Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

TCF-1 controls Treg cell functions that regulate inflammation, CD8+ T cell cytotoxicity and severity of colon cancer.

The transcription factor TCF-1 is essential for the development and function of regulatory T (Treg) cells; however, its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-sequencing analysis identified effector memory T cells and central memory Treg cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg cell transcriptional signature, but promoted alternative signaling pathways whereby Treg cells became activated and gained gut-homing properties and characteristics of the TH17 subset of helper T cells. TCF-1-deficient Treg cells strongly suppressed T cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of patients with colorectal cancer showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T cell cytotoxicity, and can determine colorectal cancer outcome.

NF-κB-inducing kinase maintains T cell metabolic fitness in antitumor immunity.

Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.

Distinct metabolic programs established in the thymus control effector functions of γδ T cell subsets in tumor microenvironments.

Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-γ+ γδ T cells were almost exclusively dependent on glycolysis, IL-17+ γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17+ γδ T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-γ+ γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy.

A Vulnerability of a Subset of Colon Cancers with Potential Clinical Utility.

BRAF(V600E) mutant colon cancers (CCs) have a characteristic gene expression signature that is also found in some tumors lacking this mutation. Collectively, they are referred to as "BRAF-like" tumors and represent some 20% of CCs. We used a shRNA-based genetic screen focused on genes upregulated in BRAF(V600E) CCs to identify vulnerabilities of this tumor subtype that might be exploited therapeutically. Here, we identify RANBP2 (also known as NUP358) as essential for survival of BRAF-like, but not for non-BRAF-like, CC cells. Suppression of RANBP2 results in mitotic defects only in BRAF-like CC cells, leading to cell death. Mechanistically, RANBP2 silencing reduces microtubule outgrowth from the kinetochores, thereby inducing spindle perturbations, providing an explanation for the observed mitotic defects. We find that BRAF-like CCs display far greater sensitivity to the microtubule poison vinorelbine both in vitro and in vivo, suggesting that vinorelbine is a potential tailored treatment for BRAF-like CCs.

Mutant KRAS Enhances Tumor Cell Fitness by Upregulating Stress Granules.

There is growing evidence that stress-coping mechanisms represent tumor cell vulnerabilities that may function as therapeutically beneficial targets. Recent work has delineated an integrated stress adaptation mechanism that is characterized by the formation of cytoplasmic mRNA and protein foci, termed stress granules (SGs). Here, we demonstrate that SGs are markedly elevated in mutant KRAS cells following exposure to stress-inducing stimuli. The upregulation of SGs by mutant KRAS is dependent on the production of the signaling lipid molecule 15-deoxy-delta 12,14 prostaglandin J2 (15-d-PGJ2) and confers cytoprotection against stress stimuli and chemotherapeutic agents. The secretion of 15-d-PGJ2 by mutant KRAS cells is sufficient to enhance SG formation and stress resistance in cancer cells that are wild-type for KRAS. Our findings identify a mutant KRAS-dependent cell non-autonomous mechanism that may afford the establishment of a stress-resistant niche that encompasses different tumor subclones. These results should inform the design of strategies to eradicate tumor cell communities.

An essential mesenchymal function for miR-143/145 in intestinal epithelial regeneration.

Downregulation of the miR-143/145 microRNA (miRNA) cluster has been repeatedly reported in colon cancer and other epithelial tumors. In addition, overexpression of these miRNAs inhibits tumorigenesis, leading to broad consensus that they function as cell-autonomous epithelial tumor suppressors. We generated mice with deletion of miR-143/145 to investigate the functions of these miRNAs in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in the absence of these miRNAs, epithelial regeneration after injury was dramatically impaired. Surprisingly, we found that miR-143/145 are expressed and function exclusively within the mesenchymal compartment of intestine. Defective epithelial regeneration in miR-143/145-deficient mice resulted from the dysfunction of smooth muscle and myofibroblasts and was associated with derepression of the miR-143 target Igfbp5, which impaired IGF signaling after epithelial injury. These results provide important insights into the regulation of epithelial wound healing and argue against a cell-autonomous tumor suppressor role for miR-143/145 in colon cancer.

Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties.

Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.

Cell-matrix interface regulates dormancy in human colon cancer stem cells.

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.

Neoadjuvant Immunotherapy in Locally Advanced Mismatch Repair-Deficient Colon Cancer.

