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Ovarian clear cell carcinoma (OCCC) is a rare, chemo-resistant subtype of ovarian cancer. To identify novel therapeutic targets and combination therapies for OCCC, we subjected a set of patient-derived ovarian cancer cell lines to arrayed high-throughput siRNA and drug screening. The results indicated OCCC cells are vulnerable to knockdown of epigenetic gene targets such as bromodomain and extra-terminal domain (BET) proteins BRD2 and BRD3. Subsequent RNA interference assays, as well as BET inhibitor treatments, validated these BET proteins as potential therapeutic targets. Because development of resistance to single targeted agents is common, we next performed sensitizer drug screens to identify potential combination therapies with the BET inhibitor CPI0610. Several PI3K or AKT inhibitors were among the top drug combinations identified and subsequent work showed CPI0610 synergized with alpelisib or MK2206 by inducing p53-independent apoptosis. We further verified synergy between CPI0610 and PI3K–AKT pathway inhibitors alpelisib, MK2206, or ipatasertib in tumor organoids obtained directly from patients with OCCC. These findings indicate further preclinical evaluation of BET inhibitors, alone or in combination with PI3K-AKT inhibitors for OCCC, is warranted.

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Supplementary Figure 2. Gene Ontology (GO) enrichment analysis indicates OCI-C5x cells are more vulnerable to epigenetic interventions than OCI-P5x cells

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MYC-induced DNA damage is exacerbated in WRN-deficient cells, leading to replication stress and accelerated cellular senescence. To determine whether WRN deficiency impairs MYC-driven tumor development, we used both xenograft and autochthonous tumor models. Conditional silencing of WRN expression in c-MYC overexpressing non–small cell lung cancer xenografts impaired both tumor establishment and tumor growth. This inhibitory effect of WRN knockdown was accompanied by increased DNA damage, decreased proliferation, and tumor necrosis. In the Eμ-Myc mouse model of B-cell lymphoma, a germline mutation in the helicase domain of Wrn (WrnΔhel/Δhel) resulted in a significant delay in emergence of lethal lymphomas, extending tumor-free survival by more than 30%. Analysis of preneoplastic B cells from Eμ-Myc Wrn mutant mice revealed increased DNA damage, elevation of senescence markers, and decreased proliferation in comparison with cells from age-matched Eμ-Myc mice. Immunohistochemical and global gene expression analysis of overt Eμ-Myc WrnΔhel/Δhel lymphomas showed a marked increase in expression of the CDK inhibitor, p16Ink4a, as well as elevation of TAp63, a known mediator of senescence. Collectively, these studies show that in the context of Myc-associated tumorigenesis, loss of Wrn amplifies the DNA damage response, both in preneoplastic and neoplastic tissue, engaging activation of tumor suppressor pathways. This leads to inhibition of tumor growth and prolonged tumor-free survival. Targeting WRN or its enzymatic function could prove to be an effective strategy in the treatment of MYC-associated cancers. Mol Cancer Res; 10(4); 535–45. ©2012 AACR.

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Tables S9-17: Table S9. HT RNAi primary validation screen with siRNA pool deconvolution Table S10. Differential analysis of primary validation screen (CTG) N=12 Table S11. Differential analysis of primary validation screen (CTG) N=3 Pooled Table S12. Statistical analysis summary of differential viability (CTG) Table S13. Statistical analysis (absolute viability) of primary validation screen (CTG) Table S14. HT RNAi primary validation screen summary of hits (CTG) Table S15. HT caspase 3/7-dependent apoptosis AUC analysis (Apotox) N=12 Table S16. HT caspase 3/7-dependent apoptosis AUC analysis (Apotox) N=3 pooled Table S17. Statistical analysis summary caspase 3/7 dependent apoptosis (Apotox)

Supplementary Table Legends

Tables S1-8:Table S1. Characteristics of the HNSCC cell lines Table S2. Characteristics of the MSCC cell lines with germline mutations in p53 pathway genes Table S3. Human kinome (713 kinases) Sigma Table S4. Murine kinome (572 kinases) Ambion Table S5. Interspecies kinome (508 kinases) Table S6. Primary validation siRNAs Table S7. Secondary validation siRNAs Table S8. Interspecies kinase targets selected for validation

Figure S3. Correlation plots of autologous HNSCC cell line pairs utilizing kinomic profile

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next-generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS-mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TRAIL, effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy, with direct translational implications for current clinical trials on metastatic pancreatic cancer patients.Significance:This study utilizes functional genetic and pharmacological profiling of KRAS-mutant pancreatic adenocarcinoma to identify therapeutic strategies and finds that TFIIH inhibition synergizes with TRAIL to induce apoptosis in KRAS-driven pancreatic cancer.

Tables S18-20: Table S18. Secondary validation assay statistical analysis (absolute viability) Table S19. Differential viability and caspase 3/7 dependent apoptosis AUC of secondary validation assay Table S20. Statistical analysis summary differential viability and caspase 3/7 dependent apoptosis

Supplementary Data from Pharmacologic Targeting of TFIIH Suppresses KRAS-Mutant Pancreatic Ductal Adenocarcinoma and Synergizes with TRAIL

Supplementary Figure from Pharmacologic Targeting of TFIIH Suppresses KRAS-Mutant Pancreatic Ductal Adenocarcinoma and Synergizes with TRAIL

Purpose: To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC).Experimental Design: RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775.Results: Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2–M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model.Conclusions: WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. Clin Cancer Res; 20(16); 4274–88. ©2014 AACR.

Table S21: Table S21. Drug sensitivity and TP53 mutational status data on squamous cell carcinoma cell lines from COSMIC on Wee1/Chk1 Inhibitor (681460)

Ovarian clear cell carcinoma (OCCC) is a rare, chemo-resistant subtype of ovarian cancer. To identify novel therapeutic targets and combination therapies for OCCC, we subjected a set of patient-derived ovarian cancer cell lines to arrayed high-throughput siRNA and drug screening. The results indicated OCCC cells are vulnerable to knockdown of epigenetic gene targets such as bromodomain and extra-terminal domain (BET) proteins BRD2 and BRD3. Subsequent RNA interference assays, as well as BET inhibitor treatments, validated these BET proteins as potential therapeutic targets. Because development of resistance to single targeted agents is common, we next performed sensitizer drug screens to identify potential combination therapies with the BET inhibitor CPI0610. Several PI3K or AKT inhibitors were among the top drug combinations identified and subsequent work showed CPI0610 synergized with alpelisib or MK2206 by inducing p53-independent apoptosis. We further verified synergy between CPI0610 and PI3K–AKT pathway inhibitors alpelisib, MK2206, or ipatasertib in tumor organoids obtained directly from patients with OCCC. These findings indicate further preclinical evaluation of BET inhibitors, alone or in combination with PI3K-AKT inhibitors for OCCC, is warranted.

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next-generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS-mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TRAIL, effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy, with direct translational implications for current clinical trials on metastatic pancreatic cancer patients. Significance: This study utilizes functional genetic and pharmacological profiling of KRAS-mutant pancreatic adenocarcinoma to identify therapeutic strategies and finds that TFIIH inhibition synergizes with TRAIL to induce apoptosis in KRAS-driven pancreatic cancer.

The oncogene Ras and the tumor suppressor gene p53 are frequently co-mutated in human cancer and mutations in Ras and p53 can cooperate to generate a more malignant cell state. To discover novel druggable targets for cancers carrying co-mutations in Ras and p53, we performed arrayed, kinome focused siRNA and oncology drug phenotypic screening utilizing a set of syngeneic Ras mutant squamous cell carcinoma (SCC) cell lines that also carried co-mutations in selected p53 pathway genes. These cell lines were derived from SCCs from carcinogen-treated inbred mice which harbored germline deletions or mutations in Trp53, p19

Cells are complex systems in which many functions are performed by different genetically defined and encoded functional modules. To systematically understand how these modules respond to drug or genetic perturbations, we develop a functional module states framework. Using this framework, we (1) define the drug-induced transcriptional state space for breast cancer cell lines using large public gene expression datasets and reveal that the transcriptional states are associated with drug concentration and drug targets, (2) identify potential targetable vulnerabilities through integrative analysis of transcriptional states after drug treatment and gene knockdown-associated cancer dependency, and (3) use functional module states to predict transcriptional state-dependent drug sensitivity and build prediction models for drug response. This approach demonstrates a similar prediction performance as approaches using high-dimensional gene expression values, with the added advantage of more clearly revealing biologically relevant transcriptional states and key regulators.

