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Cancer cells are exposed to various intrinsic and extrinsic factors that disrupt protein homeostasis, producing endoplasmic reticulum (ER) stress. To cope with these situations, cancer cells evoke a highly conserved adaptive mechanism called the unfolded protein response (UPR) to restore the ER homeostasis. Recently, several pharmacological agents have been found to exhibit anti-tumor activity by targeting the UPR components. The development of potent and specific compounds that target the UPR components has not only shed light on the regulation of the UPR in cancer cells, but also brought the field closer to clinical drug candidates. Here we present an overview of the milestones in the field of UPR biology in cancer with a focus on new strategies for pharmacological inhibition. [ABSTRACT FROM AUTHOR]

Eukaryotes use autophagy as a mechanism for maintaining cellular homeostasis by degrading and recycling organelles and proteins. This process assists in the proliferation and survival of advanced cancers. There is mounting preclinical evidence that targeting autophagy can enhance the efficacy of many cancer therapies. Hydroxychloroquine (HCQ) is the only clinically-approved autophagy inhibitor, and this systematic review focuses on HCQ use in cancer clinical trials. Preclinical trials have shown that HCQ alone and in combination therapy leads to enhancement of tumor shrinkage. This has provided the base for multiple ongoing clinical trials involving HCQ alone and in combination with other treatments. However, due to its potency, there is still a need for more potent and specific autophagy inhibitors. There are multiple autophagy inhibitors in the pre-clinical stage at various stages of development. Additional studies on the mechanism of HCQ and other autophagy inhibitors are still required to answer questions surrounding how these agents will eventually be used in the clinic. [ABSTRACT FROM AUTHOR]

Lysosomes are membrane-bound intracellular organelles that receive macromolecules delivered by endocytosis, phagocytosis, and autophagy for degradation and recycling. Over the last decade, advances in lysosome research have established a broad role for the lysosome in the pathophysiology of disease. In this review, we highlight the recent discoveries in lysosome biology, with an emphasis on their implications for cancer therapy. We focus on targeting the lysosome in cancer by exploring lysosomal biogenesis and its role in the crosstalk between apoptosis and autophagy. We also discuss how lysosomal inhibition could emerge as a new therapeutic strategy to overcome drug resistance in cancer. [ABSTRACT FROM AUTHOR]

Autophagy is a degradative and recycling process that is upregulated in cancer cells. New advances in understanding the mechanism of autophagy have uncovered novel potential targets for drug development. Clinical trials involving hydroxychloroquine have demonstrated the safety of targeting autophagy, but efficacy could be improved. Autophagy regulates tumor immunity. Novel autophagy inhibitors are entering clinical trials. Autophagy is a cellular homeostasis mechanism that fuels the proliferation and survival of advanced cancers by degrading and recycling organelles and proteins. Preclinical studies have identified that within an established tumor, tumor cell autophagy and host cell autophagy conspire to support tumor growth. A growing body of evidence suggests that autophagy inhibition can augment the efficacy of chemotherapy, targeted therapy, or immunotherapy to enhance tumor shrinkage. First-generation autophagy inhibition trials in cancer using the lysosomal inhibitor hydroxychloroquine (HCQ) have produced mixed results but have guided the way for the development of more potent and specific autophagy inhibitors in clinical trials. In this review, we will discuss the role of autophagy in cancer, newly discovered molecular mechanisms of the autophagy pathway, the effects of autophagy modulation in cancer and host cells, and novel autophagy inhibitors that are entering clinical trials. [ABSTRACT FROM AUTHOR]

