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Background: The serine/threonine kinase STK11/LKB1 is the second most commonly altered tumor suppressor in NSCLC. Non-functional mutations or loss of LKB1 expression occur more frequently in NSCLC than other alterations; however, there are currently no effective treatment strategies for this subset of tumors. KRAS-mutant LKB1 deficient NSCLC tumors often also have alterations in KEAP1 or NRF2 gene, which activate the KEAP1/NRF2 pathway known to be involved in antioxidant response. Inhibitors of ATM and ATR, two key proteins in the DNA damage response (DDR) pathway, are currently undergoing clinical testing but there are no validated biomarkers established for identifying which subgroups of patients are more likely to benefit from treatment. Here we have identified that alterations of LKB1, and the KEAP1/NRF2 pathway, are associated with enhanced response to ATM and ATR inhibitors (ATMi and ATRi) as well as other inhibitors of the DDR and may be useful biomarkers for predicting therapeutic response. Methods: To investigate the impact of LKB1 loss and KEAP1/NRF2 pathway activation on DDR and replication stress, we first tested replication fork protection in LKB1 deficient cells (KL). DNA fiber assay showed a defect in fork protection in KL cells compared with LKB1 wild type cells (K). Also, RPPA analysis revealed an activation of ATR/Chk1/Cdk1/CyclinB1 axis as well as Wee1 activation in cells harboring LKB1 and/or KEAP1 loss (KL/KLK/KK). Therefore, to evaluate response to DDR inhibitors (DDRi) we analyzed the in vitro activity of ATM, ATR, Wee1 and PARP inhibitors in NSCLC murine cell lines with or without knock out of LKB1 and/or KEAP1. In these cells, the loss of LKB1 and/or KEAP1 significantly sensitized to AZD0156 (ATMi), AZD6738 (ATRi) and AZD1775 (Wee1i) relative to cells with intact LKB1 and KEAP1. Next, we investigated whether the activity of ATR and ATMi in KL, KK or KLK tumor cells could be enhanced by the addition of a PARP inhibitor (olaparib). Although all NSCLC cells were resistant to the PARP inhibitor olaparib when used as a single agent, treatment of LKB1, KEAP1 or LKB1 plus KEAP1 deficient cells with the combination of olaparib plus ATM or ATR inhibitors significantly enhanced the antitumor cell activity of ATM or ATR inhibitors alone in vitro. We confirmed these data in an additional panel of LKB1 deficient NSCLC human cell lines treated with a broad spectrum of ATR and ATM inhibitors. In all human cell lines re-expression of LKB1 clearly reduced the sensitivity to ATR inhibition. LKB1 loss was also associated with sensitivity to PARP and ATM inhibitors, although these effects seemed to be less significant compared with ATR inhibitors. Interestingly, in vivo experiments performed in K, KL and KLK syngeneic models as well as PDX models showed greater response to ATRi and Wee1i monotherapy only in KLK but not in K or KL tumor models. Conclussions: Tumors with LKB1 deficiency or KEAP/NRF2 mutations are typically resistant to standard chemotherapy drugs and immunotherapy. Our data indicate that LKB1 and KEAP1/NRF2 loss significantly enhance the sensitivity to ATR, ATM and Wee1 inhibitors in vitro while only ATR and Wee1 inhibitor show significant tumor growth impairment in syngeneic and PDX KLK models. Thus, we have identified that NSCLC tumors bearing STK11 and KEAP1/NRF2 mutations are highly sensitive to DDR inhibitors and that genes may serve as biomarkers for selecting appropriate patients for treatment alone or in combination, such as PARPi, chemo or immunotherapy. Citation Format: Ana Galan-Cobo, Marlese Pisegna, Kavya Ramkumar, Alissa Poteete, Sungnam Cho, Fahao Zhang, You-Hong Fan, Qi Wang, Lixia Diao, Katharina Schlacher, Jing Wang, Lauren A Byers, John V. Heymcach. LKB1 deficiency and KEAP1/NRF2 pathway alterations as biomarkers of response for ATR and ATM inhibitors and other inhibitors of DNA damage response (DDR) in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A096. doi:10.1158/1535-7163.TARG-19-A096

