Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in β-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Desmoplastic melanoma is a rare subtype of melanoma characterized by dense fibrous stroma, resistance to chemotherapy and a lack of actionable driver mutations, and is highly associated with ultraviolet light-induced DNA damage. We analysed sixty patients with advanced desmoplastic melanoma who had been treated with antibodies to block programmed cell death 1 (PD-1) or PD-1 ligand (PD-L1). Objective tumour responses were observed in forty-two of the sixty patients (70%; 95% confidence interval 57-81%), including nineteen patients (32%) with a complete response. Whole-exome sequencing revealed a high mutational load and frequent NF1 mutations (fourteen out of seventeen cases) in these tumours. Immunohistochemistry analysis from nineteen desmoplastic melanomas and thirteen non-desmoplastic melanomas revealed a higher percentage of PD-L1-positive cells in the tumour parenchyma in desmoplastic melanomas (P = 0.04); these cells were highly associated with increased CD8 density and PD-L1 expression in the tumour invasive margin. Therefore, patients with advanced desmoplastic melanoma derive substantial clinical benefit from PD-1 or PD-L1 immune checkpoint blockade therapy, even though desmoplastic melanoma is defined by its dense desmoplastic fibrous stroma. The benefit is likely to result from the high mutational burden and a frequent pre-existing adaptive immune response limited by PD-L1 expression.

Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK). Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-'addicted' human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rβ-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.