Combined PD-L1/TGFß blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors.

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Generalized Additive Mixed Modeling of Longitudinal Tumor Growth Reduces Bias and Improves Decision Making in Translational Oncology.

Scientists working in translational oncology regularly conduct multigroup studies of mice with serially measured tumors. Longitudinal data collected can feature mid-study dropouts and complex nonlinear temporal response patterns. Parametric statistical models such as ones assuming exponential growth are useful for summarizing tumor volume over ranges for which the growth model holds, with the advantage that the model's parameter estimates can be used to summarize between-group differences in tumor volume growth with statistical measures of uncertainty. However, these same assumed growth models are too rigid to recapitulate patterns observed in many experiments, which in turn diminishes the effectiveness of their parameter estimates as summary statistics. To address this problem, we generalized such models by adopting a nonparametric approach in which group-level response trends for logarithmically scaled tumor volume are estimated as regression splines in a generalized additive mixed model. We also describe a novel summary statistic for group level splines over user-defined, experimentally relevant time ranges. This statistic reduces to the log-linear growth rate for data well described by exponential growth and also has a sampling distribution across groups that is well approximated by a multivariate Gaussian, thus facilitating downstream analysis. Real-data examples show that this nonparametric approach not only enhances fidelity in describing nonlinear growth scenarios but also improves statistical power to detect interregimen differences when compared with the simple exponential model so that it generalizes the linear mixed effects paradigm for analysis of log-linear growth to nonlinear scenarios in a useful way. SIGNIFICANCE: This work generalizes the statistical linear mixed modeling paradigm for summarizing longitudinally measured preclinical tumor volume studies to encompass studies with nonlinear and nonmonotonic group response patterns in a statistically rigorous manner.

Immunization associated with primary tumor growth leads to rejection of commonly used syngeneic tumors upon tumor rechallenge.

The recent success of multiple immunomodulating drugs in oncology highlights the potential of relieving immunosuppression by directly engaging the immune system in the tumor bed to target cancer cells. Durable responses to immune checkpoint inhibitors experienced by some patients may be indicative of the formation of a T cell memory response. This has prompted the search for preclinical evidence of therapy-induced long-term immunity as part of the evaluation of novel therapeutics. A common preclinical method used to document long-term immunity is the use of tumor rechallenge experiments in which tumor growth is assessed in mice that have previously rejected tumors in response to therapy. Failure of rechallenge engraftment, typically alongside successful engraftment of the same tumor in naive animals as a control, is often presented as evidence of therapy-induced tumor immunity. Here, we present evidence that formation of tumor immunity often develops independent of therapy. We observed elevated rates of rechallenge rejection following surgical resection of primary tumors for four of five commonly used models and that such postexcision immunity could be adoptively transferred to treatment-naïve mice. We also show that tumor-specific cytolytic T cells are induced on primary tumor challenge independent of therapeutic intervention. Taken together these data call into question the utility of tumor rechallenge studies and the use of naïve animals as controls to demonstrate therapy-induced formation of long-term tumor immunity.

A cell identity switch allows residual BCC to survive Hedgehog pathway inhibition.

Despite the efficacy of Hedgehog pathway inhibitors in the treatment of basal cell carcinoma (BCC)1, residual disease persists in some patients and may contribute to relapse when treatment is discontinued2. Here, to study the effect of the Smoothened inhibitor vismodegib on tumour clearance, we have used a Ptch1-Trp53 mouse model of BCC3 and found that mice treated with vismodegib harbour quiescent residual tumours that regrow upon cessation of treatment. Profiling experiments revealed that residual BCCs initiate a transcriptional program that closely resembles that of stem cells of the interfollicular epidermis and isthmus, whereas untreated BCCs are more similar to the hair follicle bulge. This cell identity switch was enabled by a mostly permissive chromatin state accompanied by rapid Wnt pathway activation and reprogramming of super enhancers to drive activation of key transcription factors involved in cellular identity. Accordingly, treatment of BCC with both vismodegib and a Wnt pathway inhibitor reduced the residual tumour burden and enhanced differentiation. Our study identifies a resistance mechanism in which tumour cells evade treatment by adopting an alternative identity that does not rely on the original oncogenic driver for survival.

