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Purpose: Evaluate the safety, pharmacokinetic profile, pharmacodynamic effects, and antitumor activity of the first-in-class investigational NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924) in patients with relapsed/refractory lymphoma or multiple myeloma.Experimental Design: Patients with relapsed/refractory myeloma (n = 17) or lymphoma (n = 27) received intravenous pevonedistat 25 to 147 mg/m2 on days 1, 2, 8, 9 (schedule A; n = 27) or 100 to 261 mg/m2 on days 1, 4, 8, 11 (schedule B; n = 17) of 21-day cycles.Results: Maximum tolerated doses were 110 mg/m2 (schedule A) and 196 mg/m2 (schedule B). Dose-limiting toxicities included febrile neutropenia, transaminase elevations, muscle cramps (schedule A), and thrombocytopenia (schedule B). Common adverse events included fatigue and nausea. Common grade ≥3 events were anemia (19%; schedule A), and neutropenia and pneumonia (12%; schedule B). Clinically significant myelosuppression was uncommon. There were no treatment-related deaths. Pevonedistat pharmacokinetics exhibited a biphasic disposition phase and approximate dose-proportional increases in systemic exposure. Consistent with the short mean elimination half-life of approximately 8.5 hours, little-to-no drug accumulation in plasma was seen after multiple dosing. Pharmacodynamic evidence of NAE inhibition included increased skin levels of CDT-1 and NRF-2 (substrates of NAE-dependent ubiquitin ligases), and increased NRF-2-regulated gene transcript levels in whole blood. Pevonedistat–NEDD8 adduct was detected in bone marrow aspirates, indicating pevonedistat target engagement in the bone marrow compartment. Three lymphoma patients had partial responses; 30 patients achieved stable disease.Conclusions: Pevonedistat demonstrated anticipated pharmacodynamic effects in the clinical setting, a tolerable safety profile, and some preliminary evidence that may be suggestive of the potential for activity in relapsed/refractory lymphoma. Clin Cancer Res; 22(1); 34–43. ©2015 AACR.

Table A1. Prior therapies received (agents received by >5 patients); Table A2. Summary of pevonedistat plasma pharmacokinetic parameters on days 1 and 9 of cycle 1 on schedule A (administration on days 1, 2, 8, 9 of 21-day cycles); Table A3. Summary of pevonedistat plasma pharmacokinetic parameters on days 1 and 4 (or, alternatively, day 11) of cycle 1 on schedule B (administration on days 1, 4, 8, 11 of 21-day cycles); Table A4. Changes from baseline in gene expression levels for NAEregulated transcriptional targets on cycle 1, day 1 following pevonedistat administration at the MTDs on schedules A and B; Table A5. Patients achieving partial responses; Table A6. Patients achieving stable disease and receiving at least 5 cycles of pevonedistat.

Representative images (20 x magnification) of formalin-fixed paraffin-embedded clots prepared from bone marrow aspirates.

Supplementary Table 1 Legend from Reproducibility of Gene Expression Signature–Based Predictions in Replicate Experiments

Supplementary Table 1 from Reproducibility of Gene Expression Signature–Based Predictions in Replicate Experiments

Purpose: The goals of this analysis were to (a) determine concordance of gene expression results from replicate experiments, (b) examine prediction agreement of multigene predictors on replicate data, and (c) assess the robustness of prediction results in the face of noise.Patients and Methods: Affymetrix U133A gene chips were used for gene expression profiling of 97 fine-needle aspiration biopsies from breast cancer. Thirty-five cases were profiled in replicates: 17 within the same laboratory, 11 in two different laboratories, and 15 to assess manual and robotic labeling. We used data from 62 cases to develop 111 distinct pharmacogenomic predictors of response to therapy. These were tested on cases profiled in duplicates to determine prediction agreement and accuracy. To evaluate the robustness of the pharmacogenomic predictors, we also introduced random noise into the informative genes in one half of the replicates.Results: The average concordance correlation coefficient was 0.978 (range, 0.96-0.99) for intralaboratory replicates, 0.962 (range, 0.94-0.98) for between-laboratory replicates, and 0.971 (range, 0.93-0.99) for manual versus robotic labeling. The mean % prediction agreement on replicate data was 97% (95% CI, 0.96-0.98; SD, 0.006), 92% (95% CI, 0.90-0.93; SD, 0.009), and 94% (95% CI, 0.92-0.95; SD, 0.008) for support vector machines, diagonal linear discriminant analysis, and k-nearest neighbor prediction methods, respectively. Mean accuracy in the test set was 77% (95% CI, 0.74-0.79; SD, 0.014), 66% (95% CI, 0.63-0.73; SD, 0.015), and 64% (95% CI, 0.60-0.67; SD, 0.016), respectively.Conclusion: Gene expression results obtained with Affymetrix U133A chips are highly reproducible within and across two high-volume laboratories. Pharmacogenomic predictions yielded >90% agreement in replicate data.

Supplementary Figure 2 from Phase I Assessment of New Mechanism-Based Pharmacodynamic Biomarkers for MLN8054, a Small-Molecule Inhibitor of Aurora A Kinase

Supplementary Tables 1-5 from Phase I Assessment of New Mechanism-Based Pharmacodynamic Biomarkers for MLN8054, a Small-Molecule Inhibitor of Aurora A Kinase

Supplementary Figure 1 from Phase I Assessment of New Mechanism-Based Pharmacodynamic Biomarkers for MLN8054, a Small-Molecule Inhibitor of Aurora A Kinase

Three proteasome inhibitors have garnered regulatory approvals in various multiple myeloma settings; but drug resistance is an emerging challenge, prompting interest in blocking upstream components of the ubiquitin-proteasome pathway. One such attractive target is the E1 ubiquitin-activating enzyme (UAE); we therefore evaluated the activity of TAK-243, a novel and specific UAE inhibitor. TAK-243 potently suppressed myeloma cell line growth, induced apoptosis, and activated caspases while decreasing the abundance of ubiquitin-protein conjugates. This was accompanied by stabilization of many short-lived proteins, including p53, myeloid cell leukemia 1 (MCL-1), and c-MYC, and activation of the activating transcription factor 6 (ATF-6), inositol-requiring enzyme 1 (IRE-1), and protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) arms of the ER stress response pathway, as well as oxidative stress. UAE inhibition showed comparable activity against otherwise isogenic cell lines with wild-type (WT) or deleted p53 despite induction of TP53 signaling in WT cells. Notably, TAK-243 overcame resistance to conventional drugs and novel agents in cell-line models, including bortezomib and carfilzomib resistance, and showed activity against primary cells from relapsed/refractory myeloma patients. In addition, TAK-243 showed strong synergy with a number of antimyeloma agents, including doxorubicin, melphalan, and panobinostat as measured by low combination indices. Finally, TAK-243 was active against a number of in vivo myeloma models in association with activation of ER stress. Taken together, the data support the conclusion that UAE inhibition could be an attractive strategy to move forward to the clinic for patients with relapsed and/or refractory multiple myeloma.

SummaryPurpose Ixazomib is an investigational proteasome inhibitor with demonstrated antitumor activity in xenograft models of multiple myeloma (MM), lymphoma, and solid tumors. This open-label, phase 1 study investigated intravenous (IV) ixazomib, in adult patients with advanced non-hematologic malignancies. Methods Patients received IV ixazomib twice-weekly for up to twelve 21-day cycles. The 0.125 mg/m2 starting dose was doubled (one patient/dose) until 1.0 mg/m2 based on dose-limiting toxicities (DLTs) in cycle 1. This was followed by 3 + 3 dose-escalation and expansion at the maximum tolerated dose (MTD). Primary objectives included safety and MTD assessment. Secondary objectives included assessment of pharmacokinetics, pharmacodynamics, and disease response. Results Ixazomib was escalated from 0.125 to 2.34 mg/m2 to determine the MTD (n = 23); patients were then enrolled to MTD expansion (n = 73) and pharmacodynamic (n = 20) cohorts. Five patients experienced DLTs (1.0 and 1.76 mg/m2: grade 3 pruritic rash; 2.34 mg/m2: grade 3 and 4 thrombocytopenia, and grade 3 acute renal failure); thus, the MTD was 1.76 mg/m2. Drug-related grade ≥3 adverse events (AEs) included thrombocytopenia (23 %), skin and subcutaneous (SC) tissue disorders (16 %), and fatigue (9 %). Among 92 evaluable patients, one (head and neck cancer) had a partial response and 30 had stable disease. Ixazomib terminal half-life was 3.8–7.2 days; plasma exposures increased dose-proportionally and drug was distributed to tumors. Inhibition of whole-blood 20S proteasome activity and upregulation of ATF-3 in tumor biopsies demonstrated target engagement. Conclusions In patients with solid tumors, ixazomib was associated with a manageable safety profile, limited antitumor activity, and evidence of downstream proteasome inhibition effects.

