A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation.

Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it "constitutively" phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FXXF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FXXF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FXXW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.

Metabolic reprogramming of murine cardiomyocytes during autophagy requires the extracellular nutrient sensor decorin.

The extracellular matrix is a master regulator of tissue homeostasis in health and disease. Here we examined how the small, leucine-rich, extracellular matrix proteoglycan decorin regulates cardiomyocyte metabolism during fasting in vivo First, we validated in Dcn-/- mice that decorin plays an essential role in autophagy induced by fasting. High-throughput metabolomics analyses of cardiac tissue in Dcn-/- mice subjected to fasting revealed striking differences in the hexosamine biosynthetic pathway resulting in aberrant cardiac O-ß-N-acetylglycosylation as compared with WT mice. Functionally, Dcn-/- mice maintained cardiac function at a level comparable with nonfasted animals whereas fasted WT mice showed reduced ejection fraction. Collectively, our results suggest that reduced sensing of nutrient deprivation in the absence of decorin preempts functional adjustments of cardiac output associated with metabolic reprogramming.

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response.

The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr(308) in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr(308) dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser(473)) increased phosphatase resistance of the phosphorylated activation loop (pThr(308)) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr(308) phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin.

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

A novel preclinical strategy for identifying cardiotoxic kinase inhibitors and mechanisms of cardiotoxicity.

RATIONALE: Despite intense interest in strategies to predict which kinase inhibitor (KI) cancer therapeutics may be associated with cardiotoxicity, current approaches are inadequate. Sorafenib is a KI of concern because it inhibits growth factor receptors and Raf-1/B-Raf, kinases that are upstream of extracellular signal-regulated kinases (ERKs) and signal cardiomyocyte survival in the setting of stress. OBJECTIVES: To explore the potential use of zebrafish as a preclinical model to predict cardiotoxicity and to determine whether sorafenib has associated cardiotoxicity, and, if so, define the mechanisms. METHODS AND RESULTS: We find that the zebrafish model is readily able to discriminate a KI with little or no cardiotoxicity (gefitinib) from one with demonstrated cardiotoxicity (sunitinib). Sorafenib, like sunitinib, leads to cardiomyocyte apoptosis, a reduction in total myocyte number per heart, contractile dysfunction, and ventricular dilatation in zebrafish. In cultured rat cardiomyocytes, sorafenib induces cell death. This can be rescued by adenovirus-mediated gene transfer of constitutively active MEK1, which restores ERK activity even in the presence of sorafenib. Whereas growth factor-induced activation of ERKs requires Raf, α-adrenergic agonist-induced activation of ERKs does not require it. Consequently, activation of α-adrenergic signaling markedly decreases sorafenib-induced cell death. Consistent with these in vitro data, inhibition of α-adrenergic signaling with the receptor antagonist prazosin worsens sorafenib-induced cardiomyopathy in zebrafish. CONCLUSIONS: Zebrafish may be a valuable preclinical tool to predict cardiotoxicity. The α-adrenergic signaling pathway is an important modulator of sorafenib cardiotoxicity in vitro and in vivo and appears to act through a here-to-fore unrecognized signaling pathway downstream of α-adrenergic activation that bypasses Raf to activate ERKs.

Skin toxicity of targeted cancer agents: mechanisms and intervention.

In recent years, targeted agents have rapidly evolved as effective tools in the clinical management of a broad range of malignant diseases. These agents disrupt molecular mechanisms and signaling modules that drive the malignant phenotype in defined subsets of malignancies. Beyond the intended cellular targets crucial to tumor growth and progression, these agents also affect signal transduction in normal cells and tissues. The resulting adverse events and their clinical management continue to change, as newer agents with an ever-increasing target spectrum are developed. We provide a succinct overview of dermatologic toxicities arising from the targeting of receptor tyrosine kinases and downstream effectors. Emergent insights into the pathomechanisms involved and the use of this knowledge base to alleviate cutaneous adverse events are discussed.

XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 monoclonal antibody, demonstrates tumor-growth inhibition and tumor-selective pharmacodynamics in mouse models of cancer.

INTRODUCTION: The clinical benefit of the anti-CTLA-4 monoclonal antibody (mAb) ipilimumab has been well established but limited by immune-related adverse events, especially when ipilimumab is used in combination with anti-PD-(L)1 mAb therapy. To overcome these limitations, we have developed XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 mAb. METHODS: XTX101 consists of an anti-human CTLA-4 mAb covalently linked to masking peptides that block the complementarity-determining regions, thereby minimizing the mAb binding to CTLA-4. The masking peptides are designed to be released by proteases that are typically dysregulated within the tumor microenvironment (TME), resulting in activation of XTX101 intratumorally. Mutations within the Fc region of XTX101 were included to enhance affinity for FcγRIII, which is expected to enhance potency through antibody-dependent cellular cytotoxicity. RESULTS: Biophysical, biochemical, and cell-based assays demonstrate that the function of XTX101 depends on proteolytic activation. In human CTLA-4 transgenic mice, XTX101 monotherapy demonstrated significant tumor growth inhibition (TGI) including complete responses, increased intratumoral CD8+T cells, and regulatory T cell depletion within the TME while maintaining minimal pharmacodynamic effects in the periphery. XTX101 in combination with anti-PD-1 mAb treatment resulted in significant TGI and was well tolerated in mice. XTX101 was activated in primary human tumors across a range of tumor types including melanoma, renal cell carcinoma, colon cancer and lung cancer in an ex vivo assay system. CONCLUSIONS: These data demonstrate that XTX101 retains the full potency of an Fc-enhanced CTLA-4 antagonist within the TME while minimizing the activity in non-tumor tissue, supporting the further evaluation of XTX101 in clinical studies.

Gene expression signatures modulated by epidermal growth factor receptor activation and their relationship to cetuximab resistance in head and neck squamous cell carcinoma.

BACKGROUND: Aberrant activation of signaling pathways downstream of epidermal growth factor receptor (EGFR) has been hypothesized to be one of the mechanisms of cetuximab (a monoclonal antibody against EGFR) resistance in head and neck squamous cell carcinoma (HNSCC). To infer relevant and specific pathway activation downstream of EGFR from gene expression in HNSCC, we generated gene expression signatures using immortalized keratinocytes (HaCaT) subjected to ligand stimulation and transfected with EGFR, RELA/p65, or HRASVal12D. RESULTS: The gene expression patterns that distinguished the HaCaT variants and conditions were inferred using the Markov chain Monte Carlo (MCMC) matrix factorization algorithm Coordinated Gene Activity in Pattern Sets (CoGAPS). This approach inferred gene expression signatures with greater relevance to cell signaling pathway activation than the expression signatures inferred with standard linear models. Furthermore, the pathway signature generated using HaCaT-HRASVal12D further associated with the cetuximab treatment response in isogenic cetuximab-sensitive (UMSCC1) and -resistant (1CC8) cell lines. CONCLUSIONS: Our data suggest that the CoGAPS algorithm can generate gene expression signatures that are pertinent to downstream effects of receptor signaling pathway activation and potentially be useful in modeling resistance mechanisms to targeted therapies.

Small GTPases and tyrosine kinases coregulate a molecular switch in the phosphoinositide 3-kinase regulatory subunit.

Phosphoinositide 3-kinase (PI3K) type IA is a heterodimer of a catalytic subunit, p110, and a regulatory subunit, p85. Here we show that p85 contains a GTPase-responsive domain and an inhibitory domain, which together form a molecular switch that regulates PI3K. H-Ras and Rac1 activate PI3K by targeting the GTPase-responsive domain. The stimulatory effect of these molecules, however, is blocked by the inhibitory domain, which functions by binding to tyrosine-phosphorylated molecules and is neutralized by tyrosine phosphorylation. The complementary effects of tyrosine kinases and small GTPases on the p85 molecular switch result in synergy between these two classes of molecules toward the activation of the PI3K/Akt pathway.

Synthetic Triterpenoid RTA-408: Limits Radiation Damage to Normal Tissue.

OBJECTIVES/HYPOTHESIS: To assess the efficacy and mechanism of action of a novel approach to mitigate acute and chronic radiation toxicity in a validated animal model. STUDY DESIGN: Randomized, prospective study using an in vivo rat model. METHODS: Experimental animal study utilizing Sprague-Dawley rats divided into three cohorts: 1) radiation + dimethyl sulfoxide (DMSO) (inert vehicle); 2) radiation + RTA-408 (therapeutic drug); and 3) no radiation + DMSO. All animals in the radiation cohorts underwent 40 Gy of radiation with subsequent inferior epigastric axial rotational flap 30 days later in all cohorts with percentage of flap necrosis and vascular density calculated by blinded observers. In a second experiment, an additional three cohorts, underwent serial punch biopsies of the abdominal skin before, during, and after radiation and drug/vehicle control treatment. Transcriptome analysis utilizing gene set enrichment analysis and digital polymerase chain reaction were performed at various time points. RESULTS: The first experiment revealed average flap necrosis of 20% (95% confidence interval [CI] 16-45) in the radiation control group, 3% (95% CI 0-11) in the nonirradiated control, and 3% (95% CI 0.2-10) in the radiation group treated with RTA-408. Vascular density was preserved in the treatment group as compared to the radiated control. Nine rats were included in the second experiment, and transcriptome analyses in the treatment group revealed robust activation of antioxidant pathways with induced expression of genes associated with hypoxia and adipogenesis/angiogenesis. CONCLUSIONS: Administration of RTA-408 during radiation treatment in a rat model resulted in transcriptome changes which appear to mitigate the toxic effects of radiation, preserving capillary networks and improving flap survival and tissue healing after subsequent surgery. LEVEL OF EVIDENCE: Foundational Evidence, Animal Research Laryngoscope, 132:1196-1204, 2022.

Tadalafil Enhances Immune Signatures in Response to Neoadjuvant Nivolumab in Resectable Head and Neck Squamous Cell Carcinoma.

PURPOSE: We hypothesize that the addition of the phosphodiesterase-5 inhibitor tadalafil to the PD-1 inhibitor nivolumab, is safe and will augment immune-mediated antitumor responses in previously untreated squamous cell carcinoma of the head and neck (HNSCC). PATIENTS AND METHODS: We conducted a two-arm multi-institutional neoadjuvant randomized trial in any-stage resectable HNSCC (NCT03238365). Patients were stratified at randomization by human papillomavirus (HPV) status. Patients in both arms received nivolumab 240 mg intravenously on days 1 and 15 followed by surgery on day 28. Those in the combination therapy arm also received tadalafil 10 mg orally once daily for 4 weeks. Imaging, blood, and tumor were obtained pretreatment and posttreatment for correlative analysis. RESULTS: Neoadjuvant therapy was well-tolerated with no grade 3 to 5 adverse events and no surgical delays. Twenty-five of 46 (54%) evaluable patients had a pathologic treatment response of ≥20%, including three (7%) patients with a complete pathologic response. Regardless of HPV status, tumor proliferation rate was a negative predictor of response. A strong pretreatment T-cell signature in the HPV-negative cohort was a predictor of response. Tadalafil altered the immune microenvironment, as evidenced by transcriptome data identifying enriched B- and natural killer cell gene sets in the tumor and augmented effector T cells in the periphery. CONCLUSIONS: Preoperative nivolumab ± tadalafil is safe in HNSCC and results in more than 50% of the patients having a pathologic treatment response of at least 20% after 4 weeks of treatment. Pretreatment specimens identified HPV status-dependent signatures that predicted response to immunotherapy while posttreatment specimens showed augmentation of the immune microenvironment with the addition of tadalafil.

Caloric Restriction Impairs Regulatory T cells Within the Tumor Microenvironment After Radiation and Primes Effector T cells.

