Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Crosstalk between cellular metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to degenerative disease, including cancer. Here, we investigated whether maintenance of circadian rhythms depends upon specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to overall levels of a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function in an in vitro mouse model of pancreatic adenocarcinoma. Metabolic profiling of a library of congenic tumor cell clones revealed significant differences in levels of lactate, pyruvate, ATP, and other crucial metabolites that we used to identify candidate clones with which to generate circadian reporter lines. Despite the shared genetic background of the clones, we observed diverse circadian profiles among these lines that varied with their metabolic phenotype: the most hypometabolic line had the strongest circadian rhythms while the most hypermetabolic line had the weakest rhythms. Treatment of these tumor cell lines with bezafibrate, a peroxisome proliferator-activated receptor (PPAR) agonist shown to increase OxPhos, decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, treatment with the Complex I antagonist rotenone enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function, and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Intratumoral Administration of High-Concentration Nitric Oxide and Anti-mPD-1 Treatment Improves Tumor Regression Rates and Survival in CT26 Tumor-Bearing Mice.

BACKGROUND: Immune checkpoint inhibitors have transformed clinical oncology. However, their use is limited as response is observed in only ~20-50% of patients. Previously, we demonstrated that treating CT26 tumor-bearing mice with ultra-high-concentration gaseous nitric oxide (UNO) followed by tumor resection stimulated antitumor immune responses. Accordingly, UNO may improve tumor response to immune checkpoint inhibitors. Here, we investigated the ability of UNO to improve the efficacy of a programmed cell death protein-1 (PD-1) antibody in vitro and in treating CT26 tumor-bearing mice. METHODS: CT26 cells were injected into the flank of Balb/c mice (n = 15-16 per group). On day 6, CT26 cells were injected into the contralateral flank, and anti-mPD-1 injections commenced. Primary tumors were treated with intratumoral UNO on day 8. Tumor volume, response rates, toxicity, and survival were monitored. RESULTS: (1) Short exposure to 25,000-100,000 parts per million (ppm) UNO in vitro resulted in significant upregulation of PD-L1 expression on CT26 cells. (2) UNO treatment in vivo consistently reduced cell viability in CT26 tumors. (3) Treatment reduced regulatory T-cell (Treg) levels in the tumor and increased levels of systemic M1 macrophages. UNO responders had increased CD8+ T-cell tumor infiltration. (4) Nine days after treatment, primary tumor growth was significantly lower in the combination arm vs. anti-mPD-1 alone (p = 0.0005). (5) Complete tumor regression occurred in 8/15 (53%) of mice treated with a combination of 10 min UNO and anti-mPD-1, 100 days post-treatment, compared to 4/16 (25%) of controls treated with anti-mPD-1 alone (p = 0.1489). (6) There was no toxicity associated with UNO treatment. (7) Combination treatment showed a trend toward increased survival 100 days post-treatment compared to anti-mPD-1 alone (p = 0.0653). CONCLUSION: Combining high-concentration NO and immune checkpoint inhibitors warrants further assessment especially in tumors resistant to checkpoint inhibitor therapy.

SWIP-a stabilized window for intravital imaging of the murine pancreas.

Pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are grave illnesses with high levels of morbidity and mortality. Intravital imaging (IVI) is a powerful technique for visualizing physiological processes in both health and disease. However, the application of IVI to the murine pancreas presents significant challenges, as it is a deep, compliant, visceral organ that is difficult to access, easily damaged and susceptible to motion artefacts. Existing imaging windows for stabilizing the pancreas during IVI have unfortunately shown poor stability for time-lapsed imaging on the minutes to hours scale, or are unable to accommodate both the healthy and tumour-bearing pancreata. To address these issues, we developed an improved stabilized window for intravital imaging of the pancreas (SWIP), which can be applied to not only the healthy pancreas but also to solid tumours like PDAC. Here, we validate the SWIP and use it to visualize a variety of processes for the first time, including (1) single-cell dynamics within the healthy pancreas, (2) transformation from healthy pancreas to acute pancreatitis induced by cerulein, and (3) the physiology of PDAC in both autochthonous and orthotopically injected models. SWIP can not only improve the imaging stability but also expand the application of IVI in both benign and malignant pancreas diseases.

Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer.

Solid tumors possess heterogeneous metabolic microenvironments where oxygen and nutrient availability are plentiful (fertile regions) or scarce (arid regions). While cancer cells residing in fertile regions proliferate rapidly, most cancer cells in vivo reside in arid regions and exhibit a slow-cycling state that renders them chemoresistant. Here, we developed an in vitro system enabling systematic comparison between these populations via transcriptome analysis, metabolomic profiling, and whole-genome CRISPR screening. Metabolic deprivation led to pronounced transcriptional and metabolic reprogramming, resulting in decreased anabolic activities and distinct vulnerabilities. Reductions in anabolic, energy-consuming activities, particularly cell proliferation, were not simply byproducts of the metabolic challenge, but rather essential adaptations. Mechanistically, Bcl-xL played a central role in the adaptation to nutrient and oxygen deprivation. In this setting, Bcl-xL protected quiescent cells from the lethal effects of cell-cycle entry in the absence of adequate nutrients. Moreover, inhibition of Bcl-xL combined with traditional chemotherapy had a synergistic antitumor effect that targeted cycling cells. Bcl-xL expression was strongly associated with poor patient survival despite being confined to the slow-cycling fraction of human pancreatic cancer cells. These findings provide a rationale for combining traditional cancer therapies that target rapidly cycling cells with those that target quiescent, chemoresistant cells associated with nutrient and oxygen deprivation. SIGNIFICANCE: The majority of pancreatic cancer cells inhabit nutrient- and oxygen-poor tumor regions and require Bcl-xL for their survival, providing a compelling antitumor metabolic strategy.

MYC Hyperactivates Wnt Signaling in APC/CTNNB1-Mutated Colorectal Cancer Cells through miR-92a-Dependent Repression of DKK3.

Activation of Wnt signaling is among the earliest events in colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding ß-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations affecting APC, a major ß-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct ß-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that MYC suppresses DKK3 transcription through one of miR-17-92 cluster miRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in Apc-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration/invasion in vitro and metastasis in a syngeneic orthotopic mouse colon cancer model. IMPLICATIONS: Our results suggest that the MYC→miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feed-forward oncogenic loop.

Dissecting phenotypic transitions in metastatic disease via photoconversion-based isolation.

Cancer patients often harbor occult metastases, a potential source of relapse that is targetable only through systemic therapy. Studies of this occult fraction have been limited by a lack of tools with which to isolate discrete cells on spatial grounds. We developed PIC-IT, a photoconversion-based isolation technique allowing efficient recovery of cell clusters of any size - including single-metastatic cells - which are largely inaccessible otherwise. In a murine pancreatic cancer model, transcriptional profiling of spontaneously arising microcolonies revealed phenotypic heterogeneity, functionally reduced propensity to proliferate and enrichment for an inflammatory-response phenotype associated with NF-κB/AP-1 signaling. Pharmacological inhibition of NF-κB depleted microcolonies but had no effect on macrometastases, suggesting microcolonies are particularly dependent on this pathway. PIC-IT thus enables systematic investigation of metastatic heterogeneity. Moreover, the technique can be applied to other biological systems in which isolation and characterization of spatially distinct cell populations is not currently feasible.

Epigenetic and Transcriptional Control of the Epidermal Growth Factor Receptor Regulates the Tumor Immune Microenvironment in Pancreatic Cancer.

Although immunotherapy has revolutionized cancer care, patients with pancreatic ductal adenocarcinoma (PDA) rarely respond to these treatments, a failure that is attributed to poor infiltration and activation of T cells in the tumor microenvironment (TME). We performed an in vivo CRISPR screen and identified lysine demethylase 3A (KDM3A) as a potent epigenetic regulator of immunotherapy response in PDA. Mechanistically, KDM3A acts through Krueppel-like factor 5 (KLF5) and SMAD family member 4 (SMAD4) to regulate the expression of the epidermal growth factor receptor (EGFR). Ablation of KDM3A, KLF5, SMAD4, or EGFR in tumor cells altered the immune TME and sensitized tumors to combination immunotherapy, whereas treatment of established tumors with an EGFR inhibitor, erlotinib, prompted a dose-dependent increase in intratumoral T cells. This study defines an epigenetic-transcriptional mechanism by which tumor cells modulate their immune microenvironment and highlights the potential of EGFR inhibitors as immunotherapy sensitizers in PDA. SIGNIFICANCE: PDA remains refractory to immunotherapies. Here, we performed an in vivo CRISPR screen and identified an epigenetic-transcriptional network that regulates antitumor immunity by converging on EGFR. Pharmacologic inhibition of EGFR is sufficient to rewire the immune microenvironment. These results offer a readily accessible immunotherapy-sensitizing strategy for PDA.This article is highlighted in the In This Issue feature, p. 521.

Global Regulation of the Histone Mark H3K36me2 Underlies Epithelial Plasticity and Metastatic Progression.

Epithelial plasticity, reversible modulation of a cell's epithelial and mesenchymal features, is associated with tumor metastasis and chemoresistance, leading causes of cancer mortality. Although different master transcription factors and epigenetic modifiers have been implicated in this process in various contexts, the extent to which a unifying, generalized mechanism of transcriptional regulation underlies epithelial plasticity remains largely unknown. Here, through targeted CRISPR/Cas9 screening, we discovered two histone-modifying enzymes involved in the writing and erasing of H3K36me2 that act reciprocally to regulate epithelial-to-mesenchymal identity, tumor differentiation, and metastasis. Using a lysine-to-methionine histone mutant to directly inhibit H3K36me2, we found that global modulation of the mark is a conserved mechanism underlying the mesenchymal state in various contexts. Mechanistically, regulation of H3K36me2 reprograms enhancers associated with master regulators of epithelial-to-mesenchymal state. Our results thus outline a unifying epigenome-scale mechanism by which a specific histone modification regulates cellular plasticity and metastasis in cancer. SIGNIFICANCE: Although epithelial plasticity contributes to cancer metastasis and chemoresistance, no strategies exist for pharmacologically inhibiting the process. Here, we show that global regulation of a specific histone mark, H3K36me2, is a universal epigenome-wide mechanism that underlies epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition in carcinoma cells. These results offer a new strategy for targeting epithelial plasticity in cancer.This article is highlighted in the In This Issue feature, p. 747.

Tumor cell-intrinsic EPHA2 suppresses anti-tumor immunity by regulating PTGS2 (COX-2).

Resistance to immunotherapy is one of the biggest problems of current oncotherapeutics. WhileT cell abundance is essential for tumor responsiveness to immunotherapy, factors that define the T cell inflamed tumor microenvironment are not fully understood. We conducted an unbiased approach to identify tumor-intrinsic mechanisms shaping the immune tumor microenvironment(TME), focusing on pancreatic adenocarcinoma because it is refractory to immunotherapy and excludes T cells from the TME. From human tumors, we identified EPHA2 as a candidate tumor intrinsic driver of immunosuppression. Epha2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy. We found that PTGS2, the gene encoding cyclooxygenase-2, lies downstream of EPHA2 signaling through TGFß and is associated with poor patient survival. Ptgs2 deletion reversed T cell exclusion and sensitized tumors to immunotherapy; pharmacological inhibition of PTGS2 was similarly effective. Thus, EPHA2-PTGS2 signaling in tumor cells regulates tumor immune phenotypes; blockade may represent a novel therapeutic avenue for immunotherapy-refractory cancers. Our findings warrant clinical trials testing the effectiveness of therapies combining EPHA2-TGFß-PTGS2 pathway inhibitors with anti-tumor immunotherapy, and may change the treatment of notoriously therapy-resistant pancreatic adenocarcinoma.

Oncogenic Notch Promotes Long-Range Regulatory Interactions within Hyperconnected 3D Cliques.

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

Acetyl-CoA Metabolism Supports Multistep Pancreatic Tumorigenesis.

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis, and new strategies for prevention and treatment are urgently needed. We previously reported that histone H4 acetylation is elevated in pancreatic acinar cells harboring Kras mutations prior to the appearance of premalignant lesions. Because acetyl-CoA abundance regulates global histone acetylation, we hypothesized that altered acetyl-CoA metabolism might contribute to metabolic or epigenetic alterations that promote tumorigenesis. We found that acetyl-CoA abundance is elevated in KRAS-mutant acinar cells and that its use in the mevalonate pathway supports acinar-to-ductal metaplasia (ADM). Pancreas-specific loss of the acetyl-CoA-producing enzyme ATP-citrate lyase (ACLY) accordingly suppresses ADM and tumor formation. In PDA cells, growth factors promote AKT-ACLY signaling and histone acetylation, and both cell proliferation and tumor growth can be suppressed by concurrent BET inhibition and statin treatment. Thus, KRAS-driven metabolic alterations promote acinar cell plasticity and tumor development, and targeting acetyl-CoA-dependent processes exerts anticancer effects. SIGNIFICANCE: Pancreatic cancer is among the deadliest of human malignancies. We identify a key role for the metabolic enzyme ACLY, which produces acetyl-CoA, in pancreatic carcinogenesis. The data suggest that acetyl-CoA use for histone acetylation and in the mevalonate pathway facilitates cell plasticity and proliferation, suggesting potential to target these pathways.See related commentary by Halbrook et al., p. 326.This article is highlighted in the In This Issue feature, p. 305.

Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy.

The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

Human embryonic stem cells exhibit increased propensity to differentiate during the G1 phase prior to phosphorylation of retinoblastoma protein.

While experimentally induced arrest of human embryonic stem cells (hESCs) in G1 has been shown to stimulate differentiation, it remains unclear whether the unperturbed G1 phase in hESCs is causally related to differentiation. Here, we use centrifugal elutriation to isolate and investigate differentiation propensities of hESCs in different phases of their cell cycle. We found that isolated G1 cells exhibit higher differentiation propensity compared with S and G2 cells, and they differentiate at low cell densities even under self-renewing conditions. This differentiation of G1 cells was partially prevented in dense cultures of these cells and completely abrogated in coculture with S and G2 cells. However, coculturing without cell-to-cell contact did not rescue the differentiation of G1 cells. Finally, we show that the subset of G1 hESCs with reduced phosphorylation of retinoblastoma has the highest propensity to differentiate and that the differentiation is preceded by cell cycle arrest. These results provide direct evidence for increased propensity of hESCs to differentiate in G1 and suggest a role for neighboring cells in preventing differentiation of hESCs as they pass through a differentiation sensitive, G1 phase.

Intra-arterial injection of human embryonic stem cells in athymic rat hind limb ischemia model leads to arteriogenesis.

UNLABELLED: Shear stress can enhance differentiation of human embryonic stem cells (hESC) to vascular cells. We tested the hypothesis that intra-arterial hESC injection will lead to arteriogenesis while intramuscular injection will have no effect on vascularization. METHODS AND RESULTS: The superficial femoral arteries were excised on both hind limbs in athymic rats. hESC (2×10(6)) were injected intra-arterially (shear stress) or intramuscular (no shear stress) in one limb after arterial excision. Blood flow, muscle perfusion, and number of arteries/mm(2) muscle were studied at 10 and 21 days after injection. Blood flow in the common iliac artery improved significantly at 10 days after intra-arterial injection of hESC (22±9%, P<.02), and tight muscle perfusion improved significantly both at 10 and 21 days (9±2%, 16±5% respectively, both P<.02). In comparison, intramuscular injection of hESC did not affect blood flow at 10 and 21 days (-3±10% and 4±6%, respectively), while perfusion showed no significant effect of hESC injection after 10 days (1±8%) and was increased 21 days after hESC injection (11±5%, P=.03). Arterial density did not improve after intra-arterial hESC injection at 10 days (15±13%, P=.15) and significantly improved at 21 days (13±4%, P<.05). No significant change was demonstrated after intramuscular injection. SUMMARY: Intra-arterial injection of hESC resulted in moderate improvement of flow and perfusion and increased number of arteries in the ischemic hind limb. No consistent change in perfusion, flow, and number of arteries was observed after intramuscular injection.