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Tumor suppressor mutations in head and neck squamous cell carcinoma (HNSCC) dominate the genomic landscape, hindering the development of effective targeted therapies. Truncating and missense mutations in NOTCH1 are frequent in HNSCC, and inhibition of PI3K can selectively target NOTCH1 mutant (NOTCH1MUT) HNSCC cells. In this study, we identify several proteins that are differentially regulated in HNSCC cells after PI3K inhibition based on NOTCH1MUT status. Expression of Aurora kinase B (Aurora B), AKT, and PDK1 following PI3K inhibition was significantly lower in NOTCH1MUT cell lines than in wild-type NOTCH1 (NOTCH1WT) cells or NOTCH1MUT cells with acquired resistance to PI3K inhibition. Combined inhibition of PI3K and Aurora B was synergistic, enhancing apoptosis in vitro and leading to durable tumor regression in vivo. Overexpression of Aurora B in NOTCH1MUT HNSCC cells led to resistance to PI3K inhibition, while Aurora B knockdown increased sensitivity of NOTCH1WT cells. In addition, overexpression of Aurora B in NOTCH1MUT HNSCC cells increased total protein levels of AKT and PDK1. AKT depletion in NOTCH1WT cells and overexpression in NOTCH1MUT cells similarly altered sensitivity to PI3K inhibition, and manipulation of AKT levels affected PDK1 but not Aurora B levels. These data define a novel pathway in which Aurora B upregulates AKT that subsequently increases PDK1 selectively in NOTCH1MUT cells to mediate HNSCC survival in response to PI3K inhibition. These findings may lead to an effective therapeutic approach for HNSCC with NOTCH1MUT while sparing normal cells. Significance: Aurora B signaling facilitates resistance to PI3K inhibition in head and neck squamous cell carcinoma, suggesting that combined inhibition of PI3K and Aurora kinase is a rational therapeutic strategy to overcome resistance.

Movie video files of a) H1299 and b) HOP62 cells were treated with 25 or 50 nM dinaciclib and continuously imaged as described in the Methods.

Supplementary figure 3. Correlation of PI3K mutation, amplification, PI3K score, and mTOR score with sensitivity to GDC0941

PDF file, 102K, Supplementary Material and Methods for gene expression analysis are provided.

PDF file, 77K, Multicovariate Cox model fitted to estimate the effect of age, gender, stage, institution and EphA4 on time to event endpoint, for overall survival.

Legend for Supplementary Tables:1,2,3,4

PDF file, 37K, Ephs and eprins in vitro.

Supplementary Tables and Methods

Supplementary figure 2. Representative FISH data

PDF file, 726K, EPHA4 and morphology.

PDF file, 85K, Distribution of Eph receptors gene mutations in NSCLC.

PDF file, 48K, Ephs and eprins in vivo.

PDF file, 116K.

PDF file, 39K, EPHA4 effect on other EPHs and EFNs.

Movie video files of HOP62 cells as described in the Methods.

Supplementary figure 4. GDC0941 effect on cell cycle

PDF file, 31K, EPHA4 in the Boston cohort.

Leukoplakia is the most common premalignant lesion of the oral cavity. Epidermal growth factor receptor (EGFR) abnormalities are associated with oral tumorigenesis and progression. We hypothesized that EGFR expression and gene copy number changes are predictors of the risk of an oral premalignant lesion (OPL) progressing to oral squamous cell carcinoma (OSCC). A formalin-fixed, paraffin-embedded OPL biopsy specimen was collected from each of 162 patients in a randomized controlled clinical trial. We assessed EGFR expression by immunohistochemistry with two methods: a semiquantitative analysis (145 evaluable specimens) and an automated quantitative analysis (127 evaluable specimens). EGFR gene copy number was assessed by fluorescence in situ hybridization (FISH) in a subset of 49 OPLs with high EGFR expression defined by the semiquantitative analysis. We analyzed EGFR abnormalities for associations with OSCC development. High EGFR expression occurred in 103 (71%) of the 145 OPLs and was associated with a nonsignificantly higher risk of OSCC (P = 0.10). Twenty (41%) of 49 OPLs assessed by FISH had an increased EGFR gene copy number (FISH-positive). Patients with FISH-positive lesions had a significantly higher incidence of OSCC than did patients with FISH-negative (a normal copy number) lesions (P = 0.0007). Of note, 10 of 11 OSCCs that developed at the site of the examined OPL were in the FISH-positive group, leaving only one FISH-negative OPL that did so (P < 0.0001). Our data indicate that an increased EGFR gene copy number is common in and associated with OSCC development in patients with OPLs expressing high EGFR, particularly OSCC developing at the site of a high-expression OPL; they also suggest that EGFR inhibitors may prevent oral cancer in patients with OPLs having an increased EGFR gene copy number. Cancer Prev Res; 3(7); 800–9. ©2010 AACR.

PDF file, 50K, Sequences of Primers for PCR.

PDF file, 68K, EPHA4 effect on drug sensitivity.

Suppl. Table 1. Binding mode of Debio 0617B to ABL. Suppl. Table 2. Physico-chemical and early ADME profile of Debio 0617B. Suppl. Table 3. Debio 0617B activity on a panel of 21 cancer cell lines. Suppl. Table 4. PK/PD Profile of Debio 0617B in mice with A549 tumors.

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.

PDF file, 48K, EFNA1, EFNB2, EPHB2 mRNA levels.

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.

PDF file, 99K, Detailed mutations identified in the Eph receptors family in NSCLC.

PDF file, 105K, EPHA4, apoptosis and cell cycle.

Suppl. Fig. 1 Characterization for pSTAT3 activation in cancer cell lines responding to Debio 0617B in vitro by Western blot analysis.Suppl. Fig. 2 A-C. Efficacy studies with Debio 0617B in A549, A2058 and A431 models: body weight loss/gain (%). Suppl. Fig. 3A. Histopathology of primary tumors in the 4T1 mammary tumor model. 3B. Efficacy of Debio 0617B in the 4T1 neoadjuvant mammary tumor model: body weight loss/gain (%).

Perspective on this Article from Epidermal Growth Factor Receptor Expression and Gene Copy Number in the Risk of Oral Cancer

Supplementary figure 5. MEK162 and GDC0941 effect on cell cycle

PDF file, 87K, Multicovariate analysis showing the association between EphA4 gene expression and overall survival in an independent cohort of patients resected for a lung adenocarcinoma.

Legend for Supplementary Figures:1A,1B,1C,1D 2A,2B,2C;3A,3B

Supplementary figure 1. Genetic alterations in the PIK3CA, PTEN, AKT, MTOR, and HRAS genes from 279 HNSCC tumors from the TCGA.

Supplementary Tables 1-4 from Epidermal Growth Factor Receptor Expression and Gene Copy Number in the Risk of Oral Cancer

Purpose:Human papillomavirus (HPV) causes >5% of cancers, but no therapies uniquely target HPV-driven cancers.Experimental Design:We tested the cytotoxic effect of 864 drugs in 16 HPV-positive and 17 HPV-negative human squamous cancer cell lines. We confirmed apoptosis in vitro and in vivo using patient-derived xenografts. Mitotic pathway components were manipulated with drugs, knockdown, and overexpression.Results:Aurora kinase inhibitors were more effective in vitro and in vivo in HPV-positive than in HPV-negative models. We hypothesized that the mechanism of sensitivity involves retinoblastoma (Rb) expression because the viral oncoprotein E7 leads to Rb protein degradation, and basal Rb protein expression correlates with Aurora inhibition–induced apoptosis. Manipulating Rb directly, or by inducing E7 expression, altered cells’ sensitivity to Aurora kinase inhibitors. Rb affects expression of the mitotic checkpoint genes MAD2L1 and BUB1B, which we found to be highly expressed in HPV-positive patient tumors. Knockdown of MAD2L1 or BUB1B reduced Aurora kinase inhibition–induced apoptosis, whereas depletion of the MAD2L1 regulator TRIP13 enhanced it. TRIP13 is a potentially druggable AAA-ATPase. Combining Aurora kinase inhibition with TRIP13 depletion led to extensive apoptosis in HPV-positive cancer cells but not in HPV-negative cancer cells.Conclusions:Our data support a model in which HPV-positive cancer cells maintain a balance of MAD2L1 and TRIP13 to allow mitotic exit and survival in the absence of Rb. Because it does not affect cells with intact Rb function, this novel combination may have a wide therapeutic window, enabling the effective treatment of Rb-deficient cancers.

Supplementary Data from Combined TRIP13 and Aurora Kinase Inhibition Induces Apoptosis in Human Papillomavirus–Driven Cancers

Figure S8. Effect of the inhibition of multiple components of the PI3K/mTOR pathway in head and neck squamous cell carcinoma (HNSCC) cell lines. A. NOTCH1MUT cells (red text) and NOTCH1WT cells (black text) were treated with increasing concentrations (0-200 nM, 2-fold dilution) of GSK2126458 (a dual PI3K/mTOR inhibitor), BAY806946 (a pan-PI3K inhibitor), rapamycin and ridaforolimus (two mTOR inhibitors), and MK2206 (an AKT inhibitor) for 72 hours, and cell viability was assessed using the CellTiter-Glo assay. B. The protein p-S6 (S240/244) bands in Figure 2D were quantified using ImageJ software and normalized to β-actin expression, and the fold change from the control was calculated. Data are the means {plus minus} standard errors for three cell lines. *p

E-cadherin knockdown does not affect NSCLC cell lines' sensitivity to PLK1 inhibition. Two epithelial NSCLC cell lines were transfected with siRNA against E-cadherin and then incubated with volasertib 48 hours later. Knockdown of E-cadherin expression did not affect cell-cycle progression (left panels), cell number as measured using an MTT assay (right panels), or apoptosis as measured using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (bottom panel).

Tumor suppressor mutations in head and neck squamous cell carcinoma (HNSCC) dominate the genomic landscape, hindering the development of effective targeted therapies. Truncating and missense mutations in NOTCH1 are frequent in HNSCC, and inhibition of PI3K can selectively target NOTCH1 mutant (NOTCH1MUT) HNSCC cells. In this study, we identify several proteins that are differentially regulated in HNSCC cells after PI3K inhibition based on NOTCH1MUT status. Expression of Aurora kinase B (Aurora B), AKT, and PDK1 following PI3K inhibition was significantly lower in NOTCH1MUT cell lines than in wild-type NOTCH1 (NOTCH1WT) cells or NOTCH1MUT cells with acquired resistance to PI3K inhibition. Combined inhibition of PI3K and Aurora B was synergistic, enhancing apoptosis in vitro and leading to durable tumor regression in vivo. Overexpression of Aurora B in NOTCH1MUT HNSCC cells led to resistance to PI3K inhibition, while Aurora B knockdown increased sensitivity of NOTCH1WT cells. In addition, overexpression of Aurora B in NOTCH1MUT HNSCC cells increased total protein levels of AKT and PDK1. AKT depletion in NOTCH1WT cells and overexpression in NOTCH1MUT cells similarly altered sensitivity to PI3K inhibition, and manipulation of AKT levels affected PDK1 but not Aurora B levels. These data define a novel pathway in which Aurora B upregulates AKT that subsequently increases PDK1 selectively in NOTCH1MUT cells to mediate HNSCC survival in response to PI3K inhibition. These findings may lead to an effective therapeutic approach for HNSCC with NOTCH1MUT while sparing normal cells.Significance:Aurora B signaling facilitates resistance to PI3K inhibition in head and neck squamous cell carcinoma, suggesting that combined inhibition of PI3K and Aurora kinase is a rational therapeutic strategy to overcome resistance.

Supplementary Figure S5. Downregulation of MCl-1, XIAP and their cleavage by AZD-1775 in HPV+ HNSCC cells are caspase-dependent;and Supplementary Figure S6. mRNA levels of MCl-1, XIAP and stable forced expression of MCl-1 in HPV+ HNSCC cells.

Purpose: c-Src inhibition in cancer cells leads to an abrogation of invasion but a variable effect on apoptosis. The pathways downstream of c-Src promoting survival are not well characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal, we sought to characterize the mechanisms of resistance to c-Src inhibition.Experimental Design: c-Src was inhibited in a panel of oral cancer cell lines and subsequent survival and signaling measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and an in vitro kinase assay. Cytotoxicity was measured and the Chou–Talalay combination index calculated. An orthotopic model of oral cancer was used to assess the effects of c-Met and c-Src inhibitors.Results: Inhibition of c-Src resulted in c-Met inhibition in sensitive cells lines, but not in resistant cell lines. Isolated c-Met was a c-Src substrate in both sensitive and resistant cells, but there was no interaction of c-Src and c-Met in intact resistant cells. To examine the biological consequences of this mechanism, we demonstrated synergistic cytotoxicity, enhanced apoptosis, and decreased tumor size with the combination of c-Src and c-Met inhibitors.Conclusions: Sustained c-Met activation can mediate resistance to c-Src inhibition. These data suggest that the differences between c-Met and c-Src signaling in sensitive and resistant cells are due to distinct factors promoting or inhibiting interactions, respectively, rather than to intrinsic structural changes in c-Src or c-Met. The synergistic cytotoxic effects of c-Src and c-Met inhibition may be important for the treatment of head and neck cancers. Clin Cancer Res; 17(3); 514–24. ©2010 AACR.

AZD-1775 as a single agent shows antitumor activity in HPV+ patient derived xenografts xenograft (PDX).