Kevin A. Heist

Metastatic Pancreatic Cancer Is Dependent on Oncogenic Kras in Mice

Pancreatic cancer is one of the deadliest human malignancies, and its prognosis has not improved over the past 40 years. Mouse models that spontaneously develop pancreatic adenocarcinoma and mimic the progression of the human disease are emerging as a new tool to investigate the basic biology of this disease and identify potential therapeutic targets. Here, we describe a new model of metastatic pancreatic adenocarcinoma based on pancreas-specific, inducible and reversible expression of an oncogenic form of Kras, together with pancreas-specific expression of a mutant form of the tumor suppressor p53. Using high-resolution magnetic resonance imaging to follow individual animals in longitudinal studies, we show that both primary and metastatic lesions depend on continuous Kras activity for their maintenance. However, re-activation of Kras* following prolonged inactivation leads to rapid tumor relapse, raising the concern that Kras*-resistance might eventually be acquired. Thus, our data identifies Kras* as a key oncogene in pancreatic cancer maintenance, but raises the possibility of acquired resistance should Kras inhibitors become available for use in pancreatic cancer.

DW-MRI as a biomarker to compare therapeutic outcomes in radiotherapy regimens incorporating temozolomide or gemcitabine in glioblastoma.

The effectiveness of the radiosensitizer gemcitabine (GEM) was evaluated in a mouse glioma along with the imaging biomarker diffusion-weighted magnetic resonance imaging (DW-MRI) for early detection of treatment effects. A genetically engineered murine GBM model [Ink4a-Arf−/− PtenloxP/loxP/Ntv-a RCAS/PDGF(+)/Cre(+)] was treated with gemcitabine (GEM), temozolomide (TMZ) +/− ionizing radiation (IR). Therapeutic efficacy was quantified by contrast-enhanced MRI and DW-MRI for growth rate and tumor cellularity, respectively. Mice treated with GEM, TMZ and radiation showed a significant reduction in growth rates as early as three days post-treatment initiation. Both combination treatments (GEM/IR and TMZ/IR) resulted in improved survival over single therapies. Tumor diffusion values increased prior to detectable changes in tumor volume growth rates following administration of therapies. Concomitant GEM/IR and TMZ/IR was active and well tolerated in this GBM model and similarly prolonged median survival of tumor bearing mice. DW-MRI provided early changes to radiosensitization treatment warranting evaluation of this imaging biomarker in clinical trials.

Multi-site clinical evaluation of DW-MRI as a treatment response metric for breast cancer patients undergoing neoadjuvant chemotherapy

Purpose To evaluate diffusion weighted MRI (DW-MR) as a response metric for assessment of neoadjuvant chemotherapy (NAC) in patients with primary breast cancer using prospective multi-center trials which provided MR scans along with clinical outcome information. Materials and Methods A total of 39 patients with locally advanced breast cancer accrued from three different prospective clinical trials underwent DW-MR examination prior to and at 3–7 days (Hull University), 8–11 days (University of Michigan) and 35 days (NeoCOMICE) post-treatment initiation. Thirteen patients, 12 of which participated in treatment response study, from UM underwent short interval (<1hr) MRI examinations, referred to as “test-retest” for examination of repeatability. To further evaluate stability in ADC measurements, a thermally controlled diffusion phantom was used to assess repeatability of diffusion measurements. MRI sequences included contrast-enhanced T1-weighted, when appropriate, and DW images acquired at b-values of 0 and 800 s/mm2. Histogram analysis and a voxel-based analytical technique, the Parametric Response Map (PRM), were used to derive diffusion response metrics for assessment of treatment response prediction. Results Mean tumor apparent diffusion coefficient (ADC) values generated from patient test-retest examinations were found to be very reproducible (|ΔADC|<0.1x10-3mm2/s). This data was used to calculate the 95% CI from the linear fit of tumor voxel ADC pairs of co-registered examinations (±0.45x10-3mm2/s) for PRM analysis of treatment response. Receiver operating characteristic analysis identified the PRM metric to be predictive of outcome at the 8–11 (AUC = 0.964, p = 0.01) and 35 day (AUC = 0.770, p = 0.05) time points (p<.05) while whole-tumor ADC changes where significant at the later 35 day time interval (AUC = 0.825, p = 0.02). Conclusion This study demonstrates the feasibility of performing a prospective analysis of DW-MRI as a predictive biomarker of NAC in breast cancer patients. In addition, we provide experimental evidence supporting the use of sensitive analytical tools, such as PRM, for evaluating ADC measurements.

Phosphorylation of FADD by the kinase CK1α promotes KRASG12D-induced lung cancer

Mutant KRAS promotes mitosis by stimulating phosphorylation of the adaptor protein FADD. Conversion of a death adaptor to a proliferation mediator Activating mutations in the protein RAS drive cell proliferation and tumor growth. Although best known for mediating cell death signaling through its death domain, when phosphorylated, the adaptor protein FADD promotes cell survival and proliferation. Bowman et al. found that, compared with KRAS mutant mice, KRAS mutant mice engineered to lack FADD or its upstream kinase CK1α developed fewer lung tumors. Lung tissue and cells from KRAS mutant mice had increased abundance of CK1α, phosphorylated FADD, and proliferative markers. In lung tumor samples from patients, expression of FADD was greater in tumors that had mutant KRAS. A CK1α inhibitor prevented FADD from physically interacting with mitotic kinases and suppressed cell proliferation in culture. Thus, blocking the phosphorylation of FADD may be a new strategy for patients with KRAS mutant lung tumors. Genomic amplification of the gene encoding and phosphorylation of the protein FADD (Fas-associated death domain) is associated with poor clinical outcome in lung cancer and in head and neck cancer. Activating mutations in the guanosine triphosphatase RAS promotes cell proliferation in various cancers. Increased abundance of phosphorylated FADD in patient-derived tumor samples predicts poor clinical outcome. Using immunohistochemistry analysis and in vivo imaging of conditional mouse models of KRASG12D-driven lung cancer, we found that the deletion of the gene encoding FADD suppressed tumor growth, reduced the proliferative index of cells, and decreased the activation of downstream effectors of the RAS–MAPK (mitogen-activated protein kinase) pathway that promote the cell cycle, including retinoblastoma (RB) and cyclin D1. In mouse embryonic fibroblasts, the induction of mitosis upon activation of KRAS required FADD and the phosphorylation of FADD by CK1α (casein kinase 1α). Deleting the gene encoding CK1α in KRAS mutant mice abrogated the phosphorylation of FADD and suppressed lung cancer development. Phosphorylated FADD was most abundant during the G2/M phase of the cell cycle, and mass spectrometry revealed that phosphorylated FADD interacted with kinases that mediate the G2/M transition, including PLK1 (Polo-like kinase 1), AURKA (Aurora kinase A), and BUB1 (budding uninhibited by benzimidazoles 1). This interaction was decreased in cells treated with a CKI-7, a CK1α inhibitor. Therefore, as the kinase that phosphorylates FADD downstream of RAS, CK1α may be a therapeutic target for KRAS-driven lung cancer.

Evaluation of Concurrent Radiation, Temozolomide and ABT-888 Treatment Followed by Maintenance Therapy with Temozolomide and ABT-888 in a Genetically Engineered Glioblastoma Mouse Model

Despite the use of ionizing radiation (IR) and temozolomide (TMZ), outcome for glioblastoma (GBM) patients remains dismal. Poly (ADP-ribose) polymerase (PARP) is important in repair pathways for IR-induced DNA damage and TMZ-induced alkylation at N7-methylguanine and N3-methyldenine. However, optimized protocols for administration of PARP inhibitors have not been delineated. In this study, the PARP inhibitor ABT-888 was evaluated in combination with and compared to current standard-of-care in a genetically engineered mouse GBM model. Results demonstrated that concomitant TMZ/IR/ABT-888 with adjuvant TMZ/ABT-888 was more effective in inducing apoptosis and reducing proliferation with significant tumor growth delay and improved overall survival over concomitant TMZ/IR with adjuvant TMZ. Diffusion-weighted MRI, an early translatable response biomarker detected changes in tumors reflecting response at 1 day post TMZ/IR/ABT-888 treatment. This study provides strong scientific rationale for the development of an optimized dosing regimen for a PARP inhibitor with TMZ/IR for upfront treatment of GBM.

