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No targeted treatments are currently approved for HER2 exon 20 insertion–mutant lung adenocarcinoma patients. Mobocertinib (TAK-788) is a potent irreversible tyrosine kinase inhibitor (TKI) designed to target human epidermal growth factor receptor 2 (HER2/ERBB2) exon 20 insertion mutations. However, the function of mobocertinib on HER2 exon 20 insertion–mutant lung cancer is still unclear. Here we conducted systematic characterization of preclinical models to understand the activity profile of mobocertinib against HER2 exon 20 insertions. In HER2 exon 20 insertion–mutant cell lines, the IC50 of mobocertinib was higher than poziotinib and comparable with or slightly lower than afatinib, neratinib, and pyrotinib. Mobocertinib had the lowest HER2 exon 20 insertion IC50/wild-type (WT) EGFR IC50 ratio, indicating that mobocertinib displayed the best selectivity profile in these models. Also, mobocertinib showed strong inhibitory activity in HER2 exon 20YVMA allograft and patient-derived xenograft models. In genetically engineered mouse models, HER2 exon 20G776>VC lung tumors exhibited a sustained complete response to mobocertinib, whereas HER2 exon 20YVMA tumors showed only partial and transient response. Combined treatment with a second antibody–drug conjugate (ADC) against HER2, ado-trastuzumab emtansine (T-DM1), synergized with mobocertinib in HER2 exon 20YVMA tumors. In addition to the tumor cell autonomous effect, sustained tumor growth control derived from M1 macrophage infiltration and CD4+ T-cell activation. These findings support the ongoing clinical development of mobocertinib (NCT02716116) and provide a rationale for future clinical evaluation of T-DM1 combinational therapy in HER2 exon 20YVMA insertion–mutant lung adenocarcinoma patients. Significance: This study elucidates the potent inhibitory activity of mobocertinib against HER2 exon 20 insertion–mutant lung cancer and the synergic effect of combined mobocertinib and T-DM1, providing a strong rationale for clinical investigation.

Mobocertinib (TAK‐788) is a tyrosine kinase inhibitor under investigation for treatment of non–small cell lung cancer with activating EGFR exon 20 insertions. This study examined the safety; tolerability; pharmacokinetics (PK), including food effects; and bioavailability of mobocertinib in healthy volunteers. In part 1, fasted volunteers were randomized to placebo or mobocertinib in single‐ascending‐dose cohorts (20‐160 mg). In part 2, mobocertinib (120/160 mg) was administered on day 1 of periods 1 and 2 under fasted or low‐fat meal conditions (2‐period, 2‐sequence crossover design). In part 3, fasted volunteers received mobocertinib 160 mg in 1 of 2 capsule products on day 1 of periods 1 and 2 with 7‐day washout. Safety and PK parameters were assessed. Sixty‐nine volunteers were enrolled (mean age, 29 years; 75% male). The most common adverse events (AEs; ≥10% of volunteers) were gastrointestinal AEs (25%‐50%) and headache (8%‐31%). No serious AEs were reported. A low‐fat meal did not affect the PK of mobocertinib or its active metabolites. The geometric mean terminal disposition phase half‐life (20 hours) supported once‐daily dosing. The 2 capsule products were bioequivalent. These data guided dosing and supported administration of mobocertinib without regard to low‐fat meal intake in ongoing and planned clinical studies.

Advanced bladder cancer is associated with a poor prognosis and limited treatment options. The PI3K/AKT/mTOR pathway is frequently activated in this disease and can be a potential therapeutic target for treatment intervention. We studied the antitumor efficacy of a new targeted therapy, TAK-228 (oral mTORC1/2 inhibitor), in preclinical models of bladder cancer. We evaluated the effects of TAK-228 in combination with a PI3Kα inhibitor (TAK-117) or with chemotherapy (paclitaxel). We used six bladder cancer cell lines and in vivo xenografts models. TAK-228 strongly inhibited cell proliferation in vitro, and reduced tumor growth and angiogenesis in vivo. Three possible biomarkers of response to TAK-228 (basal levels of 4E-BP1, p-4E-BP1/4E-BP1 ratio, or eIF4E/4E-BP1 ratio) were identified. The combination of TAK-228 and TAK-117 had synergistic effects in vitro and in vivo. Furthermore, TAK-228 demonstrated greater efficiency when combined with paclitaxel. TAK-228 also showed ex vivo activity in tumor tissue from patients with treatment-naïve bladder cancer. TAK-228 is a promising investigational agent that induces a strong effect on cell proliferation, tumor growth, and angiogenesis in bladder cancer models. High synergistic effects were observed with TAK-228 combined with a PI3K inhibitor or with chemotherapy. These results are currently being investigated in a clinic trial of TAK-228 plus paclitaxel in patients with metastatic bladder cancer (NCT03745911). Implications: Strong synergistic effects were observed when combining TAK-228 with TAK-117 (a PI3Kα inhibitor) or with paclitaxel chemotherapy. A phase II study at our institution is currently evaluating the efficacy of TAK-228 combined with paclitaxel in patients with metastatic bladder cancer.

Purpose: Recommended phase II dose (RP2D) determination for combination therapy regimens is a constrained optimization problem of maximizing antitumor activity within the constraint of clinical tolerability to provide a wide therapeutic index. A methodology for addressing this problem was developed and tested using clinical and preclinical data from combinations of the investigational drugs TAK-117, a PI3Kα inhibitor, and TAK-228, a TORC1/2 dual inhibitor. Experimental Design: Utilizing free fraction-corrected average concentrations, C_{117}^{avg}\ and C_{228}^{avg}, which are the primary pharmacokinetic predictors of single-agent preclinical antitumor activity, a preclinical exposure-efficacy surface was characterized, allowing for nonlinear interactions between growth rate inhibition of the agents on a MDA-MB-361 cell line xenograft model. Logistic regression was used to generate an exposure-effect surface for C_{117}^{avg}\ and C_{228}^{avg} versus clinical toxicity outcomes [experiencing a dose-limiting toxicity (DLT)] in single-agent and combination dose-escalation studies. A maximum tolerated exposure curve was defined at which DLT probability was 25%; predicted antitumor activity along this curve was used to determine optimal RP2D. Results: The toxicity constraint curve determined from early clinical data predicted that any clinically tolerable combination was unlikely to result in greater antitumor activity than either single-agent TAK-117 or TAK-228 administered at their respective MTDs. Similar results were obtained with 10 other cell lines, with one agent or the other predicted to outperform the combination. Conclusions: This methodology represents a general, principled way of evaluating and selecting optimal RP2D combinations in oncology. The methodology will be retested upon availability of clinical data from TAK-117/TAK-228 combination phase II studies. See related commentary by Mayawala et al., p. 6564