The Hippo pathway effector TAZ induces TEAD-dependent liver inflammation and tumors.

The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.

Translational pharmacokinetic/pharmacodynamic analysis in cancer pharmacology: a tool to maximize the value of antitumor efficacy from tumor bearing mice.

Translational pharmacokinetic/pharmacodynamic (PK/PD) analysis is becoming an increasingly important tool for the identification and selection of new anticancer agents. There are two important elements of effectively using PK/PD analysis to translate preclinical antitumor efficacy from tumor bearing mice (xenografts and allografts) to cancer patients. These two sometimes overlapping elements are termed translation 'WITHIN' and 'ACROSS' species. Translating 'WITHIN' species refers to the quantitative characterization of drug action and disease behavior within tumor bearing mice using PK/PD modeling in order to use this information to make predictions of drug response in humans. Translating 'ACROSS' species refers to use of PK/PD modeling to quantify species similarities and differences in drug response in order to understand the clinical relevance of preclinical efficacy data.

Brain Distribution and Efficacy of the Brain Penetrant PI3K Inhibitor GDC-0084 in Orthotopic Mouse Models of Human Glioblastoma.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Limited treatment options have only marginally impacted patient survival over the past decades. The phophatidylinositol 3-kinase (PI3K) pathway, frequently altered in GBM, represents a potential target for the treatment of this glioma. 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) is a PI3K inhibitor that was specifically optimized to cross the blood-brain barrier. The goals of our studies were to characterize the brain distribution, pharmacodynamic (PD) effect, and efficacy of GDC-0084 in orthotopic xenograft models of GBM. GDC-0084 was tested in vitro to assess its sensitivity to the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and in vivo in mice to evaluate its effects on the PI3K pathway in intact brain. Mice bearing U87 or GS2 intracranial tumors were treated with GDC-0084 to assess its brain distribution by matrix-assisted laser desorption ionization (MALDI) imaging and measure its PD effects and efficacy in GBM orthotopic models. Studies in transfected cells indicated that GDC-0084 was not a substrate of P-gp or BCRP. GDC-0084 markedly inhibited the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. MALDI imaging showed GDC-0084 distributed evenly in brain and intracranial U87 and GS2 tumors. GDC-0084 achieved significant tumor growth inhibition of 70% and 40% against the U87 and GS2 orthotopic models, respectively. GDC-0084 distribution throughout the brain and intracranial tumors led to potent inhibition of the PI3K pathway. Its efficacy in orthotopic models of GBM suggests that it could be effective in the treatment of GBM. GDC-0084 is currently in phase I clinical trials.

Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development.

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity.

Discovery of Clinical Development Candidate GDC-0084, a Brain Penetrant Inhibitor of PI3K and mTOR.

Inhibition of phosphoinositide 3-kinase (PI3K) signaling is an appealing approach to treat brain tumors, especially glioblastoma multiforme (GBM). We previously disclosed our successful approach to prospectively design potent and blood-brain barrier (BBB) penetrating PI3K inhibitors. The previously disclosed molecules were ultimately deemed not suitable for clinical development due to projected poor metabolic stability in humans. We, therefore, extended our studies to identify a BBB penetrating inhibitor of PI3K that was also projected to be metabolically stable in human. These efforts required identification of a distinct scaffold for PI3K inhibitors relative to our previous efforts and ultimately resulted in the identification of GDC-0084 (16). The discovery and preclinical characterization of this molecule are described within.

MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD-L1 Checkpoint Blockade.

Targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) can induce regression of tumors bearing activating mutations in the Ras pathway but rarely leads to tumor eradication. Although combining MEK inhibition with T-cell-directed immunotherapy might lead to more durable efficacy, T cell responses are themselves at least partially dependent on MEK activity. We show here that MEK inhibition did profoundly block naive CD8(+) T cell priming in tumor-bearing mice, but actually increased the number of effector-phenotype antigen-specific CD8(+) T cells within the tumor. MEK inhibition protected tumor-infiltrating CD8(+) T cells from death driven by chronic TCR stimulation while sparing cytotoxic activity. Combining MEK inhibition with anti-programmed death-ligand 1 (PD-L1) resulted in synergistic and durable tumor regression even where either agent alone was only modestly effective. Thus, despite the central importance of the MAP kinase pathway in some aspects of T cell function, MEK-targeted agents can be compatible with T-cell-dependent immunotherapy.

Structure-Based Design of GNE-495, a Potent and Selective MAP4K4 Inhibitor with Efficacy in Retinal Angiogenesis.

Diverse biological roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated the identification of potent inhibitors in order to study its function in various disease contexts. In particular, compounds that can be used to carry out such studies in vivo would be critical for elucidating the potential for therapeutic intervention. A structure-based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into the CNS led to an advanced compound 13 (GNE-495) that showed excellent potency and good PK and was used to demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were observed in the inducible Map4k4 knockout mice.

Translational value of mouse models in oncology drug development.

Much has been written about the advantages and disadvantages of various oncology model systems, with the overall finding that these models lack the predictive power required to translate preclinical efficacy into clinical activity. Despite assertions that some preclinical model systems are superior to others, no single model can suffice to inform preclinical target validation and molecule selection. This perspective provides a balanced albeit critical view of these claims of superiority and outlines a framework for the proper use of existing preclinical models for drug testing and discovery. We also highlight gaps in oncology mouse models and discuss general and pervasive model-independent shortcomings in preclinical oncology work, and we propose ways to address these issues.

Discovery of highly potent, selective, and efficacious small molecule inhibitors of ERK1/2.

Using structure-based design, a novel series of pyridone ERK1/2 inhibitors was developed. Optimization led to the identification of (S)-14k, a potent, selective, and orally bioavailable agent that inhibited tumor growth in mouse xenograft models. On the basis of its in vivo efficacy and preliminary safety profiles, (S)-14k was selected for further preclinical evaluation.

Prediction of drug distribution in subcutaneous xenografts of human tumor cell lines and healthy tissues in mouse: application of the tissue composition-based model to antineoplastic drugs.

Advanced tissue composition-based models can predict the tissue-plasma partition coefficient (Kp ) values of drugs under in vivo conditions on the basis of in vitro and physiological input data. These models, however, focus on healthy tissues and do not incorporate data from tumors. The objective of this study was to apply a tissue composition-based model to six marketed antineoplastic drugs (docetaxel, DOC; doxorubicin, DOX; gemcitabine, GEM; methotrexate, MTX; topotecan, TOP; and fluorouracil, 5-FU) to predict their Kp values in three human tumor xenografts (HCT-116, H2122, and PC3) as well as in healthy tissues (brain, muscle, lung, and liver) under steady-state in vivo conditions in female NCR nude mice. The mechanisms considered in the tissue/tumor composition-based model are the binding to lipids and to plasma proteins, but the transporter effect was also investigated. The method consisted of analyzing tissue composition, performing the pharmacokinetics studies in mice, and calculating the corresponding in vivo Kp values. Analyses of tumor composition indicated that the tumor xenografts contained no or low amounts of common transporters by contrast to lipids. The predicted Kp values were within twofold and threefold of the measured values in 77% and 93% of cases, respectively. However, predictions for brain for each drug, for liver for MTX, and for each tumor xenograft for GEM were disparate from the observed values, and, therefore, not well served by the model. Overall, this study is the first step toward the mechanism-based prediction of Kp values of small molecules in healthy and tumor tissues in mouse when no transporter and permeation limitation effect is evident. This approach will be useful in selecting compounds based on their abilities to penetrate human cancer xenografts with a physiologically based pharmacokinetic (PBPK) model, thereby increasing therapeutic index for chemotherapy in oncology study.

