Scott Peterson

Requirement for the Kinase Activity of Human DNA- Dependent Protein Kinase Catalytic Subunit in DNA Strand Break Rejoining

ABSTRACT The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.

Stimulation of the DNA-dependent Protein Kinase by Poly(ADP-Ribose) Polymerase

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1,5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.

Disruption of DNA-PK in Ku80 mutant xrs-6 and the implications in DNA double-strand break repair.

The Chinese hamster ovary (CHO) mutant cell line xrs-6C is highly sensitive to radiation and is deficient in DNA double-strand break (DSB) repair. The repair defect of xrs-6C is complemented by the human DSB repair gene designated as XRCC5. This gene was recently identified as Ku80, which encodes the human autoantigen protein Ku p80. Ku80 protein forms heterodimer with the Ku70 subunit to form a complex that possesses a DNA end-binding activity. Ku70/Ku80 heterodimer can recruit the catalytic p350 subunit of the DNA-dependent protein kinase. It is demonstrated here that, while the Ku70 mRNA expression is normal in the xrs-6C mutant, Ku70 protein is undetectable. However, introduction of human Ku80 gene into the mutant lead to increased expression of Ku70 protein and restored Ku70 binding to DNA ends, suggesting that mutation of the Ku80 gene affected the formation of Ku70/Ku80 dimers and the stability of the Ku70 protein. We also demonstrated that, although p350 protein expression in the mutants was unaffected, the capacity of p350 to bind to DNA ends was impaired in the mutants. After introduction of the human Ku80 into the mutant, the association of p350 with DNA end was restored, accompanied by recovery in cell survival and DNA double-strand break repair. The results in this report show that mutation of the Ku80 gene disrupts formation of the Ku70/Ku80 dimer and compromises the ability of Ku protein to recruit the DNA-PK p350 subunit to DNA double-strand breaks, causing a dysfunction of DNA DSB repair in the cell.

Self-association of human RAD52 protein

The yeast RAD52 protein is required for both homologous DNA recombination and repair of DNA double-strand breaks. RAD52 can bind to the yeast RAD51 protein, which shares a functional similarity with the bacterial RecA protein. The gene encoding the human homolog of the yeast RAD52 protein shares significant N-terminus amino acid homology with the yeast RAD52 protein. Using a yeast two hybrid system and purified GST-RAD52 fusion protein, we demonstrate that the human RAD52 protein self-associates both in vivo and in vitro. The region of RAD52 required for its self-interaction, mapped here as amino acid residues 65-165, has significant homology with the yeast RAD52 (52% identity, and 89% similarity), suggesting the importance of self-association for RAD52s function.

Digitally collected cryo‐electron micrographs for single particle reconstruction

Several advantages and disadvantages have been cited for image collection with a slow‐scan CCD camera. Here we explore its use for cryo‐EM single particle reconstruction and present two practical examples. The icosahedral adenovirus (Ad) type 2 (∼150 MDa) was reconstructed from 396 particle images. The Fourier shell correlation (FSC) 0.5 threshold and the Fourier shell phase residual (FSPR) 45°criterion yielded 17 ÅA resolution for the ordered viral capsid. Visual comparison with the filtered Ad2 crystallographic hexon confirmed a resolution range of 15–17 Å. The asymmetric DNA‐PKcs protein (470 kDa) was reconstructed from 9,473 particle images, using a previously published reconstruction based on class‐sum images as an orientational search model [Chiu et al. (1998) J. Mol. Biol. 284:1075–1081]. FSC and FSPR methods yielded 17 Å resolution for the new DNA‐PKcs reconstruction, indicating a small but noticeable improvement over that of the class‐sum based reconstruction. Despite the lack of symmetry for DNA‐PKcs and its lower image contrast compared to Ad2 (0.8% vs. 2.5%), the same resolution was obtained for both particles by averaging significantly more DNA‐PKcs images. Use of the CCD camera enables the microscopist to adjust the electron beam strength interactively and thereby maximize the image contrast for beam sensitive samples. On‐line Fourier transformation also allows routine monitoring of drift and astigmatism during image collection, resulting in a high percentage of micrographs suitable for image processing. In conclusion, our results show that digital image collection with the YAG‐scintillator slow‐scan CCD camera is a viable approach for 3D reconstruction of both symmetric and asymmetric particles. Microsc. Res. Tech. 49:224–232, 2000.

