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PURPOSE Some gastric cancers harbor MET gene amplifications that can be targeted by selective MET inhibitors to achieve tumor responses, but resistance eventually develops. Savolitinib, a selective MET inhibitor, is beneficial for treating patients with MET-driven gastric cancer. Understanding the resistance mechanisms is important for optimizing postfailure treatment options. PATIENTS AND METHODS Here, we identified the mechanisms of acquired resistance to savolitinib in 3 patients with gastric cancer and MET-amplified tumors who showed a clinical response and then cancer progression. Longitudinal circulating tumor DNA (ctDNA) is useful for monitoring resistance during treatment and progression when rebiopsy cannot be performed. RESULTS Using a next-generation sequencing 100-gene panel, we identified the target mechanisms of resistance MET D1228V/N/H and Y1230C mutations or high copy number MET gene amplifications that emerge when resistance to savolitinib develops in patients with MET-amplified gastric cancer. CONCLUSION We demonstrated the utility of ctDNA in gastric cancer and confirmed this approach using baseline tumor tissue or rebiopsy.

PURPOSE Some gastric cancers harbor MET gene amplifications that can be targeted by selective MET inhibitors to achieve tumor responses, but resistance eventually develops. Savolitinib, a selective MET inhibitor, is beneficial for treating patients with MET-driven gastric cancer. Understanding the resistance mechanisms is important for optimizing postfailure treatment options. PATIENTS AND METHODS Here, we identified the mechanisms of acquired resistance to savolitinib in 3 patients with gastric cancer and MET-amplified tumors who showed a clinical response and then cancer progression. Longitudinal circulating tumor DNA (ctDNA) is useful for monitoring resistance during treatment and progression when rebiopsy cannot be performed. RESULTS Using a next-generation sequencing 100-gene panel, we identified the target mechanisms of resistance MET D1228V/N/H and Y1230C mutations or high copy number MET gene amplifications that emerge when resistance to savolitinib develops in patients with MET-amplified gastric cancer. CONCLUSION We demonstrated the utility of ctDNA in gastric cancer and confirmed this approach using baseline tumor tissue or rebiopsy.

Background: Rucaparib, a poly(ADP-ribose) polymerase inhibitor (PARPi), is approved in the US as monotherapy for patients (pts) with BRCA1/2-mutated mCRPC after androgen receptor (AR)-directed therapy and a taxane. Synthetic lethality has been observed in studies combining AR-directed therapy (eg, enzalutamide) and a PARPi, independent of DNA-damage repair (DDR) gene defects. The RAMP study (NCT04179396) is investigating the safety and potential drug-drug interactions (DDIs) between rucaparib and enzalutamide in unselected mCRPC pts. In this exploratory analysis, we evaluated the association between treatment response and gene alterations of known clinical significance in mCRPC. Methods: Enrolled pts were previously treated with 0-2 lines of AR-directed therapy and ≤2 lines of chemotherapy for mCRPC. A 1-week run-in of rucaparib monotherapy (600 mg BID) was followed by rucaparib (600 mg BID) + enzalutamide (160 mg QD). Primary endpoints were pharmacokinetics (PK) and safety, including dose-limiting toxicities (DLTs). Change in prostate-specific antigen (PSA) levels from baseline was a secondary endpoint. As an exploratory objective, plasma and/or tissue samples were profiled for genomic alterations using a Foundation Medicine, Inc., assay to evaluate the relationship between genomics and clinical outcome. Results: As of Oct 1, 2020, 8 pts had received treatment; 7 were evaluable for PK and 6 for DLTs. The median age was 68.5 years and 7/8 (88%) pts had received 1 or 2 prior AR-directed therapies. The PK and overall safety profiles of rucaparib + enzalutamide were consistent with each as a single agent, and no DLTs were reported. Overall, 6/8 (75%) pts had a PSA decline, with 4/8 (50%) pts meeting criteria for a confirmed PSA response (≥50% decrease from baseline). Genomic data were available for 6/8 (75%) pts, all of whom had received prior AR-directed therapy. Of the 4/6 (66%) pts who had one or more somatic AR alterations, 3 had a PSA decline (best PSA change of -99%, -22%, and -17% from baseline) with the combination treatment. One pt had a DDR gene mutation, a subclonal ( Conclusions: A combination of enzalutamide and rucaparib had an acceptable safety profile and no clinically significant DDIs. In this small pt set, pts previously treated with AR-directed therapies showed responses to the combination even in the presence of AR alterations and in the absence of DDR gene alterations. These genomic and early efficacy data support further studies of rucaparib and enzalutamide at the recommended combination dose (rucaparib 600 mg BID + enzalutamide 160 mg QD), including in the phase 3 CASPAR study in biomarker-unselected mCRPC pts (NCT04455750). Citation Format: Arpit Rao, Charles J. Ryan, David Morris, Vasileios Assikis, Gautam Jha, Adriel-John Ablaza, Jenn Habeck, Andrea Loehr, Jim Xiao, Esha A. Gangolli. Genomic characteristics and response to rucaparib and enzalutamide in the phase 1b RAMP study of metastatic castration-resistant prostate cancer (mCRPC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 445.

