The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic disease. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and inflammation, which fosters multiple hallmark functions. Conceptual progress in the last decade has added two emerging hallmarks of potential generality to this list-reprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors exhibit another dimension of complexity: they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of hallmark traits by creating the "tumor microenvironment." Recognition of the widespread applicability of these concepts will increasingly affect the development of new means to treat human cancer.

The discovery of cancer-associated alterations has primarily focused on genetic variants. Nonetheless, altered epigenomes contribute to deregulate transcription and promote oncogenic pathways. Here, we designed an algorithmic approach (RESET) to identify aberrant DNA methylation and associated cis-transcriptional changes across >6,000 human tumors. Tumors exhibiting mutations of chromatin remodeling factors and Wnt signaling displayed DNA methylation instability, characterized by numerous hyper- and hypo-methylated loci. Most silenced and enhanced genes coalesced in specific pathways including apoptosis, DNA repair, and cell metabolism. Cancer-germline antigens (CG) were frequently epigenomically enhanced and their expression correlated with response to anti-PD-1, but not anti-CTLA4, in skin melanoma. Finally, we demonstrated the potential of our approach to explore DNA methylation changes in pediatric tumors, which frequently lack genetic drivers and exhibit epigenomic modifications. Our results provide a pan-cancer map of aberrant DNA methylation to inform functional and therapeutic studies.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease. Overall survival is typically 6 months from diagnosis. Numerous phase 3 trials of agents effective in other malignancies have failed to benefit unselected PDA populations, although patients do occasionally respond. Studies in other solid tumors have shown that heterogeneity in response is determined, in part, by molecular differences between tumors. Furthermore, treatment outcomes are improved by targeting drugs to tumor subtypes in which they are selectively effective, with breast and lung cancers providing recent examples. Identification of PDA molecular subtypes has been frustrated by a paucity of tumor specimens available for study. We have overcome this problem by combined analysis of transcriptional profiles of primary PDA samples from several studies, along with human and mouse PDA cell lines. We define three PDA subtypes: classical, quasimesenchymal and exocrine-like, and we present evidence for clinical outcome and therapeutic response differences between them. We further define gene signatures for these subtypes that may have utility in stratifying patients for treatment and present preclinical model systems that may be used to identify new subtype specific therapies.

The Arf tumor suppressor acts as a sensor of oncogenic signals, countering aberrant proliferation in large part via activation of the p53 transcriptional program, though a number of p53-independent functions have been described. Mounting evidence suggests that, in addition to promoting tumorigenesis via disruptions in the homeostatic balance between cell proliferation and apoptosis of overt cancer cells, genetic alterations leading to tumor suppressor loss of function or oncogene gain of function can also incite tumor development via effects on the tumor microenvironment. In a transgenic mouse model of multi-stage pancreatic neuroendocrine carcinogenesis (PNET) driven by inhibition of the canonical p53 and Rb tumor suppressors with SV40 large T-antigen (Tag), stochastic progression to tumors is limited in part by a requirement for initiation of an angiogenic switch. Despite inhibition of p53 by Tag in this mouse PNET model, concomitant disruption of Arf via genetic knockout resulted in a significantly accelerated pathway to tumor formation that was surprisingly not driven by alterations in tumor cell proliferation or apoptosis, but rather via earlier activation of the angiogenic switch. In the setting of a constitutional p53 gene knockout, loss of Arf also accelerated tumor development, albeit to a lesser degree. These findings demonstrate that Arf loss of function can promote tumorigenesis via facilitating angiogenesis, at least in part, through p53-independent mechanisms.

