Wnt signaling dynamics in head and neck squamous cell cancer tumor-stroma interactions.

Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti-cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial-stromal boundary. To investigate these observations, we generated three pairs of cancer-cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co-culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (eg, WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP-18R5 and OMP-54F28, significantly reduced growth of HNSCC patient-derived xenografts and suppressed Wnt activation at the tumor epithelial-stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.

Dual use of hematopoietic and mesenchymal stem cells enhances engraftment and immune cell trafficking in an allogeneic humanized mouse model of head and neck cancer.

In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.

Molecular characterisation of murine acute myeloid leukaemia induced by 56Fe ion and 137Cs gamma ray irradiation.

Exposure to sparsely ionising gamma- or X-ray irradiation is known to increase the risk of leukaemia in humans. However, heavy ion radiotherapy and extended space exploration will expose humans to densely ionising high linear energy transfer (LET) radiation for which there is currently no understanding of leukaemia risk. Murine models have implicated chromosomal deletion that includes the hematopoietic transcription factor gene, PU.1 (Sfpi1), and point mutation of the second PU.1 allele as the primary cause of low-LET radiation-induced murine acute myeloid leukaemia (rAML). Using array comparative genomic hybridisation, fluorescence in situ hybridisation and high resolution melt analysis, we have confirmed that biallelic PU.1 mutations are common in low-LET rAML, occurring in 88% of samples. Biallelic PU.1 mutations were also detected in the majority of high-LET rAML samples. Microsatellite instability was identified in 42% of all rAML samples, and 89% of samples carried increased microsatellite mutant frequencies at the single-cell level, indicative of ongoing instability. Instability was also observed cytogenetically as a 2-fold increase in chromatid-type aberrations. These data highlight the similarities in molecular characteristics of high-LET and low-LET rAML and confirm the presence of ongoing chromosomal and microsatellite instability in murine rAML.

The programmed death ligand 1 interactome demonstrates bidirectional signaling coordinating immune suppression and cancer progression in head and neck squamous cell carcinoma.

BACKGROUND: The programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) are validated cancer targets; however, emerging mechanisms and impact of PD-L1 intracellular signaling on cancer behavior are poorly understood. METHODS: We investigated the cancer cell intrinsic role of PD-L1 in multiple patient-derived models in vitro and in vivo. PD-L1 overexpression, knockdown, and PD-L1 intracellular domain (PD-L1-ICD) deletion (Δ260-290PD-L1) models were assessed for key cancer properties: clonogenicity, motility, invasion, and immune evasion. To determine how PD-L1 transduces signals intracellularly, we used the BioID2 platform to identify the PD-L1 intracellular interactome. Both human papillomavirus-positive and negative patient-derived xenografts were implanted in NOD-scid-gamma and humanized mouse models to investigate the effects of recombinant PD-1, anti-PD-L1, and anti-signal transducer and activator of transcription 3 (STAT3) in vivo. RESULTS: PD-L1 intracellular signaling increased clonogenicity, motility, and invasiveness in multiple head and neck squamous cell carcinoma (HNSCC) models, and PD-1 binding enhanced these effects. Protein proximity labeling revealed the PD-L1 interactome, distinct for unbound and bound PD-1, which initiated cancer cell-intrinsic signaling. PD-L1 binding partners interleukin enhancer binding factors 2 and 3 (ILF2-ILF3) transduced their effect through STAT3. Δ260-290PD-L1 disrupted signaling and reversed pro-growth properties. In humanized HNSCC in vivo models bearing T-cells, PD-1 binding triggered PD-L1 signaling, and dual PD-L1 and STAT3 inhibition were required to achieve tumor control. CONCLUSIONS: Upon PD-1 binding, the PD-L1 extracellular and intracellular domains exert a synchronized effect to promote immune evasion by inhibiting T-cell function while simultaneously enhancing cancer cell-invasive properties.

Studying Immunotherapy Resistance in a Melanoma Autologous Humanized Mouse Xenograft.

