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Supplementary Table S1: Differentially expressed genes (List A and List B combined) identified as E2F7-dependent cytotoxic sensitivity genes

Figure S2. Adenovirus infection of murine keratinocytes does not alter normal cell responses.

The file contains figure legends for 6 supplementary figures

Purpose: Head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have a mortality rate of 45%. We have previously shown that E2F7 may contribute to drug resistance in SCC cells. However, the mechanism and pathways involved remain unknown.Experimental Design: We used transcriptomic profiling to identify candidate pathways that may contribute to E2F7-dependent resistance to anthracyclines. We then manipulated the activity/expression of the candidate pathway using overexpression, knockdown, and pharmacological inhibitors in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and a downstream effector in a tissue microarray (TMA) generated from HNSCC patient samples.Results: E2F7-deficient keratinocytes were selectively sensitive to doxorubicin and this was reversed by overexpressing E2F7. Transcriptomic profiling identified Sphingosine kinase 1 (Sphk1) as a potential mediator of E2F7-dependent drug resistance. Knockdown and overexpression studies revealed that Sphk1 was a downstream target of E2F7. TMA studies showed that E2F7 overexpression correlated with Sphk1 overexpression in human HNSCC. Moreover, inhibition of Sphk1 by shRNA or the Sphk1-specific inhibitor, SK1-I (BML-EI411), enhanced the sensitivity of SCC cells to doxorubicin in vitro and in vivo. Furthermore, E2F7-induced doxorubicin resistance was mediated via Sphk1-dependent activation of AKT in vitro and in vivo.Conclusion: We identify a novel drugable pathway in which E2F7 directly increases the transcription and activity of the Sphk1/S1P axis resulting in activation of AKT and subsequent drug resistance. Collectively, this novel combinatorial therapy can potentially be trialed in humans using existing agents. Clin Cancer Res; 21(2); 417–27. ©2014 AACR.

PDF file - 1290K, Supplementary Figure 2. siRNA against EZH2 does not induce apoptosis. Detroit 562 cells and primary keratinocytes (HEK) were treated with siRNA against EZH2 for 48 hours or 96 hours and stained with Annexin V/7 aminoactinomycin D and subjected to FACS analysis to determine the proportion of apoptotic cells. a) The ability of siRNA to inhibit EZH2 was determined in the HEKs. β-actin is a loading control. b) EZH2 siRNA did not induce apoptosis after 48 hours or c) 96 hours. Data is mean � SEM, n=2

Purpose: Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers diagnosed worldwide and is associated with a 5-year survival rate of 55%. EZH2, a component of the polycomb repressor complex 2, trimethylates H3K27 (H3K27me3), which has been shown to drive squamous differentiation in normal keratinocytes. This study determined whether inhibition of EZH2-mediated epigenetic silencing could induce differentiation or provide therapeutic benefit in HNSCC.Experimental Design: We determined the effects of inhibiting EZH2, by either RNA interference or pharmacologically, on HNSCC growth, viability, and differentiation in vitro. Xenografts of HNSCC cell lines were used to assess efficacy of 3-deazaneplanocin A (DZNep), an inhibitor of H3K27 trimethylation, in vivo.Results: EZH2 was highly expressed in HNSCC cell lines in vitro and tissue microarray analysis revealed high expression in (n = 59) in situ relative to normal oral epithelium (n = 12). Inhibition of EZH2 with siRNA could induce expression of differentiation genes in differentiation-refractory squamous cell carcinoma cell lines. Differentiation-refractory HNSCC cell lines displayed persistent H3K27me3 on the promoters of differentiation genes. DZNep caused cancer-cell–specific apoptosis in addition to a profound reduction in colony-forming efficiency and induction of some squamous differentiation genes. Furthermore, in vivo, DZNep attenuated tumor growth in two different xenograft models, caused intratumor inhibition of EZH2, and induction of differentiation genes in situ.Conclusions: Collectively, these data suggest that aberrant differentiation in HNSCC may be attributed to epigenetic dysregulation and suggest that inhibition of PRC2-mediated gene repression may represent a potential therapeutic target. Clin Cancer Res; 19(2); 428–41. ©2012 AACR.

