Michael D. Pickard

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

Purpose: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Experimental Design: Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease. Results: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Conclusions: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. Clin Cancer Res; 17(23); 7313–23. ©2011 AACR.

Sequence analysis of β-subunit genes of the 20S proteasome in patients with relapsed multiple myeloma treated with bortezomib or dexamethasone

Variations within proteasome β (PSMB) genes, which encode the β subunits of the 20S proteasome, may affect proteasome function, assembly, and/or binding of proteasome inhibitors. To investigate the potential association between PSMB gene variants and treatment-emergent resistance to bortezomib and/or long-term outcomes, in the present study, PSMB gene sequence variation was characterized in tumor DNA samples from patients who participated in the phase 3 Assessment of Proteasome Inhibition for Extending Remissions (APEX) study of bortezomib versus high-dose dexamethasone for treatment of relapsed multiple myeloma. Twelve new PSMB variants were identified. No associations were found between PSMB single nucleotide polymorphism genotype frequency and clinical response to bortezomib or dexamethasone treatment or between PSMB single nucleotide polymorphism allelic frequency and pooled overall survival or time to progression. Although specific PSMB5 variants have been identified previously in preclinical models of bortezomib resistance, these variants were not detected in patient tumor samples collected after clinical relapse from bortezomib, which suggests that alternative mechanisms underlie bortezomib insensitivity.

Potential biomarkers of bortezomib activity in mantle cell lymphoma from the phase 2 PINNACLE trial

Immunohistochemical analyses of archival tumor specimens were used for pre-planned exploratory analyses of protocol-specified candidate biomarkers of bortezomib activity in 73 patients with relapsed/refractory mantle cell lymphoma in the phase 2 PINNACLE study. Consistent with other studies, elevated Ki-67 was a marker of poor prognosis, demonstrating significant associations with shorter time to progression and overall survival. Elevated NF-κB p65 and low PSMA5 expression demonstrated a trend for better response and were significantly associated with longer time to progression; elevated NF-κB p65 demonstrated a trend toward longer overall survival. This is consistent with myeloma clinical genomics research, suggesting biomarker relevance across tumor types. Elevated p27 was significantly associated with longer overall survival. Overall survival analyses by International Prognostic Index and Mantle Cell Lymphoma International Prognostic Index confirmed differential prognosis by both scores. These biomarkers data begin to illuminate bortezomibs mechanism of action in lymphoma.

A Quantitative Framework and Strategies for Management and Evaluation of Metabolic Drug-Drug Interactions in Oncology Drug Development New Molecular Entities as Object Drugs

This article outlines general strategies for the management and evaluation of pharmacokinetic drug-drug interactions (DDIs) resulting from perturbation of clearance of investigational anticancer drug candidates by concomitantly administered agents in a drug development setting, with a focus on drug candidates that cannot be evaluated in first-in-human studies in healthy subjects. A risk level classification is proposed, based on quantitative integration of knowledge derived from preclinical drug-metabolism studies evaluating the projected percentage contribution [fi(%)] of individual molecular determinants (e.g. cytochrome P450 isoenzymes) to the overall human clearance of the investigational agent. The following classification is proposed with respect to susceptibility to DDIs with metabolic inhibitors: a projected maximum DDI expected to result in a ≤1.33-fold increase in exposure, representing a low level of risk; a projected maximum DDI expected to result in a >1.33-fold but <2-fold increase in exposure, representing a moderate level of risk; and a projected maximum DDI expected to result in a ≥2-fold increase in exposure, representing a potentially high level of risk.For DDIs with metabolic inducers, the following operational classification is proposed, based on the sum of the percentage contributions of enzymes that are inducible via a common mechanism to the overall clearance of the investigational drug: ≪25%, representing a low level of risk; <50%, representing a moderate level of risk; and ≥50%, representing a potentially high level of risk. To ensure patient safety and to minimize bias in determination of the recommended phase II dose (RP2D), it is recommended that strong and moderate inhibitors and inducers of the major contributing enzyme are excluded in phase I dose-escalation studies of high-risk compounds, whereas exclusion of strong inhibitors and inducers of the contributing enzyme (s) is recommended as being sufficient for moderate-risk compounds. For drugs that will be investigated in diseases such as glioblastoma, where there may be relatively frequent use of enzyme-inducing antiepileptic agents (EIAEDs), a separate dose-escalation study in this subpopulation is recommended to define the RP2D. For compounds in the high-risk category, if genetic deficiencies in the activity of the major drug-metabolizing enzyme are known, it is recommended that poor metabolizers be studied separately to define the RP2D for this subpopulation.Whereas concomitant medication exclusion criteria that are utilized in the phase I dose-escalation studies will probably also need to be maintained for high-risk compounds in phase II studies unless the results of a clinical DDI study indicate the absence of a clinically relevant interaction, these exclusion criteria can potentially be relaxed beyond phase I for moderate-risk compounds, if supported by the nature of clinical toxicities and the understanding of the therapeutic index in phase I. Adequately designed clinical DDI studies will not only inform potential relaxation of concomitant medication exclusion criteria in later-phase studies but, importantly, will also inform the development of pharmacokinetically derived dose-modification guidelines for use in clinical practice when coupled with adequate safety monitoring, as illustrated in the prescribing guidance for many recently approved oncology therapeutics.

