Circadian clock perturbation frequently occurs in cancer and facilitates tumor progression by regulating malignant growth and shaping the immune microenvironment. Emerging evidence has indicated that clock genes are disrupted in melanoma and linked to immune escape. Herein, we found that the expression of retinoic acid receptor–related orphan receptor-α (RORA) is downregulated in melanoma patients and that patients with higher RORA expression have a better prognosis after immunotherapy. Additionally, RORA was significantly positively correlated with T-cell infiltration and recruitment. Overexpression or activation of RORA stimulated cytotoxic T-cell–mediated antitumor responses. RORA bound to the CD274 promoter and formed an inhibitory complex with HDAC3 to suppress PD-L1 expression. In contrast, the DEAD-box helicase family member DDX3X competed with HDAC3 for binding to RORA, and DDX3X overexpression promoted RORA release from the suppressive complex and thereby increased PD-L1 expression to generate an inhibitory immune environment. The combination of a RORA agonist with an anti-CTLA4 antibody synergistically increased T-cell antitumor immunity in vivo. A score based on the combined expression of HDAC3, DDX3X, and RORA correlated with immunotherapy response in melanoma patients. Together, this study elucidates a mechanism of clock component–regulated antitumor immunity, which will help inform the use of immunotherapy and lead to improved outcomes for melanoma patients receiving combined therapeutic treatments. Significance: RORA forms a corepressor complex to inhibit PD-L1 expression and activate antitumor T-cell responses, indicating that RORA is a potential target and predictive biomarker to improve immunotherapy response in melanoma patients.

A large population in the world has been infected by COVID-19. Understanding the mechanisms of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is important for the management and treatment of COVID-19. When it comes to the infection process, one of the most important proteins in SARS-CoV-2 is the spike (S) protein, which is able to bind to human Angiotensin-Converting Enzyme 2 (ACE2) and initializes the entry of the host cell. In this study, we implemented multiscale computational approaches to study the electrostatic features of the interfaces of the SARS-CoV-2 S protein receptor binding domain and ACE2. The simulations and analyses were performed on high-performance computing resources in the Texas Advanced Computing Center. Our study identified key residues on SARS-CoV-2, which can be used as targets for future drug design. The results shed light on future drug design and therapeutic targets for COVID-19.

The family of Janus Kinases (JAKs) associated with the JAK–signal transducers and activators of transcription signaling pathway plays a vital role in the regulation of various cellular processes. The conformational change of JAKs is the fundamental steps for activation, affecting multiple intracellular signaling pathways. However, the transitional process from inactive to active kinase is still a mystery. This study is aimed at investigating the electrostatic properties and transitional states of JAK1 to a fully activation to a catalytically active enzyme. To achieve this goal, structures of the inhibited/activated full-length JAK1 were modelled and the energies of JAK1 with Tyrosine Kinase (TK) domain at different positions were calculated, and Dijkstra’s method was applied to find the energetically smoothest path. Through a comparison of the energetically smoothest paths of kinase inactivating P733L and S703I mutations, an evaluation of the reasons why these mutations lead to negative or positive regulation of JAK1 are provided. Our energy analysis suggests that activation of JAK1 is thermodynamically spontaneous, with the inhibition resulting from an energy barrier at the initial steps of activation, specifically the release of the TK domain from the inhibited Four-point-one, Ezrin, Radixin, Moesin-PK cavity. Overall, this work provides insights into the potential pathway for TK translocation and the activation mechanism of JAK1.

Thrombocythemia and related disorders are primarily driven by persistent activation of the JAK-STAT signaling pathway, which is frequently associated with valine-to-phenylalanine (VF) point mutations in the JAK2 and TYK2 proteins. However, the precise mechanisms underlying the sustained downstream pathway activation induced by these mutations remain unclear. To investigate this, we employed molecular dynamics simulations and computational biophysical approaches to examine how VF mutations sustain JAK-STAT pathway activation. Our analyses, encompassing energetics, interaction forces, and surface electrostatic charge distributions, revealed that these mutations predominantly alter the spatial conformations and stability of the proteins by modulating inter-dimeric interaction forces and binding free energies. Specifically, the V617F mutation in JAK2 enhanced the stability of the JAK2 dimer and suppressed the activity of the JH2 pseudokinase domain. Similarly, the V678F mutation in TYK2 reducing the binding strength of the JH1 kinase domain and the binding free energy, increasing the binding affinity at the dimer interface. Both mutations ultimately resulted in heightened JH1 kinase domain activity and aberrant activation of downstream signaling pathways. Electrostatic analyses further demonstrated that JAK2V617F strengthened attractive forces within the dimer and TYK2V678F mutation mitigated repulsive forces. Decreased binding free energy confirmed enhanced dimer stability in JAK2V617F and TYK2V678F mutants, driven by distinct mechanisms: reinforced JH2 domain interactions in JAK2V617F and domain compaction with reoriented interfaces in TYK2V678F. These insights delineate the mutation-specific allosteric networks that underpin the pathogenesis of thrombocythemia. Furthermore, they establish a molecular foundation for designing isoform-selective inhibitors that target conformational vulnerabilities, thereby presenting novel therapeutic opportunities. Author Summary: The VF mutations located in JAK2 and TYK2 stabilizes the JH2 pseudokinase domain and enhances dimer interfaces to disrupt autoinhibition in JAK2, while it induces structural reorganization of the dimer FERM/SH2 domains and JH1 kinase conformation to promote substrate accessibility in TYK2. We developed a novel multiscale analyses framework, integrating conformational analysis, electrostatic analysis, hydrogen bond and salt bridges, and free energy decomposition, to collaboratively elucidate the effects of VF mutations on the protein multimers. These findings highlights divergent allosteric networks underlying pathogenic signaling, offering a molecular mechanisms for designing isoform-selective inhibitors targeting conformational vulnerabilities in myeloproliferative disorders.

Circadian clock perturbation frequently occurs in cancer and facilitates tumor progression by regulating malignant growth and shaping the immune microenvironment. Emerging evidence has indicated that clock genes are disrupted in melanoma and linked to immune escape. Herein, we found that the expression of retinoic acid receptor–related orphan receptor-α (RORA) is downregulated in melanoma patients and that patients with higher RORA expression have a better prognosis after immunotherapy. Additionally, RORA was significantly positively correlated with T-cell infiltration and recruitment. Overexpression or activation of RORA stimulated cytotoxic T-cell–mediated antitumor responses. RORA bound to the CD274 promoter and formed an inhibitory complex with HDAC3 to suppress PD-L1 expression. In contrast, the DEAD-box helicase family member DDX3X competed with HDAC3 for binding to RORA, and DDX3X overexpression promoted RORA release from the suppressive complex and thereby increased PD-L1 expression to generate an inhibitory immune environment. The combination of a RORA agonist with an anti-CTLA4 antibody synergistically increased T-cell antitumor immunity in vivo. A score based on the combined expression of HDAC3, DDX3X, and RORA correlated with immunotherapy response in melanoma patients. Together, this study elucidates a mechanism of clock component–regulated antitumor immunity, which will help inform the use of immunotherapy and lead to improved outcomes for melanoma patients receiving combined therapeutic treatments.Significance:RORA forms a corepressor complex to inhibit PD-L1 expression and activate antitumor T-cell responses, indicating that RORA is a potential target and predictive biomarker to improve immunotherapy response in melanoma patients.