HOX11 is a proto-oncogene, which is silent in normal mature T-cells, while being aberrantly activated in T-cell acute lymphoblastic leukaemia (T-ALL) by translocations t(10;14)(q24;q11) or t(7;10)(q35;q24). Although many oncogenes are expressed in alternative forms in cancer, thus far, only one form of the human HOX11 transcript has been reported. We describe here the identification of three alternative transcripts of the HOX11 proto-oncogene, expressed in primary T-ALL specimens. Using rapid amplification of cDNA ends (RACE) and targeted RT-PCR, we have sequenced 23 individual cDNA clones characterising these novel transcripts. Northern hybridisation identified particular novel exons expressed in T-ALL, which are not expressed in normal T-cells. To date, aberrant expression of HOX11 has only been associated with leukaemia. Our survey of a range of neuroblastoma and primitive neuroectodermal tumour (PNET) cell lines demonstrated the expression of these novel HOX11 transcripts in tumours of neural origin, while their expression was not detected in normal brain tissues. Strikingly, the dominant transcript in these neural tumour cell lines is more than 1 kb larger than the dominant transcript in T-ALL. These observations, combined with sequence data from several EST clones derived from medulloblastoma cDNA libraries, support a new hypothesis that HOX11 may also function as a neural oncogene or brain tumour marker.

Compound 1 [(E)-4-fluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1S,4S)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], a new, potent, selective anaplastic lymphoma kinase (ALK) inhibitor with potential application for the treatment of cancer, was selected as candidate to advance into efficacy studies in mice. However, the compound underwent mouse-specific enzymatic hydrolysis in plasma to a primary amine product (M1). Subsequent i.v. pharmacokinetics studies in mice showed that compound 1 had high clearance (CL) and a short half-life. Oral dose escalation studies in mice indicated that elimination of compound 1 was saturable, with higher doses achieving sufficient exposures above in vitro IC(50). Chemistry efforts to minimize hydrolysis resulted in the discovery of several analogs that were stable in mouse plasma. Three were taken in vivo into mice and showed decreased CL corresponding to increased in vitro stability in plasma. However, the more stable compounds also showed reduced potency against ALK. Kinetic studies in NADPH-fortified and unfortified microsomes and plasma produced submicromolar K(m) values and could help explain the saturation of elimination observed in vivo. Predictions of CL based on kinetics from hydrolysis and NADPH-dependent pathways produced predicted hepatic CL values of 3.8, 3.0, 1.6, and 1.2 l/h⋅kg for compound 1, compound 2 [(E)-3,5-difluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], compound 3 [(E)-3-chloro-5-fluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], and compound 4 [(E)-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)-3-(trifluoromethyl)benzamide], respectively. The in vivo observed CLs for compounds 1, 2, 3, and 4 were 5.52, 3.51, 2.14, and 2.66 l/h⋅kg, respectively. These results indicate that in vitro metabolism kinetic data, incorporating contributions from both hydrolysis and NADPH-dependent metabolism, could be used to predict the systemic CL of compounds cleared via hydrolytic pathways provided that the in vitro assays thoroughly investigate the processes, including the contribution of other metabolic pathways and the possibility of saturation kinetics.

A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure–activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.

Piperidine carboxamide 1 was identified as a novel inhibitor of anaplastic lymphoma kinase (ALK enzyme assay IC(50) = 0.174 μM) during high throughput screening, with selectivity over the related kinase insulin-like growth factor-1 (IGF1R). The X-ray cocrystal structure of 1 with the ALK kinase domain revealed an unusual DFG-shifted conformation, allowing access to an extended hydrophobic pocket. Structure-activity relationship (SAR) studies were focused on the rapid parallel optimization of both the right- and left-hand side of the molecule, culminating in molecules with improved potency and selectivity over IGF1R.

Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY(1604) ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen(®)) and phosflow platforms were used to detect modulation of pY(1604) ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY(1604) ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen(®) assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen(®) and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY(1604) ALK.

Damage to the gastrointestinal mucosa is a common dose-limiting toxicity of several anticancer therapies. Until recently, adequate control of oral mucositis was considered a significant unmet medical need, with most available treatments providing only palliative benefits without protecting the gastrointestinal epithelium from the damaging effects of cancer therapy. In 2005, palifermin [recombinant human keratinocyte growth factor (KGF)] was approved to decrease the incidence and duration of severe oral mucositis in patients with hematologic malignancies receiving myelotoxic therapy requiring hematopoietic stem cell support. Current trials are investigating the use of palifermin in solid tumor settings. The objective of this study was to determine whether combining palifermin with different chemotherapeutic or biological agents affected the antitumor activity of these agents in human head and neck (FaDu) and colorectal (HT29) carcinoma xenograft models. Nude CD1 mice were injected with 1 × 107 of either FaDu or HT29 cells, which express both KGF and epithelial growth factor receptors. Animals were treated with palifermin in various combinations with chemotherapeutic (5-fluorouracil and cisplatin) and/or biological (bevacizumab, cetuximab, and panitumumab) agents. Palifermin alone had no effect on either FaDu or HT29 tumor growth. Palifermin did not affect the therapeutic efficacy of 5-fluorouracil, cisplatin, cetuximab, bevacizumab, or panitumumab in any of the two- or three-way drug combinations tested in either model. The results of this study showed that palifermin did not promote the growth of two carcinoma cell lines that express functional KGF receptors and did not protect these tumor cells from the antitumor effects of several chemotherapeutic and biological agents. (Mol Cancer Res 2008;6(8):1337–46)

