Mitochondrial DNA drives abscopal responses to radiation that are inhibited by autophagy.

Autophagy supports both cellular and organismal homeostasis. However, whether autophagy should be inhibited or activated for cancer therapy remains unclear. Deletion of essential autophagy genes increased the sensitivity of mouse mammary carcinoma cells to radiation therapy in vitro and in vivo (in immunocompetent syngeneic hosts). Autophagy-deficient cells secreted increased amounts of type I interferon (IFN), which could be limited by CGAS or STING knockdown, mitochondrial DNA depletion or mitochondrial outer membrane permeabilization blockage via BCL2 overexpression or BAX deletion. In vivo, irradiated autophagy-incompetent mammary tumors elicited robust immunity, leading to improved control of distant nonirradiated lesions via systemic type I IFN signaling. Finally, a genetic signature of autophagy had negative prognostic value in patients with breast cancer, inversely correlating with mitochondrial abundance, type I IFN signaling and effector immunity. As clinically useful autophagy inhibitors are elusive, our findings suggest that mitochondrial outer membrane permeabilization may represent a valid target for boosting radiation therapy immunogenicity in patients with breast cancer.

Cellular senescence in the response of HR+ breast cancer to radiotherapy and CDK4/6 inhibitors.

BACKGROUND: Preclinical evidence from us and others demonstrates that the anticancer effects of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors can be enhanced with focal radiation therapy (RT), but only when RT is delivered prior to (rather than after) CDK4/6 inhibition. Depending on tumor model, cellular senescence (an irreversible proliferative arrest that is associated with the secretion of numerous bioactive factors) has been attributed beneficial or detrimental effects on response to treatment. As both RT and CDK4/6 inhibitors elicit cellular senescence, we hypothesized that a differential accumulation of senescent cells in the tumor microenvironment could explain such an observation, i.e., the inferiority of CDK4/6 inhibition with palbociclib (P) followed by RT (P→RT) as compared to RT followed by palbociclib (RT→P). METHODS: The impact of cellular senescence on the interaction between RT and P was assessed by harnessing female INK-ATTAC mice, which express a dimerizable form of caspase 8 (CASP8) under the promoter of cyclin dependent kinase inhibitor 2A (Cdkn2a, coding for p16Ink4), as host for endogenous mammary tumors induced by the subcutaneous implantation of medroxyprogesterone acetate (MPA, M) pellets combined with the subsequent oral administration of 7,12-dimethylbenz[a]anthracene (DMBA, D). This endogenous mouse model of HR+ mammary carcinogenesis recapitulates key immunobiological aspects of human HR+ breast cancer. Mice bearing M/D-driven tumors were allocated to RT, P or their combination in the optional presence of the CASP8 dimerizer AP20187, and monitored for tumor growth, progression-free survival and overall survival. In parallel, induction of senescence in vitro, in cultured human mammary hormone receptor (HR)+ adenocarcinoma MCF7 cells, triple negative breast carcinoma MDA-MB-231 cells and mouse HR+ mammary carcinoma TS/A cells treated with RT, P or their combination, was determined by colorimetric assessment of senescence-associated ß-galactosidase activity after 3 or 7 days of treatment. RESULTS: In vivo depletion of p16Ink4-expressing (senescent) cells ameliorated the efficacy of P→RT (but not that of RT→P) in the M/D-driven model of HR+ mammary carcinogenesis. Accordingly, P→RT induced higher levels of cellular senescence than R→TP in cultured human and mouse breast cancer cell lines. CONCLUSIONS: Pending validation in other experimental systems, these findings suggest that a program of cellular senescence in malignant cells may explain (at least partially) the inferiority of P→RT versus RT→P in preclinical models of HR+ breast cancer.

Nicotinamide drives T cell activation in the mammary tumor microenvironment.

Nicotinamide (NAM, a variant of vitamin B3) has recently been shown to accelerate the activation of human CD4+ and CD8+ T cells exposed to repeated CD3/CD28 agonism in vitro. Here, we demonstrate that T cells infiltrating mouse mammary carcinomas that are therapeutically controlled by NAM also express multiple markers of late-stage activation. Taken together, these findings lend additional support to the notion that the antineoplastic effects of NAM involve at least some degree of restored cancer immunosurveillance.

Immunogenic stress and death of cancer cells: Contribution of antigenicity vs adjuvanticity to immunosurveillance.