Outcomes for triple negative breast cancer (TNBC) are poor and may be improved by increasing CD8+ tumor infiltrating lymphocytes (TIL) to augment antitumor immunity. Radiation (RT) can promote immunogenic cell death with increased antitumor T cell activity but also stimulates suppressive regulatory T cells (Tregs). Because metabolic alterations affect immune homeostasis and prior studies show caloric restriction (CR) combined with RT improves preclinical TNBC outcomes, we hypothesized that CR augments RT, in part, by altering intratumoral immunity. Using an in vivo model of TNBC, we treated mice with ad libitum (AL) diet, radiation, a CR diet, or CR + RT, and demonstrated an immune suppressive environment with a significant increase in CD4+ CD25+Foxp3+ Tregs after RT but not in CR-fed mice. CD8:Treg ratio in CR + RT TIL increased 4-fold compared with AL + RT mice. In vivo CD8 depletion was performed to assess the role of effector T cells in mitigating the effects of CR, and it was found that in mice undergoing CR, depletion of CD8 T cells resulted in increased tumor progression and decreased median survival compared with isotype control-treated mice. In addition, PD-1 expression on CD3+CD8+ T cells within the tumor microenvironment was significantly increased in CR + RT versus AL + RT treated mice as per immunofluorescence. Serum from breast cancer patients undergoing RT alone or CR and RT was collected pre- and postintervention, and a cytokine array demonstrated that patients treated with CR + RT had notable decreases in immunosuppressive cytokines such as IL-2Rγ, IL-10Rß, and TGF-ß2 and 3 compared with patients receiving RT alone. In conclusion, combining CR with RT decreases intratumoral Tregs, increases CD8:Treg, and increases PD-1 expression via a process dependent on CD8 T cells in a TNBC model. Breast cancer patients undergoing CR concurrently with RT also had significant reduction in immunosuppressive cytokine levels compared with those receiving RT alone.

Differential regulation of p53 function by the N-terminal ΔNp53 and Δ113p53 isoforms in zebrafish embryos.

BACKGROUND: The p53 protein family coordinates stress responses of cells and organisms. Alternative promoter usage and/or splicing of p53 mRNA gives rise to at least nine mammalian p53 proteins with distinct N- and C-termini which are differentially expressed in normal and malignant cells. The human N-terminal p53 variants contain either the full-length (FL), or a truncated (ΔN/Δ40) or no transactivation domain (Δ133) altogether. The functional consequences of coexpression of the different p53 isoforms are poorly defined. Here we investigated functional aspects of the zebrafish ΔNp53 ortholog in the context of FLp53 and the zebrafish Δ133p53 ortholog (Δ113p53) coexpressed in the developing embryo. RESULTS: We cloned the zebrafish ΔNp53 isoform and determined that ionizing radiation increased expression of steady-state ΔNp53 and Δ113p53 mRNA levels in zebrafish embryos. Ectopic ΔNp53 expression by mRNA injection caused hypoplasia and malformation of the head, eyes and somites, yet partially counteracted lethal effects caused by concomitant expression of FLp53. FLp53 expression was required for developmental aberrations caused by ΔNp53 and for ΔNp53-dependent expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A, p21, Cip1, WAF1). Knockdown of p21 expression markedly reduced the severity of developmental malformations associated with ΔNp53 overexpression. By contrast, forced Δ113p53 expression had little effect on ΔNp53-dependent embryonal phenotypes. These functional attributes were shared between zebrafish and human ΔNp53 orthologs ectopically expressed in zebrafish embryos. All 3 zebrafish isoforms could be coimmunoprecipitated with each other after transfection into Saos2 cells. CONCLUSIONS: Both alternative N-terminal p53 isoforms were expressed in developing zebrafish in response to cell stress and antagonized lethal effects of FLp53 to different degrees. However, in contrast to Δ113p53, forced ΔNp53 expression itself led to developmental defects which depended, in part, on p21 transactivation. In contrast to FLp53, the developmental abnormalities caused by ΔNp53 were not counteracted by concomitant expression of Δ113p53.

Is Host Metabolism the Missing Link to Improving Cancer Outcomes?

For the past 100 years, oncologists have relentlessly pursued the destruction of tumor cells by surgical, chemotherapeutic or radiation oncological means. Consistent with this focus, treatment plans are typically based on key characteristics of the tumor itself such as disease site, histology and staging based on local, regional and systemic dissemination. Precision medicine is similarly built on the premise that detailed knowledge of molecular alterations of tumor cells themselves enables better and more effective tumor cell destruction. Recently, host factors within the tumor microenvironment including the vasculature and immune systems have been recognized as modifiers of disease progression and are being targeted for therapeutic gain. In this review, we argue that-to optimize the impact of old and new treatment options-we need to take account of an epidemic that occurs independently of-but has major impact on-the development and treatment of malignant diseases. This is the rapidly increasing number of patients with excess weight and its' attendant metabolic consequences, commonly described as metabolic syndrome. It is well established that patients with altered metabolism manifesting as obesity, metabolic syndrome and chronic inflammation have an increased incidence of cancer. Here, we focus on evidence that these patients also respond differently to cancer therapy including radiation and provide a perspective how exercise, diet or pharmacological agents may be harnessed to improve therapeutic responses in this patient population.

Cancer-Associated Fibroblast Density, Prognostic Characteristics, and Recurrence in Head and Neck Squamous Cell Carcinoma: A Meta-Analysis.

INTRODUCTION: The progression and clinical course of head and neck squamous cell carcinoma (HNSCC) relies on complex interactions between cancer and stromal cells in the tumor microenvironment (TME). Among the most abundant of these stromal cells are cancer-associated fibroblasts (CAFs). While their contribution to tumor progression is widely acknowledged, and various CAF-targeted treatments are under development, the relationship between CAF density and the clinicopathologic course of HNSCC has not been clearly defined. Here we examine the published evidence investigating the relationship of cancer-associated fibroblasts to local recurrence and indicators of prognostic significance in HNSCC. METHODS: We conducted a meta-analysis of existing publications that compare the relationship between CAF density, local recurrence, and clinically significant pathologic criteria of disease development (T stage, nodal positivity, clinical stage, vascular invasion, perineural invasion, Ki67 expression, and differentiation). Thirteen studies met the selection criteria, providing a total study population of 926 patients. Forest plots and risk ratios were generated to illustrate overall relationships. RESULTS: Higher CAF density within the tumor microenvironment is associated with advanced T stage, nodal infiltration, clinical stage, vascular invasion, perineural invasion, Ki67 expression, and differentiation (p <0.05). High CAF density is also associated with increased rates of local recurrence (p <0.001). CONCLUSIONS: Across multiple studies, increased CAF density is correlated with histopathological criteria of poor prognosis in HNSCC. These findings highlight that CAFs may play a pivotal role in HNSCC development and progression. Staining for CAFs may represent a valuable addition to current pathologic analysis and help to guide prognosis and treatment. Understanding the mechanisms by which CAFs reciprocally interact with cancer cells will be crucial for optimization of TME-focused treatment of HNSCC.

Metformin Effects on FOXP3+ and CD8+ T Cell Infiltrates of Head and Neck Squamous Cell Carcinoma.

OBJECTIVES: Alterations of cellular metabolism have been implicated in immune dysfunction in the tumor microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC). Metformin has recently emerged as a candidate of interest for combination with immunotherapy in HNSCC. This study investigated the effect of metformin on immune cell infiltrates of HNSCC. METHODS: Retrospective analysis of T cell infiltrates in primary tumor specimens from patients enrolled in a clinical window of opportunity trial of presurgical metformin. Metformin was titrated to a standard diabetic dose (2000 mg/day) for a minimum of 9 days (mean 13.6 days) prior to surgical resection. Pre and posttreatment surgical specimens from 36 patients (16 HPV+ , 20 HPV- ) were comparatively analyzed. FOXP3+ and CD8+ immune cell infiltrates in the tumor and peritumoral stroma of pre and posttreatment HNSCC specimens were quantified by digital image analysis using Visiopharm software. RESULTS: Metformin treatment was associated with a 41.4% decrease in FOXP3+ T cells in intratumor regions of interest (P = .004) and a 66.5% increase in stromal CD8+ T cells at the leading edge of the tumor (P = .021) when compared to pretreatment biopsies. This was reflected in increased CD8+ /FOXP3+ cell ratios within the tumor (P < .001) and stromal compartments (P < .001). The effects of metformin occurred independently of HPV status. CONCLUSION: Metformin treatment may favorably alter the immune TME in HNSCC independent of HPV status. LEVEL OF EVIDENCE: 1b. This study is most accurately described as a non-randomized controlled trial and therefore may reflect a level of evidence below 1b but above 2a from the provided "levels of evidence" chart. Laryngoscope, 130:E490-E498, 2020.

Mutant BRAF and MEK Inhibitors Regulate the Tumor Immune Microenvironment via Pyroptosis.

Combinations of BRAF inhibitors and MEK inhibitors (BRAFi + MEKi) are FDA-approved to treat BRAF V600E/K-mutant melanoma. Efficacy of BRAFi + MEKi associates with cancer cell death and alterations in the tumor immune microenvironment; however, the links are poorly understood. We show that BRAFi + MEKi caused durable melanoma regression in an immune-mediated manner. BRAFi + MEKi treatment promoted cleavage of gasdermin E (GSDME) and release of HMGB1, markers of pyroptotic cell death. GSDME-deficient melanoma showed defective HMGB1 release, reduced tumor-associated T cell and activated dendritic cell infiltrates in response to BRAFi + MEKi, and more frequent tumor regrowth after drug removal. Importantly, BRAFi + MEKi-resistant disease lacked pyroptosis markers and showed decreased intratumoral T-cell infiltration but was sensitive to pyroptosis-inducing chemotherapy. These data implicate BRAFi + MEKi-induced pyroptosis in antitumor immune responses and highlight new therapeutic strategies for resistant melanoma. SIGNIFICANCE: Targeted inhibitors and immune checkpoint agents have advanced the care of patients with melanoma; however, detailed knowledge of the intersection between these two research areas is lacking. We describe a molecular mechanism of targeted inhibitor regulation of an immune-stimulatory form of cell death and provide a proof-of-principle salvage therapy concept for inhibitor-resistant melanoma.See related commentary by Smalley, p. 176.This article is highlighted in the In This Issue feature, p. 161.

Effect of Hypercapnia, an Element of Obstructive Respiratory Disorder, on Pancreatic Cancer Chemoresistance and Progression.

BACKGROUND: Chronic obstructive respiratory disorders (ORDs) are linked to increased rates of cancer-related deaths. Little is known about the effects of hypercapnia (elevated CO2) on development of pancreatic ductal adenocarcinoma (PDAC) and drug resistance. STUDY DESIGN: Two PDAC cell lines were exposed to normocapnic (5% CO2) and hypercapnic (continuous/intermittent 10% CO2) conditions, physiologically similar to patients with active ORD. Cells were assessed for proliferation rate, colony formation, and chemo-/radiotherapeutic efficacy. In a retrospective clinical study design, patients with PDAC who had undergone pancreatic resection between 2002 and 2014 were reviewed. Active smokers were excluded to remove possible smoking-related protumorigenic influence. Clinical data, pathologic findings, and survival end points were recorded. Kaplan-Meier and Cox regression analyses were performed. RESULTS: Exposure to hypercapnia resulted in increased colony formation and proliferation rates in vitro in both cell lines (MIA-PaCa-2: 111% increase and Panc-1: 114% increase; p < 0.05). Hypercapnia exposure induced a 2.5-fold increase in oxaliplatin resistance (p < 0.05) in both cell lines and increased resistance to ionizing radiation in MIA-PaCa-2 cells (p < 0.05). Five hundred and seventy-eight patients were included (52% were male, median age was 68.7 years [interquartile range 60.6 to 76.8 years]). Cox regression analysis, assessing TNM staging, age, sex, and ORD status, identified ORD as an independent risk factor for both overall survival (hazard ratio 1.64; 95% CI, 1.2 to 2.3; p < 0.05) and disease-free survival (hazard ratio 1.68; 95% CI, 1.06 to 2.67). CONCLUSIONS: PDAC cells exposed to hypercapnic environments, which is common in patients with ORD, showed tumor proliferation, radioresistance, and chemoresistance. Patients with a history of ORD had a worse overall prognosis, suggesting that hypercapnic conditions play a role in the development and progression of PDAC and stressing the need for patient-tailored care.

Discordant Responses Between Primary Head and Neck Tumors and Nodal Metastases Treated With Neoadjuvant Nivolumab: Correlation of Radiographic and Pathologic Treatment Effect.

PD-1 blockade represents a promising treatment in patients with head and neck squamous cell carcinoma (HNSCC). We analyzed results of a neoadjuvant randomized window-of-opportunity trial of nivolumab plus/minus tadalafil to investigate whether immunotherapy-mediated treatment effects vary by site of involvement (primary tumor, lymph nodes) and determine how radiographic tumor shrinkage correlates with pathologic treatment effect. PATIENTS AND METHODS: Forty-four patients enrolled in trial NCT03238365 were treated with nivolumab 240 mg intravenously on days 1 and 15 with or without oral tadalafil, as determined by random assignment, followed by surgery on day 31. Radiographic volumetric response (RVR) was defined as percent change in tumor volume from pretreatment to posttreatment CT scan. Responders were defined as those with a 10% reduction in the volume of the primary tumor or lymph nodes (LN). Pathologic treatment effect (PTE) was defined as the area showing fibrosis or lymphohistiocytic inflammation divided by total tumor area. RESULTS: Sixteen of 32 patients (50%) with pathologic evidence of LN involvement exhibited discordant PTE between primary sites and LN. In four patients with widely discordant adjacent LN, increased PTE was associated with increased infiltration of tumor CD8+ T cells and CD163+ macrophages, whereas stromal regulatory T cells were associated with low nodal PTE. RVR correlated with PTE at both primary tumor (slope = 0.55, p < 0.001) and in LN (slope = 0.62, p < 0.05). 89% (16/18) of radiographic non-responders with T1-T3 primary sites had no (n = 7) or minimal PTE (n = 9), whereas 15/17 (88%) of radiographic responders had moderate (n = 12) or complete (n = 3) PTE. CONCLUSION: Nivolumab often induces discordant treatment effects between primary tumor sites and metastatic lymph nodes within subjects. This treatment discordance was also demonstrated in adjacent lymph nodes, which may correlate with local immune cell makeup. Finally, although these data were generated by a relatively small population size, our data support the use of early radiographic response to assess immunotherapy treatment effect in HNSCC.

miRNA- and cytokine-associated extracellular vesicles mediate squamous cell carcinomas.

Exosomes, or small extracellular vesicles (sEVs), serve as intercellular messengers with key roles in normal and pathological processes. Our previous work had demonstrated that Dsg2 expression in squamous cell carcinoma (SCC) cells enhanced both sEV secretion and loading of pro-mitogenic cargo. In this study, using wild-type Dsg2 and a mutant form that is unable to be palmitoylated (Dsg2cacs), we investigated the mechanism by which Dsg2 modulates SCC tumour development and progression through sEVs. We demonstrate that palmitoylation was required for Dsg2 to regulate sub-cellular localisation of lipid raft and endosomal proteins necessary for sEV biogenesis. Pharmacological inhibition of the endosomal pathway abrogated Dsg2-mediated sEV release. In murine xenograft models, Dsg2-expressing cells generated larger xenograft tumours as compared to cells expressing GFP or Dsg2cacs. Co-treatment with sEVs derived from Dsg2-over-expressing cells increased xenograft size. Cytokine profiling revealed, Dsg2 enhanced both soluble and sEV-associated IL-8 and miRNA profiling revealed, Dsg2 down-regulated both cellular and sEV-loaded miR-146a. miR-146a targets IRAK1, a serine-threonine kinase involved in IL-8 signalling. Treatment with a miR-146a inhibitor up-regulated both IRAK1 and IL-8 expression. RNAseq analysis of HNSCC tumours revealed a correlation between Dsg2 and IL-8. Finally, elevated IL-8 plasma levels were detected in a subset of HNSCC patients who did not respond to immune checkpoint therapy, suggesting that these patients may benefit from prior anti-IL-8 treatment. In summary, these results suggest that intercellular communication through cell-cell adhesion, cytokine release and secretion of EVs are coordinated, and critical for tumour growth and development, and may serve as potential prognostic markers to inform treatment options. ABBREVIATIONS: Basal cell carcinomas, BCC; Betacellulin, BTC; 2-bromopalmitate, 2-Bromo; Cluster of differentiation, CD; Cytochrome c oxidase IV, COX IV; Desmoglein 2, Dsg2; Early endosome antigen 1, EEA1; Epidermal growth factor receptor substrate 15, EPS15; Extracellular vesicle, EV; Flotillin 1, Flot1; Glyceraldehyde-3-phosphate dehydrogenase, GAPH; Green fluorescent protein, GFP; Head and neck squamous cell carcinoma, HNSCC; Interleukin-1 receptor-associated kinase 1, IRAK1; Interleukin 8, IL-8; Large EV, lEV; MicroRNA, miR; Palmitoylacyltransferase, PAT; Ras-related protein 7 Rab7; Small EV, sEV; Squamous cell carcinoma, SCC; Tissue inhibitor of metalloproteinases, TIMP; Tumour microenvironment, TME.