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Background In RAS-mutant tumors, combined MEK and autophagy inhibition using chloroquine demonstrated synthetic lethality in preclinical studies. This phase II trial evaluated the safety and activity of the MEK inhibitor binimetinib combined with hydroxychloroquine (HCQ) in patients with advanced KRAS-mutant non-small cell lung cancer (NSCLC). Methods Eligibility criteria included KRAS-mutant NSCLC, progression after first-line therapy, ECOG PS 0-1, and adequate end-organ function. Binimetinib 45 mg was administered orally (p.o.) bid with HCQ 400 mg p.o. bid. The primary endpoint was objective response rate (ORR). A Simon’s 2-stage phase II clinical trial design was used, with an α error of 5% and a power β of 80%, anticipating an ORR of 30% to proceed to the 2-stage expansion. Results Between April 2021 and January 2022, 9 patients were enrolled to stage I: median age 64 years, 44.4% females, 78% smokers. The best response was stable disease in one patient (11.1%). The median progression free survival (PFS) was 1.9 months, and median overall survival (OS) was 5.3 months. Overall, 5 patients (55.6%) developed a grade 3 adverse event (AE). The most common grade 3 toxicity was rash (33%). Pre-specified criteria for stopping the trial early due to lack of efficacy were met. Conclusion The combination of B + HCQ in second- or later-line treatment of patients with advanced KRAS-mutant NSCLC did not show significant antitumor activity. (ClinicalTrials.gov Identifier: NCT04735068).

Gamma delta (γδ) T lymphocytes are promising candidate for adoptive T cell therapy, however, their treatment efficacy is not satisfactory. Vδ2 T cells are unique to primates and few suitable models are available to assay their anti-tumour function.We tested human γδ T cell activation, tumour infiltration, and tumour-killing in four three-dimensional (3D) models, including unicellular, bicellular and multicellular melanoma spheroids, and patient-derived melanoma organoids. We studied the effects of checkpoint inhibitors on γδ T cells and performed a small molecule screen using these platforms.γδ T cells rapidly responded to melanoma cells and infiltrated melanoma spheroids better than αβ T cells in PBMCs. Cancer-associated fibroblasts (CAFs) in bicellular spheroids, stroma cells in multicellular melanoma spheroids and inhibitory immune cells in organoids significantly inhibited immune cell infiltrates including γδ T cells and lessened their cytotoxicity to tumour cells. Tumour-infiltrating γδ T cells showed exhausted immunophenotypes with high checkpoints expression (CTLA-4, PD-1 and PD-L1). Immune checkpoint inhibitors increased γδ T cell infiltration of 3D models and killing of melanoma cells in all four 3D models. Our small molecule screen assay and subsequent mechanistic studies demonstrated that epigenetic modifiers enhanced the chemotaxis and cytotoxicity of γδ T cells through upregulating MICA/B, inhibiting HDAC6/7 pathway and downregulating the levels of PD-L1 and PD-L2 in CAFs and tumour cells. These compounds increased CXCR4 and CD107a expression, IFN-γ production and decreased PD-1 expression of γδ T cells.Tumour-infiltrating γδ T cells show exhausted immunophenotypes and limited anti-tumour capacity in melanoma 3D models. Checkpoint inhibitors and epigenetic modifiers enhance anti-tumour functions of γδ T cells. These four 3D models provided valuable preclinical platforms to test γδ T cell functions for immunotherapy.

Tumor-derived extracellular vesicles (TEVs) suppress the proliferation and cytotoxicity of CD8(+) T cells, thereby contributing to tumor immune evasion. Here we report that the adhesion molecule ICAM-1 co-localizes with PD-L1 on the exosomes; both ICAM-1 and PD-L1 are upregulated by interferon-γ. Exosomal ICAM-1 interacts with LFA-1, which is upregulated on activated T cells. Blocking ICAM-1 on TEVs reduces the interaction of TEVs with CD8(+) T cells, and attenuates PD-L1-mediated suppressive effects of TEVs. During this study, we have established an Extracellular vesicle-Target cell Interaction Detection through SorTagging (ETIDS) system to assess the interaction between a TEV ligand and its target cell receptor. Using this system, we demonstrate that the interaction of TEV PD-L1 with PD-1 on T cells is significantly reduced in the absence of ICAM-1. Our study demonstrates that ICAM-1-LFA-1 mediated adhesion between TEVs and T cells is a prerequisite for exosomal PD-L1 mediated immune suppression.