In a transgenic model of multi-stage squamous carcinogenesis induced by human papillomavirus (HPV) oncogenes, infiltrating CD4+ T cells can be detected in both premalignant and malignant lesions. The lymph nodes that drain sites of epidermal neoplasia contain activated CD4+ T cells predominantly reactive toward Staphylococcal bacterial antigens. HPV16 mice deficient in CD4+ T cells were found to have delayed neoplastic progression and a lower incidence of tumors. This delay in carcinogenesis is marked by decreased infiltration of neutrophils, and reduced activity of matrix metalloproteinase-9, an important cofactor for tumor progression in this model. The data reveal an unexpected capability of CD4 T cells, whereby, proinflammatory CD4+ T cells, apparently responding to bacterial infection of dysplastic skin lesions, can inadvertently enhance neoplastic progression to invasive cancer.

Poor penetration of drugs into tumors is a major obstacle in tumor treatment. We describe a strategy for peptide-mediated delivery of compounds deep into the tumor parenchyma that uses a tumor-homing peptide, iRGD (CRGDK/RGPD/EC). Intravenously injected compounds coupled to iRGD bound to tumor vessels and spread into the extravascular tumor parenchyma, whereas conventional RGD peptides only delivered the cargo to the blood vessels. iRGD homes to tumors through a three-step process: the RGD motif mediates binding to alphav integrins on tumor endothelium and a proteolytic cleavage then exposes a binding motif for neuropilin-1, which mediates penetration into tissue and cells. Conjugation to iRGD significantly improved the sensitivity of tumor-imaging agents and enhanced the activity of an antitumor drug.

Tumor cell death is modulated by an intrinsic cell death pathway controlled by the pro- and anti-apoptotic members of the Bcl-2 family. Up-regulation of anti-apoptotic Bcl-2 family members has been shown to suppress cell death in pre-clinical models of human cancer and is implicated in human tumor progression. Previous gain-of-function studies in the RIP1-Tag2 model of pancreatic islet carcinogenesis, involving uniform or focal/temporal over-expression of Bcl-x(L), demonstrated accelerated tumor formation and growth. To specifically assess the role of endogenous Bcl-x in regulating apoptosis and tumor progression in this model, we engineered a pancreatic beta-cell-specific knockout of both alleles of Bcl-x using the Cre-LoxP system of homologous recombination. Surprisingly, there was no appreciable effect on tumor cell apoptosis rates or on tumor growth in the Bcl-x knockout mice. Other anti-apoptotic Bcl-2 family members were expressed but not substantively altered at the mRNA level in the Bcl-x-null tumors, suggestive of redundancy without compensatory transcriptional up-regulation. Interestingly, the incidence of invasive carcinomas was reduced, and tumor cells lacking Bcl-x were impaired in invasion in a two-chamber trans-well assay under conditions mimicking hypoxia. Thus, while the function of Bcl-x in suppressing apoptosis and thereby promoting tumor growth is evidently redundant, genetic ablation implicates Bcl-x in selectively facilitating invasion, consistent with a recent report documenting a pro-invasive capability of Bcl-x(L) upon exogenous over-expression.

Genetic linkage analysis previously suggested that GKAP, a scaffold protein of the N-methyl-D-aspartate receptor (NMDAR), was a potential modifier of invasion in a mouse model of pancreatic neuroendocrine tumor (PanNET). Here, we establish that GKAP governs invasive growth and treatment response to NMDAR inhibitors of PanNET via its pivotal role in regulating NMDAR pathway activity. Combining genetic knockdown of GKAP and pharmacological inhibition of NMDAR, we implicate as downstream effectors FMRP and HSF1, which along with GKAP demonstrably support invasiveness of PanNET and pancreatic ductal adenocarcinoma cancer cells. Furthermore, we distilled genome-wide expression profiles orchestrated by the NMDAR-GKAP signaling axis, identifying transcriptome signatures in tumors with low/inhibited NMDAR activity that significantly associate with favorable patient prognosis in several cancer types.

Immunotherapies have shown remarkable, albeit tumor-selective, therapeutic benefits in the clinic. Most patients respond transiently at best, highlighting the importance of understanding mechanisms underlying resistance. Herein, we evaluated the effects of the engineered immunocytokine PD1-IL2v in a mouse model of de novo pancreatic neuroendocrine cancer that is resistant to checkpoint and other immunotherapies. PD1-IL2v utilizes anti-PD-1 as a targeting moiety fused to an immuno-stimulatory IL-2 cytokine variant (IL2v) to precisely deliver IL2v to PD-1+ T cells in the tumor microenvironment. PD1-IL2v elicited substantial infiltration by stem-like CD8+ T cells, resulting in tumor regression and enhanced survival in mice. Combining anti-PD-L1 with PD1-IL2v sustained the response phase, improving therapeutic efficacy both by reprogramming immunosuppressive tumor-associated macrophages and enhancing T cell receptor (TCR) immune repertoire diversity. These data provide a rationale for clinical trials to evaluate the combination therapy of PD1-IL2v and anti-PD-L1, particularly in immunotherapy-resistant tumors infiltrated with PD-1+ stem-like T cells.

High-risk human papillomavirus (HPV) are responsible for genital and oral cancers associated with the expression of the E6/E7 HPV oncogenes. Therapeutic vaccines targeting those oncogenes can only partially control tumor progression, highlighting the necessity to investigate different treatment strategies. Using the genital orthotopic HPV16 TC-1 model, herein we sequentially investigated in progressively more stringent settings the effects of systemic administration of carboplatin/paclitaxel (C + P) chemotherapy combined with HPV16-E7 synthetic long peptide (E7LP) vaccination, followed by intravaginal immunostimulation with the synthetic toll-like-receptor-9 agonist CpG. Our data show that systemic delivery of C + P prior to E7LP vaccination significantly increased mice survival. This survival benefit was associated with both reduced genital tumor growth at the time of vaccination, and a decreased infiltration of Ly6G myeloid cells and tumor-associated macrophages. Adding intravaginal CpG, which results in increased E7-specific CD8 T cells locally, to E7LP vaccination and the chemotherapy formed a tri-therapy, which significantly increased mice survival as compared to any of the dual treatments. When the tri-therapy was further refined by using a recently optimized nanoparticle-conjugated E7LP vaccine, even larger end-stage genital-TC-1 tumors responded, with 90% of mice showing a survival benefit as compared to 30% of mice with the tri-therapy involving the traditional E7LP 'liquid' vaccine. C + P is commonly used to treat cervical cancer patients and its combination with E7/E6 vaccination is currently being tested in a phase I/II trial (NCT02128126). Our data suggests that new vaccine formulations combined with local immunostimulation and standard-of-care chemotherapy have promise to further benefit patients with HPV-associated cancer.

Factors associated with tumor sensitivity to epidermal growth factor receptor (EGFR) inhibitors in the context of wild-type EGFR remain elusive. This study investigates the mechanistic basis of responsiveness to EGFR inhibitors in the RIP1-Tag2 (RT2) mouse model of pancreatic neuroendocrine tumorigenesis (PNET). Upon treatment of RT2 mice with EGFR inhibitors, PNET tumors harboring wild-type, nonamplified alleles of Egfr grow at a markedly reduced rate and display a significant increase in tumor cell apoptosis, as well as reduced neovascularization. The authors identify Tgf-α and Hb-egf as key limiting mediators of separable pathological functions of Egfr in neuroendocrine tumor progression: Tgf-α mutant tumors present with an elevated apoptotic index, whereas Hb-egf mutant lesions exhibit decreased angiogenic switching and neovascularization. This study not only associates Tgf-α and Hb-egf expression with wild-type Egfr oncogenicity but also ascribes the proangiogenic activity of Egfr in this tumor model to a novel mesenchymal Hb-egf/Egfr signaling axis, whereby endothelial and pericyte-derived Hb-egf activates Egfr specifically in tumor-associated perivascular cells, leading to increased pericyte coverage of the tumor endothelium and enhanced angiogenesis.

Inhibition of platelet-derived growth factor-B (PDGF-B) has multiple effects on tumors, including loss of pericytes, regression of some vessels, normalization of other vessels, and reduction of interstitial pressure. PDGF-B inhibition also increases the efficacy of cancer therapeutics, but the role on tumor vessel efficiency and drug delivery is unclear. We sought to determine whether inhibition of PDGF-B signaling can increase delivery and efficacy of cyclophosphamide in Lewis lung carcinomas or RIP-Tag2 tumors. PDGF-B blockade in Lewis lung carcinoma tumors by the DNA aptamer AX102 for 14 days increased the number of perfused tumor vessels marked by lectin in the bloodstream by 50%. AX102 also increased the width of sleeves of viable tumor cells around blood vessels by 66%, increased tumor cell proliferation by 90%, and increased intratumoral delivery of Hoechst 33342 by 78%. A low dose of cyclophosphamide (20 mg/kg) reduced tumor cell proliferation by 31% when combined with AX102 but not when given alone. Synergy of cyclophosphamide and AX102 on tumor cell proliferation also was found in RIP-Tag2 tumors. Similarly, the PDGF receptor signaling inhibitor imatinib increased delivery of cyclophosphamide and reduced tumor burden in RIP-Tag2 mice, without evidence of tumor cell sensitization to chemotherapy. Together, these findings indicate that inhibition of PDGF-B signaling promotes the delivery and efficacy of chemotherapeutic agents by increasing the efficiency of tumor blood vessels.

It is not uncommon to observe circulating tumor antigen-specific T lymphocytes in cancer patients despite a lack of significant infiltration and destruction of their tumors. Thus, an important goal for tumor immunotherapy is to identify ways to modulate in vivo anti-tumor immunity to achieve clinical efficacy. We investigate this proposition in a spontaneous mouse tumor model, Rip1-Tag2.

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycERTAM;Bcl-xL model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycERTAM;Bcl-xL tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.

Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMSs) with distinguishing features: CMS1 (microsatellite instability immune, 14%), hypermutated, microsatellite unstable and strong immune activation; CMS2 (canonical, 37%), epithelial, marked WNT and MYC signaling activation; CMS3 (metabolic, 13%), epithelial and evident metabolic dysregulation; and CMS4 (mesenchymal, 23%), prominent transforming growth factor-β activation, stromal invasion and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intratumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC-with clear biological interpretability-and the basis for future clinical stratification and subtype-based targeted interventions.

The associations of tricyclic antidepressants (TCAs) with reduced incidence of gliomas and elevated autophagy in glioma cells motivated investigation in mouse models of gliomagenesis. First, we established that imipramine, a TCA, increased autophagy and conveyed modest therapeutic benefit in tumor-bearing animals. Then we screened clinically approved agents suggested to affect autophagy for their ability to enhance imipramine-induced autophagy-associated cell death. The anticoagulant ticlopidine, which inhibits the purinergic receptor P2Y12, potentiated imipramine, elevating cAMP, a modulator of autophagy, reducing cell viability in culture, and increasing survival in glioma-bearing mice. Efficacy of the combination was obviated by knockdown of the autophagic regulatory gene ATG7, implicating cell-lethal autophagy. This seemingly innocuous combination of TCAs and P2Y12 inhibitors may have applicability for treating glioma.

