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Unleashing the immune anti-tumor response through immune checkpoint inhibitors (ICIs) is a promising strategy to combat many solid-tumor malignancies, including metastatic melanoma. When successful, the anti-tumor response is potent; however, around half of melanoma patients fail to respond to ICIs. Patient responsiveness to ICIs has been characterized by increases in MHC class I (MHC-I) expression driven by increases in oxidative metabolism. Though the harsh conditions of the tumor microenvironment (TME) are known to be immunosuppressive, the specific mechanisms connecting metabolic stress and antigen presentation are not well understood. To recapitulate and investigate the ICI-responsive phenotype in vitro, a model of metabolic remodeling was developed which forces melanoma cells (A375, A101D, B16F10) to metabolically adapt to the absence of glucose. Proteomic profiling indicates a reversible, adaptive phenotype reminiscent of published ICI-responders, and pathway enrichment shows analogous increases in oxidative metabolism and restoration of MHC-I expression. This phenotype, and its impact on MHC-I expression, significantly increases tumor cell sensitivity to T-cell-mediated killing in vitro. Additionally, successful adaptive remodeling of JAK1 and IFNAR1 KO cell lines suggests the metabolism-mediated induction of MHC-I is independent of IFN signaling. Proteomic analysis of three metabolically conditioned melanoma cell lines identified downregulation of the histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2) and the activating transcription factor ATF6 as key determinants of MHC expression. In these studies, we mechanistically explore the control of MHC-I antigen presentation through the transcriptional and post-translational dysregulation of ATF6 and EZH2. Here, we demonstrate that ATF6 overexpression, and thereby activation of the adaptive unfolded protein response (UPR), prevents induction of MHC by EZH2 inhibition. These data suggest EZH2 and ATF6 act to balance MHC-I antigen presentation during metabolic stress whereby loss of EZH2 increases and activation of ATF6 prevents MHC antigen presentation. Additional and ongoing studies coupling EZH2 and ATF6 inhibition will provide crucial insight into this mechanism and have the potential to influence adjuvant therapy development. Citation Format: Jacob L. Edmondson, Megan R. Reed, Lauren C. Morehead, Billie Heflin, Brian Koss, Alan J. Tackett. EZH2 and ATF6 sense metabolic stress to balance MHC class I antigen presentation in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB041.

T-cell exhaustion in cancer is linked to poor clinical outcomes, where evidence suggests T-cell metabolic changes precede functional exhaustion. Direct competition between tumor-infiltrating lymphocytes (TIL) and cancer cells for metabolic resources often renders T cells dysfunctional. Environmental stress produces epigenome remodeling events within TIL resulting from loss of the histone methyltransferase EZH2. Here, we report an epigenetic mechanism contributing to the development of metabolic exhaustion in TIL. A multiomics approach revealed a Cdkn2a.Arf-mediated, p53-independent mechanism by which EZH2 inhibition leads to mitochondrial dysfunction and the resultant exhaustion. Reprogramming T cells to express a gain-of-function EZH2 mutant resulted in an enhanced ability of T cells to inhibit tumor growth in vitro and in vivo. Our data suggest that manipulation of T-cell EZH2 within the context of cellular therapies may yield lymphocytes that are able to withstand harsh tumor metabolic environments and collateral pharmacologic insults. Significance: These findings demonstrate that manipulation of T-cell EZH2 in cellular therapies may yield cellular products able to withstand solid tumor metabolic–deficient environments.

Introduction: Accelerated aging is associated with lifetime stress and frailty, and has been reported in aging-related disease and mortality, including lung cancer, postmenopausal breast cancer, childhood cancer, and coronary heart disease. However, current knowledge on the impact of epigenetic aging in anti-tumor immunity remains limited. Pre-existing immune status heavily contributes to cancer diagnosis and the success of immunotherapy treatment, therefore, the relationship between epigenetic aging and immune function, and how it may lead to racial health disparity needs to be further explored. Methods: Genome-wide DNA methylation analysis were conducted in a feasibility test of 16 participants with equal number of African Americans (AAs) and European Americans (EAs), and epigenetic age (mAge) were calculated using R package methylclock for the established multi-tissue epigenetic clock algorithms which consists of 353 CpGs by Horvath. Correlation analysis was performed between mAge and in vitro T cell activation tests. Results: As expected, high correlation of calculated mAge versus chronological age was observed from 16 participants using the Horvath calculator, with higher mAge in AAs than EAs. Significant separation in DNA methylation profiles between AAs and EAs existed based on the top 1,000 CpG sites from the principal component analysis. To understand the effects of aging on T cell function, a series of functional assays were performed from different age groups, and significant (P Citation Format: Ping-Ching Hsu, Michael Bauer, Tung-Chin Chiang, Lora J. Rogers, A. Murat Aydin, L. Joseph Su, Brian Koss. Racial differences in epigenetic aging and its impact on expression of T-cell inhibitory receptors [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B015.