Introduction Vemurafenib induces tumour regression in most patients with BRAF V600E -mutant melanoma; eventually, most experience progressive disease (PD). Long-term follow-up of patients with BRAF V600E melanoma treated in the phase 1 vemurafenib trial is reported. Methods Patients received vemurafenib 240–1120 mg (dose escalation cohort) or 960 mg (extension cohort) orally twice daily. Clinical response was evaluated every 8 weeks by Response Evaluation Criteria In Solid Tumors (RECIST). Patients with PD amenable to local therapy (surgery or radiotherapy) were allowed to continue vemurafenib after progression. Overall survival (OS) from time of treatment initiation and from PD was estimated. Sites of PD were recorded. Results Forty-eight patients (escalation cohort, n = 16; extension cohort, n = 32) received therapeutic doses of vemurafenib (⩾240 mg twice daily). Forty-four patients had PD by the time of this analysis and four remained progression free (follow-up time, 1.2–56.1 months). Median OS was 14 months (range, 1.2–56.1); 3- and 4-year melanoma-specific survival rate in the extension cohort was 26% and 19%, respectively. Median OS was 26.0 months (range, 7.7–56.1) among 20 patients who continued vemurafenib after local therapy. Median treatment duration beyond initial PD was 3.8 months (range, 1.1–26.6). In the extension cohort, six and five patients were alive after 3 and 4 years, respectively, on vemurafenib monotherapy. Conclusions Some patients with melanoma achieved long-term survival with vemurafenib monotherapy. Continuation of vemurafenib after PD might be beneficial in some patients because remaining disease might continue to respond to BRAF inhibition. [ABSTRACT FROM AUTHOR]

Cancer cells display several features of aberrant cellular metabolism. Two consequences of this dysregulated metabolism are rapid depletion of intracellular nutrients and a buildup of aggregated proteins and damaged organelles. Autophagy provides a mechanism for recycling proteins, lipids, and organelles. In cancer cells, oncogenes and conditions of severe stress drive profound upregulation of autophagy. In this setting, autophagy ameliorates the ill effects of dysregulated cellular metabolism, allowing a steady supply of nutrients and removal of damaged organelles. Although therapeutic strategies targeting cancer cell metabolism and autophagy are already entering clinical trials, further study of the precise mechanisms of interplay between oncogenic signaling, cellular metabolism, and autophagy will provide more effective strategies in the future. [ABSTRACT FROM AUTHOR]

Autophagy--a process of "self-eating" that involves enzymatic digestion and recycling of cellular constituents in response to stress--contributes to both cancer cell death and survival. In this issue of the JCI, Lu et al. report that controlled induction of tumor suppressor gene aplasia Ras homolog member I (ARHI) results in autophagic cell death of human ovarian cancer cells in vitro (see the related article beginning on page 3917). However, within xenograft tumors in mice, multiple factors within the tumor microenvironment switched ARHI-induced autophagy to a mechanism of tumor cell survival, leading to tumor dormancy. Since ARHI expression is suppressed in the majority of breast and ovarian cancers but is high in premalignant lesions, ARHI-induced autophagy could be manipulated for therapeutic benefit. [ABSTRACT FROM AUTHOR]

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Whether autophagy observed in treated cancer cells represents a mechanism that allows tumor cells to survive therapy or a mechanism for initiating a nonapoptotic form of programmed cell death remains controversial. To address this issue, the role of autophagy in a Myc-induced model of lymphoma generated from cells derived from p53ER(TAM)/p53ER(TAM) mice (with ER denoting estrogen receptor) was examined. Such tumors are resistant to apoptosis due to a lack of nuclear p53. Systemic administration of tamoxifen led to p53 activation and tumor regression followed by tumor recurrence. Activation of p53 was associated with the rapid appearance of apoptotic cells and the induction of autophagy in surviving cells. Inhibition of autophagy with either chloroquine or ATG5 short hairpin RNA (shRNA) enhanced the ability of either p53 activation or alkylating drug therapy to induce tumor cell death. These studies provide evidence that autophagy serves as a survival pathway in tumor cells treated with apoptosis activators and a rationale for the use of autophagy inhibitors such as chloroquine in combination with therapies designed to induce apoptosis in human cancers. [ABSTRACT FROM AUTHOR]

RAS-driven cancers exhibit variable dependency on autophagy for survival; however, it is not fully understood how. In this issue of the JCI, Cheong and colleagues demonstrate that RAS-dependent elevation of casein kinase 1α (CK1α) negatively regulates autophagy at the level of autophagy gene transcription. Moreover, combined inhibition of both CK1α and autophagy reduced proliferation of RAS-driven tumors. The results of this study provide insight into the connection between mutant RAS and autophagy, and suggest targeting CK1α as a potential therapeutic strategy to modulate autophagy in RAS-driven cancers. [ABSTRACT FROM AUTHOR]

