Stephen S. Yoo

Cellular Inhibition of Checkpoint Kinase 2 (Chk2) and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor PV1019 [7-Nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide]

Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4′-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) activity by small molecule GMX1778 regulates reactive oxygen species (ROS)-mediated cytotoxicity in a p53- and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-dependent manner.

Background: GMX1778 is an inhibitor of nicotinamide phosphoribosyltransferase for the regeneration of NAD+ from nicotinamide. Results: GMX1778 increases intracellular ROS in cancer cells but does not induce ROS in normal cells. Conclusion: Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumor cells. Significance: Selectively modulating intracellular ROS in cancers by GMX1778 provides a useful therapeutic opportunity. Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD+ from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD+ level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.

Opportunities and Challenges in the Era of Molecularly Targeted Agents and Radiation Therapy

The first annual workshop for preclinical and clinical development of radiosensitizers took place at the National Cancer Institute on August 8-9, 2012. Radiotherapy is one of the most commonly applied and effective oncologic treatments for solid tumors. It is well recognized that improved clinical efficacy of radiotherapy would make a substantive impact in clinical practice and patient outcomes. Advances in genomic technologies and high-throughput drug discovery platforms have brought a revolution in cancer treatment by providing molecularly targeted agents for various cancers. Development of predictive biomarkers directed toward specific subsets of cancers has ushered in a new era of personalized therapeutics. The field of radiation oncology stands to gain substantial benefit from these advances given the concerted effort to integrate this progress into radiation therapy. This workshop brought together expert clinicians and scientists working in various disease sites to identify the exciting opportunities and expected challenges in the development of molecularly targeted agents in combination with radiation therapy.

Decreasing the Adverse Effects of Cancer Therapy: An NCI Workshop on the Preclinical Development of Radiation Injury Mitigators/Protectors

The study of radiation mitigators/protectors for use in cancer treatment and management is complex as they must reduce normal tissue toxicity without reducing tumor cell kill. The known toxicology, pharmacokinetic, and mechanistic data for the proposed agent will direct investigators to the appropriate stage of preclinical development. Overall, we hope that this Workshop commentary facilitates the transition of radiation mitigators/protectors into clinical trials.

Lessons Learned from Radiation Oncology Clinical Trials

A workshop entitled “Lessons Learned from Radiation Oncology Trials” was held on December 7–8, 2011, in Bethesda, MD, to present and discuss some of the recently conducted radiation oncology clinical trials with a focus on those that failed to refute the null hypothesis. The objectives of this workshop were to summarize and examine the questions that these trials provoked, to assess the quality and limitations of the preclinical data that supported the hypotheses underlying these trials, and to consider possible solutions to these challenges for the design of future clinical trials. Several themes emerged from the discussions: (i) opportunities to learn from null-hypothesis trials through tissue and imaging studies; (ii) value of preclinical data supporting the design of combinatorial therapies; (iii) significance of validated biomarkers; (iv) necessity of quality assurance in radiotherapy delivery; (v) conduct of sufficiently powered studies to address the central hypotheses; and (vi) importance of publishing results of the trials regardless of the outcome. The fact that well-designed hypothesis-driven clinical trials produce null or negative results is expected given the limitations of trial design and complexities of cancer biology. It is important to understand the reasons underlying such null results, however, to effectively merge the technologic innovations with the rapidly evolving biology for maximal patient benefit through the design of future clinical trials. Clin Cancer Res; 19(22); 6089–100. ©2013 AACR.