Identifying controlling features of responsiveness to checkpoint blockade therapies is an urgent goal in oncology research. Our group and others have previously shown melanoma tumors resistant to checkpoint blockade display features of mesenchymal transition, including E-cadherin loss. Here, we present the first in vivo evidence that E-cadherin from tumor cells facilitate immune attack, using a B16F10 melanoma mouse model in which E-cadherin is exogenously expressed (B16.Ecad). We find, compared with vector control, B16.Ecad exhibits delayed tumor growth, reduced metastatic potential, and increased overall survival in vivo. Transplantation of B16.Ecad into Rag1−/− and CD103−/− mice abrogated the tumor growth delay. This indicates the anti-melanoma response against B16.Ecad is both immune and CD103+ mediated. Moreover, B16.Ecad showed increased responsiveness to combination immune checkpoint blockade (ICB) compared with vector control. This work establishes a rationale for ICB responses observed in high E-cadherin–expressing tumors and suggests therapeutic advancement through amplifying CD103+ immune cell subsets. Significance: These findings identify the mechanism behind checkpoint blockade resistance observed in melanoma that has undergone mesenchymal transition and suggest activation of CD103+ immune cells as a therapeutic strategy against other E-cadherin–expressing malignancies.

Genetic obesity increases in liver phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio, inducing endoplasmic reticulum (ER) stress without concomitant increase of ER chaperones. Here, it is found that exposing mice to a palm oil-based high fat (HF) diet induced obesity, loss of liver PE, and loss of the ER chaperone Grp78/BiP in pericentral hepatocytes. In Hepa1–6 cells treated with elevated concentration of palmitate to model lipid stress, Grp78/BiP mRNA was increased, indicating onset of stress-induced Unfolded Protein Response (UPR), but Grp78/BiP protein abundance was nevertheless decreased. Exposure to elevated palmitate also induced in hepatoma cells decreased membrane glycosylation, nuclear translocation of pro-apoptotic C/EBP-homologous-protein-10 (CHOP), expansion of ER-derived quality control compartment (ERQC), loss of mitochondrial membrane potential (MMP), and decreased oxidative phosphorylation. When PE was delivered to Hepa1–6 cells exposed to elevated palmitate, effects by elevated palmitate to decrease Grp78/BiP protein abundance and suppress membrane glycosylation were blunted. Delivery of PE to Hepa1–6 cells treated with elevated palmitate also blunted expansion of ERQC, decreased nuclear translocation of CHOP and lowered abundance of reactive oxygen species (ROS). Instead, delivery of the chemical chaperone 4-phenyl-butyrate (PBA) to Hepa1–6 cells treated with elevated palmitate, while increasing abundance of Grp78/BiP protein and restoring membrane glycosylation, also increased ERQC, expression and nuclear translocation of CHOP, non-mitochondrial oxygen consumption, and generation of ROS. Data indicate that delivery of PE to hepatoma cells under lipid stress recovers cell function by targeting the secretory pathway and by blunting pro-apoptotic branches of the UPR.

Since 2014, immune checkpoint inhibitors (ICIs) have led the charge against metastatic melanoma as the most commonly utilized first-line treatment. While ICIs have dramatically increased the 5-year overall survival rate, only half of patients respond to treatment. Recently, a proteomic study correlated high levels of tumor mitochondrial metabolic proteins and MHC-related proteins to responsiveness to anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitor treatment. In a study of 91 melanoma cell lines in the European Searchable Tumor Cell and Databank, 67% of the cell lines exhibited HLA class antigen alterations, most commonly being down-regulation of HLA-B. Loss of MHC expression is a mechanism proposed to aid in evading the immune system by decreasing T-cell recognition and killing. Approaches to reverse tumor MHC loss is needed as it is a known mechanism utilized by melanoma and increased MHC expression has been linked to ICI responders. The connection between metabolism and MHC correlation has not been fully explored, and this study sought to further explore that relationship through a proteomic analysis of a metabolically conditioned B16F10 murine melanoma model. To expand our understanding of the metabolic control of MHC Class I antigen presentation, we cultured B16F10 melanoma cells in galactose media (B16.Gal), forcing the cells into an oxidative phosphorylative state relative to B16F10 melanoma cells cultured in glucose media (B16.Glu) which exhibit a glycolytic metabolic profile. We show that enforcing mitochondrial respiration restores the lost basal MHC expression and enhances the response to interferon gamma. Further, B16.Gal cells show higher rates of T-cell killing and increased PD-L1 expression. This phenotype proved reversible as shown by moving B16.Gal cells back into glucose media (B16.GalGlu), supporting the formation of a metabolically conditioned cell model rather than a favorable selection of compatible cells. We used proteomics to identify the driving forces behind the restoration of MHC expression. The identification of these forces could provide novel therapeutic targets for enhancing immunotherapy of melanoma. Citation Format: Alyson McKinnon, Nathan Avaritt, Alan Tackett, Brian Koss. Proteomic interrogation of the metabolic control of MHC class I antigen presentation in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1892.