SummaryCells are complex systems in which many functions are performed by different genetically-defined and encoded functional modules. To systematically understand how these modules respond to drug or genetic perturbations, we developed a Functional Module States framework. Using this framework, we 1) defined the drug induced transcriptional state space for breast cancer cell lines using large public gene expression datasets, and revealed that the transcriptional states are associated with drug concentration and drug targets; 2) identified potential targetable vulnerabilities through integrative analysis of transcriptional states after drug treatment and gene knockdown associated cancer dependency; and 3) used functional module states to predict transcriptional state-dependent drug sensitivity and built prediction models using the functional module states for drug response. This approach demonstrates a similar prediction performance as do approaches using high dimensional gene expression values, with the added advantage of more clearly revealing biologically relevant transcriptional states and key regulators.

Ionizing radiation (IR) is one of the most widely used treatments for cancer. However, acute damage to the gastrointestinal tract or gastrointestinal acute radiation syndrome (GI-ARS) is a major dose-limiting side effect, and the mechanisms that underlie this remain unclear. Here we use mouse models to explore the relative roles of DNA repair, apoptosis, and cell cycle arrest in radiation response. IR induces DNA double strand breaks and DNA-PK mutant Prkdcscid/scid mice are sensitive to GI-ARS due to an inability to repair these breaks. IR also activates the tumor suppressor p53 to trigger apoptotic cell death within intestinal crypt cells and p53 deficient mice are resistant to apoptosis. To determine if DNA-PK and p53 interact to govern radiosensitivity, we compared the response of single and compound mutant mice to 8 Gy IR. Compound mutant Prkdcscid/scid/Trp53−/−mice died earliest due to severe GI-ARS. While both Prkdcscid/scid and Prkdcscid/scid/Trp53−/−mutant mice had higher levels of IR-induced DNA damage, particularly within the stem cell compartment of the intestinal crypt, in Prkdcscid/scid/Trp53−/−mice these damaged cells abnormally progressed through the cell cycle resulting in mitotic cell death. This led to a loss of Paneth cells and a failure to regenerate the differentiated epithelial cells required for intestinal function. IR-induced apoptosis did not correlate with radiosensitivity. Overall, these data reveal that DNA repair, mediated by DNA-PK, and cell cycle arrest, mediated by p53, cooperate to protect the stem cell niche after DNA damage, suggesting combination approaches to modulate both pathways may be beneficial to reduce GI-ARS. As many cancers harbor p53 mutations, this also suggests targeting DNA-PK may be effective to enhance sensitivity of p53 mutant tumors to radiation.

SummaryEpigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf+/− tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf+/− tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.

Background and Objectives: Clear cell carcinoma of the ovary (OCCC) is more resistant to platinum-based chemotherapy than high-grade serous ovarian cancer (HGSOC). Also, the indication for PARP inhibitors seems to be limited in OCCC because of the lower frequency of homologous recombination deficiency. As genomic background significantly differs among subtypes, therapeutic strategies should be optimized in response to each subtype or unique gene alterations. This study was designed to find novel therapeutic targets for OCCC by utilizing siRNA screens and unique patient-derived ovarian cancer cells that phenocopy original tumors. Materials and Methods: Novel patient-derived ovarian cancer cells were established and kindly provided by Dr. Tan Ince at University of Miami. Genomic alterations of these cells were determined by DNA sequencing. siRNA screens targeting 2,172 unique genes were performed with ARID1A-mutated OCCC cell line OCI C5x, and high-grade serous ovarian cancer cell line OCI P5x. Genes inhibition of which significantly suppressed cell proliferation were selected as hits for each subtype independently. The difference of cell viability between OCI C5x and P5x was also compared, and subtype-specific hits were additionally determined. Hits were applied to Gene Ontology (GO) enrichment analysis to comprehend the gene set enrichment. Several prioritized targets for OCCC were validated with additional RNAi experiments and small molecule inhibitors. Results: 114 and 108 genes were selected as hits specific to OCI C5x and OCI P5x, respectively. GO enrichment analysis revealed OCCC is potentially more vulnerable to epigenetic interventions than HOGSC. The following inspection revealed bromodomain-containing proteins were enriched in the hits for OCI C5x. Among these, we highlighted bromodomain and extra-terminal domain (BET) family proteins BRD2 and BRD3. Subsequent RNAi experiments validated BRD2 and BRD3 are highly potent therapeutic targets for OCCC. Conclusions: siRNA screening results suggest different approaches are required according to subtypes for the targeted therapy of ovarian cancer. We propose that epigenetic intervention such as BET protein inhibition as a potential therapeutic option for OCCC, 50-60% of which harbor ARID1A mutation. Citation Format: Shogo Shigeta, Reid Shaw, Russell Moser, Kay Gurley, Goldie Lui, Carla Grandori, Christopher Kemp. TARGETING BROMODOMAIN PROTEINS AS A THERAPEUTIC STRATEGY FOR CLEAR CELL CARCINOMA OF THE OVARY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-108.

