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Despite advances in targeted therapy, lung cancer remains the most common cause of cancer-related mortality in the United States. Chromosomal instability is a prominent feature in lung cancer and, because it rarely occurs in normal cells, it represents a potential therapeutic target. Our prior work discovered that lung cancer cells undergo anaphase catastrophe in response to inhibition of cyclin-dependent kinase 2 (CDK2), followed by apoptosis and reduced growth. In this study, the effects and mechanisms of the multi-CDK inhibitor dinaciclib on lung cancer cells were investigated. We sought to determine the specificity of CDK-dependent induction of anaphase catastrophe. Live cell imaging provided direct evidence that dinaciclib caused multipolar cell divisions resulting in extensive chromosome missegregation. Genetic knockdown of dinaciclib CDK targets revealed that repression of CDK2 and CDK1, but not CDK5 or CDK9, triggered anaphase catastrophe in lung cancer cells. Overexpression of CP110, which is a mediator of CDK2 inhibitor–induced anaphase catastrophe (and a CDK1 and 2 phosphorylation substrate), antagonized anaphase catastrophe and apoptosis following dinaciclib treatment. Consistent with our previous findings, acquisition of activated KRAS sensitized lung cancer cells to dinaciclib-mediated anaphase catastrophe and cell death. Combining dinaciclib with the mitotic inhibitor taxol augmented anaphase catastrophe induction and reduced cell viability of lung cancer cells. Thus, the multi-CDK inhibitor dinaciclib causes anaphase catastrophe in lung cancer cells and should be investigated as a potential therapeutic for wild-type and KRAS-mutant lung cancer, individually or in combination with taxanes. Mol Cancer Ther; 15(11); 2758–66. ©2016 AACR.

Tumor survival, metastases, chemoresistance, and escape from immune responses have been associated with inappropriate activation of STAT3 and/or STAT5 in various cancers, including solid tumors. Debio 0617B has been developed as a first-in-class kinase inhibitor with a unique profile targeting phospho-STAT3 (pSTAT3) and/or pSTAT5 in tumors through combined inhibition of JAK, SRC, ABL, and class III/V receptor tyrosine kinases (RTK). Debio 0617B showed dose-dependent inhibition of pSTAT3 in STAT3-activated carcinoma cell lines; Debio 0617B also showed potent antiproliferative activity in a panel of cancer cell lines and in patient-derived tumor xenografts tested in an in vitro clonogenic assay. Debio 0617B showed in vivo efficacy by inhibiting tumor growth in several mouse xenograft models. To increase in vivo efficacy and STAT3 inhibition, Debio 0617B was tested in combination with the EGFR inhibitor erlotinib in a non–small cell lung cancer xenograft model. To evaluate the impact of in vivo STAT3 blockade on metastases, Debio 0617B was tested in an orthotopic tumor model. Measurement of primary tumor weight and metastatic counts in lung tissue demonstrated therapeutic efficacy of Debio 0617B in this model. These data show potent activity of Debio 0617B on a broad spectrum of STAT3-driven solid tumors and synergistic activity in combination with EGFR inhibition. Mol Cancer Ther; 15(10); 2334–43. ©2016 AACR.

Background: Bimiralisib is a balanced dual panPI3K/mTOR inhibitor, which demonstrated anti-cancer activity in a number of preclinical models. Pharmacokinetic (PK) data suggested that intermittent targeting of the PI3K/mTOR pathway could be preferable to continuous daily dosing. Preclinical models in squamous cell head and neck cancer (SCCHN) demonstrated that NOTCH1 Loss of Function (LoF) mutations were associated with efficacy of bimiralisib and other PI3K inhibitors. Methods: We conducted a first-in-human dose-escalation study (3+3 design) to identify the maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), dose-limiting toxicities (DLT) and overall safety (CTCAE v4) of bimiralisib administered either as a continuous (80 mg – 120 mg daily) or one of two intermittent schedules (schedule A: twice weekly 80 mg – 200 mg on Day 1 and 2, or schedule B: twice weekly 80mg – 200 mg on Day 1 and 4) until disease progression or unacceptable adverse events (AEs). Eligible patients had histologically confirmed solid cancers refractory to standard therapies, ECOG 12 weeks, adequate bone marrow, liver, and renal functions, HgbA1c 6 months was observed in five pts (23.8%) in continuous schedule, 12 (48%) in intermittent schedule A, and 13 (54.2%) in intermittent schedule B. Results on patients, who consented to the serial circulating tumor DNA collection will be presented. Conclusions: Bimiralisib was well tolerated and RP2D of 140 mg given orally twice weekly on Days 1 and 2 was selected for further studies. In agreement with our preclinical models, there was an encouraging activity in a SCCHN patient with NOTCH1 LoF mutation and this observation is now being validated in a proof-of-concept phase 2 study (NCT03740100). Citation Format: Filip Janku, Faye M. Johnson, Mateusz Opyrchal, Afshin Dowlati, Cinta Hierro, Martin Forester, Sarah P. Blagden, Andreas Wicki, Debora Schmitz, Alex A. Adjei. Oral dual PI3K/mTOR inhibitor bimiralisib demonstrates tolerability and a signal of activity in head and neck squamous cell cancer with NOTCH1 loss-of-function mutation [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 B109. doi:10.1158/1535-7163.TARG-19-B109

The c-Met receptor tyrosine kinase has been implicated in cellular transformation induced by mutant Ras, a commonly activated proto-oncogene in non-small cell lung cancer (NSCLC). However, the role of c-Met has not been defined in K-ras-mutant NSCLC, a disease for which no effective targeted therapeutic options currently exist. To acquire a greater understanding of its role, we used genetic and pharmacologic approaches to inhibit c-Met in mice and cultured cells. In KrasLA1 mice, which develop premalignant lung lesions that progress to multifocal lung adenocarcinomas owing to somatic mutations in K-ras, c-Met was expressed in multiple cell types within premalignant lung lesions, and high concentrations of HGF were detected in bronchoalveolar lavage samples. Short-term treatment with PHA-665752, a c-Met inhibitor, decreased the numbers of premalignant lung lesions and induced apoptosis in tumor cells and vascular endothelial cells within lesions. In cell culture, PHA-665752 induced apoptosis of a lung adenocarcinoma cell line derived from KrasLA1 mice (LKR-13) and a murine lung endothelial cell line (MEC). c-Met depletion by siRNA transfection induced apoptosis of MECs but not LKR-13 cells. Collectively, these findings suggest that apoptosis was an on-target effect of PHA-665752 in MECs but not in LKR-13 cells. We conclude that PHA-665752 inhibited lung tumorigenesis in KrasLA1 mice and may provide a novel therapeutic approach to the prevention of K-ras-mutant NSCLC. [Mol Cancer Ther 2008;7(4):952–60]

Malignant pleural mesothelioma (MPM) is a deadly disease with few systemic treatment options. One potential therapeutic target, the non–receptor tyrosine kinase c-Src, causes changes in proliferation, motility, invasion, survival, and angiogenesis in cancer cells and may be a valid therapeutic target in MPM. To test this hypothesis, we determined the effects of c-Src inhibition in MPM cell lines and examined c-Src expression and activation in tissue samples. We analyzed four MPM cell lines and found that all expressed total and activated c-Src. Three of the four cell lines were sensitive by in vitro cytotoxicity assays to the c-Src inhibitor dasatinib, which led to cell cycle arrest and increased apoptosis. Dasatinib also inhibited migration and invasion independent of the cytotoxic effects, and led to the rapid and durable inhibition of c-Src and its downstream pathways. We used immunohistochemical analysis to determine the levels of c-Src expression and activation in 46 archived MPM tumor specimens. The Src protein was highly expressed in tumor cells, but expression did not correlate with survival. However, expression of activated Src (p-Src Y419) on the tumor cell membrane was higher in patients with advanced-stage disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Y419. Lower levels of membrane expression of inactive c-Src (p-Src Y530) correlated with advanced N stage (P = 0.02). Activated c-Src may play a role in survival, metastasis, and invasion of MPM, and targeting c-Src may be an important therapeutic strategy. [Mol Cancer Ther 2007;6(7):1962–72]

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: 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: A variety of methods for capture of rare CTCs of epithelial origin are available; most employ antibodies to epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK). Using a classic phenotypic definition, a CTC is nucleated, CK(+), CD45(-) cell. However, some CTCs may elude capture as they originate from primary tumor cells which have undergone epithelial-mesenchymal transition (EMT). We report here the use of ApoStream™, a novel dielectrophoresis field-flow-assisted, antibody-free method to isolate CTCs from blood. Methods: Blood was collected from consented NSCLC patients and processed using ApoStream™. For CTC enumeration comparison, the CellSearch® FDA-approved kit was used. Isolated cells were evaluated with a multiplexed immunofluorescent assay and laser scanning cytometry was applied to identify multiple combinations of positive and/or negative staining for CK/CD45/DAPI and EpCAM. To determine specific EGFR mutations from captured CTCs, samples were analyzed using Improved and Complete Enrichment with CO-amplification at Lower Denaturation temperature (ICE COLD-PCR). Results: Blood samples from 37 NSCLC patients and 8 healthy volunteers were processed and the results of the cell populations isolated using ApoStream™ are summarized in table. EGFR mutations [exon 19 deletion and exon 21 L858R] were determined and found to be concordant when compared to tumor tissue analysis by Sanger sequencing. Conclusions: The ApoStream™ platform enriched EpCAM(+) and EpCAM(-) CTCs from the blood of NSCLC patients demonstrating utility in recovering cancer cells with multiple phenotypes. From recovered CTCs, detection of EGFR mutations was possible indicating the clinical relevance and potential utility of CTCs as an alternative to tissue biopsy. Additional sensitivity and complete mutation analysis will be presented. (a portion of this data was presented at 2013 ASCO Annual Meeting, Chicago, IL, USA) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C29. Citation Format: Hai T. Tran, Vladislava Melnikova, Anne S. Tsao, Frank V. Fossella, Faye M. Johnson, Vali Papadimitrakoupoulou, Katherine Richardson, Marcia E. Lewis, Ben Legendre, Kenna L. Anderes, Darren W. Davis, John V. Heymach. Characterization and identification of specific EGFR mutations in circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients using an antibody independent method, ApoStream™: An update report. [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 C29.