A high content clonogenic survival drug screen identifies MEK inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer

Introduction: Traditional clonogenic survival and high throughput colorimetric assays are inadequate as drug screens to identify novel radiation sensitizers. We developed a method that we call the high content clonogenic survival assay (HCSA) that will allow screening of drug libraries to identify candidate radiation sensitizers. Methods: Drug screen using HCSA was done in 96 well plates. After drug treatment, irradiation, and incubation, colonies were stained with crystal violet and imaged on the INCell 6000 (GE Health). Colonies achieving 50 or more cells were enumerated using the INCell Developer image analysis software. A proof-of-principle screen was done on the KRAS mutant lung cancer cell line H460 and a Custom Clinical Collection (146 compounds). Results: Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several PARP inhibitors were identified as good radiation sensitizers in the HCSA screen. However, there were also a few PARP inhibitors not found to be sensitizing that have either not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor. We discovered that inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation. Furthermore, the potent MEK1/2 inhibitor tramenitib selectively enhanced radiation effects in KRAS mutant but not wild-type lung cancer cells. Conclusions: Drug screening for novel radiation sensitizers is feasible using the HCSA approach. This is an enabling technology that will help accelerate the discovery of novel radiosensitizers for clinical testing.

Abstract 2506: Palomid 529, a PI3K/Akt/mTOR dual TORC1/2 inhibitor, is a radiosensitizer with effect in both subcutaneous and orthotopic U251 glioblastoma tumor xenograft models

Palomid 529 (P529) is a small molecule drug created through three generations of computational design. P529 is an anti-tumor agent able to target and inhibit the PI3K/Akt/mTOR signal transduction pathway, specifically as an allosteric dual TORC1/TORC2 inhibitor causing the dissociation of the TORC complexes. Furthermore, P529 has been shown to broadly inhibit cell lines of the NCI 60 cell screen, inhibit HIF-1α, and show tumor growth delay in murine tumor xenograft models. P529 is able to cross the blood-brain barrier and show little or no effect of two transporters (P-gp and Bcrp1) in brain uptake of P529. Here we show that P529 has a more than additive radiation sensitizing activity inhibiting tumor growth in U251 glioblastoma tumor xenograft models both subcutaneous and orthotopic. P529 was administered in a dose-dependent manner at 25, 50 and 95 mg/kg oral dosing every day for 5 days with 4 Gy radiation given on day three. Tumor xenografts in both subcutaneous and orthotopic models were evaluated daily for tumor size or survival respectively up to 40 days post tumor implantation. P529 treatment alone showed marked decrease in tumor growth (subcutaneous) and increase in survival (orthotopic). With addition of radiation, there was a synergistic increase in activity of P529 on the inhibition of U251 tumor growth and survival. All in all, work described here provides evidence that P529 has activity as a radiosensitizer in murine models of glioblastoma and the possibility of efficacious activity in treatment of human patients under a novel mechanism of inhibition of the PI3K/Akt/mTOR pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2506.

Cellular Inhibition of Chk2 Kinase and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor Pv1019.

Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4′-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

Abstract 4898: High content clonogenic survival assay for drug library screening identifies potent radiation sensitizers for non-small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Radiotherapy is a critical component in the definitive treatment of locally-advanced non-small cell lung cancer (LA-NSCLC), and while concurrent platinum-based chemoradiation is the standard approach, improvements have been minimal. Molecularly-targeted therapy to further enhance radiation effects beyond platinum-sensitization is needed to further improve radiotherapy efficacy. A systematic approach needs to be developed that can rapidly identify novel drugs that can be combined to enhance radiation effects. However, current approaches using traditional clonogenic survival assay (tCSA) and colorimetric assays are inadequate for high throughput drug screens to identify novel radiation sensitizers. To enable screens adapted for radiation drug screening, we have developed a method which we coin the High Throughput Clonogenic Survival Assay (HCSA) that allows rapid screening of drug libraries to identify potent radiation sensitizers. As a proof-of-principle, we used the Kras mutated lung cancer cell line H460, and validated the hits using traditional assays. Drugs at various concentrations or vehicle were added to cells seeded overnight in 96 well plates at low densities, and after 6 hours, plates were irradiated at various doses. After incubation, colonies were stained and imaged on the INCell 6000® (GE Health), and colony count determined using the INCell Developer image analysis software. Drug screens were done using a Custom Clinical Collection of 150 compounds. We compared the HCSA method to tCSA and found that HCSA recapitulates a similar cell survival curve and survival fraction at 2 Gy (SF2) values for several cancer cell types. Drug screens identified several compounds that were cytotoxic to the KRAS mutant H460 cells alone at 1 µM (inhibitors to Src/Abl, IGF1R, ROCK1, AKT, PI3K, c-MET) and at low nM concentrations (HSP90 and HDAC inhibitors). Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several late generation PARP inhibitors were found to be good sensitizers in the HCSA, but the ones that have not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor, do not come out in the screen. We also discovered that several inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation, which is biologically relevant for this Kras mutant cell line. We believe these results demonstrate the promise of HCSA as a high throughput drug screening platform to identify novel radiation sensitizers. This will greatly expedite the discovery of novel targets and drugs for clinical translation to enhance the effects of radiotherapy in lung cancer. Citation Format: Steven H. Lin, Hui Liu, Yawei Qiao, Uma Giri, Jing Zhang, Clifford Stephan, Mary Sobieski, John V. Heymach, Stephen S. Yoo. High content clonogenic survival assay for drug library screening identifies potent radiation sensitizers for non-small cell lung cancer. [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 4898. doi:10.1158/1538-7445.AM2014-4898