BACKGROUND: Mismatch repair-deficient (dMMR) tumors can be found in 10 to 15% of patients with nonmetastatic colon cancer. In these patients, the efficacy of chemotherapy is limited. The use of neoadjuvant immunotherapy has shown promising results, but data from studies of this approach are limited. METHODS: We conducted a phase 2 study in which patients with nonmetastatic, locally advanced, previously untreated dMMR colon cancer were treated with neoadjuvant nivolumab plus ipilimumab. The two primary end points were safety, defined by timely surgery (i.e., ≤2-week delay of planned surgery owing to treatment-related toxic events), and 3-year disease-free survival. Secondary end points included pathological response and results of genomic analyses. RESULTS: Of 115 enrolled patients, 113 (98%; 97.5% confidence interval [CI], 93 to 100) underwent timely surgery; 2 patients had surgery delayed by more than 2 weeks. Grade 3 or 4 immune-related adverse events occurred in 5 patients (4%), and none of the patients discontinued treatment because of adverse events. Among the 111 patients included in the efficacy analysis, a pathological response was observed in 109 (98%; 95% CI, 94 to 100), including 105 (95%) with a major pathological response (defined as ≤10% residual viable tumor) and 75 (68%) with a pathological complete response (0% residual viable tumor). With a median follow-up of 26 months (range, 9 to 65), no patients have had recurrence of disease. CONCLUSIONS: In patients with locally advanced dMMR colon cancer, neoadjuvant nivolumab plus ipilimumab had an acceptable safety profile and led to a pathological response in a high proportion of patients. (Funded by Bristol Myers Squibb; NICHE-2 ClinicalTrials.gov number, NCT03026140.).

ß-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.

Wnt/ß-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic ß-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of ß-catenin in transformation, we classified ß-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that ß-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with ß-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of ß-catenin-dependent cancers in both cell lines and animal models. These observations define a ß-catenin-YAP1-TBX5 complex essential to the transformation and survival of ß-catenin-driven cancers.

RIPK1 and Caspase-8 Ensure Chromosome Stability Independently of Their Role in Cell Death and Inflammation.

Receptor-interacting protein kinase (RIPK) 1 functions as a key mediator of tissue homeostasis via formation of Caspase-8 activating ripoptosome complexes, positively and negatively regulating apoptosis, necroptosis, and inflammation. Here, we report an unanticipated cell-death- and inflammation-independent function of RIPK1 and Caspase-8, promoting faithful chromosome alignment in mitosis and thereby ensuring genome stability. We find that ripoptosome complexes progressively form as cells enter mitosis, peaking at metaphase and disassembling as cells exit mitosis. Genetic deletion and mitosis-specific inhibition of Ripk1 or Caspase-8 results in chromosome alignment defects independently of MLKL. We found that Polo-like kinase 1 (PLK1) is recruited into mitotic ripoptosomes, where PLK1's activity is controlled via RIPK1-dependent recruitment and Caspase-8-mediated cleavage. A fine balance of ripoptosome assembly is required as deregulated ripoptosome activity modulates PLK1-dependent phosphorylation of downstream effectors, such as BUBR1. Our data suggest that ripoptosome-mediated regulation of PLK1 contributes to faithful chromosome segregation during mitosis.

Memory effects of prior subculture may impact the quality of multiomic perturbation profiles.

Mass spectrometry-based omics technologies are increasingly used in perturbation studies to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by fold change and variation of each molecular parameter, but also by multiple testing corrections. While the fold change is largely determined by the biological system, the variation is determined by experimental workflows. Here, it is shown that memory effects of prior subculture can influence the variation of perturbation profiles using the two colon carcinoma cell lines SW480 and HCT116. These memory effects are largely driven by differences in growth states that persist into the perturbation experiment. In SW480 cells, memory effects combined with moderate treatment effects amplify the variation in multiple omics levels, including eicosadomics, proteomics, and phosphoproteomics. With stronger treatment effects, the memory effect was less pronounced, as demonstrated in HCT116 cells. Subculture homogeneity was controlled by real-time monitoring of cell growth. Controlled homogeneous subculture resulted in a perturbation network of 321 causal conjectures based on combined proteomic and phosphoproteomic data, compared to only 58 causal conjectures without controlling subculture homogeneity in SW480 cells. Some cellular responses and regulatory events were identified that extend the mode of action of arsenic trioxide (ATO) only when accounting for these memory effects. Controlled prior subculture led to the finding of a synergistic combination treatment of ATO with the thioredoxin reductase 1 inhibitor auranofin, which may prove useful in the management of NRF2-mediated resistance mechanisms.

IL-6 promotes tumor growth through immune evasion but is dispensable for cachexia.

Various cytokines have been implicated in cancer cachexia. One such cytokine is IL-6, deemed as a key cachectic factor in mice inoculated with colon carcinoma 26 (C26) cells, a widely used cancer cachexia model. Here we tested the causal role of IL-6 in cancer cachexia by knocking out the IL-6 gene in C26 cells. We found that the growth of IL-6 KO tumors was dramatically delayed. More strikingly, while IL-6 KO tumors eventually reached the similar size as wild-type tumors, cachexia still took place, despite no elevation in circulating IL-6. In addition, the knockout of leukemia inhibitory factor (LIF), another IL-6 family cytokine proposed as a cachectic factor in the model, also affected tumor growth but not cachexia. We further showed an increase in the infiltration of immune cell population in the IL-6 KO tumors compared with wild-type controls and the defective IL-6 KO tumor growth was rescued in immunodeficient mice while cachexia was not. Thus, IL-6 promotes tumor growth by facilitating immune evasion but is dispensable for cachexia.

