Periodic Reporting for period 3 - TrojanDC (Harnessing dendritic cell reprogramming for cancer immunotherapy)
Okres sprawozdawczy: 2023-10-01 do 2025-03-31
Each year, 17 million people are diagnosed with solid cancers. Immunotherapy employs the immune system against cancer and revolutionized cancer treatment leading to unprecedented long-term survival. However, immunotherapies are still ineffective for >80% of patients. The ability of cancer cells to avoid recognition by the patient’s immune system and the suppressive environment for immune cells within the tumor underlie resistance mechanisms to immunotherapy. Effective response to immunotherapies relies on the presence of a rare subset of immune cells, conventional dendritic cells type 1 (cDC1) that set in motion the immune system against cancer. In cancer, genetic mutations result in the production of tumor antigens, allowing the immune system to distinguish cancer cells from healthy cells. To kick-start a potent immune response, tumor antigens need to be presented by cDC1 to effector immune cells. We hypothesized that the cell fate reprogramming of other cell-types – fibroblasts or tumor cells - to become antigen-presenting cDC1 offers an attractive new cancer immunotherapy strategy. This proposal aimed to test a cancer immunotherapy concept based on cDC1 reprogramming and endowed antigen presenting cell (APC) function in tumor cells. The proposal encompassed three main aims. First, we aimed to define optimal transcription factor combinations and external cues to efficiently reprogram human fibroblasts into cDC1. Then, we aimed to reprogram mouse and human tumor cells into tumor-APCs followed by characterization of transcriptome, chromatin accessibility, surface peptidome and ability to present antigens to T cells. We evaluated whether reprogrammed cells mount an attack against tumors in mouse models. Finally, we aimed to further test the hypothesis that intratumoral delivery of reprogramming factors elicits in vivo antigen presentation, immune cell recruitment and tumor regression.
We identified PU.1 IRF8, and BATF3 (PIB) transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts, but reprogramming of human somatic cells was limited by low efficiency. Here, we investigated single-cell transcriptional dynamics during human cDC1 reprogramming. Human induced cDC1s (hiDC1s) generated from embryonic fibroblasts gradually acquired a global cDC1 transcriptional profile and expressed antigen presentation signatures, whereas other DC subsets were not induced at the single-cell level during the reprogramming process. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the cDC1-inducing transcription factor network as key drivers of the process. Combining IFN-γ, IFN-β, and TNF-α with constitutive expression of cDC1-inducing transcription factors led to improvement of reprogramming efficiency by 190-fold. Mechanistically, PU.1 showed dominant and independent chromatin targeting at early phases of reprogramming, recruiting IRF8 and BATF3 to shared binding sites. The cooperative binding at open enhancers and promoters led to silencing of fibroblast genes and activation of a cDC1 program.
We then employed the minimal cDC1 gene regulatory network to reprogram cancer cells into professional antigen presenting cells (tumor-APCs). Enforced expression of PIB was sufficient to induce cDC1 phenotype in 36 cell lines derived from human and mouse hematological and solid tumors. Within 9 days of reprogramming, tumor-APCs acquired transcriptional and epigenetic programs associated with cDC1 cells. Reprogramming restored the expression of antigen presentation complexes and costimulatory molecules on the surface of tumor cells, allowing the presentation of endogenous tumor antigens on MHC-I, and facilitating targeted killing by CD8+ T cells. Functionally, tumor-APCs engulfed and processed proteins and dead cells, secreted inflammatory cytokines and cross-presented antigens to naïve CD8+ T cells. Human primary tumor cells could also be reprogrammed to increase their capability to present antigen and to activate patient-specific tumor-infiltrating lymphocytes. In addition to acquiring improved antigen presentation, tumor-APCs had impaired tumorigenicity in vitro and in vivo. Injection of in vitro generated melanoma-derived tumor-APCs into subcutaneous melanoma tumors delayed tumor growth and increased survival in mice and was synergistic with immune checkpoint inhibitors.
We then employed the minimal cDC1 gene regulatory network to reprogram cancer cells into professional antigen presenting cells (tumor-APCs). Enforced expression of PIB was sufficient to induce cDC1 phenotype in 36 cell lines derived from human and mouse hematological and solid tumors. Within 9 days of reprogramming, tumor-APCs acquired transcriptional and epigenetic programs associated with cDC1 cells. Reprogramming restored the expression of antigen presentation complexes and costimulatory molecules on the surface of tumor cells, allowing the presentation of endogenous tumor antigens on MHC-I, and facilitating targeted killing by CD8+ T cells. Functionally, tumor-APCs engulfed and processed proteins and dead cells, secreted inflammatory cytokines and cross-presented antigens to naïve CD8+ T cells. Human primary tumor cells could also be reprogrammed to increase their capability to present antigen and to activate patient-specific tumor-infiltrating lymphocytes. In addition to acquiring improved antigen presentation, tumor-APCs had impaired tumorigenicity in vitro and in vivo. Injection of in vitro generated melanoma-derived tumor-APCs into subcutaneous melanoma tumors delayed tumor growth and increased survival in mice and was synergistic with immune checkpoint inhibitors.
Our findings provide mechanistic insights into human cDC1 specification and reprogramming and represent a platform for generating patient-tailored cDC1s, a long-sought DC subset for vaccination strategies in cancer immunotherapy. In addition, our innovative approach serves as a platform for the development of immunotherapies that endow cancer cells with the capability to process and present endogenous tumor antigens.
This proposal had the ambitious goal to develop an entirely new cancer gene therapy based on cDC1 reprogramming for hard-to-treat solid tumors, with the advantages of an off-the-shelf therapy (same therapy for all patients) yet personalized (based on the presentation of unique tumor antigens in each patient and tumor). We will now focus on the third aim of the project that aims to induce reprogramming of tumor cells to cDC1 in vivo, within tumors. We aim to characterize the in vivo reprogramming process and its impact on anti-tumor immunity in mice and human tumor spheroids, select the best delivery system and assess efficacy and safety of this new gene therapy approach. This therapeutic solution may be particularly important in cutaneous cDC1-excluded solid tumors such as melanoma, head and neck cancer, breast cancer and sarcomas that are accessible to intra-tumoral injection. By the end of the project we aim to provide proof of principle that delivering reprogramming factors intratumorally reprograms cancer cells to become antigen-presenting cDC1. We envision to preclinically develop our approach into a gene therapy for the treatment of solid tumors, providing evidence for its mechanism of action, efficacy and safety.
This proposal had the ambitious goal to develop an entirely new cancer gene therapy based on cDC1 reprogramming for hard-to-treat solid tumors, with the advantages of an off-the-shelf therapy (same therapy for all patients) yet personalized (based on the presentation of unique tumor antigens in each patient and tumor). We will now focus on the third aim of the project that aims to induce reprogramming of tumor cells to cDC1 in vivo, within tumors. We aim to characterize the in vivo reprogramming process and its impact on anti-tumor immunity in mice and human tumor spheroids, select the best delivery system and assess efficacy and safety of this new gene therapy approach. This therapeutic solution may be particularly important in cutaneous cDC1-excluded solid tumors such as melanoma, head and neck cancer, breast cancer and sarcomas that are accessible to intra-tumoral injection. By the end of the project we aim to provide proof of principle that delivering reprogramming factors intratumorally reprograms cancer cells to become antigen-presenting cDC1. We envision to preclinically develop our approach into a gene therapy for the treatment of solid tumors, providing evidence for its mechanism of action, efficacy and safety.