Fragment-based identification and optimization of a class of potent pyrrolo[2,1-f][1,2,4]triazine MAP4K4 inhibitors.

MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. In an effort to generate small molecule MAP4K4 inhibitors, a fragment-based screen was carried out and a pyrrolotriazine fragment with excellent ligand efficiency was identified. Further modification of this fragment guided by X-ray crystal structures and molecular modeling led to the discovery of a series of promising compounds with good structural diversity and physicochemical properties. These compounds exhibited single digit nanomolar potency and compounds 35 and 44 achieved good in vivo exposure.

Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine inhibitors of Erk2.

The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.

Discovery and preclinical development of vismodegib.

INTRODUCTION: Vismodegib is the first Hedgehog (Hh) pathway inhibitor approved in the US for the treatment of adults with metastatic or locally advanced basal cell carcinoma (BCC). It was approved by the US FDA on 30 January 2012, and by the European Commission on 12 July 2013, for the treatment of adult patients with symptomatic metastatic BCC, or locally advanced BCC inappropriate for surgery or radiotherapy. Vismodegib selectively inhibits the Hh signaling pathway, binding to and inhibiting a critical signal-transducing component of the pathway, Smoothened (SMO). Vismodegib was discovered by Genentech, Inc., under a collaboration agreement with Curis, Inc. AREAS COVERED: This article reviews the development of vismodegib from its discovery, preclinical pharmacology and validation to the clinical pharmacokinetics and validation in Phase I and II clinical investigations. We also provide a survey of other Hh pathway inhibitors in clinical development. EXPERT OPINION: The authors' experience in target-based drug discovery suggests that vismodegib's path to the clinic deserves some reflection to identify key steps that have contributed to its success. Targeting the Hh pathway with vismodegib blocks the abberant signaling caused by mutational inactivation of the negative regulator PTCH1 or mutational activation of SMO. Vismodegib gives physicians a treatment option for patients with locally advanced or metastatic BCC for whom surgery or radiation is not recommended.

Discovery of selective 4-Amino-pyridopyrimidine inhibitors of MAP4K4 using fragment-based lead identification and optimization.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido[3,2-d]pyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.

PTEN loss mitigates the response of medulloblastoma to Hedgehog pathway inhibition.

Medulloblastoma is a cancer of the cerebellum, for which there is currently no approved targeted therapy. Recent transcriptomics approaches have demonstrated that medulloblastoma is composed of molecularly distinct subgroups, one of which is characterized by activation of the Hedgehog pathway, which in mouse models is sufficient to drive medulloblastoma development. There is thus considerable interest in targeting the Hedgehog pathway for therapeutic benefit in medulloblastoma, particularly given the recent approval of the Hedgehog pathway inhibitor vismodegib for metastatic and locally advanced basal cell carcinoma. Like other molecularly targeted therapies, however, there have been reports of acquired resistance to vismodegib, driven by secondary Hedgehog pathway mutations and potentially by activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Given that acquired resistance to vismodegib may occur as a result of inappropriate PI3K pathway activation, we asked if loss of the PI3K pathway regulator, phosphatase and tensin homologue (Pten), which has been reported to occur in patients within the Hedgehog subgroup, would constitute a mechanism of innate resistance to vismodegib in Hedgehog-driven medulloblastoma. We find that Hedgehog pathway inhibition successfully restrains growth of Pten-deficient medulloblastoma in this mouse model, but does not drive tumor regression, as it does in Pten-wild-type medulloblastoma. Combined inhibition of the Hedgehog and PI3K pathways may lead to superior antitumor activity in PTEN-deficient medulloblastoma in the clinic.