Radiation therapy (RT), a mainstay treatment for head and neck squamous cell carcinoma (HNSCC), kills cancer cells and modulates the tumor immune microenvironment. We sought to assess the effect of RT in combination with PD-L1/TGF-(3 dual blockade in squamous cell carcinomas (SCC) and analyze the underlying mechanisms. We transplanted mouse SCC cells derived from keratin-15 (K15) stem cells harboring KrasG12D/Smad4- /- mutations into syngeneic recipients and irradiated tumors followed by PD-L1/TGF-(3 dual blockade. We identified a responder line and a non-responder line to this combination therapy. Responder hosts eradicated SCCs by the combined therapy and rejected re-transplanted SCC cells 6 months post tumor eradication, which correlated with clonotype expansions of splenic CD8 T cells and effector memory gene expression identified by single cell sequencing of TCR and transcriptomes, respectively. Mechanistically, RT upregulated MHC-I (major histocompatibility complex I) and its transcriptional regulators including NLRC5, in SCCs of the responders but not nonresponders. These data are consistent with the TCGA HNSCC database in which NLRC5 correlated to MHC-I genes and CD8 T cell gene expression. Functional contribution of MHC-I to PD-L1/TGF-(3 blockade response was confirmed by knocking out beta-2-microglobulin in responder cells that attenuated the response to the same therapy. Thus, the therapeutic effectiveness appeared to largely depend on cancer-cell MHC-I expression, triggering CD8 T cell effector memory-driven responses against tumor cell antigens. Identifying the differential RT response to MHC-I induction may serve as a predictive marker for stratifying patients that are most likely to benefit from this combination therapy.

Radiation therapy (RT), a mainstay treatment for head and neck squamous cell carcinoma (HNSCC), kills cancer cells and modulates the tumor immune microenvironment. We sought to assess the effect of RT in combination with PD-L1/TGF-β dual blockade in squamous cell carcinomas (SCC) and analyze the underlying mechanisms. We transplanted mouse SCC cells derived from keratin-15 (K15) stem cells harboring KrasG12D/Smad4-/- mutations into syngeneic recipients and irradiated tumors followed by PD-L1/TGF-β dual blockade. We identified a responder line and a non-responder line to this combination therapy. Responder hosts eradicated SCCs by the combined therapy and rejected re-transplanted SCC cells 6 months post tumor eradication, which correlated with clonotype expansions of splenic CD8 T cells and effector memory gene expression identified by single cell sequencing of TCR and transcriptomes, respectively. Mechanistically, RT upregulated MHC-I (major histocompatibility complex I) and its transcriptional regulators including NLRC5, in SCCs of the responders but not non-responders. These data are consistent with the TCGA HNSCC database in which NLRC5 correlated to MHC-I genes and CD8 T cell gene expression. Functional contribution of MHC-I to PD-L1/TGF-β blockade response was confirmed by knocking out beta-2-microglobulin in responder cells that attenuated the response to the same therapy. Thus, the therapeutic effectiveness appeared to largely depend on cancer-cell MHC-I expression, triggering CD8 T cell effector memory-driven responses against tumor cell antigens. Identifying the differential RT response to MHC-I induction may serve as a predictive marker for stratifying patients that are most likely to benefit from this combination therapy.

Abstract Purpose: Head and neck squamous cell carcinoma (HNSCC) is often marked by an immunosuppressive tumor microenvironment, which contributes to the dismal 10-20% response rate to immune checkpoint inhibitor (ICI) therapy in HNSCC patients. Radiotherapy (RT) is a currently standard of care and frontline therapy for HNSCC, yet resistance is common. We sought to examine the combination therapy of RT followed by ICI therapy to test if RT induces an in situ vaccination which enhances the efficacy of subsequent ICI. Methods: We utilized SCC cell lines derived from K15.KrasG12D/Smad4−/− SCCs, which mimic the progression of HPV-unrelated HNSCC, and transplanted SCC cells into immune-competent C57BL/6J mouse recipients. Mice were subjected to RT followed by anti-PD-L1/anti-TGFb therapy. CD8+ splenic T cells of responder or non-responder mice were examined by paired single-cell VDJ sequencing and single cell RNA sequencing (TCR/sc-RNAseq) to look for adaptations of the cytotoxic T cell compartment following combination therapy. Responder and non-responder tumor lines or tumors were treated with RT alone in vitro and in vivo, respectively, to examine major histocompatibility complex I (MHC-I) levels on tumor cells. Results: Responders to this combination therapy exhibited complete tumor eradication while non-responders displayed rapid tumor progression. When re-challenged with same HNSCCs cells, responders rejected implanted tumor cells while naïve recipients rapidly developed HNSCCs. Single-cell TCR sequencing of splenic CD8+ T cells revealed that responders, but not naïve recipients exhibited multiclonal CD8+ T cell expansion specifically of memory cell populations. Responder tumor cells exhibited greater levels of MHC-I protein level presentation than non-responders when stimulated with RT. Conclusions: Our data suggest that 1) elevated MHC-I by SCC tumor cells presentation driven by RT and ICI combination therapy facilitate a systemic expansion of memory CD8+ T cell clonotypes which drive anti-tumor immunity and 2) MHC-I expression at baseline or when induced by RT may serve as a predictive marker for therapeutic response to RT and ICI combination therapy in HNSCCs. Citation Format: Hanne Lind, Alexander Strait, Spencer Hall, Jack B. Goon, John D. Aleman, Samantha Chen, Philip Owens, Jing H. Wang, Christian Young, Xiao-Jing Wang. Investigating the combination of radiation and subsequent immune checkpoint inhibitor treatment in head and neck squamous cell carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6646.

Squamous cell carcinoma (SCC) is often marked by an immunosuppressive tumor microenvironment, particularly in metastatic/recurrent disease. The purpose of this study is to evaluate the anti-tumor efficacy of and mechanisms of response to the combination therapy of radiotherapy (RT) and anti-PD-L1/anti-TGFb treatment using SCC models. We transplanted tumor cells derived from either carcinogen-induced SCCs or spontaneous SCCs of K15.Kras12D/Smad4-/- mice to syngeneic mouse recipients and subjected them to RT and anti-PD-L1/anti-TGFb therapy. Responders to this combination therapy exhibited complete tumor eradiation while non-responders displayed rapid tumor progression. Response-status was not predicated upon driver mutation. When re-challenged with same SCC cells, responders rejected tumor cells while naïve recipients rapidly developed SCCs, demonstrating that tumor eradiation is associated with anti-tumor immunity and long-term memory T cells. Responder tumor cells exhibited greater levels of MHC Class-I (MHCcI) mRNA and protein-presentation than non-responders. When treated with RT, responder SCC cells induce MHCcI expression and presentation greater non-responder cells. Additionally, knocking out Beta-2-Microglobulin, a subunit of MHC Class I, by CRISPR abrogated the anti-tumor immune response driven by anti-PD-L1/anti-TGFb treatment in mice inoculated with responder SCC cells. Single cell TCR sequencing of splenic T cells reveals that responders, but not non-responders exhibit multi-clonal CD8+ T cell expansion. Our data suggest that 1) elevated MHCcI by SCC tumor cells presentation facilitate a systemic, clonal expansion of long-term memory CD8+ T cells which drives anti-tumor immunity and 2) MHCcI expression at baseline or when induced by RT may serve as a predictive marker for therapeutic response to RT and anti-PD-L1/anti-TGFb combination therapy in SCCs.

Combined TGFβ/PD‐L1 inhibition is currently undergoing clinical trials in multiple cancer types. The early reported clinical trials of bintrafusp alfa, a bifunctional fusion protein targeting both of these pathways, have had mixed results. Here, we briefly review recent preclinical advances that can be used to refine these ongoing clinical trials and improve their outcomes.

Transforming growth factor beta (TGFβ) and programmed death-ligand 1 (PD-L1) are often overproduced in refractory squamous cell carcinoma (SCC). We examined spatial patterns of PD-L1+ cells in mouse and human SCCs and found that PD-L1 was primarily expressed on infiltrating leukocytes. Although combined TGFβ and PD-L1 blockade are undergoing cancer clinical trials, there are no predictive markers for therapeutic responders. To address this, we used both a small molecule TGFβ inhibitor in combination with anti-PD-L1 and a bifunctional fusion protein targeting both TGFβ and PD-L1 to treat mouse SCCs and found TGFβ inhibition enhanced PD-L1 blockade-induced tumor eradication in multiple tumor models. Furthermore, we identified distinct cell populations of responders and non-responders to bintrafusp alfa, with responders showing a shift toward a more immune-permissive microenvironment. The cellular and molecular signatures of responders versus non-responders to combined TGFβ and PD-L1 blockade provide important insights into future personalized immunotherapy in SCC.

Despite a decline in the incidence of squamous cell carcinomas (SCCs) over the past 20 years, their survival rate has remained nearly the same, indicating that treatment options have not improved relative to other cancer types. Immunotherapies have a high potential for a sustained effect in SCC patients, but their response rate is low. Here, we review the suppressive role of transforming growth factor‐beta (TGFβ) on the antitumor immune response in SCC and present its potential as a therapeutic target in combination with the current range of immunotherapies available for SCC patients. We conclude that SCCs are an optimal cancer type to study the effectiveness of TGFβ inhibition due to the prevalence of dysregulated TGFβ signaling in them.

Smad4 is frequently lost or downregulated in squamous cell carcinomas (SCCs). A spontaneous mouse model of Smad4-/- SCC derived from K15+ stem cells exhibited elevated TGFβ secretion in tumors compared to normal stratified epithelial tissues. In TCGA data, the mRNA expression of Smad4 is negatively correlated with TGFβ ligand mRNA expression in head and neck SCCs. Long-term treatment of mice bearing Smad4-null SCCs with a TGFβ-receptor (TGFβR) small molecule inhibitor modestly reduced tumor growth in immune-competent mice relative to a vehicle control (p<0.05), but had no effect under the same conditions in nude mice, implying that the effectiveness of TGFβ inhibition is due to its modulation of the immune microenvironment. Because these tumor cells express detectable levels of the immune-suppressive checkpoint ligand PD-L1 when co-cultured with CD8 T cells in vitro and because the tumors have high expression of PD-L1 in both tumor cells and infiltrating myeloid cells in vivo, we hypothesized that they would be susceptible to dual inhibition of TGFβR and PD-L1. We treated mice with a short-term (one week) treatment regimen of a TGFβR inhibitor and a PD-L1 blockade antibody. TGFβR inhibition greatly enhanced the effect of PD-L1 blockade on both tumor growth and survival in mice bearing Smad4-null tumors relative to the both the single agents alone and a vehicle control (p<0.01). Furthermore, the same SCC cell line failed to grow when surviving mice were rechallenged 6 months after the primary injection, indicating that they acquired and retained a memory T cell response to the tumor cells. Taken together, this indicates that a short-term treatment with a combination of TGFβR inhibition and PD-L1 blockade has dramatic long-term antitumor effects.

Cancer stem cells (CSCs) are found in many cancer types, including squamous cell carcinoma (SCC). CSCs initiate cancer formation and are linked to metastasis and resistance to therapies. Studies have revealed that several distinct CSC populations coexist in SCC and that tumor initiation and metastatic potential of these populations can be uncoupled. Therefore, it is critical to understand CSC biology to develop novel CSC-targeted therapies for patients with SCC with poor prognoses. This review compares the properties of CSCs in SCC with normal stem cells in the skin, summarizes current advances and characteristics of CSCs, and considers the challenges for CSC-targeted treatment of SCC.

Phosphorylation-dependent protein ubiquitylation and degradation provides an irreversible mechanism to terminate protein kinase signaling. Here, we report that mammary epithelial cells require cullin-5-RING-E3-ubiquitin-ligase complexes (Cul5-CRLs) to prevent transformation by a Src-Cas signaling pathway. Removal of Cul5 stimulates growth-factor-independent growth and migration, membrane dynamics and colony dysmorphogenesis, which are all dependent on the endogenous tyrosine kinase Src. Src is activated in Cul5-deficient cells, but Src activation alone is not sufficient to cause transformation. We found that Cul5 and Src together stimulate degradation of the Src substrate p130Cas (Crk-associated substrate). Phosphorylation stimulates Cas binding to the Cul5-CRL adaptor protein SOCS6 and consequent proteasome-dependent degradation. Cas is necessary for the transformation of Cul5-deficient cells. Either knockdown of SOCS6 or use of a degradation-resistant Cas mutant stimulates membrane ruffling, but not other aspects of transformation. Our results show that endogenous Cul5 suppresses epithelial cell transformation by several pathways, including inhibition of Src-Cas-induced ruffling through SOCS6.

Regulation of protein turnover mediated by ZEITLUPE (ZTL) constitutes an important mechanism of the circadian clock in Arabidopsis thaliana. Here, we report that FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) and LOV KELCH PROTEIN2 (LKP2) play similar roles to ZTL in the circadian clock when ZTL is absent. In contrast with subtle circadian clock defects in fkf1, the clock in ztl fkf1 has a considerably longer period than in ztl. In ztl fkf1 lkp2, several clock parameters were even more severely affected than in ztl fkf1. Although LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression levels are lower in ztl than in the wild type, introducing both fkf1 and lkp2 mutations into the ztl mutant dramatically diminished LHY expression without further affecting CCA1 expression. This demonstrates different contributions of ZTL, FKF1, and LKP2 in the regulation of LHY and CCA1 expression. In addition, FKF1 and LKP2 also interacted with TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATOR5 (PRR5), and both proteins were further stabilized in ztl fkf1 and ztl fkf1 lkp2 compared with in ztl. Our results indicate that ZTL, FKF1, and LKP2 together regulate TOC1 and PRR5 degradation and are major contributors to determining the period of circadian oscillation and enhancing robustness.