The acquired activation of stem cell leukemia (SCL) during T lymphopoiesis is a common event in T-cell acute lymphoblastic leukemia (T-ALL). Here, we generated tamoxifen (TAM)–inducible transgenic mice (lck-ERT2-SCL) to study the consequences of acquired SCL activation during T-cell development. Aberrant activation of SCL in thymocytes resulted in the accumulation of immature CD4+CD8+ (double-positive, DP) cells by preventing normal surface expression of the T-cell receptor αβ (TCRαβ) complex. SCL-induced immature DP cells were further characterized by up-regulated NOTCH1 and generated noncycling polyclonal CD8+TCRβlow cells. The prevalence of these cells was SCL dependent because TAM withdrawal resulted in their disappearance. Furthermore, we observed that SCL activation led to a dramatic up-regulation of NOTCH1 target genes (Hes-1, Deltex1, and CD25) in thymocytes. Strikingly, NOTCH1 target gene up-regulation was already observed after short-term SCL induction, implying that enhanced NOTCH signaling is mediated by SCL and is not dependent on secondary genetic events. These data represent the basis for a novel pathway of SCL-induced leukemogenesis and provide a functional link between SCL and NOTCH1 during this process.

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.

369 Background: PI3K/AKT/mTOR pathway is a promising target for cancer treatment being commonly deregulated in human bladder tumors and resulting in the promotion of tumor cell growth, survival, and resistance to chemotherapy. The aim of this study is to characterize the effects of MLN0128, a novel mammalian target of rapamycin (mTOR) complex 1 and 2 (mTORC1/2) dual inhibitor, and MLN1117, an orally bioavailable inhibitor of the class I phosphoinositide 3-kinase (PI3K) alpha isoform that may be more efficacious and less toxic than pan-PI3K inhibitors as bladder cancer therapies. Methods: We evaluated the effects of MLN0128 and MLN1117 both as single agents and in combination with each other or with a SOC chemotherapy agent (paclitaxel). The effects of the agents alone or in combination were analysed in a panel of six bladder cancer cell lines and in tumor xenografts. These models were selected based on specific genomic alterations that could be considered as potential therapeutic targets (PIK3CA and TSC mutations). Molecular effects of both agents and the combinations on cell-cycle, apoptosis, autophagy and on cell viability were tested in the bladder cancer cell lines. The in vivo effects on tumor growth inhibition were also assessed. Results: Both MLN0128 and MLN1117 inhibit the PI3K/AKT/mTOR pathway and reduce cell proliferation in bladder cancer cell lines with diverse genetic backgrounds. Combination of MLN0128 + MLN1117 produced synergistic antiproliferative effects in cell lines and improved the effect of each drug alone in vitro and in vivo, with no signs of toxicity in these models. Similar effects were observed with the combination of paclitaxel + MLN0128. Conclusions: Our results show that MLN0128 and MLN1117 are promising investigational agents that might be of value for bladder cancer patients. Further investigation as novel anti-cancer agents alone or in combination with chemotherapy in clinical trials in humans is warranted.

Inducible transgenic mouse models that impose a constraint on both temporal and spatial expression of a given transgene are invaluable. These animals facilitate experiments that can address the role of a specific cell or group of cells within an animal or in a particular window of time. A common approach to achieve inducibility involves the site-specific recombinase 'Cre', which is linked to a modified version of one of various steroid hormone-binding domains. Thus, the expression of Cre is regulated such that a functional nuclear transgene product can only be generated with the addition of an exogenous ligand. However, critical requirements of this system are that the nuclear localization of the transgene product be tightly regulated, that the dosage of the inducing agent remains consistent among experimental animals and that the transgene cassette cannot express in the absence of the inducing agent. We used the Cre ER(T2) cassette, which is regulated by the addition of the estrogen antagonist tamoxifen to determine whether cross-contamination of tamoxifen between animals housed together can be a significant source of spurious results. We found that cross-contamination of exogenous tamoxifen does occur. It occurred in all animals tested. We suggest that the mechanism of contamination is through exposure to tamoxifen in the general environment and/or to coprophagous behavior. These results have important implications for the interpretation and design of experiments that use 'inducible' transgenic animals.

Mapping of transcriptional control elements normally depends on the generation of a series of deletion mutants. The consequences of particular deletions are then functionally assessed by their ability to alter gene expression. The information derived from such investigations provides a general regulatory profile of the gene of interest, as well as generating a focus for future experiments. Due to the limitations of conventional DNA cloning methods, it has previously not been possible to use such an approach to rapidly assess the role of long-range regulatory elements that frequently lie further than 20 kb away from the coding region. In order to identify regulatory elements of the proto-oncogene HOX11 that may be mutated in a subset of childhood T-cell acute lymphoblastic leukaemia specimens, we generated nested deletions from a P1 artificial chromosome (PAC). This clone contained 95 kilobases (kb) of the HOX11 locus at 10q24; including 63 kb of 5' regulatory DNA. The deletion series was produced by the use of a recombination based cloning system and clones were subsequently transfected into mammalian cells. We have identified several long-range regulatory elements that mediate transcriptional control of HOX11. This approach is simple, rapid, and inexpensive. Furthermore, it generates multiple deletion clones in a single experiment. This novel approach opens up a new avenue for investigating long-range transcription control. Additionally, by allowing analysis of these elements in the natural context of large integrants the approach does not require the use of artificial extrachromosomal elements. This methodology can be applied to any gene cloned into a PAC or BAC vector and could also be useful in identifying appropriately sized deletion mutants for functional testing in transgenic models.