Human papillomaviruses (HPV) are among the most common viral causes of cancer; infections account for a majority of cervical squamous cell carcinomas and endocervical adenocarcinomas (CESCs) and oropharyngeal squamous cell carcinomas. HPV-positive cancers demonstrate well-established differences in histopathology and clinical outcome compared to HPV-negative cancers, yet the biological mechanisms underlying these differences are not fully characterized. In this analysis, we highlight key pathways and candidate targets differentially expressed in HPV-positive head and neck squamous cell carcinomas (HNSCCs) and CESCs and investigate potential contributors to these differences, including microRNA (miRNA). Methods: We conducted an analysis of mRNA and protein expression in HPV-positive and HPV-negative HNSCCs and CESCs from the Cancer Genome Atlas (TCGA) to investigate the potential pathways that are differentially expressed between HPV-positive and HPV-negative cancers. mRNA levels were measured by RNA Seq and protein levels by reverse phase protein arrays (RPPA). We also analyzed miRNA levels for both HNSCCs and CESCs, then used TargetScan to identify potential targets for miRNA that were differentially expressed. Results: Proteins involved in extracellular matrix (ECM) adherence including Paxillin and Fibronectin were higher in HPV-negative HNSCCs and CESCs (Paxillin’s p-value= 0.04 for CESCs and p-value Citation Format: Claudia Heymach, Robert Cardnell, Lixia Diao, Jing Wang, Kwok-Shing Ng, Lauren Byers. Proteomic analysis reveals alterations in apoptotic signaling machinery in HPV-associated cancers and identifies BRD4 and BCL2 as potential therapeutic targets in HPV-positive HNSCCs [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B032. doi:10.1158/1535-7163.TARG-19-B032

Therapeutic resistance limits effective treatment of non-small cell lung cancer (NSCLC) and a better understanding of mechanisms contributing to resistance and strategies to overcome these are urgently needed. AXL, a TAM family receptor tyrosine kinase, has emerged as a key determinant of resistance to chemotherapy, radiation and targeted therapies in NSCLC and other cancers, through its roles in mediating epithelial-mesenchymal transition (EMT) and immune escape. As several small-molecule AXL inhibitors and anti-AXL biologics are currently in clinical testing, AXL has emerged as a target of interest in treatment-resistant NSCLC. Here, we investigated AXL expression in NSCLC by analyzing genomic, transcriptomic and proteomic profiles across 3 treatment-naïve (1095 tumors) and 2 previously treated (245 tumors) clinical cohorts of lung adenocarcinoma and squamous cell carcinoma. While AXL expression did not vary with disease stage, tumor samples from patients with prior systemic treatment (and subsequent relapse) had significantly higher AXL expression and were more likely to have undergone EMT (based on our published EMT score), as compared to treatment-naïve NSCLC patients (p 0.3; p Citation Format: Kavya Ramkumar, Carminia M. Della Corte, Lixia Diao, Robert J. Cardnell, Vali A. Papadimitrakopoulou, John V. Heymach, Jing Wang, Don L. Gibbons, Lauren A. Byers. Clinical, genomic, and immune landscape of the receptor tyrosine kinase AXL in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B024. doi:10.1158/1535-7163.TARG-19-B024

