activity of cisplatin in head and neck cancer models to yield long-term survival by modulating key DNA damage and redox pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5211.

A Genome-Wide CRISPR/Cas9 Knockout Screen Identifies BEND3 As a Determinant of Sensitivity to UBA1 Inhibition in Acute Myeloid Leukemia

UBA1 is the major ubiquitin-activating enzyme that initiates the ubiquitylation cascade whereby proteins are tagged with mono- or polyubiquitin to mark them for proteasomal degradation or modify their functions. Despite having equal levels of UBA1 protein, AML cell lines and primary AML cells are more dependent on UBA1 activity compared to normal hematopoietic cells, rendering them more vulnerable to UBA1 inhibition. Recently, we demonstrated that inhibiting UBA1 with the small-molecule inhibitor TAK-243 was selectively cytotoxic to a subset of AML cells and stem cells in vitro and in vivo through a mechanism at least partly dependent on inducing ER stress (Leukemia, 2018).To identify potential determinants of sensitivity/resistance to TAK-243 (Millennium Pharmaceuticals, Takeda) in AML, we conducted a genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with cytotoxic TAK-243 concentrations corresponding to the IC90 and IC99. By next-generation sequencing and enrichment analysis, we then identified genes whose knockout renders AML cells resistant to TAK-243. We identified 34 hits in the IC90 and 11 hits in the IC99 arms of the screen (cut off FDR < 0.2). These hits are involved in signaling pathways including transcriptional regulation, histone methylation, ubiquitin conjugation, cell cycle progression, mTOR and NF-κB signaling pathways, consistent with the broad range of pathways regulated by UBA1-mediated ubiquitylation. We focused our investigation on BEN domain-containing protein 3 (BEND3) that ranked as a top hit in both arms (FDR = 0.0012). Compared to control, all 6 BEND3-targeting gRNAs were enriched up to 10,000 times. BEND3 is a transcriptional repressor that regulates heterochromatin organization.To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells using the 4 top performing gRNAs in the screen. We confirmed target knockout by immunoblotting. BEND3 knockout did not alter the basal proliferation rate of the cells. However, knockout of BEND3 rendered OCI-AML2 cells resistant to TAK-243 with up to a 4-fold increase in the IC50 by the MTS assay. Resistance to TAK-243 was confirmed by Annexin V/PI staining, PARP cleavage, and colony-forming assays. Cells were cross resistant to the NEDD8-activating enzyme inhibitor pevonedistat (2-fold IC50 increase), but not bortezomib or daunorubicin. As assessed by immunoblotting, BEND3 knockout did not change expression of UBA1, or the related enzymes UBA2, UBA3, or UBA6. BEND3 knockout was associated with reduced induction of ER stress as assessed by levels of CHOP and ATF4 after TAK-243 treatment.ConclusionsThrough a genome-wide CRISPR screen, we identified BEND3 as a determinant of sensitivity to TAK-243 in AML. Mechanistically, lack of BEND3 expression dampens the ER stress response to UBA1 inhibition. Thus, these results may highlight a new mechanism of sensitivity to TAK-243. DisclosuresSchimmer:Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The Neddylation Inhibitor Pevonedistat (MLN4924) Suppresses and Radiosensitizes Head and Neck Squamous Carcinoma Cells and Tumors.

The cullin RING E3 ubiquitin ligase 4 (CRL4) with its substrate receptor CDT2 (CRL4-CDT2) is emerging as a critical regulator of DNA replication through targeting CDT1, SET8, and p21 for ubiquitin-dependent proteolysis. The aberrant increased stability of these proteins in cells with inactivated CRL4-CDT2 results in DNA rereplication, which is deleterious to cells due to the accumulation of replication intermediates and stalled replication forks. Here, we demonstrate that CDT2 is overexpressed in head and neck squamous cell carcinoma (HNSCC), and its depletion by siRNA inhibits the proliferation of human papilloma virus-negative (HPV-ve) HNSCC cells primarily through the induction of rereplication. Treatment of HNSCC with the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924), which inhibits all cullin-based ligases, induces significant rereplication and inhibits HNSCC cell proliferation in culture and HNSCC xenografts in mice. Pevonedistat additionally sensitizes HNSCC cells to ionizing radiation (IR) and enhances IR-induced suppression of xenografts in mice. Induction of rereplication via CDT2 depletion, or via the stabilization or activation of CDT1, also radiosensitizes HNSCC cells. Collectively, these results demonstrate that induction of rereplication represents a novel approach to treating radioresistant HNSCC tumors and suggest that pevonedistat may be considered as an adjuvant for IR-based treatments. Mol Cancer Ther; 17(2); 368-80. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

TAK-243 Is a Selective UBA1 Inhibitor That Displays Preclinical Activity in Acute Myeloid Leukemia (AML)

Introduction:Ubiquitin-like Modifier Activating Enzyme 1 (UBA1; UAE) is the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Compared to normal hematopoietic cells, AML cell lines and primary AML cells have equal levels of UBA1 protein, but increased requirement for this enzyme. TAK-243 is a potent and selective inhibitor of UBA1 and we determined the preclinical activity, biological effects and mechanisms of resistance to the drug in AML.Results:TAK-243 reduced growth and viability of human AML cell lines (OCI-AML2, TEX, U937 and NB4) in a concentration- and time-dependent manner with IC50's ranging from 15-40 nM after treatment for 48 hours. In primary AML samples, most (n=18/21) were sensitive to TAK-243 with an IC50<75 nM at 48 hours of incubation. These samples included patients with high-risk cytogenetics, FLT3 mutations, and patients refractory to induction chemotherapy. We also compared the effects of TAK-243 on primary AML cells (n=6) and normal hematopoietic cells (n=6) and demonstrated that TAK-243 preferentially inhibited the clonogenic growth of AML cells over normal (19-fold reduction CFU-leukemia vs CFU-GM (normal), p ≤ 0.01).Binding of TAK-243 to UBA1 and related E1 enzymes was measured in intact AML cells using the cellular thermal shift assay (CETSA). In AML cell lines and primary AML samples, TAK-243 bound UBA1 at concentrations associated with cell death, but bound other E1 enzymes UBA3 and UBA6 only at much higher concentrations.Next, we evaluated the biological effects of UBA1 inhibition by TAK-243. At concentrations associated with cell death, TAK-243 decreased the abundance of poly- and mono-ubiquitylated proteins in OCI-AML2 cells and primary AML samples. In addition, TAK-243 treatment increased PERK phosphorylation, CHOP, XBP1s and ATF4 which are markers of proteotoxic stress and unfolded protein response. TAK-243 inhibited DNA double strand break (DSB) repair as evidenced by reduced recruitment of 53BP1 to DSBs and sustained γH2AX foci after 3 Gy of irradiation.We assessed the preclinical efficacy and toxicity of TAK-243 in mouse models of AML. OCI-AML2 cells were injected subcutaneously (sc) into SCID mice, and when tumors were palpable, mice were treated with TAK-243 (20 mg/kg sc twice weekly). TAK-243 significantly delayed tumor growth in mice (T/C=0.02) with no toxicity as evidenced by no changes in mouse body weight, serum chemistry, or organ histology. In tumors and organs isolated from the above treated mice, TAK-243 preferentially reduced levels of mono- and poly-ubiquitylated proteins in tumors over normal tissues. As an additional model, primary AML cells from 2 patients were injected into the femurs of NOD-SCID mice. Two weeks after injection, mice were treated with TAK-243 (20 mg/kg sc twice weekly). After 3 weeks of treatment, mice were sacrificed, and AML engraftment in the non-injected femur was measured by flow cytometry. TAK-243 reduced primary AML tumor burden in both tested samples without toxicity. Using secondary transplantations, we demonstrated that TAK-243 had targeted the leukemic stem cells.To understand mechanisms of resistance to TAK-243, we selected a population of TAK-243-resistant OCI-AML2 by culturing cells with increasing concentrations of the drug. Persisting cells were 33-fold more resistant to TAK-243 compared to wild-type cells (IC50 757 vs 23 nM), but had a normal rate of proliferation and remained equally sensitive to bortezomib, daunorubicin, mitoxantrone and the NEDD8-activating enzyme inhibitor pevonedistat. Using CETSA, we showed reduced binding of TAK-243 to UBA1 in the resistant cells. We sequenced UBA1 exons 12-16 and 23-24 that span the adenylation domain. Resistant cells had a missense mutation in exon 16 resulting in substitution of tyrosine with cysteine at codon 583 (Y583C). Y583 in human UBA1 corresponds to Y551 in yeast Uba1, which makes a favorable interaction with TAK-243 in its Uba1 binding site. Therefore, Y583C substitution is predicted to interfere with TAK-243 binding to UBA1.Conclusions:TAK-243 is a potent and selective UBA1 inhibitor that displays preferential activity towards AML cells over normal hematopoietic cells. Acquired mutations affect drug binding and may be a clinically relevant mechanism of resistance. These data support conducting a clinical trial of TAK-243 in patients with AML. DisclosuresHyer:Takeda Pharmaceuticals International Co.: Employment. Berger:Takeda Pharmaceuticals International Co.: Employment. Traore:Takeda Pharmaceuticals International Co.: Employment. Sintchak:Takeda Pharmaceuticals International Co.: Employment. Milhollen:Takeda Pharmaceuticals International Co.: Employment. Schimmer:Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.

Synergistic anti-AML effects of the LSD1 inhibitor T-3775440 and the NEDD8-activating enzyme inhibitor pevonedistat via transdifferentiation and DNA rereplication

Lysine-specific demethylase 1A (LSD1, KDM1A) specifically demethylates di- and monomethylated histones H3K4 and K9, resulting in context-dependent transcriptional repression or activation. We previously identified an irreversible LSD1 inhibitor T-3775440, which exerts antileukemic activities in a subset of acute myeloid leukemia (AML) cell lines by inducing cell transdifferentiation. The NEDD8-activating enzyme inhibitor pevonedistat (MLN4924, TAK-924) is an investigational drug with antiproliferative activities in AML, and is also reported to induce cell differentiation. We therefore tested the combination of these two agents in AML models. The combination treatment resulted in synergistic growth inhibition of AML cells, accompanied by enhanced transdifferentiation of an erythroid leukemia lineage into granulomonocytic-like lineage cells. In addition, pevonedistat-induced rereplication stress during the S phase was greatly augmented by concomitant treatment with T-3775440, as reflected by the increased induction of apoptosis. We further demonstrated that the combination treatment was markedly effective in subcutaneous tumor xenograft models as well as in a disseminated model of AML, leading to tumor eradication or prolonged survival in T-3775440/pevonedistat cotreated mice. Our findings indicate the therapeutic potential of the combination of LSD1 inhibitors and pevonedistat for the treatment of AML.

Abstract 1423: Nedd8-activating enzyme inhibitor pevonedistat synergizes with cisplatin and carboplatin through interference with nucleotide excision repair and interstrand cross-link repair mechanisms in non-small cell lung cancer

Abstract Platins are one of the most widely used classes of chemotherapeutic drugs. Although used as part of first-line standard of care regimens for advanced non-small cell lung cancer (NSCLC), their effectiveness remains limited, with a 5-year survival rate of 18%. Therefore, combination therapy to overcome resistance and improve survival is needed. Pevonedistat (TAK-924/MLN4924) is an investigational first-in-class Nedd8-activating enzyme (NAE) inhibitor that blocks activation of the cullin family of E3 ubiquitin ligases by preventing cullin conjugation with Nedd8, a ubiquitin-like protein. In vitro, pevonedistat is synergistic with carboplatin in a cell viability assay in 6 out of 20 NSCLC cell lines either with or without paclitaxel. In vivo studies using patient-derived xenograft (PDX) models showed a combination effect in a carboplatin-insensitive but not carboplatin-sensitive model. To evaluate the mechanism of the synergy between pevonedistat and platinum, RNAi of 320 DNA-damage response genes was performed in 4 cell lines in vitro. Depletion of genes involved in TC-NER (transcription-coupled nucleotide excision repair) and ICR (interstrand crosslink repair) reduced the synergy between pevonedistat and platinum, with the contribution of each pathway varying by cell line. Since TC-NER and ICR are also involved in the repair of DNA damage by platins, the results suggest that delaying completion of platin induced DNA repair by pevonedistat results in enhanced cell death. Pevonedistat may impede TC-NER by inhibition of neddylation of the E3 ubiquitin ligase CUL4-RBX1-DDB1-ERCC8, thereby possibly providing a direct reversal of resistance to platins. Citation Format: Xiaozhen Liu, David Bouck, Khristofer Garcia, Jonathan Blank, Hugues Bernard, Allison Berger, Mike Kuranda, Erik Koenig, Eric Lightcap. Nedd8-activating enzyme inhibitor pevonedistat synergizes with cisplatin and carboplatin through interference with nucleotide excision repair and interstrand cross-link repair mechanisms in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1423. doi:10.1158/1538-7445.AM2017-1423

Targeting the protein ubiquitination machinery in melanoma by the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924).

Background The neddylation pathway conjugates NEDD8 to cullin-RING ligases and controls the proteasomal degradation of specific proteins involved in essential cell processes. Pevonedistat (MLN4924) is a selective small molecule targeting the NEDD8-activating enzyme (NAE) and inhibits an early step in neddylation, resulting in DNA re-replication, cell cycle arrest and death. We investigated the anti-tumor potential of pevonedistat in preclinical models of melanoma. Methods Melanoma cell lines and patient-derived tumor xenografts (PDTX) treated with pevonedistat were assessed for viability/apoptosis and tumor growth, respectively, to identify sensitive/resistant models. Gene expression microarray and gene set enrichment analyses were performed in cell lines to determine the expression profiles and pathways of sensitivity/resistance. Pharmacodynamic changes in treated-PDTX were also characterized. Results Pevonedistat effectively inhibited cell viability (IC50<0.3μM) and induced apoptosis in a subset of melanoma cell lines. Sensitive and resistant cell lines exhibited distinct gene expression profiles; sensitive models were enriched for genes involved in DNA repair, replication and cell cycle regulation, while immune response and cell adhesion pathways were upregulated in resistant models. Pevonedistat also reduced tumor growth in melanoma cell line xenografts and PDTX with variable responses. An accumulation of pevonedistat-NEDD8 adduct and CDT1 was observed in sensitive tumors consistent with its mechanism of action. Conclusions This study provided preclinical evidence that NAE inhibition by pevonedistat has anti-tumor activity in melanoma and supports the clinical benefits observed in recent Phase 1 trials of this drug in melanoma patients. Further investigations are warranted to develop rational combinations and determine predictive biomarkers of pevonedistat.

An inhibitor of apoptosis protein antagonist T-3256336 potentiates the antitumor efficacy of the Nedd8-activating enzyme inhibitor pevonedistat (TAK-924/MLN4924)

Inhibitors of apoptosis proteins (IAPs) are antiapoptotic regulators that block cell death, and are frequently overexpressed in several human cancers, where they facilitate evasion of apoptosis and promote cell survival. IAP antagonists are also known as second mitochondria-derived activator of caspase (SMAC)-mimetics, and have recently been considered as novel therapeutic agents for inducing apoptosis, alone and in combination with other anticancer drugs.In this study, we showed that T-3256336, the orally available IAP antagonist has synergistically enhances the antiproliferative effects of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924), and these effects were attenuated by a TNFα-neutralizing antibody. In the present mechanistic analyses, pevonedistat induced TNFα mRNA and triggered IAP antagonist-dependent extrinsic apoptotic cell death in cancer cell lines. Furthermore, synergistic effects of the combination of T-3256336 and pevonedistat were demonstrated in a HL-60 mouse xenograft model. Our findings provide mechanistic evidence of the effects of IAP antagonists in combination with NAE inhibitors, and demonstrate the potential of a new combination therapy for cancer.

The NEDD8-activating enzyme inhibitor pevonedistat activates the eIF2α and mTOR pathways inducing UPR-mediated cell death in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for adults remain dismal. Further, effective treatment strategies for relapsed/refractory ALL remain elusive. We previously uncovered that ALL cells are prone to apoptosis via endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated mechanisms. We investigated the antineoplastic activity of pevonedistat®, a novel NEDD8-activating enzyme inhibitor that targets E3 cullin-RING ligases (CRLs) dependent proteasomal protein degradation, in ALL. Herein, we report that pevonedistat induces apoptosis in ALL cells by dysregulating the translational machinery leading to induction of proteotoxic/ER stress and UPR-mediated cell death. Mechanistically, pevonedistat led to P-eIF2a dephosphorylation causing atypical proteotoxic/ER stress from failure to halt protein translation via the UPR and upregulation of mTOR/p70S6K. Additional studies revealed that pevonedistat re-balanced the homeostasis of pro- and anti-apoptotic proteins to favor cell death through altered expression and/or activity of Mcl-1, NOXA, and BIM, suggesting that pevonedistat has a “priming” effect on ALL by altering the apoptotic threshold through modulation of Mcl-1 activity. Further, we demonstrated that pevonedistat synergizes with selected anti-leukemic agents in vitro, and prolongs survival of NSG mice engrafted with ALL cells, lending support for the use of pevonedistat as part of a multi-agent approach.

Abstract 4547: Rebalancing of Bcl-2 family proteins mediate the vulnerability of pevonedistat-treated acute lymphoblastic leukemia cells towards MEK/ERK pathway inhibition

Abstract Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children and the relapse rate in adult ALL patients is about 50%, highlighting the need for new therapeutic strategies. Data from our laboratory and others showed that ALL cells are sensitive to drugs that induce endoplasmic reticulum (ER) stress/unfolded protein response (UPR). In search for novel strategies to target the ER stress/UPR in ALL, we tested the efficacy of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924, pevo) in ALL. We found ALL cells exhibited significant in vitro and in vivo sensitivity to pevo-induced ER stress/UPR. Specifically, proteotoxic/ER stress was observed in pevo-treated ALL cells secondary to their inability to halt protein translation following pevo-induced activation of the mTOR pathway and concomitant de-phosphorylation of p-eIF2α (S51). In addition, aberrant activation of MEK/ERK has been correlated with resistance/relapse in pediatric ALL (Blood 2014; 124: 3420-3430). In our Bp- and T-ALL cell line models, we found consistent induction of p-ERK1/2 (T202/Y204) following pevo treatment, suggesting phosphorylation of ERK1/2 as a compensatory survival mechanism in response to pevo's cytotoxicity. Supporting this hypothesis, we observed significant in vitro synergy between the MEK inhibitor selumetinib (SEL) and pevo (CI = 0.017). On this basis, we tested the in vivo efficacy of pevo + SEL in NSG mice injected with NALM6 cells expressing the luciferase gene (NALM6/LUC). Engrafted NSG mice were treated with pevo (s.c., 66 mg/kg) and SEL (p.o., 50 mg/kg) twice daily on weekdays and once per day on weekends. Bioluminescence analysis of animals 21 days post ALL injection revealed significant reduction of tumor burden in mice treated with pevo alone or pevo + SEL (p&amp;lt;0.05 for both vs. control). Kaplan-Meier curves showed a significant survival advantage for mice treated with pevo + SEL compared with the control group (p&amp;lt;0.05). Mechanistic studies showed that pevo led to induction of NOXA and BIM whereas Mcl-1 levels were stabilized, suggesting sequestration of the pro-survival activity of Mcl-1 by NOXA/BIM. Indeed, co-IP analysis demonstrated that binding between NOXA and/or BIM with Mcl-1 was enhanced in pevo-treated ALL cells. Further, significant downregulation of Mcl-1 was observed in ALL cells co-treated with pevo + SEL. We found that the synergy of this combination was prevented by co-treatment with the pan-caspase inhibitor Z-VAD, but observed persistence of Mcl-1downregulation whereas BIM expression remained unchanged. We conclude that MEK/ERK pathway inhibition rebalances the Bcl-2 family proteins in favor of synergistic apoptotic death in ALL cells treated in vitro and in vivo with the NAE inhibitor pevonedistat. Our data supports further investigations of agents targeting NAE and the MEK/ERK pathway in relapsed/refractory ALL. Citation Format: Shuhua Zheng, Gilles M. Leclerc, Guy J. Leclerc, Joanna DeSalvo, Ronan T. Swords, Julio C. Barredo. Rebalancing of Bcl-2 family proteins mediate the vulnerability of pevonedistat-treated acute lymphoblastic leukemia cells towards MEK/ERK pathway inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4547.

The NAE inhibitor pevonedistat interacts with the HDAC inhibitor belinostat to target AML cells by disrupting the DDR

AbstractTwo classes of novel agents, NEDD8-activating enzyme (NAE) and histone deacetylase (HDAC) inhibitors, have shown single-agent activity in acute myelogenous leukemia (AML)/myelodysplastic syndrome (MDS). Here we examined mechanisms underlying interactions between the NAE inhibitor pevonedistat (MLN4924) and the approved HDAC inhibitor belinostat in AML/MDS cells. MLN4924/belinostat coadministration synergistically induced AML cell apoptosis with or without p53 deficiency or FLT3–internal tandem duplication (ITD), whereas p53 short hairpin RNA (shRNA) knockdown or enforced FLT3-ITD expression significantly sensitized cells to the regimen. MLN4924 blocked belinostat-induced antiapoptotic gene expression through nuclear factor–κB inactivation. Each agent upregulated Bim, and Bim knockdown significantly attenuated apoptosis. Microarrays revealed distinct DNA damage response (DDR) genetic profiles between individual vs combined MLN4924/belinostat exposure. Whereas belinostat abrogated the MLN4924-activated intra-S checkpoint through Chk1 and Wee1 inhibition/downregulation, cotreatment downregulated multiple homologous recombination and nonhomologous end-joining repair proteins, triggering robust double-stranded breaks, chromatin pulverization, and apoptosis. Consistently, Chk1 or Wee1 shRNA knockdown significantly sensitized AML cells to MLN4924. MLN4924/belinostat displayed activity against primary AML or MDS cells, including those carrying next-generation sequencing–defined poor-prognostic cancer hotspot mutations, and CD34+/CD38−/CD123+ populations, but not normal CD34+ progenitors. Finally, combined treatment markedly reduced tumor burden and significantly prolonged animal survival (P < .0001) in AML xenograft models with negligible toxicity, accompanied by pharmacodynamic effects observed in vitro. Collectively, these findings argue that MLN4924 and belinostat interact synergistically by reciprocally disabling the DDR in AML/MDS cells. This strategy warrants further consideration in AML/MDS, particularly in disease with unfavorable genetic aberrations.

The NEDD8 Activating Enzyme Inhibitor Pevonedistat Induces ER Stress/UPR-Mediated Cell Death and Rebalances Homeostasis of Pro- and Anti-Apoptotic Proteins in Favor of Cell Death in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children. Most adults will die from the disease. Hence, current therapy in ALL is inadequate. We previously showed that ALL cells were differentially sensitive to agents inducing endoplasmic reticulum (ER) stress (Mol Cancer Res 2012; 10:969-978; PLoS One 2013; 8:e74420). In this regard, we investigated the anti-leukemic activity of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924). Pevonedistat prevents neddylation of cullin-RING E3 ligases, leading to accumulation of cullin dependent substrates with anti-proliferative effects. We demonstrated that pevonedistat induced dose-dependent cell growth inhibition (IC50 between 159-300 nM) and cell death (EC50 between 317-463 nM) in both T- and B-ALL cells (cell lines and primary patient material). We found that neddylated cullin levels were significantly decreased confirming inhibition of the NEDD8 conjugation pathway by pevonedistat. Pevonedistat induced ER stress and unfolded protein response (UPR) mediated cell death in ALL cell lines and primary cells as evidenced by increased expression of UPR markers (GRP78 and CHOP), and cleaved-PARP. Mechanistically, pevonedistat caused proteotoxic/ER stress from failure to halt protein translation mediated by p-eIF2α (Ser51) de-phosphorylation via up-regulation of the PERK inhibitor p58IPK. We also observed up-regulation of mTOR/p70S6K, further increasing protein synthesis and augmenting proteotoxic/ER stress. Indeed, we demonstrated that pevonedistat induced nascent protein synthesis, and that co-treatment with protein synthesis inhibitors (rapamycin, cycloheximide) rescued ALL cells from pevonedistat induced cytotoxicity (p < 0.01 for combination treatment vs. pevonedistat alone). The observed rescue correlated with decreased UPR markers and P-p70S6K (Thr389) expression, suggesting that proteotoxic stress is a central determinant in pevonedistat-induced ALL cell death. Additional studies into the mechanism of pevonedistat-induced apoptosis revealed that homeostasis of pro- and anti-apoptotic proteins was rebalanced in favor of cell death through decreased Mcl-1 pro-survival activity, via sequestration by NOXA and BIM. Co-immunoprecipitation experiments showed that pevonedistat increased the interaction between NOXA and Mcl-1, and BIM and Mcl-1 proteins, resulting in the reduction of Mcl-1 pro-survival activity. siRNA-mediated down-regulation of NOXA and BIM expression decreased the sensitivity of ALL cells to pevonedistat, supporting the sequestration by NOXA and BIM of Mcl-1's pro-survival activity. Activation of the MEK/ERK/Mcl-1 pathway following pevonedistat-induced cell death was also noted, possibly as a compensatory mechanism. We found that pevonedistat plus a MEK inhibitor (PD98059, selumetinib) induced significant cell death compared to each drug alone (p < 0.0001), which was associated with decreased Mcl-1 expression and increased cleaved-PARP cleavage, supporting a critical role of Mcl-1 in ALL cell survival. In addition, we demonstrated synergy between pevonedistat and effective anti-leukemic agents such as dexamethasone, doxorubicin and cytarabine in ALL cell line models, with CI values of 0.17, 0.46, and 0.23, respectively. More importantly, we showed that NSG mice engrafted with human ALL cells had statistically significant increased survival when treated with pevonedistat (pevo) plus dexamethasone (dex) compared to single agent therapy (p = 0.0076 for pevo+dex vs. dex alone; p = 0.0182 pevo+dex vs. pevo alone), lending support for the use of pevonedistat as part of a multi-agent approach. Taken together, our data demonstrate that the NAE inhibitor pevonedistat alters cellular translational machinery leading to ER stress/UPR-mediated cell death, and suggest that pevonedistat may have a "priming" effect on ALL cells by altering the apoptotic threshold through modulation of Mcl-1's pro-survival activity. DisclosuresNo relevant conflicts of interest to declare.