Resistance to immunotherapy is a significant challenge, and the scarcity of human models hinders the identification of the underlying mechanisms. To address this limitation, we constructed an autologous humanized mouse (aHM) model with hematopoietic stem and progenitor cells (HSPC) and tumors from 2 melanoma patients progressing to immunotherapy. Unlike mismatched humanized mouse (mHM) models, generated from cord blood-derived HSPCs and tumors from different donors, the aHM recapitulates a patient-specific tumor microenvironment (TME). When patient tumors were implanted on aHM, mHM, and NOD/SCID/IL2rg-/- (NSG) cohorts, tumors appeared earlier and grew faster on NSG and mHM cohorts. We observed that immune cells differentiating in the aHM were relatively more capable of circulating peripherally, invading into tumors and interacting with the TME. A heterologous, human leukocyte antigen (HLA-A) matched cohort also yielded slower growing tumors than non-HLA-matched mHM, indicating that a less permissive immune environment inhibits tumor progression. When the aHM, mHM, and NSG cohorts were treated with immunotherapies mirroring what the originating patients received, tumor growth in the aHM accelerated, similar to the progression observed in the patients. This rapid growth was associated with decreased immune cell infiltration, reduced interferon gamma (IFNγ)-related gene expression, and a reduction in STAT3 phosphorylation, events that were replicated in vitro using tumor-derived cell lines. IMPLICATIONS: Engrafted adult HSPCs give rise to more tumor infiltrative immune cells, increased HLA matching leads to slower tumor initiation and growth, and continuing immunotherapy past progression can paradoxically lead to increased growth.

Cancer Cell CD44 Mediates Macrophage/Monocyte-Driven Regulation of Head and Neck Cancer Stem Cells.

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction, whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K-4EBP1-SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery.

Inhibiting Translation Elongation with SVC112 Suppresses Cancer Stem Cells and Inhibits Growth in Head and Neck Squamous Carcinoma.

Cancer stem cells (CSC) drive growth, therapy resistance, and recurrence in head and neck squamous cell carcinoma (HNSCC). Regulation of protein translation is crucial for normal stem cells and CSCs; its inhibition could disrupt stemness properties, but translation inhibitors are limited clinically due to toxicity. SVC112 is a synthetic derivative of bouvardin, a plant-derived translation elongation inhibitor. SVC112 had greater antiproliferative effects on HNSCC cells compared with the FDA-approved translation inhibitor omacetaxine mepesuccinate (HHT). SVC112 preferentially inhibited cancer cells compared with patient-matched cancer-associated fibroblasts, whereas HHT was equally toxic to both. SVC112 reduced sphere formation by cell lines and CSCs. SVC112 alone inhibited the growth of patient-derived xenografts (PDX), and SVC112 combined with radiation resulted in tumor regression in HPV-positive and HPV-negative HNSCC PDXs. Notably, CSC depletion after SVC112 correlated with tumor response. SVC112 preferentially impeded ribosomal processing of mRNAs critical for stress response and decreased CSC-related proteins including Myc and Sox2. SVC112 increased cell-cycle progression delay and slowed DNA repair following radiation, enhancing colony and sphere formation radiation effects. In summary, these data demonstrate that SVC112 suppresses CSC-related proteins, enhances the effects of radiation, and blocks growth of HNSCC PDXs by inhibiting CSCs. SIGNIFICANCE: Inhibiting protein elongation with SVC112 reduces tumor growth in head and neck squamous cell carcinoma and increases the effects of radiation by targeting the cancer stem cell pool.

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-XL Inhibitor.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.

Leading edge or tumor core: Intratumor cancer stem cell niches in oral cavity squamous cell carcinoma and their association with stem cell function.

OBJECTIVES: To describe differences in cancer stem cell (CSC) presence and behavior associated with their intratumor compartment of origin using a patient-derived xenograft (PDX) model of oral cavity squamous cell carcinoma (OCSCC). MATERIALS AND METHODS: Four HPV-negative OCSCC PDX cases were selected (CUHN004, CUHN013, CUHN096, CUHN111) and the percentage of CSCs (ALDH+CD44high) was measured in the tumor Leading Edge (LE) and Core compartments of each PDX tumor case via fluorescence activated cell sorting (FACS). The fraction of cells in the proliferative phase was measured by Ki-67 labelling index of paraffin embedded tissue. The proliferation and invasion of LE versus Core CSCs were compared using sphere and Matrigel invasion assays, respectively. RESULTS: Both CUHN111 and CUHN004 demonstrate CSC enrichment in their LE compartments while CUHN013 and CUHN096 show no intratumor difference. Cases with LE CSC enrichment demonstrate greater Ki-67 labelling at the LE. CSC proliferative potential, assessed by sphere formation, reveals greater sphere formation in CUHN111 LE CSCs, but no difference between CUHN013 LE and Core CSCs. CUHN111 CSCs do not demonstrate an intratumor difference in invasiveness while CUHN013 LE CSCs are more invasive than Core CSCs. CONCLUSION: A discrete intratumor CSC niche is present in a subset of OCSCC PDX tumors. The CSC functional phenotype with regard to proliferation and invasion is associated with the intratumor compartment of origin of the CSC: LE or Core. These individual functional characteristics appear to be modulated independently of one another and independently of the presence of an intratumor CSC niche.

