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Adjuvant ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) both improve relapse-free survival of stage III melanoma patients 1 , 2 . In stage IV disease, the combination of ipilimumab + nivolumab is superior to ipilimumab alone and also appears to be more effective than nivolumab monotherapy 3 . Preclinical work suggests that neoadjuvant application of checkpoint inhibitors may be superior to adjuvant therapy 4 . To address this question and to test feasibility, 20 patients with palpable stage III melanoma were 1:1 randomized to receive ipilimumab 3 mg kg −1 and nivolumab 1 mg kg −1 , as either four courses after surgery (adjuvant arm) or two courses before surgery and two courses postsurgery (neoadjuvant arm). Neoadjuvant therapy was feasible, with all patients undergoing surgery at the preplanned time point. However in both arms, 9/10 patients experienced one or more grade 3/4 adverse events. Pathological responses were achieved in 7/9 (78%) patients treated in the neoadjuvant arm. None of these patients have relapsed so far (median follow-up, 25.6 months). We found that neoadjuvant ipilimumab + nivolumab expand more tumor-resident T cell clones than adjuvant application. While neoadjuvant therapy appears promising, with the current regimen it induced high toxicity rates; therefore, it needs further investigation to preserve efficacy but reduce toxicity. Neoadjuvant combination immunotherapy in patients with advanced melanoma shows favorable activity over adjuvant treatment and warrants future evaluation with modified dosing schedules to reduce treatment-related adverse events.

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.

Krüppel-like factors are transcriptional regulators that function both as tumour suppressors and oncogenes through their control of p21 expression. This might be an important nodal point of cell control for other factors that have opposing functions in cancer. Key Points Krüppel-like transcription factors (KLFs) regulate the expression of many genes, including those that are involved in differentiation and cell-cycle arrest. One KLF family member, KLF4, possesses tumour-suppressor-like properties, as the gene that encodes it is deleted or methylated (silenced) in human gastrointestinal tract tumours. In mice, deletion of Klf4 in the gastric compartment induces hyperplasia and polyps. Ectopic expression of KLF4 induces cell-cycle arrest in a p21-dependent manner. KLF4 also seems to function as a dominant oncogene, as it is often overexpressed in human breast tumours and squamous cell carcinomas, and can also contribute to oncogenic transformation of cultured cells. Loss of p21 is sufficient to convert KLF4 from an inhibitor of proliferation into a transforming oncogene in vitro . p21 might represent a nodal point for signals from multiple factors with opposing, context-dependent functions in cancer, including transforming growth factor-β, Notch, Runx and Ras. Although CDKN1A (cyclin-dependent kinase inhibitor 1A, the gene that encodes p21) is a target gene of many key tumour-suppressor pathways, it is not often lost or mutated in human tumours. p21 itself also possesses opposing functions that might both counteract and contribute to cancer progression. Partial, but not complete, loss of p21 might provide a selective advantage to tumour cells, which could be related to the proposed roles for p21 in suppressing apoptosis and inducing cell-cycle arrest. Krüppel-like factors are transcriptional regulators that influence several cellular functions, including proliferation. Recent studies have shown that one family member, KLF4 , can function both as a tumour suppressor and an oncogene. The ability of KLF4 to affect the levels of expression of the cell-cycle regulator p21 seems to be involved, in that this protein might function as a switch that determines the outcome of KLF4 signalling. Is this role of p21 restricted to KLF4, or does p21 represent a nodal point for signals from multiple other factors with opposing functions in cancer?

KLF4 ( GKLF / EZF ) encodes a transcription factor that is associated with both tumour suppression and oncogenesis. We describe the identification of KLF4 in a functional genomic screen for genes that bypass RAS V12 -induced senescence. However, in untransformed cells, KLF4 acts as a potent inhibitor of proliferation. KLF4-induced arrest is bypassed by oncogenic RAS V12 or by the RAS target cyclin-D1. Remarkably, inactivation of the cyclin-D1 target and the cell-cycle inhibitor p21 CIP1 not only neutralizes the cytostatic action of KLF4, but also collaborates with KLF4 in oncogenic transformation. Conversely, KLF4 suppresses the expression of p53 by directly acting on its promoter, thereby allowing for RAS V12 -mediated transformation and causing resistance to DNA-damage-induced apoptosis. Consistently, KLF4 depletion from breast cancer cells restores p53 levels and causes p53-dependent apoptosis. These results unmask KLF4 as a regulator of p53 that oncogenically transforms cells as a function of p21 CIP1 status. Furthermore, they provide a mechanistic explanation for the context-dependent oncogenic or tumour-suppressor functions of KLF4.

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.

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.

Cell senescence and cancer Cellular senescence, a growth-arrest program that limits the lifespan of mammalian cells and prevents unlimited cell proliferation, is attracting considerable interest because of its links to tumour suppression. Using a mouse model in which the oncogene Ras is activated in the haematopoietic compartment of bone marrow, Braig et al . show that cellular senescence can block lymphoma development. Genetic inactivation of the histone methyltransferase Suv39h1 that controls senescence by ‘epigenetic’ modification of DNA-associated proteins, or a pharmacological approach that mimics loss of this enzyme, allow the formation of malignant lymphomas in response to oncogenic Ras . This work has important implications for both tumour development and tumour therapy. Michaloglou et al . report that oncogene-induced senescence may be a physiologically important process in humans, keeping moles in a benign state for many years: unchecked they develop into malignant melanomas. Chen et al . also find that cellular senescence blocks tumorigenesis in vivo : they show that acting together, the p53 tumour suppressor and the cellular senescence system can prevent prostate cancer induction in mice by the PTEN mutation. Collado et al . show that cellular senescence is a defining feature of Ras-initiated premalignant tumours; this could prove valuable in the diagnosis and prognosis of cancer. See the web focus . Most normal mammalian cells have a finite lifespan 1 , thought to constitute a protective mechanism against unlimited proliferation 2 , 3 , 4 . This phenomenon, called senescence, is driven by telomere attrition, which triggers the induction of tumour suppressors including p16 INK4a (ref. 5 ). In cultured cells, senescence can be elicited prematurely by oncogenes 6 ; however, whether such oncogene-induced senescence represents a physiological process has long been debated. Human naevi (moles) are benign tumours of melanocytes that frequently harbour oncogenic mutations (predominantly V600E, where valine is substituted for glutamic acid) in BRAF 7 , a protein kinase and downstream effector of Ras. Nonetheless, naevi typically remain in a growth-arrested state for decades and only rarely progress into malignancy (melanoma) 8 , 9 , 10 . This raises the question of whether naevi undergo BRAF V600E -induced senescence. Here we show that sustained BRAF V600E expression in human melanocytes induces cell cycle arrest, which is accompanied by the induction of both p16 INK4a and senescence-associated acidic β-galactosidase (SA-β-Gal) activity, a commonly used senescence marker. Validating these results in vivo , congenital naevi are invariably positive for SA-β-Gal, demonstrating the presence of this classical senescence-associated marker in a largely growth-arrested, neoplastic human lesion. In growth-arrested melanocytes, both in vitro and in situ , we observed a marked mosaic induction of p16 INK4a , suggesting that factors other than p16 INK4a contribute to protection against BRAF V600E -driven proliferation. Naevi do not appear to suffer from telomere attrition, arguing in favour of an active oncogene-driven senescence process, rather than a loss of replicative potential. Thus, both in vitro and in vivo , BRAF V600E -expressing melanocytes display classical hallmarks of senescence, suggesting that oncogene-induced senescence represents a genuine protective physiological process.

Metastasis is a major factor in the malignancy of cancers, and is often responsible for the failure of cancer treatment. Anoikis (apoptosis resulting from loss of cell–matrix interactions) has been suggested to act as a physiological barrier to metastasis; resistance to anoikis may allow survival of cancer cells during systemic circulation, thereby facilitating secondary tumour formation in distant organs 1 , 2 , 3 . In an attempt to identify metastasis-associated oncogenes, we designed an unbiased, genome-wide functional screen solely on the basis of anoikis suppression. Here, we report the identification of TrkB, a neurotrophic tyrosine kinase receptor 4 , 5 , as a potent and specific suppressor of caspase-associated anoikis of non-malignant epithelial cells. By activating the phosphatidylinositol-3-OH kinase/protein kinase B pathway, TrkB induced the formation of large cellular aggregates that survive and proliferate in suspension. In mice, these cells formed rapidly growing tumours that infiltrated lymphatics and blood vessels to colonize distant organs. Consistent with the ability of TrkB to suppress anoikis, metastases—whether small vessel infiltrates or large tumour nodules—contained very few apoptotic cells. These observations demonstrate the potent oncogenic effects of TrkB and uncover a specific pro-survival function that may contribute to its metastatic capacity, providing a possible explanation for the aggressive nature of human tumours that overexpress TrkB.

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.

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.