Tumors develop through multiple stages, implicating multiple effectors, but the tools to assess how candidate genes contribute to stepwise tumor progression have been limited. We have developed a novel system in which progression of phenotypes in a mouse model of pancreatic islet cell tumorigenesis can be used to measure the effects of genes introduced by cell-type-specific infection with retroviral vectors. In this system, bitransgenic mice, in which the rat insulin promoter (RIP) drives expression of both the SV40 T antigen (RIP-Tag) and the receptor for subgroup A avian leukosis virus (RIP-tva), are infected with avian viral vectors carrying cDNAs encoding candidate progression factors. Like RIP-Tag mice, RIP-Tag; RIP-tva bitransgenic mice develop isolated carcinomas by approximately 14 wk of age, after progression through well-defined stages that are similar to aspects of human tumor progression, including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. When avian retroviral vectors carrying a green fluorescent protein marker were introduced into RIP-Tag; RIP-tva mice by intra-cardiac injection at the hyperplastic or early dysplastic stage of tumorigenesis, approximately 20% of the TVA-positive cells were infected and expressed green fluorescent proteins as measured by flow cytometry. Similar infection with vectors carrying cDNA encoding either of two progression factors, a dominant-negative version of cadherin 1 (dnE-cad) or Bcl-xL, accelerated the formation of islet tumors with invasive properties and pancreatic lymph node metastasis. To begin studying the mechanism by which Bcl-xL, an anti-apoptotic protein, promotes invasion and metastasis, RIP-Tag; RIP-tva pancreatic islet tumor cells were infected in vitro with RCASBP-Bcl-xL. Although no changes were observed in rates of proliferation or apoptosis, Bcl-xL altered cell morphology, remodeled the actin cytoskeleton, and down-regulated cadherin 1; it also induced cell migration and invasion, as evaluated using two-chamber transwell assays. In addition, myosin Va was identified as a novel Bcl-xL-interacting protein that might mediate the effects of Bcl-xL on tumor cell migration and invasion.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor β- and γ-chain (IL-2Rβγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rβγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rβγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

Pancreatic neuroendocrine tumors (PanNET) comprise two molecular subtypes, relatively benign islet tumors (IT) and invasive, metastasis-like primary (MLP) tumors. Until now, the origin of aggressive MLP tumors has been obscure. Herein, using multi-omics approaches, we revealed that MLP tumors arise from IT via dedifferentiation following a reverse trajectory along the developmental pathway of islet β cells, which results in the acquisition of a progenitor-like molecular phenotype. Functionally, the miR-181cd cluster induces the IT-to-MLP transition by suppressing expression of the Meis2 transcription factor, leading to upregulation of a developmental transcription factor, Hmgb3. Notably, the IT-to-MLP transition constitutes a distinct step of tumorigenesis and is separable from the classic proliferation-associated hallmark, temporally preceding accelerated proliferation of cancer cells. Furthermore, patients with PanNET with elevated HMGB3 expression and an MLP transcriptional signature are associated with higher-grade tumors and worse survival. Overall, our results unveil a new mechanism that modulates cancer cell plasticity to enable malignant progression. SIGNIFICANCE: Dedifferentiation has long been observed as a histopathologic characteristic of many cancers, albeit inseparable from concurrent increases in cell proliferation. Herein, we demonstrate that dedifferentiation is a mechanistically and temporally separable step in the multistage tumorigenesis of pancreatic islet cells, retracing the developmental lineage of islet β cells.This article is highlighted in the In This Issue feature, p. 2355.

Glioblastoma (GBM) is poorly responsive to therapy and invariably lethal. One conceivable strategy to circumvent this intractability is to co-target distinctive mechanistic components of the disease, aiming to concomitantly disrupt multiple capabilities required for tumor progression and therapeutic resistance. We assessed this concept by combining vascular endothelial growth factor (VEGF) pathway inhibitors that remodel the tumor vasculature with the tricyclic antidepressant imipramine, which enhances autophagy in GBM cancer cells and unexpectedly reprograms immunosuppressive tumor-associated macrophages via inhibition of histamine receptor signaling to become immunostimulatory. While neither drug is efficacious as monotherapy, the combination of imipramine with VEGF pathway inhibitors orchestrates the infiltration and activation of CD8 and CD4 T cells, producing significant therapeutic benefit in several GBM mouse models. Inclusion up front of immune-checkpoint blockade with anti-programmed death-ligand 1 (PD-L1) in eventually relapsing tumors markedly extends survival benefit. The results illustrate the potential of mechanism-guided therapeutic co-targeting of disparate biological vulnerabilities in the tumor microenvironment.