A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response.

Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors, has not been fully explored. Here we use genetically engineered mouse models to conduct a 'co-clinical' trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244) increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

[(18)F]-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography of LAPC4-CR Castration-Resistant Prostate Cancer Xenograft Model in Soft Tissue Compartments.

Preclinical xenograft models have contributed to advancing our understanding of the molecular basis of prostate cancer and to the development of targeted therapy. However, traditional preclinical in vivo techniques using caliper measurements and survival analysis evaluate the macroscopic tumor behavior, whereas tissue sampling disrupts the microenvironment and cannot be used for longitudinal studies in the same animal. Herein, we present an in vivo study of [(18)F]-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) designed to evaluate the metabolism within the microenvironment of LAPC4-CR, a unique murine model of castration-resistant prostate cancer. Mice bearing LAPC4-CR subcutaneous tumors were administered [(18)F]-FDG via intravenous injection. After a 60-minute distribution phase, the mice were imaged on a PET/CT scanner with submillimeter resolution; and the fused PET/CT images were analyzed to evaluate tumor size, location, and metabolism across the cohort of mice. The xenograft tumors showed [(18)F]-FDG uptake that was independent of tumor size and was significantly greater than uptake in skeletal muscle and liver in mice (Wilcoxon signed-rank P values of .0002 and .0002, respectively). [(18)F]-FDG metabolism of the LAPC4-CR tumors was 2.1 ± 0.8 ID/cm(3)*wt, with tumor to muscle ratio of 7.4 ± 4.7 and tumor to liver background ratio of 6.7 ± 2.3. Noninvasive molecular imaging techniques such as PET/CT can be used to probe the microenvironment of tumors in vivo. This study showed that [(18)F]-FDG-PET/CT could be used to image and assess glucose metabolism of LAPC4-CR xenografts in vivo. Further work can investigate the use of PET/CT to quantify the metabolic response of LAPC4-CR to novel agents and combination therapies using soft tissue and possibly bone compartment xenograft models.

Using glow stick chemistry for biological imaging.

PURPOSE: This study describes an imaging strategy based on glow stick chemistry to non-invasively image oxidative stress and reactive oxygen species (ROS) production in living animals. PROCEDURES: Upon stimulation, phagocytes produce toxic levels of ROS to kill engulfed microorganisms. The mitochondrial respiratory chain continually generates low levels of superoxide (O2·(-)) that serve as a source for generation of downstream ROS, which function as regulatory signaling intermediaries. A ROS-reacting substrate, 2-methyl-6-[4-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one hydrochloride, was used as the chemical energy donor for generating energy transfer luminescence in phagosomes and mitochondria. RESULTS: Using targeted photoluminescent dyes with specific subcellular localization that serve as chemical energy recipients, our imaging data demonstrate proof-of-concept for using glow stick chemistry to visualize ROS production associated with phagocytosis and mitochondrial respiration in living mice. CONCLUSIONS: Glow stick imaging is a complementary hybrid of chemiluminescence and photoluminescence imaging, capable of generating red or far-red emission for deep tissue imaging.

Targeted imaging of Ewing sarcoma in preclinical models using a 64Cu-labeled anti-CD99 antibody.

PURPOSE: Ewing sarcoma is a tumor of the bone and soft tissue characterized by diffuse cell membrane expression of CD99 (MIC2). Single-site, surgically resectable disease is associated with an excellent 5-year event-free survival; conversely, patients with distant metastases have a poor prognosis. Noninvasive imaging is the standard approach to identifying sites of metastatic disease. We sought to develop a CD99-targeted imaging agent for staging Ewing sarcoma and other CD99-expressing tumors. EXPERIMENTAL DESIGN: We identified a CD99 antibody with highly specific binding in vitro and labeled this antibody with (64)Cu. Mice with either subcutaneous Ewing sarcoma xenograft tumors or micrometastases were imaged with the (64)Cu-labeled anti-CD99 antibody and these results were compared with conventional MRI and 2[18F]fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) imaging. RESULTS: (64)Cu-labeled anti-CD99 antibody demonstrated high avidity for the CD99-positive subcutaneous tumors, with a high tumor-to-background ratio, greater than that demonstrated with FDG-PET. Micrometastases, measuring 1 to 2 mm on MRI, were not detected with FDG-PET but were readily visualized with the (64)Cu-labeled anti-CD99 antibody. Probe biodistribution studies demonstrated high specificity of the probe for CD99-positive tumors. CONCLUSIONS: (64)Cu-labeled anti-CD99 antibody can detect subcutaneous Ewing sarcoma tumors and metastatic sites with high sensitivity, outperforming FDG-PET in preclinical studies. This targeted radiotracer may have important implications for the diagnosis, surveillance, and treatment of Ewing sarcoma. Similarly, it may impact the management of other CD99 positive tumors.

Co-clinical trials demonstrate superiority of crizotinib to chemotherapy in ALK-rearranged non-small cell lung cancer and predict strategies to overcome resistance.

PURPOSE: To extend the results of a phase III trial in patients with non-small cell lung cancer with adenocarcinomas harboring EML4-ALK fusion. EXPERIMENTAL DESIGN: We conducted a co-clinical trial in a mouse model comparing the ALK inhibitor crizotinib to the standard-of-care cytotoxic agents docetaxel or pemetrexed. RESULTS: Concordant with the clinical outcome in humans, crizotinib produced a substantially higher response rate compared with chemotherapy, associated with significantly longer progression-free survival. Overall survival was also prolonged in crizotinib- compared with chemotherapy-treated mice. Pemetrexed produced superior overall survival compared with docetaxel, suggesting that this agent may be the preferred chemotherapy in the ALK population. In addition, in the EML4-ALK-driven mouse lung adenocarcinoma model, HSP90 inhibition can overcome both primary and acquired crizotinib resistance. Furthermore, HSP90 inhibition, as well as the second-generation ALK inhibitor TAE684, demonstrated activity in newly developed lung adenocarcinoma models driven by crizotinib-insensitive EML4-ALK L1196M or F1174L. CONCLUSIONS: Our findings suggest that crizotinib is superior to standard chemotherapy in ALK inhibitor-naïve disease and support further clinical investigation of HSP90 inhibitors and second-generation ALK inhibitors in tumors with primary or acquired crizotinib resistance.

Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM, and ATR.

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC(50) of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases including ATM (EC(50), 28 nmol/L), ATR (EC(50), 35 nmol/L), and DNA-PK (EC(50), 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role.