P345: AN IMMUNE SCREEN IDENTIFIES 5-NONLOXYTRYPTAMINE AS A NOVEL ANTI-CANCER AGENT CAPABLE OF IMPROVING ANTI-TUMOR IMMUNITY IN COMBINATION WITH ANTI-PD1 THERAPY

Abstract

Background: Exploiting the immune system, particularly CD8+ T cells to eliminate tumors has revolutionized the treatment landscape. However, despite the evident successes of CD8+ T cell-targeting immunotherapies such as anti-PD1 checkpoint inhibitors, many patients fail to respond especially those with poorly immunogenic tumors. Additional obstacles to successful immunotherapy responses include therapy-induced toxicity, lack of bio-markers of response, an immunosuppressive environment leading to T cell dysfunction and exhaustion and poor immune infiltration highlighting a need for novel combinatorial approaches to augment immunotherapeutic activity. Aims: We aimed to uncover novel therapeutic agents capable of augmenting the anti-tumoral responses of CD8+ T cells in ex vivo systems and in vivo syngenetic poorly immunogenic tumor models. Methods: To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen, using Lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells that were combined tumor cells expressing the LCMV gp33 (B16) peptide CTL epitope and incubated with an NIH 770 compound library. Hits that increased tumor-cell killing when combined with LCMV-primed splenic T cells were further validated ex vivo. The key candidate was assessed in vivo in C57BL/6J syngeneic tumor models. Immunocompromised NSG mice as well as antibody depletion were used validate CD8+ T cell dependence. In vitro and in vivo immunomodulatory effects were deciphered using flow cytometry, immunoblot, CRISPR/Cas9 and histological analyses. Results: We identified 5-Nonyloxytryptamine (5-NL), a serotonin receptor (HTR) agonist, as increasing the ability of T cells to target tumor cells. In vitro, 5-NL induced apoptosis in melanoma and childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) at low micromolar levels. 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts’ immune system, specifically CD8+ T cells as their depletion abrogated the phenotype (Figure 1A-B). 5-NL’s pro-immune effects were attributed to the upregulation of antigen presenting machinery in melanoma, BCP-ALL and other poorly immunogenic tumors with low MHC-I/HLAA-C expression (Figure 1C). Importantly, the upregulation of MHC-I/HLAA-C occurred without concomitant increases in PD-L1 expression and was linked to upregulation of cAMP Response Element-Binding Protein (CREB) in vitro and in vivo. As demonstrated through CRISPR/Cas9 HTR1D knockout, the immunomodulatory and pro-apoptotic effects of 5-NL occurred independently of receptor target signaling. 5-NL was successfully combined with an anti-PD1 antibody in vivo for maximal tumor growth inhibition. Figure 1: The serotonin agonist 5-Nonyloxytryptamine (5-NL) was identified as potentiating T cell mediated anti-tumor immunity. (A) C57BL/6J or (B) CD8 depleted C57BL/6J mice were subcutaneously injected with 5 x 105 gp33 expressing tumor cells. 7 days post-tumor injection, mice were randomized and treated daily 6.25 mg/kg of 5-NL or vehicle for five consecutive days and tumor volume was measured (n = 9-12). H2-Db (MHC-I, murine cell lines) and HLAA-C (human cells lines) were assessed by FACS analysis following treatment with 5-NL for 24 hours (n = 3-9). Error bars in all experiments indicate SEM; *P < 0.05 as determined by a Student´s t-test (unpaired, 2 tailed). Image:Summary/Conclusion: This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors and increasing their responsiveness to immunotherapies.