Abstract B08: High-throughput synthetic lethal drug screen to identify novel radiation sensitizers for KRAS mutant non-small cell lung cancer

Traditional clonogenic survival and high throughput colorimetric assays are inadequate for drug screens to identify novel radiation sensitizers. To bridge this gap in knowledge, we have developed a method which we coin the High Throughput Clonogenic Survival Screen (HCS) that will allow high volume screening of drug libraries to identify potent radiation sensitizers, particularly for KRAS mutant lung cancer, a current unmet need. Drugs at various concentrations or vehicle were added to cells seeded overnight in 96 well plates at low densities, and after 6 hours, plates were irradiated at various doses. Plates were kept in culture 4-6 days depending on the cell line. After incubation, colonies were stained with cresyl-violet, imaged on the INCell 6000® (GE Health), and colony count determined using the INCell Developer image analysis software. Colonies achieving 50 or more cells were tallied. Traditional clonogenic survival assays (tCSA) were performed by seeding cells into 6-well plates overnight in triplicate and adding drugs or vehicle for 6 hours prior to irradiation. Media was changed after 3 days, and plates were kept in culture for 10-14 days. Drug screens were done using the KRAS mutant lung cancer cell line H460 and the Custom Clinical Collection (145 compounds). We compared the HCS method to tCSA and found that HCS recapitulates a similar cell survival curve and survival fraction at 2 Gy (SF2) values for several cancer cell types. As a proof-of-principle to demonstrate that HCS could identify radiation sensitizers, we tested a known sensitizer vorinostat (SAHA) in both the tCSA and HCS assays. As expected, 1 μM vorinostat caused a significant radiation sensitizing effect using tCSA, and was recapitulated, albeit at a lower concentration (100 nM), using the HCS assay. Drug screens (repeated 3 times) identified several compounds that were cytotoxic to the KRAS mutant H460 cells alone at 1 μM (inhibitors to Src/Abl, IGF1R, ROCK1, AKT, PI3K, c-MET) and at low nM concentrations (HSP90 and HDAC inhibitors). The entire class of HDAC inhibitors (5 drugs) had some sensitizing effect to radiation (a leftward shift in the IC50 curve by ~10 fold). However, many drugs such as inhibitors to PARP, SRC, Chk1/2, DNA-PK, JNK, and MEK1/2, exhibited significant radiation sensitizing effect with minimal to no activity by themselves. We validated the sensitizing effect of the potent MEK1/2 inhibitor trametinib using tCSA, and found the effect was specific for KRAS mutant and not KRAS wild type cells. From these results, we believe high throughput drug screening for novel radiation sensitizers for KRAS mutant lung cancer is feasible using the HCS approach. This is an enabling technology that will allow synthetic lethal drug screens with radiation and accelerate the translation of drugs into clinical testing. Citation Format: Steven H. Lin, Jing Zhang, Uma Giri, Clifford Stephan, Mary Sobieski, Stephen S. Yoo, John V. Heymach. High-throughput synthetic lethal drug screen to identify novel radiation sensitizers for KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B08.