Ectopic expression of the homeobox gene HOX11 is associated with a significant proportion of childhood T-cell acute lymphoblastic leukaemias (T-ALLs). We hypothesise that one mechanism of gene deregulation involves overcoming the silencing mechanism(s) of gene expression present in normal cells. Here, we describe a search for trans-acting factors that control transcriptional activity from a distal 5 ′ region of the HOX11 promoter. We have identified a region of this promoter which contributes significantly to HOX11 activation and two distinct regulatory elements are involved. First, a PBX2 Regulatory Element PRE-1048 has been identified which contains a novel DNA-binding sequence and mediates significant activation of the HOX11 gene in K562 cells. This is the first report of a homeobox gene being specifically regulated by PBX2 and the second report of a vertebrate homeobox target gene of a PBX protein. The PREP1 protein was also shown to be part of the PRE-1048 -binding complex. The other regulatory element we describe here RE-1019 contains little sequence conservation to known transcription control elements. It appears that this element is a novel sequence that binds an as yet unidentified factor, mediating significant activation of the HOX11 gene in K562 cells. This is the first detailed report of elements that mediate regulation of the proto-oncogene HOX11 .

The design of an optimal combination therapy can be thought of as a set of tradeoffs designed to maximize the efficacy of the combination for a given toxicity budget. As anticancer agents (even targeted ones) often have overlapping toxicities, an efficient approach to making efficacy-toxicity tradeoffs is critical. In this context, a major impediment is that the search space grows exponentially with each additional drug while in vivo studies have a fixed upper size limit. Here, we model the efficacy and toxicity landscapes of multidrug cocktails, and describe an approach to visualize and design efficient studies for three-drug combinations. We further demonstrate the application of our approach in a practical drug development context by showing its impact on actual study design, and validation with in vivo datasets. As our approach relies on equations from first principles, it is in theory extensible to an arbitrary number of drugs, subject to the practical constraints of drug dosing. First, we demonstrate an approach to visually determine the optimal dose combination for a three-drug combination with fully overlapping toxicity. Efficacy and toxicity isoboles visualized as surfaces in three-drug space demonstrate the optimal dose combination, which corresponds to the point of tangency between the MTD and efficacy isobole surfaces. Next, we derive an analytical method to efficiently estimate this point with a minimum of data, and without using graphical methods (important for extending the work beyond three dimensions). The combined efficacy and toxicity of a three-drug combination were modeled from first principles using isobolograms as a sum of the single-agent PK/Efficacy (PK/E) relationships and four combination terms (three binary and one ternary) to account for potential interactions between the drugs. Borrowing from Microeconomic Utility Theory, we found the point of greatest efficacy along the Maximum Tolerated Dose (MTD) toxicity contour, which corresponds to the efficacy isobole tangentially intersecting the MTD contour. Through both analytical and numerical approaches, we determined the the single- and double-agent efficacy parameters uniquely determine the dose escalation path which provides the best estimate of combination efficacy. Simulated experiments demonstrate the optimal observation path up to MTD performs acceptably compared to a uniformly-gridded exposure space. Finally, we demonstrate the application of this approach in a proposed experimental design for three-drug combinations, and validate it with experimental data. While developing multi-drug cocktails poses many scientific and operational challenges, the approach presented here provides a straightforward route to preclinical testing and validation through the design of parsimonious studies that can explicitly define the contribution of each individual drug (and each two drug combination) to the overall efficacy and toxicity landscape of the cocktail. Citation Format: Christopher J. Zopf, Andrew Chen, Santhosh Palani, Rachael Brake, Mark Manfredi, Jeffrey Ecsedy, Wen Chyi Shyu, Arijit Chakravarty. Rational dose optimization for multi-drug cocktails. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 791. doi:10.1158/1538-7445.AM2014-791

8580 Background: Spleen Tyrosine Kinase (SYK) is a non-receptor cytoplasmic tyrosine kinase that is a common member of various signal transduction cascades in cells of the hematopoietic lineage inc...

A component of heterogeneity of human cancers is the presence of multiple mutations that can activate a number of signaling pathways that leads to growth and survival including both MAPK (RAS/RAF/MEK/ERK) and PI3K/Akt/mTOR. Previous preclinical and clinical studies have found that KRAS mutant cancers demonstrate a heterogeneous response to inhibitors targeting the MAPK pathway. The molecular mechanisms responsible for this heterogeneous response remain unclear. KRAS mutations are observed in more than 25% of NSCLC and can occur concurrently with LKB1 or PIK3CA and LKB mutations. Due to the presence of these complex genotypes and the resultant concomitant activation of different signaling pathways, it has become evident that inhibiting a single node remains insufficient to achieve a sustained anti-tumor response. These findings suggest the need to combine different targeted agents to overcome pathway cross talk and feedback activation that has been observed in the single agent setting. Using KRAS driven NSCLC as a model we have postulated that the combination of a mTORC1 / 2 inhibitor and a MEK inhibitor (MLN0128 + TAK-733) could provide improved activity over either agent alone in preclinical models. The effect of this combination on in vitro growth in a panel of NSCLC cell lines was assessed. Synergistic to additive inhibition of proliferation was observed with administration of the combination MLN0128 + TAK-733 over single agent effects in cell lines with KRAS and LKB1, or KRAS combined with PIK3CA and LKB mutations, while no additive inhibition was observed in wild-type cells. An increase in apoptosis, as measured by cleaved PARP, and reduced expression of cyclin D1 relative to single agents alone was also demonstrated after administration of MLN0128 +TAK-733, which likely contributes to the observed decreased tumor cell proliferation observed with the combination. The in vivo anti-tumor activity of MLN0128 + TAK-733 was examined in 2 human tumor xenograft models of NSCLC. Administration of the combination of MLN0128 + TAK-733 improved anti-tumor activity in NCI-H460 (KRAS, PIK3CA and LKB) over single agent administration. In the A549 (KRAS and LKB1) model, the administration of the combination of MLN0128 + TAK-733 using various dosing regimens resulted in reduction in tumor volume in all schedules examined compared to tumor stasis following single agent administration. Overall, these data support further research of the combination of MLN0128 (mTORC1/2) and MEK inhibitors for KRAS plus LKB1 or KRAS plus PIK3CA and LKB mutated NSCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C264. Citation Format: Robyn Fabrey, Joy Atienza, David Briere, Rachael Brake, Patrick W. Vincent. Effective preclinical treatment of KRAS mutant lung cancers with a combination of MLN0128, an investigational novel mTORC1/2 inhibitor, and a selective MEK inhibitor. [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 C264.

