Pathway level subtyping identifies a slow-cycling biological phenotype associated with poor clinical outcomes in colorectal cancer.

Molecular stratification using gene-level transcriptional data has identified subtypes with distinctive genotypic and phenotypic traits, as exemplified by the consensus molecular subtypes (CMS) in colorectal cancer (CRC). Here, rather than gene-level data, we make use of gene ontology and biological activation state information for initial molecular class discovery. In doing so, we defined three pathway-derived subtypes (PDS) in CRC: PDS1 tumors, which are canonical/LGR5+ stem-rich, highly proliferative and display good prognosis; PDS2 tumors, which are regenerative/ANXA1+ stem-rich, with elevated stromal and immune tumor microenvironmental lineages; and PDS3 tumors, which represent a previously overlooked slow-cycling subset of tumors within CMS2 with reduced stem populations and increased differentiated lineages, particularly enterocytes and enteroendocrine cells, yet display the worst prognosis in locally advanced disease. These PDS3 phenotypic traits are evident across numerous bulk and single-cell datasets, and demark a series of subtle biological states that are currently under-represented in pre-clinical models and are not identified using existing subtyping classifiers.

Targeting stromal cell sialylation reverses T cell-mediated immunosuppression in the tumor microenvironment.

Immunosuppressive tumor microenvironments (TMEs) reduce the effectiveness of immune responses in cancer. Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote tumor progression by enhancing immune cell suppression in colorectal cancer (CRC). Hyper-sialylation of glycans promotes immune evasion in cancer through binding of sialic acids to their receptors, Siglecs, expressed on immune cells, which results in inhibition of effector functions. The role of sialylation in shaping MSC/CAF immunosuppression in the TME is not well characterized. In this study, we show that tumor-conditioned stromal cells have increased sialyltransferase expression, α2,3/6-linked sialic acid, and Siglec ligands. Tumor-conditioned stromal cells and CAFs induce exhausted immunomodulatory CD8+ PD1+ and CD8+ Siglec-7+/Siglec-9+ T cell phenotypes. In vivo, targeting stromal cell sialylation reverses stromal cell-mediated immunosuppression, as shown by infiltration of CD25 and granzyme B-expressing CD8+ T cells in the tumor and draining lymph node. Targeting stromal cell sialylation may overcome immunosuppression in the CRC TME.

Epithelial TGFß engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features.

The pro-tumourigenic role of epithelial TGFß signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFß signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFß signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFß signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFß signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC's with born to be bad traits.

Rerouting the drug response: Overcoming metabolic adaptation in KRAS-mutant cancers.

Mutations in guanosine triphosphatase KRAS are common in lung, colorectal, and pancreatic cancers. The constitutive activity of mutant KRAS and its downstream signaling pathways induces metabolic rewiring in tumor cells that can promote resistance to existing therapeutics. In this review, we discuss the metabolic pathways that are altered in response to treatment and those that can, in turn, alter treatment efficacy, as well as the role of metabolism in the tumor microenvironment (TME) in dictating the therapeutic response in KRAS-driven cancers. We highlight metabolic targets that may provide clinical opportunities to overcome therapeutic resistance and improve survival in patients with these aggressive cancers.

Activation of innate-adaptive immune machinery by poly(I:C) exposes a therapeutic vulnerability to prevent relapse in stroma-rich colon cancer.

OBJECTIVE: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. DESIGN: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. RESULTS: By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). CONCLUSION: This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC.

Clinical Positioning of the IAP Antagonist Tolinapant (ASTX660) in Colorectal Cancer.

Inhibitors of apoptosis proteins (IAPs) are intracellular proteins, with important roles in regulating cell death, inflammation, and immunity. Here, we examined the clinical and therapeutic relevance of IAPs in colorectal cancer. We found that elevated expression of cIAP1 and cIAP2 (but not XIAP) significantly correlated with poor prognosis in patients with microsatellite stable (MSS) stage III colorectal cancer treated with 5-fluorouracil (5FU)-based adjuvant chemotherapy, suggesting their involvement in promoting chemoresistance. A novel IAP antagonist tolinapant (ASTX660) potently and rapidly downregulated cIAP1 in colorectal cancer models, demonstrating its robust on-target efficacy. In cells co-cultured with TNFα to mimic an inflammatory tumor microenvironment, tolinapant induced caspase-8-dependent apoptosis in colorectal cancer cell line models; however, the extent of apoptosis was limited because of inhibition by the caspase-8 paralogs FLIP and, unexpectedly, caspase-10. Importantly, tolinapant-induced apoptosis was augmented by FOLFOX in human colorectal cancer and murine organoid models in vitro and in vivo, due (at least in part) to FOLFOX-induced downregulation of class I histone deacetylases (HDAC), leading to acetylation of the FLIP-binding partner Ku70 and downregulation of FLIP. Moreover, the effects of FOLFOX could be phenocopied using the clinically relevant class I HDAC inhibitor, entinostat, which also induced acetylation of Ku70 and FLIP downregulation. Further analyses revealed that caspase-8 knockout RIPK3-positive colorectal cancer models were sensitive to tolinapant-induced necroptosis, an effect that could be exploited in caspase-8-proficient models using the clinically relevant caspase inhibitor emricasan. Our study provides evidence for immediate clinical exploration of tolinapant in combination with FOLFOX in poor prognosis MSS colorectal cancer with elevated cIAP1/2 expression.

Metabolic Reprogramming: A Friend or Foe to Cancer Therapy?

Drug resistance is a major cause of cancer treatment failure, effectively driven by processes that promote escape from therapy-induced cell death. The mechanisms driving evasion of apoptosis have been widely studied across multiple cancer types, and have facilitated new and exciting therapeutic discoveries with the potential to improve cancer patient care. However, an increasing understanding of the crosstalk between cancer hallmarks has highlighted the complexity of the mechanisms of drug resistance, co-opting pathways outside of the canonical "cell death" machinery to facilitate cell survival in the face of cytotoxic stress. Rewiring of cellular metabolism is vital to drive and support increased proliferative demands in cancer cells, and recent discoveries in the field of cancer metabolism have uncovered a novel role for these programs in facilitating drug resistance. As a key organelle in both metabolic and apoptotic homeostasis, the mitochondria are at the forefront of these mechanisms of resistance, coordinating crosstalk in the event of cellular stress, and promoting cellular survival. Importantly, the appreciation of this role metabolism plays in the cytotoxic response to therapy, and the ability to profile metabolic adaptions in response to treatment, has encouraged new avenues of investigation into the potential of exploiting metabolic addictions to improve therapeutic efficacy and overcome drug resistance in cancer. Here, we review the role cancer metabolism can play in mediating drug resistance, and the exciting opportunities presented by imposed metabolic vulnerabilities.

The pseudo-caspase FLIP(L) regulates cell fate following p53 activation.

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

Metabolic rewiring in mutant Kras lung cancer.

Lung cancer is the leading cause of cancer-related death worldwide, reflecting an unfortunate combination of very high prevalence and low survival rates, as most cases are diagnosed at advanced stages when treatment efficacy is limited. Lung cancer comprises several disease groups with non small cell lung cancer (NSCLC) accounting for ~ 85% of cases and lung adenocarcinoma being its most frequent histological subtype. Mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) affect ~ 30% of lung adenocarcinomas but unlike other commonly altered proteins (EGFR and ALK, affected in ~ 14% and 7% of cases respectively), mutant KRAS remains untargetable. Therapeutic strategies that rely instead on the inhibition of mutant KRAS functional output or the targeting of mutant KRAS cellular dependencies (i.e. synthetic lethality) are an appealing alternative approach. Recent studies focused on the metabolic properties of mutant KRAS lung tumours have uncovered unique metabolic features that can potentially be exploited therapeutically. We review these findings here with a particular focus on in vivo, physiologic, mutant KRAS activity.

Lung tumors with distinct p53 mutations respond similarly to p53 targeted therapy but exhibit genotype-specific statin sensitivity.

Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in TP53 (p53), making these mutants appealing targets for lung cancer therapy. As mutant p53 remains untargetable, mutant p53-dependent phenotypes represent alternative targeting opportunities, but the prevalence and therapeutic relevance of such effects (gain of function and dominant-negative activity) in lung adenocarcinoma are unclear. Through transcriptional and functional analysis of murine KrasG12D -p53null , -p53R172H (conformational), and -p53R270H (contact) mutant lung tumors, we identified genotype-independent and genotype-dependent therapeutic sensitivities. Unexpectedly, we found that wild-type p53 exerts a dominant tumor-suppressive effect on mutant tumors, as all genotypes were similarly sensitive to its restoration in vivo. These data show that the potential of p53 targeted therapies is comparable across all p53-deficient genotypes and may explain the high incidence of p53 loss of heterozygosity in mutant tumors. In contrast, mutant p53 gain of function and their associated vulnerabilities can vary according to mutation type. Notably, we identified a p53R270H -specific sensitivity to simvastatin in lung tumors, and the transcriptional signature that underlies this sensitivity was also present in human lung tumors, indicating that this therapeutic approach may be clinically relevant.

KRAS Allelic Imbalance: Strengths and Weaknesses in Numbers.

The identification of therapeutic vulnerabilities in mutant KRAS tumors has proven difficult to achieve. Burgess and colleagues recently reported in Cell that mutant/wild-type Kras allelic dosage determines clonal fitness and MEK inhibitor sensitivity in a leukemia model, demonstrating that KRAS allelic imbalance is likely an important and overlooked variable.

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. METHODS: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. RESULTS: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. CONCLUSIONS: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease.