Erin McNamara

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.

Antitumor Activity of Targeted and Cytotoxic Agents in Murine Subcutaneous Tumor Models Correlates with Clinical Response

Purpose: Immunodeficient mice transplanted with subcutaneous tumors (xenograft or allograft) are widely used as a model of preclinical activity for the discovery and development of anticancer drug candidates. Despite their widespread use, there is a widely held view that these models provide minimal predictive value for discerning clinically active versus inactive agents. To improve the predictive nature of these models, we have carried out a retrospective population pharmacokinetic–pharmacodynamic (PK–PD) analysis of relevant xenograft/allograft efficacy data for eight agents (molecularly targeted and cytotoxic) with known clinical outcome. Experimental Design: PK–PD modeling was carried out to first characterize the relationship between drug concentration and antitumor activity for each agent in dose-ranging xenograft or allograft experiments. Next, simulations of tumor growth inhibition (TGI) in xenografts/allografts at clinically relevant doses and schedules were carried out by replacing the murine pharmacokinetics, which were used to build the PK–PD model with human pharmacokinetics obtained from literature to account for species differences in pharmacokinetics. Results: A significant correlation (r = 0.91, P = 0.0008) was observed between simulated xenograft/allograft TGI driven by human pharmacokinetics and clinical response but not when TGI observed at maximum tolerated doses in mice was correlated with clinical response (r = 0.36, P = 0.34). Conclusions: On the basis of these analyses, agents that led to greater than 60% TGI in preclinical models, at clinically relevant exposures, are more likely to lead to responses in the clinic. A proposed strategy for the use of murine subcutaneous models for compound selection in anticancer drug discovery is discussed. Clin Cancer Res; 18(14); 3846–55. ©2012 AACR.

AXL Inhibition Sensitizes Mesenchymal Cancer Cells to Antimitotic Drugs

Molecularly targeted drug therapies have revolutionized cancer treatment; however, resistance remains a major limitation to their overall efficacy. Epithelial-to-mesenchymal transition (EMT) has been linked to acquired resistance to tyrosine kinase inhibitors (TKI), independent of mutational resistance mechanisms. AXL is a receptor tyrosine kinase associated with EMT that has been implicated in drug resistance and has emerged as a candidate therapeutic target. Across 643 human cancer cell lines that were analyzed, elevated AXL was strongly associated with a mesenchymal phenotype, particularly in triple-negative breast cancer and non-small cell lung cancer. In an unbiased screen of small-molecule inhibitors of cancer-relevant processes, we discovered that AXL inhibition was specifically synergistic with antimitotic agents in killing cancer cells that had undergone EMT and demonstrated associated TKI resistance. However, we did not find that AXL inhibition alone could overcome acquired resistance to EGFR TKIs in the EMT setting, as previously reported. These findings reveal a novel cotreatment strategy for tumors displaying mesenchymal features that otherwise render them treatment refractory.

MAP4K4 regulates integrin-FERM binding to control endothelial cell motility

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4–moesin–talin–β1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to β1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5β1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.

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.

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.

Abstract A11: Antitumor activity of targeted and cytotoxic agents in xenograft models correlates with clinical response: A pharmacokinetic-pharmacodynamic analysis.

Immunodeficient mice transplanted subcutaneously with human tumors (xenografts) are a widely used model of preclinical efficacy in the discovery and development of anti-cancer agents. We performed a retrospective population pharmacokinetic-pharmacodynamic (PK-PD) analysis of xenograft efficacy data that accounts for differences in clinical and preclinical drug exposure in order to improve their predictive value. In addition, we investigated the ability of xenograft studies to predict clinical response with newer molecular targeted agents. Data from preclinical and clinical studies with three cytotoxic chemotherapeutic agents (docetaxel, carboplatin, and 5-fluorouracil) and five molecular targeted agents (erlotinib, sunitnib, dasatinib, trastuzumab, and vismodegib) were included in this analysis. Anti-cancer agents were administered at a range of doses to xenograft mice bearing tumors relevant to the cancer of interest. No correlation was observed when comparing clinical response to % tumor growth inhibition (%TGI) at the maximum tolerated dose (MTD) in mice. PK-PD modeling was used to simulate %TGI in xenografts using human exposures at clinically relevant doses. These simulated %TGI in xenografts driven by human drug exposures were found to correlate (r=0.92; p=0.0005) with clinical response, illustrating the importance of taking into account species differences in drug exposure when interpreting xenograft data. Distinct differences were observed in the anti-tumor activity of sunitinib in Colo205 colorectal (simulated %TGI = 64%) and 786-O renal cell adenocarcinoma (simulated %TGI = 81%) xenografts, and vismodegib in medulloblastoma allografts (simulated %TGI =102%) and D5123 colorectal xenografts (simulated %TGI = 33%). These differences were consistent with the underlying biology of these models and demonstrate the importance of understanding tumor biology when selecting relevant xenograft models for molecular targeted agents. Based on these analyzes, agents that lead to greater than 60% tumor growth inhibition in preclinical models at clinically relevant exposures are more likely to lead to responses in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A11.