Objectives A diverse intratumoral T‐cell receptor (TCR) repertoire is associated with improved survival in diffuse large B‐cell lymphoma (DLBCL) treated with rituximab/cyclophosphamide/doxorubicin/prednisolone/vincristine (R‐CHOP) chemoimmunotherapy. We explored the impact of intratumoral TCR repertoire on interim PET (iPET) done after four cycles of R‐CHOP, the relationships between intratumoral and circulating repertoire, and the phenotypes of expanded clonotypes. Methods We sequenced the third complementarity‐determining region of TCRβ in tumor samples, blood at pre‐therapy and after four cycles of R‐CHOP in 35 patients enrolled in ALLGNHL21 trial in high‐risk DLBCL. We correlated the TCR diversity metrics with iPET status, gene expression profiles and HLA‐class I genotypes. We then sequenced the FACS‐sorted peripheral blood T cells in six patients, and pentamer‐sorted EBV‐specific CD8+ T cells in one patient from this cohort. Results Compared with iPET− patients, the intratumoral TCR repertoire in iPET+ patients was characterised by higher cumulative frequency of abundant clonotypes and higher productive clonality. There was a variable overlap between circulating and intratumoral repertoires, with the dominant intratumoral clonotypes more likely to be detected in the blood. The majority of shared clonotypes were CD8+ PD‐1HI T cells, and CD8+ T cells had the largest clonal expansions in tumor and blood. In a patient with EBV+ DLBCL, EBV‐specific intratumoral clonotypes were trackable in the blood. Conclusion This study demonstrates that clonally expanded intratumoral TCR repertoires are associated with iPET+ and that the blood can be used to track tumor‐associated antigen‐specific clonotypes. These findings assist the rationale design and therapeutic monitoring of immunotherapeutic strategies in DLBCL., In a cohort of high‐risk diffuse large B‐cell lymphoma patients enrolled in phase 2 clinical trial, clonally expanded intratumoral TCR repertoires were associated with a positive interim PET scans. Abundant intratumoral clonotypes were identified in peripheral blood at baseline and during chemoimmunotherapy.

Hodgkin lymphoma (HL) comprises two distinct disease entities based on clinical, morphologic and genotypic characteristics. Relative to classical Hodgkin Lymphoma (cHL), nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is rare, and its tumor microenvironment (TME) is very poorly characterized. With the exception of rituximab, there are no targeted treatments nor advances in the treatment of NLPHL for decades. Unlike cHL, the utility of immune-checkpoint blockade (ICB) has not been evaluated in NLPHL. Diagnostic samples were collected from 49 NLPHL patients from 4 Australian centres and compared with stage-matched cHL patients (and with normal lymph nodes). An integrative transcriptomic, proteomic, T-cell clonal and functional analysis was performed to enable a comparison of the composition of the TME and its contribution to immune-evasion in NLPHL with cHL. 730 cancer-immune related genes were digitally quantified. Relative to cHL, gene set enrichment analysis identified T-cell receptor (TCR) and immune-checkpoint signaling pathway dysregulation in NLPHL. Most striking was prominent differential expression of the immune-transcriptome, particularly enrichment for programmed cell death-1 (PD-1) and T-cell Ig and ITIM domain (TIGIT) in NLPHL versus cHL. These were also over expressed compared to normal lymph nodes. Consistent with this, there was also upregulation of numerous T-cell markers (CD247, CD3D, GZMK, CD28, EOMES) in NLPHL. In contrast, immunosuppressive macrophage (CD163, CD36, CD68, COLEC12, MARCO) and regulatory T-cell genes (FOXP3) were higher in cHL. Importantly, PD-L1 and CD155 (respective ligands for PD-1 and TIGIT) were expressed at the surface of NLPHL and cHL malignant B-cells. Multispectral immunofluorescent microscopy showed intratumoral TIGIT+CD4+ and PD-1+CD4+ T-cells were markedly increased in NLPHL versus cHL and localised within NLPHL follicles. Expanded populations of intratumoral CD4+ T-cell clones were predominantly PD-1+ and frequently also TIGIT+. Multi-parameter flow cytometry and dimensionality reduction was used to establish the distribution of immune checkpoints within circulating T-cell subsets. PD-1+TIGIT+CD4+ T-cells were raised in circulating Treg, TH1 and TH2 subsets in NLPHL versus cHL, and PD-1+TIGIT+ TH2 T-cells displayed raised levels of the exhaustion marker EOMES, collectively indicating systemic T-cell dysfunction. To functionally demonstrate the utility of ICB to stimulate T-cells, an assay using cHL and/or NLPHL cell-lines co-cultured with a genetically engineered effector T-cell-line was developed. This showed that TIGIT/PD-1 dual-blockade was more effective than mono-blockade in inducing NLPHL and cHL tumor-directed CD4+ T-cell activation. Overall, our results indicate that immune-evasion mechanisms in NLPHL are distinct to those operative in cHL, with markedly greater T-cell, PD-1 and TIGIT dysregulation. PD-1 and/or TIGIT blockade warrants evaluation in NLPHL. Citation Format: Jay Gunawardana, Soi C. Law, Muhammed B. Sabdia, Karolina Bednarska, Sandra Brosda, Andreea Zaharia, Hennes Tsang, Lilia M. de Long, Melinda Burgess, Dipti Talaulikar, Justina N. Lee, Emily Jude, Eliza A. Hawkes, Sanjiv Jain, Karthik Nath, Clare Gould, Fiona Swain, Joshua W. D. Tobin, Colm Keane, Simone Birch, Mohamed Shanavas, Cameron Snell, Maher K. Gandhi. The immune checkpoints TIGIT and PD-1 are markedly upregulated in NLPHL compared to classical Hodgkin Lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A17.

Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma. Twenty to twenty-five percent of FL patients have progression of disease within 24 months. These patients may benefit from immunotherapy if intact antigen presentation is present. Molecular mechanisms impairing major histocompatibility complex class-I (MHC-I) in FL remain undefined. Here, by sequencing of 172 FL tumours, we found the MHC-I transactivator NLRC5 was the most frequent gene abnormality in the MHC-I pathway. Pyrosequencing showed that epigenetic silencing of the

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) comprises 5% of all Hodgkin lymphomas (HL). Its biology remains poorly characterized. Like classical HL (cHL), it contains minimal malignant cells embedded within a T cell rich intra-tumoral microenvironment (TME). Unlike cHL, it can transform to diffuse large B cell lymphoma (DLBCL). Immune-checkpoint blockade is effective in cHL but has minimal activity in DLBCL. No data is currently available regarding the potential to reactivate host anti-tumoral activity via immune-checkpoint blockade in NLPHL. Diagnostic FFPE samples from 49 NLPHL patients retrospectively collected from 4 Australian centres were interrogated. Inclusion criteria were sample availability and centrally confirmed histological NLPHL. Characteristics were in line with the literature: median age 45 years, range 13-82 years; F:M 1:3.5; stage I/II 55%, III/IV 35% (10% stage unknown) with the majority of cases were of immuno-architectural types A or C. RNA was digitally quantified using the NanoString 770-gene PanCancer Immune panel. Multi-spectral immunofluorescent (mIF) microscopy, plasma soluble PD-1 quantification, cell sorting, T cell receptor (TCR) repertoire analysis and functional immuno-assays were also performed. Results were compared with samples from 38 cHL and 35 DLBCL patients. We initially compared gene expression of NLPHL and cHL, looking for molecular similarities and differences. Ten non-lymphomatous nodes (NLN) were included as controls. Unsupervised clustering showed all but 3 NLPHL cases segregated from the cHL cluster. All NLN congregated in a discrete sub-cluster. As expected, RNA analysis showed significant enrichment for CD20 in NLPHL and CD30 in HL. Volcano plots (Fig. 1a), corrected for false-discovery showed marked variation in gene expression. For NLPHL (vs. cHL) there were 105 upregulated and 337 down regulated genes. Strikingly, the most significantly differentially over-expressed genes in NLPHL were all T cell related (CD247: CD3 zeta chain; CD3D: CD3 delta chain; GZMK: granzyme K; EOMES: marker of CD8 + T cell tolerance; and the immune checkpoints PDCD1: encodes for PD-1; and TIGIT). CD8B expression was increased in NLPHL. For cHL, the most over-expressed genes included macrophage-derived chemokines CCL17 and CCL22. Gene set enrichment analysis revealed activation of the PD-L1 expression and PD-1 checkpoint pathway and 9 of the top 10 Gene Ontology (GO) term enrichment scores involved lymphocyte signalling in NLPHL (Fig. 1b). To better appreciate the impact of the relevant immune checkpoints on their signalling axis, we compared gene ratios for PD-1 and TIGIT receptors with their ligands (PD-L1/L2 and PVR, respectively). NLPHL showed the highest enrichment ratios of these signalling pathways vs. cHL, DLBCL and NLN (Fig. 1c). Although it is known that CD4 +PD-1 +T cells form rosettes around NLPHL cells, the differential cellular localization of immune proteins has not been compared between HL entities. Using mIF, the proportion of intra-tumoral PD-1 + was markedly higher for CD4 + (~7-fold; p Finally, we developed a functional assay using PD-L1/PD-L2 expressing NLPHL and cHL cell lines. These were co-cultured with genetically engineered PD-1 +CD4 + T cells that express a luciferase reporter. Similar levels of heightened T cell activation were seen with immune-checkpoint blockade for both HL entities, indicating that immune-checkpoint inhibition may also be of benefit in NLPHL. In conclusion, our multi-faceted analysis of the immunobiological features of the TME in NLPHL, provides a compelling rationale for early phase clinical studies that incorporate immune-checkpoint blockade in NLPHL. Figure 1 Figure 1. Disclosures Hawkes: Bristol Myers Squib/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Specialised Therapeutics: Consultancy; Merck KgA: Research Funding; Merck Sharpe Dohme: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Antigene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Speakers Bureau; Janssen: Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel and accommodation expenses, Research Funding, Speakers Bureau. Swain: Janssen: Other: Travel expenses paid; Novartis: Other: Travel expenses paid. Keane: BMS: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Karyopharm: Consultancy; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Talaulikar: Takeda: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Jansenn: Honoraria, Research Funding; Roche: Honoraria, Research Funding; EUSA Pharma: Honoraria, Research Funding. Gandhi: janssen: Research Funding; novartis: Honoraria.

