C Cullin-1 inhibition stabilizes CD19 surface expression, preventing its loss under immunotherapeutic pressure and improving the efficiency of CD19-targeting immunotherapies, thus arguing for potential incorporation of pevonedistat into novel combination therapies.
Abstract
Cancer immunotherapy targeting B-cell specific CD19 antigen meant a major breakthrough in the treatment of B-cell malignancies. Yet, vast proportion of treated patients experience relapse and failure of the therapy. Although multiple mechanisms of CD19-immunotherapy failure have been described, CD19-negative relapses represent the major hurdle in achieving higher and durable response rates. Our established in vitro co-culture models revealed that suboptimal CAR-T cell performance, inefficient to mediate target cell killing, results in robust downregulation of CD19 target antigen. Using genome-wide CRISPR screening, we addressed the mechanisms responsible for such CD19 downregulation and identified Cullin-1 and CD81 playing instrumental role in negative and positive regulation of CD19 expression, respectively. Inhibiting Cullin-1 activity with pevonedistat prevents the loss of CD19 under immunotherapeutic pressure, results in higher CD19 surface levels and consequently enhances the efficacy of target cell killing by CD19-CAR-T cells, CD19-CAR-NK cells and CD19-targeting antibody treatment. Mechanistically, we show that pevonedistat blocks the degradation of CD19 upon its internalization and allows its recycling back to the plasma membrane. CD81 chaperone protein is critically involved in this process as the absence of CD81 abrogates the effect of pevonedistat. In summary, we identify Cullin-1 as a novel and druggable regulator of CD19 protein stability. Cullin-1 inhibition augments CD19 surface expression, thereby improving the efficiency of CD19-targeting immunotherapies, thus arguing for potential incorporation of pevonedistat into novel combination therapies. Key Points Cullin-1 inhibition stabilizes CD19 surface expression, preventing its loss under immunotherapeutic pressure Pevonedistat treatment enhances the efficacy of CD19-CAR-T cells, CAR-NK cells and CD19-targeting antibodies
A recent major breakthrough in cancer immunotherapy is the Chimeric antigen receptor-T cell (CAR-T cell) therapy, which has shown significant clinical efficacy in haematological malignancy treatment. Nonetheless, its application in a more general way is limited by a number of challenges, such as T-cell exhaustion, off-target associated toxicities, and the difficulty of personalised manufacturing. Recently, new opportunities have come into solving these problems with the introduction of clustered regularly interspaced short tandem repeats (CRISPR)-Cas9 genome editing, which has made it possible to perform precise and combinatorial genetic editing in CAR-T cells. The important applications of CRISPR in CAR-T cell engineering, which include the disruption of inhibitory immune checkpoints to enhance antitumour activity, the generation of universal allogeneic CAR-T cells by deletion of T-cell receptor and human leukocyte antigen (HLA) genes, and the modulation of cytokine signalling pathways to reduce toxicity are discussed in this review. Moreover, novel approaches, including targeted CAR integration and multiplex gene editing, are discussed as having the potential to enhance the therapeutic efficacy and scalability. In spite of these improvements, there are issues of off-target effect, delivery efficacy, genomic instability, and unaddressed issues of long-term safety. CAR-T cell therapies are likely to be improved further in future through advancements in genome editing technology, delivery methods, and synthetic biology. In general, CRISPR/Cas9-based engineering is a promising way of developing the next generation of precision cancer immunotherapy.
Meng-Ying Liu· International Journal of Bio...· 0 citations
Immune aging impairs T cell-mediated tumor control as well as cancer immunotherapy outcomes. The most important drivers of T cell dysfunction in aged tumors remain unknown. We performed single-cell CRISPR screens to identify Dusp5 and Zfp219 as key regulators of CD8+ T cell persistence and effector differentiation within aged tumors. Loss of Dusp5 increased extracellular signal-regulated kinase (ERK) phosphorylation and globally enhanced T cell proliferation. Conversely, Zfp219 deletion induced epigenetic reprogramming and increased expression of cytotoxic molecules, enhancing antitumor immunity specifically in aging. Levels of the human ortholog ZNF219 were higher within intratumoral CD8+ T cells from older cancer patients, which correlates with worse survival following immunotherapy. Zfp219 ablation synergized with immune checkpoint inhibitors to expand effector-like CD8+ T cells, leading to tumor clearance in aged mice. Our findings highlight Dusp5 and Zfp219 as critical drivers of age-related T cell dysfunction that can be targeted to rejuvenate antitumor immunity in older cancer patients.
Alex C. Y. Chen, Keely Y. Ji, C. Yerinde et al.· Cell· 0 citations
Natural killer (NK) cell-based immunotherapies are promising for cancer treatment due to their ability to eliminate cancer cells independently of antigen presentation and “off-the-shelf” utility. However, molecular determinants governing tumor susceptibility to NK cytotoxicity remain incompletely understood. Here we employ CRISPR activation (CRISPRa) screening to identify cancer cell surface regulators of NK killing. Using a surfaceome-focused library, we screen human and murine cancer cell lines co-cultured with NK cells, identifying known and novel ligands modulating NK cytotoxicity. Screens reveal established factors including CD43 and previously uncharacterized regulators CD44, PDPN, and Siglec-1/CD169. Validation with orthogonal approaches confirm that disruption of these factors alters NK killing susceptibility in vitro and in humanized mouse models. Mechanistically, we find that CD43-mediated NK resistance operates independently of its proposed interaction with Siglec-7, and that targeting CD43 on NK cells or CAR T cells substantially enhances cytotoxic activity against leukemia. These results establish gain-of-function surfaceome screening as a powerful tool for identifying therapeutic targets for NK cell-based immunotherapy. NK cell determinants of tumor susceptibility remain poorly understood. CRISPRa surfaceome screening identifies CD43, CD44, PDPN, and Siglec-1 as modulators of NK cytotoxicity, and reveals that CD43 targeting enhances NK and CAR T cell killing of leukemia.