Hepatic haemangiosarcoma is a deadly malignancy whose aetiology remains poorly understood. Inactivation of the CDKN2A locus, which houses the ARF and p16INK4a tumour suppressor genes, is a common event in haemangiosarcoma patients, but the precise role of ARF in vascular tumourigenesis is unknown. To determine the extent to which ARF suppresses vascular neoplasia, we examined the incidence of hepatic vascular lesions in Arf-deficient mice exposed to the carcinogen urethane [intraperitoneal (i.p.), 1 mg/g]. Loss of Arf resulted in elevated morbidity and increased the incidence of both haemangiomas and incipient haemangiosarcomas. Suppression of vascular lesion development by ARF was heavily dependent on both Arf gene-dosage and the genetic strain of the mouse. Trp53-deficient mice also developed hepatic vascular lesions after exposure to urethane, suggesting that ARF signals through a p53-dependent pathway to inhibit the development of hepatic haemangiosarcoma. Our findings provide strong evidence that inactivation of Arf is a causative event in vascular neoplasia and suggest that the ARF pathway may be a novel molecular target for therapeutic intervention in haemangiosarcoma patients. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MYC-induced DNA damage is exacerbated in WRN-deficient cells, leading to replication stress and accelerated cellular senescence. To determine whether WRN deficiency impairs MYC-driven tumor development, we used both xenograft and autochthonous tumor models. Conditional silencing of WRN expression in c-MYC overexpressing non–small cell lung cancer xenografts impaired both tumor establishment and tumor growth. This inhibitory effect of WRN knockdown was accompanied by increased DNA damage, decreased proliferation, and tumor necrosis. In the Eμ-Myc mouse model of B-cell lymphoma, a germline mutation in the helicase domain of Wrn (WrnΔhel/Δhel) resulted in a significant delay in emergence of lethal lymphomas, extending tumor-free survival by more than 30%. Analysis of preneoplastic B cells from Eμ-Myc Wrn mutant mice revealed increased DNA damage, elevation of senescence markers, and decreased proliferation in comparison with cells from age-matched Eμ-Myc mice. Immunohistochemical and global gene expression analysis of overt Eμ-Myc WrnΔhel/Δhel lymphomas showed a marked increase in expression of the CDK inhibitor, p16Ink4a, as well as elevation of TAp63, a known mediator of senescence. Collectively, these studies show that in the context of Myc-associated tumorigenesis, loss of Wrn amplifies the DNA damage response, both in preneoplastic and neoplastic tissue, engaging activation of tumor suppressor pathways. This leads to inhibition of tumor growth and prolonged tumor-free survival. Targeting WRN or its enzymatic function could prove to be an effective strategy in the treatment of MYC-associated cancers. Mol Cancer Res; 10(4); 535–45. ©2012 AACR.

p53 is required for DNA damage-induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53-deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA-dependent protein kinase (DNA-PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation-induced apoptosis, whereas apoptosis in DNA-PK(cs)/p53, Ku80/p53 and Ku70/p53 double-null mice was quantitatively equivalent to that seen in wild-type mice. This p53-independent apoptotic response was specific to the loss of DNA-PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double-null mice. Furthermore, it was associated with an increase in phospho-checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA-PK, does not require p53.

