Nupura Kale

Engineered T-cell therapies are limited by progressive dysfunction under chronic tumor antigen exposure, and genetic perturbation of negative regulators has emerged as a promising strategy to sustain effector activity. We reanalyzed a genome-wide CRISPR knockout (KO) screen in primary human T cells under repetitive tumor stimulation; protein–protein interaction mapping revealed seven members of the cullin–RING ligase 5 (CRL5) complex as an enriched hit cluster. KO of any of these members [cullin-5 (CUL5), ARIH2, RNF7, UBE2F, UBE2L3, CISH, and DCUN1D3] enhanced tumor serial killing, and CUL5, the complex scaffold, emerged as the strongest target. CUL5 KO T cells exhibited sustained cytotoxicity, elevated effector cytokine production, and fewer exhaustion marker–positive cells while retaining antigen specificity and cytokine dependence. Quantitative ubiquitylation proteomics in resting and acutely and chronically stimulated T cells mapped CRL5-dependent candidate substrates beyond the canonical JAK/STAT axis, including nutrient transporters, receptor trafficking proteins, and T-cell receptor (TCR) signaling components. The benefit of CUL5 KO extended across chimeric antigen receptor (CAR) targeting CD19, B-cell maturation antigen (BCMA), and human epidermal growth factor receptor 2 (HER2) with different costimulatory domains and to 1XX-modified CAR and human leukocyte antigen–independent TCR architectures. Combining CUL5 ablation with a tumor-induced IL2 circuit produced tumor control across nine rounds of repetitive stimulation, whereas either modification alone eventually failed. In vivo, CUL5 KO BCMA CAR T cells were enriched in the bone marrow following repeated tumor rechallenge and exhibited enhanced ex vivo cytotoxicity after nearly 90 days in tumor-bearing mice. These findings position CUL5 perturbation as a strategy to improve T-cell persistence and the efficacy of adoptive cell therapies, potentially with engineered cytokine support. Significance: Knockout of CLR5 complex members, especially CUL5, attenuates T-cell exhaustion during prolonged tumor exposure, enabling CAR- and TCR-engineered T cells to sustain cytotoxicity during repeated tumor challenge. See related commentary by Hanina and Maus, p. XX . See related article by Saco et al., p. XX .

T-cell exhaustion limits durable antitumor responses and remains a barrier to adoptive cell therapies (ACT) in solid tumors. To identify regulators of T-cell persistence, we performed genome-wide CRISPR knockout screens in primary human CD8+ T cells expressing an NY-ESO-1–specific T-cell receptor and subjected them to repeat tumor cell rechallenges. These screens revealed an enrichment of single-guide RNAs targeting components of cullin–RING E3 ubiquitin ligases, particularly cullin-5–RING E3 ligase (CRL5) assemblies comprising CUL5, RNF7, and ARIH2. Disruption of CRL5 genes in T cells preserved cytotoxic activity and enhanced cytokine production while sustaining effector-associated transcriptional programs and suppressing exhaustion-related signatures. Pharmacologic inhibition of cullin neddylation with the NEDD8-activating enzyme inhibitor MLN4924 recapitulated CRL5 loss and enhanced T-cell effector function in vitro. In a human melanoma xenograft model, ACT with CRL5-deficient T cells improved tumor control. Together, these findings identify CRL5 as a negative regulator of T-cell fitness and a potential therapeutic target to enhance ACT durability in solid tumors. Significance: The CRL5 complex negatively regulates CD8+ T-cell persistence during chronic antigen stimulation, identifying CRL5 disruption as a potential strategy to sustain T-cell effector function and improve ACT. See related commentary by Hanina and Maus, p. XX . See related article by Bachl et al., p. XX .

Targeted epigenetic engineering of gene expression in cell therapies would allow programming of desirable phenotypes without many of the challenges and safety risks associated with double-strand break-based genetic editing approaches. Here, we develop an all-RNA platform for efficient, durable and multiplexed epigenetic programming in primary human T cells, stably turning endogenous genes off or on using CRISPRoff and CRISPRon epigenetic editors. We achieve epigenetic programming of diverse targeted genomic elements without the need for sustained expression of CRISPR systems. CRISPRoff-mediated gene silencing is maintained through numerous cell divisions, T cell stimulations and in vivo adoptive transfer, avoiding cytotoxicity or chromosomal abnormalities inherent to multiplexed Cas9-mediated genome editing. Lastly, we successfully combined genetic and epigenetic engineering using orthogonal CRISPR Cas12a–dCas9 systems for targeted chimeric antigen receptor (CAR) knock-in and CRISPRoff silencing of therapeutically relevant genes to improve preclinical CAR-T cell-mediated in vivo tumor control and survival.

