No abstract

Overexpression of S100B is routinely used for disease-staging and for determining prognostic outcomes in patients with malignant melanoma. Intracellular interactions between S100B and wild-type (WT)-p53 have been demonstrated to limit the availability of free WT-p53 in tumor cells, inhibiting the apoptotic signaling cascade. Herein, we demonstrate that, while oncogenic overexpression of S100B is poorly correlated (R < 0.3; p > 0.05) to alterations in S100B copy number or DNA methylation in primary patient samples, the transcriptional start site and upstream promoter of the gene are epigenetically primed in melanoma cells with predicted enrichment of activating transcription factors. Considering the regulatory role of activating transcription factors in S100B upregulation in melanoma, we stably suppressed S100b (murine ortholog) by using a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor, Krüppel-associated box (KRAB). Selective combination of S100b-specific single-guide RNAs and the dCas9-KRAB fusion significantly suppressed expression of S100b in murine B16 melanoma cells without noticeable off-target effects. S100b suppression resulted in recovery of intracellular WT-p53 and p21 levels and concomitant induction of apoptotic signaling. Expression levels of apoptogenic factors (i.e., apoptosis-inducing factor, caspase-3, and poly-ADP ribose polymerase) were altered in response to S100b suppression. S100b-suppressed cells also showed reduced cell viability and increased susceptibility to the chemotherapeutic agents, cisplatin and tunicamycin. Targeted suppression of S100b therefore offers a therapeutic vulnerability to overcome drug resistance in melanoma.

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.

Microtubule targeting agents (MTAs) have been used for the treatment of cancer for many decades and are among the most successful chemotherapeutic agents. However, their application and effectiveness are limited because of toxicity and resistance as well as a lack of knowledge of molecular mechanisms downstream of microtubule inhibition. Insights into key pathways that link microtubule disruption to cell death is critical for optimal use of these drugs, for defining biomarkers useful in patient stratification, and for informed design of drug combinations. Although MTAs characteristically induce death in mitosis, microtubule destabilizing agents such as vincristine also induce death directly in G1 phase in primary acute lymphoblastic leukemia (ALL) cells. Because many signaling pathways regulating cell survival and death involve changes in protein expression and phosphorylation, we undertook a comprehensive quantitative proteomic study of G1 phase ALL cells treated with vincristine. The results revealed distinct alterations associated with c-Jun N-terminal kinase signaling, anti-proliferative signaling, the DNA damage response, and cytoskeletal remodeling. Signals specifically associated with cell death were identified by pre-treatment with the CDK4/6 inhibitor palbociclib, which caused G1 arrest and precluded death induction. These results provide insights into signaling mechanisms regulating cellular responses to microtubule inhibition and provide a foundation for a better understanding of the clinical mechanisms of MTAs and for the design of novel drug combinations. The mass spectrometry proteomics data have been deposited to the PRIDE Archive () via the PRIDE partner repository with the data set identifier PXD027190 and 10.6019/PXD027190.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Despite numerous clinical trials, the standard of care therapy has remained unchanged for the last decade. One of the main problems contributing to the limited effective treatment options and poor overall survival is the lack of models which can reliably recapitulate tumor heterogeneity. Organoids are 3D self‐organized structures which mimic tumor architecture, microenvironment, and cellular interactions which makes them an improved model for anti‐cancer drug discovery. Monensin (MON) is a polyether ionophore antibiotic characterized by wide range of biological properties including anti‐cancer activity. In order to identify more potent compounds based on the scaffold of MON, we investigated the anti‐GBM activity of 14 novel esters and urethanes of MON. In 3D mini‐ring cell viability assays, we identified seven analogues (IC50 = 91.5 ± 54.4 ‐ 291.7 ± 68.8 nM) more potent towards GBM than the parental MON (IC50 = 612.6 ± 184.4 nM). These analogues induced DNA fragmentation in an organoid model of GBM, suggestive of apoptotic cell death. Furthermore, the most potent analog, compound 1, significantly reduced GBM cell migration, induced PARP cleavage and degradation, increased ɣH2AX signaling and increased expression of the autophagy marker LCII. To investigate the activity of these novel compounds in a tumor microenvironment, we have developed a host:tumor hybrid 3D organoid system. For host tissue, we generated human cerebral organoids (COs) from hiPSCs. The COs displayed multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1+), primitive ventricular system (SOX2+/Ki67+), intermediate zone (TBR2+) and cortical plate (MAP2+). These findings suggest the level of differentiation and development of our COs as equivalent to early stage human fetal brain. We then co‐cultured RFP‐labeled U87MG cells with fully formed COs. After establishing U87MG tumor formation, hybrid organoids were treated with MON or compound 1. Compound 1 significantly reduced U87MG tumor size after four days of treatment. Our findings highlight the therapeutic potential of MON analogues towards GBM and support further research and clinical development of these compounds.