Immune checkpoint inhibitors (ICIs) have revolutionized melanoma therapy. Still, the 5-year survival for advanced melanoma is only 50%. This leads to two questions: what makes some tumors less responsive to ICIs, and what can be done to increase responsiveness? Prior research suggests that hypoxia in the tumor microenvironment (TME) decreases the efficacy of ICIs, and metformin, an antihyperglycemic and caloric restriction mimetic (CRM), is able to reverse this condition. We hypothesize that other CRMs will also increase responsiveness to ICIs through hypoxia, and potentially other mechanisms. We chose three CRMs to study alongside metformin: hydroxycitrate, resveratrol, and sulforaphane. To identify pathways and understand more about CRMs effect on tumor hypoxia, we performed a proteomics experiment on B16F10 murine melanoma cells treated with all four CRMs. Proteomics analysis was done with a tandem mass tag labeling. To determine effect of caloric restriction mimetics on immunotherapy efficacy, B16F10 cells will be injected subcutaneously in immunocompetent male and female C57BL/6J mice, separated into four groups: 1) no treatment control, 2) ICI therapy only, 3) CRM therapy only, 4) ICI + CRM combination. Our preliminary data shows that, like metformin, additional CRMs decrease oxidative phosphorylation in cancer cells. This supports the hypothesis that the other CRMs may function similarly to metformin in the TME. We started testing the effect of four CRMs on tumor growth and hypoxia in an animal model. Our ultimate goal is to improve responsiveness to immune checkpoint inhibitors in patients with metastatic melanoma. Citation Format: Lauren C. Morehead, Brian S. Koss, Katherine F. Wallis, Alan J. Tackett, Isabelle Racine Miousse. Caloric restriction mimetics as adjuvant to immune checkpoint inhibitors for treatment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1577.

UV exposure-induced oxidative stress is implicated as the driving mechanism of melanoma development. Increased oxidative stress results in imbalanced reactive oxygen species (ROS) that damage DNA and dysregulated epigenetic modifications and leads to melanoma progression. Sulforaphane (SFN) is the natural bioactivated product of the cruciferous vegetable family (e.g., broccoli and Brussels sprouts) and is known to play a dual role in cytoprotection and to induce apoptosis in the tumor via antioxidant regulation and histone modification of epigenetic enzymes. We investigated the combinational effect of 5-aza-2’-deoxycytidine (DAC), an FDA-approved DNA-modification epigenetic drug and SFN in melanoma cells. We found further cell growth inhibition and an increased number of altered gene expression profiles, increased survival-related cytokine expression, as well as unique histones post-translational modifications (PTMs) upon combinational treatments compared to single treatment in vitro. These results set the stage for animal studies. The long-term goal of the current study is to find an optimal dietary dose of antioxidants from SFN to reduce the burden of oxidative stress, which may contribute to melanoma recurrence, a constant problem with current treatments. In addition to recurrence, a reduced dose of epigenetic drugs applied may minimize the side effects of single-high-dose treatment. Citation Format: Tung-chin Chiang, L. Joseph Su, Brian S. Koss, Charity Washam, Stephanie Byrum, Aaron Storey, Alan J. Koss. Combinational effect of sulforaphane (SFN) and epigenetic demethylation agent 5-aza-2’-deoxycytidine (DAC) on metastatic melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B26.