Chloroquine (CQ) can induce cell death in a subset of cancer cell lines, and some melanoma cell lines are quite susceptible. Although it is well known that CQ impairs lysosomal function and can serve as an autophagy inhibitor, the molecular target of CQ and the subsequent cascade of events that lead to cell death are not fully understood. Recent evidence indicates that in melanoma cell lines, CQ induces apoptosis by preventing degradation of the pro-apoptotic BH3-only protein p53-upregulated modulator of apoptosis. This finding adds to the unfolding story of CQ's mechanism of action as a cancer therapeutic agent. [ABSTRACT FROM AUTHOR]

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy‐related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders. [ABSTRACT FROM AUTHOR]

The article discusses a report within the issue regarding cancer research by Michaud and colleagues who show chemotherapy-induced autophagy causes the release of the co-enzyme adenosine 5'-triphosphate (ATP) from tumor cells, which stimulates anti-tumor immune responses. Michaud's team found that when the expression of the genes that encode essential autophagy proteins, ATG5 and ATG7, was reduced in immunogenic allograft mouse tumors, it blunted the release of ATP by the tumor cells. Also discussed is how tumor cell autophagy limits immune-mediated cytotoxicity and the usefulness of transgenic mouse models, deficient in autophagy, in exploring the impact of the tumor microenvironment and chemotherapy protocols on anti-tumor immune responses.

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. [ABSTRACT FROM AUTHOR]

RAS (rat sarcoma virus) mutant cancers remain difficult to treat despite the advances in targeted therapy and immunotherapy. Targeted therapies against the components of mitogen-activated protein kinase (MAPK) pathways, including RAS, RAF, MEK, and ERK, have demonstrated activity in BRAF mutant and, in limited cases, RAS mutant cancer. RAS mutant cancers have been found to activate adaptive resistance mechanisms such as autophagy during MAPK inhibition. Here, we review the recent clinically relevant advances in the development of the MAPK pathway and autophagy inhibitors and focus on their application to RAS mutant cancers. We provide analysis of the preclinical rationale for combining the MAPK pathway and autophagy and highlight the most recent clinical trials that have been launched to capitalize on this potentially synthetic lethal approach to cancer therapy. [ABSTRACT FROM AUTHOR]

Background: Sentinel lymph node biopsy (SLNB) has not been studied for invasive melanomas treated with Mohs micrographic surgery using frozen‐section MART‐1 immunohistochemical stains (MMS‐IHC). The primary objective of this study was to assess the accuracy and compliance with National Comprehensive Cancer Network (NCCN) guidelines for SLNB in a cohort of patients who had invasive melanoma treated with MMS‐IHC. Methods: This retrospective cohort study included all patients who had primary, invasive, cutaneous melanomas treated with MMS‐IHC at a single academic center between March 2006 and April 2018. The primary outcomes were the rates of documenting discussion and performing SLNB in patients who were eligible based on NCCN guidelines. Secondary outcomes were the rate of identifying the sentinel lymph node and the percentage of positive lymph nodes. Results: In total, 667 primary, invasive, cutaneous melanomas (American Joint Committee on Cancer T1a‐T4b) were treated with MMS‐IHC. The median patient age was 69 years (range, 25‐101 years). Ninety‐two percent of tumors were located on specialty sites (head and/or neck, hands and/or feet, pretibial leg). Discussion of SLNB was documented for 162 of 176 (92%) SLNB‐eligible patients, including 127 of 127 (100%) who had melanomas with a Breslow depth >1 mm. SLNB was performed in 109 of 176 (62%) SLNB‐eligible patients, including 102 of 158 melanomas (65%) that met NCCN criteria to discuss and offer SLNB and 7 of 18 melanomas (39%) that met criteria to discuss and consider SLNB. The sentinel lymph node was successfully identified in 98 of 109 patients (90%) and was positive in 6 of those 98 patients (6%). Conclusions: Combining SLNB and MMS‐IHC allows full pathologic staging and confirmation of clear microscopic margins before reconstruction of specialty site invasive melanomas. SLNB can be performed accurately and in compliance with consensus guidelines in patients with melanoma using MMS‐IHC. Sentinel lymph node biopsy has not been studied for patients who have invasive melanomas treated with Mohs micrographic surgery using frozen‐section MART‐1 immunostains. Sentinel lymph node biopsy can be performed accurately and in compliance with consensus guidelines in these patients. [ABSTRACT FROM AUTHOR]

