Sumita Trivedi

Identification of the transforming STRN-ALK fusion as a potential therapeutic target in the aggressive forms of thyroid cancer

Significance Thyroid cancer is common and has an excellent outcome in many cases, although a proportion of these tumors have a progressive clinical course and high mortality. Using whole-transcriptome (RNA-sequencing) analysis, we discovered previously unknown genetic events, anaplastic lymphoma kinase (ALK) gene fusions, in thyroid cancer and demonstrate that they occur more often in aggressive cancers. The most common fusion identified in these tumors involved the striatin (STRN) gene, and we show that it is transforming and tumorigenic in vivo. Finally, we demonstrate that the kinase activity of STRN-ALK can be blocked by ALK inhibitors, raising a possibility that ALK fusions may be used as a therapeutic target for patients with the most aggressive and frequently lethal forms of thyroid cancer. Thyroid cancer is a common endocrine malignancy that encompasses well-differentiated as well as dedifferentiated cancer types. The latter tumors have high mortality and lack effective therapies. Using a paired-end RNA-sequencing approach, we report the discovery of rearrangements involving the anaplastic lymphoma kinase (ALK) gene in thyroid cancer. The most common of these involves a fusion between ALK and the striatin (STRN) gene, which is the result of a complex rearrangement involving the short arm of chromosome 2. STRN-ALK leads to constitutive activation of ALK kinase via dimerization mediated by the coiled-coil domain of STRN and to a kinase-dependent, thyroid-stimulating hormone–independent proliferation of thyroid cells. Moreover, expression of STRN-ALK transforms cells in vitro and induces tumor formation in nude mice. The kinase activity of STRN-ALK and the ALK-induced cell growth can be blocked by the ALK inhibitors crizotinib and TAE684. In addition to well-differentiated papillary cancer, STRN-ALK was found with a higher prevalence in poorly differentiated and anaplastic thyroid cancers, and it did not overlap with other known driver mutations in these tumors. Our data demonstrate that STRN-ALK fusion occurs in a subset of patients with highly aggressive types of thyroid cancer and provide initial evidence suggesting that it may represent a therapeutic target for these patients.

Identification of the Cell-Intrinsic and -Extrinsic Pathways Downstream of EGFR and IFNγ That Induce PD-L1 Expression in Head and Neck Cancer

Many cancer types, including head and neck cancers (HNC), express programmed death ligand 1 (PD-L1). Interaction between PD-L1 and its receptor, programmed death 1 (PD-1), inhibits the function of activated T cells and results in an immunosuppressive microenvironment, but the stimuli that induce PD-L1 expression are not well characterized. Interferon gamma (IFNγ) and the epidermal growth factor receptor (EGFR) utilize Janus kinase 2 (JAK2) as a common signaling node to transmit tumor cell-mediated extrinsic or intrinsic signals, respectively. In this study, we investigated the mechanism by which these factors upregulate PD-L1 expression in HNC cells in the context of JAK/STAT pathway activation, Th1 inflammation, and HPV status. We found that wild-type, overexpressed EGFR significantly correlated with JAK2 and PD-L1 expression in a large cohort of HNC specimens. Furthermore, PD-L1 expression was induced in an EGFR- and JAK2/STAT1-dependent manner, and specific JAK2 inhibition prevented PD-L1 upregulation in tumor cells and enhanced their immunogenicity. Collectively, our findings suggest a novel role for JAK2/STAT1 in EGFR-mediated immune evasion, and therapies targeting this signaling axis may be beneficial to block PD-L1 upregulation found in a large subset of HNC tumors.

PD-1/SHP-2 Inhibits Tc1/Th1 Phenotypic Responses and the Activation of T Cells in the Tumor Microenvironment

Immune rejection of tumors is mediated by IFNγ production and T-cell cytolytic activity. These processes are impeded by PD-1, a coinhibitory molecule expressed on T cells that is elevated in tumor-infiltrating lymphocytes (TIL). PD-1 elevation may reflect T-cell exhaustion marked by decreased proliferation, production of type I cytokines, and poor cytolytic activity. Although anti-PD-1 antibodies enhance IFNγ secretion after stimulation of the T-cell receptor (TCR), the mechanistic link between PD-1 and its effects on T-cell help (Tc1/Th1 skewing) remains unclear. In prospectively collected cancer tissues, we found that TIL exhibited dampened Tc1/Th1 skewing and activation compared with peripheral blood lymphocytes (PBL). When PD-1 bound its ligand PD-L1, we observed a marked suppression of critical TCR target genes and Th1 cytokines. Conversely, PD-1 blockade reversed these suppressive effects of PD-1:PD-L1 ligation. We also found that the TCR-regulated phosphatase SHP-2 was expressed higher in TIL than in PBL, tightly correlating with PD-1 expression and negative regulation of TCR target genes. Overall, these results defined a PD-1/SHP-2/STAT1/T-bet signaling axis mediating the suppressive effects of PD-1 on Th1 immunity at tumor sites. Our findings argue that PD-1 or SHP-2 blockade will be sufficient to restore robust Th1 immunity and T-cell activation and thereby reverse immunosuppression in the tumor microenvironment.

CD137 stimulation enhances cetuximab induced natural killer (NK): dendritic cell (DC) priming of anti-tumor T cell immunity in head and neck cancer patients

Purpose: Cetuximab, an EGFR-specific antibody (mAb), modestly improves clinical outcome in patients with head and neck cancer (HNC). Cetuximab mediates natural killer (NK) cell:dendritic cell (DC) cross-talk by cross-linking FcγRIIIa, which is important for inducing antitumor cellular immunity. Cetuximab-activated NK cells upregulate the costimulatory receptor CD137 (4-1BB), which, when triggered by agonistic mAb urelumab, might enhance NK-cell functions, to promote T-cell–based immunity. Experimental design: CD137 expression on tumor-infiltrating lymphocytes was evaluated in a prospective cetuximab neoadjuvant trial, and CD137 stimulation was evaluated in a phase Ib trial, in combining agonistic urelumab with cetuximab. Flow cytometry and cytokine release assays using NK cells and DC were used in vitro, testing the addition of urelumab to cetuximab-activated NK, DC, and cross presentation to T cells. Results: CD137 agonist mAb urelumab enhanced cetuximab-activated NK-cell survival, DC maturation, and tumor antigen cross-presentation. Urelumab boosted DC maturation markers, CD86 and HLA DR, and antigen-processing machinery (APM) components TAP1/2, leading to increased tumor antigen cross-presentation. In neoadjuvant cetuximab-treated patients with HNC, upregulation of CD137 by intratumoral, cetuximab-activated NK cells correlated with FcγRIIIa V/F polymorphism and predicted clinical response. Moreover, immune biomarker modulation was observed in an open label, phase Ib clinical trial, of patients with HNC treated with cetuximab plus urelumab. Conclusions: These results suggest a beneficial effect of combination immunotherapy using cetuximab and CD137 agonist in HNC. Clin Cancer Res; 23(3); 707–16. ©2016 AACR.

STAT1-Induced HLA Class I Upregulation Enhances Immunogenicity and Clinical Response to Anti-EGFR mAb Cetuximab Therapy in HNC Patients

Srivastava and colleagues identify that increase in STAT-1–mediated HLA class I upregulation after cetuximab therapy in patients with head and neck cancer correlates with clinical outcome based on results from a prospective clinical trial investigating neoadjuvant treatment with cetuximab; they suggest that the increase may be a biomarker of response to cetuximab treatment. The goal of this study was to characterize the molecular mechanisms underlying cetuximab-mediated upregulation of HLA class I antigen-processing machinery components in head and neck cancer (HNC) cells and to determine the clinical significance of these changes in cetuximab-treated HNC patients. Flow cytometry, signaling studies, and chromatin immunoprecipitation (ChIP) assays were performed using HNC cells treated with cetuximab alone or with Fcγ receptor (FcγR)-bearing lymphocytes to establish the mechanism of EGFR-dependent regulation of HLA APM expression. A prospective phase II clinical trial of neoadjuvant cetuximab was used to correlate HLA class I expression with clinical response in HNC patients. EGFR blockade triggered STAT1 activation and HLA upregulation, in a src homology-containing protein (SHP)-2–dependent fashion, more prominently in HLA-B/C than in HLA-A alleles. EGFR signaling blockade also enhanced IFNγ receptor 1 (IFNAR) expression, augmenting induction of HLA class I and TAP1/2 expression by IFNγ, which was abrogated in STAT1−/− cells. Cetuximab enhanced HNC cell recognition by EGFR853–861-specific CTLs, and notably enhanced surface presentation of a non-EGFR peptide (MAGE-3271–279). HLA class I upregulation was significantly associated with clinical response in cetuximab-treated HNC patients. EGFR induces HLA downregulation through SHP-2/STAT1 suppression. Reversal of HLA class I downregulation was more prominent in clinical responders to cetuximab therapy, supporting an important role for adaptive immunity in cetuximab antitumor activity. Abrogating EGFR-induced immune escape mechanisms and restoring STAT1 signaling to reverse HLA downregulation using cetuximab should be combined with strategies to enhance adaptive cellular immunity. Cancer Immunol Res; 3(8); 936–45. ©2015 AACR.

Immune biomarkers of anti-EGFR monoclonal antibody therapy

The tumor antigen (TA)-targeted monoclonal antibodies (mAb) cetuximab and panitumumab target the human epidermal growth factor receptor and have been integrated into treatment regimens for advanced squamous cell carcinoma of the head and neck (SCCHN). The therapeutic efficacy of these mAbs has been found to be enhanced when combined with radiotherapy and chemotherapy. However, clinical trials indicate that these findings are limited to fewer than 20% of treated patients. Therefore, identifying patients who are likely to benefit from these agents is crucial to improving therapeutic strategies. Interestingly, it has been noted that TA-targeted mAbs mediate their effects by contributing to cell-mediated cytotoxicity in addition to inhibition of downstream signaling pathways. Here, we describe the potential immunogenic mechanisms underlying these clinical findings, their role in the varied clinical response and identify the putative biomarkers of antitumor activity. We review potential immunological biomarkers that affect mAb therapy in SCCHN patients, the implications of these findings and how they translate to the clinical scenario, which are critical to improving patient selection and ultimately outcomes for patients undergoing therapy.The tumor antigen (TA)-targeted monoclonal antibodies (mAb) cetuximab and panitumumab target the human epidermal growth factor receptor and have been integrated into treatment regimens for advanced squamous cell carcinoma of the head and neck (SCCHN). The therapeutic efficacy of these mAbs has been found to be enhanced when combined with radiotherapy and chemotherapy. However, clinical trials indicate that these findings are limited to fewer than 20% of treated patients. Therefore, identifying patients who are likely to benefit from these agents is crucial to improving therapeutic strategies. Interestingly, it has been noted that TA-targeted mAbs mediate their effects by contributing to cell-mediated cytotoxicity in addition to inhibition of downstream signaling pathways. Here, we describe the potential immunogenic mechanisms underlying these clinical findings, their role in the varied clinical response and identify the putative biomarkers of antitumor activity. We review potential immunological biomarkers that affect mAb therapy in SCCHN patients, the implications of these findings and how they translate to the clinical scenario, which are critical to improving patient selection and ultimately outcomes for patients undergoing therapy.

Anti-EGFR Targeted Monoclonal Antibody Isotype Influences Antitumor Cellular Immunity in Head and Neck Cancer Patients

Purpose: EGF receptor (EGFR) is highly overexpressed on several cancers and two targeted anti-EGFR antibodies which differ by isotype are FDA-approved for clinical use. Cetuximab (IgG1 isotype) inhibits downstream signaling of EGFR and activates antitumor, cellular immune mechanisms. As panitumumab (IgG2 isotype) may inhibit downstream EGFR signaling similar to cetuximab, it might also induce adaptive immunity. Experimental Design: We measured in vitro activation of cellular components of the innate and adaptive immune systems. We also studied the in vivo activation of components of the adaptive immune system in patient specimens from two recent clinical trials using cetuximab or panitumumab. Results: Both monoclonal antibodies (mAb) primarily activate natural killer (NK) cells, although cetuximab is significantly more potent than panitumumab. Cetuximab-activated neutrophils mediate antibody-dependent cellular cytotoxicity (ADCC) against head and neck squamous cell carcinomas (HNSCC) tumor cells, and interestingly, this effect was FcγRIIa- and FcγRIIIa genotype–dependent. Panitumumab may activate monocytes through CD32 (FcγRIIa); however, monocytes activated by either mAb are not able to mediate ADCC. Cetuximab enhanced dendritic cell (DC) maturation to a greater extent than panitumumab, which was associated with improved tumor antigen cross-presentation by cetuximab compared with panitumumab. This correlated with increased EGFR-specific cytotoxic CD8+ T cells in patients treated with cetuximab compared with those treated with panitumumab. Conclusions: Although panitumumab effectively inhibits EGFR signaling to a similar extent as cetuximab, it is less effective at triggering antitumor, cellular immune mechanisms which may be crucial for effective therapy of HNSCC. Clin Cancer Res; 22(21); 5229–37. ©2016 AACR.

Regulation of HPV16 E6 and MCL1 by SF3B1 inhibitor in head and neck cancer cells

ABT-737 inhibits the anti-apoptotic proteins B-cell lymphoma 2 (BCL-2) and BCL-XL. Meayamycin B switches the splicing pattern of myeloid cell leukemia factor 1 (MCL1) pre-mRNA. Specifically, inhibition of splicing factor 3B subunit 1 (SF3B1) with meayamycin B promotes the generation of the proapoptotic, short splicing variant (MCL1-S) and diminishes the antiapoptotic, long variant (MCL1-L). This action was previously associated with the cytotoxicity of meayamycin B in non-small cell lung carcinoma cell lines. ABT-737 induced apoptosis in response to an ablation of MCL1-L by meayamycin B. In this study, we further exploited this synergistic combination in head and neck squamous cell carcinoma (HNSCC), up to 90% of which overexpress MCL1 and BCL-XL. In a panel of seven HNSCC cell lines, the combination of meayamycin B and ABT-737 rapidly triggered a Bax/Bak-mediated apoptosis that overcame the resistance from HPV16-positive HNSCC against each agent alone. Both RT-PCR and Western blotting showed that meayamycin B up-regulated MCL1-S and down-regulated MCL1-L. Significantly, we discovered that SF3B1 was involved in the splicing of oncogenic HPV16 E6 to produce non-oncogenic HPV16 E6*, indicating that SF3B1 may inhibit HPV16-induced tumorigenesis.

EGFR-targeted mAb therapy modulates autophagy in head and neck squamous cell carcinoma through NLRX1–TUFM protein complex

Epidermal growth factor receptor (EGFR)-targeted therapy in head and neck squamous cell carcinoma (HNSCC) patients frequently results in tumor resistance to treatment. Autophagy is an emerging underlying resistance mechanism, however, the molecular autophagy machinery in HNSCC cells and potential biomarkers of patient response to EGFR-targeted therapy remain insufficiently characterized. Here we show that the EGFR blocking with cetuximab leads to varied autophagic responses, which modulate cancer cell susceptibility to EGFR inhibition. Inhibition of autophagy sensitizes HNSCC cells to EGFR blockade. Importantly, we identify a novel signaling hub centering on the NLRX1 (nucleotide-binding, lots of leucine-rich repeats-containing protein member X1)–TUFM (Tu translation elongation factor mitochondrial) protein complex, promoting autophagic flux. Defects in the expression of either NLRX1 or TUFM result in compromised autophagy when treated with EGFR inhibitors. As a previously undefined autophagy-promoting mechanism, we found that TUFM serves as a novel anchorage site, recruiting Beclin-1 to mitochondria, promoting its polyubiquitination, and interfering with its interaction with Rubicon. This protein complex is also essential for endoplasmic reticulum stress signaling induction, possibly as an additional mechanism to promote autophagy. Utilizing tumor specimens from a novel neoadjuvant clinical trial, we show that increased expression of the autophagy adaptor protein, SQSTM1/p62, is associated with poor response to cetuximab therapy. These findings expand our understanding of the components involved in HNSCC autophagy machinery that responds to EGFR inhibitors, and suggest potential combinatorial approaches to enhance its therapeutic efficacy.

Characterization of human papillomavirus antibodies in individuals with head and neck cancer

BACKGROUND Human papillomavirus type 16 (HPV16) E6 antibodies are a promising biomarker of oropharyngeal cancer (OPC); however, seropositivity among non-OPC cases is not well characterized. METHODS Pre-treatment sera from 260 (38 OPC, 222 non-OPC) incident head and neck cancers diagnosed at the University of Pittsburgh between 2003 and 2006 were tested for HPV16 (L1,E1,E2,E4,E6,E7) and non-HPV16 E6 (HPV6,11,18,33) antibodies. Sensitivity and specificity of HPV16 E6 antibodies for HPV-driven tumors was evaluated among tumors with known HPV status (n=25). RESULTS 63.2% of OPC versus 27.5% of non-OPC cases were HPV16 seropositive; HPV16 E6 seroprevalence was 60.5% and 6.3% respectively, odds ratio 22.8 (95% confidence interval [CI] 9.8-53.1). Sensitivity and specificity of HPV16 E6 antibodies for HPV-driven OPC was 100% [95% CI: 50-100%; n=6] and 100% [95% CI: 60-100%, n=4] compared to 0% (n=2) and 0% (n=13) for non-OPC cases. CONCLUSIONS HPV16 antibodies were significantly more common in OPC versus non-OPC cases, particularly HPV16 E6 antibodies.