Realization of high temperature physical measurement sensors, which are needed in many of the current and emerging technologies, is challenging due to the degradation of their electrical stability by drift currents, material oxidation, thermal strain, and creep. In this paper, for the first time, we demonstrate that 3D printed sensors show a metamaterial-like behavior, resulting in superior performance such as high sensitivity, low thermal strain, and enhanced thermal stability. The sensors were fabricated using silver (Ag) nanoparticles (NPs), using an advanced Aerosol Jet based additive printing method followed by thermal sintering. The sensors were tested under cyclic strain up to a temperature of 500 °C and showed a gauge factor of 3.15 ± 0.086, which is about 57% higher than that of those available commercially. The sensor thermal strain was also an order of magnitude lower than that of commercial gages for operation up to a temperature of 500 °C. An analytical model was developed to account for the ...

Purpose: To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC). Experimental Design: RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Results: Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2–M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model. Conclusions: WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. Clin Cancer Res; 20(16); 4274–88. ©2014 AACR.

Non-small-cell lung carcinoma (NSCLC) is among the deadliest of human cancers. The CDKN2A locus, which houses the INK4a and ARF tumor suppressor genes, is frequently altered in NSCLC. However, the specific role of ARF in pulmonary tumorigenesis remains unclear. KRAS and other oncogenes induce the expression of ARF, thus stabilizing p53 activity and arresting cell proliferation. To address the role of ARF in Kras-driven NSCLC, we compared the susceptibility of NIH/Ola strain wild-type and Arf-knockout mice to urethane-induced lung carcinogenesis. Lung tumor size, malignancy and associated morbidity were significantly increased in Arf(-/-) compared with Arf(+/+) animals at 25 weeks after induction. Pulmonary tumors from Arf-knockout mice exhibited increased cell proliferation and DNA damage compared with wild-type mice. A subgroup of tumors in Arf(-/-) animals presented as dedifferentiated and metastatic, with many characteristics of pulmonary sarcomatoid carcinoma, a neoplasm previously undocumented in mouse models. Our finding of a role for ARF in NSCLC is consistent with the observation that benign adenomas from Arf(+/+) mice robustly expressed ARF, while ARF expression was markedly reduced in malignant adenocarcinomas. ARF expression also frequently colocalized with the expression of p21(CIP1), a transcriptional target of p53, arguing that ARF induces the p53 checkpoint to arrest cell proliferation in vivo. Taken together, these findings demonstrate that induction of ARF is an early response in lung tumorigenesis that mounts a strong barrier against tumor growth and malignant progression.

The effects of chronic lead administration on renal function and cytoarchitecture and on the immunohistochemical localization of glutathione S-transferase (GST) isoenzymes were determined. Pregnant rats were given 250 ppm lead acetate in drinking water from conception until weaning and mothers and pups received 500 ppm of lead acetate from weaning until termination at either 3 or 7 weeks of age. Light and electron microscopic studies after 3 weeks of lead administration showed tubular injury with frequent mitoses noted in proximal tubular cells and, after 7 weeks of treatment, interstitial fibrosis, characteristic intranuclear inclusions, and tubular injury characterized by both nuclear and cytoplasmic pleomorphism. Rats treated with lead for 7 weeks showed significantly lower body weights and creatinine clearances than age-matched control animals. Immunohistochemical studies of glutathione transferase subunits in control rats showed unique isoform localization in each segment of the nephron; treatment with lead caused large increases in immunoreactive protein of Yc, Yk, Yb1, and Yp GST subunits in proximal tubules. No increases in the antioxidant enzymes copper-zinc superoxide dismutase, catalase, and glutathione peroxidase were found in lead-treated rats, but there was a diffuse lead-related increase in immunoreactive protein for manganese superoxide dismutase throughout the renal cortex. Our results demonstrate large lead-induced increases of specific isoforms of glutathione S-transferase in specific kidney cell types and show that these increases preceded irreversible renal damage.

