Showing papers by "Brian Ruffell published in 2023"

Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunotherapy efficacy

Abstract: Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.

Cell death, therapeutics, and the immune response in cancer.

Abstract: Induction of cell death is inexorably linked with cancer therapy, but this can also initiate wound-healing processes that have been linked to cancer progression and therapeutic resistance. Here we describe the contribution of apoptosis and the lytic cell death pathways in the response to therapy (including chemotherapy and immunotherapy). We also discuss how necroptosis, pyroptosis, and ferroptosis function to promote tumor immunogenicity, along with emerging findings that these same forms of death can paradoxically contribute to immune suppression and tumor progression. Understanding the duality of cell death in cancer may allow for the development of therapeutics that shift the balance towards regression.

Combination IFNβ and membrane-stable CD40L maximize tumor dendritic cell activation and lymph node trafficking to elicit systemic T-cell immunity.

Abstract: Oncolytic viral therapies induce direct killing of tumor cells and activation of conventional dendritic cells (cDCs); however, cDC activation has not been optimized with current therapies. We evaluated adenoviral delivery of engineered membrane-stable CD40L (MEM40) and IFNβ to locally activate cDCs in mouse tumor models. Combined tumor MEM40 and IFNβ expression induced the highest cDC activation coupled with increased lymph node migration, increased systemic antitumor CD8+ T-cell responses, and regression of established tumors in a cDC1-dependent manner. MEM40+IFNβ combined with checkpoint inhibitors led to effective control of distant tumors and lung metastases. An oncolytic adenovirus (MEM-288) expressing MEM40+IFNβ in phase 1 clinical testing induced cancer cell loss concomitant with enhanced T-cell infiltration and increased systemic presence of tumor T-cell clonotypes in NSCLC patients. This approach to simultaneously target two major DC-activating pathways has potential to significantly impact the solid tumor immunotherapy landscape.

Abstract 678: Investigating the mechanisms involved in HMGB1-dependent DNA uptake and STING activation in dendritic cells

Abstract: Background: The cGAS-STING pathway is critical for the development of anti-tumor immunity. Activation of host STING can occur by sensing of extracellular DNA by intratumoral dendritic cells (DCs). After chemotherapy and under conditions of chromosomal instability, tumor cells release double-stranded DNA. We have previously shown DCs are able to internalize DNA in a dynamin-dependent manner, suggesting that a membrane receptor might mediate DNA endocytosis. DNA uptake also requires the presence of the high mobility group box protein 1 (HMGB1). HMGB1 has multiple known receptors such as TLR2, TLR4, RAGE and CD24. However, whether these receptors are involved in DNA engulfment by DCs remains unknown. Here, we sought to elucidate the mechanisms facilitating DNA uptake and subsequent STING activation in DCs. Methods: Bone marrow derived (BM)DCs obtained from wild type (WT), RAGE, TLR2 or TLR4-deficient mice, as well as and WT or CD24-deficient MutuDC1940 cells, were cultured in the presence or absence of Cy5-labeled plasmid DNA, a recombinant Flag-tagged HMGB1 protein (rHMGB1) and/or DMXAA. Cy5-DNA uptake, Flag-HMGB1 binding and IRF3 phosphorylation was analyzed by flow cytometry or immunofluorescence microscopy. MutuDC1940 cells were also treated with plasmid DNA, rHMGB1, tumor debris (HS) and/or L-Leucyl-L-Leucine methyl ester (LLOMe) and galectin-3 clustering was quantified by immunofluorescence. Statistically significant differences were determined by ANOVA test. Results: DCs take-up Cy5-DNA when rHMGB1 was added, and it was HMGB1 dose-dependent, supporting HMGB1 as a key factor for DNA uptake. We found that TLR2, TLR4, RAGE or CD24 deficient DCs were capable of internalizing Cy5-DNA in the presence of rHMGB1 to the same extent as WT DCs, indicating that none of these receptors are necessary for HMGB1-mediated DNA uptake. DNA-HMGB1 stimulation triggered IRF3 phosphorylation in DCs, suggesting extracellular DNA enters the cytosol and is sensed by cGAS, leading to STING activation. In support of this, DNA-HMGB1 treatment induced an increase in lysosomal membrane permeabilization, as measured by the extent of galectin-3 clusters within cells. Conclusion: HMGB1 promotes DNA uptake by DCs independently of TLR2, TLR4, RAGE or CD24, suggesting other receptor(s) mediate the process of HMGB1-dependent DNA internalization. Further, DNA-HMGB1 induced lysosomal membrane destabilization which may facilitate DNA release into the cytosol for sensing and trigger activation of the cGAS-STING pathway. Citation Format: Daiana P. Celias, Kay Hänggi, Brian Ruffell. Investigating the mechanisms involved in HMGB1-dependent DNA uptake and STING activation in dendritic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 678.

Abstract 6401: HMGB1 release predicts chemotherapy combinatorial efficacy with TIM-3 blockade

Abstract: We have recently shown that TIM-3 blockade promotes response to paclitaxel chemotherapy in preclinical breast cancer models through its ability to suppress uptake of HMGB1-DNA complexes by dendritic cells. However, it remains unclear why paclitaxel is necessary for response to TIM-3 blockade. Here we demonstrate that the taxanes paclitaxel and docetaxel induce the loss of HMGB1 from breast cancer cells in vitro and in vivo. Interestingly, this does not occur through loss of cellular integrity, but rather through an active secretion process involving ADP-ribosylation of HMGB1 by PARP1. PARP1 was activated by docetaxel treatment, as measured by detection of its cleaved form in the cytoplasm, and HMGB1 release was prevented by the use of the PARP inhibitor niraparib. Both PARP1 activation and ADP-ribosylation of HMGB1 were dependent upon the activity of TLR4, a known receptor for paclitaxel, as demonstrated with CRISPR/Cas9-generated knockout cells and TLR4 inhibitors. However, TLR4 stimulation alone was insufficient to induce HMGB1 release. By understanding how taxanes promote the release of HMGB1 we may be able to develop biomarkers for the selection of patients most likely to respond to the combination of chemotherapy and immunotherapy with TIM-3 blocking antibodies. Citation Format: Alexis Onimus, Jessica Mandula, Paulo Rodriguez, Brian Ruffell. HMGB1 release predicts chemotherapy combinatorial efficacy with TIM-3 blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6401.

Abstract 6398: Targeting ENPP1 to boost anti-tumor immunity during PARP inhibition

Abstract: Homologous recombination (HR) deficiency confers sensitivity to poly (ADP-ribose) polymerase inhibitors (PARPi), which have been approved for use in ovarian and triple negative breast cancers (TNBC) carrying loss-of-function mutations in HR pathway genes, most commonly BRCA1/2. Preclinical studies in BRCA1-deficient tumor models have demonstrated that the immune compartment is required for PARPi efficacy through the ability of PARPi to cause micronuclei formation and activate cGAS in tumors cells. The production of the 2’3’-cGAMP secondary intermediate stimulates STING in neighboring tumor-infiltrating immune cells to promote an anti-tumor response. Despite this evidence of immune stimulation, PARPi does not extend overall survival in TNBC patients, suggesting that immune suppression remains a significant barrier for efficacy. Here we sought to evaluate the role of ectonucleotide pyrophosphatase/phosphodiesterase 1(ENPP1), a type II transmembrane glycoprotein that hydrolyzes extracellular 2’3’-cGAMP. We generated Brca1/Pten/Trp53-deficient TNBC cell lines from a genetically engineered mouse model and found that these expressed higher levels of ENPP1 than Brca1-proficient TNBC and non-TNBC cell lines. Knocking out Enpp1 via CRISPR/Cas9 increased 2’3’-cGAMP production during Niraparib treatment but did not affect cellular proliferation in vitro. Critically, knocking out Enpp1 significantly reduced the growth of orthotopic Brca1-deficient tumors, with no change in growth observed for orthotopic E0771 or PyMT models, suggesting an important role for ENPP1 in HR-deficient tumors. Therapeutic dosing of Niraparib further reduced the growth of Enpp1 knockout tumors in a manner that was dependent upon host STING and correlated with higher CD8+ T cell infiltration and effector function. These findings demonstrate that ENPP1 suppresses anti-tumor immunity in Brca1-deficient tumors, suggesting that blocking the enzymatic activity of ENPP1 may provide therapeutic benefits during PARPi treatment for TNBC patients. Citation Format: Jie Li, Charlotte Mason, Kay Hanggi, Vincent Luca, Brian Ruffell. Targeting ENPP1 to boost anti-tumor immunity during PARP inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6398.

Abstract 2881: Investigating cholesterol transfer between macrophages and prostate cancer cells

Abstract: Background: Androgens stimulate androgen receptor (AR) signaling, driving prostate cancer (PC) cell proliferation and progression. Androgen deprivation therapy (ADT) effectively treats primary disease, but 20-30% of patients relapse after ~5 years, resulting in castration-resistant prostate cancer (CRPC), which has limited treatment options. We have previously shown in a murine model of CRPC that macrophages promote AR signaling in CRPC and that depleting tumor macrophages extends survival in response to ADT with Lupron. This correlates with reduced androgen levels in macrophage-depleted tumors and the ability of macrophages to transfer cholesterol, the anabolic precursor of androgens, to prostate cancer cells. However, the mechanism by which this transfer occurs, and whether cholesterol exchange mediates resistance to ADT, is currently unknown. Methods: To assess cholesterol transfer from macrophages to cancer cells, we established a culture system involving prostate cancer cell lines and either the RAW264.7 macrophage cell line or bone marrow-derived macrophages (BMDMs). Macrophages were pre-loaded with cholesterol using fluorescently labeled low-density lipoprotein (LDL) and were co-cultured or placed in a transwell system with tumor cells for 24 hours. Cholesterol transfer was then measured by flow cytometry. Results: Fluorescent signal from LDL was detected in 50-70% of tumor cells after 24 hours of co-culture. Cell contact was required for cholesterol transfer, as no fluorescence was detected in prostate cancer cells during transwell experiments. Macrophages were also able to internalize and transfer acetylated LDL, which is poorly taken up by tumor cells compared to unmodified LDL. Macrophage polarization with interleukin-4 did not impact cholesterol transfer and uptake of acetylated LDL occurred independently of the scavenger receptor CD36. The scavenger receptor SR-B1, which is often upregulated in prostate cancer cells, was dispensable for transfer, as knocking out SR-B1 via CRISPR/Cas9 in cancer cell lines did not reduce LDL uptake. Conclusion: Cholesterol transfer between macrophages and prostate cancer cells occurs in a contact-dependent manner, independently of the scavenger receptors most commonly associated with cholesterol accumulation. Citation Format: Olabisi Osunmakinde, Brian Ruffell. Investigating cholesterol transfer between macrophages and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2881.

Abstract 655: Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells

Abstract: Programmed cell death (PCD) was originally limited to apoptosis, but now includes pyroptosis, ferroptosis, necroptosis, and paraptosis. Necroptosis has attracted particular interest as an approach for immunotherapy as activation of the RIPK1/RIPK3 necrosome phosphorylates MLKL to form membrane pores, leading to loss of integrity and enhanced ability to induce an antigen-specific immune response in models of vaccination. Here we sought to determine if necroptosis within tumor cells was an important regulatory of anti-tumor immunity. Deficiency in Ripk3 or Mlkl did not impact tumor incidence or growth in genetic models of mammary carcinoma or lung adenocarcinoma. Similarly, the growth of syngeneic cell lines was not altered by loss of Ripk3 or Mlkl in vitro or in vivo. We next examined the induction of apoptosis or necroptosis in syngeneic tumor models using genetic constructs with doxycycline-controlled expression. In line with published data, subcutaneous vaccination demonstrated that RIPK3-induced necroptosis promoted an antigen-specific CD8+ T cell response greater than that observed with caspase 8-induced apoptosis. Surprisingly however, when necroptosis was induced within established PyMT-B6 orthotopic mammary tumors, we observed significantly reduced survival, which correlated with higher neutrophil and macrophage recruitment, along with lower CD8+ T cell infiltration and IFN-γ expression. In vitro, necroptosis induced with either RIPK3 or MLKL greatly enhanced expression of Cxcl1 and Cxcl2, a property that could be recapitulated by transferring dead cell supernatant to live cells, suggesting the presence of an alarmin or damage-associated molecular pattern (DAMP). We therefore knocked out MyD88, which serves as a critical adapter protein to a number of surface receptors, including the TLR family, the IL1 receptor, and RAGE. The loss of MyD88 completely eliminated the ability of tumor cells to express Cxcl1 in response to supernatant from necrotic cells. Expression was not driven by release of HMGB1 or signaling through RAGE or TLRs. Instead, we found that blocking the interleukin (IL)1 receptor or IL1α, but not IL1β, prevented Cxcl1 expression. IL1α is a constitutively expressed by some tumors, suggesting a potential role for IL1α in regulating immune responses to therapies that induce necrotic-like forms of cell death. In support of this, Il1a expression was strongly predictive of progression free survival in triple-negative breast cancer. These findings suggest that IL1α has an unexplored role in regulating the tumor microenvironment. By understanding how IL1α regulates the immune response during cytotoxic therapy, our findings may explain why certain therapies are immunogenic (or not) in different cancers. Citation Format: Kay Hänggi, Aysenur Keske, Alycia Gardner, Jie Li, Olabisi Osunmakinde, Daiana Celias, Amer Beg, Brian Ruffell. Necroptosis promotes tumor growth through IL1-alpha release and recruitment of immunosuppressive myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 655.