Lin28A and Lin28B inhibit let-7 microRNA biogenesis by distinct mechanisms.

Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUT4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a Zcchc11-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts are restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors, whereas Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function and have implications for the development of new strategies for cancer therapy.

Immune receptor inhibition through enforced phosphatase recruitment.

Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

Biodegradable lipophilic polymeric mRNA nanoparticles for ligand-free targeting of splenic dendritic cells for cancer vaccination.

Nanoparticle (NP)-based mRNA cancer vaccines hold great promise to realize personalized cancer treatments. To advance this technology requires delivery formulations for efficient intracellular delivery to antigen-presenting cells. We developed a class of bioreducible lipophilic poly(beta-amino ester) nanocarriers with quadpolymer architecture. The platform is agnostic to the mRNA sequence, with one-step self-assembly allowing for delivery of multiple antigen-encoding mRNAs as well as codelivery of nucleic acid-based adjuvants. We examined structure-function relationships for NP-mediated mRNA delivery to dendritic cells (DCs) and identified that a lipid subunit of the polymer structure was critical. Following intravenous administration, the engineered NP design facilitated targeted delivery to the spleen and preferential transfection of DCs without the need for surface functionalization with targeting ligands. Treatment with engineered NPs codelivering antigen-encoding mRNA and toll-like receptor agonist adjuvants led to robust antigen-specific CD8+ T cell responses, resulting in efficient antitumor therapy in in vivo models of murine melanoma and colon adenocarcinoma.

ATXN3 functions as a tumor suppressor through potentiating galectin-9-mediated apoptosis in human colon adenocarcinoma.

While deubiquitinase ATXN3 has been implicated as a potential oncogene in various types of human cancers, its role in colon adenocarcinoma remains understudied. Surprisingly, our findings demonstrate that ATXN3 exerts an antitumor effect in human colon cancers through potentiating Galectin-9-induced apoptosis. CRISPR-mediated ATXN3 deletion unexpectedly intensified colon cancer growth both in vitro and in xenograft colon cancers. At the molecular level, we identified ATXN3 as a bona fide deubiquitinase specifically targeting Galectin-9, as ATXN3 interacted with and inhibited Galectin-9 ubiquitination. Consequently, targeted ATXN3 ablation resulted in reduced Galectin-9 protein expression, thereby diminishing Galectin-9-induced colon cancer apoptosis and cell growth arrest. The ectopic expression of Galectin-9 fully reversed the growth of ATXN3-null colon cancer in mice. Furthermore, immunohistochemistry staining revealed a significant reduction in both ATXN3 and Galectin-9 protein expression, along with a positive correlation between them in human colon cancer. Our study identifies the first Galectin-9-specific deubiquitinase and unveils a tumor-suppressive role of ATXN3 in human colon cancer.

Circulating Tumor DNA Analysis Guiding Adjuvant Therapy in Stage II Colon Cancer.

BACKGROUND: The role of adjuvant chemotherapy in stage II colon cancer continues to be debated. The presence of circulating tumor DNA (ctDNA) after surgery predicts very poor recurrence-free survival, whereas its absence predicts a low risk of recurrence. The benefit of adjuvant chemotherapy for ctDNA-positive patients is not well understood. METHODS: We conducted a trial to assess whether a ctDNA-guided approach could reduce the use of adjuvant chemotherapy without compromising recurrence risk. Patients with stage II colon cancer were randomly assigned in a 2:1 ratio to have treatment decisions guided by either ctDNA results or standard clinicopathological features. For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery prompted oxaliplatin-based or fluoropyrimidine chemotherapy. Patients who were ctDNA-negative were not treated. The primary efficacy end point was recurrence-free survival at 2 years. A key secondary end point was adjuvant chemotherapy use. RESULTS: Of the 455 patients who underwent randomization, 302 were assigned to ctDNA-guided management and 153 to standard management. The median follow-up was 37 months. A lower percentage of patients in the ctDNA-guided group than in the standard-management group received adjuvant chemotherapy (15% vs. 28%; relative risk, 1.82; 95% confidence interval [CI], 1.25 to 2.65). In the evaluation of 2-year recurrence-free survival, ctDNA-guided management was noninferior to standard management (93.5% and 92.4%, respectively; absolute difference, 1.1 percentage points; 95% CI, -4.1 to 6.2 [noninferiority margin, -8.5 percentage points]). Three-year recurrence-free survival was 86.4% among ctDNA-positive patients who received adjuvant chemotherapy and 92.5% among ctDNA-negative patients who did not. CONCLUSIONS: A ctDNA-guided approach to the treatment of stage II colon cancer reduced adjuvant chemotherapy use without compromising recurrence-free survival. (Supported by the Australian National Health and Medical Research Council and others; DYNAMIC Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583.).