Abstract 762: ONT-10, a liposomal vaccine targeting hypoglycosylated MUC1, induces a potent cellular and humoral response and suppresses the growth of MUC1 expressing tumors

MUC1 is highly expressed in multiple tumor histologies and is hypoglycosylated relative to normal tissue, with prematurely terminated carbohydrate modifications including αN-acetylgalactosamine (Tn). The aberrant glycosylation state of MUC1 makes this antigen immunologically distinct in tumor relative to normal tissues and an attractive vaccine target. ONT-10 is a liposomal formulated vaccine that incorporates a synthetic glycopeptide antigen that includes two complete 20 amino acid tandem repeats from human MUC1, including six glycosylated sites modified by Ser- or Thr-O- Tn, and is incorporated into the liposomal drug product via two C-terminal lipid serines. The ONT-10 drug product is formulated with the novel, fully synthetic lipid A adjuvant, penta erythritol lipid A (PET Lipid A). PET Lipid A displays a similar spatial distribution of lipid chains as natural Lipid A, but is composed of pure 6-acyl moieties, resulting in enhanced potency as an activator of Toll-like Receptor 2 and 4 (TLR2, TLR4). In preclinical studies, immunization of mice with ONT-10 induces a prominent T cell and antibody response to MUC1 and results in a substantial growth inhibition of MUC1 expressing tumors. T cell recall assays show a Th1 cytokine pattern with strong IFNγ production in spleen derived CD4 and CD8 cells in response to Tn-MUC1. At doses as low as 5μg/mouse ONT-10 results in a high titer anti-MUC1 antibody response composed principally of IgG2b and IgG3 that show preferential binding to Tn-MUC1 and selectively target tumor cells that express human MUC1. In a preventative B16-MUC1 tumor model, vaccination with 5μg ONT-10 potently suppresses tumor growth, resulting in 99% mean tumor growth inhibition relative to controls with 9/12 animals tumor free four weeks after tumor cell implantation. Similarly, in a MC38-MUC1 tumor model 5μg ONT-10 results in an 90% reduction in tumor growth relative to controls. Based on these preclinical data, we propose that the combination of the glycopeptide antigen structure and improved Lipid A adjuvant in ONT-10 offer advantages relative to previous MUC1 targeted vaccines by potently activating both humoral and cellular immunity against hypoglycosylated MUC1 and that these features may provide additional therapeutic benefit to patients with MUC1 expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 762. doi:10.1158/1538-7445.AM2011-762

Abstract 652: PI3K inhibition combined with either cetuximab or docetaxel in a direct patient tumor model of HPV-positive and negative head and neck cancers

Purpose: Data suggest that human papilloma virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) are distinct from HPV negative tumors. We have generated a direct patient tumor xenograft model (DPTM) of HNSCC. DPTM maintain tumor histology better than cell lines and are optimally positioned to investigate in vivo tumor sensitivity and changes in tumor initiating cell (TIC) biology in response to therapy based upon HPV status. Phosphatidyl inositol 3 kinase (PI3K) is a downstream signal of epidermal growth factor receptor (EGFR) that may play a critical role in TIC biology and resistance to chemotherapy or anti-EGFR monoclonal antibodies in HNSCC. PX-866 is an irreversible pan-isoform inhibitor of class 1 PI3K currently in early clinical trials. We aimed to test the in vivo efficacy of PX-866, cetuximab, docetaxel, or a combination of PX-866 and cetuximab or docetaxel. Methods: DPTM were left untreated or exposed to PX-866, docexatel, cetuximab, or a combination of PX-866 plus cetuximab or docetaxel for 25-29 days. Tumor measurements were taken twice weekly during therapy and thereafter until study conclusion. TIC population were determined by flow cytometry. Results: We have treated three DPTM (CUHN015, CUHN022, CUHN027) with PX-866, cetuximab, or PX-866 plus cetuximab, and three DPTM (CUHN004, CUHN011, CUHN015) with PX-866, docetaxel, or the combination of PX-866 plus docetaxel. Neither combination had increased toxicity compared with the single agents. All six untreated DPTM had rapid tumor growth. In two DPTM (CUHN015 and CUHN022) cetuximab was similar to no treatment, whereas single agent PX-866 was slightly more effective than no treatment in CUHN022 and significantly more effective in CUHN015. In CUHN027 both PX-866 and cetuximab arrested growth but the combination caused greater than 50% tumor reduction. The combination of cetuximab and PX-866 was more effective than cetuximab alone in all DPTM. Two xenografts treated with PX-866, docetaxel, or the combination showed decreased tumor growth with PX-866 compared to either docetaxel or no treatment (CUHN004 and CUHN015). In all three DTPM the combination of PX-866 and docetaxel was more effective than either single agent. In the HPV-positive xenograft (CUHN022) cetuximab had no effect. Treatment with chemotherapeutic agents induced TIC accumulation that was partially reversed by combination therapy. Conclusions: PX-866 was equal or superior to cetuximab or docetaxel at slowing tumor growth in 5 of 6 DPTM of HNSCC. The combination of PX-866 plus another agent was superior to single agent therapy in 5 of 6 DPTM. Cetuximab was more effective in HPV-negative cases. Correlations with pathway gene mutations and pharmacodynamic events, including pathway inhibition and TIC subpopulation dynamics, will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 652. doi:10.1158/1538-7445.AM2011-652