The effects of acute and chronic exposure to lead on glutathione S-transferase (GST) isoforms were determined in developing kidney in the rat. The ontogeny of glutathione S-transferase isoforms was characterized as were the effects of depletion of dietary calcium on glutathione S-transferase isoform profiles in control and lead-treated rats. In the acute exposure experiments, rats of 14 and 50 days of age received three daily injections of lead acetate (114 mg/kg) and in the chronic exposure studies, rats received lead acetate at doses ranging from 50 to 500 ppm in their drinking water. Lead acetate administration in these chronic studies began 1 day after conception. Acute and chronic lead exposure had similar effects, causing increases in all but one glutathione S-transferase isoform (Yb3); these increases were markedly exacerbated by dietary calcium depletion. In all lead paradigms, GST subunits Yb1 and Yp showed the largest increases--greater than 25-fold in rats fed a low-calcium diet. GST subunit Yb3 showed small increases in the 14-day acute lead and the 4 week low-calcium animals and did not increase in other groups. Lead-related increases in GSTs were partially reversed by transferring animals previously receiving lead to lead-free water for a 4-week period. Kidneys of rats fed the low-calcium diet did not have detectable GST Yk, but in rats on this low-calcium diet that received 500 ppm lead; this GST isoform was found at levels comparable to those in control rats fed lab chow.

p27(kip1) is a cyclin-dependent kinase inhibitor and a tumor suppressor. In some tumors, p27 suppresses tumor growth by inhibition of cell proliferation. However, this is not universally observed, implying additional mechanisms of tumor suppression by p27. p27-deficient mice are particularly susceptibility to genotoxin-induced tumors, suggesting a role for p27 in the DNA damage response. To test this hypothesis, we measured genotoxin-induced mutations and chromosome damage in p27-deficient mice. Both p27(+/-) and p27(-/-) mice displayed a higher N-ethyl-N-nitrosourea-induced mutation frequency in the colon than p27(+/+) littermates. Furthermore, cells from irradiated p27-deficient mice exhibited a higher number of chromatid breaks and showed modestly increased micronucleus formation compared to cells from wild-type littermates. To determine if this mutator phenotype was related to the cell cycle-inhibitory function of p27, we measured cell cycle arrest in response to DNA damage. Both normal and tumor cells from p27-deficient mice showed impaired G(2)/M arrest following low doses of ionizing radiation. Thus, p27 may inhibit tumor development through two mechanisms. The first is by reducing the proliferation of cells that have already sustained an oncogenic lesion. The second is by transient inhibition of cell cycle progression following genotoxic insult, thereby minimizing chromosome damage and fixation of mutations.

Since the groundbreaking studies in the middle part of the last century showing liver cancer in rodents exposed to aromatic amines, the liver has been widely used as a model target organ of chemical carcinogenesis. This protocol describes a method for inducing liver tumors by injecting mice with the widely used alkylating agents N-ethyl-N-nitrosourea (ENU) and N-nitrosodiethylamine (DEN). ENU does not require metabolic activation and readily induces tumors in a number of tissues, including the lungs, stomach, and ovaries, as well as inducing lymphomas. Mice injected with DEN can also develop other tumors, including those of the gastrointestinal tract, skin, lungs, and lymphocytes, but because DEN is metabolized in the liver, it is most effective at inducing liver tumors.

In this protocol, colon cancer is induced in mice through a series of injections with 1,2-dimethylhydrazine. Mice will develop primarily colon tumors starting at about 3 mo after the first injection. Tumors in the lung, uterus, and small intestine may also be seen, as well as lymphomas.

In this protocol, urethane (ethyl carbamate) is used to induce lung tumors in mice. The use of urethane as an experimental carcinogen is especially attractive as it is inexpensive, relatively safe to handle, stable, and water soluble, and the protocol involves simple intraperitoneal (i.p.) injections in young mice. Urethane typically induces bronchioalveolar adenomas and, to a lesser extent, adenocarcinomas that resemble the adenocarcinoma subtype of non–small cell lung carcinoma. On a sensitive genetic background such as A/J, mice develop multiple adenomas visible on the lung surface by 25 wk, followed by the appearance of adenocarcinomas by 40 wk. Less-sensitive strains such as B6/129 develop tumors with a longer latency.

