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.

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.

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.

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.

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.

We conducted a phase I trial evaluating pembrolizumab+hypofractionated radiotherapy (HFRT) for patients with metastatic cancers.

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.

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.

"Day-7" myeloid DCs are commonly used in the clinic. However, there is a strong need to develop DCs faster that have the same potent immunostimulatory capacity as "Day-7" myeloid DCs and at the same time minimizing time, labor and cost of DC preparations. Although "2 days" DCs can elicit peptide-specific responses, they have not been demonstrated to engulf, process and present complex whole tumor lysates, which could be more convenient and personalized source of tumor antigens than defined peptides. In this preclinical study, we evaluated the T-cell stimulatory capacity of Day-2, Day-4, and Day-7 cultured monocyte-derived DCs loaded with SKOV3 cell whole lysate prepared by freeze-thaw or by UVB-irradiation followed by freeze-thaw, and matured with lipopolysaccharide (LPS) and interferon (IFN)-gamma. DCs were evaluated for antigen uptake, and following maturation with LPS and IFN-gamma, DCs were assessed for expression of CD80, CD40, CD86, ICAM-1 and CCR7, production of IL-12p70 and IP-10, and induction of tumor-specific T-cell responses. Day-4 and Day-7 DCs exhibited similar phagocytic abilities, which were superior to Day-2 DCs. Mature Day-7 DCs expressed the highest CD40 and ICAM-1, but mature Day-4 DCs produced the most IL-12p70 and IP-10. Importantly, Day-4 and Day-7 DCs derived from ovarian cancer patients stimulated equally strongly tumor-specific T-cell responses. This is the first study demonstrating the highly immunogenic and strong T-cell stimulatory properties of Day-4 myeloid DCs, and provided important preclinical data for rapid development of potent whole tumor lysate-loaded DC vaccines that are applicable to many tumor types.

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.

T cells are important for controlling ovarian cancer (OC). We previously demonstrated that combinatorial use of a personalized whole-tumor lysate-pulsed dendritic cell vaccine (OCDC), bevacizumab (Bev), and cyclophosphamide (Cy) elicited neoantigen-specific T cells and prolonged OC survival. Here, we hypothesize that adding acetylsalicylic acid (ASA) and low-dose interleukin (IL)-2 would increase the vaccine efficacy in a recurrent advanced OC phase I trial (NCT01132014). By adding ASA and low-dose IL-2 to the OCDC-Bev-Cy combinatorial regimen, we elicited vaccine-specific T-cell responses that positively correlated with patients' prolonged time-to-progression and overall survival. In the ID8 ovarian model, animals receiving the same regimen showed prolonged survival, increased tumor-infiltrating perforin-producing T cells, increased neoantigen-specific CD8+ T cells, and reduced endothelial Fas ligand expression and tumor-infiltrating T-regulatory cells. This combinatorial strategy was efficacious and also highlighted the predictive value of the ID8 model for future ovarian trial development.

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 previously performed animal studies and Phase I-II human trials, Bowman-Birk inhibitor concentrate (BBIC) appeared to be a promising cancer chemopreventive agent. The present study describes the results of two phase I randomized double-blind placebo-controlled trials performed in male subjects to assess the safety and toxicity of the original and new formulations of BBIC administered in a single dose as a suspension in orange juice. The dose of BBIC varied from 800-2,000 chymotrypsin inhibitor (CI) units. The BBI concentration in the serum samples collected from the subjects was analyzed by a dot-blot analysis procedure using the 5G2 monoclonal antibody, which is specific for reduced BBI. A total of 41 subjects were enrolled, 20 in the initial BBIC study and 21 in the second BBIC study. In these human trials, no clinically relevant changes in hematological or biochemical parameters were observed. Overall, BBIC was found to be well-tolerated. For these BBIC single-dose phase I trials, there was no dose-limiting toxicity for BBIC, even at the highest dose evaluated, and there were no apparent differences between the clinical trial results for the two formulations of BBIC. The bioavailability of BBI in the second clinical trial, which used the new BBIC formulation, was approximately 40 to 43% of the BBI bioavailability reached in the first clinical trial, which used the original BBIC formulation. The observed bioavailability difference was attributed to the different BBIC formulations used in these two clinical trials. These trials demonstrated that BBIC is safe when administered in a single dose of up to 2,000 CI units. Therefore, the results from the two trials indicate that a multi-dose trial of BBIC may be safely performed with doses of up to 2,000 CI units per day.

CD40 is a cell-surface molecule that critically regulates immune responses. CP-870,893 is a fully human, CD40-specific agonist monoclonal antibody (mAb) exerting clinical antineoplastic activity. Here, the safety of CP-870,893 combined with carboplatin and paclitaxel was assessed in a Phase I study. Patients with advanced solid tumors received standard doses of paclitaxel and carboplatin on day 1 followed by either 0.1 mg/Kg or 0.2 mg/Kg CP-870,893 on day 3 (Schedule A) or day 8 (Schedule B), repeated every 21 d. The primary objective was to determine safety and maximum-tolerated dose (MTD) of CP-870,893. Secondary objectives included the evaluation of antitumor responses, pharmacokinetics and immune modulation. Thirty-two patients were treated with CP-870,893, 16 patients on each schedule. Two dose-limiting toxicities were observed (grade 3 cytokine release and transient ischemic attack), each at the 0.2 mg/Kg dose level, which was estimated to be the MTD. The most common treatment-related adverse event was fatigue (81%). Of 30 evaluable patients, 6 (20%) exhibited partial responses constituting best responses as defined by RECIST. Following CP-870,893 infusion, the peripheral blood manifested an acute depletion of B cells associated with upregulation of immune co-stimulatory molecules. T-cell numbers did not change significantly from baseline, but transient tumor-specific T-cell responses were observed in a small number of evaluable patients. The CD40 agonist mAb CP-870,893, given on either of two schedules in combination with paclitaxel and carboplatin, was safe for patients affected with advanced solid tumors. Biological and clinical responses were observed, providing a rationale for Phase II studies.

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.

Dendritic cell (DC) function will likely be important for immunological cancer therapies, but our knowledge of the roles of DCs in tumors is limited, in part because most studies were performed before DC subtypes were defined. We studied the distributions of immature epidermal, immature dermal, mature, and plasmacytoid DCs in 63 primary tumors and eight lymph node metastases from oral squamous cell carcinoma patients without evidence of distant metastasis. Immature CD207/Langerin+ and CD209/DC-SIGN+ DCs were present in the primary tumor region, but CD209/DC-SIGN+ cells rarely infiltrated the tumor. Mature DCs were rare, and presence of CD123+ plasmacytoid DCs was associated with poorer outcome. Unexpectedly, migration and maturation of DCs was associated with a worse outcome. Overall, the distribution of DC subtypes indicated that ineffective DC response to tumor is a failure of DC function rather than recruitment, suggesting that a strategy of shifting the balance of secreted factors in the tumor milieu may be more effective in restoring anti-tumor immune function than current methods restoring only one population of DCs to the immunosuppressive tumor region.