Abstract 578: Discovery and characterization of novel antagonistic antibodies that bind with high affinity to human, cynomolgus, and murine TIGIT, an immune checkpoint receptor

TIGIT is a recently identified coinhibitory immune checkpoint receptor expressed on NK, effector T, and regulatory T cells. In the oncology setting, TIGIT is upregulated on tumor infiltrating immune cells and is co-expressed with exhaustion markers including PD-1, TIM-3 and LAG-3 on infiltrates. TIGIT binds at least two ligands, CD155 and CD112, which are expressed on antigen presenting cells and other tissues, including tumor cells. These ligands also bind the activating receptor CD226, often co-expressed with TIGIT, creating a network that modulates adaptive and innate immune response in a manner analogous to the CD28-CTLA4-CD80-CD86 network. The absence of TIGIT signaling, resulting from genetic deficiency or blockade, enhances anti-tumor immunity in murine models, suggesting that disruption of TIGIT signaling may have clinical utility. To explore this concept, yeast antibody display was used to identify fully human, anti-TIGIT antibodies that block binding to ligands. Multiple rounds of selection with human and mouse TIGIT protein were performed to promote species cross-reactivity, diversity and affinity. A pool of 695 unique clones were screened for binding to TIGIT protein; 65 clones were then selected for further evaluation. Of the 65, 63 competed with CD155 for binding to TIGIT in a ForteBio screen. Fifty-three clones bound cyno TIGIT and 25 bound TIGIT from all three species (human, cyno, mouse). Antibodies bound endogenous TIGIT on primary T cells and blocked binding of ligands to cell surface expressed TIGIT in a dose dependent manner. Twelve clones showed functional activity in a TIGIT blockade bioassay and showed synergy with anti-PD-1 antibody in a PD-1/TIGIT combination bioassay. Activity in the bioassays correlated with affinity for recombinant and cell surface expressed TIGIT. Based on species cross-reactivity, binding affinity and activity in the bioassays, a lead candidate antibody was selected and produced as mouse IgG1 and IgG2a chimeras for testing in mouse tumor models. The chimeric antibodies behaved similarly to the parent clone in vitro exhibiting high affinity for TIGIT, competition with ligand for binding to TIGIT, and functional blockade of CD155-TIGIT interaction. Evaluation of the chimeric anti-TIGIT candidates alone and in combination with anti-PD-1 antibody in mouse syngeneic tumor models is ongoing, and results will be reported at the meeting. Antibody mediated blockade of coinhibitory immunoreceptors has proven clinically efficacious and supports the development of antibodies that target TIGIT. The unique human, non-human primate, and murine cross-reactive TIGIT-specific antibodies described here offer a simplified preclinical development path and the functional activity of these molecules supports their consideration as candidates for therapeutic development. Citation Format: Julia C. Piasecki, Kenneth Brasel, Robert Rosler, Kevin M. Klucher, Scott R. Peterson. Discovery and characterization of novel antagonistic antibodies that bind with high affinity to human, cynomolgus, and murine TIGIT, an immune checkpoint receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 578. doi:10.1158/1538-7445.AM2017-578

Abstract 4090: Preclinical pharmacokinetics of CASC-578, a novel, selective, potent, and orally bioavailable small molecule checkpoint kinase 1 inhibitor