Neoantigens are generated by mutations acquired in malignant cells. To be immunogenic, neoantigen peptides must be processed and then presented by HLA molecules on the tumor cell surface to evoke a T cell response. Emerging evidence suggests neoantigens are promising new targets of personalised cancer immunotherapies, provided that antigen presentation remains intact. Recently, we (Tobin, JCO 2019) demonstrated the link between the tumor microenvironment (TME) and clinical outcome in patients with advanced-stage Follicular Lymphoma (ASFL). In particular, ASFL patients with favorable outcomes had high levels of immune infiltration (best stratified by PD-L2 expression) with increased intratumoral CD8+ T cell clonotypes suggestive of expanded antigen-specific T cells in response to presentation by HLA class I neoantigens. This is supported by HLA class I pathway genetic aberrations being infrequent (unlike HLA class II) in FL. However, to date there is minimal data on the frequency and nature of putative neoantigens, their relationship with HLA class I antigen presentation and with the TME in any stage of FL. In particular, the immunobiology of early-stage FL (ESFL) has been largely neglected. Here, we present detailed characterization of these parameters in patients with early-stage FL (ESFL) from the TROG99.03 prospective clinical trial (MacManus, JCO 2018). Digital multiplex gene expression (770 gene PanCancer Immune Panel, NanoString) and targeted NGS (365 genes recurrently mutated in hematological malignancies) was performed on 97 FFPE diagnostic tissue samples. Intratumoral CD8A expression cut-off derived by maximally selected rank statistics was associated with >2-fold differential median PFS (CD8Ahi: 11.5 years vs. CD8Alo: 4.9 years, p=0.0042, Figure 1A). In keeping with a relationship between HLA class I immune effector infiltration within the TME and FL tumor antigen presentation/processing, expression of NLRC5 (a transcriptional activator of HLA class I) was also associated with differential median PFS (NLRC5hi: 10.8 years vs. NLRC5lo: 4.9 years, p=0.021, Figure 1B). The impact of expression of CD8A and NLRC5 were independent of mutations in BCL-2,TP53 and all but of one the m7-FLIPI genes. The exception was EZH2, mutations of which were enriched in cases with reduced CD8A (p=0.0066) and NLRC5 (p Next, we investigated the expression of HLA class I and predicted neoantigens. HLA genotyping was performed using a custom sequencing panel on matched germline peripheral blood DNA for 71 TROG99.03 diagnostic samples. For the corresponding somatic coding variants, the strongest HLA class I binding efficiencies were calculated using 8 epitope prediction algorithms. 41% of samples had no detectable neoantigens, whereas 59% of samples had ≥1 neoantigens detected, with 116 neoantigens total. Importantly, unsupervised hierarchical clustering showed 2-fold enrichment of samples with neoantigens among those with elevated HLA A/B expression vs. samples with low HLA A/B (p=0.0023, Figure 1C). Interestingly, roughly one third of neoantigens were due to indels causing frameshift events resulting in the generation of 'non-self' peptides with strong predicted binding affinity. Recently TCGA data has been used to show that frameshift variants occurring at different sites in a gene can converge to generate a handful neo open reading frame peptides ('NOPs') common to multiple cancer patients. Therefore, it has been postulated that these NOPs could have a considerable therapeutic potential as a novel pan-cancer 'semi-personalized' immunotherapy. In keeping with data derived in solid tumors, in ESFL we observed frameshift variants were most frequently observed in KMT2D. Finally, 5 relapsed/transformed FL samples were compared to their paired diagnostic tissue samples. Not only did neoantigens present at diagnosis typically remain detectable, but in several cases additional neoantigens were also found. In summary, we demonstrate for the first time in ESFL that neoantigens are typically associated with intact HLA class I presentation; CD8 infiltration within the TME is related to tumor antigen processing and associated with favorable outcomes; and that a subset of FL tumors have NOPs. These data have important implications for the design of innovative next-generation immunotherapies in FL. Disclosures Keane: BMS: Research Funding; Celgene: Honoraria, Other: Travel; Roche: Honoraria, Other: Travel, Speakers Bureau; MSD Oncology: Honoraria, Other: Travel; Gilead: Honoraria, Other: Travel, Speakers Bureau. Blombery:Amgen: Consultancy; Novartis: Consultancy; Janssen: Honoraria; Invivoscribe: Honoraria. Tobin:Gilead: Research Funding. Seymour:Nurix: Honoraria; Morphosys: Consultancy, Honoraria; Mei Pharma: Consultancy, Honoraria; Gilead: Consultancy; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Gandhi:Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Merck Sharp & Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Gilead Sciences: Honoraria; Genentech: Honoraria; Mater Research: Current Employment; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Other: Travel, accommodation, expenses .

