Embracing cancer complexity: Hallmarks of systemic disease.

The last 50 years have witnessed extraordinary developments in understanding mechanisms of carcinogenesis, synthesized as the hallmarks of cancer. Despite this logical framework, our understanding of the molecular basis of systemic manifestations and the underlying causes of cancer-related death remains incomplete. Looking forward, elucidating how tumors interact with distant organs and how multifaceted environmental and physiological parameters impinge on tumors and their hosts will be crucial for advances in preventing and more effectively treating human cancers. In this perspective, we discuss complexities of cancer as a systemic disease, including tumor initiation and promotion, tumor micro- and immune macro-environments, aging, metabolism and obesity, cancer cachexia, circadian rhythms, nervous system interactions, tumor-related thrombosis, and the microbiome. Model systems incorporating human genetic variation will be essential to decipher the mechanistic basis of these phenomena and unravel gene-environment interactions, providing a modern synthesis of molecular oncology that is primed to prevent cancers and improve patient quality of life and cancer outcomes.

A Multivalent Peptoid Conjugate Modulates Androgen Receptor Transcriptional Activity to Inhibit Therapy-resistant Prostate Cancer.

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance due to ongoing AR expression and function. To counter this, we developed a new approach to modulate the AR and inhibit castration-resistant prostate cancer (CRPC) using multivalent peptoid conjugates (MPC) that contain multiple copies of the AR-targeting ligand ethisterone attached to a peptidomimetic scaffold. Here, we investigated the antitumor effects of compound MPC309, a trivalent display of ethisterone conjugated to a peptoid oligomer backbone that binds to the AR with nanomolar affinity. MPC309 exhibited potent antiproliferative effects on various enzalutamide-resistant prostate cancer models, including those with AR splice variants, ligand-binding mutations, and noncanonical AR gene expression programs, as well as mouse prostate organoids harboring defined genetic alterations that mimic lethal human prostate cancer subtypes. MPC309 is taken up by cells through macropinocytosis, an endocytic process more prevalent in cancer cells than in normal ones, thus providing an opportunity to target tumors selectively. MPC309 triggers a distinct AR transcriptome compared with DHT and enzalutamide, a clinically used antiandrogen. Specifically, MPC309 enhances the expression of differentiation genes while reducing the expression of genes needed for cell division and metabolism. Mechanistically, MPC309 increases AR chromatin occupancy and alters AR interactions with coregulatory proteins in a pattern distinct from DHT. In xenograft studies, MPC309 produced significantly greater tumor suppression than enzalutamide. Altogether, MPC309 represents a promising new AR modulator that can combat resistant disease by promoting an AR antiproliferative gene expression program.

Cytoskeletal association of ATP citrate lyase controls the mechanodynamics of macropinocytosis.

Macropinocytosis is an actin-dependent mode of nonselective endocytosis that mediates the uptake of extracellular fluid-phase cargoes. It is now well recognized that tumor cells exploit macropinocytosis to internalize macromolecules that can be catabolized and used to support cell growth and proliferation under nutrient-limiting conditions. Therefore, the identification of molecular mechanisms that control macropinocytosis is fundamental to the understanding of the metabolic adaptive landscape of tumor cells. Here, we report that the acetyl-CoA-producing enzyme, ATP citrate lyase (ACLY), is a key regulator of macropinocytosis and describes a heretofore-unappreciated association of ACLY with the actin cytoskeleton. The cytoskeletal tethering of ACLY is required for the spatially defined acetylation of heterodimeric actin capping protein, which we identify as an essential mediator of the actin remodeling events that drive membrane ruffling and macropinocytosis. Furthermore, we identify a requirement for mitochondrial-derived citrate, an ACLY substrate, for macropinocytosis, and show that mitochondria traffic to cell periphery regions juxtaposed to plasma membrane ruffles. Collectively, these findings establish a mode of metabolite compartmentalization that supports the spatiotemporal modulation of membrane-cytoskeletal interactions required for macropinocytosis by coupling regional acetyl-CoA availability with dynamic protein acetylation.

CD73-Dependent Adenosine Signaling through Adora2b Drives Immunosuppression in Ductal Pancreatic Cancer.

The microenvironment that surrounds pancreatic ductal adenocarcinoma (PDAC) is profoundly desmoplastic and immunosuppressive. Understanding triggers of immunosuppression during the process of pancreatic tumorigenesis would aid in establishing targets for effective prevention and therapy. Here, we interrogated differential molecular mechanisms dependent on cell of origin and subtype that promote immunosuppression during PDAC initiation and in established tumors. Transcriptomic analysis of cell-of-origin-dependent epithelial gene signatures revealed that Nt5e/CD73, a cell-surface enzyme required for extracellular adenosine generation, is one of the top 10% of genes overexpressed in murine tumors arising from the ductal pancreatic epithelium as opposed to those rising from acinar cells. These findings were confirmed by IHC and high-performance liquid chromatography. Analysis in human PDAC subtypes indicated that high Nt5e in murine ductal PDAC models overlaps with high NT5E in human PDAC squamous and basal subtypes, considered to have the highest immunosuppression and worst prognosis. Multiplex immunofluorescent analysis showed that activated CD8+ T cells in the PDAC tumor microenvironment express high levels of CD73, indicating an opportunity for immunotherapeutic targeting. Delivery of CD73 small-molecule inhibitors through various delivery routes reduced tumor development and growth in genetically engineered and syngeneic mouse models. In addition, the adenosine receptor Adora2b was a determinant of adenosine-mediated immunosuppression in PDAC. These findings highlight a molecular trigger of the immunosuppressive PDAC microenvironment elevated in the ductal cell of origin, linking biology with subtype classification, critical components for PDAC immunoprevention and personalized approaches for immunotherapeutic intervention. SIGNIFICANCE: Ductal-derived pancreatic tumors have elevated epithelial and CD8+GZM+ T-cell CD73 expression that confers sensitivity to small-molecule inhibition of CD73 or Adora2b to promote CD8+ T-cell-mediated tumor regression. See related commentary by DelGiorno, p. 977.

MACHETE identifies interferon-encompassing chromosome 9p21.3 deletions as mediators of immune evasion and metastasis.

The most prominent homozygous deletions in cancer affect chromosome 9p21.3 and eliminate CDKN2A/B tumor suppressors, disabling a cell-intrinsic barrier to tumorigenesis. Half of 9p21.3 deletions, however, also encompass a type I interferon (IFN) gene cluster; the consequences of this co-deletion remain unexplored. To functionally dissect 9p21.3 and other large genomic deletions, we developed a flexible deletion engineering strategy, MACHETE (molecular alteration of chromosomes with engineered tandem elements). Applying MACHETE to a syngeneic mouse model of pancreatic cancer, we found that co-deletion of the IFN cluster promoted immune evasion, metastasis and immunotherapy resistance. Mechanistically, IFN co-deletion disrupted type I IFN signaling in the tumor microenvironment, leading to marked changes in infiltrating immune cells and escape from CD8+ T-cell surveillance, effects largely driven by the poorly understood interferon epsilon. These results reveal a chromosomal deletion that disables both cell-intrinsic and cell-extrinsic tumor suppression and provide a framework for interrogating large deletions in cancer and beyond.

α2,6-Sialylation Is Upregulated in Severe COVID-19, Implicating the Complement Cascade.

Better understanding of the molecular mechanisms underlying COVID-19 severity is desperately needed in current times. Although hyper-inflammation drives severe COVID-19, precise mechanisms triggering this cascade and what role glycosylation might play therein are unknown. Here we report the first high-throughput glycomic analysis of COVID-19 plasma samples and autopsy tissues. We find that α2,6-sialylation is upregulated in the plasma of patients with severe COVID-19 and in autopsied lung tissue. This glycan motif is enriched on members of the complement cascade (e.g., C5, C9), which show higher levels of sialylation in severe COVID-19. In the lung tissue, we observe increased complement deposition, associated with elevated α2,6-sialylation levels, corresponding to elevated markers of poor prognosis (IL-6) and fibrotic response. We also observe upregulation of the α2,6-sialylation enzyme ST6GAL1 in patients who succumbed to COVID-19. Our work identifies a heretofore undescribed relationship between sialylation and complement in severe COVID-19, potentially informing future therapeutic development.

Exercise-induced engagement of the IL-15/IL-15Rα axis promotes anti-tumor immunity in pancreatic cancer.

Aerobic exercise is associated with decreased cancer incidence and cancer-associated mortality. However, little is known about the effects of exercise on pancreatic ductal adenocarcinoma (PDA), a disease for which current therapeutic options are limited. Herein, we show that aerobic exercise reduces PDA tumor growth, by modulating systemic and intra-tumoral immunity. Mechanistically, exercise promotes immune mobilization and accumulation of tumor-infiltrating IL15Rα+ CD8 T cells, which are responsible for the tumor-protective effects. In clinical samples, an exercise-dependent increase of intra-tumoral CD8 T cells is also observed. Underscoring the translational potential of the interleukin (IL)-15/IL-15Rα axis, IL-15 super-agonist (NIZ985) treatment attenuates tumor growth, prolongs survival, and enhances sensitivity to chemotherapy. Finally, exercise or NIZ985 both sensitize pancreatic tumors to αPD-1, with improved anti-tumor and survival benefits. Collectively, our findings highlight the therapeutic potential of an exercise-oncology axis and identify IL-15 activation as a promising treatment strategy for this deadly disease.

α2,6-Sialylation is Upregulated in Severe COVID-19 Implicating the Complement Cascade.

Better understanding of the mechanisms of COVID-19 severity is desperately needed in current times. Although hyper-inflammation drives severe COVID-19, precise mechanisms triggering this cascade and what role glycosylation might play therein is unknown. Here we report the first high-throughput glycomic analysis of COVID-19 plasma samples and autopsy tissues. We find α2,6-sialylation is upregulated in plasma of patients with severe COVID-19 and in the lung. This glycan motif is enriched on members of the complement cascade, which show higher levels of sialylation in severe COVID-19. In the lung tissue, we observe increased complement deposition, associated with elevated α2,6-sialylation levels, corresponding to elevated markers of poor prognosis (IL-6) and fibrotic response. We also observe upregulation of the α2,6-sialylation enzyme ST6GAL1 in patients who succumbed to COVID-19. Our work identifies a heretofore undescribed relationship between sialylation and complement in severe COVID-19, potentially informing future therapeutic development.

Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia.

Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.

Metabolic reprogramming of tumor-associated macrophages by collagen turnover promotes fibrosis in pancreatic cancer.

A hallmark of pancreatic tumors is their highly desmoplastic stroma composed of fibroblasts, immune cells, and a dense network of collagen fibers. Tumor-associated macrophages are one of the most abundant immune cell populations in the pancreatic tumor stroma. Their protumorigenic function has been attributed predominantly to their capacity to promote immune evasion and metastasis. Tumor-assoc iated macrophages are also well known for their role in the remodeling of the stroma via collagen production and degradation, with the latter being mediated by mannose receptor (MRC1)-dependent endocytosis of collagen. Here we show that MRC1-mediated collagen internalization and subsequent lysosomal degradation by macrophages harboring a tumor-associated phenotype are accompanied by the accumulation of collagen-derived intracellular free amino acids and increased arginine biosynthesis. The resulting increase in intracellular arginine levels leads to the up-regulation of inducible nitric oxide synthase and the production of reactive nitrogen species. Furthermore, reactive nitrogen species derived from internalized and degraded collagen promotes a profibrotic phenotype in pancreatic stellate cells resulting in enhanced intratumoral collagen deposition. Overall, our findings identify a role for extracellular matrix remodeling in the functional modulation of tumor-associated macrophages via metabolic rewiring.

EMSY inhibits homologous recombination repair and the interferon response, promoting lung cancer immune evasion.

Non-small cell lung cancers (NSCLCs) harboring KEAP1 mutations are often resistant to immunotherapy. Here, we show that KEAP1 targets EMSY for ubiquitin-mediated degradation to regulate homologous recombination repair (HRR) and anti-tumor immunity. Loss of KEAP1 in NSCLC induces stabilization of EMSY, producing a BRCAness phenotype, i.e., HRR defects and sensitivity to PARP inhibitors. Defective HRR contributes to a high tumor mutational burden that, in turn, is expected to prompt an innate immune response. Notably, EMSY accumulation suppresses the type I interferon response and impairs innate immune signaling, fostering cancer immune evasion. Activation of the type I interferon response in the tumor microenvironment using a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of KEAP1-mutant tumors. Our results suggest that targeting PARP and STING pathways, individually or in combination, represents a therapeutic strategy in NSCLC patients harboring alterations in KEAP1.

Exploiting cancer's drinking problem: regulation and therapeutic potential of macropinocytosis.

Macropinocytosis, an evolutionarily conserved endocytic mechanism that mediates non-specific fluid-phase uptake, is potently upregulated by various oncogenic pathways. It is now well appreciated that high macropinocytic activity is a hallmark of many human tumors, which use this adaptation to scavenge extracellular nutrients for fueling cell growth. In the context of the nutrient-scarce tumor microenvironment, this process provides tumor cells with metabolic flexibility. However, dependence on this scavenging mechanism also illuminates a potential metabolic vulnerability. As such, there is a great deal of interest in understanding the molecular underpinnings of macropinocytosis. In this review, we will discuss the most recent advances in characterizing macropinocytosis: the pathways that regulate it, its contribution to the metabolic fitness of cancer cells, and its therapeutic potential.

Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Glycans, such as carbohydrate antigen 19-9, are biomarkers of PDAC and are emerging as important modulators of cancer phenotypes. Herein, we used a systems-based approach integrating glycomic analysis of the well-established KC mouse, which models early events in transformation, and analysis of samples from human pancreatic cancer patients to identify glycans with potential roles in cancer formation. We observed both common and distinct patterns of glycosylation in pancreatic cancer across species. Common alterations included increased levels of α-2,3-sialic acid and α-2,6-sialic acid, bisecting GlcNAc and poly-N-acetyllactosamine. However, core fucose, which was increased in human PDAC, was not seen in the mouse, indicating that not all human glycomic changes are observed in the KC mouse model. In silico analysis of bulk and single-cell sequencing data identified ST6 beta-galactoside alpha-2,6-sialyltransferase 1, which underlies α-2,6-sialic acid, as overexpressed in human PDAC, concordant with histological data showing higher levels of this enzyme at the earliest stages. To test whether ST6 beta-galactoside alpha-2,6-sialyltransferase 1 promotes pancreatic cancer, we created a novel mouse in which a pancreas-specific genetic deletion of this enzyme overlays the KC mouse model. The analysis of our new model showed delayed cancer formation and a significant reduction in fibrosis. Our results highlight the importance of a strategic systems approach to identifying glycans whose functions can be modeled in mouse, a crucial step in the development of therapeutics targeting glycosylation in pancreatic cancer.

Gain-of-function p53R172H mutation drives accumulation of neutrophils in pancreatic tumors, promoting resistance to immunotherapy.

Tumor genotype can influence the immune microenvironment, which plays a critical role in cancer development and therapy resistance. However, the immune effects of gain-of-function Trp53 mutations have not been defined in pancreatic cancer. We compare the immune profiles generated by KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDECs) engineered genetically to express the Trp53R172H mutation with their p53 wild-type control. KrasG12D/+;Trp53R172H/+ tumors have a distinct immune profile characterized by an influx of CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 cells. Knockdown of CXCL2, a neutrophil chemokine, in the tumor epithelial compartment of CRISPR KrasG12D/+;Trp53R172H/+ PDEC tumors reverses the neutrophil phenotype. Neutrophil depletion of mice bearing CRISPR KrasG12D/+;Trp53R172H/+ tumors augments sensitivity to combined CD40 immunotherapy and chemotherapy. These data link Trp53R172H to the presence of intratumoral neutrophils in pancreatic cancer and suggest that tumor genotypes could inform selection of affected individuals for immunotherapy.

A novel target for combination immunotherapy in pancreatic cancer: IL-1ß mediates immunosuppression in the tumour microenvironment.

Immune checkpoint blockade (ICB) has demonstrated efficacy in multiple cancers, offering the potential of long-term disease control not achievable with cytotoxic or targeted therapies. However, the field has not yet achieved the crucial next steps - the expansion of the response rate and achievement of clinical efficacy in so-called "cold tumours". Mechanistic studies of tumour-type specific immunosuppressive pathways can reveal underlying biological hurdles to immunotherapy and offer new therapeutic insights. Our finding that tumour-derived IL-1ß mediates immunosuppression in pancreatic cancer has precipitated a new clinical trial.

Macropinocytosis as a Key Determinant of Peptidomimetic Uptake in Cancer Cells.

Peptides and peptidomimetics represent the middle space between small molecules and large proteins-they retain the relatively small size and synthetic accessibility of small molecules while providing high binding specificity for biomolecular partners typically observed with proteins. During the course of our efforts to target intracellular protein-protein interactions in cancer, we observed that the cellular uptake of peptides is critically determined by the cell line-specifically, we noted that peptides show better uptake in cancer cells with enhanced macropinocytic indices. Here, we describe the results of our analysis of cellular penetration by different classes of conformationally stabilized peptides. We tested the uptake of linear peptides, peptide macrocycles, stabilized helices, ß-hairpin peptides, and cross-linked helix dimers in 11 different cell lines. Efficient uptake of these conformationally defined constructs directly correlated with the macropinocytic activity of each cell line: high uptake of compounds was observed in cells with mutations in certain signaling pathways. Significantly, the study shows that constrained peptides follow the same uptake mechanism as proteins in macropinocytic cells, but unlike proteins, peptide mimics can be readily designed to resist denaturation and proteolytic degradation. Our findings expand the current understanding of cellular uptake in cancer cells by designed peptidomimetics and suggest that cancer cells with certain mutations are suitable mediums for the study of biological pathways with peptide leads.

Covalent Targeting of Ras G12C by Rationally Designed Peptidomimetics.

Protein-protein interactions (PPIs) play a critical role in fundamental biological processes. Competitive inhibition of these interfaces requires compounds that can access discontinuous binding epitopes along a large, shallow binding surface area. Conformationally defined protein surface mimics present a viable route to target these interactions. However, the development of minimal protein mimics that engage intracellular targets with high affinity remains a major challenge because mimicry of a portion of the binding interface is often associated with the loss of critical binding interactions. Covalent targeting provides an attractive approach to overcome the loss of noncovalent contacts but have the inherent risk of dominating noncovalent contacts and increasing the likelihood of nonselective binding. Here, we report the iterative design of a proteolytically stable α3ß chimeric helix mimic that covalently targets oncogenic Ras G12C as a model system. We explored several electrophiles to optimize preferential alkylation with the desired C12 on Ras. The designed lead peptide modulates nucleotide exchange, inhibits activation of the Ras-mediated signaling cascade, and is selectively toxic toward mutant Ras G12C cancer cells. The relatively high frequency of acquired cysteines as missense mutations in cancer and other diseases suggests that covalent peptides may offer an untapped therapeutic approach for targeting aberrant protein interactions.

Selective Alanine Transporter Utilization Creates a Targetable Metabolic Niche in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) evolves a complex microenvironment comprised of multiple cell types, including pancreatic stellate cells (PSC). Previous studies have demonstrated that stromal supply of alanine, lipids, and nucleotides supports the metabolism, growth, and therapeutic resistance of PDAC. Here we demonstrate that alanine cross-talk between PSCs and PDAC is orchestrated by the utilization of specific transporters. PSCs utilize SLC1A4 and other transporters to rapidly exchange and maintain environmental alanine concentrations. Moreover, PDAC cells upregulate SLC38A2 to supply their increased alanine demand. Cells lacking SLC38A2 fail to concentrate intracellular alanine and undergo a profound metabolic crisis resulting in markedly impaired tumor growth. Our results demonstrate that stromal-cancer metabolic niches can form through differential transporter expression, creating unique therapeutic opportunities to target metabolic demands of cancer. SIGNIFICANCE: This work identifies critical neutral amino acid transporters involved in channeling alanine between pancreatic stellate and PDAC cells. Targeting PDAC-specific alanine uptake results in a metabolic crisis impairing metabolism, proliferation, and tumor growth. PDAC cells specifically activate and require SLC38A2 to fuel their alanine demands that may be exploited therapeutically.This article is highlighted in the In This Issue feature, p. 890.

Tumor Cell-Derived IL1ß Promotes Desmoplasia and Immune Suppression in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy typified by a highly stromal and weakly immunogenic tumor microenvironment that promotes tumor evolution and contributes to therapeutic resistance. Here, we demonstrate that PDA tumor cell-derived proinflammatory cytokine IL1ß is essential for the establishment of the protumorigenic PDA microenvironment. Tumor cell-derived IL1ß promoted the activation and secretory phenotype of quiescent pancreatic stellate cells and established an immunosuppressive milieu mediated by M2 macrophages, myeloid-derived suppressor cells, CD1dhiCD5+ regulatory B cells, and Th17 cells. Loss of tumor cell-derived IL1 signaling in tumor stroma enabled intratumoral infiltration and activation of CD8+ cytotoxic T cells, attenuated growth of pancreatic neoplasia, and conferred survival advantage to PDA-bearing mice. Accordingly, antibody-mediated neutralization of IL1ß significantly enhanced the antitumor activity of α-PD-1 and was accompanied by increased tumor infiltration of CD8+ T cells. Tumor cell expression of IL1ß in vivo was driven by microbial-dependent activation of toll-like receptor 4 (TLR4) signaling and subsequent engagement of the NLRP3 inflammasome. Collectively, these findings identify a hitherto unappreciated role for tumor cell-derived IL1ß in orchestrating an immune-modulatory program that supports pancreatic tumorigenesis. SIGNIFICANCE: These findings identify a new modality for immune evasion in PDA that depends on IL1ß production by tumor cells through TLR4-NLRP3 inflammasome activation. Targeting this axis might provide an effective PDA therapeutic strategy.