Aims: We evaluated the potential effect of sonidegib at an oral dose of 800 mg once daily (QD) on the pharmacokinetics (PK) of the probe drugs warfarin (CYP2C9) and bupropion (CYP2B6). Methods: This was a multicentre, open‐label study to evaluate the effect of sonidegib on the PK of the probe drugs warfarin and bupropion in patients with advanced solid tumours. Cohort 1 patients received a single warfarin 15‐mg dose on Day 1 of the run‐in period and on Cycle 2 Day 22 (C2D22) of sonidegib administration. Cohort 2 patients received a single bupropion 75‐mg dose on Day 1 of run‐in period and on C2D22 of sonidegib administration. Sonidegib 800 mg QD oral dosing began on Cycle 1 Day 1 of a 28‐day cycle after the run‐in period in both cohorts. Results: The geometric means ratios [90% confidence interval] for (S)‐warfarin with and without sonidegib were: area under the concentration–time curve from time 0 to infinity (AUCinf) 1.15 [1.07, 1.24] and maximum plasma concentration (Cmax) 0.88 [0.81, 0.97]; and for (R)‐warfarin were: AUCinf 1.10 [0.98, 1.24] and Cmax 0.93 [0.87, 1.0]. The geometric means ratios [90% confidence interval] of bupropion with and without sonidegib were: AUCinf 1.10 [0.99, 1.23] and Cmax 1.16 [0.95, 1.42]. Sonidegib 800 mg had a safety profile that was similar to that of lower dose sonidegib 200 mg and was unaffected by single doses of the probe drugs. Conclusions: Sonidegib dosed orally at 800 mg QD (higher than the Food and Drug Administration‐approved dose) did not impact the PK or pharmacodynamics of warfarin (CYP2C9 probe substrate) or the PK of bupropion (CYP2B6 probe substrate). [ABSTRACT FROM AUTHOR]

Background: Gut microbial diversity is associated with improved response to immune checkpoint inhibitors (ICI). Based on the known detrimental impact that antibiotics have on microbiome diversity, we hypothesized that antibiotic receipt prior to ICI would be associated with decreased survival.Methods: Patients with stage III and IV melanoma treated with ICI between 2008 and 2019 were selected from an institutional database. A window of antibiotic receipt within 3 months prior to the first infusion of ICI was prespecified. The primary outcome was overall survival (OS), and secondary outcomes were melanoma-specific mortality and immune-mediated colitis requiring intravenous steroids. All statistical tests were two-sided.Results: There were 568 patients in our database of which 114 received antibiotics prior to ICI. Of the patients, 35.9% had stage III disease. On multivariable Cox proportional hazards analysis of patients with stage IV disease, the antibiotic-exposed group had statistically significantly worse OS (hazard ratio [HR] = 1.81, 95% confidence interval [CI] = 1.27 to 2.57; P <.001). The same effect was observed among antibiotic-exposed patients with stage III disease (HR = 2.78, 95% CI = 1.31 to 5.87; P =.007). When limited to only patients who received adjuvant ICI (n = 89), antibiotic-exposed patients also had statistically significantly worse OS (HR = 4.84, 95% CI = 1.09 to 21.50; P =.04). The antibiotic group had a greater incidence of colitis (HR = 2.14, 95% CI = 1.02 to 4.52; P =.046).Conclusion: Patients with stage III and IV melanoma exposed to antibiotics prior to ICI had statistically significantly worse OS than unexposed patients. Antibiotic exposure was associated with greater incidence of moderate to severe immune-mediated colitis. Given the large number of antibiotics prescribed annually, physicians should be judicious with their use in cancer populations likely to receive ICI. [ABSTRACT FROM AUTHOR]