Direct competition between tumor-infiltrating lymphocytes and cancer cells for metabolic resources often renders T cells dysfunctional. It is becoming clearer that T cell metabolic changes can precede functional exhaustion. In the case of solid tumors, the driving force of this dysfunction is thought to be a result of inhibitory metabolism (e.g. glucose deprivation and mitochondrial dysfunction). The mechanisms connecting inhibitory metabolism and T cell metabolic exhaustion are largely unknown. Interestingly, T cells undergo a loss of the histone methyltransferase EZH2 (H3K27me3) during tumor infiltration. Methyltransferases, like EZH2, depend on mitochondria to supply S-Adenosyl-L-Methionine (SAM), the primary methyl donor. We sought to determine if there was a link between EZH2 and mitochondrial function. In this work, we utilized highly specific EZH2 inhibitors to model acute inhibition of EZH2 in CD8+ and CD4+ T cells. Taking an unbiased, multi-omics approach (proteomics, RNAseq, ChIPseq) we fully interrogate the downstream consequences of EZH2 inhibition in T cells. The loss of H3K27me3 does indeed lead to an induction of mitochondrial dysfunction, which in turn, drives a dependency on glycolytic metabolism and sensitivity to glucose withdrawal. The metabolic shift phenotype was confirmed using extracellular metabolic flux analysis. In T cells, we found the loss of H3K27me3 repression of the Cdkn2aArf locus to be a major contributor to mitochondrial dysfunction, independent of the canonical role to stabilize p53. Furthermore, we show Arf-/- mice are resistant to EZH2-inhibition induced mitochondrial dysfunction. Reprogramming tumor-specific T cells to exogenously express a gain-of-function EZH2 mutant (Y641F) resulted in an enhanced ability of T cells to inhibit solid tumor growth. This work demonstrates the potential for manipulation of EZH2 in cellular therapies for solid tumors with harsh metabolic conditions and sheds light on the dynamic interplay of epigenetics and metabolic sufficiency. Citation Format: Brian Koss, Bradley D. Shields, Erin M. Taylor, Aaron J. Storey, Stephanie D. Byrum, Allen J. Gies, Charity L. Washam, Samrat Roy Choudhury, Jeong Hyun Ahn, Hidetaka Uryu, Kimberly J. Krager, Tung-Chin Chiang, Samuel G. Mackintosh, Rick D. Edmondson, Nukhet Aykin-Burns, Gang Greg Wang, Alan J. Tackett. Epigenetic control of tumor-infiltrating lymphocyte metabolic-exhaustion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1029.

UV exposure-induced oxidative stress is implicated as a driving mechanism for melanoma. Increased oxidative stress results in DNA damage and epigenetic dysregulation. We previously found that a low dose of the antioxidant sulforaphane (SFN) in combination with the epigenetic drug 5 –aza –2 ′–deoxycytidine (DAC) slowed cell growth in melanoma at a greater rate than DAC alone. Here, we report a multi –omics analysis with combination and single drug treatment. We observed a significant increase in global protein abundance with an altered level of expression in the combination treatment as compared to individual treatment. Proteins associated with methylation patterns and with reactive oxidative species were found dysregulated in combination treatment. A comparison analysis of proteomic and transcriptomic data with Ingenuity Pathway Analysis showed common canonical pathways involved in phosphorylation (ERK/MAPK signaling) and immune response with combination treatment. The global DNA methylation pattern was analyzed to explore the impact of combination and single drug treatment on methylation status. Interestingly, we observed an increase in the levels of a critical immuno-regulator cytokine, C –C motif ligand 5 (CCL5), which functions as a chemoattractant for natural killer (NK) cells. Animal studies showed the level of CCL5in blood and tumor was increased with DAC and SFN combination treatment compared to that of control, which coincided with tumor reduction. Our data demonstrate the impact of SFN on the effectiveness of the FDA –approved drug DAC to reduce metastatic melanoma tumors. Citation Format: Tung-Chin Chiang, Brian Koss, Aaron J. Storey, Jovanny Zabaleta, Samrat Roy Choudhury, Bradley D. Shields, Elizabeth C. Draper, L Joseph Su, Alan J. Tackett. Combination of sulforaphane and 5-aza-2′-deoxycytidine slows the growth and upregulates the chemoattractant CCL5 in metastatic melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-182.

Background: UV exposure-induced oxidative stress is implicated as a driving mechanism for melanoma. Increased oxidative stress results in DNA damage and epigenetic dysregulation. Accordingly, we explored whether a low dose of the antioxidant sulforaphane (SFN) in combination with the epigenetic drug 5-aza-2&rsquo, deoxycytidine (DAC) could slow melanoma cell growth. SFN is a natural bioactivated product of the cruciferous family, while DAC is a DNA methyltransferase inhibitor. Methods: Melanoma cell growth characteristics, gene transcription profiles, and histone epigenetic modifications were measured after single and combination treatments with SFN and DAC. Results: We detected melanoma cell growth inhibition and specific changes in gene expression profiles upon combinational treatments with SFN and DAC, while no significant alterations in histone epigenetic modifications were observed. Dysregulated gene transcription of a key immunoregulator cytokine&mdash, C-C motif ligand 5 (CCL-5)&mdash, was validated. Conclusions: These results indicate a potential combinatorial effect of a dietary antioxidant and an FDA-approved epigenetic drug in controlling melanoma cell growth.

Anti-apoptotic Bcl-2 family proteins are central to the regulation of the intrinsic apoptotic pathway, and as such constitute an important group of targets with great potential as oncology therapeutics. The Bcl-2 family protein Mcl-1 has been demonstrated to facilitate survival and chemoresistance in multiple myeloma, AML, and other cancers, and agents which affect this pathway have become highly sought after. Currently, however, no therapies exist which directly target Mcl-1. We have identified compounds that target Mcl-1 which may be characterized as both Mcl-1-selective and pan-Mcl-1/Bcl-2 inhibitors. This effort has been facilitated by utilization of the BH3 profiling technology to guide SAR. This assay allows for determination of the mitochondrial priming state of both cell culture samples and primary patient samples. We have demonstrated a correlation between myeloma and leukemia cell line response to treatment with our inhibitors and the mitochondrial priming state of such cell lines. Such correlations have also been shown with respect to the extent of cytochrome C release. In the case of the selective Mcl-1 inhibitor, we have shown that cytochrome C release occurs preferentially in leukemia cell lines which are highly primed for Mcl-1 rather than Bcl-2. In addition, our Mcl-1 selective inhibitor demonstrates enhanced cell killing ability in leukemia cells which have been engineered to selectively express Mcl-1, Bcl-2, and Bcl-xL. Our current lead candidate possesses excellent drug-like properties and displays impressive efficacy in a multiple myeloma disseminated xenograft model. This work demonstrates the utility of the BH3 profiling assay as providing a functional biomarker for drug discovery tool and its ability to validate the on-target activity of Mcl-1 and Bcl-2 inhibitors. Citation Format: David J. Richard, Nicole Carlson, William Pierceall, Ryan Lena, Thomas Bannister, Peter Hodder, Timothy Spicer, Michael Andreeff, Joseph Opferman, Brian Koss, Andrew Kung, Michael Cardone. Characterization and development of on-target Mcl-1 inhibitors; BH3 profiling provides a valuable drug discovery tool. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2466. doi:10.1158/1538-7445.AM2013-2466

BAX is a pro-apoptotic BCL-2 family member that lies dormant in the cytosol until triggered by cellular stress to translocate to the mitochondria and form toxic oligomeric pores. The majority of cancer cells retain wild-type BAX and instead neutralize the death pathway by overexpressing anti-apoptotic BCL-2 family proteins. Thus, direct activation of BAX represents a pharmacologic opportunity to lower the apoptotic threshold in the setting of chemoresistance. Using a Stabilized Alpha-Helix of BCL-2 domain (SAHB) modeled after the BIM BH3 helix, we previously identified the “trigger site” for direct BAX activation at the confluence of alpha-helices 1 and 6. In silico screening for small molecule modulators of BAX led to our identification of BAX activator molecule 7 (BAM7), which selectively engages the trigger site and promotes BAX-mediated cell death (Gavathiotis et al, Nat Chem Biol, 2012). Here, we applied BAM7 to a panel of acute lymphoblastic leukemia cells and observe dose-responsive killing in the micromolar range. To improve the potency of anti-ALL activity, we undertook a systematic medicinal chemistry-based iteration of BAM7’s molecular features. A series of analogs demonstrate improved BAX binding activity based on a FITC-BIM SAHB/BAX competitive fluorescence polarization assay. The most potent binders were advanced to testing in a cellular model of anti-apoptotic protein-dependent chemoresistance. Whereas select BAM7 derivatives exhibited a potency similar to or greater than the BCL-2/BCL-XL inhibitor ABT-737 in a BCL-XL-dependent ALL cell line, the compounds significantly outperformed ABT-737 in the isogenic MCL-1-dependent ALL cells. Preliminary studies revealed favorable pharmacokinetic profiles for lead BAM7 derivatives, including oral bioavailability and central nervous system penetration. These early encouraging structure-activity relationship and pharmacokinetic data on chemically-optimized BAM7 analogs suggest that direct and selective activation of BAX may be a viable strategy for therapeutic induction of apoptosis in the context of cancer chemoresistance. Citation Format: Joseph A. Bellairs, Denis Reyna, Divakaramenon Sethumadhavan, Madhavi Neelagiri, Alex Broadhead, Christopher Baccei, Brian Koss, Joseph T. Opferman, Evripidis Gavathiotis, Loren D. Walensky. Chemical optimization of direct and selective molecular activators of pro-apoptotic BAX for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2013-2471