2562 Background: Tumors produce high levels of extracellular adenosine which suppress anti-tumor immune responses. Blocking A2A receptors, predominantly expressed on tumor-infiltrating immune cells, can reverse the immunosuppressive effect of adenosine. Inupadenant is a non brain-penetrant, potent and highly selective small molecule antagonist of the A2A receptor that remains active even at the high adenosine concentrations found in tumors. Methods: This is the phase I portion of an ongoing first-in-human, clinical trial (NCT02740985) to evaluate safety/tolerability, pharmacokinetic, pharmacodynamic and anti-tumor activity of inupadenant in adult patients with solid tumors who have exhausted standard treatment options. In addition, tumor biomarkers, including adenosine-pathway markers by immunohistochemistry (IHC), are being evaluated. We present updated results of the dose escalation, new results from the monotherapy expansion and new analysis of tumor biomarkers. Results: Overall, 42 patients (21 patients in the dose escalation and an additional 21 patients in a monotherapy expansion) with a median of 3 prior regimens were treated as of the data cut off (DCO, 30Nov20).The dose levels investigated, along with the most frequent (>20%) treatment-emergent adverse events (TEAEs) across all dose levels are presented in the table. 7 AEs led to discontinuation; 2 (atrial fibrillation and myocardial infarction) were considered possibly study drug-related by the investigator. No dose reductions were required. Two partial responses (PRs) were reported: melanoma (NRAS-mutant; received prior immunotherapy), and prostate cancer (received antiandrogen and chemotherapy). At the DCO, both PRs were ongoing with a duration of response >230 days. 12 patients had stable disease (SD) as best response and SD >6 months was observed in 3 patients. Response and stable disease were associated with a higher number of cells expressing the A2A receptor within the tumor at baseline, as measured by IHC. Conclusions: Inupadenant monotherapy was generally well-tolerated as of the DCO at a dose of 80 mg twice daily with initial evidence of clinical benefit, including 2 durable PRs in patients who have exhausted standard treatment options. Analysis of pre-treatment tumor biopsies has identified the A2A receptor as a biomarker which may be associated with clinical benefit. Clinical trial information: NCT03873883. [Table: see text]

79 Background: Rucaparib, a poly(ADP-ribose) polymerase inhibitor (PARPi), is approved in the US as monotherapy for BRCA1/2 mutated mCRPC that has been treated with androgen receptor (AR)-directed therapy and a taxane. In previous studies, synthetic lethality was observed with a combination of AR-directed therapy (eg, enzalutamide) and a PARPi, independent of DNA-damage repair gene defects. The potential for drug-drug interaction between rucaparib and enzalutamide was recognized as enzalutamide is a strong clinical inducer of CYP3A4, a mediator of the formation of rucaparib metabolite M324. RAMP (NCT04179396) is investigating the combination of rucaparib and enzalutamide in unselected pts with mCRPC to assess PK and safety and inform subsequent studies. Methods: Eligible pts had 0–2 lines of AR-directed therapy and ≤2 lines of chemotherapy for mCRPC. Prior PARPi treatment was not allowed. A 1-week run-in of rucaparib monotherapy (600 mg BID) was followed by rucaparib (600 mg BID) + enzalutamide (160 mg QD) in continuous 28-day cycles. Trough PK was evaluated for rucaparib and enzalutamide and their metabolites (M324 and N-desmethyl enzalutamide). Dose-limiting toxicities (DLTs) were assessed during the first 2 cycles. Primary endpoints were PK and safety for the combination. Secondary endpoints were change from baseline in prostate-specific antigen (PSA) levels and objective response rate (per modified RECIST/PCWG3). Results: As of Oct 1, 2020, 8 pts had received treatment; 7 were evaluable for PK and 6 for DLTs. The median age was 68.5 years, and 6/8 (75%) pts had ≥2 prior mCRPC therapies. Rucaparib and M324 reached apparent steady-state PK at the end of the run-in period. Concomitant treatment with enzalutamide had no significant effect on the PK of either rucaparib or M324. The PK of enzalutamide and N-desmethyl enzalutamide in the combination were also consistent with monotherapy enzalutamide PK data. No DLTs were reported. The most common grade ≥3 TEAEs among all 8 pts were anemia (n = 5) and fatigue (n = 4). Seven of 8 (87.5%) pts required a dose modification (treatment interruption or dose reduction). Four of 8 (50%) pts had a confirmed PSA response (reduction ≥50% from baseline); 1/1 (100%) pt with measurable disease achieved a confirmed complete radiographic response. At the time of data cutoff, 1/8 (12.5%) pts was on study for > 6 cycles. Conclusions: A combination of enzalutamide and rucaparib was not associated with a clinically significant effect on PK profiles of either drug. No DLTs were observed. The overall safety profile of rucaparib 600 mg BID + enzalutamide 160 mg QD was consistent with that observed in monotherapy. These PK, safety, and early efficacy data support further studies of rucaparib and enzalutamide at the recommended combination dose, including in the phase 3 CASPAR study in pts with mCRPC (NCT04455750). Clinical trial information: NCT04179396.

Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKI's physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKIalpha, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RIalpha subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling.

The human neuroblastoma cell line SK-N-SH has been used as a model system to study the interactions of the human estrogen receptor (hER) with neurotransmitters. We have successfully transfected these cells using an adenoviral delivery system and have reconstituted ligand-dependent responses to estradiol and ligand-independent responses to a series of dopamine D1 receptor agonists. The full agonist for the D1 receptor, SKF 82958, shows a robust activation of hER, comparable to that induced by estradiol. This activation is blocked by the protein kinase A inhibitor H-89, is mimicked by forskolin, and is therefore thought to be mediated in part through the cAMP/protein kinase A pathway. We have examined deletion mutants of hER for activation by SKF 82958 and find that both its transactivation domains, AF-1 and AF-2, must cooperate to impact the full response to the agonist. Significantly, an agonist of the muscarinic acetylcholine receptor, carbachol, though not active by itself, synergistically activates hER in conjunction with suboptimal doses of SKF 82958. This is the first reported instance of two neurotransmitters synergizing to activate a member of the nuclear receptor superfamily, and might predict a role for multiple neural inputs modulating the effects of these receptors in the central nervous system.

The protein kinase inhibitor (PKI) family includes three genes encoding small, heat-stable inhibitors of the cyclic AMP-dependent kinase PKA. Each PKI isoform contains a PKA inhibitory domain and a nuclear export domain, enabling PKI to both inhibit PKA and remove it from the nucleus. The PKIbeta isoform, also known as testis PKI, is highly expressed in germ cells of the testis and is found at more modest levels in other tissues. In order to investigate its physiological role, we have generated PKIbeta knockout mice by gene targeting. These mice exhibit a partial loss of PKI activity in testis but remain fertile with normal testis development and function. PKIbeta knockout females also reproduce normally. The PKIbeta mutants were crossed with our previously derived PKIalpha mutants to obtain double-knockout mice. Remarkably, these mice are also viable and fertile with no obvious physiological defects in either males or females.

Combination therapy in oncology drug development has the potential to overcome resistance against single agent-based therapy. In many cancers, the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways confer major cell growth and survival mechanisms. Therefore, a greater treatment effect may be achieved by inhibiting both pathways simultaneously through drug combination. In this study, the enhanced anti-proliferative activity of investigational drug MLN0128 (mTORC1/2 investigational agent) when combined with TAK733 (MEK1/2 investigational agent) was assessed using an in-vitro cell viability assay on 240 cancer cell lines. Cells were administered with MLN0128 and TAK-733 in DMSO in ten 2-fold serial dilutions and cell viability was measured using Promega's CellTiter-GLO® assay after a 72 hour incubation period. The data points were fit to a 9-parameter model to obtain a response surface and the effect of the combined administration for each cell line was measured using a non-linear blending score estimated from the response surface. Statistical analysis was conducted to determine if the estimated combination effect was associated with known mutated oncogenes, tumor suppressor genes, or cancer histology reports of the 240 cell lines. Interestingly, the synergy of combined agents appeared to be mainly driven by TAK733, as cell lines sensitive to TAK733 as a single agent demonstrated beneficial combination effect with the addition of MLN0128. Furthermore, cell lines with mutations in KRAS, NRAS, and BRAF showed greater synergy scores of the combination. Likewise, cell lines of colon, pancreatic, and skin origins were more sensitive to the combination. Of particular interest are cell lines with mutated KRAS/NRAS or colon origins since they were generally resistant to single agent MLN0128, but became sensitized to the combination with TAK733. In conclusion, our study of drug combination assays on a large-scale in-vitro cell line panel revealed that mutant KRAS/NRAS or colorectal cancers may be sensitive to combined MLN0128 with TAK733. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B28. Citation Format: Hyunjin Shin, Derek Blair, Bin Li, Greg Hather, William L. Trepicchio, Jeffrey Ecsedy, Esha Gangolli. Identification of drug-response biomarkers for combined mTORC1/2 and MEK1/2 investigational agents using a large-scale cancer cell line screen. [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 B28.