In many cancers, monotherapy targeted against signal transduction can lead to pathway reactivation by feedback mechanisms. The phosphoinositide 3-kinase (PI3K) pathway has been studied in this regard. Allosteric inhibitors of mammalian target of rapamycin complex 1 (mTORC1), such as rapamycin analogs, interrupt the normal negative regulation of insulin receptor substrate-1 (IRS-1) mediated by ribosomal protein S6 kinase (S6K), leading to pathway reactivation, as demonstrated by AKT phosphorylation and activation of its downstream substrates. Recent data suggest that administration of catalytic mTOR inhibitors can lead to similar feedback. Using in vitro models we have shown that the investigational catalytic mTORC1/2 inhibitor MLN0128 can lead to pathway reactivation, as measured by increases in AKT phosphorylation at threonine 308 (T308) over time. Furthermore we have shown pre-clinically that administration of the investigational isoform-selective PI3Kα inhibitor MLN1117 mitigates this pathway reactivation. In in vitro studies the combination of MLN0128 plus MLN1117 resulted in greater inhibition of key pathway regulators (pAKT, pS6, and p4EBP1) than either single agent alone, and a sustained decrease in AKT (T308) phosphorylation. The combination also induced greater apoptosis pre-clinically, as indicated by increases in cleaved poly (ADP-ribose) polymerase (PARP). The antiproliferative effects of this combination were assessed in 14 human tumor cell lines known to demonstrate sensitivity to single-agent MLN0128. In all settings (across tumor types and genotypes), administration of MLN0128 plus MLN1117 resulted in additive antiproliferative activity, except in two breast cancer models in which the activity of the combination was considered to show synergistic activity. In in vivo studies, concurrent oral administration of MLN0128 plus MLN1117 resulted in additive or synergistic antitumor activity in human tumor xenograft models. The combined antitumor activity correlated with modulation of pharmacodynamic markers and was observed irrespective of the dosing schedules tested or genotypes of the models, which included PI3K-mutant breast, PIK3CA/KRAS-mutant colon, PTEN-null breast, and EGFR-mutant non-small-cell lung cancer.The results from these preclinical experiments suggest that, in certain patient populations, co-administration of MLN0128 plus MLN1117 may lead to greater therapeutic effect than either single agent alone. The flexibility of being able to schedule these compounds independently and the isoform-selective profile of MLN1117 could provide distinct clinical advantages over other mechanisms of PI3K pathway inhibition. Together these data support the rationale that has led to the investigation of the safety of this combination clinically in an ongoing phase I study ([NCT01899053][1]). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C176. Citation Format: Rachael L. Brake, Robyn Fabrey, Jeff Szwaya, Michael Fitzgerald, Natasha Iartchouk, Xin Guo, Keisuke Kuida, Fabian Zohren, Mark Manfredi. The combination of mTORC1/2 and PI3Kα inhibition alleviates PI3K pathway reactivation and leads to significant antitumor activity in multiple preclinical xenograft models. [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 C176. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01899053&atom=%2Fmolcanther%2F12%2F11_Supplement%2FC176.atom

The PI3K-Akt-mTOR signaling pathway is the most dysregulated pathway in cancer. Specifically, helical and kinase domain mutations in the p110α subunit (E542K, E545K, H1047R) account for greater than 15% mutations in many cancers. In addition, copy number gain of the PIK3CA locus (>2.5 copies) also occur at a high frequency suggesting that these events are also associated with the oncogenic process. MLN1117 is an investigational PI3Kα selective inhibitor that is currently in Phase I dose escalation in subjects with advanced cancer (NCT01449370). It is widely hypothesized that PI3Kα isoform selective inhibitors will demonstrate considerable therapeutic benefit in PI3K “addicted” tumors; the work presented here is focused on identifying the association of genetic alterations with sensitivity to MLN1117. We have assessed the sensitivity profile both in vitro (n=47) and in vivo (n=29) using human tumor cell lines and primary human tumor xenografts, representing 9 different tumor types. PIK3CA gene point mutations are associated with sensitivity to MLN1117, in both in vitro and in vivo settings. 14 of 17 (82%) PIK3CA mutant cell lines demonstrate an in vitro IC50 of ≤ 5.0 μM and all non mutant models demonstrate an IC50 of > 5.0 μM. In vivo 12 of 15 PIK3CA mutant models including two non-hotspot alterations (R88Q and G106_R108del) demonstrate a TGI value of > 50%. Although copy number gain of PIK3CA is a frequent genetic event and could also be considered as a potential marker of sensitivity to PI3Kα inhibitors, in our assessment this was not apparent. We assessed 25 in vitro models and 16 in vivo models with a gain in the PIK3CA copy number greater than 2.5. Using a cut-off of IC50 of 50% in both settings we did not identify a significant association between PIK3CA amplification and MLN1117 sensitivity. Thus, PIK3CA mutation status is a significant predictor in classifying sensitive vs. non-sensitive models to MLN1117 whereas PIK3CA copy number status is not (compare p=0.00002 vs p = 0.7668). Our assessment of models with PTEN alterations found all models to be insensitive to MLN1117 using our established in vitro criteria whereas several of these models were found to have borderline sensitivity (range 41-50% TGI) in vivo. These results suggest the need for additional exploration of the PTEN role and MLN1117 sensitivity. Additional trends observed in this data set are 1) PIK3CA co-mutated with KRAS is insensitive to MLN1117 in both in vitro and in vivo systems; and 2) complex mutations involving PIK3CA and ARID1A or EIF4B demonstrate a distribution of sensitivities. We present data that support the use of PIK3CA mutation as a patient selection strategy for clinical studies of MLN1117 while the utility of identifying patients with increases in PIK3CA copy number for clinical studies of MLN1117 may be limited. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A169. Citation Format: Natasha Iartchouk, Erik Koenig, Pooja Shah, Jeff Szwaya, Michael Fitzgerald, Dong Mei Zhang, Yueying Cao, Keli Song, Rachael Brake. Assessment of genotype-correlated sensitivity to the investigational PI3Kα selective inhibitor MLN1117 in preclinical models. [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 A169.

Schedule selection for novel combination therapy in oncology poses a daunting array of choices, such as whether to optimize each agent's schedule individually or to select dosing schedules to enhance the concomitant overlap in exposures. While combination dose schedules could be optimized empirically in the clinic, this would typically require large and expensive trials to accurately determine differences between response rates. Here we describe the utilization of preclinical in vivo tumor growth models combined with mathematical modeling techniques to translate dose schedule selection of two in-pathway inhibitors. We applied these techniques to determine the contribution of concomitant dosing of the novel combination of MLN0128 and MLN1117, investigational inhibitors of mTORC1/2 and PI3Kα respectively. Tumor Growth Inhibition (TGI) assessment of the combination was made in a diverse panel of human tumor xenografts. The combination and single agents were delivered on schedules with different degrees of dosing overlap. Isobolograms were first constructed from the in vivo TGI data and this analysis demonstrated that in most models, the overall effect of the combination on growth rate across dose schedule was additive, suggesting that co-dosing would not be a significant factor on the overall effect. To test this directly, multiple linear regressions were used to assess the degree of efficacy derived from exposure to each agent alone in contrast to the amount derived from concomitance in exposure. This regression analysis also showed that the combination efficacy did not require concomitant exposure. Finally, the lack of requirement for concomitance was corroborated using a dynamic model capturing the rates of tumor growth. This parsimonious model provided a clear fit to the observed tumor volumes, and demonstrated no requirement for concomitant dosing for combination efficacy to be obtained in the preponderance of xenograft models. Taken together, these analyses suggest that in a variety of genetic xenograft backgrounds, the effect of combining a mTORC1/2 and a PI3Kα inhibitor on efficacy is greater than each single agent alone, is additive with respect to tumor growth rate, and that concomitance of dosing is not needed to achieve maximal efficacy of the combination. This affords future trials with this combination to focus on optimization of schedule by minimizing overlapping on-mechanism tolerability concerns to achieve the greatest therapeutic benefit. These techniques illustrate a quantitative approach to using preclinical TGI data to inform early clinical dose and schedule selection of novel-novel combinations. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A216. Citation Format: Christopher Zopf, Mayank Patel, Ekta Kadakia, Robyn Fabry, Arijit Chakravarty, Swapan Chowdhury, Jing-Tao Wu, Wen Chyi Shyu, Mark Manfredi, Fabian Zohren, Chirag Patel, Keisuke Kuida, Natasha Iartchouk, Rachael Brake, Jerome Mettetal. Combination schedule optimization through preclinical PK-efficacy modeling of an investigational novel-novel combination: A case study of MLN0128 an mTORC1/2 inhibitor with MLN1117 a PI3Kα isoform selective inhibitor. [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 A216.

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

Background: MLN0128 is an investigational, potent, and highly selective inhibitor of mTORC1/2, which are integral to cell proliferation, angiogenesis, and cellular metabolism. This first-in-human study (NCT01058707) aimed to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of oral MLN0128. Methods: Pts aged ≥18 years with advanced solid tumors were enrolled in a 3+3 dose-escalation design initially to receive MLN0128 once daily (QD), and then enrolled in escalating intermittent schedules of once weekly (QW), 3 days/week (QDx3d QW), or 5 days/week (QDx5d QW) dosing, in 28-day cycles. Blood samples were collected at multiple timepoints for PK analysis. PD endpoints were evaluated in skin to determine the effect on mTORC1/2-dependent biomarkers. A preclinical PK-efficacy model was generated (Phoenix NLME v1.1) with tumor xenograft efficacy data, and implemented using clinical PK parameters to simulate tumor volume-time curves for various MLN0128 doses/schedules. Response was assessed by RECIST v1.1. Results: 115 pts received MLN0128 doses in the ranges 2-7 mg QD (n=30), 7-40 mg QW (n=30), 6-20 mg QDx3d QW (n=33), and 7-13 mg QDx5d QW (n=22). Median age was 60 years (range 24-89); 40% were male. The most common tumor types were colorectal (22%), renal (9%), and ovarian (8%) cancer. The MTDs were 6 mg QD, 40 mg QW, 16 mg QDx3d QW, and 10 mg QDx5d QW. Based on the overall safety profiles, the recommended phase 2 doses were 5 mg QD, 40 mg QW, 9 mg QDx3d QW, and 7 mg QDx5d QW. Pts received a median of 2 cycles (range 1-23). The most common drug-related adverse events (AEs) were hyperglycemia (65%), nausea (60%), vomiting (44%), decreased appetite (36%), diarrhea (33%), asthenia (30%), and mucosal inflammation (30%). The most common drug-related grade ≥3 AEs were hyperglycemia (12%), asthenia (9%), and mucosal inflammation (5%). MLN0128 exhibited dose-linear PK with a plasma half-life of ∼8 h, and did not accumulate in plasma with QD dosing. Modeling of preclinical PK-efficacy data and simulation of human tumor volume-time curves using clinical PK parameters suggested potential greater antitumor effect with schedules using more frequent dosing (i.e. QD, QDx5d QW). There was a treatment-related inhibition of mTORC1/2 biomarkers in skin. Two of 10 pts (20%) with renal cancer receiving MLN0128 at 15 and 40 mg QW had a partial response; 5 pts (6%) had stable disease for ≥6 cycles. Conclusions: Based on the safety profile, PK/PD, simulated tumor volume-time curves, and responses, MLN0128 5 mg QD and 40 mg QW were selected for further evaluation in an expansion phase in pts with renal, endometrial, or urothelial cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C252. Citation Format: Jeffrey R. Infante, Josep Tabernero, Andres Cervantes, Shadia Jalal, Howard A. Burris, Teresa Macarulla, J. Alejandro Perez-Fidalgo, Rachel Neuwirth, Chirag Patel, Esha Gangolli, Rachael Brake, Jeffrey Sturm, Eric H. Westin, Michael Gordon. A phase 1, dose-escalation study of MLN0128, an investigational oral mammalian target of rapamycin complex 1/2 (mTORC1/2) catalytic inhibitor, in patients (pts) with advanced non-hematologic malignancies. [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 C252.