Introduction: Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division in response to genotoxic stress by arresting cell cycle progression in the S & G2 phases. Pharmacological inhibition of Chk1 is proposed to selectively uncouple the completion of DNA replication from G2/M phase transition in tumor cells that have impaired DNA damage response networks, resulting in mitotic catastrophe and cell death. CASC-578 is a novel small molecule inhibitor of Chk1 that is selective, highly potent and orally bioavailable in multiple preclinical species. Methods: The in vitro ADME properties of CASC-578 were evaluated, including in vitro intrinsic microsomal clearance, Caco2 permeability, plasma protein binding and stability, blood to plasma partitioning, cytochrome P450 inhibition and induction, and transporter inhibition. Pharmacokinetic studies of CASC-578 were conducted in mice, rats, and cynomolgus monkeys as either single dose (IV and/or PO) or repeat dose (PO only). Multiple oral dose pharmacokinetic studies were conducted in mice, rats and cynomolgus monkeys for 5-7 days. Results: CASC-578 is highly bound in plasma protein across all species. The apparent permeability is high in Caco2 bi-directional transport study and correlated well with a rapid absorption profile observed in vivo. CASC-578 is not a substrate of P-glycoporotein (efflux =1). There was no direct nor time dependent inhibition on human CYP450 enzymes, and only a slight induction of CYP3A4 was seen at 10 uM drug concentration in a transporter cell-based induction assay. In all animal species, CASC-578 exhibited species-dependent systemic clearance resulting from both phase I and phase II metabolism, and a moderate to high volume of distribution. The elimination kinetics appeared to be monophasic. Oral bioavailability was high in all species studied (>60% F). There was no significant difference in the pharmacokinetics of the drug between genders. Maximal plasma concentration and total drug exposure (AUC) appeared to be proportional from repeat dose studies. Overall, CASC-578 has very desirable drug-like properties and ideal pharmacokinetics for an oral once daily drug, and represents a suitable candidate for clinical development as a novel potential therapeutic approach for the treatment of solid and hematological malignancies Citation Format: Dina Leviten, Teresa Sierra, Ashley Dozier, Richard Boyce, Bob Boyle, Scott Peterson, Alex C. Vo. Preclinical pharmacokinetics of CASC-578, a novel, selective, potent, and orally bioavailable small molecule checkpoint kinase 1 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4090. doi:10.1158/1538-7445.AM2017-4090

Abstract 295: CASC-578, a novel Chk1 inhibitor, is active as a single agent in solid tumors and displays synergistic anti-tumor activity in combination with Wee1 inhibition

Background: Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division in response to genotoxic stress by arresting cell cycle progression in the S & G2 phases. Pharmacological inhibition of Chk1 is proposed to target tumor cells with increased DNA replication stress, resulting in the uncoupling of DNA replication checkpoint function and the induction of DNA damage and cell death. These properties make Chk1 inhibition a novel therapeutic approach as a single agent in cancers with high replication stress that is driven by oncogenic signaling and loss of parallel DNA damage response pathway function. Methods and Results: This report highlights the activity of the orally bioavailable, selective small molecule Chk1 inhibitor, CASC-578, in solid tumor derived cell lines. CASC-578 is a sub-nanomolar enzymatic inhibitor of Chk1 with limited off-target activity against a panel of protein kinases. When evaluated in a large cell line panel in vitro, CASC-578 demonstrated a broad potency range as a single agent in solid tumor derived cells lines, with IC50s ranging from 30 nM to greater than 50 μM. Several solid tumor types demonstrated enriched sensitivity to CASC-578 in vitro, including gastric, non-small cell lung and ovarian cancers. Treatment of sensitive cell lines with CASC-578 resulted in the induction of DNA damage, as measured by phosphorylated histone H2AX, and the induction of cell death. CASC-578 was active as a single agent in SK-MES-1 and NCI-H727 NSCLC tumor xenograft models in vivo with minimal effects on body weight in treated mice. In addition to the potent single agent activity of CASC-578, combination with the Wee1 inhibitor AZD-1775 was highly synergistic in vitro in multiple solid tumor cell lines and the combination was more efficacious than either agent alone in NSCLC tumor xenograft models. These data support the advancement of CASC-578 into clinical development as a potential therapeutic agent for the treatment of solid tumor diseases. Experiments are ongoing to identify biomarkers associated with sensitivity to CASC-578 as a single agent in solid tumor cell lines to prospectively identify tumor genotypes that are more responsive to the drug. Citation Format: Alex Vo, Janelle Taylor, Robert Rosler, Julia Piasecki, Dina Leviten, Teresa Sierra, Ashley Dozier, Kevin Klucher, Bob Boyle, Rich Boyce, Scott Peterson. CASC-578, a novel Chk1 inhibitor, is active as a single agent in solid tumors and displays synergistic anti-tumor activity in combination with Wee1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2017-295