Blockade of the PD-1 axis has modest efficacy in diffuse large B-cell lymphoma (DLBCL), but data regarding LAG3 are sparse. The impact of LAG3 digital gene expression was tested in 309 patients with DLBCL treated with standard chemoimmunotherapy. Cellular distribution of LAG3 protein was determined by immunohistochemistry and flow cytometry. In tumor-infiltrating lymphocytes (TILs), LAG3 expression was highest on CD4+ regulatory T cells (Tregs) and was also highly expressed on CD8+ T cells compared with CD4+ non-Tregs (both P = .008). LAG3high TILs were enriched in PD-1 and TIM-3. LAG3 was also expressed on a proportion of malignant B cells, and these patients had significantly higher LAG3 messenger RNA in their biopsies (P = .03). LAG3high gene expression was associated with inferior survival in discovery/validation cohorts, independent of cell of origin and the international prognostic index. Patients who were PD-L1high were fivefold more likely to be LAG3high (P < .0001). Patients who were LAG3high/PD-L1high had an inferior progression-free survival (P = .011) and overall survival (P = .005) compared with patients who were LAG3low/PD-L1high. Digital spatial protein analysis confirms LAG3 expression on T cells and, surprisingly, tumor-associated macrophages (TAMs) at higher levels than found on CD20+ B cells in the tumor microenvironment. LAG3 is frequently expressed on CD4+ Tregs and CD8+ TILs, typically with other immune checkpoints, and is also present in a proportion of malignant B cells in DLBCL and in areas enriched for TAMs. LAG3high expression is associated with poor outcome independent of conventional prognosticators.

Summary A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

Recent studies have reported that epigenetic mechanisms may regulate the initiation and progress of squamous differentiation in normal and transformed keratinocytes. In particular, the role of the repressive H3K27me3 mark in the regulation of squamous differentiation has been prominent. However, there is conflicting literature showing that squamous differentiation may be dependent upon or independent of changes in H3K27me3 status. In this study we have examined the binding of trimethylated H3K27 to the promoters of proliferation or differentiation genes in keratinocytes undergoing squamous differentiation in vitro and in vivo. Initially, we examined the expression levels for EZH1, EZH2, and H3K27me3 in differentiating keratinocytes in vitro and in vivo. We extended this to include H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq). Based on these studies, we could find no evidence for an association between widespread gain or loss of H3K27me3 on the promoters of proliferation-specific or differentiation-specific target genes, respectively, during squamous differentiation in adult human keratinocytes. These data suggest that squamous differentiation may occur independent of regulation by H3K27me3 on proliferation and differentiation genes of normal adult human keratinocytes.

Patient mortality rates have remained stubbornly high (40%) for the past 35 years in head and neck squamous cell carcinoma (HNSCC) due to inherent or acquired drug resistance. Thus, a critical issue in advanced SCC is to identify and target the mechanisms that contribute to therapy resistance. We report that the transcriptional inhibitor, E2F7, is mislocalized to the cytoplasm in >80% of human HNSCCs, whereas the transcriptional activator, E2F1, retains localization to the nucleus in SCC. This results in an imbalance in the control of E2F-dependent targets such as SPHK1, which is derepressed and drives resistance to anthracyclines in HNSCC. Specifically, we show that (i) E2F7 is subject to exportin 1 (XPO1)-dependent nuclear export, (ii) E2F7 is selectively mislocalized in most of SCC and multiple other tumor types, (iii) mislocalization of E2F7 in HNSCC causes derepression of Sphk1 and drives anthracycline resistance, and (iv) anthracycline resistance can be reversed with a clinically available inhibitor of XPO1, selinexor, in xenotransplant models of HNSCC. Thus, we have identified a strategy to repurpose anthracyclines for use in SCC. More generally, we provide a strategy to restore the balance of E2F1 (activator) and E2F7 (inhibitor) activity in cancer.

