Bin Fang

Covalent inhibition continues to gain momentum as a strategy for selective protein modulation in both therapeutic and chemical biology contexts. Covalent reactive groups (CRGs) typically engage nucleophilic residues such as cysteine, resulting in targeted protein inactivation. However, common electrophiles such as acrylamides often suffer from nonselective reactivity, leading to off-target effects and toxicity. To overcome these limitations, we developed a modular sulfur(IV) reagent platform for the mild, late-stage installation of sulfonyl- and sulfonimidoyl-bicyclobutane motifs with complete cysteine selectivity. This methodology enables access to diverse sulfur(VI) CRGs with tunable strain-release reactivity. Incorporation into US Food and Drug Administration–approved covalent inhibitors demonstrated effective bioisosteric replacement of acrylamides and the potential of strain-release CRGs for selective protein targeting. Preclinical studies in mice have validated this approach, highlighting its promise for next-generation covalent drug design.

T cell receptor (TCR) signaling is precisely tuned to prevent self-reactivity while allowing protective immunity. Here we found that acetylation modulated TCR signaling. The loss of SIRT2 deacetylase activity in T cells led to amplified calcium mobilization and phosphorylation of key proximal TCR molecules in naive T cells and reversed dampened TCR signaling in anergic T cells. During thymic selection, SIRT2 deficiency lowered the TCR signaling threshold and resulted in a broader TCR repertoire diversity. Mechanistically, we identified acetyl-lysine K228 on the linker region of LCK as a substrate specific for SIRT2 that governed LCK conformation and activity. SIRT2 inhibition in exhausted mouse and human tumor-infiltrating T cells restored TCR responsiveness and antitumor immunity. These findings highlighted SIRT2-modulated protein acetylation as a regulatory mechanism that set the TCR threshold in T cells.

Cancer-associated fibroblasts (CAFs) are associated with tumor progression and drug resistance. Here, we investigated the mechanisms underlying the cross-talk between CAFs and tumor cells in non–small cell lung cancer (NSCLC). In NSCLC cell lines with EML4-ALK fusions, we observed substantial CAF-mediated drug resistance to clinically used inhibitors of the tyrosine kinase ALK. Array-based cytokine profiling of CAF-derived conditioned medium indicated that a major contributor to the phenomenon was the secreted growth factor HGF, and blocking its receptor MET overcame paracrine resistance to ALK inhibition. However, cell-selective labeling of the proteome in cocultures also revealed an equally important contribution by the fibronectin-integrin pathway, specifically integrin β ₁ , which was confirmed through pharmacological inhibition and cell-specific silencing or knockout. Concurrent targeting of MET and integrin signaling effectively abrogated ALK inhibitor resistance in EML4-ALK ⁺ NSCLC cells cocultured with CAFs. Moreover, the combination of the ALK inhibitor alectinib with the MET inhibitor capmatinib and/or the integrin inhibitor cilengitide was more effective than single-agent treatment in suppressing tumor growth in allografted mice. The findings illustrate a previously unappreciated complex nature of concurrent paracrine and juxtacrine mechanisms of CAF-driven resistance that may inform the development of more effective therapeutic approaches.

Adaptive immunity to cancer requires effective antigen presentation on Major Histocompatibility Complexes1. While recent advances in identifying cancer-specific MHC-I antigens have ushered in a new era of antigenspecific immunotherapies2-4, studies on MHC-II are less developed due to the lack of robust tools that enable isolation of MHC-II from cell types of interest in physiologically relevant models. We engineered an Cre-inducible endogenous affinity-tagged mouse MHC-II beta chain allele (H2-Ab1, “AbStrep”), the application of which to models of cancer, infectious disease, and autoimmunity has great potential for basic immunology discovery. We applied this tool to lung adenocarcinoma (LUAD), enabling precise and specific purification of MHC-II peptides from malignant LUAD cells or healthy AT2 cells in vivo. We found that numerous peptides were derived from the tumor microenvironment (TME) and systemic circulation. Leveraging these insights, we introduced synthetic antigens systemically, which were scavenged and presented by malignant cells in the TME. Moreover, LUAD cells presented a highly complex landscape of post-translationally modified MHC-II peptides, revealing additional MHC-II peptide binding modes and reflecting intracellular oncogenic signaling5. During LUAD progression, both tumor-host interactions and post-translational modifications dynamically remodel the MHC-II immunopeptidome. These observations suggest the presence of an ever-evolving landscape of MHC-II targets for CD4+ T cells during tumor progression that influences the success or failure of immunotherapy.

The bromodomain-containing protein, transcription factor IID subunit 1 (TAF1; transcription factor II-250), is the largest component of the multiprotein assembly transcription factor IID, a dynamic complex that serves as a general factor for transcription initiation. CRISPR and RNAi screens of pan-cancer cell lines revealed that TAF1 is broadly required for optimal cell growth and survival, but a subset of cell lines showed enhanced TAF1 dependence. These observations suggest that TAF1 has the potential to serve as a therapeutic target in sensitive tumors. Current approaches employed to target TAF1 are limited to monovalent small-molecule inhibitors of the bromodomain. However, recent studies showed that such inhibitors lack cancer cell kill potential. We applied a structure-guided approach to generate cereblon recruiting Proteolysis Targeting Chimera (PROTAC) degraders of TAF1 using the chemical scaffolds of ceralasertib and GNE371. We present evidence that GNE371-based PROTACs are effective in degradation of TAF1 at concentrations as low as 1 nmol/L. TAF1 depletion activated p53 and induced apoptosis in acute myeloid leukemia (AML) cell lines and certain solid tumor cells. An in vivo active TAF1 PROTAC inhibited the growth of AML tumor xenografts. The results showed that inhibition of the bromodomain is not sufficient to inactivate TAF1 functions, whereas a PROTAC approach induces strong biological effects. Furthermore, TAF1 PROTACs have therapeutic potential against AML and other sensitive tumors.

Synthetic chemistry procedures

Supplemental Table 2. RNA-seq analysis of differentially regulated genes after ZS3-046 treatment.

Supplemental Figure 5. TAF1 depletion by PROTAC activates p53.

Supplemental Figure 3. Effects of TAF1 PROTAC on cell cycle profile.

Supplemental Table 3. Proteomics analysis of proteins affected by ZS3-046 treatment.