Ravi K. Dinesh, Xiaotong Wang, Imran A. Mohammad et al.· Nature Communications· 0 citations
Abstract Immune checkpoint inhibitors have transformed cancer therapy with the programmed cell death protein 1–programmed death‐ligand 1 axis, demonstrating substantial efficacy by targeting adaptive immunity across multiple malignancies. However, the limited clinical responses observed in a considerable proportion of patients highlights the need for more effective engagement of innate immune mechanisms. In this context, the cluster of differentiation 47–signal regulatory protein α (CD47–SIRPα) axis has emerged as a next‐generation immune checkpoint that regulates phagocytosis. CD47 is a ubiquitously expressed transmembrane glycoprotein containing an N‐terminal extracellular immunoglobulin variable‐like domain and is frequently overexpressed in both solid tumors and hematological malignancies. By binding to SIRPα on macrophages, CD47 transmits a canonical “don't eat me” signal that suppresses phagocytosis and enables tumor immune evasion. Beyond this canonical role, CD47 interacts with ligands such as thrombospondin‐1 and integrins to regulate tumor cell migration, metabolic adaptation, and immune balance within the tumor microenvironment. Importantly, CD47 links innate immune clearance with antigen presentation and downstream adaptive immune activation, positioning it as an actionable node for next‐generation therapeutic designs. Although the first‐generation CD47 blockade has revealed challenges related to hematologic toxicity, antigen sink effects, and limited monotherapy durability, these challenges have also accelerated the development of more selective, controllable, and context‐responsive therapeutic strategies. In this Review, we elucidate the structural features, molecular mechanisms, pathological functions, therapeutic strategies, translational challenges, and emerging solutions of CD47, with the aim of providing a theoretical basis for overcoming current therapeutic limitations and advancing more precise cancer immunomodulatory strategies.
Personalized neoantigen (neoAg) vaccines have shown clinical promise in solid tumors1–8, yet their efficacy and mechanism of action in hematopoietic malignancies remain poorly defined9–11. Herein, we establish an immunocompetent syngeneic A20 B-cell lymphoma platform to test the efficacy of neoAg vaccines used either as mono- or combinatorial therapies with other immunotherapies12–17. Whereas subcutaneous A20 tumors were refractory to single-agent αPD-1 or αCTLA4 therapy, they were eradicated in a T cell-dependent manner in 90% of syngeneic hosts treated with dual immune checkpoint therapy (dual ICT, i.e., αPD-1 + αCTLA4). By mapping antigen specificity of dual-ICT-elicited T cells, we identified and validated dominant endogenous A20 MHC-I and MHC-II neoantigens and designed therapeutic synthetic long peptide (SLP) vaccines containing these neoepitopes. This vaccine (A20 neoVAX) promoted robust neoAg-specific CD4□ and CD8□ T cell responses in naïve syngeneic BALB/c mice and induced tumor rejection in ∼70% of subcutaneous tumor-bearing mice. In addition, nearly all mice rejected their subcutaneous A20 tumors when A20 neoVAX was combined with αPD-1. To render the results of this study more physiologic, we developed a systemic A20 lymphoma model and found that dual ICT failed to control tumor progression and A20 neoVAX delayed tumor progression and prolonged animal survival but did not induce tumor rejection. In contrast, A20 neoVAX plus dual ICT achieved durable systemic tumor elimination. Mechanistically, the combination of A20 neoVAX plus dual ICT amplified priming of A20 neoAg-specific T cells, prevented T cell dysfunction, sustained the cytotoxic capacity of tumor-specific CD8+ T cells, and induced Th1-skewing of CD4+ T cells in tumor and peripheral compartments. To increase the clinical relevance of these findings and to minimize potential adverse events in tumor-bearing, therapeutically treated individuals, we substituted CD8-targeted cytokine muteins (CD8-IL2 or CD8-IL21) for αCTLA4. These agents represent genetically modified forms of IL-2 or IL-21 that selectively stimulate CD8+ T cells but have significantly reduced capacity to activate chronic inflammation and immunosuppressive functions of other immune cells. Whereas mice bearing systemic A20 lymphoma treated with either nothing, A20 neoVAX, or A20 neoVAX + CD8-IL2 failed to control tumor outgrowth, 66.7% of tumor-bearing mice treated with A20 neoVAX + CD8-IL2 + αPD-1 rejected their tumors. In similar experiments in which CD8-IL21 was substituted for CD8-IL2, tumor clearance was also observed in two-thirds of A20-bearing mice but now rejection occurred in the absence of αPD1. Together, these data define a framework for optimal personalized neoAg vaccination in B-lymphoma and demonstrate that neoAg vaccines can safely synergize with CD8+ T cell-selective immunotherapies to prevent T-cell dysfunction and generate durable systemic anti-tumor immunity.
Yuang Song, E. Aladyeva, Ruan F. Vieira Medrano et al.· bioRxiv· 0 citations
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