We conducted a phase I dose-escalation trial of radiation with ipilimumab in patients with melanoma with ≥2 metastatic lesions. Here, we report the final full clinical analysis. Patients received RT (6 or 8 Gy x 2 or 3 doses) to a single lesion followed by 4 cycles of ipilimumab. The primary endpoint was maximum tolerated dose of RT, and secondary endpoint was response at non-radiated sites. Twenty-two patients with treatment-naïve (n = 11) or treatment-refractory (n = 11) Stage IV melanoma were enrolled. There were 31 treatment-related adverse events (AEs), of which 16 were deemed immune-related. Eleven patients had grade 3 AEs (no grade 4/5). There were no dose-limiting toxicities related to the radiation/ipilimumab combination. Five of 22 patients (22.7%, 95% CI 7.8-45.4%) had partial response as best response and three (13.6%) had stable disease. Median overall survival was 10.7 months (95% CI, 4.9 months to not-estimable) and median progression-free survival 3.6 months (95% CI, 2.9 months to 7.8 months). Seven patients were still alive at the time of last follow-up (median follow-up 89.2 months), most of whom received pembrolizumab after progression. Radiotherapy followed by ipilimumab was well tolerated and yielded a response rate that compares favorably to the objective response rate with ipilimumab alone. Furthermore, 32% of patients are long-term survivors, most of whom received pembrolizumab. Based on these results, the recommended dose that was used in subsequent Phase 2 trials was 8 Gy x 3 doses. Clinical Trial Registration: NCT01497808 (www.clinicaltrials.gov) [ABSTRACT FROM AUTHOR]

Cancer cells encounter numerous stresses that pose a threat to their survival. Tumor microenviroment stresses that perturb protein homeostasis can produce endoplasmic reticulum (ER) stress, which can be counterbalanced by triggering the unfolded protein response (UPR) which is considered the canonical ER stress response. The UPR is characterized by three major proteins that lead to specific changes in transcriptional and translational programs in stressed cells. Activation of the UPR can induce apoptosis, but also can induce cytoprotective programs such as autophagy. There is increasing appreciation for the role that UPR-induced autophagy plays in supporting tumorigenesis and cancer therapy resistance. More recently several new pathways that connect cell stresses, components of the UPR and autophagy have been reported, which together can be viewed as non-canonical ER stress responses. Here we review recent findings on the molecular mechanisms by which canonical and non-canonical ER stress responses can activate cytoprotective autophagy and contribute to tumor growth and therapy resistance. Autophagy has been identified as a druggable pathway, however the components of autophagy (ATG genes) have proven difficult to drug. It may be the case that targeting the UPR or non-canonical ER stress programs can more effectively block cytoprotective autophagy to enhance cancer therapy. A deeper understanding of these pathways could provide new therapeutic targets in cancer. [ABSTRACT FROM AUTHOR]

The article discusses a research examining efficacy of anti–PD-1/PD-L1 therapy in nonresponding cancer patients. It mentions 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. It also mentions that inhibition of Vps34 reprograms cold into hot inflamed tumors and improves anti–PD-1/PD-L1 immunotherapy.

Background: Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors exhibit promising activity against ovarian cancers, but their efficacy can be limited by acquired drug resistance. This study explores the role of autophagy in regulating the sensitivity of ovarian cancer cells to PARP inhibitors.Methods: Induction of autophagy was detected by punctate LC3 fluorescence staining, LC3I to LC3II conversion on Western blot analysis, and electron microscopy. Enhanced growth inhibition and apoptosis were observed when PARP inhibitors were used with hydroxychloroquine, chloroquine (CQ), or LYS05 to block the hydrolysis of proteins and lipids in autophagosomes or with small interfering RNA against ATG5 or ATG7 to prevent the formation of autophagosomes. The preclinical efficacy of the combination of CQ and olaparib was evaluated with a patient-derived xenograft (PDX) and the OVCAR8 human ovarian cancer cell line.Results: Four PARP inhibitors (olaparib, niraparib, rucaparib, and talazoparib) induced autophagy in a panel of ovarian cancer cells. Inhibition of autophagy with CQ enhanced the sensitivity of ovarian cancer cells to PARP inhibitors. In vivo, olaparib and CQ produced additive growth inhibition in OVCAR8 xenografts and a PDX. Olaparib inhibited PARP activity, and this led to increased reactive oxygen species (ROS) and an accumulation of γ-H2AX. Inhibition of autophagy also increased ROS and γ-H2AX and enhanced the effect of olaparib on both entities. Treatment with olaparib increased phosphorylation of ATM and PTEN while decreasing the phosphorylation of AKT and mTOR and inducing autophagy.Conclusions: PARP inhibitor-induced autophagy provides an adaptive mechanism of resistance to PARP inhibitors in cancer cells with wild-type BRCA, and a combination of PARP inhibitors with CQ or other autophagy inhibitors could improve outcomes for patients with ovarian cancer. [ABSTRACT FROM AUTHOR]

Autophagy is a conserved, self-degradation system that is critical for maintaining cellular homeostasis during stress conditions. Dysregulated autophagy has implications in health and disease. Specifically, in cancer, autophagy plays a dichotomous role by inhibiting tumor initiation but supporting tumor progression. Early results from clinical trials that repurposed hydroxychloroquine for cancer have suggested that autophagy inhibition may be a promising approach for advanced cancers. In this review of the literature, the authors present fundamental advances in the biology of autophagy, approaches to targeting autophagy, the preclinical rationale and clinical experience with hydroxychloroquine in cancer clinical trials, the potential role of autophagy in tumor immunity, and recent developments in next-generation autophagy inhibitors that have clinical potential. Autophagy is a promising target for drug development in cancer. Cancer 2018. © 2018 American Cancer Society. [ABSTRACT FROM AUTHOR]

To determine the feasibility of liquid biopsy for monitoring of patients with advanced melanoma, cell-free DNA was extracted from plasma for 25 Stage III/IV patients, most (84.0%) having received previous therapy. DNA concentrations ranged from 0.6 to 390.0 ng/ml (median = 7.8 ng/ml) and were positively correlated with tumor burden as measured by imaging (Spearman rho = 0.5435, p = .0363). Using ultra-deep sequencing for a 61-gene panel, one or more mutations were detected in 12 of 25 samples (48.0%), and this proportion did not vary significantly for patients on or off therapy at the time of blood draw (52.9% and 37.5% respectively; p = .673). Sixteen mutations were detected in eight different genes, with the most frequent mutations detected in BRAF, NRAS, and KIT. Allele fractions ranged from 1.1% to 63.2% (median = 29.1%). Among patients with tissue next-generation sequencing, nine of 11 plasma mutations were also detected in matched tissue, for a concordance of 81.8%. [ABSTRACT FROM AUTHOR]

The stress-inducible chaperone protein HSP70 (HSPA1) is implicated in melanoma development, and HSP70 inhibitors exert tumor-specific cytotoxic activity in cancer. In this study, we documented that a significant proportion of melanoma tumors express high levels of HSP70, particularly at advanced stages, and that phospho-FAK (PTK2) and BRAF are HSP70 client proteins. Treatment of melanoma cells with HSP70 inhibitors decreased levels of phospho-FAK along with impaired migration, invasion, and metastasis in vitro and in vivo. Moreover, the HSP70 inhibitor PET-16 reduced levels of mutant BRAF, synergized with the BRAF inhibitor PLX4032 in vitro, and enhanced the durability of response to BRAF inhibition in vivo. Collectively, these findings provide strong support for HSP70 inhibition as a therapeutic strategy in melanoma, especially as an adjuvant approach for overcoming the resistance to BRAF inhibitors frequently observed in melanoma patients. Cancer Res; 76(9); 2720-30. [ABSTRACT FROM AUTHOR]

Resistance to BRAF inhibitors ( BRAFi) is one of the major challenges for targeted therapies for BRAF-mutant melanomas. However, little is known about the role of micro RNAs in conferring BRAFi resistance. Herein, we demonstrate that miR-200c expression is significantly reduced whereas miR-200c target genes including Bmi1, Zeb2, Tubb3, ABCG5, and MDR1 are significantly increased in melanomas that acquired BRAFi resistance compared to pretreatment tumor biopsies. Similar changes were observed in BRAFi-resistant melanoma cell lines. Overexpression of miR-200c or knock-down of Bmi1 in resistant melanoma cells restores their sensitivities to BRAFi, leading to deactivation of the PI3K/ AKT and MAPK signaling cascades, and acquisition of epithelial-mesenchymal transition-like phenotypes, including upregulation of E-cadherin, downregulation of N-cadherin, and ABCG5 and MDR1 expression. Conversely, knock-down of miR-200c or overexpression of Bmi1 in BRAFi-sensitive melanoma cells activates the PI3K/ AKT and MAPK pathways, upregulates N-cadherin, ABCG5, and MDR1 expression, and downregulates E-cadherin expression, leading to BRAFi resistance. Together, our data identify miR-200c as a critical signaling node in BRAFi-resistant melanomas impacting the MAPK and PI3K/ AKT pathways, suggesting miR-200c as a potential therapeutic target for overcoming acquired BRAFi resistance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Autophagy plays a key role in the maintenance of cellular homeostasis. In healthy cells, such a homeostatic activity constitutes a robust barrier against malignant transformation. Accordingly, many oncoproteins inhibit, and several oncosuppressor proteins promote, autophagy. Moreover, autophagy is required for optimal anticancer immunosurveillance. In neoplastic cells, however, autophagic responses constitute a means to cope with intracellular and environmental stress, thus favoring tumor progression. This implies that at least in some cases, oncogenesis proceeds along with a temporary inhibition of autophagy or a gain of molecular functions that antagonize its oncosuppressive activity. Here, we discuss the differential impact of autophagy on distinct phases of tumorigenesis and the implications of this concept for the use of autophagy modulators in cancer therapy. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

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. • RNA-seq and ATAC-seq atlas of 14 human T cell subsets from healthy donors • The atlas provides a reference map for interpreting signatures of T cells from 3 diseases • Prediction of cis -regulatory elements that control CD8 T cell subset gene expression • Validation of functional enhancers for CXCR3 and GZMB using CRISPRi Giles et al. generated an epigenomic T cell differentiation atlas from healthy human donor (HD) blood. This atlas was used to (1) identify transcriptional and epigenetic modules in human CD8 T cell differentiation, (2) interpret signatures of T cells from cancer and autoimmunity, and (3) predict and validate CD8 T cell enhancers. [ABSTRACT FROM AUTHOR]

This study investigates the mechanism of action behind the long-term responses (12-16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. Although single agent MK-2206 inhibited phospho- AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin was cytotoxic in long-term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase-dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti-oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs. [ABSTRACT FROM AUTHOR]

The mechanisms underlying adaptive resistance of melanoma to targeted therapies remain unclear. By combining ChIP sequencing with microarray-based gene profiling, we determined that ERBB3 is upregulated by FOXD3, a transcription factor that promotes resistance to RAF inhibitors in melanoma. Enhanced ERBB3 signaling promoted resistance to RAF pathway inhibitors in cultured melanoma cell lines and in mouse xenograft models. ERBB3 signaling was dependent on ERBB2; targeting ERBB2 with lapatinib in combination with the RAF inhibitor PLX4720 reduced tumor burden and extended latency of tumor regrowth in vivo versus PLX4720 alone. These results suggest that enhanced ERBB3 signaling may serve as a mechanism of adaptive resistance to RAF and MEK inhibitors in melanoma and that cotargeting this pathway may enhance the clinical efficacy and extend the therapeutic duration of RAF inhibitors. [ABSTRACT FROM AUTHOR]

Autophagy is a lysosome-dependent degradative process that protects cancer cells from multiple stresses. In preclinical models, autophagy inhibition with chloroquine (CQ) derivatives augments the efficacy of many anticancer therapies, but CQ has limited activity as a single agent. Clinical trials are underway combining anticancer agents with hydroxychloroquine (HCQ), but concentrations of HCQ required to inhibit autophagy are not consistently achievable in the clinic. We report the synthesis and characterization of bisaminoquinoline autophagy inhibitors that potently inhibit autophagy and impair tumor growth in vivo. The structural motifs that are necessary for improved autophagy inhibition compared with CQ include the presence of two aminoquinoline rings and a triamine linker and C-7 chlorine. The lead compound, Lys01, is a 10-fold more potent autophagy inhibitor than HCQ. Compared with HCQ, Lys05, a water-soluble salt of Lys01, more potently accumulates within and deacidifies the lysosome, resulting in impaired autophagy and tumor growth. At the highest dose administered, some mice develop Paneth cell dysfunction that resembles the intestinal phenotype of mice and humans with genetic defects in the autophagy gene ATG16L1, providing in vivo evidence that Lys05 targets autophagy. Unlike HCQ, significant single-agent antitumor activity is observed without toxicity in mice treated with lower doses of Lys05, establishing the therapeutic potential of this compound in cancer. [ABSTRACT FROM AUTHOR]

Background: Approximately 50% of melanomas harbor activating (V600) mutations in the serine–threonine protein kinase B-RAF (BRAF). The oral BRAF inhibitor vemurafenib (PLX4032) frequently produced tumor regressions in patients with BRAF V600–mutant metastatic melanoma in a phase 1 trial and improved overall survival in a phase 3 trial. Methods: We designed a multicenter phase 2 trial of vemurafenib in patients with previously treated BRAF V600–mutant metastatic melanoma to investigate the efficacy of vemurafenib with respect to overall response rate (percentage of treated patients with a tumor response), duration of response, and overall survival. The primary end point was the overall response rate as ascertained by the independent review committee; overall survival was a secondary end point. Results: A total of 132 patients had a median follow-up of 12.9 months (range, 0.6 to 20.1). The confirmed overall response rate was 53% (95% confidence interval [CI], 44 to 62; 6% with a complete response and 47% with a partial response), the median duration of response was 6.7 months (95% CI, 5.6 to 8.6), and the median progression-free survival was 6.8 months (95% CI, 5.6 to 8.1). Primary progression was observed in only 14% of patients. Some patients had a response after receiving vemurafenib for more than 6 months. The median overall survival was 15.9 months (95% CI, 11.6 to 18.3). The most common adverse events were grade 1 or 2 arthralgia, rash, photosensitivity, fatigue, and alopecia. Cutaneous squamous-cell carcinomas (the majority, keratoacanthoma type) were diagnosed in 26% of patients. Conclusions: Vemurafenib induces clinical responses in more than half of patients with previously treated BRAF V600–mutant metastatic melanoma. In this study with a long follow-up, the median overall survival was approximately 16 months. (Funded by Hoffmann–La Roche; ClinicalTrials.gov number, NCT00949702.) [ABSTRACT FROM PUBLISHER]

[Display omitted] We have previously reported the unique features of dimeric bisaminoquinolines as anticancer agents and have identified their cellular target as PPT1, a protein palmitoyl-thioesterase. We now report a systematic study on the role of the linker in these constructs, both with respect to the distance between the heterocycles, the linker hydrophobicity and the methylation status (primary vs. secondary vs. tertiary) of the central nitrogen atom on the observed biological activity. [ABSTRACT FROM AUTHOR]

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. • Ly108 and CD69 define four Tex subsets linked in a hierarchical developmental pathway • Two TCF1+ subsets, effector-like and terminally exhausted subsets, are identified • Key transcriptional, epigenetic, and biological changes define subset transitions • TCF1, T-bet, and Tox coordinate Tex subset development and dynamics Beltra et al. define a hierarchical developmental pathway for CD8+ T cell exhaustion, revealing four stages and multistep transcriptional and epigenetic dynamics underlying subset transitions and subset-associated biological changes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]