MRI-Guided Stereotactic Biopsy of Murine GBM for Spatiotemporal Molecular Genomic Assessment

Brain tumor biopsies that are routinely performed in clinical settings significantly aid in diagnosis and staging. The aim of this study is to develop and evaluate a methodological image-guided approach that would allow for routine sampling of glioma tissue from orthotopic mouse brain tumor models. A magnetic resonance imaging-guided biopsy method is presented to allow for spatially precise stereotaxic sampling of a murine glioma coupled with genome-scale technology to provide unbiased characterization of intra- and intertumoral clonal heterogeneity. Longitudinal and multiregional sampling of intracranial tumors allows for successful collection of tumor biopsy samples, thus allowing for a pathway-enrichment analysis and a transcriptional profiling of RNA sequencing data. Spatiotemporal gene expression pattern variations revealing genomic heterogeneity were found.

A Bifunctional MAPK/PI3K Antagonist for Inhibition of Tumor Growth and Metastasis

Responses to targeted therapies frequently are brief, with patients relapsing with drug-resistant tumors. For oncogenic MEK and BRAF inhibition, drug resistance commonly occurs through activation of PI3K/AKT/mTOR signaling and immune checkpoint modulation, providing a robust molecular target for concomitant therapy. Here, we evaluated the efficacy of a bifunctional kinase inhibitor (ST-162) that concurrently targets MAPK and PI3K signaling pathways. Treatment with ST-162 produced regression of mutant KRAS- or BRAF-addicted xenograft models of colorectal cancer and melanoma and stasis of BRAF/PTEN–mutant melanomas. Combining ST-162 with immune checkpoint blockers further increased efficacy in a syngeneic KRAS-mutant colorectal cancer model. Nascent transcriptome analysis revealed a unique gene set regulated by ST-162 related to melanoma metastasis. Subsequent mouse studies revealed ST-162 was a potent inhibitor of melanoma metastasis to the liver. These findings highlight the significant potential of a single molecule with multikinase activity to achieve tumor control, overcome resistance, and prevent metastases through modulation of interconnected cell signaling pathways. Mol Cancer Ther; 16(11); 2340–50. ©2017 AACR.

Ultrasmall Paramagnetic Iron Oxide Nanoprobe Targeting Epidermal Growth Factor Receptor for In Vivo Magnetic Resonance Imaging of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common worldwide cancer that is rising rapidly in incidence. MRI is a powerful noninvasive imaging modality for HCC detection, but lack of specific contrast agents limits visualization of small tumors. EGFR is frequently overexpressed in HCC and is a promising target. Peptides have fast binding kinetics, short circulatory half-life, low imaging background, high vascular permeability, and enhanced tissue diffusion for deep tumor penetration. We demonstrate a peptide specific for EGFR labeled with an ultrasmall paramagnetic iron oxide (UPIO) nanoparticle with 3.5 nm dimensions to target HCC using T1-weighted MRI. We modified the hydrophobic core with oleic acid and capped with PEGylated phospholipids DSPE-PEG and DSPE-PEG-Mal. The EGFR peptide is attached via thioether-mediated conjugation of a GGGSC linker to the maleimide-terminated phospholipids. On in vivo MR images of HCC xenograft tumors, we observed peak nanoprobe uptake at 2 h post-injection followed by a rapid return to baseline by ∼24 h. We measured significantly greater MR signal in tumor with the targeted nanoprobe versus scrambled peptide, blocked peptide, and Gadoteridol. Segmented regions on MR images support rapid renal clearance. No significant difference in animal weight, necropsy, hematology, and chemistry was found between treatment and control groups at one month post-injection. Our nanoprobe based on an EGFR specific peptide labeled with UPIO designed for high stability and biocompatibility showed rapid tumor uptake and systemic clearance to demonstrate safety and promise for clinical translation to detect early HCC.

