Despite breakthroughs achieved with cancer checkpoint blockade therapy (CBT), many patients do not respond to anti–programmed cell death-1 (PD-1) due to primary or acquired resistance. Human tumor profiling and preclinical studies in tumor models have recently uncovered transforming growth factor–β (TGFβ) signaling activity as a potential point of intervention to overcome primary resistance to CBT. However, the development of therapies targeting TGFβ signaling has been hindered by dose-limiting cardiotoxicities, possibly due to nonselective inhibition of multiple TGFβ isoforms. Analysis of mRNA expression data from The Cancer Genome Atlas revealed that TGFΒ1 is the most prevalent TGFβ isoform expressed in many types of human tumors, suggesting that TGFβ1 may be a key contributor to primary CBT resistance. To test whether selective TGFβ1 inhibition is sufficient to overcome CBT resistance, we generated a high-affinity, fully human antibody, SRK-181, that selectively binds to latent TGFβ1 and inhibits its activation. Coadministration of SRK-181-mIgG1 and an anti–PD-1 antibody in mice harboring syngeneic tumors refractory to anti–PD-1 treatment induced profound antitumor responses and survival benefit. Specific targeting of TGFβ1 was also effective in tumors expressing more than one TGFβ isoform. Combined SRK-181-mIgG1 and anti–PD-1 treatment resulted in increased intratumoral CD8+ T cells and decreased immunosuppressive myeloid cells. No cardiac valvulopathy was observed in a 4-week rat toxicology study with SRK-181, suggesting that selectively blocking TGFβ1 activation may avoid dose-limiting toxicities previously observed with pan-TGFβ inhibitors. These results establish a rationale for exploring selective TGFβ1 inhibition to overcome primary resistance to CBT.

Summary Post-translational protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as small ubiquitin like modifier (SUMO) regulates processes including protein homeostasis, the DNA damage response, and the cell cycle. Proliferating cell nuclear antigen (PCNA) is modified by Ub or poly-Ub at Lys164 after DNA damage to recruit repair factors. Yeast PCNA is modified by SUMO on Lys164 and Lys127 during S-phase to recruit the anti-recombinogenic helicase Srs2. Lys164 modification requires specialized E2/E3 enzyme pairs for SUMO or Ub conjugation. For SUMO, Lys164 modification is strictly dependent on the E3 ligase Siz1, suggesting the E3 alters E2 specificity to promote Lys164 modification. The structural basis for substrate interactions in activated E3/E2-Ub/Ubl complexes remains unclear. Here, we report an engineered E2 protein and cross-linking strategies that trap an E3/E2-Ubl/substrate complex for structure determination, illustrating how an E3 can bypass E2 specificity to force-feed a substrate lysine into the E2 active site.

Attachment of ubiquitin (Ub) and ubiquitin-like proteins (Ubls) to cellular proteins regulates numerous cellular processes including transcription, the cell cycle, stress responses, DNA repair, apoptosis, immune responses, and autophagy, to name a few. The mechanistically parallel but functionally distinct conjugation pathways typically require the concerted activities of three types of protein: E1 Ubl-activating enzymes, E2 Ubl carrier proteins, and E3 Ubl ligases. E1 enzymes initiate pathway specificity for each cascade by recognizing and activating cognate Ubls, followed by catalyzing Ubl transfer to cognate E2 protein(s). Under certain circumstances, the E2 Ubl complex can direct ligation to the target protein, but most often requires the cooperative activity of E3 ligases. Reviewed here are recent structural and functional studies that improve our mechanistic understanding of E1-, E2-, and E3-mediated Ubl conjugation.

Background: Ubiquitin carrier protein (E2) recognition by ubiquitin activating enzyme (E1) defines fidelity in subsequent conjugation reactions. Results: E2 transthiolation kinetics identify structural features defining the specificity of E1-E2 binding. Conclusion: E2 paralogs contain a conserved E1 binding motif, and the E1 ␤-grasp domain is a specificity filter for E2 binding. Significance: This defines structural features determining ubiquitin conjugation fidelity.

Background: Targeted degradation by tripartite motif (TRIM) ligase-catalyzed polyubiquitin chain formation is critical for cell regulation and innate immunity. Results: TRIM32-catalyzed chain formation requires oligomerization and uses a cooperative allosteric mechanism. Conclusion: Kinetics suggest TRIM32 assembles polyubiquitin chains as E2-linked thioesters prior to en bloc target protein transfer. Significance: A general mechanism for degradation signal assembly is revealed for the TRIM ligase superfamily.