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This review of oncogene-induced cell senescence describes a physiological mechanism that provides protection against cancer. The mechanisms underlying the arrest of growth in benign or premalignant neoplasms have considerable clinical implications. This review of oncogene-induced cell senescence describes a physiological mechanism that provides protection against cancer. In many tissues, there are numerous small and inconspicuous neoplastic lesions that rarely become overt cancers. In these small lesions, clonality and oncogenic mutations have been identified. These lesions include established benign tumors such as melanocytic nevi, 1 some that were previously regarded to be of a “reactive” nature, 2 and even groups of cells that are histologically only marginally abnormal. 3 Once they have grown to a certain size, such lesions stop growing appreciably and do not become more aggressive over many years or even decades. For instance, few moles, which are benign tumors of cutaneous melanocytes, grow larger than 1 cm, . . .

Anoikis (detachment-induced apoptosis) prevents the survival of cells at inappropriate sites of the body and can therefore act as a barrier to metastasis. In a function-based genome-wide screen, we have previously identified the neurotrophic tyrosine kinase receptor TrkB as a potent suppressor of anoikis. Consistently, activated TrkB oncogenically transforms non-malignant epithelial cells and causes them to invade and produce metastatic tumors in vivo . Overexpression of activated TrkB also results in morphological transformation, resembling epithelial-mesenchymal transition (EMT). E-cadherin, an important EMT regulator, and two E-cadherin repressors, Twist and Snail, are critical for these TrkB functions. As Snail has been shown to induce Zeb1, another E-cadherin repressor, we hypothesized that Zeb1 could be a TrkB target, too. We show here that Zeb1 is required for TrkB-induced EMT in epithelial cells, as RNAi-mediated knockdown of Zeb1 reverted the morphological changes induced by TrkB. Furthermore, Zeb1 is involved in TrkB-induced anoikis resistance, migration and invasion. In vivo , knockdown of Zeb1 strongly reduced TrkB-induced metastasis. Finally, epistasis experiments showed that Zeb1 acts downstream of Twist and Snail. We conclude that Zeb1 is required for several TrkB-induced effects in vitro and in vivo , including metastasis.

Background The tumor immune microenvironment is a heterogeneous entity. Gene expression analysis allows us to perform comprehensive immunoprofiling and may assist in dissecting the different components of the immune infiltrate. As gene expression analysis also provides information regarding tumor cells, differences in interactions between the immune system and specific tumor characteristics can also be explored. This study aims to gain further insights in the composition of the tumor immune infiltrate and to correlate these components to histology and overall survival in non-small cell lung cancer (NSCLC). Methods Archival tissues from 530 early stage, resected NSCLC patients with annotated tumor and patient characteristics were analyzed using the NanoString nCounter Analysis system. Results Unsupervised clustering of the samples was mainly driven by the overall level of inflammation, which was not correlated with survival in this patient set. Adenocarcinoma (AD) showed a significantly higher degree of immune infiltration compared to squamous cell carcinoma (SCC). A 34-gene signature, which did not correlate with the overall level of immune infiltration, was identified and showed an OS benefit in SCC. Strikingly, this benefit was not observed in AD. This difference in OS in SCC specifically was confirmed in two independent NSCLC cohorts. The highest correlation between expression of the 34-gene signature and specific immune cell populations was observed for NK cells, but although a plausible mechanism for NK cell intervention in tumor growth could be established in SCC over AD, this could not be translated back to immunohistochemistry, which showed that NK cell infiltration is scarce irrespective of histology. Conclusions These findings suggest that the ability of immune cell infiltration and the interaction between tumor and immune cells may be different between AD and SCC histology and that a subgroup of SCC tumors seems more susceptible to Natural Killer cell recognition and killing, whereas this may not occur in AD tumors. A highly sensitive technique like NanoString was able to detect this subgroup based on a 34-gene signature, but further research will be needed to assist in explaining the biological rationale of such low-level expression signatures.

Increasing evidence implies altered signaling through the neurotrophic receptor tyrosine kinase TrkB in promoting tumor formation and metastasis. TrkB, sometimes in conjunction with its primary ligand BDNF, is often overexpressed in a variety of human cancers, ranging from neuroblastomas to pancreatic ductal adenocarcinomas, in which it may allow tumor expansion and contribute to resistance to anti-tumor agents. In vitro, TrkB acts as a potent suppressor of anoikis (detachment-induced apoptosis), which is associated with the acquisition of an aggressive tumorigenic and metastatic phenotype in vivo. In view of its predicted contribution to tumorigenicity and metastasis in humans, TrkB corresponds to a potential drug target, and preclinical models have already been established. The encouraging results of pharmacological Trk inhibitors in tumor xenograft models suggest that TrkB inhibition may represent a promising novel anti-tumor therapeutic strategy. This hypothesis is currently being evaluated in clinical trials. Here, we will discuss the latest developments on TrkB in these contexts as well as highlight some critical questions that remain to be addressed for evaluating TrkB as a therapeutic target in cancer.