The HOX11 proto-oncogene is normally expressed in embryogenesis where it directs the synthesis of the spleen. In adult tissues, HOX11 expression is silenced by an unknown mechanism. Aberrant expression of HOX11 occurs in T-cell acute lymphoblastic leukaemia (T-ALL), where it is thought to be involved in T-cell immortalization. The deregulated expression of HOX11 is frequently associated with chromosomal translocations which juxtapose a T-cell receptor (TCR) gene upstream of the HOX11 gene. In these cases, it is presumed that the activation of HOX11 expression results from bringing the gene under the control of TCR enhancer elements. However, activation of HOX11 also occurs in the absence of an associated translocation in both T-ALL and erythroleukaemia cells, implying that an alternative activation mechanism may exist. We hypothesized that HOX11 may be repressed in normal T-cells and erythroid cells by the action of negative elements which may be deleted or mutated in leukaemia. We therefore conducted a search for negative elements in the human HOX11 promoter which may function to silence its expression in normal cells of the haematopoietic lineages. Since little sequence of the HOX11 promoter was available, we began our investigation by sequencing over 4.5 kilobases of untranslated DNA from upstream of HOX11. The human sequence that overlaps with the 2.1 kb of murine Hox11 is highly conserved, suggesting that a large region of DNA upstream of HOX11 may have a regulatory function. We then used transfection assays to test the ability of portions of the promoter to drive transcription of a reporter gene. These studies identified four negative elements. Two of them (NRE2 and NRE4) function in all cell lines tested, while the remaining two (NRE1 and NRE3) appear to be cell-type specific. The DNA sequences of three elements are conserved between the human and mouse HOX11/Hox11 promoters. We propose a model in which the combined action of these negative elements contributes to the overall repression of HOX11 expression in normal blood cells.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that has been implicated as a driving oncogene in a number of cancers, including non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma and inflammatory myofibroblastic tumors (IMT). Numerous genetic aberrations at the ALK locus are observed in cancer including point mutations, amplifications, translocations and inversions. Inversions are exemplified by inv(2)(p21;p23), which leads to the constitutively active oncogenic fusion protein EML4-ALK present in ∼5% of NSCLC. Crizotinib, a dual cMet/ALK kinase inhibitor, was recently approved by the FDA for locally advanced or metastatic NSCLC that is ALK-positive, thereby validating ALK as therapeutic target. Here we describe the pharmacological characterization of a novel series of potent, selective and orally bioavailable ALK kinase inhibitors. Members of this series inhibit wild type ALK, NPM-ALK fusion and crizotinib resistant ALK[L1196M] kinase activity at sub-nanomolar concentrations, displaying up to ∼200 fold increased inhibitory activity over crizotinib. Kinase profiling indicate that members of this series display increased selectivity scores relative crizotinib. In Karpas-299 cells, selected compounds inhibited both pY1604 ALK activation (IC50 = 2 nM) and cell proliferation (IC50 = 1 nM). Members of this series were also evaluated in the EML4-ALK expressing NSCLC cell line H3122, and displayed equipotent inhibition of pY1604 ALK activation and inhibition of cell proliferation (both IC50 = 1 nM). Members of this class did not inhibit growth of an ALK negative lymphoma cell line (HT). The in vivo activity of this series was examined in the Karpas-299 ALCL xenograft model. Compound was dosed daily (PO) at 10, 30 and 60 mg/kg. Tumor growth inhibition was observed at all dose levels, and the highest dose level resulted in significant tumor regression (96%, p80% pALK inhibition was observed at the lowest doses tested, and complete inhibition was seen at doses of 30 mg/kg and above. In a direct comparison, members of this series achieved ALK inhibition in these tumors at a ∼15 fold lower plasma concentration than crizotinib. A PK/PD time course study was performed in the Karpas-299 model. A single 60 mg/kg dose of an inhibitor was able to maintain >90% ALK inhibition in tumors up to 24 hours post-dose, indicating significant tumor penetration and sustained ALK kinase inhibition. In conclusion, the described compounds are potent and selective inhibitors of ALK kinase, possess an impressive efficacy profile and drug-like pharmacokinetic properties. These features together indicate the potential for significant advantages over crizotinib. There is a compelling case for their clinical evaluation in patients with ALK-driven cancers. 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 1795. doi:1538-7445.AM2012-1795

Abstract 4964 Immune receptors such as the B cell receptor (BCR) require key signaling intermediate phosphatidylinositol-3 kinase delta (PI3Kδ) for normal immune cell survival, development and function. PI3Kδ is a class IA lipid kinase, is expressed primarily within the hematopoietic system and is composed of a catalytic subunit p110δ and a regulatory subunit p85. Recently, deregulated BCR-PI3Kδ signaling has been reported to play a role in B-cell malignancies such as chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL) by mediating abnormal B-cell growth and survival. Indeed, the constitutive phosphorylation of downstream signaling intermediate AKT is associated with poor prognosis in several B cell malignancies. Here, we have investigated the potential of a novel small molecule inhibitor of PI3Kδ, AMG 319, to suppress PI3K signaling in human B cell lines and assessed the subsequent effects on viability as a single agent and in combination with chemotherapeutic drugs in preclinical models. Small molecule AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent preclinical pharmacokinetic (PK) properties. AMG 319 was found to potently inhibit PI3Kδ in enzyme assays (IC50 5000 fold). Furthermore, AMG 319 was considered inactive at 10 μM on non-PI3K class I kinases in a broader kinome screen of 402 kinases. In preclinical PK studies, AMG 319 had low systemic clearance, T1/2 range of 2–4 hr, oral bioavailability of >45% and unbound fractions in plasma of 5–19%. Here, we have investigated the potential for AMG 319 to inhibit constitutive PI3K mediated signaling and effects on human B cell line viability. In a broad screen of >20 cell lines derived from B cell malignancies, the majority of lines were found to express PI3Kδ protein, all cells lines expressed the PI3Kα and β isoforms and variable levels of constitutive pAKTS473 were detected. AMG 319 was found to potently suppress constitutive pAKTS473 in the cell lines with IC50 in the low single to double digit nM range. Cellular viability was inhibited by AMG 319 though lines were variably sensitive to drug (range low double digit nM to μM IC50). As cell lines were variably sensitive to AMG 319 as a single agent, we examined if AMG 319 could enhance the efficacy of chemotherapeutic agents in vitro and in vivo. These studies focused on a DLBCL cell line HT which was relatively insensitive to AMG 319 as a single agent (IC50 ∼10 μM) in viability assays even though pAKTS473 was potently suppressed (IC50 ∼ 0.030 μM). Treatment with AMG 319 was found to synergize with the effects of vincristine to reduce cell viability in vitro using a 72 hr viability assay. Next we examined whether the enhanced cytotoxicity using these drugs in combination could be observed in vivo. Using the human B-cell lymphoma HT xenograft model, we found that AMG 319 in combination with vincristine enhanced tumor growth inhibition above that observed with either agent alone. Taken together, these findings suggest that the inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancies. In conclusion, AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent PK properties. AMG 319 inhibited constitutive pAKTS473, reduced the viability of B cell lines and synergized with vincristine in vitro and in vivo. The safety, PK and preliminary efficacy of AMG 319 are currently being investigated in a Phase I trial in patients with relapsed or refractory lymphoid malignancies. Disclosures: Sinclair: Amgen: Employment, Stock and Options. Metz:Amgen, Inc: Employment, Stock and Options. Cushing:Amgen, Inc: Employment, Stock and Options. Liu:Amgen, Inc: Employment, Stock and Options. Brake:Amgen, Inc: Employment, Stock and Options. Starnes:Amgen, Inc: Employment, Stock and Options. Means:Amgen, Inc: Employment, Stock and Options. Henne:Amgen, Inc: Employment, Stock and Options. Archibeque:Amgen: Employment, Stock and Options. Mattson:Amgen, Inc: Employment, Stock and Options. Drew:Amgen, Inc: Employment, Stock and Options. Busse:Amgen, Inc: Employment, Stock and Options. Wang:Amgen, Inc: Employment, Stock and Options. Al-Assaad:Amgen, Inc: Employment, Stock and Options. Molineux:Amgen: Employment, Stock and Options.

Anaplastic lymphoma kinase (ALK) is predominantly expressed in the brain and spinal cord during neonatal development and is subsequently down-regulated in the adult. In cancer, ALK is ectopically expressed as a consequence of one of several different cytogenetic translocations or inversions that lead to the expression of an oligomerized, constitutively active, intracellular tyrosine kinase. The resulting fusion protein has been identified in anaplastic large cell lymphomas, non small cell lung carcinomas and more recently breast and colorectal carcinomas. Preclinical data convincingly demonstrates that fusion transcripts can drive tumorigenesis and inhibition of ALK in vitro and in vivo leads to inhibition of cellular growth. Full length (fl) ALK is also expressed in a variety of other tumor types, and recent data suggest that at least in neuroblastoma, the flALK transcript contains activating point mutations in the tyrosine kinase domain. These data suggest that flALK may also be a therapeutic target in cancer. Here we demonstrate that a significant subset of ovarian carcinoma cell lines and biopsy specimens aberrantly express ALK. Gene expression analysis in 126 ovarian carcinoma samples demonstrated a prevalence of ALK expression of 19%. Analysis of the histological subtypes within this study, showed specific enrichment of ALK transcript in papillary ovarian carcinoma (15 of 42 samples). This subtype showed a range of ALK expression between 2 - 4 fold increase above normal ovarian tissue. We have also confirmed that in a subset of ovarian tumor cell lines and primary tumor samples that ALK transcript is converted into ALK protein. DNA sequence analysis has revealed that in these patient samples and cell lines, ALK is predominantly wild type. Single nucleotide polymorphism (SNP) analysis and fluorescent in situ hybridization (FISH) suggest that over expression may occur as a consequence of low copy number amplification. These preliminary findings suggest that ALK expression in cancer has now been observed in ovarian carcinomas by a mechanism distinct from cytogenetic translocations or inversions. Currently, we are attempting to correlate the aberrant expression of ALK with ovarian cancer prognosis as well as understand the value of inhibiting ALK expression in this cell type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3142.

The acquired activation of stem cell leukemia (SCL) during T-lymphopoiesis is a common event in T-cell acute lymphoblastic leukemia. Here, we generated a novel tamoxifen-inducible transgenic mouse model (lck-ERT2-SCL) to study the cellular targets of acquired SCL activation during T-cell development. Upon tamoxifen treatment we observed the thymic emergence of abnormal, non-cycling CD8+TCRβlow and immature CD4+CD8+ (double-positive, DP) cells displaying increased viability. Unexpectedly, fetal thymic organ culture analysis of lck-ERT2-SCL thymi revealed the development of DP cells before the emergence of CD8+TCRβlow cells, which implied the derivation of CD8+TCRβlow cells from DPs rather than immature CD8 single-positive (SP) thymocytes. Interestingly, histone deacetylase (HDAC) inhibition with trichostatin A (TSA) had a divergent effect on SCL perturbed thymopoiesis: TSA increased T-cell receptor surface expression within DP and CD8 SP cells however did not alter the CD8 shifted CD4/CD8-ratio. Furthermore, we studied the expression of NOTCH1 in SCL induced TCRβlow thymocytes. Strikingly, we found that SCL induced NOTCH1-upregulation in DP TCRβlow cells. We therefore conclude that SCL promotes the emergence of abnormal CD8+TCRβlow cells by an only partially HDAC dependent mechanism from DP TCRβlow cells. Moreover, SCL induced DP TCRβlow cells are characterized by upregulated NOTCH1, which in turn might promote the effect of acquired NOTCH1 mutations during T-leukemogenesis.