Endometrial cancers are the most common gynecologic cancers in the US, and are a heterogeneous group of malignancies with distinct molecular characteristics. The phosphatidylinositol-3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway is frequently dysregulated, being activated in 70-80% of hormone-dependent endometrioid carcinomas (Type I) and 40% of non-endometrioid carcinomas (Type II). Loss of PTEN or activation of PIK3CA results in constitutive activation of AKT and up-regulation of mTOR. We evaluated MLN0128, an investigational catalytic mTORC1/2 inhibitor, in preclinical models of endometrial cancer to assess its activity as a single agent and in combination with paclitaxel across various dose schedules and sequences. In vitro MLN0128 showed antiproliferative activity with IC50 values of 5-100 nM across cell line models (n=9) with various genetic aberrations including complex combinations of PTEN loss and mutations in PIK3CA, KRAS and/or FGFR. This antiproliferative activity was associated with inhibition of pathway markers, including phosphorylated AKT, S6, and 4EBP1. In vivo MLN0128 demonstrated tumor growth inhibition (TGI) in a PIK3CA/KRAS model (HEC-1A) and a PTEN/FGFR model (AN3CA) with TGI values of 62% and 73%, respectively. Similar TGI values were achieved regardless of dosing schedule (once daily [QD], 3 days a week [QDx3D], or once weekly [QW]). TGI could be improved by using a combination approach. In the AN3CA model, the in vitro combination of MLN0128 and paclitaxel suppressed the PI3K pathway activation (phosphorylation of AKT, S6, and 4EBP1) that occurred when paclitaxel was administered alone; the combination also suppressed S6 phosphorylation at 24 hours. This finding correlated with an increase in the induction of cleaved PARP, a marker of apoptosis, and accumulation of cells in the sub-G1 phase of the cell cycle. The combination was also observed to add benefit in vivo, most prominently when tumor regrowth was assessed. In the HEC-1A model, TGI only increased by 10% with concomitant administration of MLN0128 and paclitaxel daily vs MLN0128 alone. However, when monitoring re-growth 18 days after administration, this difference in TGI (combination vs MLN0128 alone) increased to 44%. Furthermore pretreatment with paclitaxel before MLN0128 was also associated with significant enhancement of antitumor activity relative to the concomitant administration of both agents; this effect was observed with the QDx3D and QW MLN0128 schedules. Results from these preclinical studies support further investigation of MLN0128 in endometrial cancer, either as a single agent or in combination with current standards of care. Phase 1 studies are ongoing to investigate single-agent MLN0128 ([NCT01058707][1]) and MLN0128 plus paclitaxel ([NCT01351350][2]) in advanced solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B189. Citation Format: Karuppiah Kannan, Robyn Fabrey, Jodi Cooper, Jessica Huck, Esha Gangolli, Eric Westin, Rachael Brake. MLN0128, an investigational mTORC1/2 inhibitor, demonstrates potent antitumor activity alone and in combination with paclitaxel in preclinical models of endometrial cancer. [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 B189. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01058707&atom=%2Fmolcanther%2F12%2F11_Supplement%2FB189.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01351350&atom=%2Fmolcanther%2F12%2F11_Supplement%2FB189.atom

2547 Background: MLN2480 is an investigational pan-RAF kinase inhibitor. In vivo, MLN2480 showed antitumor activity in melanoma, colon, lung, and pancreatic cancer xenograft models. This first-in-human study aimed to evaluate the safety of MLN2480, determine the MTD/recommended phase 2 dose (RP2D), and evaluate pharmacokinetics (PK) and preliminary efficacy. Methods: Pts aged ≥18 yrs with advanced solid tumors who had failed/were not candidates for standard therapy received oral MLN2480 every other day (Q2D) in 22-d cycles, with dose escalation (3+3 design) based on DLTs in cycle 1. AEs were graded per NCI-CTCAE v4.03. Blood samples for plasma PK assessment were taken pre-dose and at multiple times post-dose, d 1 and 21, cycle 1. Results: 24 pts (10 male, median age 64.5 yrs [range 37–83]) have been treated at 20, 40, 80, 135, 200, and 280 mg (n=4, 3, 3, 3, 4, and 7), respectively. The most common tumors included colorectal cancer in 11 pts and non-small-cell lung cancer in 2 pts. Pts received a median of 2 (range 1–6) cycles. 2 pts treated at 280 mg had DLTs: grade 3 macular rash and grade 3 periorbital edema. 20 pts had drug-related AEs, including fatigue 46%, arthralgia 25%, maculopapular rash 21%, and myalgia 17%. 4 pts had drug-related grade ≥3 AEs, which included the 2 DLTs listed above, anemia, dyspnea, and fatigue. No keratocanthomas/ squamous cutaneous carcinomas have been seen to date. 4 pts discontinued due to AEs. There were 3 on-study deaths (1 treatment-related per investigator; dyspnea and respiratory failure). At 20–200 mg MLN2480 PK data (13 pts) exhibited rapid absorption (median Tmax 2 hr), low fluctuation at steady state (mean peak to trough ratio 2.1), and mean accumulation half-life of 67 hr. Overall mean accumulation was 2.6-fold following repeated Q2D dosing for 21 d. Steady-state (d 21) exposures increased in an approximately dose-proportional manner over 20–200 mg range. No pts had an objective response to date; no pts with BRAF mutation enrolled to date. Conclusions: In this first-in-human study (n=24), the safety profile of MLN2480 up to 200 mg Q2D was acceptable. Accrual continues at 200 mg to confirm the MTD. Melanoma expansion cohorts are planned at the RP2D using a Q2D 28-d cycle. Clinical trial information: NCT01425008.

2567 Background: MLN0128 (INK128) is an investigational oral, potent, and highly selective inhibitor of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) currently in clinical investigation. In the phase1 study INK128-001, MLN0128 was administered once daily (QD), once weekly (QW), QDx3D/week, and QDx5D/week, with respective MTDs of 6, 40, 16, and 10 mg. To guide selection of dose/schedule for further investigation, PD modulation in skin (pS6, p4EBP1, pNDRG1, pPRAS40) was put into context of clinical PK in INK128-001. A preclinical translational dynamic-PK efficacy model was used to describe the relationship and determine PK drivers of efficacy in tumor xenograft models. This model was implemented using human PK parameters to predict tumor volume-time curves, which was utilized to help determine the optimal MLN0128 dose/schedule. Methods: Phoenix NLME v1.1 was used for compartmental modeling of clinical and preclinical PK data, and modeling the preclinical PK-efficacy relationship of MLN0128. PD activity in skin was measured by immunohistochemistry, reported as H scores. Tumor growth curves were simulated using NONMEM v7.2; predicted tumor growth curves were plotted in S-Plus v8.1. Results: Clinical skin PD data suggests exposure dependent inhibition of pS6, and p4EBP1. A two compartment PK model adequately described the PK characteristics of MLN0128 [mean (%CV) ka: ~5.305 h-1 (114), k12: ~0.490 h-1(85), k21: ~0.67 h-1(69), V/F: ~180 L (44), Tlag: 0.317 h (73)]. Simulation of human tumor volume-time curves suggest efficacy is dependent on schedule and that MLN0128 administered in more frequent schedules (QD, QDx5D) provides stronger antitumor effect vs less frequent schedules (QW, QDx3D). Conclusions: The results indicate that per unit MLN0128 plasma exposure, QD and QDx5D may be optimal in comparison with QDx3D and QW dosing. However, these results will also need to be put into context with the overall safety profile and respective MTDs and RP2Ds for each schedule with their resultant achievable total cycle dose by schedule. Clinical trial information: NCT01058707.

MLN0128 is an investigational, orally bioavailable, potent and highly selective, ATP-competitive inhibitor of TORC1/2. A phase 1 study was initiated to evaluate the safety and maximum tolerated dose (MTD) of single-agent MLN0128 at different dose levels and schedules (QW, QD, 3-on/4-off and 5-on/2-off) in patients with advanced nonhematologic tumors. As multiple MTDs were achieved, we used exposure-efficacy modeling to interpret the achieved MTDs in the context of preclinical antitumor efficacy, thus supporting the selection of dosing schedule in the clinic. To this end, we conducted xenograft efficacy studies using the 786-0 renal cell carcinoma xenograft model (PTENmut,VHLdel) in athymic nude mice. MLN0128 was orally administered at a range of different doses under several different schedules (QD, bid QW, 3-on/4-off and 5-on/2-off). Single and repeat-dose PK data was also obtained for a range of different time-points and a linear two-compartment PK model was built. This PK model was used to simulate pharmacokinetic profiles corresponding to the arms of the efficacy studies, for the study duration (504 hours, or 21 days). The average MLN0128 concentrations (Cavg, 0-504) for each arm of the efficacy study were then compared against the tumor growth rate, and found to be broadly proportional, with the exception of the most infrequent (QW) dose schedule, for which efficacy was sub-exposure proportional. A dynamic tumor growth model was found to provide an accurate description of antitumor effect for all schedules, and to predict the schedule effect from the most infrequent schedule. A mathematical analysis demonstrated that the schedule effect occurs due to the saturability of the underlying PK/kill curve, with high infrequent doses resulting in large portions of the PK profile at concentrations where the marginal benefit of additional drug levels approaches zero. A sensitivity analysis conducted on this fitted model found that it was insensitive to tumor growth rate and tumor sensitivity to drug, but not to half-life. This dynamic model will be extended in a translational context, to account for the differences in half-life between mice and humans, as well as to account for the uncertainty in parameter estimation and the human PK variability. Our work thus provides a fundamental basis for the understanding of the PK/efficacy relationship for mTOR inhibitors, which, contrary to some reports in the literature, appears to be mostly AUC-proportional. Highly infrequent dosing was found to be sub-exposure proportional, and our work provides a rational explanation for this effect. Taken together, the approach of constructing a dynamic PK/efficacy model and eventually extending this model into a human setting by adjusting the PK half-life provides a rational basis for the preclinical-to-clinical translation of schedule effects. Citation Format: Mayankbhai Patel, Jocelyn Staunton, Jerome Mettetal, Santhosh Palani, Jeffrey Ecsedy, Chuang Lu, Elly Barry, Eric Westin, Mark Manfredi, Wej Chyi Shyu, Katayoun Jessen, Esha A. Gangolli, Arijit Chakravarty. Preclinical-to-clinical translation of optimal schedule for MLN0128, an investigational novel small-molecule selective mTOR1/2 inhibitor, based on exposure-efficacy modeling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3532. doi:10.1158/1538-7445.AM2013-3532

Background TAK-733 is an investigational, orally available, selective, non-ATP-competitive, allosteric inhibitor of MEK1/2 shown to have anti-tumor activity in multiple xenograft models. In this first-in-human ph 1 study (NCT00948467), we evaluated safety, pharmacokinetics (PK), pharmacodynamics (PD), maximum tolerated dose (MTD) and efficacy of TAK-733 in pts with advanced solid tumors. Methods Pts aged ≥18 y, with ECOG PS 0–2 and radiographically or clinically evaluable tumors were eligible. Pts received TAK-733 QD on d 1–21 in 28-d cycles; doses escalated in a modified 3 + 3 design based on dose-limiting toxicities (DLTs) in cycle 1 to determine MTD. Plasma and blood samples for PK and PD analysis were collected pre-dose on d 1, 8, 15 and 21 and post-dose d 1 and 21 in cycle 1. Results As of March 16, 2012, 44 pts median age 58 (range 24 - 75) and 50% male, received TAK-733 (dose mg, [n]: 0.2, [1]; 0.4, [1]; 0.8, [2]; 1.6, [2]; 3.2, [4]; 4.4, [4]; 6, [4]; 8.4, [9]; 11.8, [8]; 16, [9]). 2 pts had DLTs (11.8 and 16.0 mg; both Grade 3 acneiform rash); MTD has not been reached. Pts received a median of 2 cycles (range 1-11, 6 pts ≥6 cycles). Safety and PK data are shown in the table. Maximum inhibition (Emax) of ERK phosphorylationin peripheral blood lymphocytes ranged from 21-95% (TAK-733; 0.2-16 mg QD), median Emax were 63%, 78%, and 92% at 8.4, 11.8, and 16 mg, respectively. In 32 evaluable pts, 1 pt (16 mg dose) with melanoma (BRAF L597R) had partial response confirmed at cycle 6 (RECIST v1.1) and is still on treatment at cycle 8; 12 pts had a best response of stable disease. Conclusions These preliminary data indicate that TAK-733 is generally well tolerated and pharmacodynamically active with signs of anti-tumor activity in pts with advanced non-hematologic malignancies. Dose escalation is continuing to determine the MTD. AEs, NCI-CTCAE v4.0 N = 44 Drug-related AE, n (%) 37 (84) ≥15% Dermatitis acneiform 19 (43) Diarrhoea 8 (18) Grade ≥3 drug-related AE, n (%) 7 (16) ≥5% Dermatitis acneiform 2 (5) Creatine phosphokinase evaluation 2 (5) PK data N = 41 Tmax, h 3 T1/2, h 53 Disclosure P.M. LoRusso: Consultancy or advisory board (Millennium Pharmaceuticals Inc.), Research Funding (Millennium Pharmaceuticals Inc.). A. Ribas: Consultancy or advisory board (Millennium Pharmaceuticals Inc.), Honoraria (Millennium Pharmaceuticals Inc.). J.A. Sosman: Consultancy or advisory board (Millennium Pharmaceuticals Inc.), Honoraria (Millennium Pharmaceuticals Inc.). B. Chmielowski: Consultancy within past 2 years (GSK, Genentech, Prometheus, CytRx, Morphotek). Membership on Board of Directors, Speakers Bureau, Advisory Committee (BMS, Genentech, Prometheus). P. Lipman: Employment (Millennium Pharmaceuticals Inc.). X. Zhou: Employment (Millennium Pharmaceuticals Inc.). V. Bozon: Employment (Millennium Pharmaceuticals Inc.). All other authors have declared no conflicts of interest.