Salivary Gland Cancer Patient-Derived Xenografts Enable Characterization of Cancer Stem Cells and New Gene Events Associated with Tumor Progression.

Purpose: Salivary gland cancers (SGC) frequently present with distant metastases many years after diagnosis, suggesting a cancer stem cell (CSC) subpopulation that initiates late recurrences; however, current models are limited both in their availability and suitability to characterize these rare cells.Experimental Design: Patient-derived xenografts (PDX) were generated by engrafting patient tissue onto nude mice from one acinic cell carcinoma (AciCC), four adenoid cystic carcinoma (ACC), and three mucoepidermoid carcinoma (MEC) cases, which were derived from successive relapses from the same MEC patient. Patient and PDX samples were analyzed by RNA-seq and Exome-seq. Sphere formation potential and in vivo tumorigenicity was assessed by sorting for Aldefluor (ALDH) activity and CD44-expressing subpopulations.Results: For successive MEC relapses we found a time-dependent increase in CSCs (ALDH+CD44high), increasing from 0.2% to 4.5% (P=0.033), but more importantly we observed an increase in individual CSC sphere formation and tumorigenic potential. A 50% increase in mutational burden was documented in subsequent MEC tumors, and this was associated with increased expression of tumor-promoting genes (MT1E, LGR5, and LEF1), decreased expression of tumor-suppressor genes (CDKN2B, SIK1, and TP53), and higher expression of CSC-related proteins such as SOX2, MYC, and ALDH1A1. Finally, genomic analyses identified a novel NFIB-MTFR2 fusion in an ACC tumor and confirmed previously reported fusions (NTRK3-ETV6 and MYB-NFIB)Conclusions: Sequential MEC PDX models preserved key patient features and enabled the identification of genetic events putatively contributing to increases in both CSC proportion and intrinsic tumorigenicity, which mirrored the patient's clinical course. Clin Cancer Res; 24(12); 2935-43. ©2018 AACR.

Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2.

Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. Conclusions: The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.

A pilot study of cetuximab and the hedgehog inhibitor IPI-926 in recurrent/metastatic head and neck squamous cell carcinoma.

BACKGROUND: This phase 1, dose-finding study determined the safety, maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), antitumor activity, and molecular correlates of IPI-926, a Hedgehog pathway (HhP) inhibitor, combined with cetuximab in patients with relapsed/metastatic squamous cell carcinoma of the head and neck. PATIENTS AND METHODS: Cetuximab was given with a 400mg/m(2) loading dose followed by 250mg/m(2) weekly. IPI-926 was given daily starting two weeks after cetuximab initiation. A "3+3" study design was used. Prior therapy with cetuximab was allowed. Tumor biopsies occurred prior to cetuximab initiation, prior to IPI-926 initiation, and after treatment with both drugs. RESULTS: Nine patients were enrolled. The RP2D was 160mg, the same as the single-agent IPI-926 MTD. Among 9 treated, 8 evaluable patients, the best responses were 1 partial response (12.5%), 4 stable disease (50%), and 3 disease progressions (37.5%). The median progression free survival was 77days (95% confidence interval 39-156). Decreases in tumor size were seen in both cetuximab-naïve patients (one HPV-positive, one HPV-negative). The most frequent treatment-emergent adverse events were fatigue, muscle cramps, and rash. No DLTs were observed. Tumor shrinkage and progression free survival were associated with intra-tumoral ErbB and HhP gene expression down-regulation during therapy, supporting the preclinical hypothesis. CONCLUSION: Treatment with IPI-926 and cetuximab yielded expected toxicities with signs of anti-tumor activity. Serial tumor biopsies were feasible and revealed proof-of-concept biomarkers.

Targeting the Wnt pathway in human cancers: therapeutic targeting with a focus on OMP-54F28.