Introduction: Identifying molecular aberrations that drive carcinogenesis remains a daunting problem. Uncovering important combinations of such aberrations, e.g. overexpression of a gene in concordance with copy number amplification, pose additional challenges. Publicly available large-scale cell line panels profiled for drug response often do not reflect the true tumor heterogeneity and therefore predictions made solely based on such data may be difficult to translate into therapeutic targets. Integrating predicted associations with drug sensitivity (resistance) on one hand, with siRNA screens with the same drug (patient-derived samples and cell lines) on the other hand, should allow for the identification of therapeutically relevant molecular traits. Methods: We propose a novel Bayesian model for multi-task learning with a gene-wise prior, which accounts for multiple aberrations in a given gene (expression, mutation and copy number). Our method allows for the simultaneous learning of drug response for multiple drugs in a given panel (N_Sanger=138, N_CCLE=24 drugs). Our model aims to identifying sets of important aberrations, which individually occur at low frequencies. To train and evaluate the performance of our model we use the CCLE and Sanger datasets. Further, we use the Cisplatin and Doxorubicin drug responses from the Sanger dataset to rank genome-wise aberrations to find features best predictive of response to the chemotherapeutic agents. We then consider our findings in the context of high-throughput siRNA synthetic lethal screens in head and neck, ovarian and breast cancer with Cisplatin and Doxorubicin in patient-derived samples and cell lines. Results: Our approach improves the quality of drug sensitivity prediction (Pearson correlation in five-fold cross validation) over methods that model individual drug responses (e.g. Elastic Net). It further allows for the subtyping of drugs by target/mechanism of action commonalities, which we recover closely for drugs with known mechanism of action. Our model recovers reasonably the targets of consensus drug interactions (e.g. Erlotinib and EGFR) and in addition identifies other known associations currently undergoing preclinical/clinical validation (i.e. Cisplatin and JAK2 mutation, and RUNX3 expression and Doxorubicin). Integrated analysis of the results from our predictive model in conjunction with the siRNA screens are currently under investigation. Conclusions: We present an integrated approach that combines a novel Bayesian multi-task learning model with high-throughput siRNA screens. Our approach aims to uncover sets of important aberrations and allows for the subtyping of drugs based on similarities in targets and mechanisms of action. We integrate our results with high-throughput RNAi experiments to identify synthetic lethal events in specific therapeutic context. Citation Format: Olga H. Nikolova, Mehmet Gönen, Rodrigo Dienstmann, In Sock Jang, Russell Moser, Silvia Cermelli, Chang Xu, Ryan M. Mitchell, Eduardo Mendez, Carla Grandori, Christopher Kemp, Stephen Friend, Justin Guinney, Adam Margolin. Integrated computational cell-line modeling of drug sensitivity and high-throughput siRNA screening reveals novel molecular biomarkers for conventional chemotherapy. [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 5323. doi:10.1158/1538-7445.AM2014-5323