The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Background MicroRNAs (miRNA) are varied in length, under 25 nucleotides, single-stranded noncoding RNA that regulate post-transcriptional gene expression via translational repression or mRNA degradation. Elevated levels of miRNAs can be detected in systemic circulation after tissue injury, suggesting that miRNAs are released following cellular damage. Because of their remarkable stability, ease of detection in biofluids, and tissue specific expression patterns, miRNAs have the potential to be specific biomarkers of organ injury. The identification of miRNA biomarkers requires a systematic approach: 1) determine the miRNA tissue expression profiles within a mammalian species via next generation sequencing; 2) identify enriched and/or specific miRNA expression within organs of toxicologic interest, and 3) in vivo validation with tissue-specific toxicants. While miRNA tissue expression has been reported in rodents and humans, little data exists on miRNA tissue expression in the dog, a relevant toxicology species. The generation and evaluation of the first dog miRNA tissue atlas is described here. Results Analysis of 16 tissues from five male beagle dogs identified 106 tissue enriched miRNAs, 60 of which were highly enriched in a single organ, and thus may serve as biomarkers of organ injury. A proof of concept study in dogs dosed with hepatotoxicants evaluated a qPCR panel of 15 tissue enriched miRNAs specific to liver, heart, skeletal muscle, pancreas, testes, and brain. Dogs with elevated serum levels of miR-122 and miR-885 had a correlative increase of alanine aminotransferase, and microscopic analysis confirmed liver damage. Other non-liver enriched miRNAs included in the screening panel were unaffected. Eli Lilly authors created a complimentary Sprague Dawely rat miRNA tissue atlas and demonstrated increased pancreas enriched miRNA levels in circulation, following caerulein administration in rat and dog. Conclusion The dog miRNA tissue atlas provides a resource for biomarker discovery and can be further mined with refinement of dog genome annotation. The 60 highly enriched tissue miRNAs identified within the dog miRNA tissue atlas could serve as diagnostic biomarkers and will require further validation by in vivo correlation to histopathology. Once validated, these tissue enriched miRNAs could be combined into a powerful qPCR screening panel to identify organ toxicity during early drug development. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2958-x) contains supplementary material, which is available to authorized users.

Objective. Use of tyramide signal amplification (TSA) to detect autophagy biomarkers in formalin fixed and paraffin embedded (FFPE) xenograft tissue. Materials and Methods. Autophagy marker regulation was studied in xenograft tissues using Amp HQ IHC and standard IHC methods. Results. The data demonstrate the feasibility of using high sensitivity TSA IHC assays to measure low abundant autophagy markers in FFPE xenograft tissue.

TAK-659 is a highly potent, reversible inhibitor of spleen tyrosine kinase (SYK) and fms related tyrosine kinase 3 (FLT3) that is currently being investigated in Phase I and II clinical trials. SYK is expressed ubiquitously in hematopoietic cells, and abnormal function of SYK has been implicated in several types of hematopoietic malignancies, including diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), and peripheral T-cell lymphoma (PTCL). Although there are considerable data supporting a role for SYK signaling in hematological cancers, data on the relevance of SYK in solid tumors are emerging. The literature suggests that SYK expression plays a role in the growth of breast and ovarian cancers, and the pancreatic adenocarcinoma microenvironment has been shown to be B-cell rich, giving us a basis to explore the activity of TAK-659 in these indications. Furthermore, ovarian cancer patients with recurrent disease have been shown to have an increase in pSYK at the time of relapse versus at diagnosis, suggesting a role for SYK in chemoresistance. The ability of TAK-659 to inhibit ex vivo colony formation of cells with the ability to grow anchorage independently in semi-solid medium was examined in 31 different human tumor xenografts. TAK-659 showed concentration-dependent inhibition of tumor colony growth in 5/31 tumor models tested, including gastric, breast, and ovarian cancers. Pre-clinically, TAK-659 has exhibited significant antitumor activity in a number of mouse xenograft models of solid tumors. We demonstrate here that daily oral administration of TAK-659 at 60mg/kg over 21 days leads to significant antitumor activity in primary human tumor models of gastric, pancreatic, triple negative breast (TNBC), and ovarian cancers, all of which express pSYK. Significant anti-tumor activity was also observed in the HCC70 TNBC xenograft model which does not express total SYK, demonstrating that TAK-659 may have anti-tumor effects beyond the inhibition of pSYK signaling. A decrease in myeloid derived suppressor cells was seen following TAK-659 treatment in this xenograft model, suggesting an immunomodulatory response may be driving the antitumor activity. Taken together, these data along with published information support the rationale for the investigation of TAK-659 in solid tumor cancer indications. Citation Format: Jessica J. Sappal, Matthew Theisen, Zhongmin Xiang, Stephen Tirrell, Rudy Christmas, Jie Yu, Mengkun Zhang, Karuppiah Kannan. TAK-659, a SYK kinase inhibitor, demonstrates preclinical antitumor activity in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3844.

Estrogen and progestins are essential for mammary growth and differentiation but also enhance the activity of the p53 tumor suppressor protein in the mammary epithelium. However, the pathways by which these hormones regulate p53 activity are unknown. Microarrays were used to profile the transcriptional changes within the mammary gland after administration of either vehicle, 17-estradiol (E), or progesterone (P) individually and combined (EP). Treatment with EP yielded 1182 unique genes that were differentially expressed compared to the vehicle-treated group. Although 30% of genes were responsive to either E or P individually, combined treatment with both EP had a synergistic effect accounting for 60% of the differentially regulated genes. Analysis of protein-proteininteractionsidentifiedp53,RelA,Snw1,andIgfalsascommon targets of genes regulated by EP. RelA and p53 form hubs within a network connected by genes that are regulated by EP and that may coordinate the competing functions of RelA and p53 in proliferation and survival of cells. Induction of early growth response 1 (Egr1) and Stratifin (Sfn) (also known as 14–3-3) by EP was confirmed by reverse transcription-quantitative PCR and shown to be p53 independent. In luciferase reporter assays, Egr1 was shown to enhance transcriptional activation by p53 and inhibit nuclear factor B activity. These results identify a gene expression network that provides redundant activation of RelA to support proliferation as well as sensitize p53 to ensure proper surveillance and integration of their competing functions through factors such as Egr1, which both enhance p53 and inhibit RelA. (Endocrinology 149: 4809–4820, 2008)

Purpose: Evaluate the safety, pharmacokinetic profile, pharmacodynamic effects, and antitumor activity of the first-in-class investigational NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924) in patients with relapsed/refractory lymphoma or multiple myeloma. Experimental Design: Patients with relapsed/refractory myeloma (n = 17) or lymphoma (n = 27) received intravenous pevonedistat 25 to 147 mg/m2 on days 1, 2, 8, 9 (schedule A; n = 27) or 100 to 261 mg/m2 on days 1, 4, 8, 11 (schedule B; n = 17) of 21-day cycles. Results: Maximum tolerated doses were 110 mg/m2 (schedule A) and 196 mg/m2 (schedule B). Dose-limiting toxicities included febrile neutropenia, transaminase elevations, muscle cramps (schedule A), and thrombocytopenia (schedule B). Common adverse events included fatigue and nausea. Common grade ≥3 events were anemia (19%; schedule A), and neutropenia and pneumonia (12%; schedule B). Clinically significant myelosuppression was uncommon. There were no treatment-related deaths. Pevonedistat pharmacokinetics exhibited a biphasic disposition phase and approximate dose-proportional increases in systemic exposure. Consistent with the short mean elimination half-life of approximately 8.5 hours, little-to-no drug accumulation in plasma was seen after multiple dosing. Pharmacodynamic evidence of NAE inhibition included increased skin levels of CDT-1 and NRF-2 (substrates of NAE-dependent ubiquitin ligases), and increased NRF-2-regulated gene transcript levels in whole blood. Pevonedistat–NEDD8 adduct was detected in bone marrow aspirates, indicating pevonedistat target engagement in the bone marrow compartment. Three lymphoma patients had partial responses; 30 patients achieved stable disease. Conclusions: Pevonedistat demonstrated anticipated pharmacodynamic effects in the clinical setting, a tolerable safety profile, and some preliminary evidence that may be suggestive of the potential for activity in relapsed/refractory lymphoma. Clin Cancer Res; 22(1); 34–43. ©2015 AACR.

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.

We examined how well differentially expressed genes and multigene outcome classifiers retain their class-discriminating values when tested on data generated by different transcriptional profiling platforms. RNA from 33 stage I-III breast cancers was hybridized to both Affymetrix GeneChip and Millennium Pharmaceuticals cDNA arrays. Only 30% of all corresponding gene expression measurements on the two platforms had Pearson correlation coefficient ror= 0.7 when UniGene was used to match probes. There was substantial variation in correlation between different Affymetrix probe sets matched to the same cDNA probe. When cDNA and Affymetrix probes were matched by basic local alignment tool (BLAST) sequence identity, the correlation increased substantially. We identified 182 genes in the Affymetrix and 45 in the cDNA data (including 17 common genes) that accurately separated 91% of cases in supervised hierarchical clustering in each data set. Cross-platform testing of these informative genes resulted in lower clustering accuracy of 45 and 79%, respectively. Several sets of accurate five-gene classifiers were developed on each platform using linear discriminant analysis. The best 100 classifiers showed average misclassification error rate of 2% on the original data that rose to 19.5% when tested on data from the other platform. Random five-gene classifiers showed misclassification error rate of 33%. We conclude that multigene predictors optimized for one platform lose accuracy when applied to data from another platform due to missing genes and sequence differences in probes that result in differing measurements for the same gene.