Introduction. Intra-tumoral T-cell infiltration is associated with R-CHOP responsiveness in aggressive B-cell lymphoma (Keane, Lancet Haem 2015). These patients also have a broad (i.e. diverse) intra-tumoral T-cell receptor (TCR) repertoire with a ~20% superior survival compared to those with a narrow (i.e. clonal) repertoire after R-CHOP therapy. Here, the major contributor to the TCR clonal expansion were CD8+ T cells (Keane, CCR 2017). Paradoxically, our recent results in Follicular Lymphoma (FL) (Tobin, JCO in press) found that clonal T-cell expansions were markedly enriched in those patients that experienced progression of disease within 24 months (POD24). Given that FL is a histological subtype associated with a tumor microenvironment distinct from DLBCL including numerous CD4+ T-follicular helper cells (TFH), we now expand upon these findings by comparing TCR repertoires across histological subtypes. We then established whether the TCR repertoire in FL is related to differential TCR clonal expansions between different T-cell subsets and immune checkpoints. Finally, the overlap between tissue and blood TCR repertoires was investigated. Methods. Firstly, unbiased, high-throughput TCRβ sequencing (ImmunoSEQ, Adaptive Biotechnologies) was compared in 164 FFPE tissues (12 healthy nodes, 40 FL, 88 DLBCL, and as a comparator tumor known to be sensitive to checkpoint blockade and to have a high neoantigen burden, 24 melanoma tissues). Next, to determine the contribution of individual T-cell subsets to overall clonality, a further 21 fresh de-aggregated/cryopreserved FL tumor samples were FACS sorted into four T-cell groupings: CD8+ cytotoxic T-lymphocytes (CTLs), CD4+ TFH, CD4+ regulatory T-cells (TREGs) and 'other' (non-TFH/TREG) CD4+ T-cells. Flow cytometry quantified the expression of the checkpoints LAG3, TIM3 and PD1. Then, 5 FL paired tissue/blood samples were tested for shared TCR clones. Results. FL exhibited strikingly reduced TCR repertoire clonality (higher diversity) compared to DLBCL, melanoma and healthy lymph nodes (Fig 1A). Analysis of de-aggregated sorted nodal T-cells revealed a more complex TCR repertoire. The outcome measure was median clonality index (CIx ranging from '0' or minimal, to '1' or maximal clonality). Large T-cell clones in FL (CIx=0.12) predominantly resided within the CTL subset (34% all T-cells). By contrast, there was marked T-cell diversity in TFH (CIx=0.04; 27% all T-cells), TREG (CIx=0.02; 7% all T-cells) and 'other' CD4+ T-cells (CIx=0.02; 32% all T-cells) (Fig 1B). The CTL population had a bimodal expression for PD1 (+51%/-49%), a marker in FL that has been shown to remain functionally active unless co-expressed with LAG3 and/or TIM3 (Yang, Oncotarget 2017). These dual-checkpoint expressing CTLs have reduced capacity to produce cytokines or lytic granules (i.e. they are 'exhausted'). Notably, 54% of the PD1+ CTLs co-expressed either LAG3 or TIM3. Put together, these results are consistent with expanded CTL clones that are frequently functionally exhausted. In contrast, TFH, TREG and 'other' CD4+ T-cells had a low expression of LAG3 and TIM3, although PD1 was frequently found (as expected, particularly in the TFH cells). Finally, in paired tissue/blood samples, there was weak overlap between the circulating and intra-tumoral TCR repertoire in CTLs and TFH T-cells. Conclusion. Although FL has a markedly less clonal TCR repertoire compared to DLBCL, melanoma and even healthy nodes, this result is misleading. Detailed analysis on sorted intra-tumoral T-cell subsets in FL revealed large clonal expansions in CTLs, with approximately half of these classified as functionally exhausted (dual-positive for PD1 and LAG3 and/or TIM3), a state potentially amenable to reversal by dual-checkpoint blockade. The explanation for TCR repertoire diversity lies in CD4+ T-cells (representing approximately two-thirds of T-cells, including the large TFH subset). T-cells in blood did not reflect FL tissue T-cell clones, further highlighting the need for sorted intra-tumoral nodal tissues to accurately assess TCR repertoires in FL. Further characterization of the neo-antigenic targets that CTL clones potentially recognize is required. These results have implications for therapeutic vaccine design and selective recruitment of patients for immune checkpoint blockade. Disclosures Keane: MSD: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Roche: Consultancy, Other: Travel Grant; BMS: Research Funding. Gandhi:Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding.

Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10-20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci.

We have previously shown that E2F7 contributes to drug resistance in head and neck squamous cell carcinoma (HNSCC) cells. Considering that dysregulation of responses to chemotherapy-induced cytotoxicity is one of the major reasons for treatment failure in HNSCC, identifying the downstream effectors that regulate E2F7-dependent sensitivity to chemotherapeutic agents may have direct clinical impact. We used transcriptomic profiling to identify candidate pathways that contribute to E2F7-dependent resistance to doxorubicin. We then manipulated the expression of the candidate pathway using overexpression and knockdown in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and RacGAP1 in a custom tissue microarray (TMA) generated from HNSCC patient samples. Transcriptomic profiling identified RacGAP1 as a potential mediator of E2F7-dependent drug resistance. We validated E2F7-dependent upregulation of RacGAP1 in doxorubicin-insensitive SCC25 cells. Extending this, we found that selective upregulation of RacGAP1 induced doxorubicin resistance in previously sensitive KJDSV40. Similarly, stable knockdown of RacGAP1 in insensitive SCC25 cells induced sensitivity to doxorubicin in vitro and in vivo. RacGAP1 expression was validated in a TMA, and we showed that HNSCCs that overexpress RacGAP1 are associated with a poorer patient overall survival. Furthermore, E2F7-induced doxorubicin resistance was mediated via RacGAP1-dependent activation of AKT. Finally, we show that SCC cells deficient in RacGAP1 grow slower and are sensitized to the cytotoxic actions of doxorubicin in vivo. These findings identify RacGAP1 overexpression as a novel prognostic marker of survival and a potential target to sensitize SCC to doxorubicin. Mol Cancer Ther; 14(8); 1939–50. ©2015 AACR.

Purpose: Head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have a mortality rate of 45%. We have previously shown that E2F7 may contribute to drug resistance in SCC cells. However, the mechanism and pathways involved remain unknown. Experimental Design: We used transcriptomic profiling to identify candidate pathways that may contribute to E2F7-dependent resistance to anthracyclines. We then manipulated the activity/expression of the candidate pathway using overexpression, knockdown, and pharmacological inhibitors in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and a downstream effector in a tissue microarray (TMA) generated from HNSCC patient samples. Results: E2F7-deficient keratinocytes were selectively sensitive to doxorubicin and this was reversed by overexpressing E2F7. Transcriptomic profiling identified Sphingosine kinase 1 (Sphk1) as a potential mediator of E2F7-dependent drug resistance. Knockdown and overexpression studies revealed that Sphk1 was a downstream target of E2F7. TMA studies showed that E2F7 overexpression correlated with Sphk1 overexpression in human HNSCC. Moreover, inhibition of Sphk1 by shRNA or the Sphk1-specific inhibitor, SK1-I (BML-EI411), enhanced the sensitivity of SCC cells to doxorubicin in vitro and in vivo. Furthermore, E2F7-induced doxorubicin resistance was mediated via Sphk1-dependent activation of AKT in vitro and in vivo. Conclusion: We identify a novel drugable pathway in which E2F7 directly increases the transcription and activity of the Sphk1/S1P axis resulting in activation of AKT and subsequent drug resistance. Collectively, this novel combinatorial therapy can potentially be trialed in humans using existing agents. Clin Cancer Res; 21(2); 417–27. ©2014 AACR.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers diagnosed worldwide and is associated with a 5-year survival rate of 55%. EZH2, a component of the polycomb repressor complex 2, trimethylates H3K27 (H3K27me3), which has been shown to drive squamous differentiation in normal keratinocytes. This study determined whether inhibition of EZH2-mediated epigenetic silencing could induce differentiation or provide therapeutic benefit in HNSCC. Experimental Design: We determined the effects of inhibiting EZH2, by either RNA interference or pharmacologically, on HNSCC growth, viability, and differentiation in vitro. Xenografts of HNSCC cell lines were used to assess efficacy of 3-deazaneplanocin A (DZNep), an inhibitor of H3K27 trimethylation, in vivo. Results: EZH2 was highly expressed in HNSCC cell lines in vitro and tissue microarray analysis revealed high expression in (n = 59) in situ relative to normal oral epithelium (n = 12). Inhibition of EZH2 with siRNA could induce expression of differentiation genes in differentiation-refractory squamous cell carcinoma cell lines. Differentiation-refractory HNSCC cell lines displayed persistent H3K27me3 on the promoters of differentiation genes. DZNep caused cancer-cell–specific apoptosis in addition to a profound reduction in colony-forming efficiency and induction of some squamous differentiation genes. Furthermore, in vivo, DZNep attenuated tumor growth in two different xenograft models, caused intratumor inhibition of EZH2, and induction of differentiation genes in situ. Conclusions: Collectively, these data suggest that aberrant differentiation in HNSCC may be attributed to epigenetic dysregulation and suggest that inhibition of PRC2-mediated gene repression may represent a potential therapeutic target. Clin Cancer Res; 19(2); 428–41. ©2012 AACR.

Background Overexpression of CEACAM6 has been reported for a number of malignancies. However, the mechanism of how CEACAM6 contributes to cancer formation and its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. Therefore, we examined the role of CEACAM6 in head and neck squamous cell carcinoma (HNSCC). Methods CEACAM6 expression was examined in normal squamous epithelia as well as a number of patient HNSCC samples and tumours derived from HNSCC cell lines injected into NOD/SCID mice. CEACAM6 expression was manipulated in HNSCC cell lines by shRNA-mediated CEACAM6 knockdown or virally-delivered overexpression of CEACAM6. The role of CEACAM6 in tumour growth and chemotherapeutic sensitivity was then assessed in vivo and in vitro respectively. Results CEACAM6 expression was significantly increased in highly tumourigenic HNSCC cell lines when compared to poorly tumourigenic HNSCC cell lines. Moreover, HNSCC patient tumours demonstrated focal expression of CEACAM6. Functional investigation of CEACAM6, involving over-expression and knock down studies, demonstrated that CEACAM6 over-expression could enhance tumour initiating activity and tumour growth via activation of AKT and suppression of caspase-3 mediated cell death. Conclusion We report that CEACAM6 is focally overexpressed in a large fraction of human HNSCCs in situ. We also show that over-expression of CEACAM6 increases tumour growth and tumour initiating activity by suppressing PI3K/AKT-dependent apoptosis of HNSCC in a xenotransplant model of HNSCC. Finally, our studies indicate that foci of CEACAM6 expressing cells are selectively ablated by treatment of xenotransplant tumours with pharmacological inhibitors of PI3K/AKT in vivo.

Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers diagnosed worldwide and is associated with a 5-year survival rate of 55%. EZH2, a component of the polycomb repressor complex 2, trimethylates H3K27 (H3K27me3); which has been shown to drive squamous differentiation in normal keratinocytes. This study determined whether inhibition of EZH2-mediated epigenetic silencing could induce differentiation or provide therapeutic benefit in HNSCC. We determined the effects of inhibiting EZH2, by either RNA interference or pharmacologically, on HNSCC growth, viability and differentiation in vitro. Xenografts of HNSCC cell lines were used to assess efficacy of 3-Deazaneplanocin A (DZNep), an inhibitor of H3K27 trimethylation, in vivo. EZH2 was highly expressed in HNSCC cell lines in vitro and tissue microarray analysis revealed high expression in (n= 59) in situ relative to normal oral epithelium (n=12). Inhibition of EZH2 with siRNA could induce expression of differentiation genes in differentiation-refractory squamous cell carcinoma cell lines. Differentiation-refractory HNSCC cell lines displayed persistent H3K27me3 on the promoters of differentiation genes. DZNep caused cancer-cell specific apoptosis in addition to a profound reduction in colony forming efficiency and induction of some squamous differentiation genes. Furthermore, in vivo, DZNep attenuated tumour growth in two different xenograft models, caused intra-tumour inhibition of EZH2 and induction of differentiation genes in situ. Collectively, these data suggest that aberrant differentiation in HNSCC may be attributed to epigenetic dysregulation and suggests that inhibition of PRC2-mediated gene repression may represent a potential therapeutic target. Citation Format: Orla M. Gannon, Lilia Merida de Long, Liliana Endo Munoz, Mehlika Hazar Rethinam, Nicholas Saunders. Dysregulation of the repressive H3K27 trimethylation mark in head and neck squamous cell carcinoma contributes to dysregulated squamous differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5352. doi:10.1158/1538-7445.AM2013-5352

In this study we have examined the potential value of a series of clinically relevant PI3K-mTOR inhibitors alone, or in combination with histone deacetylase inhibitors, in a model of head and neck squamous cell carcinoma (HNSCC). We present data relating to in vitro and in vivo pharmacological effects and efficacy for both these classes of drugs. HNSCC cell lines, human keratinocyte and HNSCC xenograft models were treated with histone deacetylase inhibitors (HDACIs) and new generation PI3K and dual PI3K-mTOR inhibitors either alone or in combination. Cell and tumour tissue viability and proliferation were determined in vitro and in vivo following treatments. Effect upon activation status of signalling pathways was also investigated. We report that PI3K, AKT, and dual PI3K-mTOR inhibitors (LY294002, Wortmannin, AKT VIII, BEZ235, BKM120 and BGT226) caused a marked in vitro enhancement of cytotoxicity induced by HDACIs (vorinostat, depsipeptide, valproic acid and LBH589) in HNSCC cancer cells (SCC25, Cal27, Detroit 562). This effect correlates with intense and duration of AKT inhibition, reactive oxygen species (ROS) accumulation, and is attenuated by expression of constitutively active AKT. LBH589, BEZ235, BGT226 and BKM120 inhibited tumour growth alone and in combination in a xenograft model of HNSCC. However, we saw no evidence of improved efficacy with an HDACI / PI3KI combination. Importantly, we observed intratumoural HDAC inhibition and PI3K inhibition as assessed by histone H3 acetylation status and phospho-AKT staining respectively. Finally, further studies indicated that the PI3KI/mTOR inhibitor, BGT226, could significantly enhance cisplatin or taxane-induced cytotoxicity in HNSCC cell lines in vitro.This study indicates, for the first time, that new generation clinically relevant PI3K and dual PI3K-mTOR inhibitors possess antitumour effect against SCC in vivo. These findings provide a rational basis for pursuing clinical trials of PI3KIs in HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2559. doi:10.1158/1538-7445.AM2011-2559