p53 deficient cells are resistant to apoptosis, which contributes to malignant progression and therapy resistance of p53 mutant tumors. For this reason, identifying and targeting p53 independent cell death pathways is an important therapeutic goal. We previously reported that the DNA damage kinases Atm and DNA-PK exhibit p53 independent synthetic lethal interactions (Current Biology 11:191-194, 2001) and that DNA-PK regulates a DNA damage induced apoptotic response that is p53 independent (EMBO Reports, 10:87-93, 2009). To identify additional synthetic lethal interactions with the p53 pathway, we used high throughput siRNA screens to interrogate the kinomes of both human and mouse tumor cell cultures stratified for p53 status and metastatic potential. Mouse cells included low passage squamous cell carcinoma (SCC) cells isolated from tumors from five genotypes of mice: wild type, Atm-/-, DNA-PKcs-/-, p19/Arf -/- and p53+/-. Human cells were derived from both primary and metastatic head and neck squamous cell carcinomas (HNSCC). siRNA kinome screens were run in the presence of three concentrations of the chemotherapy agent doxorubicin to identify genotype specific sensitizers. We identified numerous additional synthetic partners with each p53 pathway mutation: Atm, DNA-PKcs, p19/Arf, and p53. These synthetic lethal interactions varied with doxorubicin exposure, thus defining gene targets that sensitize tumor cells of defined genotype to genotoxic therapy. Cross species comparison and independent validation was used to prioritize 20 survival kinases specific to p53 mutant metastatic HNSCC. These kinases are involved in phosphatidylinositol, Src, focal adhesion and integrin signaling pathways, and G2/M cell cycle transition. We selected the G2/M regulatory kinase, WEE1, for preclinical testing as a small molecule inhibitor is available with potential for clinical application. Relative to p53 wild type SCC cells, p53 deficient cells were more sensitive to the WEE1 inhibitor MK-1775, as well as the CHK1 inhibitor AZD7762 providing chemical confirmation of our siRNA screen results. Examination of 42 additional SCC cell lines revealed that p53 mutation conferred increased sensitivity to a dual WEE1/CHK1 inhibitor. Treatment of mice bearing xenografts of p53 mutant HNSCC cells with MK-1775, either alone or in conjunction with cisplatin, impaired tumor growth and in some cases led to tumor regression. These results demonstrate the utility of high throughput siRNA screens applied to genetically defined early passage tumor cells combined with cross species comparisons to efficiently pinpoint novel drug targets for p53 mutant cancers. This abstract is also presented as Poster A04. Citation Format: Russell Moser, Chang Xu, Michael Kao, In Sock Jang, Kay Gurley, Adam Margolin, Carla Grandori, Eddie Mendez, Chris Kemp. Construction of synthetic lethal networks for p53 tumor suppressor pathways identifies candidate therapeutic targets for metastatic, chemotherapy resistant HNSCC. [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 PR05.

While changes in the epigenetic landscape are ubiquitous in cancer, the origin and causal effects of most of these changes on neoplastic progression are largely unknown. The DNA binding protein Ctcf, regulates a diverse array of epigenetic processes, including DNA methylation spreading, but is role in cancer is unclear. Here we show that Ctcf heterozygous knockout mice are remarkably susceptible to spontaneous, ionizing radiation, and chemically-induced neoplasia in a broad range of tissues. Analysis of lung tumors from Ctcf+/− mice establishes Ctcf as a haploinsufficient tumor suppressor gene whose loss cooperates with mutant Kras to accelerate tumor growth and malignant progression. Increased DNA methylation at Ctcf binding sites in normal tissues from Ctcf+/− mice indicates that reduction of Ctcf leads to epigenetic deregulation and this occurs prior to overt neoplastic transformation. This indicates epigenetic instability can predispose a broad range of tissues to neoplastic transformation. A mouse model of Ctcf regulated epigenetic stability provides a useful setting to dissect the mechanistic link between environmental exposure, and risk of cancer or other diseases with an epigenetic component. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 118. doi:1538-7445.AM2012-118

Non-small cell lung carcinoma (NSCLC) is among the deadliest of human cancers, with an overall five-year survival rate of 10–20%. Activating mutations in the Kras proto-oncogene occur in approximately one-third of NSCLC patients, but tumor suppression in the lung remains poorly elucidated. The Cdkn2a locus, which houses both the p16lnk4a and Arf tumor suppressor genes, is frequently altered in NSCLC; however, the specific role of Arf in lung tumorigenesis has not been well characterized. Several lines of evidence indicate that Kras and other oncogenes induce the expression of Art, which in turn stabilizes p53 activity and arrests the growth of transformed cells. To determine the extent to which Arf suppresses NSCLC development in vivo, we employed the urethane chemical carcinogenesis model of Kras-driven NSCLC. We treated both wild-type and Arf-deficient mice with urethane and found that early-stage lung adenomas in wild-type mice robustly expressed Arf, In contrast to adenomas, malignant lung adenocarcinomas exhibited little induction of Art. Moreover, Arf-deficient mice exposed to urethane displayed accelerated lung tumor-associated morbidity and significantly increased lung tumor size compared to Arf wild-type and heterozygous littermates. Malignant progression of adenomas to adenocarcinomas was also markedly elevated in Arf-deficient mice. These data demonstrate that Arf functions as a barrier to tumor promotion and progression in a carcinogen model of Kras-driven NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A34.