Abstract B92: Nedd8-activating enzyme inhibitor MLN4924 provides synergy in nonclinical models with mitomycin C through interactions with ATR, BRCA1/BRCA2 and chromatin dynamics pathways.

MLN4924 is an investigational small molecule inhibitor of the Nedd8-activating enzyme (NAE) currently in Phase 1 clinical trials. MLN4924 induces DNA damage via rereplication in most cell lines. This distinct mechanism of DNA damage may affect its ability to combine with standards of care, including other DNA damaging agents. We studied the interaction of MLN4924 with other DNA damaging agents in a panel of 4 cell lines and found that mitomycin C, cisplatin, carboplatin, cytarabine, ultraviolet radiation, SN-38, and gemcitabine demonstrated synergy in combination with MLN4924 in at least 1 cell line. Further testing in xenograft-bearing mice demonstrated synergy of MLN4924 with mitomycin C and with carboplatin, and additivity with gemcitabine. Based in part on this data, MLN4924 is currently being evaluated in a Phase 1b trial ([NCT01862328][1]) with 3 combination arms: MLN4924 + carboplatin and paclitaxel, MLN4924 + gemcitabine, and MLN4924 + docetaxel. To evaluate the mechanism of synergy between MLN4924 and mitomycin C, in vitro experiments with RNAi were performed. Depletion of genes within the ATR and BRCA1/BRCA2 pathways, chromatin modification, and transcription-coupled repair reduced the synergy between mitomycin C and MLN4924. Our data suggest that mitomycin C causes stalled replication forks, which when combined with rereplication induced by MLN4924, results in frequent replication fork collisions, leading to cell death. This study provides a straightforward approach to understand the mechanism of synergy, which may be applied to additional combinations currently under clinical evaluation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B92. Citation Format: Eric S. Lightcap, Khristofer Garcia, Jonathan L. Blank, David C. Bouck, Xiaozhen J. Liu, Greg Hather, Allison Berger, Katherine Cosmopoulos, Michael P. Thomas, Mike Kuranda, Michael D. Pickard, Ray Liu, Syamala Bandi, Peter G. Smith. Nedd8-activating enzyme inhibitor MLN4924 provides synergy in nonclinical models with mitomycin C through interactions with ATR, BRCA1/BRCA2 and chromatin dynamics pathways. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B92. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01862328&atom=%2Fmolcanther%2F12%2F11_Supplement%2FB92.atom

Abstract A196: Development, validation, and clinical implementation of a peripheral blood RT-PCR pharmacodynamic assay for MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE).

The ubiquitin proteasome system regulates the degradation of intracellular proteins that are involved in many cellular processes. These processes, such as cell cycle, DNA replication, and response to oxidative stress play a key role in tumor growth and survival. The ubiquitination and proteasomal degradation of a subset of proteins involved in these processes is controlled by Cullin-RING ubiquitin ligases (CRLs). NEDD8-activating enzyme (NAE) activates the ubiquitin-like protein NEDD8 for conjugation to CRLs and therefore regulates the proteasomal destruction of CRL substrate proteins. NAE inhibition prevents degradation of CRL substrates (e.g. NRF2, CDT1), leading to their accumulation, subsequent apoptosis and cell death. MLN4924 is an investigational small molecule NAE inhibitor with antitumor activity in preclinical models of several tumor types that is currently in Phase I clinical development. 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 a pharmacodynamic (PD) RT-PCR assay that quantifies levels of gene transcripts regulated in response to NAE inhibition in human whole blood. To develop a prototype PD assay, Affymetrix gene expression profiling studies on HCT116 cells treated in-vitro with an NAE inhibitor were performed. Genes regulated greater than two-fold by NAE inhibition were identified. The list of genes was observed to be enriched for NRF2 regulated transcripts, consistent with the mechanism of action for MLN4924. Twenty-seven of the most robustly regulated genes were tested by RT-PCR (TaqMan) using RNA samples extracted from MLN4924 treated HCT-116 cells and human PBMCs treated ex-vivo with MLN4924 in order to validate the microarray results and develop a RT-PCR based PD assay in human blood. Transcripts showing a robust response to NAE inhibition in both HCT116 cells and PBMCs were identified for further evaluation in whole blood as a preferred sample matrix for clinical studies. To explore the relationship of MLN4924 regulated transcripts with time and drug concentration, blood samples from multiple healthy volunteers were treated ex-vivo with a range of MLN4924 concentrations for three durations (4, 8, 24 hours). Eight genes (NQO1, SLC7A11, TXNRD1, SRXN1, GSR, GCLM, ATF3, and MAG1) that displayed a robust induction (>3-fold) in whole blood were selected for the PD biomarker panel. Inter- and intra-assay variability was established via testing two pools of cDNA control samples (RNA from MLN4924 ex-vivo treated and untreated blood) in replicates on the same plate, and on multiple plates run separately. All genes had inter- and intra-assay CV values of 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 A196.

Abstract A38: Development and implementation of immunohistochemistry (IHC)-based pharmacodynamic (PD) biomarkers demonstrate NAE pathway inhibition in MLN4924 solid tumor clinical trials.

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.

Abstract A31: Development of an immunohistochemistry-based pharmacodynamic biomarker program for phase I trials of MLN4924, an inhibitor of the NEDD8 activating enzyme

The novel small molecule MLN4924 is an inhibitor of the NEDD8 activating enzyme (NAE) and is currently in Phase I trials. As part of the ubiquitin-proteasome system, NAE is required for activity of cullin ring ligases (CRLs), a category of ubiquitin E3 ligases. When NAE is inhibited, levels of CRL substrates increase due to impaired ubiquitination and proteasomal degradation; CRL substrates can therefore serve as pharmacodynamic (PD) biomarkers of NAE pathway inhibition. We describe the development of Immunohistochemistry (IHC) assays for two CRL substrates: NRF2 (a transcription factor involved in the oxidative stress response) and CDT1 (a protein that regulates DNA replication). CDT1 and NRF2 IHC assays were initially evaluated in PD studies of xenograft tumors grown in immunocompromised mice and showed dose-dependent regulation. The IHC assays were then adapted for use in clinical samples, including skin and tumor biopsies from patients in Phase I trials of MLN4924. Sample quality and morphology were assessed by pathologist review of hematoxylin and eosin pathology stain. A set of formalin-fixed, paraffin-embedded cell pellets was evaluated with each IHC run to detect technical variability and establish pass/fail criteria for each experimental IHC run. The QC samples were generated from the HCT116 cell line treated with various concentrations of MLN4924. The QC controls were assayed multiple times to generate an expected range as a percent positive area above a threshold for each concentration of MLN4924. The ranges were then applied to each experimental run to pass or fail the experiment. In cases of assay failure, the clinical samples were re-stained. Skin and melanoma tumors are inherently challenging for IHC analysis due to the population variability of melanin pigment, which can be indistinguishable from brown DAB chromagen. Adaption of the assays to a blue chromagen (NBT/BCIP) enabled a contrast between the chromagen stain and endogenous pigments. A region of interest (ROI) for skin was manually drawn to select the epidermal area. Semi-automated image analysis was used to measure percent positive area for CDT1 and NRF2 within the total epidermal region. To determine the baseline levels for CDT1 and NRF2 signal, 10 benign skin samples were studied. Staining of six sections at 10-micron increments was performed on each sample to investigate both the inter- and intra-patient variability. Overall, the percent positive area of both substrates was below 1% in benign skin and variation from section to section was minimal. To survey the baseline levels of NRF2 and CDT1 across a variety of tumor types, we evaluated archival tumors of various types including melanoma, head & neck squamous cell carcinoma, prostate, lung, and breast cancer. A manual ROI was identified by pathologist review to select for tumor area, and semi-automated image analysis was used to measure the percent positive area for NRF2 and CDT1 within the total tumor area. Unlike skin, the tumor samples showed variable levels of NRF2 and CDT1. However, most samples showed a percent positive area less than 40%, indicating the feasibility of detecting a PD effect by comparing pre- and post-dose tumor biopsies. In summary, reliable IHC PD biomarker assays have been established for the NAE inhibitor MLN4924. The assays are currently being used to measure PD effects of NAE inhibition in skin and solid tumors from Phase I trials of MLN4924. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A31

Abstract B34: Clinical pharmacodynamic assay development for the first small molecule inhibitor of Nedd8‐activating enzyme (NAE), MLN4924

NEDD8‐Activating Enzyme (NAE) initiates the conjugation of the ubiquitin‐like protein NEDD8 to its cellular targets, members of the cullin protein family. NEDD8 conjugation to cullins is known to be essential for the ubiquitination activity of cullin‐RING ubiquitin ligases (CRLs). CRLs control the timely ubiquitination and subsequent degradation of many proteins with important roles in cell cycle progression and signal transduction. MLN4924 is a first in class, small molecule inhibitor of NAE. Inhibition of NAE with MLN4924 disrupts the conjugation of NEDD8 to CRLs. This subsequently prevents ubiquitination and proteasomal degradation of CDL substrates involved in cell cycle regulation (p27), signal transduction (pIkBa), DNA replication (Cdt‐1), stress response (Nrf‐2), and other processes crucial to tumor cell growth and survival. The regulation of these markers of NAE inhibition is well characterized across of a variety of cancer cell types grown in culture and as xenografts implanted in immunocompromised mice. Here we describe the development of clinical pharmacodynamic (PD) assays to evaluate two markers of NAE inhibition in the blood compartment, measurement of neddylated cullins and pIkBa levels in peripheral blood mononuclear cells (PBMCs). In vivo administration of a single dose of MLN4924 to nude mice harboring subcutaneous HCT‐116 colon carcinoma xenografts results in a dose‐dependent decrease in neddylated cullin (CUL‐N8) levels and elevation of the CRL substrate pIkBa. In addition, we demonstrate a dose‐dependent inhibition of CUL‐N8 levels in PBMCs isolated from the same mice. We expanded our PD analysis to human PBMCs treated ex vivo with MLN4924 to assess CUL‐N8 levels by quantitativeWestern blot and pIKBa levels by ELISA. PBMCs isolated from healthy volunteers were used to assess the technical and biological variability of theWestern blot and ELISA assays. In these experiments, whole blood was treated ex vivo with increasing concentrations of MLN4924 and PBMCs were subsequently isolated with VACUTAINER® CPT™ tubes that are often utilized in Phase I trials. Repeated CUL‐N8 or pIkBa measurements of replicate samples on different days demonstrated coefficient of variation values of less than 12% for both assays. Biological variability of baseline (i.e. untreated) and MLN4924‐regulated levels of CUL‐N8 and pIKBa in PBMCs was assessed by performing ex vivo treatment of whole blood obtained from the same donors on three separate occasions. Statistical analysis of this data demonstrated good biological reproducibility for both assays. Plasma concentrations measured from the ex vivo treated blood samples indicate that both the CUL‐N8 Western and pIkBa ELISA assays detect MLN4924‐induced regulation within the predicted range for human plasma exposures in a clinical setting. The demonstrated PD response and anti‐tumor activity of MLN4924 in preclinical models has supported its ongoing evaluation for safety and PD in patients with hematological and solid tumor disease in multiple Phase I clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B34.

Abstract A70: A genome‐wide siRNA screen for modulators of cell death induced by the proteasome inhibitor bortezomib

Multiple pathways have been proposed as the mechanism by which proteasome inhibition induces cell death. To clarify their relative importance, we performed a genome‐wide siRNA screen to evaluate the genetic determinants that confer sensitivity of the HCT‐116 colon cancer cell line to bortezomib (VELCADE®, PS‐341). The screen identified 100 genes whose knock‐down affects the lethality of bortezomib. From this list, the accumulation of the proteins ASF1B, Myc, ODC1, PMAIP1 (Noxa), BNIP3, Gadd45α, p‐SMC1A, SREBF1, and p53 by proteasome inhibition was linked to the induction of cell death. Fifty‐nine genes in the A375 melanoma cell line and 56 genes in the HeLa cervical cancer cell line showed similar interactions with bortezomib to those seen in HCT‐116 and a subset of 39 genes were common to all three cell lines. Finally, knockdown of these 100 genes in HCT‐116 cells similarly affected their responsiveness to a structurally diverse set of proteasome inhibitors. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A70.