BackgroundAn increasing body of evidence suggests that in addition to the type, density, and state of immune cells in the tumor microenvironment (TME), also their proximity to cancer cells influences immunotherapy outcome. For example, favorable responses to immune checkpoint inhibitors in melanoma are associated with higher densities of CD8+ tumor-infiltrating lymphocytes (TIL) within 20 μm distance of melanoma cells. This notion is in line with the understanding that upon specific antigen recognition, cytotoxic T cells physically engage with their target cells through their TCRs followed by immunological synapse formation. Indeed, structural and functional avidity of cytotoxic CD8+ T cells correlates strongly with their activity against cancer cells. Together, these observations point to the importance of direct interactions between cytotoxic T cells and tumor cells in the TME. This led us to investigate whether tumor-specific CD8+ T cells can be isolated from clinical cancer specimens as heterotypic clusters.Materials and MethodsWe employed a tumor cell-T cell co-culture in vitro model, patient samples and ex vivo assays. To evaluate functional interactions between human T cells and tumor cells, we made use of a system we engineered previously, comprising melanoma cells expressing both HLA-A*02:01 and the MART-1 tumor antigen. They were challenged with CD8+ T cells from PBMCs that were retrovirally transduced with a MART-1-specific TCR. To asses these interactions in patient material, upon surgical removal tissue was cut into small fragments, digested and analyzed by (image-based) flow cytometry. Interacting (cluster) and not-interacting (singlets) T cells were isolated and expanded in vitro. To characterize tumor cell:T cell interactions single cell TCR and RNA sequencing is used, as well as ex vivo co-cultures with autologous tumor cells.ResultsWe found that in defined co-cultures, tumor antigen-recognizing T cells were commonly enriched over non-specific T cells in heterotypic clusters with tumor cells, prompting us to investigate whether such specific clusters could be isolated also from cancer specimens. We observed that from 10/10 human melanoma metastases, we were able to isolate heterotypic clusters, comprising CD8+ T cells interacting with one or more tumor cells and/or antigen-presenting cells (APCs), which was validated by imaging flow cytometry. Upon expansion, CD8+ T cells from tumor cell clusters and APC clusters exerted on average 7.6-fold increased melanoma-killing activity over T cell singlets, which was associated with enhanced cytokine production. CD8+ T cells from clusters were enriched for tumor-reactive and exhausted gene signatures. Integration with T cell receptor (TCR)-sequencing showed increased clonality of clustered T cells, indicative of expansion upon antigen recognition.ConclusionsTogether, these results demonstrate that tumor-reactive CD8+ T cells are enriched in functional clusters with tumor cells and/or APCs, and that they can be isolated and expanded from clinical samples. Being often excluded in cell sorting procedures, these distinct heterotypic CD8+ T cell clusters serve as a valuable source amenable to deciphering functional tumor-immune cell interactions, while they may also be therapeutically explored. S. Ibáñez Molero: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; P097110NL. J. Veldman: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; P097110NL. J. J H Traets: None. A. George: None. K. Hoefakker: None. S. Pack: None. L. Tas: None. P. Alóndiga-Mérida: None. B. van den Broek: None. R. Harkes: None. M. Nieuwland: None. M. van Baalen: None. E. Mul: None. S. Tol: None. J.B.A.G. Haanen: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Amgen, Asher Bio, BioNTech, BMS, MSD, Novartis, Sastra Cell Therapy. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Neogene Tx. F. Consultant/Advisory Board; Modest; BMS, CureVac, GSK, Imcyse, Iovance Bio, Instil Bio, Immunocore, Ipsen, Merck Serono, MSD, Molecular Partners, Novartis, Pfizer, Roche/Genentech, Sanofi, Scenic, Third Rock Ventures, Achilles Tx, BioNTech US, Instil Bio, PokeAcell, T-Knife, Scenic, Neogene Therapeutics. W.J.V. Houdt: None. D.S. Peeper: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Oncode Institute, Dutch Cancer Society KWF. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; P097110NL, Immagene. F. Consultant/Advisory Board; Modest; Immagene.

Of the RAF family of protein kinases, BRAF is the only member to be frequently activated by mutation in cancer. A single amino acid substitution (V600E) accounts for the vast majority and results in constitutive activation of BRAF kinase function. Its expression is required to maintain the proliferative and oncogenic characteristics of BRAF(E600)-expressing human tumour cells. Although BRAF(E600) acts as an oncogene in the context of additional genetic lesions, in primary cells it appears to be associated rather with transient stimulation of proliferation. Eventually, BRAF(E600) signalling triggers cell cycle arrest with the hallmarks of cellular senescence, as is illustrated by several recent studies in cultured cells, animal models and benign human lesions. In this review, we will discuss recent advances in our understanding of the role of BRAF(E600) in benign and malignant human tumours and the implications for therapeutic intervention.

Neoadjuvant ipilimumab plus nivolumab showed high pathologic response rates (pRRs) in patients with macroscopic stage III melanoma in the phase 1b OpACIN ( NCT02437279 ) and phase 2 OpACIN-neo ( NCT02977052 ) studies 1 , 2 . While the results are promising, data on the durability of these pathologic responses and baseline biomarkers for response and survival were lacking. After a median follow-up of 4 years, none of the patients with a pathologic response ( n  = 7/9 patients) in the OpACIN study had relapsed. In OpACIN-neo ( n  = 86), the 2-year estimated relapse-free survival was 84% for all patients, 97% for patients achieving a pathologic response and 36% for nonresponders ( P  < 0.001). High tumor mutational burden (TMB) and high interferon-gamma-related gene expression signature score (IFN-γ score) were associated with pathologic response and low risk of relapse; pRR was 100% in patients with high IFN-γ score/high TMB; patients with high IFN-γ score/low TMB or low IFN-γ score/high TMB had pRRs of 91% and 88%; while patients with low IFN-γ score/low TMB had a pRR of only 39%. These data demonstrate long-term benefit in patients with a pathologic response and show the predictive potential of TMB and IFN-γ score. Our findings provide a strong rationale for a randomized phase 3 study comparing neoadjuvant ipilimumab plus nivolumab versus standard adjuvant therapy with antibodies against the programmed cell death protein-1 (anti-PD-1) in macroscopic stage III melanoma. Long-term outcomes and biomarker analyses of two neoadjuvant immunotherapy clinical trials in melanoma patients support the clinical benefit of this treatment approach and uncover prognostic correlates of response.

Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy‐resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX‐derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF‐MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression. Synopsis Proteomics and phosphoproteomics analyses in melanoma cells identify CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors. Its expression is regulated by the transcription factor MITF and dinaciclib, a CDK2 inhibitor, overcomes the resistance to both classes of inhibitors. Proteome and phosphoproteome profiles of resistant versus sensitive melanoma cell lines were compared upon BRAFi, Hsp90i and combination thereof. Hsp90i resistance is driven by CDK2 upregulation, mediated by MITF, in melanoma cells. CDK2i, i.e. dinaciclib, overcomes BRAFi and Hsp90i resistance in melanoma cells. Graphical Abstract Proteomics and phosphoproteomics analyses in melanoma cells identify CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors. Its expression is regulated by the transcription factor MITF and dinaciclib, a CDK2 inhibitor, overcomes the resistance to both classes of inhibitors.

The Ras proto-oncogene is a central component of mitogenic signal-transduction pathways, and is essential for cells both to leave a quiescent state (G0) and to pass through the G1/S transition of the cell cycle 1–6 . The mechanism by which Ras signalling regulates cell-cycle progression is unclear, however. Here we report that the retinoblastoma tumour-suppressor protein (Rb), a regulator of G1 exit 7 , functionally links Ras to passage through the G1 phase. Inactivation of Ras in cycling cells caused a decline in cyclin D1 protein levels, accumulation of the hypophosphorylated, growth-suppressive form of Rb, and G1 arrest. When Rb was disrupted either genetically or biochemically, cells failed to arrest in G1 following Ras inactivation. In contrast, inactivation of Ras in quiescent cells prevented growth-factor induction of both immediate-early gene transcription and exit from G0 in an Rb-independent manner. These data suggest that Rb is an essential G1-specific mediator that links Ras-dependent mitogenic signalling to cell-cycle regulation.

Resistance of primary cells to transformation by oncogenic Ras has been attributed to the induction of replicative growth arrest 1 , 2 , 3 . This irreversible 'fail-safe mechanism' resembles senescence and requires induction by Ras of p19 ARF and p53 (refs 3 – 5 ). Mutation of either p19ARF or p53 alleviates Ras-induced senescence and facilitates oncogenic transformation by Ras 3 , 6 , 7 . Here we report that, whereas Rb and p107 are each dispensable for Ras-induced replicative arrest, simultaneous ablation of both genes disrupts Ras-induced senescence and results in unrestrained proliferation. This occurs despite activation by Ras of the p19 ARF /p53 pathway, identifying pRb and p107 as essential mediators of Ras-induced antiproliferative p19 ARF /p53 signalling. Unexpectedly, in contrast to p19ARF or p53 deficiency, loss of Rb/p107 function does not result in oncogenic transformation by Ras, as Ras-expressing Rb −/− /p107 −/− fibroblasts fail to grow anchorage-independently in vitro and are not tumorigenic in vivo . These results demonstrate that in the absence of both Rb and p107 cells are resistant to p19 ARF /p53-dependent protection against Ras-induced proliferation, and uncouple escape from Ras-induced premature senescence from oncogenic transformation.