We report long-term clinical outcomes and immune responses observed from a phase 1 trial of agonist CD40 monoclonal antibody (mAb) and blocking CTLA-4 mAb in patients with metastatic melanoma. Twenty-four patients previously untreated with checkpoint blockade were enrolled. The agonistic CD40 mAb CP-870,893 and the CTLA-4 blocking mAb tremelimumab were dosed concomitantly every 3 weeks and 12 weeks, respectively, across four dose combinations. Two patients developed dose-limiting grade 3 immune-mediated colitis that led to the definition of the maximum tolerated dose (MTD). Other immune-mediated toxicity included uveitis (n = 1), hypophysitis (n = 1), hypothyroidism (n = 2), and grade 3 cytokine release syndrome (CRS) (n = 1). The estimated MTD was 0.2 mg/kg of CP-870,893 and 10 mg/kg of tremelimumab. In 22 evaluable patients, the objective response rate (ORR) was 27.3%: two patients (9.1%) had complete responses (CR) and four (18.2%) patients had partial responses (PR). With a median follow-up of 45 months, the median progression-free survival (PFS) was 3.2 months (95% CI, 1.3-5.1 months) and median overall survival (OS) was 23.6 months (95% CI, 11.7-35.5 months). Nine patients are long-term survivors (> 3 years), 8 of whom subsequently received other therapy including PD-1 mAb, surgery, or radiation therapy. Elevated baseline soluble CD25 was associated with shorter OS. Immunologically, treatment was associated with evidence of T cell activation and increased tumor T cell infiltration that was accomplished without therapeutic PD-1/PD-L1 blockade. These results suggest opportunities for immune activation and cancer immunotherapy beyond PD-1.

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.

Deregulation of the cell cycle machinery is a hallmark of cancer. While CDK4/6 inhibitors are FDA approved (palbociclib) for treating advanced estrogen receptor-positive breast cancer, two major clinical challenges remain: (i) adverse events leading to therapy discontinuation and (ii) lack of reliable biomarkers. Here we report that breast cancer cells activate autophagy in response to palbociclib, and that the combination of autophagy and CDK4/6 inhibitors induces irreversible growth inhibition and senescence in vitro, and diminishes growth of cell line and patient-derived xenograft tumours in vivo. Furthermore, intact G1/S transition (Rb-positive and low-molecular-weight isoform of cyclin E (cytoplasmic)-negative) is a reliable prognostic biomarker in ER positive breast cancer patients, and predictive of preclinical sensitivity to this drug combination. Inhibition of CDK4/6 and autophagy is also synergistic in other solid cancers with an intact G1/S checkpoint, providing a novel and promising biomarker-driven combination therapeutic strategy to treat breast and other solid tumours.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and β-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.

Autophagy is a resistance mechanism to BRAF/MEK inhibition in BRAFV600-mutant melanoma. Here we used hydroxychloroquine (HCQ) to inhibit autophagy in combination with dabrafenib 150 mg twice daily and trametinib 2 mg every day (D+T).

The lack of tumor infiltration by CD8+ T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8+ T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8+ T cells into tumors. In tissue samples from patients with melanoma, CD8+ T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1+ immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.

Substantial COVID-19 research investment has been allocated to randomized clinical trials (RCTs) on hydroxychloroquine/chloroquine, which currently face recruitment challenges or early discontinuation. We aim to estimate the effects of hydroxychloroquine and chloroquine on survival in COVID-19 from all currently available RCT evidence, published and unpublished. We present a rapid meta-analysis of ongoing, completed, or discontinued RCTs on hydroxychloroquine or chloroquine treatment for any COVID-19 patients (protocol: https://osf.io/QESV4/ ). We systematically identified unpublished RCTs (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform, Cochrane COVID-registry up to June 11, 2020), and published RCTs (PubMed, medRxiv and bioRxiv up to October 16, 2020). All-cause mortality has been extracted (publications/preprints) or requested from investigators and combined in random-effects meta-analyses, calculating odds ratios (ORs) with 95% confidence intervals (CIs), separately for hydroxychloroquine and chloroquine. Prespecified subgroup analyses include patient setting, diagnostic confirmation, control type, and publication status. Sixty-three trials were potentially eligible. We included 14 unpublished trials (1308 patients) and 14 publications/preprints (9011 patients). Results for hydroxychloroquine are dominated by RECOVERY and WHO SOLIDARITY, two highly pragmatic trials, which employed relatively high doses and included 4716 and 1853 patients, respectively (67% of the total sample size). The combined OR on all-cause mortality for hydroxychloroquine is 1.11 (95% CI: 1.02, 1.20; I² = 0%; 26 trials; 10,012 patients) and for chloroquine 1.77 (95%CI: 0.15, 21.13, I² = 0%; 4 trials; 307 patients). We identified no subgroup effects. We found that treatment with hydroxychloroquine is associated with increased mortality in COVID-19 patients, and there is no benefit of chloroquine. Findings have unclear generalizability to outpatients, children, pregnant women, and people with comorbidities.

Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in "immune hot" tumors but not in "immune cold" tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Therapeutic blocking of the PD1 pathway results in significant tumor responses, but resistance is common. We demonstrate that prolonged interferon signaling orchestrates PDL1-dependent and PDL1-independent resistance to immune checkpoint blockade (ICB) and to combinations such as radiation plus anti-CTLA4. Persistent type II interferon signaling allows tumors to acquire STAT1-related epigenomic changes and augments expression of interferon-stimulated genes and ligands for multiple T cell inhibitory receptors. Both type I and II interferons maintain this resistance program. Crippling the program genetically or pharmacologically interferes with multiple inhibitory pathways and expands distinct T cell populations with improved function despite expressing markers of severe exhaustion. Consequently, tumors resistant to multi-agent ICB are rendered responsive to ICB monotherapy. Finally, we observe that biomarkers for interferon-driven resistance associate with clinical progression after anti-PD1 therapy. Thus, the duration of tumor interferon signaling augments adaptive resistance and inhibition of the interferon response bypasses requirements for combinatorial ICB therapies.

One of the major challenges limiting the efficacy of anti-PD-1/PD-L1 therapy in nonresponding patients is the failure of T cells to penetrate the tumor microenvironment. We showed that genetic or pharmacological inhibition of Vps34 kinase activity using SB02024 or SAR405 (Vps34i) decreased the tumor growth and improved mice survival in multiple tumor models by inducing an infiltration of NK, CD8+, and CD4+ T effector cells in melanoma and CRC tumors. Such infiltration resulted in the establishment of a T cell-inflamed tumor microenvironment, characterized by the up-regulation of pro-inflammatory chemokines and cytokines, CCL5, CXCL10, and IFNγ. Vps34i treatment induced STAT1 and IRF7, involved in the up-regulation of CCL5 and CXCL10. Combining Vps34i improved the therapeutic benefit of anti-PD-L1/PD-1 in melanoma and CRC and prolonged mice survival. Our study revealed that targeting Vps34 turns cold into hot inflamed tumors, thus enhancing the efficacy of anti-PD-L1/PD-1 blockade.

New strategies are needed to enhance the efficacy of anti-programmed cell death protein antibody (anti-PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-β in macrophages, the latter being a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.

The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas.

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin-Sca-1+c-kit+CD48-CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.

Atg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.

CD8+ T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8+ T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at "re-invigorating" Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1+ progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy.

Here, we report that functional heterogeneity of macrophages in cancer could be determined by the nature of their precursors: monocytes (Mons) and monocytic myeloid-derived suppressor cells (M-MDSCs). Macrophages that are differentiated from M-MDSCs, but not from Mons, are immune suppressive, with a genomic profile matching that of M-MDSCs. Immune-suppressive activity of M-MDSC-derived macrophages is dependent on the persistent expression of S100A9 protein in these cells. S100A9 also promotes M2 polarization of macrophages. Tissue-resident- and Mon-derived macrophages lack expression of this protein. S100A9-dependent immune-suppressive activity of macrophages involves transcription factor C/EBPβ. The presence of S100A9-positive macrophages in tumor tissues is associated with shorter survival in patients with head and neck cancer and poor response to PD-1 antibody treatment in patients with metastatic melanoma. Thus, this study reveals the pathway of the development of immune-suppressive macrophages and suggests an approach to their selective targeting.

Autophagy, a multistep lysosomal degradation pathway that supports nutrient recycling and metabolic adaptation, has been implicated as a process that regulates cancer. Although autophagy induction may limit the development of tumors, evidence in mouse models demonstrates that autophagy inhibition can limit the growth of established tumors and improve response to cancer therapeutics. Certain cancer genotypes may be especially prone to autophagy inhibition. Different strategies for autophagy modulation may be needed depending on the cancer context. Here, we review new advances in the molecular control of autophagy, the role of selective autophagy in cancer, and the role of autophagy within the tumor microenvironment and tumor immunity. We also highlight clinical efforts to repurpose lysosomal inhibitors, such as hydroxychloroquine, as anticancer agents that block autophagy, as well as the development of more potent and specific autophagy inhibitors for cancer treatment, and review future directions for autophagy research. SIGNIFICANCE: Autophagy plays a complex role in cancer, but autophagy inhibition may be an effective therapeutic strategy in advanced cancer. A deeper understanding of autophagy within the tumor microenvironment has enabled the development of novel inhibitors and clinical trial strategies. Challenges and opportunities remain to identify patients most likely to benefit from this approach.

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor-Lys05-with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.