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Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that has been implicated in numerous oncogenic processes. GSK-3 inhibitor elraglusib (9-ING-41) has shown promising preclinical and clinical antitumor activity across multiple tumor types. Despite promising early-phase clinical trial results, there have been limited efforts to characterize the potential immunomodulatory properties of elraglusib. We report that elraglusib promotes immune cell-mediated tumor cell killing of microsatellite stable colorectal cancer (CRC) cells. Mechanistically, elraglusib sensitized CRC cells to immune-mediated cytotoxicity and enhanced immune cell effector function. Using western blots, we found that elraglusib decreased CRC cell expression of NF-κB p65 and several survival proteins. Using microarrays, we discovered that elraglusib upregulated the expression of proapoptotic and antiproliferative genes and downregulated the expression of cell proliferation, cell cycle progression, metastasis, TGFβ signaling, and anti-apoptotic genes in CRC cells. Elraglusib reduced CRC cell production of immunosuppressive molecules such as VEGF, GDF-15, and sPD-L1. Elraglusib increased immune cell IFN-γ secretion, which upregulated CRC cell gasdermin B expression to potentially enhance pyroptosis. Elraglusib enhanced immune effector function resulting in augmented granzyme B, IFN-γ, TNF-α, and TRAIL production. Using a syngeneic, immunocompetent murine model of microsatellite stable CRC, we evaluated elraglusib as a single agent or combined with immune checkpoint blockade (anti-PD-1/L1) and observed improved survival in the elraglusib and anti-PD-L1 group. Murine responders had increased tumor-infiltrating T cells, augmented granzyme B expression, and fewer regulatory T cells. Murine responders had reduced immunosuppressive (VEGF, VEGFR2) and elevated immunostimulatory (GM-CSF, IL-12p70) cytokine plasma concentrations. To determine the clinical significance, we then utilized elraglusib-treated patient plasma samples and found that reduced VEGF and BAFF and elevated IL-1 beta, CCL22, and CCL4 concentrations correlated with improved survival. Using paired tumor biopsies, we found that tumor-infiltrating immune cells had a reduced expression of inhibitory immune checkpoints (VISTA, PD-1, PD-L2) and an elevated expression of T-cell activation markers (CTLA-4, OX40L) after elraglusib treatment. These results address a significant gap in knowledge concerning the immunomodulatory mechanisms of GSK-3 inhibitor elraglusib, provide a rationale for the clinical evaluation of elraglusib in combination with immune checkpoint blockade, and are expected to have an impact on additional tumor types, besides CRC.

Supplementary Table S2: qPCR primer for subcluster E genes

Increasing evidence suggests the presence of minor cell subpopulations in prostate cancer that are androgen independent and poised for selection as dominant clones after androgen deprivation therapy. In this study, we investigated this phenomenon by stratifying cell subpopulations based on transcriptome profiling of 144 single LNCaP prostate cancer cells treated or untreated with androgen after cell-cycle synchronization. Model-based clustering of 397 differentially expressed genes identified eight potential subpopulations of LNCaP cells, revealing a previously unappreciable level of cellular heterogeneity to androgen stimulation. One subpopulation displayed stem-like features with a slower cell doubling rate, increased sphere formation capability, and resistance to G2–M arrest induced by a mitosis inhibitor. Advanced growth of this subpopulation was associated with enhanced expression of 10 cell-cycle–related genes (CCNB2, DLGAP5, CENPF, CENPE, MKI67, PTTG1, CDC20, PLK1, HMMR, and CCNB1) and decreased dependence upon androgen receptor signaling. In silico analysis of RNA-seq data from The Cancer Genome Atlas further demonstrated that concordant upregulation of these genes was linked to recurrent prostate cancers. Analysis of receiver operating characteristic curves implicates aberrant expression of these genes and could be useful for early identification of tumors that subsequently develop biochemical recurrence. Moreover, this single-cell approach provides a better understanding of how prostate cancer cells respond heterogeneously to androgen deprivation therapies and reveals characteristics of subpopulations resistant to this treatment.Significance: Illustrating the challenge in treating cancers with targeted drugs, which by selecting for drug resistance can drive metastatic progression, this study characterized the plasticity and heterogeneity of prostate cancer cells with regard to androgen dependence, defining the character or minor subpopulations of androgen-independent cells that are poised for clonal selection after androgen-deprivation therapy. Cancer Res; 78(4); 853–64. ©2017 AACR.

Supplementary Figure S1: Time-course expression analysis of LNCaP cells reveals dynamic and heterogeneous responses to androgen stimulation. Supplementary Figure S2: Cell cycle progression of LNCaP cells treated with or without R1881 after cell synchronization by double thymidine block (DTB). Supplementary Figure S3: The summary of aligned and unaligned single-cell RNA-seq reads counts. Supplementary Figure S4: Correlation of single-cell RNA-seq ensemble reads with representative bulk RNA-seq reads. Supplementary Figure S5: Venn diagrams illustrating the comparison of differentially expressed genes found in single- and bulk-cell LNCaP experiments. Supplementary Figure S6: The Mclust algorithm defines likely subgroups using a "model-based" approach. Supplementary Figure S7: Each cell within each subcluster exhibits a similar gene expression profile. Supplementary Figure S8: Gleason score and biochemical recurrence status compared to each subcluster''s gene expression profile. Supplementary Figure S9: Differential expression of 10 genes in LNCaP''s 8.1 subpopulation of cells. Supplementary Figure S10: High single-cell expression of 10 genes in PC3 similar to 12hr-R1881 LNCaP cells.

This review focuses on the role of palliative surgery in the care of the palliative care patient, and the appropriate role of the surgeon. The surgeon has much to bring to the palliative care team. The surgeon's role goes beyond the technical requirements of the palliative procedure, which itself must be strictly defined, and has recognized utility for improving quality of life in selected patients. These benefits may be substantial, but come at significant risk; requiring careful balancing of risks and benefits that is most completely understood by the surgeon. The surgeon's judgement can help determine which procedure best meets a patient's goals. The complex dialogue involved in the decision to undergo a palliative operation requires excellent communication between the palliative care team, the patient, and their family. Integrating the surgeon into the palliative care team could help with earlier initiation of those palliative discussions, and assist deliberation of palliative surgery. Surgeons also understand the importance of communication around palliative surgical interventions and have adapted several teaching models to the specifics of this crucial communication. A palliative team combining both surgeons and palliative care physicians may promote goal-concordant decision-making and remove barriers to communication and team engagement. The future of palliative surgery research will involve measures of success that go beyond physiology or mortality, to include more evaluations of communication and patient goals.

Inhibition of GSK-3 using small-molecule elraglusib has shown promising preclinical antitumor activity. Using in vitro systems, we found that elraglusib promotes immune cell-mediated tumor cell killing, enhances tumor cell pyroptosis, decreases tumor cell NF-κB-regulated survival protein expression, and increases immune cell effector molecule secretion. Using in vivo systems, we observed synergy between elraglusib and anti-PD-L1 in an immunocompetent murine model of colorectal cancer. Murine responders had more tumor-infiltrating T-cells, fewer tumor-infiltrating Tregs, lower tumorigenic circulating cytokine concentrations, and higher immunostimulatory circulating cytokine concentrations. To determine the clinical significance, we utilized human plasma samples from patients treated with elraglusib and correlated cytokine profiles with survival. Using paired tumor biopsies, we found that CD45+ tumor-infiltrating immune cells had lower expression of inhibitory immune checkpoints and higher expression of T-cell activation markers in post-elraglusib patient biopsies. These results introduce several immunomodulatory mechanisms of GSK-3 inhibition using elraglusib, providing a rationale for the clinical evaluation of elraglusib in combination with immunotherapy.Statement of significancePharmacologic inhibition of GSK-3 using elraglusib sensitizes tumor cells, activates immune cells for increased anti-tumor immunity, and synergizes with anti-PD-L1 immune checkpoint blockade. These results introduce novel biomarkers for correlations with response to therapy which could provide significant clinical utility and suggest that elraglusib, and other GSK-3 inhibitors, should be evaluated in combination with immune checkpoint blockade.

Inflammatory cytokines, chemokines, and growth factors are molecular messengers that circulate and have the capability to modify the tumor microenvironment and impact therapeutic response. The characterization of soluble mediators as biomarkers for diagnosis and prognosis is of interest in oncology. We utilize the cytokinome to characterize the response of colorectal tumor cell lines to selected small-molecules in oncology as a proof-of-concept dataset with immunomodulatory analyte heat map rankings for drug and cell line combinations. We observed overall trends in drug class effects with MEK-, BRAF-, PARP-inhibitors, and Imipridones in cytokine, chemokine, and growth factor responses that may help guide therapy selection. MEK-inhibitor treatment downregulated analytes VEGF, CXCL9/MIG, and IL-8/CXCL8 and upregulated CXCL14/BRAK, Prolactin, and CCL5/RANTES. BRAF-inhibitor treatment downregulated VEGF and IL-8/CXCL8, while increasing soluble TRAIL-R2. Treatment with PARP-inhibitors decreased CXCL9/MIG, IL-8/CXCL8, CCL3/MIP-1 alpha, VEGF, and CXCL14/BRAK, while treatment increased soluble TRAIL-R2 and prolactin. Treatment with Imipridones decreased CCL3/MIP-1 alpha, VEGF, CXCL14/BRAK, IL-8/CXCL8, and Prolactin and increased CXCL5/ENA-78. We also observed differential responses to therapeutics depending on the mutational profile of the cell line. In the future, a similar but larger dataset may be utilized in the clinic to aid in the prediction of patient response to immunomodulatory therapies based on tumor genotype.

People experiencing homelessness are particularly vulnerable when diagnosed with pancreatic cancer. Patients with lower socioeconomic status have worse outcomes from pancreatic cancer as the result of disparities in access to treatment and barriers to navigation of the health care system. Patients with lower socioeconomic status, or who are vulnerably housed, are less likely to receive surgical treatment even when it is recommended by National Comprehensive Cancer Network guidelines. This disparity in access to surgical care explains much of the gap in pancreatic cancer outcomes. There are many factors that contribute to this disparity in surgical management of pancreatic cancer in people experiencing homelessness. These include a lack of reliable transportation, feeling unwelcome in the medical setting, a lack of primary care and health insurance, and implicit biases of health care providers, including racial bias. Solutions that focus on rectifying these problems include utilizing patient navigators, addressing implicit biases of all health care providers and staff, creating an environment that caters to the needs of patients experiencing homelessness, and improving their access to insurance and regional support networks. Implementing these potential solutions all the way from the individual provider to national safety nets could improve outcomes for patients with pancreatic cancer who are experiencing homelessness.

ONC201 was originally discovered as TNF-Related Apoptosis Inducing Ligand (TRAIL)-inducing compound TIC10. ONC201 appears to act as a selective antagonist of the G protein coupled receptor (GPCR) dopamine receptor D2 (DRD2), and as an allosteric agonist of mitochondrial protease caseinolytic protease P (ClpP). Downstream of target engagement, ONC201 activates the ATF4/CHOP-mediated integrated stress response leading to TRAIL/Death Receptor 5 (DR5) activation, inhibits oxidative phosphorylation via c-myc, and inactivates Akt/ERK signaling in tumor cells. This typically results in DR5/TRAIL-mediated apoptosis of tumor cells; however, DR5/TRAIL-independent apoptosis, cell cycle arrest, or antiproliferative effects also occur. The effects of ONC201 extend beyond bulk tumor cells to include cancer stem cells, cancer associated fibroblasts and immune cells within the tumor microenvironment that can contribute to its efficacy. ONC201 is orally administered, crosses the intact blood brain barrier, and is under evaluation in clinical trials in patients with advanced solid tumors and hematological malignancies. ONC201 has single agent clinical activity in tumor types that are enriched for DRD2 and/or ClpP expression including specific subtypes of high-grade glioma, endometrial cancer, prostate cancer, mantle cell lymphoma, and adrenal tumors. Synergy with radiation, chemotherapy, targeted therapy and immune-checkpoint agents has been identified in preclinical models and is being evaluated in clinical trials. Structure-activity relationships based on the core pharmacophore of ONC201, termed the imipridone scaffold, revealed novel potent compounds that are being developed. Imipridones represent a novel approach to therapeutically target previously undruggable GPCRs, ClpP, and innate immune pathways in oncology.

Introduction: An immunosuppressed, apoptosis-resistant phenotype characterizes pancreatic ductal adenocarcinoma (PDAC). Both inflammation and tumorigenesis are influenced by the Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) pathway. TLY012 is pegylated recombinant TRAIL protein and is an orphan drug for systemic sclerosis and chronic pancreatitis. The potential synergy between TLY012 and anti-mouse PD-1 antibody was investigated in a syngeneic immunocompetent heterotopic mouse model. Methods: KPC-luc PDAC cells were subcutaneously implanted into C57Bl/6 mice. Treatment combined 10 mg/kg TLY012 three times per week with 10 mg/kg anti-mouse PD-1 antibody twice weekly and were compared to individual treatment and control groups. Cytokine profiling via 32-plex Luminex immunoassay was completed after 7 days and after three months of treatment. Flow cytometry at three months evaluated NK and T cell profiles in the spleen and tumors. Results: PDAC tumor-bearing mice treated with the combination of anti-mouse PD-1 and TLY012 had slower tumor growth and reduced tumor volume at 70 days compared to either drug alone (all p Conclusions: The combination of anti-mouse PD-1 and TLY012 prevents growth of PDAC in an immunocompetent mouse model. The combination also increases tumor-infiltrating CD8+ T cells, decreases circulating Treg cells and alters cytokine expression. CCL5 is a T-cell chemokine, and both interferon gamma and IL-3 are produced by activated T cells and macrophages, and have proinflammatory, antitumor effects. Combining TLY012 and anti-mouse PD-1 creates these changes in immune cell and cytokine levels, suggesting induction of a more proinflammatory immune environment. The proinflammatory state may contribute to decreased tumor growth. Citation Format: Anna D. Louie, Kelsey E. Huntington, Young Lee, Jared Mompoint, Jinxuan Wu, Seulki Lee, Wafik S. El-Deiry. Combining PD-1 inhibition with the TRAIL receptor agonist TLY012 promotes pancreatic ductal adenocarcinoma tumor regression in an immunocompetent mouse model [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 5607.

Although the range of immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is variable, cytokine storm is observed in a subset of symptomatic individuals. To further understand the disease pathogenesis and, consequently, to develop an additional tool for clinicians to evaluate patients for presumptive intervention, we sought to compare plasma cytokine levels between a range of donor and patient samples grouped by a COVID-19 Severity Score (CSS) based on the need for hospitalization and oxygen requirement. Here we utilize a mutual information algorithm that classifies the information gain for CSS prediction provided by cytokine expression levels and clinical variables. Using this methodology, we found that a small number of clinical and cytokine expression variables are predictive of presenting COVID-19 disease severity, raising questions about the mechanism by which COVID-19 creates severe illness. The variables that were the most predictive of CSS included clinical variables such as age and abnormal chest x-ray as well as cytokines such as macrophage colony-stimulating factor, interferon-inducible protein 10, and interleukin-1 receptor antagonist. Our results suggest that SARS-CoV-2 infection causes a plethora of changes in cytokine profiles and that particularly in severely ill patients, these changes are consistent with the presence of macrophage activation syndrome and could furthermore be used as a biomarker to predict disease severity.

ONC212 is a fluorinated imipridone with preclinical efficacy against pancreatic and other malignancies. Although mitochondrial protease ClpP was identified as an ONC212-binding target, the mechanism leading to cancer cell death is incompletely understood. We investigated mitochondrial dysfunction and metabolic rewiring triggered by ONC212 in pancreatic cancer, a deadly malignancy with an urgent need for novel therapeutics. We found ClpP is expressed in pancreatic cancer cells and is required for ONC212 cytotoxicity. ClpX, the regulatory binding partner of ClpP, is suppressed upon ONC212 treatment. Immunoblotting and extracellular flux analysis showed ONC212 impairs oxidative phosphorylation (OXPHOS) with decrease in mitochondrial-derived ATP production. Although collapse of mitochondrial function is observed across ONC212-treated cell lines, only OXPHOS-dependent cells undergo apoptosis. Cells relying on glycolysis undergo growth arrest and upregulate glucose catabolism to prevent ERK1/2 inhibition and apoptosis. Glucose restriction or combination with glycolytic inhibitor 2-deoxy-D-glucose synergize with ONC212 and promote apoptosis in vitro and in vivo. Thus, ONC212 is a novel mitocan targeting oxidative metabolism in pancreatic cancer, leading to different cellular outcomes based on divergent metabolic programs.

Introduction: Breast cancer metastases (BCM), which cause most breast cancer (BC)-associated mortality, have increased genetic complexity compared to early-stage disease. However, the contribution of genetic alterations to site of BCM is not well-understood. Different breast cancer subtypes have varying patterns of BCM, e.g., lobular carcinoma more frequently spreads to gynecologic (Gyn) organs and the GI tract, perhaps hinting at selection pressures wherein some organs are hospitable to tumors with certain genetic alterations. Methods: Relationships between BCM site and mutations detected by DNA next-generation sequencing (NGS; NextSeq 592 gene panels or NovaSeq whole exome sequencing) were investigated using 12,464 BC samples sequenced at Caris Life Sciences (sample sizes, Table 1). PD-L1 expression was tested through IHC (Clone SP-142 (cut-off ≥1, 1%)). Tumor mutational burden (TMB) was measured by totaling somatic mutations per tumor (high ≥ 10 mutations per MB). Immune cell fractions were calculated by deconvolution of whole-transcriptome data (NovaSeq) using Quantiseq (reference). Statistical significance was determined using chi-square and Wilcoxon rank sum tests adjusted for multiple comparisons. Results: Compared to primary breast tumors, BCM had increased frequency of TMB-H (10.08% vs. 4.94%), decreased PD-L1 positivity (21.09% vs. 35.82%), and were enriched for PIK3CA (34.62% vs 30.53%) and ESR1 mutations (13.34% vs 2.17%) (all P Table 1.Breast Cancer TumorsTumor SiteTotalPredominant Breast Cancer SubtypeAll12464HR+/HER2- (51.6%)Breast5014HR+/HER2- (46.5%)Liver2003HR+/HER2- (63.3%)Bone1132HR+/HER2- (69.4%)Axilla1051HR+/HER2- (47.7%)Lung823HR+/HER2- (46.1%)Lymph Node647HR+/HER2- (43.4%)Chest/Chest Wall375HR+/HER2- (44.8%)Brain359TNBC (38.2%)Other315HR+/HER2- (57.1%)Skin282HR+/HER2- (50.7%)Connective Tissue193HR+/HER2- (51.8%)GI Organs143HR+/HER2- (69.9%)Gynecologic Organs127HR+/HER2- (76.4%) Citation Format: Anna D Louie, Rani Chudasama, Sharon Wu, Marzia Capelletti, Daniel Magee, W. Michael Korn, Virginia Kaklamani, Antoinette R Tan, Pavani Chalasani, Wafik S El-Deiry, Don Dizon, Stephanie L. Graff. Mutational landscape and immune infiltration of breast cancer metastases to gynecologic and other organs [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-04.

Increasing evidence suggests the presence of minor cell subpopulations in prostate cancer that are androgen independent and poised for selection as dominant clones after androgen deprivation therapy. In this study, we investigated this phenomenon by stratifying cell subpopulations based on transcriptome profiling of 144 single LNCaP prostate cancer cells treated or untreated with androgen after cell-cycle synchronization. Model-based clustering of 397 differentially expressed genes identified eight potential subpopulations of LNCaP cells, revealing a previously unappreciable level of cellular heterogeneity to androgen stimulation. One subpopulation displayed stem-like features with a slower cell doubling rate, increased sphere formation capability, and resistance to G2–M arrest induced by a mitosis inhibitor. Advanced growth of this subpopulation was associated with enhanced expression of 10 cell-cycle–related genes (CCNB2, DLGAP5, CENPF, CENPE, MKI67, PTTG1, CDC20, PLK1, HMMR, and CCNB1) and decreased dependence upon androgen receptor signaling. In silico analysis of RNA-seq data from The Cancer Genome Atlas further demonstrated that concordant upregulation of these genes was linked to recurrent prostate cancers. Analysis of receiver operating characteristic curves implicates aberrant expression of these genes and could be useful for early identification of tumors that subsequently develop biochemical recurrence. Moreover, this single-cell approach provides a better understanding of how prostate cancer cells respond heterogeneously to androgen deprivation therapies and reveals characteristics of subpopulations resistant to this treatment. Significance: Illustrating the challenge in treating cancers with targeted drugs, which by selecting for drug resistance can drive metastatic progression, this study characterized the plasticity and heterogeneity of prostate cancer cells with regard to androgen dependence, defining the character or minor subpopulations of androgen-independent cells that are poised for clonal selection after androgen-deprivation therapy. Cancer Res; 78(4); 853–64. ©2017 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer with a 4.2% 5-year survival rate. There is an urgent need for innovative treatments for patients suffering from PDAC. Patients with PDAC often have immunosuppressed tumors that are apoptosis-resistant and have limited immune cell infiltration and/or activation. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces cancer cell apoptosis during innate immunity, while ONC212 is a potent fluorinated second-generation imipridone currently in IND-enabling studies that induces TRAIL signaling and the integrated stress response (ISR). TLY012 is a novel PEG’ylated trimeric TRAIL receptor agonist being clinically developed to overcome the short half-life of TRAIL. We hypothesized that combining a TRAIL pathway inducer and a TRAIL receptor agonist may be efficacious in PDAC and may activate immune cell killing based on prior work with imipridone ONC201. Immune cell co-culture experiments were conducted using PANC1 PDAC cells and TALL-104 CD8+ cytotoxic T-cells at a 1:1 effector-to-target cell ratio with or without ONC212. We observed a significant increase in T-cell killing of PDAC cells in the co-culture assay following treatment with ONC212 at several doses as compared to controls. We noted an increase in tumor-immune cell interactions as measured by the number of T-cells that were directly touching tumor cells at each timepoint. The BxPC-3 immunodeficient murine PDAC model revealed an increase in natural killer (NK) cell tumor infiltration by immunohistochemistry (IHC) staining for NK1.1 after 30 days of 50 mg/kg ONC212 treatment three times a week. The combination of ONC212 and TLY012 was evaluated using subcutaneously-implanted KPC-Luc PDAC cells in a syngeneic immunocompetent C57BL/6 mouse model. Mice treated with 25 mg/kg ONC212 alone twice a week, 10 mg/kg TLY012 alone twice a week, or combination therapy with ONC212 and TLY012 showed statistically significant decreases in tumor growth compared to controls after 5 weeks of treatment (n=6 mice per treatment condition) by tumor volume and in vivo bioluminescent imaging. We are analyzing murine plasma cytokine profiles, changes in tumor-infiltrating NK- and T-cells by flow cytometric and IHC analysis of murine spleen and KPC-Luc tumor samples. We report a novel immune stimulation of the TRAIL pathway and T-cell activation by ONC212 plus TLY012 leading to cytotoxic anti-PDAC responses in vivo. Citation Format: Kelsey E. Huntington, Anna Louie, Young Lee, Jared Mompoint, Isacco Ferrarini, Aakash Jhaveri, Varun V. Prabhu, Allen Melemed, Seulki Lee, Wafik S. El-Deiry. ONC212 stimulates cytotoxic T-cell killing, increases tumor-immune cell interactions, and promotes tumor regression in combination with TLY012 in a PDAC murine model [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-064.

Tumor cells upregulate a plethora of proteins that are important for pH regulation, resulting in the acidification of the extracellular tumor microenvironment. Abnormal pH is known to dampen immune function, resulting in a worsened anti-tumor immune response. Understanding how alterations in pH modulate the interactions between immune cells and tumors cells could help elucidate opportunities for new therapeutic approaches. To determine how pH modification impacts immune cell-mediated killing of tumor cells, co-culture experiments were conducted with GFP+ SW480 colorectal cancer cells and either NK-92 natural killer cells or TALL-104 T cells at various pHs in a 48-well plate. Manipulation of pH occurred using the compounds lactic acid and sodium bicarbonate. We observed that both NK-92 cells and TALL-104 cells exhibited decreasing amounts of cell killing in response to decreasing pH. Next, to determine if therapeutic manipulation of pH via alteration of pH pump activity could overcome the negative changes in immune cell activity associated with pH, we added Cariporide, a selective NHE1 inhibitor, to the co-culture experiments. Treatment with Cariporide bolstered T cell killing in a dose-dependent manner, however the doses used did not impact natural killer cell-mediated killing. Given the importance of cell signaling on immune function, and because we saw potentially significant changes in immune killing with Cariporide, we then evaluated how changes in pH using Cariporide affected these signaling molecules. Colorectal cancer cells (HCT116, HT29, KM12C) were treated with Cariporide at 5 µm and 10 µm doses and cancer cell culture supernatants were collected and frozen down at -80 °C. Cytokine, chemokine, and growth factor levels were analyzed using a Luminex 200 multiplexing instrument. Overall cytokine levels showed a decreasing trend in response to increasing doses of Cariporide. There were notable decreases in the levels of IL-8/CXCL8, VEGF, CCL3/MIP-1 alpha, and M-CSF with increasing doses of Cariporide. All of these factors, as well as extracellular acidosis, are known to promote epithelial mesenchymal transition (EMT), thus, promoting metastatic dissemination. A therapeutic that targets factors that lead to EMT could help prevent metastasis. Moreover, we saw that pH impacts the function of immune cells and this is relevant in the context of a highly acidic TME. Therapeutically modifying the pH of the TME using Cariporide treatment may be a way to improve immune response and prevent EMT. These experiments can help inform future investigations into how the pH of the tumor microenvironment may be extrinsically modulated to improve anti-tumor immune response. Citation Format: Kelsey E. Huntington, Aaron W. Maxwell, Anna Louie, Shengliang Zhang, Lanlan Zhou, Wafik S. El-Deiry. Decreasing colorectal cancer extracellular pH dampens immune cell killing and is partially ameliorated by treatment with NHE1 inhibitor Cariporide [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 2736.

ONC212 is a fluorinated imipridone shown to have preclinical efficacy against multiple cancers, including pancreatic cancer. One of the binding targets of ONC212 is the mitochondrial peptidase, ClpP, but its role in the anticancer activity of ONC212 is incompletely understood. Understanding the effects of ONC212 on the bioenergetics of pancreatic cancer could facilitate understanding and amplification of its anti-tumor effects. On extracellular flux analysis, ONC212 impairs mitochondrial ATP production and oxidative phosphorylation in multiple pancreatic cancer lines. However, pancreatic adenocarcinoma uses both glycolysis and mitochondrial function to meet its energy demands. Some pancreatic cancer cell lines are resistant to ONC212 inhibition of oxidative phosphorylation alone, but co-treatment with glycolytic inhibitor 2-deoxy-D-glucose (2-DG) synergizes with ONC212 and increases tumor cell death in vitro. Suppression of ClpX, the regulatory binding protein of ClpP, occurs with ONC212 treatment and was used as a biomarker of ONC212 activity. This suppression was demonstrated in multiple pancreatic cancer lines in vitro and in vivo. In a xenograft mouse tumor model of pancreatic cancer using both the ONC212 resistant Bxpc3 pancreatic cancer cell line and the more sensitive HPAF-II cell line, the combination of ONC212 and 2-DG treatment significantly decreased tumor growth and reduced the intertumoral variability. Immunohistochemistry also revealed increased apoptotic markers in these tumors. There was no significant toxicity from dual treatment in mice, which suggests that tumor energy deprivation from dual inhibition of oxidative phosphorylation and glycolysis via ONC212 and 2-DG has a therapeutic window and should be explored further as a potential combination therapy. In addition, if this effect holds true across other models and cancer types, baseline mitoATP percentage could be used as a functional biomarker of sensitivity to ONC212. Citation Format: Anna Donovan Louie, Isacco Ferrarini, Lanlan Zhou, Wafik S. El-Deiry. ONC212-induced impairment of oxidative phosphorylation is synergistic with glycolysis inhibition in treatment of pancreatic cancer in vitro and in vivo [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 2329.

BACKGROUND Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR). METHODS Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses. RESULTS Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci—SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy. CONCLUSIONS Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. Prostate 75:1790–1801, 2015. © 2015 Wiley Periodicals, Inc.

Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR).Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses.Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci-SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy.Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. .

Approximately 30% of prostate cancer (PCa) patients who receive androgen-deprivation therapy experience disease progression, including bone metastasis, in 18-36 months. Clinical diagnosis of this castration resistance is primarily based on a continuous rise in serum PSA levels during therapy. However, the underlying disease progression predates the clinical onset of castration resistance by many months. The clinical challenge is to distinguish indolent vs aggressive cancer for better disease management. During the prostate cancer progression, malignant circulating tumor cells (CTC) shed from the primary site into the bloodstream through epigenetic modifications and an epithelial-to-mesenchymal transition (EMT). Evidence suggests that increased CTCs are present in the blood of castration-resistant PCa patients and can be obtained through routine phlebotomy. EMT changes of CTCs present a great potential for disease prognosis, treatment stratification and subsequent disease monitoring. We have recently established the combined microfiltration-micromanipulation system (CM2S) to enrich prostate CTCs from blood as published in The Prostate. Our preliminary studies based on a relatively small sample size show that CTCs isolated from advanced PCa patients often lose the typical features of prostate epithelial cells and display incremental EMT-related gene expression signatures, higher elasticity and smoother membrane features. Furthermore, incremental expression of these genes and particular nanomechanical features in CTCs are associated with castration-resistant and metastatic PCa. In this study, we would like expand the sample size to clinically develop this methodology. CTCs were isolated from patients' blood (∼15 ml) using microfiltration system (ScreenCell). We analyzed castration-sensitive (CS) and castration-resistant (CR) PCa cells and CTCs using high throughput microfluidic single-cell RT-PCR. The data were subject to hierarchical clustering, violin plot and Ingenuity Pathway Analysis (IPA). Increased expression of EMT genes was found in CR PCa cells and CTCs as compared to CS counterparts. IPA indicated that these EMT genes are closely related to AKT, β-catenin, Myc and NFκB pathways. Some of CTCs isolated from patients were subject to nanomechanical and nanochemical analysis using PeakForce QNM Catalyst atomic force microscopy (AFM) (Bruker). CTCs of CR patients are about 3 fold more elastic and 7 fold more adherent than CTCs of CS patients. Additionally, CTCs of CR patients are about 3 fold more deformable than CTCs of CS patients. In conclusion, we confirmed our previous study with a larger patient sample size. The increased expression of EMT-related genes and nanomechanical and nanochemical phenotypes in CTCs are potential biomarkers for detection of the castration-resistant PCa, treatment stratification and subsequent disease monitoring. Citation Format: Chun-Liang Chen, Pawel Osmulski, Devalingam Mahalingam, Aaron M. Horning, Rohit R. Jadhav, Anna D. Louie, Chiou-Miin Wang, Tim H.-M. Huang. Epithelial-to-mesenchymal markers of circulating tumor cells for detection of castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5588. doi:10.1158/1538-7445.AM2014-5588