Assessment of the hepatic CYP reductase null mouse model and its potential application in drug discovery.

CYP Oxidoreductase (Por) is the essential electron donor for all CYP enzymes and is responsible for the activation of CYP. The Taconic Hepatic CYP Reductase Null (HRN) mouse model possesses a targeted mutation that results in liver-specific deletion of the Por gene thereby resulting in a disruption of CYP metabolism in the liver. The objectives of these studies were to further characterize the HRN mouse using probe drugs metabolized by CYP. In addition, tumor exposure in xenograft tumor bearing HRN immune-compromised (nude) mice was also determined. In HRN mice following intravenous (iv) administration of midazolam, clearance (CL) was reduced by ∼ 80% compared to wild-type mice (WT). After oral administration, the AUC of midazolam was increased by ∼ 20-fold in HRN mice compared to WT mice; this greater effect suggests that hepatic first pass plays a role in the oral CL of midazolam. A 50% and an 80% decrease in CL were also observed in HRN mice following iv administration of docetaxel and theophylline, respectively, compared to WT mice. In addition, a 2- to 3-fold increase in tumor concentrations of G4222, a tool compound, were observed in tumor bearing HRN nude mice compared to tumor bearing nude WT mice. The observations from these experiments demonstrate that, for compounds that are extensively metabolized by hepatic CYP, the HRN mouse model could potentially be valuable for evaluating in vivo efficacy of tool compounds in drug discovery where high hepatic CL and low exposure may prevent in vivo evaluation of a new chemical entity.

Chemotherapy Combines Effectively with Anti–PD-L1 Treatment and Can Augment Antitumor Responses

Immunotherapy with checkpoint inhibitors has proved to be highly effective, with durable responses in a subset of patients. Given their encouraging clinical activity, checkpoint inhibitors are increasingly being tested in clinical trials in combination with chemotherapy. In many instances, there is little understanding of how chemotherapy might influence the quality of the immune response generated by checkpoint inhibitors. In this study, we evaluated the impact of chemotherapy alone or in combination with anti–PD-L1 in a responsive syngeneic tumor model. Although multiple classes of chemotherapy treatment reduced immune cell numbers and activity in peripheral tissues, chemotherapy did not antagonize but in many cases augmented the antitumor activity mediated by anti–PD-L1. This dichotomy between the detrimental effects in peripheral tissues and enhanced antitumor activity was largely explained by the reduced dependence on incoming cells for antitumor efficacy in already established tumors. The effects of the various chemotherapies were also agent specific, and synergy with anti–PD-L1 was achieved by different mechanisms that ultimately helped establish a new threshold for response. These results rationalize the combination of chemotherapy with immunotherapy and suggest that, despite the negative systemic effects of chemotherapy, effective combinations can be obtained through distinct mechanisms acting within the tumor.

Abstract A114: Effect of different standard of care chemotherapeutics on anti-PD-L1 responses in syngeneic mouse tumor models

Immunotherapy with checkpoint inhibitors has proven to be highly effective, with durable responses in a subset of patients. In an effort to broaden the number of responding individuals, overcome resistance to single-agent therapy and extend the duration of responses, combination of immunotherapy with other treatments such as chemotherapy is currently being tested in multiple clinical trials. Importantly, combination of immunotherapy with standard of care chemotherapy could bring the benefits of immunotherapy into earlier lines of treatment. Here, we have evaluated the effects of different classes of chemotherapeutic agents alone or in combination with anti-PD-L1 treatment in syngeneic murine tumor models. We have found that combination of anti-PD-L1 with various alkylating agents, taxanes and platins caused differential changes on the frequency and number of intratumoral immune cell subsets, but did not antagonize the functional changes mediated by anti-PD-L1 treatment. Importantly, by comparing the effects of these chemotherapies in different tumor models, we have observed that their effects are model-specific. These differences may be due to tumor-specific immune composition and tumor intrinsic responses to different chemotherapies. Our results demonstrate that in the models we have tested, chemotherapeutic agents do not appear to directly antagonize the activity of anti-PD-L1 as measured by pharmacodynamic changes in immune cell activation although some differences can be observed in the frequency and number of certain intratumoral immune cell subsets. Additionally, our findings also reveal that tumor models might respond differently to the same chemotherapeutic treatment adding complexity to how we evaluate the effect of combination treatments with chemotherapy. Nonetheless, our results suggest a rationale for combining immunotherapy with chemotherapy in the clinic, although consideration should be taken when generalizing results from mouse tumor models. Citation Format: Rafael Cubas, Marina Moskalenko, Jeanne Cheung, Shiuh-Ming Luoh, Erin McNamara, Marcia Belvin, Jeong Kim, Stephen Gould. Effect of different standard of care chemotherapeutics on anti-PD-L1 responses in syngeneic mouse tumor models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A114.