Abstract 106: A requirement for FADD and its phosphorylation for KRAS-driven oncogenesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Lung cancer accounts for 27% of all cancer deaths worldwide. 80% of these deaths are due to a specific subtype Non-small cell lung cancer (NSCLC). Almost half of all NSCLC patients carry gatekeeper mutations in the EGFR/KRAS pathway. Although KRAS mutations are the most prevalent mutations in human cancers, therapies targeting KRAS have fallen short. Tyrosine kinase inhibitors have been successful in NSCLC patients with mutant or amplified EGFR, however resistance to these inhibitors is common. Thus therapies targeting mutant KRAS or its downstream effectors are desperately needed for the treatment of NSCLC. FAS Associated Death Domain (FADD) is a well characterized adaptor protein involved in death receptor mediated apoptosis, but more recently has been implicated in the regulation of other cellular functions such as cell cycle and proliferation. Increasing evidence suggests that it is FADDs phosphorylated state which regulates these non-apoptotic signaling pathways. We and others have previously identified phosphorylated FADD as a predictor of aggressiveness and clinical stage in lung and head and neck cancer patients, respectively. Using non-invasive bioluminescence and µCT imaging in a Kras driven mouse model of NSCLC, we demonstrate the requirement for FADD in tumor progression. Lung-specific activation of KrasG12D and simultaneous FADD deletion resulted in reduced lung tumor burden. Furthermore lung lesions of KrasG12D FADD null mice were significantly smaller with a decrease in proliferative index. Similar findings were obtained in MEFs, where siRNA mediated Kras knockdown or the use of MEK inhibitors resulted in a Kras dependent decrease in FADD phosphorylation, decrease in cell cycle progression and cell proliferation. These data propound the phosphorylation of FADD as a downstream event of Kras signaling and demonstrate its requirement for Kras dependent neoplasia in a mouse model for NSCLC. In summary our findings demonstrate that elevated FADD and phosphorylated FADD levels are predictive of poor survival in an animal model of NSCLC agreeing with clinical findings. Furthermore, it implicates phosphorylation of FADD downstream of KRAS activation and thus suggests FADD phosphorylation as a valid target in the treatment of NSCLC patients. Citation Format: Brittany M. Bowman, Stefanie Galban, Benjamin A. Hoff, Kevin A. Heist, Jennifer L. Boes, Craig J. Galban, Rajiv M. Patel, Jianke Zhang, Brian D. Ross, Alnawaz Rehemtulla. A requirement for FADD and its phosphorylation for KRAS-driven oncogenesis. [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 106. doi:10.1158/1538-7445.AM2014-106

Abstract 4433: DW-MR imaging as a predictive biomarker of radiosensitization of GBM through targeted inhibition of checkpoint kinases.

Purpose: The inherent treatment resistance of glioblastoma (GBM) can involve multiple mechanisms including checkpoint kinase (Chk1/2) mediated increased DNA repair capability which can attenuate the effects of genotoxic chemotherapies and radiation. The goal of this study was to evaluate DW-MRI as a biomarker for Chk1/2 inhibitors in combination with radiation for enhancement of treatment efficacy in GBM. Experimental Design: We evaluated a specific small molecule inhibitor of Chk1/2, AZD7762, in combination with radiation using in vitro human cell lines and in vivo using a genetically engineered GBM mouse model. Diffusion-weighted and T1-contrast MRI were used to follow treatment effects on intracranial tumor cellularity and growth rates, respectively. Results: AZD7762 inhibited clonal proliferation in a panel of GBM cell lines and increased radiosensitivity in p53-mutated GBM cell lines to a greater extent compared to p53-wild type cells. In vivo efficacy of AZD7762 demonstrated a dose-dependent inhibitory effect on GBM tumor growth rate and a reduction in tumor cellularity based on DW-MRI scans along with enhancement of radiation efficacy. Conclusion: DW-MRI was found to be a useful imaging biomarker for the detection of radiosensitization through inhibition of checkpoint kinases. Chk1/2 inhibition resulted in antiproliferative activity, prevention of DNA-damage induced repair, and radiosensitization in preclinical GBM tumor models, both in vitro and in vivo. The effects were found to be maximal in p53-mutated GBM cells. These results provide the rationale for integration of DW-MRI in clinical translation of Chk1/2 inhibition with radiation for the treatment of GBM. Citation Format: Terence Williams, Stefanie Galban, Fei Li, Kevin Heist, Craig Galban, Theodore S. Lawrence, Eric C. Holland, Tami Thomae, Thomas Chenevert, Alnawaz Rehemtulla, Brian D. Ross. DW-MR imaging as a predictive biomarker of radiosensitization of GBM through targeted inhibition of checkpoint kinases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4433. doi:10.1158/1538-7445.AM2013-4433