The Wnt signaling pathways are a group of signal transduction pathways that play an important role in cell fate specification, cell proliferation and cell migration. Aberrant signaling in these pathways has been implicated in the development and progression of multiple cancers by allowing increased proliferation, angiogenesis, survival and metastasis. Activation of the Wnt pathway also contributes to the tumorigenicity of cancer stem cells (CSCs). Therefore, inhibiting this pathway has been a recent focus of cancer research with multiple targetable candidates in development. OMP-54F28 is a fusion protein that combines the cysteine-rich domain of frizzled family receptor 8 (Fzd8) with the immunoglobulin Fc domain that competes with the native Fzd8 receptor for its ligands and antagonizes Wnt signaling. Preclinical models with OMP-54F28 have shown reduced tumor growth and decreased CSC frequency as a single agent and in combination with other chemotherapeutic agents. Due to these findings, a phase 1a study is nearing completion with OMP-54F28 in advanced solid tumors and 3 phase 1b studies have been opened with OMP-54F28 in combination with standard-of-care chemotherapy backbones in ovarian, pancreatic and hepatocellular cancers. This article will review the Wnt signaling pathway, preclinical data on OMP-54F28 and other Wnt pathway inhibitors and ongoing clinical trials.

Hedgehog signaling drives radioresistance and stroma-driven tumor repopulation in head and neck squamous cancers.

Local control and overall survival in patients with advanced head and neck squamous cell cancer (HNSCC) remains dismal. Signaling through the Hedgehog (Hh) pathway is associated with epithelial-to-mesenchymal transition, and activation of the Hh effector transcription factor Gli1 is a poor prognostic factor in this disease setting. Here, we report that increased GLI1 expression in the leading edge of HNSCC tumors is further increased by irradiation, where it contributes to therapeutic inhibition. Hh pathway blockade with cyclopamine suppressed GLI1 activation and enhanced tumor sensitivity to radiotherapy. Furthermore, radiotherapy-induced GLI1 expression was mediated in part by the mTOR/S6K1 pathway. Stroma exposed to radiotherapy promoted rapid tumor repopulation, and this effect was suppressed by Hh inhibition. Our results demonstrate that Gli1 that is upregulated at the tumor-stroma intersection in HNSCC is elevated by radiotherapy, where it contributes to stromal-mediated resistance, and that Hh inhibitors offer a rational strategy to reverse this process to sensitize HNSCC to radiotherapy.

TERRA, hnRNP A1, and DNA-PKcs Interactions at Human Telomeres.

Maintenance of telomeres, repetitive elements at eukaryotic chromosomal termini, and the end-capping structure and function they provide, are imperative for preserving genome integrity and stability. The discovery that telomeres are transcribed into telomere repeat containing RNA (TERRA) has revolutionized our view of this repetitive, rather unappreciated region of the genome. We have previously shown that the non-homologous end-joining, shelterin associated DNA dependent protein kinase catalytic subunit (DNA-PKcs) participates in mammalian telomeric end-capping, exclusively at telomeres created by leading-strand synthesis. Here, we explore potential roles of DNA-PKcs and its phosphorylation target heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in the localization of TERRA at human telomeres. Evaluation of co-localized foci utilizing RNA-FISH and three-dimensional (3D) reconstruction strategies provided evidence that both inhibition of DNA-PKcs kinase activity and siRNA depletion of hnRNP A1 result in accumulation of TERRA at individual telomeres; depletion of hnRNP A1 also resulted in increased frequencies of fragile telomeres. These observations are consistent with previous demonstrations that decreased levels of the nonsense RNA-mediated decay factors SMG1 and UPF1 increase TERRA at telomeres and interfere with replication of leading-strand telomeres. We propose that hTR mediated stimulation of DNA-PKcs and subsequent phosphorylation of hnRNP A1 influences the cell cycle dependent distribution of TERRA at telomeres by contributing to the removal of TERRA from telomeres, an action important for progression of S-phase, and thereby facilitating efficient telomere replication and end-capping.

Hedgehog signaling alters reliance on EGF receptor signaling and mediates anti-EGFR therapeutic resistance in head and neck cancer.

The EGF receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of squamous cell carcinoma of the head and neck (HNSCC) but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here, we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacologic or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four of six HNSCC patient-derived xenografts; however, they eventually regrew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed regrowth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts, suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state, delaying or completely blocking tumor recurrence.

A patient tumor transplant model of squamous cell cancer identifies PI3K inhibitors as candidate therapeutics in defined molecular bins.

Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV-, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine.