Supplemental Appendix
Supplemental methodology
Immunohistochemical (IHC) assay methodology
IHC assays were performed on 5-micron formalin-fixed, paraffin-embedded (FFPE) tumor sections using the Ventana XT® auto-stainer (Ventana Medical Systems, Tucson, AZ). Antigen retrieval consisted of incubation with Ventana’s CC1 antigen retrieval solution for 20 minutes. Primary antibody to pevonedistat-NEDD8 adduct (Millennium Pharmaceuticals, Inc., Cambridge, MA) was incubated for 1 hour at 37ºC and labeled with biotinylated goat anti-rabbit IgG secondary antibody (Vector Laboratories, Burlingame, CA) for 32 minutes at room temperature and detected with horseradish peroxidase (HRP) DAB Map system (Ventana Medical Systems) followed by hematoxylin and bluing reagent (Ventana Medical Systems) counter-stain. For melanoma tumors, antibodies to CDT1 (Millennium Pharmaceuticals, Inc.) and NRF2 (Epitomics Catalog #2178) were incubated for 1 hour at room temperature, labeled with UltraMap anti-mouse alkaline phosphatase or UltraMap ant-rabbit phosphatase multimer secondary antibody, respectively for 32 minutes at room temperature, and detected with NBT/BCIP substrate system (Ventana Medical Systems). A blue chromagen and red counter-stain (Ventana Medical Systems) was used to avoid overlap with melanin pigmentation. In all other tumor biopsies, antibodies to CDT1 and NRF2 were labeled with UltraMap anti-mouse or UltraMap anti-rabbit HRP multimer secondary antibodies (Ventana Medical Systems) and detected with the ChromoMap DAB Kit (Ventana Medical Systems), followed by hematoxylin and bluing reagent (Ventana Medical Systems) counter-stain. Images were captured at 20x magnification using the ScanScope XT (Aperio Technologies, Vista, CA) whole-slide imaging system.
Statistical methodology
The dose-toxicity relationship was modeled by a single-parameter logistic model:
where the variable “dose” refers to the dose level administered. A truncated beta prior distribution was used to estimate the posterior distribution of the parameter θ. The prior distributions used were Beta(1, 37) for θ values in the interval (0.0039, 0.1956) for Schedule A and Beta(0.29, 10.71) for θ values in the interval (0.0039, 0.1057) for both Schedules B and C. These parameter values were set to reflect current understanding of the compound at the start of each schedule’s dose escalation, while ensuring good operating characteristics of the model (via simulation studies) under various assumptions for the location of the MTD.
Supplemental results
Schedule A
Safety and treatment duration
Patients in schedule A were treated for a median of 2 (range 1–7) cycles (Figure 1 in main manuscript). All patients were evaluable for toxicity. Seven (58%) patients experienced grade ≥3 adverse events (AEs) and only elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were reported at grade ≥3 in more than one patient overall (Table 2 in main manuscript). One patient discontinued due to multi-organ failure. Six (50%) patients experienced at least 1 treatment-related ≥ grade 3 AE (supplemental table 1). Three (25%) patients experienced treatment-related ≥ grade 3 serious AEs (SAEs): grade 3 nausea and vomiting, grade 5 multi-organ failure, and grade 5 disease progression.
Pharmacodynamics
Pharmacodynamic results for schedule A were similar to those for schedules B and C, and are presented together in Figure 3C in the main manuscript.
Antitumor activity
One of 10 (10%) evaluable patients on schedule A achieved a best response of partial response that lasted for 1 cycle. This melanoma patient was treated at the 25 mg/m2 dose level. Seven patients treated at the 25, 50, 61, and 83 mg/m2 dose levels (n=2, 5, 1, and 2, respectively) had a best response of stable disease. This lasted for 4.6 and 3.3 months, respectively, in two patients, one with small-cell lung cancer treated at 50 mg/m2 and one with breast cancer treated at 83 mg/m2.
Supplemental Table 1
Treatment-related AEs experienced by ≥10% of patients on any schedule, and treatment-related grade ≥3 AEs reported in at least 1 patient.
