Abstract Background: Metastatic uveal melanoma (UM) is an aggressive cancer, with a 1-year overall survival of < 50%. While the bispecific T-cell engager tabentafusp is approved for HLA-A2+ patients with gp100+ UM, there are limited treatment options for patients who do not express HLA-A2. We present a strategy and proof-of-concept for designing and manufacturing a tumor-selective endogenous T-cell therapy (ETC) for a patient with metastatic UM. Methods: PBMC were collected by apheresis in 2013 upon first diagnosis with uveal melanoma; 8 years later, following progression, cryopreserved PBMC were used to generate autologous antigen-specific T cells for adoptive therapy. The validated tumor antigen SLC45A2 was found to be overexpressed in the patient’s metastatic tumor based on RNA expression profiling. Peripheral blood derived SLC45A2-specific HLA-A2402-restricted central memory CD8+ T cells were generated via a process of in vitro priming, IL-21 treatment, tetramer-guided sorting and cell expansion. Clinical response was assessed via longitudinal CT imaging and ctDNA-based MRD monitoring. Persistence of transferred T cells was measured via serial flow cytometry and RNA-sequencing of PBMCs, along with TCR and RNA sequencing of tumor tissue collected post infusion. Results: A 39 year old male received SLC45A2-specific ETC therapy and checkpoint inhibitor treatment for advanced metastatic UM (GNAQ (Q209P), BAP1 (E398*, LOF). Bulk RNAseq data from baseline biopsies were queried for known tumor-associated antigens, and SLC45A2 was expressed at high levels. The patient experienced widespread disease stabilization with minimal toxicity. SLC45A2-specific CD8+ T cells persisted in circulation up to 181 days after the first infusion and 43 days after the second infusion, ranging from 6.7% - 1.5% of total circulating CD3 T cells. T cell clones from the infusion product included the dominating TCR subclone in tumor tissue collected 6 weeks post infusion, comprising 33% of the TCRá and 26% of TCRâ population. SLC45A2-specific T cells were identified in the tumor infiltrating lymphocytes in a separate lesion collected 10 months after the first infusion. A duodenum lesion progressed during treatment, and RNAseq demonstrated reduced expression of genes associated with HLA presentation and SLC45A2 and reduced TIL infiltration, suggesting tissue-specific immune editing of the tumor cell population. Conclusions: 1. Tumor-reactive antigen-specific T cells can be isolated and expanded for adoptive therapy using PBMCs cryopreserved for > 8 years. 2. Following infusion, peripheral blood and tumor profiling support the persistence of transferred T cells in a patient with metastatic uveal melanoma. 3. Disease stabilization in aggressive metastatic UM was achieved with minimal toxicity. Overall, our study provides evidence and rationale for utilizing autologous antigen-specific T cells for treating rare cancers. Citation Format: Shailbala Singh, Esther Ryu, Katie M. Campbell, Amber Smith, Suzanne Phillips, Luisa Soto, Ivy Lai, Gregory Lizee, Benjamin Vincent, Marshall Thompson, William Hoos, Sapna Patel, Cassian Yee. Case study: Efficacy and persistence of autologous T cell therapy targeting SLC45A2 in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3607.
Adoptive cell therapy (ACT) can address an unmet clinical need for patients with relapsed/refractory acute myeloid leukemia (AML), but its effect is often modest in the setting of high tumor burden. In this study, we postulated that strategies to lower the AML apoptotic threshold will augment T cell killing of AML cells. BH3 mimetics, such as venetoclax, are a clinically approved class of compounds that predispose cells to intrinsic apoptosis by inhibiting anti-apoptotic mitochondrial proteins. We explored the anti-leukemic efficacy of BH3 mimetics combined with WT1-specific CD8 + T cells on AML cell lines and primary samples from patients with a diverse array of disease characteristics to evaluate if lowering the cellular apoptotic threshold via inhibition of anti-apoptotic mitochondrial proteins can increase leukemic cell sensitivity to T cell therapy. We found that the combination approach of BH3 mimetic and CD8 + T cells led to significantly increased killing of established AML lines as well as of adverse-risk primary AML leukemic blast cells. In contrast to the hypothesis that enhanced killing would be due to combined activation of the intrinsic and extrinsic apoptotic pathways, we found that CTL-mediated killing of AML cells was accomplished primarily through activation of the intrinsic/mitochondrial apoptotic pathway. This highly effective combinatorial activity due to convergence on the same apoptotic pathway was conserved across multiple AML cell lines and primary samples, suggesting that mitochondrial priming may represent a novel mechanism of optimizing adoptive cell therapy for AML patients.
Visual systems have evolved to recognize and extract features from complex scenes using limited sensory information. Contour perception is essential to this process and can occur despite breaks in the continuity of neighboring features. Such robustness of the animal visual system to degraded or occluded shapes may also give rise to an interesting phenomenon of optical illusions. These illusions provide a great opportunity to decipher neural computations underlying contour integration and object detection. Kanizsa illusory contours have been shown to evoke responses in the early visual cortex despite the lack of direct receptive field activation. Recurrent processing between visual areas has been proposed to be involved in this process. However, it is unclear whether higher visual areas directly contribute to the generation of illusory responses in the early visual cortex. Using behavior, in vivo electrophysiology, and optogenetics, we first show that the primary visual cortex (V1) of male mice responds to Kanizsa illusory contours. Responses to Kanizsa illusions emerge later than the responses to the contrast-defined real contours in V1. Second, we demonstrate that illusory responses are orientation-selective. Finally, we show that top-down feedback controls the neural correlates of illusory contour perception in V1. Our results suggest that higher-order visual areas may fill in the missing information in the early visual cortex necessary for illusory contour perception. SIGNIFICANCE STATEMENT Perception of the Kanizsa illusory contours is impaired in neurodevelopmental disorders such as schizophrenia, autism, and Williams syndrome. However, the mechanism of the illusory contour perception is poorly understood. Here we describe the behavioral and neural correlates of Kanizsa illusory contours perception in mice, a genetically tractable model system. We show that top-down feedback controls the neural responses to Kanizsa illusion in V1. To our knowledge, this is the first description of the neural correlates of the Kanizsa illusion in mice and the first causal demonstration of their regulation by top-down feedback.
Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+ HNSCC) is recognized as a distinct disease with unique etiology and clinical features. Current standard of care therapeutic modalities are identical for HPV+ and HPV- HNSCC and thus, there remains an opportunity to develop innovative pharmacologic approaches to exploit the inherent vulnerabilities of HPV+ HNSCC. In this study, using an inducible HPVE6E7 knockdown system, we found that HPV+ HNSCC cells are addicted to HPVE6E7, such that loss of these viral oncogenes impaired tumorigenicity in vitro and in vivo. A number of druggable pathways, including PPAR and Wnt, were modulated in response to HPVE6E7 loss. Fenofibrate showed significant anti-proliferative effects in a panel of HPV+ cancer cell lines. Additionally, fenofibrate impaired tumor growth as monotherapy and potentiated the activity of cisplatin in a pre-clinical HPV+ animal model. Systemic fenofibrate treatment induced p53 protein accumulation, and surprisingly, re-programmed the tumor-immune microenvironment to drive immune cell infiltration. Since fenofibrate is FDA-approved with a favorable long-term safety record, repositioning of this drug, as a single agent or in combination with cisplatin or checkpoint blockade, for the HPV+ HNSCC setting should be prioritized.
Neural circuits underlying brain functions are vulnerable to damage, including ischemic injury, leading to neuronal loss and gliosis. Recent technology of direct conversion of endogenous astrocytes into neurons in situ can simultaneously replenish the neuronal population and reverse the glial scar. However, whether these newly reprogrammed neurons undergo normal development, integrate into the existing neuronal circuit, and acquire functional properties specific for this circuit is not known. We investigated the effect of NeuroD1-mediated in vivo direct reprogramming on visual cortical circuit integration and functional recovery in a mouse model of ischemic injury. After performing electrophysiological extracellular recordings and two-photon calcium imaging of reprogrammed cells in vivo and mapping the synaptic connections formed onto these cells ex vivo , we discovered that NeuroD1 reprogrammed neurons were integrated into the cortical microcircuit and acquired direct visual responses. Furthermore, following visual experience, the reprogrammed neurons demonstrated maturation of orientation selectivity and functional connectivity. Our results show that NeuroD1-reprogrammed neurons can successfully develop and integrate into the visual cortical circuit leading to vision recovery after ischemic injury.
Abstract Malignant Peripheral Nerve Sheath tumors(MPNSTs) are highly aggressive soft tissue sarcomas derived Schwann cell tumors. Half of these tumors occur sporadically, while the rest occur in the context of Neurofibromatosis Type 1 Syndrome (NF1) developing from pre-existing plexiform neurofibromas. NF1 is characterized by loss-of-function mutations in the gene encoding neurofibromin, a negative regulator of the oncogenic Ras pathway. To better understand genetic factors that give rise to MPNSTs, we performed a Sleeping Beauty (SB) transposon screen in mice. The results implicated Wnt/β-catenin, PI3K-AKT-mTOR, growth factor receptor signaling, and other pathways. It is our goal to understand the role of these pathways in human Schwann cell transformation, tumorigenesis, and tumor maintenance. We next sought to validate SB screen gene hits in a human cellular model using CRISPR/Cas9 technology as a tool to induce loss-of-function mutations in tumor suppressor gene (TSG) and oncogene candidates. A total 103 genes were independently targeted with multiple guideRNAs in immortalized human Schwann and MPNST cell lines and effects on transformation assessed. Transformation was assessed by anchorage-independent colony formation in soft agar, transwell migration, and tumor formation in NRG mice. In these assays, more than 30 genes scored as TSG candidates. Our results revealed a role for the Wnt/β-catenin, Hippo/Yap, PI3K-AKT-mTOR, Rho, and growth factor receptor signaling pathways in human neurofibroma and MPNST development and maintenance. These are generating hypothesis driven pre-clinical treatment studies we're pursuing now. Citation Format: German L. Velez Reyes, Nicholas Koes, Gabriel Kaufmann, Esther Ryu, David A. Largaespada. Transposon mutagenesis and CRISPR/Cas9 screening reveal pathways driving peripheral nerve sheath tumor development and maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-197.
Ischemic injury caused by stroke is one of the leading causes of severe morbidity and mortality. Neuronal loss, proliferation of reactive astrocytes and the formation of a glial scar represent the three critical problems preventing the functional recovery. Direct reprogramming of astrocytes into neurons in vivo using NeuroD1‐containing AAV virus represents a new promising gene therapy capable of replenishing the neuronal population and reducing gliosis. We have used NeuroD1‐containing AAV virus to convert astrocytes into neurons in the mouse primary visual cortex (V1) following focal ischemic injury. We have demonstrated that this in vivo reprogramming can restore visual evoked potentials (VEPs) and individual neuronal responses impaired after ischemic injury in V1. We have used Channelrhodopsin Assisted Circuit Mapping (CRACM) to demonstrate that this functional restoration is accompanied by the integration of the newly reprogrammed neurons into the visual microcircuit and reestablishment of the specific interlaminar long‐range connections. Our work suggests that in vivo NeuroD1‐based viral therapy can convert reactive astrocytes into neurons and restore visual function lost after local ischemia Support or Funding Information Purdue Research Foundation
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)