Vianzon, Vianca V

Background Genes that enhance T-cell function represent promising targets for improving engineered T-cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T-cell persistence, employing Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-of-function (GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. Methods We developed transgenic mice carrying D oxycycline (Dox)- i nducible SB mutag e nesis s y stem (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T-cell persistence under chronic antigen exposure. Specifically, CD8 + T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 + T cells using enhanced-specificity tagmentation sequencing and RNA sequencing, respectively. Results Under chronic stimulation, SB -mutagenized CD8 + T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation. T2/Onc2 insertions into Bach2 and Elmo1 were recurrently identified at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T-cell memory formation and resistance to chronic viral infection-induced exhaustion but less characterized in engineered T cells for cancer therapy, was found to counteract exhaustion in vitro and enhance in vivo tumor persistence in the B16-Ova tumor model. Further, we showed that ectopic Bach2 expression levels influence engineered T-cell differentiation lineage, as low Bach2 overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation. Conclusions Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T-cell phenotypes important for their use as therapies. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.

Patients with advanced cancers who either do not experience initial response to or progress while on immune checkpoint inhibitors (ICIs) receive salvage radiotherapy to reduce tumor burden and tumor-related symptoms. Occasionally, some patients experience substantial global tumor regression with a rebound of cytotoxic CD8 + T cells. We have termed the rebound of cytotoxic CD8 + T cells in response to salvage therapy as T cell resilience and examined the underlying mechanisms of resilience. Resilient T cells are enriched for CX3CR1 + CD8 + T cells with low mitochondrial membrane potential, accumulate less reactive oxygen species (ROS), and express more malic enzyme 1 (ME1). ME1 overexpression enhanced the cytotoxicity and expansion of effector CD8 + T cells partially via the type I interferon pathway. ME1 also increased mitochondrial respiration while maintaining the redox state balance. ME1 increased the cytotoxicity of peripheral lymphocytes from patients with advanced cancers. Thus, preserved resilient T cells in patients rebound after salvage therapy and ME1 enhances their resiliency.

Abstract Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignancies. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysis-independent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Supported by grants from Mayo Clinic Foundation and NIH (R01CA 256927-2)

Abstract T cell–mediated cancer immunotherapies, including anti–PD-1 and CAR-T cells, are becoming standard treatments for many cancer types. One potential strategy to overcome resistance is to enhance the ability of T cells to traffic into tumors. We recently reported results from an innovative, genome-wide screen to identify endogenous T cell genes that enhance intratumoral accumulation using the Sleeping Beauty (SB) transposon system. We successfully identified many promising candidates with this in vivo approach using immunocompetent models of solid tumors. Others have performed similar independent whole-genome and targeted screens in T cells assessing tissue localization using adoptive T cell transfer approaches. To determine how our gene candidates compared with significant candidates identified in these independent screens, we performed a formal rank aggregation to generate a single, integrated ranked list in descending order of selection strength. Aggregated results yielded approximately 1500 T cell gene candidates whose biological functions are enriched in chemotaxis. We hypothesized that these candidates represent novel therapeutic targets to improve T cell migration into solid tumors. Here, we present in vivo validation of two of the most strongly selected candidates, including AP2-associated protein kinase 1 (Aak1) and Small Proline-Rich Protein 1B (SPRR1B). Importantly, in vivo migration of adoptively transferred T cells into tumors was significantly increased when the T cells overexpressed AAK1DN125 or SPRR1B. Our observations strongly support the strength of genomic screens to identify truly novel candidates for further development as immunotherapies to enhance T cell homing to the tumor microenvironment. Supported by Mayo Clinic Graduate School of Biomedical Sciences, K22 (K22CA225786, PI: LM Rogers), and Cancer Immunology and Immunotherapy Program Pilot Award (PI: LM Rogers)

Abstract The immune response to anti-PD-1 therapy in cancer patients is not completely defined due to lack of reliable biomarkers that can determine T cell function. Bim, a member of the Bcl-2 family, was recently identified as a downstream signaling molecule in the PD-1 pathway. Within CD8 T cells, Bim appears to have a dual function, and can be pro-apoptotic or promote effector activity. Our clinical data has shown that high levels of Bim in CD8 T cells are predictive of response to anti-PD-1 therapy in metastatic melanoma whereas in colorectal cancer, a low level of Bim is associated with treatment response. Thus, we sought to determine what molecular partners might influence the role of Bim. Using co-immunoprecipitation, high resolution microscopy and FRET we identified ANT2 (Adenine Nucleotide Translocase 2), an inner membrane protein of mitochondria, as a protein that interacts with Bim. Accordingly, we found CD8 T cells with low and high mitochondrial potential demonstrated different ANT2/Bim ratios, and a low ANT2/Bim ratio was present in effector T cells with low mitochondrial potential but high cytotoxic activity. Given the pro-survival function of ANT2, our results indicate that ANT2 may balance the pro-apoptotic and effector roles of Bim in mitochondria in order to preserve effector T cells before contraction. Understanding the molecular mechanism of Bim and ANT2 interactions in CD8 T cells may provide a novel marker to predict clinical response to anti-PD-1 therapy.