AE, n (%) / Schedule An=12 / Schedule B
n=17 / Schedule C
n=19 / Total
N=48 /
Any treatment-related AE / 12 (100) / 15 (88) / 16 (84) / 43 (90)
Fatigue / 6 (50) / 9 (53) / 3 (16) / 18 (38)
Nausea / 5 (42) / 1 (6) / 8 (42) / 14 (29)
Diarrhea / 4 (33) / 3 (18) / 2 (11) / 9 (19)
Elevated alanine aminotransferase / 4 (33) / 4 (24) / 1 (5) / 9 (19)
Elevated aspartate aminotransferase / 3 (25) / 4 (24) / 2 (11) / 9 (19)
Anemia / 2 (17) / 1 (6) / 4 (21) / 7 (15)
Hyperbilirubinemia / 1 (8) / 4 (24) / 2 (11) / 7 (15)
Vomiting / 2 (17) / 2 (12) / 3 (16) / 7 915)
Myalgia / 1 (8) / 0 / 5 (26) / 6 (13)
Pyrexia / 2 (17) / 2 (12) / 2 (11) / 6 (13)
Hypomagnesemia / 2 (17) / 0 / 3 (16) / 5 (10)
Constipation
Decreased appetite / 1 (8)
1 (8) / 2 (12)
1 (6) / 1 (5)
2 (11) / 4 (8)
4 (8)
Any treatment-related grade ≥3 AE / 6 (50) / 4 (24) / 0 / 10 (21)
Elevated alanine aminotransferase / 2 (17) / 1 (6) / 0 / 3 (6)
Elevated aspartate aminotransferase / 2 (17) / 0 / 0 / 2 (4)
Anemia / 0 / 1 (6) / 0 / 1 (2)
Elevated γ-glutamyltransferase / 0 / 1 (6) / 0 / 1 (2)
Fatigue / 0 / 1 (6) / 0 / 1 (2)
Hyperbilirubinemia / 1 (8) / 0 / 0 / 1 (2)
Hyperglycemia / 1 (8) / 0 / 0 / 1 (2)
Hypophosphatemia / 1 (8) / 0 / 0 / 1 (2)
Multi-organ failure / 1 (8) / 0 / 0 / 1 (2)
Nausea / 1 (8) / 0 / 0 / 1 (2)
Neutropenia / 1 (8) / 0 / 0 / 1 (2)
Transaminases increased / 1 (8) / 0 / 0 / 1 (2)
Vomiting / 1 (8) / 0 / 0 / 1 (2)
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Supplemental Table 2
Summary of hepatotoxicities reported with pevonedistat on each dosing schedule and overall.
AE, n (%) / Schedule An = 12 / Schedule B
n = 17 / Schedule C
n = 19 / Total
N = 48 /
Elevated ALT / 4 (33) / 4 (24) / 3 (16) / 11 (23)
Treatment-related / 4 (33) / 4 (24) / 1 (5) / 9 (19)
Grade ≥3 / 2 (17) / 1 (6) / 0 / 3 (6)
Treatment-related grade ≥3 / 2 (17) / 1 (6) / 0 / 3 (6)
DLTs (dose level[s]) / 1 (61 mg/m2) / 1 (50 mg/m2, MTD) / 0 / 2
Elevated AST / 3 (25) / 4 (24) / 4 (21) / 11 (23)
Treatment-related / 3 (25) / 4 (24) / 2 (11) / 9 (19)
Grade ≥3 / 2 (17) / 0 / 0 / 2 (4)
Treatment-related grade ≥3 / 2 (17) / 0 / 0 / 2 (4)
DLTs (dose level[s]) / 2 (61 & 83 mg/m2) / 1 (50 mg/m2, MTD) / 1 (89 mg/m2) / 4
Hyperbilirubinemia / 1 (8) / 4 (24) / 3 (16) / 8 (17)
Treatment-related / 1 (8) / 4 (24) / 2 (11) / 7 (15)
Grade ≥3 / 1 (8) / 0 / 1 (5) / 2 (4)
Treatment-related grade ≥3 / 1 (8) / 0 / 0 / 1 (2)
DLTs (dose level[s]) / 0 / 2 (67 & 89 mg/m2) / 1 (89 mg/m2) / 3
Increased blood alkaline phosphatase / 2 (17) / 2 (12) / 4 (21) / 8 (17)
Treatment-related / 2 (17) / 1 (6) / 1 (5) / 4 (8)
Grade ≥3 / 0 / 1 (6) / 0 / 1 (2)
Treatment-related grade ≥3 / 0 / 0 / 0 / 0
Gamma--glutamyltransferase increased / 0 / 2 (12) / 0 / 2 (4)
Treatment-related / 0 / 1 (6) / 0 / 1 (2)
Grade ≥3 / 0 / 1 (6) / 0 / 1 (2)
Treatment-related grade ≥3 / 0 / 1 (6) / 0 / 1 (2)
Transaminases increased / 1 (8) / 0 / 2 (11) / 3 (6)
Treatment-related / 1 (8) / 0 / 2 (11) / 3 (6)
Grade ≥3 / 1 (8) / 0 / 0 / 1 (2)
Treatment-related grade ≥3 / 1 (8) / 0 / 0 / 1 (2)
DLTs (dose level[s]) / 1 (50 mg/m2, MTD) / 0 / 0 / 1
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Supplemental Figure 1
Individual dose-normalized exposures of pevonedistat (as assessed by Cmax and AUC24) obtained on Day 1 and Day 5 for schedule B (with dexamethasone) and schedule C (without dexamethasone). All available patient exposure data from all 3 dose levels tested (50, 67 and 89 mg/m2) on Days 1, 3, and 5 were included; in a few instances, Cmax and/or AUC24 could not be determined on Day 5. Median (range) actual dose of pevonedistat administered was 109 mg (86-189) for schedule B and 142 mg (95-237) for schedule C. Horizontal marks represent the geometric mean dose-normalized Cmax and AUC24 values for each subgroup.
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