Mechanistics of Prothymosin alpha and Nrf2 in the Keap1-Nrf2 mediated Oxidative Stress Response

In an effort to dissect the mechanism of interaction of IDPs, in this thesis we focus on Prothymosin a (ProTa) and nuclear factor erythroid 2-related factor 2 (Nrf2), intrinsically disordered proteins, in the Nrf2 mediated oxidative stress response. Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of Nrf2, a key transcription factor of cytoprotective genes. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses Nrf2 activity. During oxidative stress, Nrf2 is released from Keap1 and is shuttled to the nucleus, where it initiates pro cell survival gene transcription. ProTa also interacts with the Kelch domain and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, the interaction between ProTa and the Kelch domain of Keap1 has been delineated using nuclear magnetic resonance spectroscopy (NMR), isothermal titration calorimetry (ITC), peptide array analysis, and site-directed mutagenesis. The results revealed that ProTa retains a high level of flexibility, even in the Kelch-bound state. Mutational analysis pinpointed that the region 38NANEENGE45 of ProTa is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding. A high yield purification protocol with complete backbone NMR resonance assignment lays the foundation for structural and biophysical studies of the full length-Neh2 domain of the human Nrf2. In this work the full-length Neh2 domain was used to investigate binding to Kelch in the presence of cancer causing somatic mutations. To understand the mechanistic links between Keap1 mutations and cancer pathogenesis, the molecular effects of a series of mutations (G333C, G364C, G379D, G350S, R413L, R415G, A427V, G430C, and G476R on the structural and target recognition properties of Keap1 are investigated. These mutations are found to exert differential effects on the protein stability and target binding. Together with the proposed Hinge-and-Latch mechanism of Nrf2/Keap1 binding, these results provide important insight into the molecular impact of different somatic mutations on Keap1’s function as an Nrf2 repressor

Molecular effects of cancer-associated somatic mutations on the structural and target recognition properties of Keap1.

Kelch-like ECH-associated protein 1 (Keap1) plays an important regulatory role in the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent oxidative stress response pathway. It functions as a repressor of Nrf2, a key transcription factor that initiates the expression of cytoprotective enzymes during oxidative stress to protect cells from damage caused by reactive oxygen species. Recent studies show that mutations of Keap1 can lead to aberrant activation of the antioxidant pathway, which is associated with different types of cancers. To gain a mechanistic understanding of the links between Keap1 mutations and cancer pathogenesis, we have investigated the molecular effects of a series of mutations (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C and G476R) on the structural and target recognition properties of Keap1 by using nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) and isothermal titration calorimetry (ITC). Depending on their locations in the protein, these mutations are found to exert differential effects on the protein stability and target binding. Together with the proposed hinge-and-latch mechanism of Nrf2-Keap1 binding in the literature, our results provide important insight into the molecular affect of different somatic mutations on Keap1\u27s function as an Nrf2 repressor

Fuzzy complex formation between the intrinsically disordered prothymosin α and the Kelch domain of Keap1 involved in the oxidative stress response.

Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription. Prothymosin α (ProTα), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, we have characterized the interaction between ProTα and the Kelch domain of Keap1 by using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, peptide array analysis, site-directed mutagenesis, and molecular dynamic simulations. The results of nuclear magnetic resonance chemical shift mapping, amide hydrogen exchange, and spin relaxation measurements revealed that ProTα retains a high level of flexibility, even in the bound state with Kelch. This finding is in agreement with the observations from the molecular dynamic simulations of the ProTα-Kelch complex. Mutational analysis of ProTα, guided by peptide array data and isothermal titration calorimetry, further pinpointed that the region (38)NANEENGE(45) of ProTα is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding

Tumor molecular differences associated with outcome disparities of Black patients with head and neck cancer

BackgroundNumerous studies of head and neck squamous cell carcinoma (HNSCC) have demonstrated disparate outcomes by race and ethnicity. Beyond known associations with socioeconomic variables, whether these are also associated with differences in tumor molecular composition has thus far been poorly explored.MethodsWe downloaded clinical and multiplatform molecular data from The Cancer Genome Atlas and other published studies. These were compared between non-Hispanic Black (n = 43) and White (n = 354) patients with non-HPV-related tumors, using multivariable models. Publicly available validation cohorts were used.ResultsBlack patients had poorer progression-free survival than White patients. Tumors of Black patients had greater copy number aberrations, and increased SFRP1 methylation and miRNA-mediated PRG4 silencing associated with poor survival. PI3K/AkT/mTOR pathway proteins were differentially expressed.ConclusionsThere are molecular differences between tumors of Black and White patients that may partially account for differences in survival. These may inform targeted treatment decisions to achieve equitable outcomes

Analysis of the TCGA Dataset Reveals that Subsites of Laryngeal Squamous Cell Carcinoma are Molecularly Distinct.

Laryngeal squamous cell carcinoma (LSCC) from different subsites have distinct presentations and prognosis. In this study, we carried out a multiomic comparison of LSCC subsites. The Cancer Genome Atlas (TCGA) LSCC cohort was analyzed in the R statistical environment for differences between supraglottic and glottic cancers in single nucleotide variations (SNVs), copy number alterations (CNAs), mRNA abundance, protein abundance, pathway overrepresentation, tumor microenvironment (TME), hypoxia status, and patient outcome. Supraglottic cancers had significantly higher overall and smoking-associated SNV mutational load. Pathway analysis revealed upregulation of muscle related pathways in glottic cancer and neural pathways in supraglottic cancer. Proteins involved in cancer relevant signaling pathways including PI3K/Akt/mTOR, the cell cycle, and PDL1 were differentially abundant between subsites. Glottic and supraglottic tumors have different molecular profiles, which may partially account for differences in presentation and response to therapy

Analysis of the TCGA Dataset Reveals that Subsites of Laryngeal Squamous Cell Carcinoma Are Molecularly Distinct

Laryngeal squamous cell carcinoma (LSCC) from different subsites have distinct presentations and prognosis. In this study, we carried out a multiomic comparison of LSCC subsites. The Cancer Genome Atlas (TCGA) LSCC cohort was analyzed in the R statistical environment for differences between supraglottic and glottic cancers in single nucleotide variations (SNVs), copy number alterations (CNAs), mRNA abundance, protein abundance, pathway overrepresentation, tumor microenvironment (TME), hypoxia status, and patient outcome. Supraglottic cancers had significantly higher overall and smoking-associated SNV mutational load. Pathway analysis revealed upregulation of muscle related pathways in glottic cancer and neural pathways in supraglottic cancer. Proteins involved in cancer relevant signaling pathways including PI3K/Akt/mTOR, the cell cycle, and PDL1 were differentially abundant between subsites. Glottic and supraglottic tumors have different molecular profiles, which may partially account for differences in presentation and response to therapy

3p Arm Loss and Survival in Head and Neck Cancer: An Analysis of TCGA Dataset

Loss of the 3p chromosome arm has previously been reported to be a biomarker of poorer outcome in both human papillomavirus (HPV)-positive and HPV-negative head and neck cancer. However, the precise operational measurement of 3p arm loss is unclear and the mutational profile associated with the event has not been thoroughly characterized. We downloaded the clinical, single nucleotide variation (SNV), copy number aberration (CNA), RNA sequencing, and reverse phase protein assay (RPPA) data from The Cancer Genome Atlas (TCGA) and The Cancer Proteome Atlas HNSCC cohorts. Survival data and hypoxia scores were downloaded from published studies. In addition, we report the inclusion of an independent Memorial Sloan Kettering cohort. We assessed the frequency of loci deletions across the 3p arm separately in HPV-positive and -negative disease. We found that deletions on chromosome 3p were almost exclusively an all or none event in the HPV-negative cohort; patients either had <1% or >97% of the arm deleted. 3p arm loss, defined as >97% deletion in HPV-positive patients and >50% in HPV-negative patients, had no impact on survival (p > 0.05). However, HPV-negative tumors with 3p arm loss presented at a higher N-category and overall stage and developed more distant metastases (p < 0.05). They were enriched for SNVs in TP53, and depleted for point mutations in CASP8, HRAS, HLA-A, HUWE1, HLA-B, and COL22A1 (false discovery rate, FDR < 0.05). 3p arm loss was associated with CNAs across the whole genome (FDR < 0.1), and pathway analysis revealed low lymphoid-non-lymphoid cell interactions and cytokine signaling (FDR < 0.1). In the tumor microenvironment, 3p arm lost tumors had low immune cell infiltration (FDR < 0.1) and elevated hypoxia (FDR < 0.1). 3p arm lost tumors had lower abundance of proteins phospho-HER3 and ANXA1, and higher abundance of miRNAs hsa-miR-548k and hsa-miR-421, which were all associated with survival. There were no molecular differences by 3p arm status in HPV-positive patients, at least at our statistical power level. 3p arm loss is largely an all or none phenomenon in HPV-negative disease and does not predict poorer survival from the time of diagnosis in TCGA cohort. However, it produces tumors with distinct molecular characteristics and may represent a clinically useful biomarker to guide treatment decisions for HPV-negative patients

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification.

BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification

Background There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. Methods We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. Findings A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 x 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. Interpretation With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. Copyright (C) 2022 The Author(s). Published by Elsevier B.V