Shaleeka Cornelius

Conditional deletion of nonmuscle myosin II-A in mouse tongue epithelium results in squamous cell carcinoma

To investigate the contribution of nonmuscle myosin II-A (NM II-A) to early cardiac development we crossed Myh9 floxed mice and Nkx2.5 cre-recombinase mice. Nkx2.5 is expressed in the early heart (E7.5) and later in the tongue epithelium. Mice homozygous for deletion of NM II-A (ANkx/ANkx) are born at the expected ratio with normal hearts, but consistently develop an invasive squamous cell carcinoma (SCC) of the tongue (32/32 ANkx/ANkx) as early as E17.5. To assess reproducibility a second, independent line of Myh9 floxed mice derived from a different embryonic stem cell clone was tested. This second line also develops SCC indistinguishable from the first (15/15). In ANkx/ANkx mouse tongue epithelium, genetic deletion of NM II-A does not affect stabilization of TP53, unlike a previous report for SCC. We attribute the consistent, early formation of SCC with high penetrance to the role of NM II in maintaining mitotic stability during karyokinesis.

SMAC Mimetic Birinapant plus Radiation Eradicates Human Head and Neck Cancers with Genomic Amplifications of Cell Death Genes FADD and BIRC2

Comparison of tumors from The Cancer Genome Atlas (TCGA) reveals that head and neck squamous cell carcinomas (HNSCC) harbor the most frequent genomic amplifications of Fas-associated death domain (FADD), with or without Baculovirus inhibitor of apoptosis repeat containing BIRC2 (cIAP1), affecting about 30% of patients in association with worse prognosis. Here, we identified HNSCC cell lines harboring FADD/BIRC2 amplifications and overexpression by exome sequencing, RT-PCR, and Western blotting. In vitro, FADD or BIRC2 siRNA knockdown inhibited HNSCC displaying amplification and increased expression of these genes, supporting their functional importance in promoting proliferation. Birinapant, a novel SMAC mimetic, sensitized multiple HNSCC lines to cell death by agonists TNFα or TRAIL and inhibited cIAP1>XIAP>IAP2. Combination of birinapant and TNFα induced sub-G0 DNA fragmentation in sensitive lines and birinapant alone also induced significant G2-M cell-cycle arrest and cell death in UM-SCC-46 cells. Gene transfer and expression of FADD sensitized resistant UM-SCC-38 cells lacking FADD amplification to birinapant and TNFα, supporting a role for FADD in sensitization to IAP inhibitor and death ligands. HNSCC varied in mechanisms of cell death, as indicated by reversal by inhibitors or protein markers of caspase-dependent apoptosis and/or RIPK1/MLKL-mediated necroptosis. In vivo, birinapant inhibited tumor growth and enhanced radiation-induced TNFα, tumor responses, and host survival in UM-SCC-46 and -11B xenograft models displaying amplification and overexpression of FADD+/- BIRC2 These findings suggest that combination of SMAC mimetics such as birinapant plus radiation may be particularly active in HNSCC, which harbor frequent FADD/BIRC2 genomic alterations. Cancer Res; 76(18); 5442-54. ©2016 AACR.

Abstract 3821: Targeted therapy for head and neck squamous cell carcinoma using the novel SMAC-mimetic birinapant

Head and neck squamous cell carcinoma (HNSCC) is the most prevalent cancer affecting the upper aerodigestive tract, with an annual incidence of 600,000 patients and a five year survival of approximately 60% worldwide. Molecular mechanisms driving the development of HNSCC have recently begun to be discovered, with The Cancer Genome Atlas (TCGA) uncovering the genomic landscape of 279 cases of HNSCC. Alterations in cell death pathways were commonly found in the TCGA analysis, with ∼30% of samples harboring 11q13/22 amplifications and overexpression of genes encoding for Fas-associated death domain (FADD) and/or cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2). While overexpression of cIAP1 has been implicated in resistance to cytotoxic therapies, the role of FADD amplification as a target for therapy and in mechanisms of cell death is not well understood. Birinapant is a novel second mitochondria-derived activator of caspases (SMAC)-mimetic that targets and promotes degradation of cIAPs. Its clinical efficacy is currently being investigated in phase II trials of patients with ovarian cancer and leukemia. However, its preclinical and clinical efficacies have not been tested in HNSCC and genomic markers of sensitivity remain to be defined. Here we hypothesized that overexpression of FADD and cIAP1/2 could modulate birinapant sensitivity in HNSCC. To test this hypothesis, we have treated a panel of 11 HPV(-) and 8 HPV(+) HNSCC cell lines with birinapant alone and in combination with death agonists TNFα or TRAIL. UMSCC-46, an HPV(-) cell line which possesses high FADD expression, was the only cell line to reach half maximal inhibitory concentration (IC50) 72 hours post treatment with birinapant alone (IC50 = 10.7 nM); however, 8 of 11 HPV(-) cell lines and all 8 HPV(+) cell lines attained an IC50 (range: 0.1 - 794 nM) when treated with birinapant in combination with either TNFα or TRAIL. We further demonstrated that forced FADD overexpression in a previously resistant cell line (UMSCC-38) led to sensitization when treated with birinapant and TNFα. In vivo, two FADD/cIAP1 overexpressing murine xenograft models of HNSCC, UMSCC-46 and UMSCC-11B, were treated with birinapant at 15 mg/kg or 30 mg/kg every 3 days for a total of 10 treatments. The single modality regimen led to tumor growth inhibition and prolonged host survival. Additionally, combination treatment with birinapant 15 mg/kg and radiation 2Gy/day M-F for 2 weeks synergistically induced TNFα and led to a cure of animals bearing UMSCC-46 xenografts. Mechanistically, birinapant enhanced degradation of cIAP1 and modulated caspase apoptotic or MLKL necroptotic cell death markers in vitro and in vivo. These results suggest that patients harboring genomic alterations in FADD and/or cIAP overexpression may be candidates for treatment with birinapant and radiation. Supported by NIDCD intramural projects ZIA-DC-000073, and 74. Citation Format: Adeeb Derakhshan, Danielle Eytan, Grace Snow, Sophie Carlson, Anthony Saleh, Hui Cheng, Stephen Schiltz, Suresh Mohan, Shaleeka Cornelius, Jamie Coupar, Anastasia Sowers, Lydia Hernandez, James Mitchell, Christina Annunziata, Zhong Chen, Carter Van Waes. Targeted therapy for head and neck squamous cell carcinoma using the novel SMAC-mimetic birinapant. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3821.

Abstract 81: RNA-seq, exome-seq, functional RNAi screening and bioinformatics analyses identify molecules promoting aberrant activation of classical and alternative NF-kB pathways in head and neck cell lines

We previously observed aberrant activation of NF-kB in head and neck squamous cell carcinoma (HNSCC), however, the signaling constitutively activating NF-kB in solid cancers has not been well defined. Recently, The Cancer Genome Atlas (TCGA) project has investigated 279 HNSCC tissue specimens, and uncovered significant genomic alterations of key molecules involved in inflammation, NF-kB, and death pathways. These genetic alterations include amplification of FADD and BIRC2/3, mutations of caspase 8 and RIPKs in HPV(-), or deletion of TRAF3 in HPV(+) HNSCC tissues. Using bioinformatics analyses and protein structural and interactive tools, we identified ∼60 proteins that potentially interact with these genetically altered molecules. More than 90% HNSCC tissues studied in TCGA exhibited expression or genetic alterations of these genes, of which FADD amplification and/or overexpression ranked first with 37%. We searched this group of genes among more than 20 major cancer types investigated by TCGA, among which HNSCC ranked the highest for these alterations. To further investigate and identify experimental models for functional studies of these genetic and phenotypic alterations, we have performed RNA-seq and exome DNA-seq in 15 HPV(-) and 11 HPV(+) HNSCC lines, and compared them with three normal human oral mucosa lines and 8 matched blood samples. Among the top 30 genes identified from TCGA with the alteration rate greater than 8% in cancer tissues, we found consistent expression patterns for ∼57% molecules in our cell lines. To further test the function of these molecules, we established HNSCC cell lines stably transfected with a vector that contains NF-kB transcription factor response elements upstream of the α-lactamase reporter gene (Gene BLAzer). Using these NF-kB reporter cell lines, genome-wide RNAi screening assays have been performed and the regulatory and signaling molecules involved in NF-κB and death pathways in responding to TNF-α have been identified. We further validated the screening results by knockdown of 35 genes individually using two siRNAs for each genes, and found that knockdown of 16 genes significantly modulated TNF-α and Lymphotoxin β (LTβ) induced NF-κB activity and/or cell survival. We observed that TNF-α and LTβ cross-activate classical and alternative NF-κB pathways and regulate key molecules that serve differential roles in proliferation, survival and migration in HNSCC. Our data help define key molecules modulating aberrant classical and alternative NF-kB activation, as candidate molecular biomarkers for diagnosis and prognosis and targets for further preclinical and clinical investigation in HNSCC. (Supported by NIDCD/NIH intramural projects ZIA-DC-000016, 73, 74; and NCI/NIH, under contract number HHSN261200800001E). Citation Format: Hui Cheng, Xinping Yang, Anthony Saleh, Shaleeka Cornelius, Carter Van Waes, Zhong Chen, Emine Guven-Maiorov, Ozlem Keskin, Attila Gursoy, Ruth Nussinov. RNA-seq, exome-seq, functional RNAi screening and bioinformatics analyses identify molecules promoting aberrant activation of classical and alternative NF-kB pathways in head and neck cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 81.

Abstract 3622: Novel SMAC-mimetic birinapant demonstrates antitumor activity in human head and neck cancer models exhibiting alterations in cell death pathways

The Cancer Genome Atlas (TCGA) uncovered frequent and prognostically important genomic alterations of cell death signaling molecules, including chromosome 11q13/22 FADD and/or BIRC2 (cIAP1) amplifications, or CASP8 mutations, in distinct head and neck squamous cell carcinoma (HNSCC) subtypes. Consistent with TCGA, we found increased FADD and/or cIAP1 expression in a panel of 11 HNSCC cell lines. Overexpression of cIAP1 has been implicated in resistance to standard of care therapies that act through the induction of apoptosis. Birinapant, a novel SMAC-mimetic that targets and promotes degradation of cIAPs, inhibited cell density and sensitized multiple HNSCC lines to death agonists TNF or TRAIL, which was reversible by inhibitors of caspase-dependent apoptosis and/or RIPK1-mediated necroptosis. In vivo, birinapant significantly inhibited tumor growth and prolonged host survival in two FADD/cIAP1-overexpressing xenograft models, UMSCC 46 and 11B. Remarkably, birinapant combined with radiation treatment, an inducer of TNF, cured animals bearing UM-SCC-46 xenografts. These results suggest that birinapant generates strong synergy with radiotherapy in HNSCC harboring cell death pathway genomic alterations linked to worse prognosis. Citation Format: Sophie Carlson, Danielle Eytan, Grace Snow, Stephen Schiltz, Suresh Mohan, Anthony Saleh, Shaleeka Cornelius, Jamie Coupar, Carter Van Waes, Zhong Chen. Novel SMAC-mimetic birinapant demonstrates antitumor activity in human head and neck cancer models exhibiting alterations in cell death pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3622. doi:10.1158/1538-7445.AM2015-3622

Abstract 1111: Altered inflammatory and death pathways in head and neck cell lines model genomic and expression signatures identified in The Cancer Genome Atlas

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The Cancer Genome Atlas (TCGA) project has investigated 279 HNSCC tissue specimens, and uncovered significant genomic alterations of key molecules involved in inflammation, NF-KB, and death pathways. These genetic alterations include amplification of FADD (11q13) and BIRC2/3 (11q22), mutations of caspase 8 and RIPKs in HPV(-), or deletion of TRAF3 in HPV(+) HNSCC tissues. Using bioinformatic analyses and protein structural and interactive tools, we identified ∼60 proteins that may potentially interact with these genetically altered molecules involved in the inflammation and death pathways. More than 90% HNSCC tissues studied in TCGA exhibited expression or genetic alterations of these genes, with FADD amplification and/or overexpression ranking first with 37%. We also searched this group of genes among more than 20 major cancer types investigated by TCGA, among which HNSCC ranked the highest for the alterations. To further investigate and identify experimental models for functional studies of these genetic and phenotypic alterations, we have performed whole transcriptome (RNA-seq) and genome-wide exome DNA sequencing (exome DNA-seq) in 15 HPV(-) and 11 HPV(+) HNSCC lines, and compared them with three normal human oral mucosa lines and 8 matched blood samples. We have identified gene amplifications and overexpression among many of the molecules involved in inflammation, NF-KB and death pathways in cell lines, which were observed in the HNSCC TCGA project. Among the top 22 genes identified from TCGA with the alteration rate greater than 8% in cancer tissues, we found consistent expression patterns of ∼77% molecules in our cell lines. To further test the function of these molecules, we established HNSCC cell lines stably transfected with a vector that contains NF-κB transcription factor response elements upstream of the β-lactamase reporter gene, to measure a fluorescence resonance energy transfer (FRET) substrate cleaved by β-lactamase, that results in blue fluorescence. Using these NF-KB reporter cell lines, large RNAi screening assays have been performed to assess the regulatory and signaling molecules involve in NF-κB and death pathways to model the findings from TCGA. The function and mechanistic validation of these molecules are under the way, which could provide precise molecular targets of diagnosis and prognosis for further preclinical and clinical investigation in HNSCC. Supported by NIDCD intramural projects ZIA-DC-000016, 73 and 74; and funded under contract HHSN261200800001E Citation Format: Xinping Yang, Hui Cheng, Anthony Saleh, Shaleeka Cornelius, Emine Guven-Maiorov, Ruth Nussinov, Carter Van Weas, Zhong Chen, Ozlem Keskin, Attila Gursoy. Altered inflammatory and death pathways in head and neck cell lines model genomic and expression signatures identified in The Cancer Genome Atlas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1111. doi:10.1158/1538-7445.AM2015-1111

Abstract 5253: LTβ receptor and NIK signaling activates the alternative NF-kB pathway in head and neck squamous cell carcinoma

Head and neck squamous cell carcinomas (HNSCC) are highly inflammatory and preferentially metastasize to lymph nodes. NF-κB, a key target and regulator of inflammatory and cancer genes is constitutively activated in HNSCC, but the upstream regulation, especially for activation of alternative pathway involving LTβ/LTβR is not clear. We hypothesized that LTβ/LTβR mediated alternative NF-kB pathway signaling is an important mechanism for activation of downstream target genes, and promotion of malignant and metastatic phenotypes in HNSCC. In this study, we have found that LTβ and LTβR are overexpressed in subsets of HNSCC (UM-SCC) tissues and cell lines. In UM-SCC cell lines, LTβ mainly activated the alternative NF-kB pathway, enhancing nuclear translocation of RELB and NF-κB2/p52, in contrast to TNFα induced activation of classical NF-κB pathway. Knockdown of LTβR by Si RNA decreased LTβR mRNA by quantitative RT-PCR and protein expression by Western blots. Knockdown of LTβR decreased its target kinase NIK, and downstream NF-κB subunits RELB and NF-κB2/p52 protein expression. Knockdown of NIK protein decreased RELB and p52 protein expression, while LTβ treatment stabilized NIK, RELB and NF-κB2/p52 expression. LTB mediated NF-κB activity was examined by transient luciferase reporter assay or using stably transfected NF-κB reporter cell line through beta lactamase Fret Blazer activity. Knockdown of LTBR and NIK functionally decreased NF-κB reporter activity, while treatment of LTB partially restored the NF-κB reporter activity. Our data have shown that constitutive and LTβ induce activation of NIK and downstream NF-κB alternate pathway proteins, RELB and NF-kB2/p52 in HNSCC. LTβ and NIK activation of the alternative NF-κB pathway may contribute to the malignant phenotype and metastasis of HNSCC. Citation Format: Rita Das, Tsu-Fang Cheng, Jamie coupar, Anthony Saleh, Xingping Yang, jialing Zhang, shaleeka Cornelius, Carter Van Waes, zhong Chen. LTβ receptor and NIK signaling activates the alternative NF-kB pathway in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5253. doi:10.1158/1538-7445.AM2015-5253