Squamous cell carcinomas (SCC) account for approximately 25-30% of all non-small cell lung cancer (NSCLC) cases. To date, a limited number of therapeutic targets have been identified for SCC NSCLC; thus there is a critical need to identify novel, effective therapeutic regimens for this patient population. We utilized reverse phase proteomic arrays (RPPA) from two cohorts of early-stage NSCLC patients to investigate potential therapeutic targets overexpressed in SCC NSCLC. We identified tropomyosin receptor kinase B (TrkB) as being overexpressed in SCC NSCLC compared to non-SCC NSCLC. Using a panel of NSCLC cell lines, we determined that phosphorylated TrkB levels were significantly elevated in SCC NSCLC cell lines compared to adenocarcinoma NSCLC cell lines. Treatment with the TrkB inhibitor, AZD7451, significantly reduced the constitutive activation of TrkB and decreased phosphorylation of downstream signaling components, including Erk and AKT in HCC95 cells (SCC). The addition of the TrkB ligand, BDNF, resulted in a modest increase in TrkB phosphorylation in H1703 cells (SCC), which was blocked with the addition of AZD7451. We next investigated the role of TrkB in facilitating tumor cell invasiveness. Treatment of A549 cells (adenocarcinoma NSCLC) with exogenous BDNF significantly enhanced tumor cell migration, which was blocked with the addition of AZD7451. However, the effects of TrkB activation on cell migration were cell line specific as BDNF did not enhance tumor cell migration in H460 or H23 cells. Given prior publications suggesting potential crosstalk between epidermal growth factor receptor (EGFR) and TrkB, we sought to determine whether crosstalk between EGFR and TrkB occurs in squamous cell carcinoma cells. Although evidence of crosstalk was heterogeneous among cell lines, EGF stimulation increased activation of TrkB in Cal27 (head and neck SCC), and this effect was blocked by the EGFR tyrosine kinase inhibitor (TKI), erlotinib. Moreover, in vitro cell viability assays demonstrated a synergistic effect of erlotinib in combination with AZD7451 in both adenocarcinoma and SCC cell lines. Taken together, these findings indicate that TrkB is a potential therapeutic target in a subset of SCC NSCLC patients and that the antitumor activity of TrkB-targeting agents may be potentiated by EGFR inhibition. In addition, our findings suggest that activation of the TrkB pathway may drive an invasive phenotype in a subset of NSCLCs. Citation Format: Mia E. Hofstad, Daniel R. Gomez, Monique B. Nilsson, Lauren A. Byers, Lixia Diao, Jing Wang, Lerong Li, Pan Tong, John V. Heymach. TrkB (NTRK2) as a potential therapeutic target in NSCLC in combination with EGFR TKIs [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B101.

The PI3K/AKT/mTOR pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), but pathway inhibition has variable efficacy. Identification of predictive biomarkers and mechanisms of resistance would allow selection of patients most likely to respond and novel therapeutic combinations. The purpose of this study was to extend recent discoveries regarding the PI3K/AKT/mTOR pathway in HNSCC by more broadly examining potential biomarkers of response, by examining pathway inhibitors with a diverse range of targets, and by defining mechanisms of resistance and potential combination therapies. We used reverse-phase protein arrays (RPPA) to simultaneously evaluate expression of 195 proteins; SNP array to estimate gene copy number; and mass array to identify mutations. We examined altered signaling at baseline and after pathway inhibition. Likewise, we examined the activation of the PI3K/AKT/mTOR pathway in HNSCC tumors by RPPA. Cell lines with PIK3CA mutations were sensitive to pathway inhibitors, whereas amplification status did not predict sensitivity. While we identified a set of individual candidate biomarkers of response to pathway inhibitors, proteomic pathway scores did not correlate with amplification or mutation and did not predict response. Several receptor tyrosine kinases, including EGFR and ERK, were activated following PI3K inhibition in resistant cells; dual pathway inhibition of PI3K and EGFR or MEK demonstrated synergy. Combined MEK and PI3K inhibition was markedly synergistic in HRAS-mutant cell lines. Our findings indicate that clinical trials of single-agent PI3K/AKT/mTOR pathway inhibitors in selected populations and of PI3K/EGFR or PI3K/MEK inhibitor combinations are warranted; we plan to conduct such trials. Mol Cancer Ther; 13(11); 2738–50. ©2014 AACR.

Background: Small cell lung cancer (SCLC) is an aggressive subtype of lung cancer, accounting for ∼15% of all lung cancer cases in US. Talazoparib (BMN 673) is a potent, specific PARP1/2 inhibitor that has demonstrated significant clinical activity in ovarian and breast cancer patients with deleterious germline BRCA1/2 mutations (ASCO 2014). Antitumor responses were also reported in SCLC patients treated with talazoparib (ASCO 2014). However, molecular determinants for SCLC response to talazoparib treatment have not been determined. Method: in vitro: 38 human SCLC cell lines were screened for their sensitivity towards single-agent treatment with talazoparib or cisplatin. Sensitive vs. resistant cell lines were categorized based on median GI50 of 38 cell lines (as measured by generational growth inhibition approach) and 90% experimental maximum inhibition. Bioinformatics analysis was carried out to search for genomic features associated with talazoparib sensitivity, using CCLE genomic profiling data. in vivo: 12 human SCLC patient-derived xenograft (PDX) tumor models were established in immunocompromised mice. These PDX models were treated with either vehicle or talazoparib, and tumor growth was monitored by standard methods. Untreated tumor samples were collected from mice for molecular analyses (Myriad HRD assay, Foundation Medicine gene mutation analysis, reverse-phase protein array (RPPA), and Western blotting) to survey for talazoparib sensitivity biomarkers. Result: The 38 SCLC lines showed a wide range of sensitivity to talazoparib (GI50 ranging from 2 nM to >2000 nM, with median GI50 = 56 nM) and cisplatin treatment (GI50 ranging from 10 nM to >10,000 nM). Sensitivity towards talazoparib and cisplatin are well correlated (Pearson r = 0.748). Differential gene expression analysis suggests sensitivity to talazoparib correlated well with high SLFN11 expression in SCLC lines (nominal p-value < 0.001). The in vitro sensitivity results were confirmed in several cell line-derived xenograft models. In the SCLC PDX tumor screen, 3 of 12 PDX models showed tumor regression during talazoparib treatment with one complete response; 3 of 12 PDX tumors exhibited stable disease (SD)-like responses, while the remainder were resistant to talazoparib treatment. Gene mutation analysis indicates that all 12 SCLC PDX tumors have TP53 and/or RB1 mutations as expected for SCLC, but a correlation between mutations in the homologous recombination DNA repair genes (e.g. BRCA1, BRCA2, ATM) and response to talazoparib was not established in these models. Furthermore, no apparent relationship was found between talazoparib response and the Myriad HRD score in these SCLC PDX models, with all HRD scores being below the cutoff score associated with response to platinum treatment in breast and ovarian cancer. Protein expression analysis of PDX tumors is currently underway, and data will be presented at the meeting. Citation Format: Ying Feng, Karen Yu, Robert Cardnell, Yuanbin (Kevin) Ru, Evelyn Wang, Jing Wang, Leonard E. Post, Lauren A. Byers, Yuqiao (Jerry) Shen. Talazoparib predictive biomarker analysis in human small cell lung cancer cells and PDX tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A37.

Background: Small cell lung cancer (SCLC) is the most lethal form of lung cancer, accounting for around 15% of lung cancers in the United States, currently having no targeted therapies with established efficacy. Using a high-throughput proteomic screen, we demonstrated overexpression of checkpoint kinase 1 (Chk1) in SCLC. Chk1 being the master regulator of the cell cycle in absence of p53, we hypothesize that Chk1 targeting may be effective SCLC because of near ubiquitous loss of p53. In the present study, we tested the effect of Chk1 targeting by shRNA-mediated knockdown and pharmacologic inhibition in an extensive panel of SCLC cell lines and in animal models. Experimental procedures: shRNA-mediated knockdown of Chk1 was done in select human SCLC lines and confirmed at the mRNA and protein level. The effect of Chk1 inhibition by LY2606368 alone and in combination with cisplatin or the PARP1/2 inhibitor talazoparib was tested in 39 SCLC cell lines. Proliferation was assessed by cell titer glo analysis and effect on cell cycle was tested by flow cytometry. Drug sensitivity (IC50) was then correlated with baseline expression level of >190 total or phosphorylated proteins measured by reverse phase protein array (RPPA) to identify potential predictive biomarkers. In vivo effect of the Chk1 inhibitor with or without cisplatin or talazoparib was tested in syngeneic and xenograft model of SCLC. Results: shRNA-mediated targeted Chk1 knockdown significantly decreased the proliferation of SCLC lines confirming Chk1 as a viable target (p90% reduction in volume) which was durable (ongoing response >50 days in 7/9 animals). The combination with cisplatin or talazoparib also resulted in tumor regression and was significantly more effective than either cisplatin or talazoparib alone. Tumor regression following LY2606368 treatment was also observed in cis-resistant H69 xenograft model of SCLC. Conclusion: The study established checkpoint kinase 1 (Chk1) targeting as a novel and active therapeutic strategy for SCLC, a disease for which the standard of care has remained unchanged for >30 years. Chk1 knockdown was sufficient to inhibit proliferation in SCLC cell lines and LY2606368, a Chk1 inhibitor in current clinical trials, showed activity as a single agent and in combination with cisplatin and PARP inhibition in vitro and in vivo. TP53-mutant cells (hallmark of SCLC) rely on Chk1 for cell cycle arrest; hence, inhibition of Chk1 in SCLC likely works by driving cells to mitotic catastrophe and apoptosis. These findings support the further investigation of Chk1 targeting and Chk1 predictive biomarkers in SCLC. Citation Format: Triparna Sen, Pan Tong, C. Allison Stewart, You Hong-Fan, Ying Feng, Jing Wang, Lauren A. Byers. Investigation of Chk1 as a novel therapeutic target for small cell lung cancer (SCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C153.

Background: Wee1 is a protein kinase that plays a key role in regulating the G2 checkpoint and homologous recombination (HR) in response to DNA damage. Recent studies have demonstrated activity of the WEE1 inhibitor, AZD1775 (previously MK1775), in cancers with loss of the tumor suppressor TP53, including colon, pancreatic and breast. Hence, we hypothesize that WEE1 would be an ideal target for monotherapy in small cell lung cancer (SCLC), which is marked by ubiquitous loss of TP53. Moreover, because Wee1 inhibition induces a HR deficient state, dual targeting of Wee1 and PARP (another promising target previously identified in our group and in clinical trial testing) may induce synthetic lethality. In the present study we evaluate the efficacy of AZD1775 alone and with the PARP inhibitor, olaparib, in SCLC and investigate potential predictive biomarkers by proteomic profiling. Experimental procedures: We evaluated the gene expression of WEE1 in SCLC tumor sample (n = 23) compared to normal lung (n = 42). We then treated with AZD1775 +/- olaparib SCLC cell lines (n = 10). Drug sensitivity (IC50) was correlated with baseline expression level of 200 total or phosphorylated proteins measured by reverse phase protein array (RPPA) to identify potential predictive markers. SCLC cell lines with AZD1775 resistance (n = 3) were treated with TP0903 (10, 20, 40, 80nM) in combination with AZD1775 (0.1nM to 1uM) to test the efficacy of AXL co targeting in augmenting WEE1 inhibitor response. Results: SCLC tumors have a significantly higher gene expression level of WEE1 as compared to normal lung (p Conclusion and significance: The therapeutic options for patients with SCLC are limited and survival is poor. The WEE1 inhibitors are currently in clinical trials for SCLC. However, as with any targeted therapy, primary and secondary drug resistance is an important barrier to clinical benefit which could potentially be addressed with therapeutic combinations. In this study we show the over expression of WEE1 in SCLC and its efficacy as a single agent and in combination with olaparib. Here we also show that the activity of the WEE1 inhibitors might be limited in cancer cells overexpressing of AXL and that AXL inhibition re-sensitized the cells to AZD1775. Our work supports further exploration of the combination of PARP and WEE1 in SCLC and also the possibility of AXL inhibition as a mechanism to overcome WEE1 inhibition resistance in SCLC. Acknowledgement: This work was supported by Uniting Against Lung Cancer grant (LB). Citation Format: Triparna Sen, Pan Tong, Jennifer Hoff, C. Allison Stewart, Jing Wang, Lauren Byers. AXL as a potential mediator of WEE1 inhibitor resistance in small cell lung cancer (SCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A03.

Tumor hypoxia regulates many cytokines and angiogenic factors (CAF) and is associated with worse prognosis in head and neck squamous cell cancer (HNSCC). Serum CAF profiling may provide information regarding the biology of the host and tumor, prognosis, and response to therapy. We investigated 38 CAFs in HNSCC patients receiving induction therapy on a phase II trial of carboplatin, paclitaxel, and cetuximab. CAFs were measured by multiplex bead assay and enzyme-linked immunosorbent assay in 32 patients. Baseline and postinduction CAF levels were correlated with disease progression (PD) and human papilloma virus (HPV) status by Wilcoxon rank sum test. Baseline levels of eight hypoxia-regulated CAFs (the “high-risk signature” including vascular endothelial growth factor, interleukins 4 and 8, osteopontin, growth-related oncogene-α, eotaxin, granulocyte-colony stimulating factor, and stromal cell–derived factor-1α) were associated with subsequent PD. Elevation in ≥6 of 8 factors was strongly associated with shorter time to progression (P = 0.001) and was 73% specific and 100% sensitive for PD. Increasing growth-related oncogene-α from baseline to week 6 was also associated with PD. Progression-free and overall survival were shorter in patients with HPV-negative tumors (P = 0.012 and 0.046, respectively), but no individual CAF was associated with HPV status. However, among 14 HPV-negative patients, the high-risk CAF signature was seen in all 6 patients with PD, but only 2 of 14 without PD. In conclusion, serum CAF profiling, particularly in HPV-negative patients, may be useful for identifying those at highest risk for recurrence. Mol Cancer Ther; 9(6); 1755–63. ©2010 AACR.

Background: NSCLC is a deadly disease. Targeted therapy is very effective; however, only ∼ 20% of patients are candidates for this approach. In a search for new therapeutic targets in NSCLC, we have identified Polo like kinase (Plk). Plk1 regulates centrosomes at the G2/M transition and orchestrates cell cycle progression. Clinical trials have shown that a subgroup of refractory NSCLC patients responded to Plki as single agents. Our aim is to validate and identify biomarkers that predict sensitivity to Plki. Methods: To identify therapeutic targets, we evaluated 2 databases with 123 NSCLC cell lines tested with 130 drugs incorporating genomic and gene expression data seeking agents that were markedly effective in a subgroup of cell lines. To identify association between sensitivity to Plki and gene expression, we acquired the datasets from the databases and tested the correlation between IC50 and gene expression. Spearman rank was used to test the association between mutation in key genes and IC50. MTT assay was used to test drug sensitivity in NSCLC cell lines in our laboratory. Cell cycle was evaluated by flow cytometry using BrdU incorporation. Immunofluorescence with anti-phospho-H2yAX antibody was used to identify mitotic catastrophe. Results: In the databases, 12/19 NSCLC lines had IC50 < Cmax of 1.6 µM for the PLKi BI2536 and 6/19 had IC50 < 30 nM. The correlation analysis found mRNA overexpression of 20 genes to be significantly associated with sensitivity to both Plki. Based upon the likelihood of their expression influencing Plk1 function, 4 genes were selected for further study: CEP250, which encodes a core centrosomal protein, and TGFA, RHBDF1 and FOSL2, which are involved with epithelial cell proliferation. KRAS mutation was also correlated with sensitivity to Plki. Our preliminary cell line screening found that 10/37 NSCLC cell lines are exquisitely sensitive to BI2536 with IC70 < 40 nM. The Plki's BI2536 and BI6727 cause complete Plk substrate (TCTP) inhibition with 25 and 100 nM respectively at 12 h as measured by Western blotting. However, in a resistant cell line, TCTP reactivation was observed following 24 h Plk inhibition, while in a sensitive cell line, there was sustained TCTP inhibition up to 120 h. Cell cycle analysis of sensitive cell lines showed a significant arrest in G2/M. Drug treatment caused mitotic catastrophe in sensitive lines. Conclusion: Our statistical analysis utilizing data from 2 databases demonstrated an association between CEP250, TGFA, RHBDF1 and FOSL2 overexpression and KRAS mutation and sensitivity to Plki. These results are being validated in a larger cell line screen that may identify additional biomarkers. Substrate reactivation was observed in one resistant cell line following 24h of Plk inhibition, demonstrating one potential mechanism for resistance. Screening of additional cell lines and mechanistic studies are ongoing to extend these findings. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A182. Citation Format: Renata Ferrarotto, Manasi Suryavanshi, Suk Young Yoo, Jing Wang, Lauren Byers, Bonnie S. Glisson, John Heymach, Faye M. Johnson. Identifying biomarkers of sensitivity to polo-like kinase inhibitors (Plki) in non small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A182.

Background: BMN 673 is the most potent and specific inhibitor of PARP1/2 in clinical development (IC50 Methods: Pharmacokinetics (PK), pharmacodynamics (PD), safety and anti-tumor activity of BMN 673 were evaluated in a 2-stage escalation/expansion study. Expansion included cohorts of pts with BRCA-related cancers, small cell lung cancer (SCLC) and Ewing sarcoma (ES). Results: (As of July 29, 2013) In escalation, 39 pts were enrolled in 9 cohorts from 25 to 1100 µg/d with 1000 µg/d being the MTD related to dose-limiting hematologic toxicity. 38 pts have been enrolled in expansion including 25 patients with BRCA related cancers. For all 77 (63F/14M) pts, median age (range) is 52 (18-81), PS, 0 (0-1) and # of prior therapies 4 (1-13). Eight ES, 7 SCLC and 48 patients with deleterious BRCA mutations and breast (n=18), ovarian (n=28) or pancreatic (n=2) cancer were enrolled. BMN 673 demonstrated good oral bioavailability and a long half-life supporting daily dosing (ASCO 2013 Abstract 2580). One related grade 4 toxicity (thrombocytopenia) occurred. Related grade 3 adverse events have included fatigue, anemia, neutropenia and thrombocytopenia, all in fewer than 15% of patients. Dose-related inhibition of PARP activity in PBMC's was observed. Response, reduction in neutrophils and platelets and the need for blood transfusions and dose reductions appear to correlate with BMN 673 exposure.Overall, the objective response (including CA-125) and clinical benefit response rates are for all BRCA patients are 60.4% and 81%, respectively. Treatment is ongoing in 5 of 7 SCLC patients and 2 of 8 ES patients with no objective responses observed yet. Conclusions: BMN 673 is well tolerated with high objective and clinical benefit response rates in heavily pre-treated ovarian and breast cancer pts with deleterious germline BRCA mutations. A phase 3 trial in metastatic breast cancer is underway. Clinicaltrial.gov ID: NCT01286987 BRCA Breast and Ovarian CancerNBRCA Tumor TypeResponse18BreastCRPRSD > 12 weeksCBR*18Breast1 (6%)9 (50%)∧14 (78%)Response - 25 RECIST evaluable; 27 CA-125 evaluable28Ovarian28OvarianCRPRCA-1251 (4%)10 (40%)19 (70%)2PancreasCRPRSD ≥ 12 weeks2Pancreas0 (0%)0 (0%)2 (100%)**∧3 breast cancer responses are not yet confirmed; *CBR: clinical benefit response; **CA19-9 normalized in 1 patient Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C295. Citation Format: Zev A. Wainberg, Johann S. de Bono, Lida Mina, Jasgit Sachdev, Lauren Averett Byers, Rashmi Chugh, Charlie Zhang, Joshua W. Henshaw, Andrew Dorr, John Glaspy, Ramesh Ramanathan. Update on first-in-man trial of novel oral PARP inhibitor BMN 673 in patients with solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C295.

Background: The discovery of driver genes such as EGFR, ALK, and ROS1 in non-small cell lung cancer has led to novel, highly active therapies for a subset of patients. However, the majority of lung adenocarcinomas do not have alterations in established driver oncogenes. Here we used an integrated proteomic and genomic analysis of the Cancer Genome Atlas (TCGA) to identify potential therapeutic targets in oncogene-negative lung adenocarcinomas. Methods: Protein expression was measured by reverse phase protein array in 181 TCGA lung adenocarcinoma tumors. Protein levels were correlated with mutational status by t-test for individual mutations and for oncogene-positive versus negative tumors. A false discovery rate of 0.10 (corresponding p-value ≤0.047) was used for these analyses. Results: Expression of 160 total and phospho-proteins were compared between oncogene-positive and negative tumors using reverse phase protein array (RPPA). Oncogene-positive tumors included those with canonical mutations in KRAS, EGFR, BRAF, ROS1, ALK, RET, MAPK1, HRAS, NRAS, AKT1, MET, or ERBB2. For the two most frequent driver oncogenes, KRAS (n=47) and EGFR (n=27), the top markers expressed in mutated tumors were pRaf/pMAPK/pERK (KRAS mutated) and pEGFR (EGFR mutated) (p Conclusion: Potentially targetable or predictive markers, including Chk1/2, Bim, and thymidylate synthase, were expressed at higher levels in oncogene-negative lung adenocarcinomas. These findings support further investigation of these targets and associated biomarkers and could provide a treatment strategy for patients without established driver mutations such as EGFR, ALK, and ROS1. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C117. Citation Format: Lixia Diao, Pan Tong, Jing Wang, You-Hong Fan, Yiling Lu, Wenbin Liu, Carmen Behrens, Ignacio I. Wistuba, John V. Heymach, John N. Weinstein, Gordon B. Mills, Lauren A. Byers. An integrated proteomic analysis of lung adenocarcinomas from The Cancer Genome Atlas (TCGA) reveals potential targets for oncogene-negative tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C117.

Background: Targeted therapy for head and neck squamous cell carcinoma (HNSCC) is currently limited to EGFR inhibition with agents such as cetuximab. We and others have previously demonstrated that epithelial to mesenchymal transition (EMT) is associated with resistance to EGFR inhibitors in HNSCC and lung cancers but greater sensitivity to other targeted drugs, such as AXL inhibitors. Here, we apply a previously published EMT signature to identify novel potential therapeutic targets (PTTs) in mesenchymal HNSCC tumors (MHNTs). Methods: A high-throughput analysis of gene and protein expression in HNSCC tumors from The Cancer Genome Atlas (TCGA) (A, n=113) was performed to identify PTTs in MHNTs. Specifically, the EMT signature score for each tumor was correlated with marker expression levels by Pearson correlation (mRNA, protein) or t-test (mutation, HPV status). PTTs were validated in an independent cohort of high-risk, resected tumors (B, n=105) and HNSCC cell lines (C, n=60). PTTs were defined as druggable targets found on recent HNSCC publications, our in-house targeted sequencing panel of 202 genes and publicly available cancer cell lines drug sensitivity datasets (DSDs). Results: At a false discovery rate of 1% (p0.5) and confirmed in our independent cohort included DDR2, FGFR1, KDR, MMP14, MMP2, PDGFRA, PDGFRB and TGFBR2. Targets that were overexpressed in both tumors and cell lines included NOTCH4 (A and B p Conclusions: An integrated analysis of EMT and expression data from two independent cohorts identifies previously unrecognized, candidate PTTs in mesenchymal HNSCC tumors. Validation of the PTTs at the protein level is ongoing. The approach described may identify patient subsets, beyond mutational status, for whom targeted therapy can be successful. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A131. Citation Format: Milena P. Mak, Lixia Diao, Jing Wang, Patrick KS Ng, You-Hong Fan, Michael D. Story, William N. William, John V. Heymach, John N. Weinstein, Kevin R. Coombes, Lauren A. Byers. Identification of candidate therapeutic targets in mesenchymal head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A131.

Background: Epithelial/mesenchymal transition (EMT) is associated with loss of cell adhesion molecules such as E-cadherin and increased invasion, migration, and proliferation in epithelial cancers. In non-small cell lung cancer (NSCLC), EMT is associated with greater resistance to EGFR inhibitors. However, its potential to predict response to other targeted drugs or chemotherapy has not been well characterized. Using a previously-derived EMT gene signature, we tested the association between EMT and drug response in a panel of >50 NSCLC cell lines. Materials/Methods: NSCLC cell lines were classified as epithelial or mesenchymal by a previously described 76-gene EMT gene expression signature. IC50s for more than 30 drugs or drug combinations were determined by MTS assay. Differences in IC50 between epithelial and mesenchymal lines were assessed by t-test (p-values ≤ 0.05 were considered significant). Results: As expected, EGFR mutated cell lines were classified by the EMT signature as epithelial and were highly sensitive to EGFR inhibition by erlotinib. Among wild-type EGFR cell lines, those that had undergone EMT were significantly more resistance to erlotinib (p=0.029). NSCLC cell lines with mesenchymal signatures were also more resistant to drugs targeting the PI3K/Akt pathway such as GDC0941 (p=0.037) and 8-amino-adenosine (p=0.009). A trend towards greater resistance was also seen in mesenchymal cells treated with the Akt inhibitor MK2206 (p=0.12). There was no association between EMT and response to cytotoxic chemotherapies, including cisplatin, carboplatin, gemcitabine, pemetrexed, docetaxel, paclitaxel, and platinum-doublets (p-values ≥0.2). Conclusion: EMT is associated with greater resistance to PI3K/Akt pathway inhibitors and may be a useful predictive marker for patients treated with these drugs. The EMT signature is being investigated prospectively in patients treated with an Akt inhibitor in an ongoing clinical trial. Supported in part by the NIH Lung Cancer SPORE grant P50 CA70907. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B156.