TAK-931 is a small molecule inhibitor of the cell division cycle 7 (CDC7) kinase. As a serine/threonine kinase that contributes to DNA replication and the DNA damage response, CDC7 is hypothesized to be a promising cancer drug target. CDC7 inhibition with TAK-931 has demonstrated antiproliferative activity with cancer cell lines and tumor growth inhibition (TGI) in murine ectopic xenograft models. Herein, the analysis of multiple models to characterize pharmacokinetic (PK) and pharmacodynamic (PD) relationships with xenograft TGI is described. TAK-931 treatment-induced TGI was dose schedule-independent and could be described using plasma drug concentrations or tumor PD inhibition. However, the efficacious doses were at least 10-fold higher for the PK-TGI relationship than for the PD-TGI relationship. This discrepancy was used to select a dynamic PK-PD-TGI modeling approach to project the minimal efficacious dose (MED) and minimal biological active dose (MBAD) for TAK-931 due to the large differences in time-concentration profiles predicted for humans versus mice. The Phase I human trial is on-going and will be used to verify the dynamic PK-PD-driven modeling approach for the CDC7 inhibitor. Citation Format: Charles Locuson, Mayank Patel, Akihiro Ohashi, Kenichi Iwai, Tadahiro Nambu, Toshiyuki Takeuchi, Akifumi Kogame, Douglas Bowman, Stephen Tirrell, Huifeng Niu, Cindy Xia. Translational pharmacokinetic-pharmacodynamic xenograft model for TAK-931, a small molecule cell division cycle 7 (CDC7) kinase inhibitor [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 5041. doi:10.1158/1538-7445.AM2017-5041

Immunohistochemistry-based biomarkers are commonly used to understand target inhibition in key cancer pathways in preclinical models and clinical studies. Automated slide-scanning and advanced high-throughput image analysis software technologies have evolved into a routine methodology for quantitative analysis of immunohistochemistry-based biomarkers. Alongside the traditional pathology H-score based on physical slides, the pathology world is welcoming digital pathology and advanced quantitative image analysis, which have enabled tissue- and cellular-level analysis. An automated workflow was implemented that includes automated staining, slide-scanning, and image analysis methodologies to explore biomarkers involved in 2 cancer targets: Aurora A and NEDD8-activating enzyme (NAE). The 2 workflows highlight the evolution of our immunohistochemistry laboratory and the different needs and requirements of each biological assay. Skin biopsies obtained from MLN8237 (Aurora A inhibitor) phase 1 clinical trials were evaluated for mitotic and apoptotic index, while mitotic index and defects in chromosome alignment and spindles were assessed in tumor biopsies to demonstrate Aurora A inhibition. Additionally, in both preclinical xenograft models and an acute myeloid leukemia phase 1 trial of the NAE inhibitor MLN4924, development of a novel image algorithm enabled measurement of downstream pathway modulation upon NAE inhibition. In the highlighted studies, developing a biomarker strategy based on automated image analysis solutions enabled project teams to confirm target and pathway inhibition and understand downstream outcomes of target inhibition with increased throughput and quantitative accuracy. These case studies demonstrate a strategy that combines a pathologist’s expertise with automated image analysis to support oncology drug discovery and development programs.

Background: The ubiquitin-proteasome system (UPS) has been validated as a target in multiple myeloma (MM) through the success of proteasome inhibitors such as bortezomib, but drug resistance is an emerging challenge. Targeting some of the upstream components of the UPS, such as the E1 ubiquitin activating enzyme (UAE), could therefore be a promising alternative. TAK-243 (MLN7243) specifically blocks the ubiquitin conjugation cascade through the formation of a TAK-243-ubiquitin adduct, thereby inhibiting the UAE. Our aim was to explore the effectiveness of TAK-243 against pre-clinical myeloma models, and to understand some its mechanisms of action. Methods: We performed pre-clinical studies in myeloma cell lines and mouse models using TAK-243. Downstream effects were evaluated using viability, apoptosis assays, western blotting, gene expression profiling (GEP), and Reverse Phase Protein Array (RPPA) techniques. Results: MM1.S and MOLP-8 TP53 wild-type cell lines were sensitive to TAK-243, with median inhibitory concentrations (IC50) of 25 nM at 24 hours based on viability assays. In otherwise isogenic cell lines in which TP53 was suppressed using genome editing techniques, the IC50 was ~40 nM, but higher TAK-243 concentrations of 100 nM overcame resistance due to TP53 inactivation. Similarly, TAK-243 was able to overcome resistance to both conventional (dexamethasone) and novel (bortezomib, lenalidomide) drugs in paired sensitive and resistant cell line models. After treatment with TAK-243, Annexin V and TO-PRO3 staining determined that viable MM1.S cells were induced into early or late apoptosis. This was accompanied by a significant increase in cleaved caspase-3, -8, and -9 as detected by flow cytometry, and in cleaved caspase-7 detected by RPPA and western blot. Exposure to TAK-243 reduced the cellular content of ubiquitin-protein conjugates, and did not enhance expression levels of a fusion protein degraded by the proteasome in a ubiquitin-independent manner, indicating the lack of direct proteasome inhibition. GEP analysis and RPPA detected enhanced expression of p53-pathway related proteins, including MDM2, TP53, and p21 in TAK-243 treated MM1.S cells. Several mRNAs and proteins in the ER stress pathway, including ATF6, ATF4, IRE1a and XBP1 were also elevated, as were many non-coding RNAs and DNA-damage related genes. Combination experiments in MM cell lines demonstrated synergy between TAK-243 and lenalidomide, pomalidomide, panobinostat, melphalan and doxorubicin. Finally, TAK-243 demonstrated in vivo antitumor activity against MM1.S and MOLP-8 xenograft models when dosed at 12.5 mg/kg IV twice-weekly for 2 weeks (tumor growth inhibition of 60% and 73%, respectively). Elevation of BiP, ATF4, XBP1s and cleaved-caspase 3 was detected within the first 24 hrs after dosing in the sensitive MM1.S xenografts. In contrast, RPMI 8226 cells, which showed a 2000 nM IC50 in cell culture, were also resistant to TAK-243 in vivo, with no tumor growth inhibition detected. Conclusions: TAK-243 is a UAE inhibitor that is active against myeloma cells in vitro and in xenograft models in vivo, overcomes conventional and novel drug resistance, and its action is associated with stimulation of the TP53 and ER stress pathways. Thus, it may deserve further evaluation as an anti-myeloma agent. Disclosures Berger: Takeda Pharmaceuticals: Employment. Hyer:Takeda Pharmaceuticals: Employment. Chattopadhyay:Takeda Pharmaceuticals: Employment. Syed:Takeda Pharmaceuticals: Employment. Shi:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. Shinde:Takeda Pharmaceuticals: Employment. Kreshock:Takeda Pharmaceuticals: Employment. Tirrell:Takeda Pharmaceuticals: Employment. Menon:Takeda Pharmaceuticals: Employment. Orlowski:Takeda Pharmaceuticals: Research Funding.

The ubiquitin-proteasome system (UPS) represents the primary mechanism by which cells degrade proteins. Proteasome inhibition results in accumulation of proteasome substrates, leading to cell cycle disruption, endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and, ultimately, the activation of apoptotic pathways and cell death. Ixazomib (NINLARO) is the first FDA-approved oral proteasome inhibitor, indicated in combination with lenalidomide and dexamethasone for treatment of patients with multiple myeloma who have received at least one prior therapy. Ixazomib has been evaluated as a single agent in relapsed/refractory lymphoma, but additional data is needed to identify combination agents for lymphoma trials. Brentuximab vedotin (ADCETRIS) is an anti-CD30 antibody drug conjugate (ADC) that is protease cleavable linker with monomethyl auristatin E (MMAE), a highly potent antimicrotubule agent. Brentuximab vedotin binds specifically to CD30, a cell surface marker expressed at high levels on Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) tumor cells and induces cell death by direct cytotoxicity and also antibody-dependent cellular phagocytosis (ADCP). Brentuximab vedotin is approved in patients with relapsed/refractory HL, relapsed/refractory ALCL, and for consolidation in in the post autologous stem cell transplant (ASCT) setting of HL. Preclinical evidence suggests that resistance to anti-CD30 therapy can be mediated via activation of the NFkB pathway, which can be reversed by proteasome inhibitor treatment, suggesting a rationale for the combination of ixazomib and brentuximab vedotin. In the current study we evaluated this combination in three CD30+ ALCL xenograft models, SR-786, Karpas-299 and SUDHL-2 grown in immunocompromised mice. In SR-786 and Karpas-299, the combination treatment using dose levels of ixazomib and brentuximab vedotin with minimal single-agent activity resulted in synergistic effect with complete tumor regression. In most animals, the tumor did not regrow as long as 60 days after the last dose of combination drug treatment. Unlike in SR-786 and Karpas-299, no combination benefit was observed in the SUDHL-2 xenograft model. Pharmacodynamic studies are ongoing to understand the mechanism of synergy. Our data suggest that combination of ixazomib and brentuximab vedotin warrants clinical evaluation in CD30+ lymphoma patients. Disclosures Chattopadhyay: Takeda Pharmaceuticals: Employment. Syed:Takeda Pharmaceuticals: Employment. Zhang:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. He McDougall:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals: Employment. Tirrell:Takeda Pharmaceuticals: Employment. Berger:Takeda Pharmaceuticals: Employment. van de Velde:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Tremblay:Takeda Pharmaceuticals: Employment. Huebner:Takeda Pharmaceuticals International Co.: Employment, Equity Ownership.

e14091Background: TAK-659 is a reversible, dual inhibitor of SYK and FLT-3 being developed for oncology indications including Acute Myelogenous Leukemia (AML), a disease driven by SYK and/or FLT-3-mediated signaling. Loss of SYK in AML cell lines has been shown to induce myeloid differentiation. FLT-3 is a Class III receptor tyrosine kinase that is normally expressed only in hematopoietic stem and progenitor cells. However, its expression has been found in the blasts of a majority of patients with AML. Activating mutations of FLT-3, including internal tandem duplication (ITDs) within the juxtamembrane region of FLT-3 and point mutations in the FLT-3 activation loop, are observed in approximately 30% of AML patients. These FLT-3 mutations, associated with early relapse and poor survival, represent a critical prognostic factor for AML. Methods: TAK-659 is an investigational inhibitor of SYK and FLT-3 that is currently being evaluated in a Phase 1b/2 clinical trial of AML patients (NCT02323113). TAK-659 inhi...

Immunohistochemistry (IHC) is a powerful tool for studying pharmacodynamic biomarkers in tissue samples. However, IHC using standard methodologies to monitor autophagy has met with limited success due to the technical difficulties involved in detecting transient and low abundance autophagy markers. To address this issue, we have applied a highly sensitive IHC method using a tyramide-based signal amplification system to detect autophagy pathway proteins in paraffin sections of human tumor cell line and xenograft tissues. We were able to detect basal levels of punctate staining LC3B and NBR1 on both HCT116 and Calu-6 xenograft tumors using the tyramide-based signal amplification method whereas standard IHC showed little to no specific staining. Consistent with in vitro observations, lipidated LC3B, as detected by puncta formation, was elevated following autophagy induction using mTOR inhibitor treatment, and decreased following ATG7 knockout or knock down in both cell lines and xenograft tumors. As a result of autophagy pathway interruption following ATG7 inhibition, accumulation of the adapter proteins NBR1 and p62 was also detected. Our data demonstrate the feasibility of using high sensitivity IHC assays to reliably measure low abundant autophagy markers in xenograft tissues and may aid the exploration of autophagy regulation in cancer development. Citation Format: Yu Yang, Natalie Roy D'Amore, Jouhara Chouitar, Jie Yu, Kristine Burke, Zhongmin Xiang, Lunyin Yu, Kerri Lasky, James Brownell, Chris Simpson, Evan Luongo, Doug Bowman, Stephen Tirrell, Helen He. A sensitive immunohistochemistry method for in vivo autophagy biomarker detection and pharmacodynamic studies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2908. doi:10.1158/1538-7445.AM2015-2908

Background: Immunohistochemistry provides visual context to protein modulation and is relied upon to elucidate mechanism of action, marker translocation and pharmacodynamics (PD) in drug discovery. Markers of the PI3K-MTOR pathway have been monitored in this way to aid in the clinical development of various inhibitors targeting this pathway. Here, assays were developed using preclinical xenograft PD studies to explore the feasibility of automated staining of pAKT 473, pS6, p4EBP1 and pRb. In preparation for clinical assessment of these markers in human skin samples; these markers were also examined in normal mouse skin. p4EBP1 was selected to examine the reproducibility of these automated assays and to explore the correlation between pathway inhibition in PD xenograft tissues and murine skin tissues. The automated staining technique was coupled with whole slide scanning and digital image analysis for quantitative comparison. Methods: Assays were chosen for evaluation based on known biology. FFPE PTEN-null breast cancer primary human tumor xenografts with high, medium and low biomarker expression, with companion skin samples, were selected for feasibility and reproducibility testing. Whole slide scanning and reproducibility analysis was executed using Aperio Scanscope XT and Spectrum software. Statistical analysis methods of mixed effect linear models and two sample t-tests were applied. Xenograft and skin samples from NCI-H1048 tumor bearing Nude mice treated with MTOR (MLN0128)and PI3Kα (MLN1117) investigational inhibitors were immunohistochemically stained for p4EBP1. Definiens object-based imaging software was used to isolate and analyze viable tumor area and exclude necrotic and stromal components in xenografts. A customized Definiens® algorithm was used on skin to identify the epithelial layer for analysis. Results: After evaluation, baseline biomarker expression in the xenograft and skin samples determined marker selection for future steps. Baseline p4EBP1 levels were high in both xenograft and skin. The reproducibility testing yielded consistent results in both xenograft and skin, validating autostaining use for PD studies. Phosphomarker p4EBP1 expression was inhibited after MLN0128 administration, with increased reduction after combination with MLN1117. Development of a novel analysis solution using Definiens software for skin samples permitted the area of interest to be restricted to the epithelial layer and excluded other epidermal features with similar morphometric characteristics. The p4EBP1 modulation was concordant between skin and tumor though more pronounced in skin, indicating that skin is a suitable surrogate tissue for determining modulation of this PI3K-MTOR pathway marker. Conclusions: Autostaining platforms provide reproducible results and stain consistently for PI3K-MTOR phosphomarkers on both xenografts and mouse skin samples. Utilizing p4EBP1, target effect was observed after MTOR and PI3Kα administration. This development and evaluation of the phosphobiomarker using automated staining technology and advanced image analysis supports preclinical PD assessment and combination clinical trials as well as influenced timepoint selection for clinical assessment. Citation Format: Anna Kreshock, Natalia Iartchouk, Yueying Cao, Jeffrey Szwaya, Feng Gao, Christopher Simpson, Evan Luongo, Kristine Burke, Esha Gangolli, Keisuke Kuida, Rachael Brake, Stephen Tirrell, Vaishali Shinde. Automated immunohistochemistry of phosphobiomarkers: Case study of MTOR (MLN0128) and PI3Kα (MLN1117) investigational inhibitors, single agent and in combination, on xenografts and mouse skin. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A41.

Purpose: The goals of this analysis were to (a) determine concordance of gene expression results from replicate experiments, (b) examine prediction agreement of multigene predictors on replicate data, and (c) assess the robustness of prediction results in the face of noise. Patients and Methods: Affymetrix U133A gene chips were used for gene expression profiling of 97 fine-needle aspiration biopsies from breast cancer. Thirty-five cases were profiled in replicates: 17 within the same laboratory, 11 in two different laboratories, and 15 to assess manual and robotic labeling. We used data from 62 cases to develop 111 distinct pharmacogenomic predictors of response to therapy. These were tested on cases profiled in duplicates to determine prediction agreement and accuracy. To evaluate the robustness of the pharmacogenomic predictors, we also introduced random noise into the informative genes in one half of the replicates. Results: The average concordance correlation coefficient was 0.978 (range, 0.96-0.99) for intralaboratory replicates, 0.962 (range, 0.94-0.98) for between-laboratory replicates, and 0.971 (range, 0.93-0.99) for manual versus robotic labeling. The mean % prediction agreement on replicate data was 97% (95% CI, 0.96-0.98; SD, 0.006), 92% (95% CI, 0.90-0.93; SD, 0.009), and 94% (95% CI, 0.92-0.95; SD, 0.008) for support vector machines, diagonal linear discriminant analysis, and k-nearest neighbor prediction methods, respectively. Mean accuracy in the test set was 77% (95% CI, 0.74-0.79; SD, 0.014), 66% (95% CI, 0.63-0.73; SD, 0.015), and 64% (95% CI, 0.60-0.67; SD, 0.016), respectively. Conclusion: Gene expression results obtained with Affymetrix U133A chips are highly reproducible within and across two high-volume laboratories. Pharmacogenomic predictions yielded >90% agreement in replicate data.

3077 Background: MLN9708 is a potent investigational proteasome inhibitor, which upon intravenous (IV) administration immediately hydrolyzes to the active form MLN2238. MLN9708 is currently being evaluated in a phase 1 trial in solid tumors (NCT00830869). This trial has a dose-escalation arm and five expansion cohorts: non-small cell lung cancer (NSCLC), soft tissue sarcoma, head and neck cancer, prostate cancer, and a tumor biopsy cohort of mixed histology. The purpose of the tumor biopsy cohort was to obtain pre- and post-dose biopsies to determine drug distribution and target engagement in post-dose tumor samples. The latter was measured by the increase in levels of ATF-3, a marker of unfolded protein response/endoplasmic reticulum stress, which is upregulated in response to proteasome inhibition. Methods: The tumor biopsy cohort included 20 patients dosed at the maximum tolerated dose who consented to core needle biopsies during screening and after either the first or second dose of MLN9708 (IV 1.76 mg/m2; 4–20 hours post-dose). Tumor biopsies were individually weighed, homogenized, and analyzed for the presence of MLN2238 using a quantified LC/MS/MS methodology. ATF-3 levels in tumors were determined by an immunohistochemical assay (IHC) on six sections for each tumor biopsy. Tumor area was identified using Aperio Genie, a machine learning program for pattern recognition, and the percentage of ATF-3 positive area in the tumor was measured. Results: Biopsies from 20 patients were collected for assessment of drug distribution and target engagement. Ten patients with paired pre- and post-dose biopsies of sufficient size were considered evaluable for PK analysis; MLN2238 was present in all 10 (100%) post-dose biopsies analyzed. Tumor pairs from 7 patients passed quality control by H&E staining for tumor content and were evaluable for ATF-3 IHC. Six of 7 paired samples (86%) showed a statistically significant (p

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.