Abstract A38: Development and implementation of immunohistochemistry (IHC)-based pharmacodynamic (PD) biomarkers demonstrate NAE pathway inhibition in MLN4924 solid tumor clinical trials.
Authors: McDonald Alice A. , Burke Kristine , Thomas Michael , Berger Alison , Smith Peter G. , Tirrell Stephen , Blakemore Stephen J. , Mulligan Geogre , Pickard Michael D. , Fleming Mark D. , Kauh John S. , Sarantopoulos John , Dezube Bruce J. , Stringer Bradley 
Published: June 2012

MLN4924 is an investigational small molecule NEDD8 activating enzyme (NAE) inhibitor with antitumor activity in preclinical models of several tumor types that is currently in Phase I clinical development in both hematological and solid tumors. NAE is an essential controller of the NEDD8 conjugation pathway that is required for cullin-RING ligase (CRL) activity. MLN4924 forms a covalent adduct with NAE, inhibiting enzyme activity and thus preventing ubiquitination and proteasomal degradation of CRL substrate proteins. CRL substrates have important roles in cell-cycle progression, DNA replication (CDT1), oxidative stress response (NRF-2), and survival signaling; interfering with their degradation ultimately leads to apoptosis. MLN4924 is the first NAE inhibitor to enter clinical development and evidence of target inhibition and/or downstream pathway modulation were key objectives of the Phase I studies. Here we describe the development, validation and clinical implementation of IHC PD assays for MLN4924 that quantify levels of CRL substrates CDT1 and NRF-2 and demonstrate MLN-4924 NEDD8 adduct formation in both skin and solid tumors.

Pre-clinical solid tumor xenograft models were treated with increasing doses of MLN4924. Tumors were collected at multiple post-dose time points (30 minutes to 48 hours) to develop clinical assays and establish the biopsy schedule. Levels of stabilized substrates CDT1 and NRF-2, and presence of MLN4924-NEDD8 adduct were measured by quantitative and semi-quantitative IHC, respectively. Slides were scanned as whole slide images using an Aperio XTscan scope and analyzed for a percent positive pixel count using Metamorph imaging software. Western blotting of xenograft material showed decreased cullin neddylation while IHC showed an increase of CRL substrates CDT1 and NRF-2. Regulation of all PD markers was dose and time dependent. Substrate levels were most robust in the highest dose groups and peaked between 2–8 hours after dosing, returning to base line levels by 24 hours. The MLN4924-NEDD8 adduct was observed in xenograft tumors within 30 minutes of treatment indicating that MLN4924 rapidly distributed to the tumor tissue and persisted up to 24 hours. These data supported the selection of a 3–6 hr window for post-dose biopsy sampling in the clinical studies.

IHC assays were successfully adapted to clinical trial fine needle tumor biopsies and skin punch biopsies. In phase I studies levels of CDT1, NRF-2 and MLN4924-NEDD8 adduct in skin and tumor were compared in biopsies obtained at screening and 3–6 hours after the second day of dosing. CDT1 and NRF-2 IHC staining was quantified exclusively in regions of tumor or skin epidermal area. Elevations in CDT1 and NRF-2 substrate levels were observed in skin and tumor biopsies. Greater than 50% of all skin (n=38) and tumor biopsies (n=16) demonstrated a robust PD response suggesting target engagement. IHC analysis of MLN4924-NEDD8 adduct showed that drug was present in 100% of the post dose tumor biopsies.

These data demonstrate evidence of inhibition of NAE activity and downstream pathway modulation by MLN4924 in skin and multiple tumor types.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A38.