Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of ...mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes-for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.
Long non-coding RNAs (lncRNAs) have been implicated in numerous physiological processes and diseases, most notably cancers. However, little is known about the mechanism of many functional lncRNAs. We ...identified an abundantly expressed lncRNA associated with decreased melanoma patient survival. Increased expression of this lncRNA, SLNCR1, mediates melanoma invasion through a highly conserved sequence similar to that of the lncRNA SRA1. Using a sensitive technique we term RATA (RNA-associated transcription factor array), we show that the brain-specific homeobox protein 3a (Brn3a) and the androgen receptor (AR) bind within and adjacent to SLNCR1’s conserved region, respectively. SLNCR1, AR, and Brn3a are specifically required for transcriptional activation of matrix metalloproteinase 9 (MMP9) and increased melanoma invasion. Our observations directly link AR to melanoma invasion, possibly explaining why males experience more melanoma metastases and have an overall lower survival in comparison to females.
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•Increased expression of SLNCR1 is associated with reduced overall melanoma survival•SLNCR1 knockdown decreases melanoma invasion•The transcription factors AR and Brn3a bind adjacent sequences in SLNCR1•SLNCR1, AR, and Brn3a are all required for upregulating the matrix metalloproteinase MMP9
Schmidt et al. identify a lncRNA, SLNCR1, associated with poor melanoma survival. The androgen receptor (AR) and the brain-specific homeobox protein 3a (Brn3a) bind to adjacent conserved sequences within SLNCR1 and increase melanoma invasion by upregulating the matrix metalloproteinase MMP9. These results may explain the female-favoring bias in melanoma.
Effective anti-tumour immunity in humans has been associated with the presence of T cells directed at cancer neoantigens, a class of HLA-bound peptides that arise from tumour-specific mutations. They ...are highly immunogenic because they are not present in normal tissues and hence bypass central thymic tolerance. Although neoantigens were long-envisioned as optimal targets for an anti-tumour immune response, their systematic discovery and evaluation only became feasible with the recent availability of massively parallel sequencing for detection of all coding mutations within tumours, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous human leukocyte antigen (HLA) molecules. We hypothesized that vaccination with neoantigens can both expand pre-existing neoantigen-specific T-cell populations and induce a broader repertoire of new T-cell specificities in cancer patients, tipping the intra-tumoural balance in favour of enhanced tumour control. Here we demonstrate the feasibility, safety, and immunogenicity of a vaccine that targets up to 20 predicted personal tumour neoantigens. Vaccine-induced polyfunctional CD4
and CD8
T cells targeted 58 (60%) and 15 (16%) of the 97 unique neoantigens used across patients, respectively. These T cells discriminated mutated from wild-type antigens, and in some cases directly recognized autologous tumour. Of six vaccinated patients, four had no recurrence at 25 months after vaccination, while two with recurrent disease were subsequently treated with anti-PD-1 (anti-programmed cell death-1) therapy and experienced complete tumour regression, with expansion of the repertoire of neoantigen-specific T cells. These data provide a strong rationale for further development of this approach, alone and in combination with checkpoint blockade or other immunotherapies.
Abstract
Desmoid tumors (DTs) are unusual neoplasms of mesenchymal origin that exhibit locally invasive behavior. Surgical resection is the initial treatment of choice for DTs. For patients with ...recurrent or unresectable disease, however, medical options are limited. Sorafenib is a multikinase inhibitor with known antitumor activity in various cancers via suppression of the PI3K/Akt/mTOR pathway. Here, we examined the effects of sorafenib on patient-derived DT cell lines, with the aim of characterizing the efficacy and molecular mechanism of action. Early passage DT-derived cells were treated with increasing doses of sorafenib (0-10 µM) and demonstrated up to 90% decrease in proliferation and invasion relative to controls. Signaling arrays identified multiple potential targets of sorafenib in the Ras/MEK/ERK and PI3K/Akt/mTOR signaling cascades. Immunoblot analysis revealed that sorafenib inhibited Akt, MEK and ERK phosphorylation, and this effect correlated with inhibition of total Akt and total MEK, while total ERK levels remained unchanged. Sorafenib also inhibited 4E-BP1 phosphorylation, and this effect correlated with decrease of p-eIF4E and total eIF4E. Finally, in combination with the mammalian target of rapamycin (mTOR) inhibitor everolimus, sorafenib decreased phosphorylation of the ribosomal protein and mTOR effector S6K in an additive manner. Taken together, our results suggest that sorafenib suppresses DT proliferation and invasion via inhibition of Ras/MEK/ERK and PI3K/Akt/mTOR signaling pathways with additional effects on translation. Sorafenib may be a promising therapeutic option in the treatment of DTs. Additional studies in DT patients are warranted to examine the efficacy of combination therapy using sorafenib.
Sorafenib is a multikinase inhibitor that demonstrates antitumor effects in patient-derived desmoid tumor (DT) cell lines including decreased proliferation and invasion. Signaling pathways targeted by sorafenib in DTs include Ras/MEK/ERK and PI3K/Akt/mTOR pathways with additional effects on translation.
To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, ...immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.
There is a need for better classification and understanding of tumor-infiltrating lymphocytes (TILs). Here, we applied advanced functional genomics to interrogate 9,000 human tumors and multiple ...single-cell sequencing sets using benchmarked T cell states, comprehensive T cell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared with other T cell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8+ TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early T cell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity.
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•Improved global TIL classification methods are required to deconvolve cell states•αPD-1 non-responder TILs and dysfunctional TILs score for T activation, not exhaustion•αPD-1 response and patient survival associates with late T cell memory/TRM scoring•Persistent programs co-express with DC maturation and IFN-γ response programs
Jaiswal et al. highlight the need for improved tumor-infiltrating lymphocyte (TIL) classification by showing that current transcriptome assignments may misclassify early activated/effector TILs as exhausted. The study surveys 9,000 solid tumors, multiple single-cell RNA sequencing sets, mouse and human models, and scoring methods to reclassify TILs and associate melanoma survival to T cell memory/resident memory.
Socioeconomic variables including sex, race, ethnicity, marital status, and insurance status are associated with survival in pancreatic cancer. It remains unknown exactly how these variables ...influence survival, including whether they affect stage at presentation or receipt of treatment or are independently associated with outcomes.
To investigate the relationship between socioeconomic factors and odds of resection in early-stage, resectable pancreatic adenocarcinoma and to determine whether these same factors were independently associated with survival in patients who underwent resection.
This was a retrospective cohort study of patients diagnosed as having T1 through T3 M0 pancreatic adenocarcinoma between January 1, 2004, and December 31, 2011, identified from the Surveillance, Epidemiology, and End Results database.
Socioeconomic and geographic variables associated with utilization of resection and disease-specific survival.
A total of 17,530 patients with localized, nonmetastatic pancreatic cancer were identified. The resection rate among these patients was 45.4% and did not change over time. Utilization of resection was independently associated with white vs African American race (odds ratio OR = 0.76; 95% CI, 0.65-0.88; P < .001), non-Hispanic ethnicity (for Hispanic, OR = 0.72; 95% CI, 0.60-0.85; P < .001), married status (OR = 1.42; 95% CI, 1.30-1.57; P < .001), insurance coverage (OR = 1.63; 95% CI, 1.22-2.18; P = .001), and the Northeast region (vs Southeast, OR = 1.67; 95% CI, 1.44-1.94; P < .001). Stage at presentation correlated with sex, race, ethnicity, marital status, and geographic region (ethnicity, P = .003; all others, P < .001); however, the factors associated with increased resection correlated with more advanced stage. Patients who underwent resection had significantly improved disease-specific survival compared with those who did not undergo resection (median, 21 vs 6 months; hazard ratio HR for disease-specific death = 0.32; 95% CI, 0.31-0.33; P < .001). Disease-specific survival among the patients who underwent surgical resection was independently associated with geographic region, with patients in the Pacific West (HR for death = 0.706; 95% CI, 0.628-0.793), Northeast (HR for death = 0.766; 95% CI, 0.667-0.879), and Midwest (HR for death = 0.765; 95% CI, 0.640-0.913) having improved survival in comparison with those in the Southeast (all P < .001).
Disparities in the utilization of surgical resection for patients with early-stage, resectable pancreatic cancer are associated with socioeconomic variables including race, ethnicity, marital status, insurance status, and geographic location. Of these factors, only geographic location is independently associated with survival in patients undergoing resection.
Most targeted cancer therapies fail to achieve complete tumor regressions or attain durable remissions. To understand why these treatments fail to induce robust cytotoxic responses despite ...appropriately targeting oncogenic drivers, here we systematically interrogated the dependence of cancer cells on the BCL-2 family of apoptotic proteins after drug treatment. We observe that multiple targeted therapies, including BRAF or EGFR inhibitors, rapidly deplete the pro-apoptotic factor NOXA, thus creating a dependence on the anti-apoptotic protein MCL-1. This adaptation requires a pathway leading to destabilization of the NOXA mRNA transcript. We find that interruption of this mechanism of anti-apoptotic adaptive resistance dramatically increases cytotoxic responses in cell lines and a murine melanoma model. These results identify NOXA mRNA destabilization/MCL-1 adaptation as a non-genomic mechanism that limits apoptotic responses, suggesting that sequencing of MCL-1 inhibitors with targeted therapies could overcome such widespread and clinically important resistance.
Patients with stage IIB or IIC melanoma who undergo surgery alone are at a substantial risk for disease recurrence. Adjuvant pembrolizumab significantly improved recurrence-free survival versus ...placebo in stage IIB or IIC melanoma in the first interim analysis of the KEYNOTE-716 trial. Here, we report results from the secondary endpoint of distant metastasis-free survival (prespecified third interim analysis), and recurrence-free survival with longer follow-up.
KEYNOTE-716 is a multicentre, double-blind, placebo-controlled, crossover or rechallenge, randomised, phase 3 trial done at 160 academic medical centres and hospitals across 16 countries. Eligible patients were aged 12 years and older with newly-diagnosed, completely resected, and histologically confirmed stage IIB (T3b or T4a) or IIC (T4b) cutaneous melanoma; negative sentinel lymph node biopsy; and an Eastern Cooperative Oncology Group performance status of 0–1. Patients were randomly assigned (1:1) to receive either 200 mg of pembrolizumab (2 mg/kg up to a maximum of 200 mg in paediatric patients) or placebo, both intravenously, every 3 weeks for 17 cycles (part 1) or until disease recurrence or unacceptable toxicity. Eligible patients with disease recurrence could receive further treatment with pembrolizumab in the part 2 crossover or rechallenge phase. Randomisation was done using an interactive response technology system and stratified by T category and paediatric status. The primary endpoint was investigator-assessed recurrence-free survival (assessed here with longer follow-up), and we report the prespecified third interim analysis of distant metastasis-free survival (secondary endpoint). Efficacy analyses were done in the intention-to-treat population (all patients who were randomly assigned, according to assigned group) and safety was assessed in all patients who were randomly assigned and received at least one dose of trial treatment, according to the treatment received. KEYNOTE-716 is registered at ClinicalTrials.gov, NCT03553836, and has completed recruitment.
Between Sept 23, 2018, and Nov 4, 2020, 976 patients were randomly assigned to receive pembrolizumab (n=487) or placebo (n=489). At a median follow-up of 27·4 months (IQR 23·1–31·7), median distant metastasis-free survival was not reached (95% CI not reached NR–NR) in either group. Pembrolizumab significantly improved distant metastasis-free survival (hazard ratio HR 0·64, 95% CI 0·47–0·88, p=0·0029) versus placebo. Median recurrence-free survival was 37·2 months (95% CI NR–NR) in the pembrolizumab group and not reached in the placebo group (95% CI NR–NR). The risk of recurrence remained lower with pembrolizumab versus placebo (HR 0·64, 95% CI 0·50–0·84). The most common grade 3 or worse adverse events were hypertension (16 3% of 483 patients in the pembrolizumab group vs 17 4% of 486 patients in the placebo group), diarrhoea (eight 2% vs one <1%), rash (seven 1% vs two <1%), autoimmune hepatitis (seven 1% vs two <1%), and increased lipase (six 1% vs eight 2%). Treatment-related serious adverse events occurred in 49 (10%) patients in the pembrolizumab group and 11 (2%) patients in the placebo group. No treatment-related deaths were reported.
Adjuvant pembrolizumab is an efficacious treatment option for resected stage IIB and IIC melanoma, with significant improvement in distant-metastasis free survival versus placebo and continued reduction in the risk of recurrence with an adverse event profile consistent with previous studies of pembrolizumab. The overall benefit–risk of pembrolizumab continues to be positive in the adjuvant setting.
Merck Sharp & Dohme, a subsidiary of Merck & Co.
Within the tumour microenvironment, CD4
T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules
, but ...how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4
T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4
T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4
T regulatory (T
) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4
T
clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4
T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4
T
cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma.
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