MEK1/2 kinases have diverse functions in cell proliferation and survival, including the regulation of cell cycle transitions between the G1, S, and G2 phases and mediation of DNA damage response pathways. Therefore, inhibition of MEK1/2 may facilitate the antiproliferative effects of other therapies that disrupt cell cycle progression leading to DNA damage, including antimitotic therapies. AAK is a serine/threonine protein kinase essential for normal mitotic progression. Inhibition of AAK leads to inappropriate progression through mitosis in the presence of misaligned chromosomes resulting in aneuploidy and DNA damage, which leads to apoptosis or senescence. Here, we evaluated the combination of the allosteric MEK1/2 inhibitor TAK-733 with alisertib, the selective AAK inhibitor. Proliferation assays in multiple tumor cell lines showed a concentration-dependent decrease in the IC50 of TAK-733 and alisertib with increasing concentrations of the second agent, suggesting increased anti-proliferative activity of these agents when combined. This result was corroborated by measuring changes in cell confluence over time, a surrogate for proliferation rate, using an IncuCyte live-cell imaging system. The decreased proliferation rate of the combination was due in part to induction of apoptosis, as demonstrated by Annexin V and sub-diploid DNA content analysis. Cell cycle progression differences of the TAK-733/alisertib combination relative to the single agents were assessed using time-lapse video microscopy by measuring the time to the first mitotic event after drug addition, time spent in mitosis, and time between mitotic events. Of these, the only observed difference in cell-cycle progression time in the A2780 ovarian carcinoma cell line was the time between mitotic events, which was 10 hours longer for the combination relative to single agent alisertib. Moreover, the combination of TAK-733 and alisertib significantly reduced the ability of the cells to re-enter the cell cycle after one mitosis compared to either single agent; 30% for the combination, compared to 90% for TAK-733 and 76% for alisertib. Flow cytometry DNA content analysis showed that TAK-733 suppressed the formation of >4N/tetraploid cells induced by alisertib alone in multiple cell lines. This observed decrease in the proportion of >4N cells is greater than the decrease that could be extrapolated solely from the cell cycle-slowing effect of the combination, suggesting that TAK-733 reduces the proliferative potential of the alisertib-generated aneuploid cells via cell-cycle arrest and apoptosis. Collectively, these findings demonstrate an added benefit of TAK-733 over alisertib alone and provide a pre-clinical rationale for further investigation of the combination of TAK-733 and alisertib for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3738. doi:1538-7445.AM2012-3738

MEK and Aurora A kinase inhibitors (AAK) have profound impacts on cell cycle progression. Inhibition of MEK leads to defects in cell-cycle progression followed by cell cycle arrest and/or apoptosis. These cell-cycle progression defects may in part be due to the role of MEK in the DNA replication or damage checkpoint responses through positive regulation of DNA repair mechanisms. AAK inhibition leads to chromosome congression and segregation defects leading to DNA damage followed by apoptosis or senesence. The overlapping biological effects of inhibition of AAK and MEK kinases in cell cycle progression and chromosomal integrity raise the possibility that inhibiting both targets would provide added benefit over the inhibition of either target alone. The in vivo antitumor activity of TAK-733, an investigational potent, selective, non-ATP-competitive allosteric inhibitor of MEK, in combination with alisertib, an investigational potent, selective, reversible, ATP-competitive inhibitor of Aurora A kinase, was examined in experimental human solid tumor xenograft models including NSCLC (NCI H23 [KRAS and LKB1 mutations]), CRC (SW620 [KRAS, APC, p53 mutations]), and pancreatic cancer (Panc 1 and Capan 1 [KRAS mutations] and BxPC-3 [No MAPK mutations]) models in immunocompromised mice. The previously established maximally efficacious doses for the single agents dosed once daily (QD) orally (PO) (10 mg/kg TAK-733 and 30-mg/kg alisertib) were examined. A lower dose of 20 mg/kg alisertib was also examined in the event the maximally efficacious dose of each agent was not tolerated in combination. The once daily (QD) concurrent oral administration of TAK-733 and alisertib resulted in additive to synergistic antitumor activity and in prolonged inhibition of tumor regrowth after terminating treatment compared to single agent treatment in all xenograft models examined. Concurrent administration of TAK-733 and alisertib daily for 21 days was well tolerated. Treatment with TAK-733 alone and in combination with alisertib clearly inhibited phosphorylated ERK and produced a slight increase in phosphorylated histone H3. The results from the nonclinical models examined demonstrate considerable improvement in nonclinical antitumor activity over either single agent alone and provide a biological rationale for clinical evaluation of a TAK-733/alisertib combination in patients with advanced malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3739. doi:1538-7445.AM2012-3739

Abstract 750 Background: DKK1 is a negative regulator of the Wnt signaling pathway in bone that is overexpressed in a subset of newly-diagnosed MM patients with osteolytic lesions as well as in refractory and relapsed patients. Expression also correlates with the number of osteolytic lesions in untreated MM patients. BHQ880 is a novel anti-DKK1 human monoclonal antibody. Alleviation of DKK1 inhibition by BHQ880 results in activation of the Wnt signaling pathway, leading to increased bone mass mediated via upregulation of osteoblasts in mice and monkeys. In murine models of MM bone disease; this anabolic activity of BHQ880 increased trabecular and cortical bone density. Lytic bone disease in MM is caused by osteoclast activation and osteoblast inhibition. Current approved therapies for the treatment of MM bone disease are focused on osteoclast inhibition (e.g., bisphosphonates) and BHQ880 therapy may be able to reverse the effects of DKK1-induced osteoblast inhibition. Therefore, dual therapy with zoledronic acid to decrease bone resorption and BHQ880 to increase new bone formation may provide an effective treatment strategy for MM bone disease. Methods: In the phase I portion of this phase I/II study, patients with relapsed or refractory MM with prior skeletal-related event (SRE) were treated with BHQ880 as an IV infusion Q28 days. Patients also received Zol (4 mg) and approved MM Tx (bortezomib not allowed). Bone markers and total DKK1 levels along with bone mineral density are being measured. Full PK profiles were collected during the first and second cycle, after which predose (trough) samples were collected to assess accumulation. Results: Ten pts (6:M, 4:F), median age: 66.5 yrs (range 41- 70), performance status-0(5 pts), 1(4 pts), 2 (1 pts) have been enrolled in the following dose levels (mg/kg) 3 (2 pts), 10 (starting dose level -4 pts), 20 (4 pts) and have been on treatment for 1 day to 5 28-day cycles. No BHQ880-related AE's have been observed to date. Bone mineral density (BMD) data from the first two patients treated at 10 mg show the following: a.) Pt 1 (hip +5.8%, spine N/A due to surgery, wrist -1.8%); b.) Pt 2 (hip -0.2%, spine +6.1%, wrist not done). The biomarker data from these patients show: a.) Pt 1 shows a maximal +98% change over baseline in PINP at day 15 post-treatment, while osteocalcin (OC) changes +17% at day 15, reaching a +56% change over baseline at cycle 4 day 1; uNTx/Cr changes – 43 % at cycle 2 day 15. b) Partial data from Pt 2 suggests a -44 % change in PINP and –73 % change in OC and a +73% change in uNTx/Cr. Baseline total DKK1 levels on 3 patients ranged from 8.8 to 21.5 ng/mL. Preliminary PK analysis is available in 1 out of 3 patients treated at 10mg/kg. The Cmax achieved after the first infusion was 165 ug/mL and 201 ug/mL after the second infusion. Calculated t1/2 was 188 hours after the first infusion and 254 hours after the second infusion. Regarding overall exposure, AUC0-672 hours after first infusion was 36081 hr*ng/mL and 48533 hr*ng/mL after the second infusion. Conclusion: BHQ880 given IV Q28 days appears to be well tolerated in combination with Zol and chosen MM Tx. Once safety of 40 mg/kg BHQ880 or the MTD dose has been determined, pts will be enrolled in the phase II portion to assess activity on a SRE endpoint. Updated safety, efficacy, bone density and biomarker data will be presented on all patients at the upcoming ASH meeting. Disclosures: Padmanabhan: Genentech: Consultancy, Honoraria; GSK: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Off Label Use: BHQ880, a novel osteoblast activating, anti-DKK1 antibody. Dzik-Jurasz:Novartis: Employment, Equity Ownership. Gangolli:Novartis: Employment, Equity Ownership. Ettenberg:Novartis: Employment, Equity Ownership. Miner:Novartis: Employment, Equity Ownership. Bilic:Novartis: Employment, Equity Ownership. Whyte:Novartis: Employment, Equity Ownership. Mehdi:Novartis: Employment, Equity Ownership. Chiang:Novartis: Employment, Equity Ownership. Rae:Novartis: Employment, Equity Ownership. Shah:Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Millenium: Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Elan: Consultancy. Giles:Novartis: Consultancy, Research Funding; BMS: Research Funding; Merck: Research Funding; Clavis: Research Funding. Stewart:Novartis: Consultancy; Amgen: Consultancy; Millenium: Consultancy, Research Funding; Proteolix: Consultancy; Celgene: Honoraria.