Radiation therapy is commonly used to treat cancers in the definitive setting, but the local control often remains suboptimal, thereby motivating us to search for novel radiosensitizers. Recently a series of novel quinacrines based upon a previously reported dimeric chloroquine has been synthesized. We studied one of these agents, DQ660. By itself, the drug upregulates both p53 protein and p53 transcriptional targets and also increases apoptosis in p53-wildtype H460 lung cancer cells. Furthermore, we found that DQ660 is a potent radiosensitizer in vitro. This radiosensitization is not contingent upon wildtype p53 as it occurs in cells null for p53 and containing mutant p53. The combination of radiation and DQ660 proded significant growth retardation of human xenografts grown in vivo compared to radiation or DQ660 alone. We saw higher levels of γ-H2AX foci with combined treatment compared to either alone, suggesting that the drug increases DNA damage induction. Using agarose gel electrophoresis and kinetoplast DNA (kDNA) as a substrate, we found that DQ660 can inhibit topoisomerase II activity. DQ660 did not potentiate the phosphorylation of ATM at S1981 seen after radiation. However, the drug did lead to an increase in Ser428 phosphorylation of ATR. This phosphorylation was not seen in response to radiation alone, suggesting that DQ660 and radiation activate different signaling pathways in response to DNA damage. We saw similar results with another dimeric quinacrine, DQ550, suggesting that this class of agents may be radiosensitizers. Our findings have uncovered a novel class of radiosensitizers that work at nanomolar concentrations and will spur investigations for its use in combination therapy for tumors treated with radiation Citation Format: Ilias V. Karagounis, Jayashree Karar, Christoforos Thomas, Constantinos Koumenis, Jeffrey Winkler, Ravi Amaravadi, Amit Maity. A novel class of dimeric quinacrine compounds induce DNA damage and increase the anticancer activity of radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 217.

Over the last few years, we have gained insights into how immunotherapy, including checkpoint blockade, modulates key CD4 and CD8 T cell subsets in anti-tumor immunity. This information can now give us insights into how immunotherapy can impact immunity in the setting of COVID-19. Indeed, we recently demonstrated that cancer patients with T cell depletion have high COVID-19 mortality despite adequate B cell and antibody production, highlighting the importance of cellular immunity. Conversely, in the setting of B cell depletion by anti-CD20 therapy, CD8 T cells can compensate for impaired humoral immunity. PD-1 blockade increases the activation and proliferation of CD4 T follicular-helper cells, which plays a key role in promoting B cell responses and quality antibody production. Thus, it is possible that PD-1 blockade enhances the efficacy of SARS-CoV-2 vaccination. However, PD-1 blockade in melanoma patients was actually associated with at 2-fold decrease in SARS-CoV-2 specific antibodies and neutralizing antibodies, compared to a healthy donor cohort. PD-1 blockade was also associated with depletion of memory CD4 T cells, suggesting there may be consequences to prolonged PD-1 blockade. Citation Format: Paulina Coutifaris, Kevin Wang, Sokratis Apostolidis, Mark Painter, Ahron Flowers, Rishi Goel, Divij Mathew, Ajinkya Pattekar, Ravi Amaravadi, Tara Mitchell, Paul Bates, Scott Hensley, Giorgos Karakousis, Lynn Schuchter, Allie Greenplate, E. John Wherry, Alexander C. Huang. Impact of immunotherapy on COVID-19 immunity: Insights from checkpoint blockade in cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr IA07.

How tumor-infiltrating T lymphocytes (TILs) adapt to the metabolic constrains within the tumor microenvironment (TME) and to what degree this affects their ability to combat tumor progression remain poorly understood. Using mouse melanoma models, we report that CD8