A major challenge in melanoma treatment is the emergence of aggressive metastases from previously dormant disseminated cancer cells (DCCs). Here we identify Midkine (MDK) as a key factor that disrupts melanoma DCC dormancy by triggering an autocrine signal that suppresses the dormancy inducer NR2F1 and alters the p-p38/p-ERK ratio, via ALK signaling. Dormant melanoma DCCs in lymph nodes and visceral tissues exhibit an MDK ^LOW /NR2F1 ^HIGH phenotype. Transient NR2F1 activation for one month with a potent agonist counteracts MDK-driven mTORC1 activity and suppresses lung metastasis in mice bearing human melanoma DCCs. Dual targeting of MDK (genetic blockade) and NR2F1 (activation) markedly limits metastatic outgrowth and extends survival for 6 months in mouse models independent of BRAF status. These findings support the MDK-NR2F1 axis as a promising therapeutic target to sustain dormancy and prevent melanoma recurrence. Statement of Significance: This work identifies Midkine as a key disruptor of tumor dormancy in melanoma, driving metastatic awakening via NR2F1 suppression. Dual targeting of Midkine (inhibition) and NR2F1 (activation) prolongs survival in preclinical models.

T cell therapies, such as chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) T cells, are a growing class of anti-cancer treatments. However, expansion to novel indications and beyond last-line treatment requires engineering cells’ dynamic population behaviors. Here we develop the tools for cellular behavior analysis of T cells from live-cell imaging, a common and inexpensive experimental setup used to evaluate engineered T cells. We first develop a state-of-the-art segmentation and tracking pipeline, Caliban , based on human-in-the-loop deep learning. We then build the Occident pipeline to collect a catalog of phenotypes that characterize cell populations, morphology, movement, and interactions in co-cultures of modified T cells and antigen-presenting tumor cells. We use Caliban and Occident to interrogate how interactions between T cells and cancer cells differ when beneficial knock-outs of RASA2 and CUL5 are introduced into TCR T cells. We apply spatiotemporal models to quantify T cell recruitment and proliferation after interactions with cancer cells. We discover that, compared to a safe harbor knockout control, RASA2 knockout T cells have longer interaction times with cancer cells leading to greater T cell activation and killing efficacy, while CUL5 knockout T cells have increased proliferation rates leading to greater numbers of T cells for hunting. Together, segmentation and tracking from Caliban and phenotype quantification from Occident enable cellular behavior analysis to better engineer T cell therapies for improved cancer treatment.

Cancer cells can disseminate during very early and sometimes asymptomatic stages of tumor progression. Though biological barriers to tumorigenesis have been identified and characterized, the mechanisms that limit early dissemination remain largely unknown. We report here that the orphan nuclear receptor nuclear receptor subfamily 2, group F, member 1 (NR2F1)/COUP-TF1 serves as a barrier to early dissemination. NR2F1 expression was decreased in patient ductal carcinoma in situ (DCIS) samples. High-resolution intravital imaging of HER2+ early-stage cancer cells revealed that loss of function of NR2F1 increased in vivo dissemination and was accompanied by decreased E-cadherin expression, activation of wingless-type MMTV integration site family, member 1 (WNT)-dependent β-catenin signaling, disorganized laminin 5 deposition, and increased expression of epithelial–mesenchymal transition (EMT) genes such as twist basic helix-loop-helix transcription factor 1 (TWIST1), zinc finger E-box binding homeobox 1 (ZEB1), and paired related homeobox 1 (PRRX1). Furthermore, downregulation of NR2F1 promoted a hybrid luminal/basal phenotype. NR2F1 expression was positively regulated by p38α signaling and repressed by HER2 and WNT4 pathways. Finally, early cancer cells with NR2F1LOW/PRRX1HIGH staining were observed in DCIS samples. Together, these findings reveal the existence of an inhibitory mechanism of dissemination regulated by NR2F1 in early-stage breast cancer cells.Significance:During early stages of breast cancer progression, HER2-mediated suppression of NR2F1 promotes dissemination by inducing EMT and a hybrid luminal/basal-like program.

Supplementary Data from NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells

Supplementary Figure from NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells

Supplementary Data from NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells