We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1. We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies. Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice. While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. .

Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy signifi cantly better than hydroxychloroquine (HCQ). Using an in situ photoaffi nity pulldown strategy, we identifi ed palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in signifi cant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives. SIGNIFICANCE:This study identifi es PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These fi ndings provide a strong rationale for targeting PPT1 in cancer.

Lysosomes serve dual roles in cancer metabolism, executing catabolic programs (i.e. autophagy and macropinocytosis), while promoting mTORC1-dependent anabolism. Antimalarial compounds such as chloroquine or quinacrine have been used as lysosomal inhibitors, but fail to inhibit mTOR signaling. Further, the molecular target of these agents has not been identified. We report a screen of novel dimeric antimalarials that identifies dimeric quinacrines (DQs) as potent anticancer compounds, which concurrently inhibit mTOR and autophagy. Central nitrogen methylation of the DQ linker enhances lysosomal localization and potency. An in situ photoaffinity pulldown identified palmitoyl-protein thioesterase 1 (PPT1) as the molecular target of DQ661. PPT1 inhibition concurrently impairs mTOR and lysosomal catabolism through the rapid accumulation of palmitoylated proteins. DQ661 inhibits the in vivo tumor growth of melanoma, pancreatic, and colorectal cancer mouse models and can be safely combined with chemotherapy. Thus, lysosome-directed PPT1 inhibitors represent a new approach to concurrently targeting mTORC1 and lysosomal catabolism in cancer.

Older melanoma patients have lower rates of sentinel lymph node (LN) metastases yet paradoxically have inferior survival. Patient age correlated with an inability to retain Technetium radiotracer during sentinel LN biopsy in over 1000 patients, and high technecium counts correlated to better survival. We hypothesized that loss of integrity in the lymphatic vasculature due to ECM degradation might play a role. We have implicated HAPLN1 in age-dependent ECM degradation in the dermis. Here we queried whether HAPLN1 could be altered in the lymphatic ECM. Lymphatic HAPLN1 expression was prognostic of long-term patient survival. Adding rHAPLN1 to aged fibroblast ECMs in vitro reduced endothelial permeability via modulation of VE-Cadherin junctions, whereas endothelial permeability was increased following HAPLN1-knockdown in young fibroblasts. In vivo, reconstitution of HAPLN1 in aged mice increased the number of LN metastases, but reduced visceral metastases. These data suggest that age-related changes in ECM can contribute to impaired lymphatics.