Cancer cells are subjected to constant selection by the immune system, meaning that tumors that become clinically manifest have managed to subvert or hide from immunosurveillance. Immune control can be facilitated by induction of autophagy, as well as by polyploidization of cancer cells. While autophagy causes the release of ATP, a chemotactic signal for myeloid cells, polyploidization can trigger endoplasmic reticulum stress with consequent exposure of the "eat-me" signal calreticulin on the cell surface, thereby facilitating the transfer of tumor antigens into dendritic cells. Hence, both autophagy and polyploidization cause the emission of adjuvant signals that ultimately elicit immune control by CD8+ T lymphocytes. We investigated the possibility that autophagy and polyploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. Mass spectrometry led to the identification of peptides that were presented on major histocompatibility complex (MHC) class I molecules in an autophagy-dependent fashion or that were specifically exposed on the surface of polyploid cells, yet lost upon passage of such cells through immunocompetent (but not immunodeficient) mice. However, the preferential recognition of autophagy-competent and polyploid cells by the innate and cellular immune systems did not correlate with the preferential recognition of such peptides in vivo. Moreover, vaccination with such peptides was unable to elicit tumor growth-inhibitory responses in vivo. We conclude that autophagy and polyploidy increase the immunogenicity of cancer cells mostly by affecting their adjuvanticity rather than their antigenicity.

eIF2α phosphorylation as a biomarker of immunogenic cell death.

Cancer cells exposed to some forms of chemotherapy and radiotherapy die while eliciting an adaptive immune response. Such a functionally peculiar variant of apoptosis has been dubbed immunogenic cell death (ICD). One of the central events in the course of ICD is the activation of an endoplasmic reticulum (ER) stress response. This is instrumental for cells undergoing ICD to emit all the signals that are required for their demise to be perceived as immunogenic by the host, and culminates with the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). In particular, eIF2α phosphorylation is required for the pre-apoptotic exposure of the ER chaperone calreticulin (CALR) on the cell surface, which is a central determinant of ICD. Importantly, phosphorylated eIF2α can be quantified in both preclinical and clinical samples by immunoblotting or immunohistochemistry using phosphoneoepitope-specific monoclonal antibodies. Of note, the phosphorylation of eIF2α and CALR exposure do not necessarily correlate with each other, and neither of these parameters is sufficient for cell death to be perceived as immunogenic. Nonetheless, accumulating data indicate that assessing the degree of phosphorylation of eIF2α provides a convenient parameter to monitor ICD. Here, we discuss the role of the ER stress response in ICD and the potential value of eIF2α phosphorylation as a biomarker for this clinically relevant variant of apoptosis.

Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (i.e., palbociclib, abemaciclib, and ribociclib) are well known for their capacity to mediate cytostatic effects by promoting cell cycle arrest in the G1 phase, thus inhibiting cancer cell proliferation. Cytostatic effects induced by CDK4/6 inhibitors can be transient or lead to a permanent state of cell cycle arrest, commonly defined as cellular senescence. Induction of senescence is often associated to metabolic modifications and to the acquisition of a senescence-associated secretory phenotype (SASP) by cancer cells, which in turn can promote or limit antitumor immunity (and thus the efficacy of CDK4/6 inhibitors) depending on SASP components. Thus, although accumulating evidence suggests that anti-cancer effects of CDK4/6 inhibitors also depend on the promotion of antitumor immune responses, assessing cell cycle arrest and progression in cells treated with palbociclib remains a key approach for investigating the efficacy of CDK4/6 inhibitors. Here, we describe a method to assess cell cycle distribution simultaneously with active DNA replication by flow cytometry in cultured hormone receptor-positive breast cancer MCF7 cells.

Quantitative assessment of mitophagy in irradiated cancer cells.

Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through lysosomal degradation. Dysfunctional mitochondria are prone to release potentially cytotoxic factors including reactive oxygen species (ROS) and caspase activators, such as cytochrome c, somatic (CYCS). Thus, proficient mitophagic responses mediate prominent cytoprotective functions. Moreover, the rapid degradation of permeabilized mitochondria limits the release of mitochondrial components that may drive inflammatory reactions, such as mitochondrial DNA (mtDNA) and transcription factor A, mitochondrial (TFAM), implying that mitophagy also mediates potent anti-inflammatory effects. Here, we detail a simple, flow cytometry-assisted protocol for the specific measurement of mitophagic responses as driven by radiation therapy (RT) in mouse hormone receptor (HR)+ mammary carcinoma TS/A cells. With some variations, this method - which relies on the mitochondria-restricted expression of a fluorescent reporter that is sensitive to pH and hence changes excitation wavelength within lysosomes (mt-mKeima) - can be adapted to a variety of human and mouse cancer cell lines and/or straightforwardly implemented on fluorescence microscopy platforms.

Leveraging immune resistance archetypes in solid cancer to inform next-generation anticancer therapies.

Anticancer immunotherapies, such as immune checkpoint inhibitors, bispecific antibodies, and chimeric antigen receptor T cells, have improved outcomes for patients with a variety of malignancies. However, most patients either do not initially respond or do not exhibit durable responses due to primary or adaptive/acquired immune resistance mechanisms of the tumor microenvironment. These suppressive programs are myriad, different between patients with ostensibly the same cancer type, and can harness multiple cell types to reinforce their stability. Consequently, the overall benefit of monotherapies remains limited. Cutting-edge technologies now allow for extensive tumor profiling, which can be used to define tumor cell intrinsic and extrinsic pathways of primary and/or acquired immune resistance, herein referred to as features or feature sets of immune resistance to current therapies. We propose that cancers can be characterized by immune resistance archetypes, comprised of five feature sets encompassing known immune resistance mechanisms. Archetypes of resistance may inform new therapeutic strategies that concurrently address multiple cell axes and/or suppressive mechanisms, and clinicians may consequently be able to prioritize targeted therapy combinations for individual patients to improve overall efficacy and outcomes.

BCL2 Inhibition Reveals a Dendritic Cell-Specific Immune Checkpoint That Controls Tumor Immunosurveillance.

We developed a phenotypic screening platform for the functional exploration of dendritic cells (DC). Here, we report a genome-wide CRISPR screen that revealed BCL2 as an endogenous inhibitor of DC function. Knockout of BCL2 enhanced DC antigen presentation and activation as well as the capacity of DCs to control tumors and to synergize with PD-1 blockade. The pharmacologic BCL2 inhibitors venetoclax and navitoclax phenocopied these effects and caused a cDC1-dependent regression of orthotopic lung cancers and fibrosarcomas. Thus, solid tumors failed to respond to BCL2 inhibition in mice constitutively devoid of cDC1, and this was reversed by the infusion of DCs. Moreover, cDC1 depletion reduced the therapeutic efficacy of BCL2 inhibitors alone or in combination with PD-1 blockade and treatment with venetoclax caused cDC1 activation, both in mice and in patients. In conclusion, genetic and pharmacologic BCL2 inhibition unveils a DC-specific immune checkpoint that restrains tumor immunosurveillance. SIGNIFICANCE: BCL2 inhibition improves the capacity of DCs to stimulate anticancer immunity and restrain cancer growth in an immunocompetent context but not in mice lacking cDC1 or mature T cells. This study indicates that BCL2 blockade can be used to sensitize solid cancers to PD-1/PD-L1-targeting immunotherapy. This article is featured in Selected Articles from This Issue, p. 2293.

Lessons for the Next Generation of Scientists from the Second Annual Arthur and Sandra Irving Cancer Immunology Symposium.

The Arthur and Sandra Irving Cancer Immunology Symposium has been created as a platform for established cancer immunologists to mentor trainees and young investigators as they launch their research career in the field. By sharing their different paths to success, the senior faculty mentors provide an invaluable resource to support the development of the next generation of leaders in the cancer immunology community. This Commentary describes some of the key topics that were discussed during the 2022 symposium: scientific and career trajectory, leadership, mentoring, collaborations, and publishing. For each of these topics, established investigators discussed the elements that facilitate success in these areas as well as mistakes that can hinder progress. Herein, we outline the critical points raised in these discussions for establishing a successful independent research career. These points are highly relevant for the broader scientific community.

Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells.

BACKGROUND: Metastatic melanoma is a lethal skin cancer and its incidence is rising every year. It represents a challenge for oncologist, as the current treatment options are non-curative in the majority of cases; therefore, the effort to find and/or develop novel compounds is mandatory. Pemetrexed (Alimta®, MTA) is a multitarget antifolate that inhibits folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase, required for de novo synthesis of nucleotides for DNA replication. It is currently used in the treatment of mesothelioma and non-small cell lung cancer (NSCLC), and has shown clinical activity in other tumors such as breast, colorectal, bladder, cervical, gastric and pancreatic cancer. However, its effect in human melanoma has not been studied yet. RESULTS: In the current work we studied the effect of MTA on four human melanoma cell lines A375, Hs294T, HT144 and MeWo and in two NSCLC cell lines H1299 and Calu-3. We have found that MTA induces DNA damage, S-phase cell cycle arrest, and caspase- dependent and -independent apoptosis. We show that an increment of the intracellular reactive oxygen species (ROS) and p53 is required for MTA-induced cytotoxicity by utilizing N-Acetyl-L-Cysteine (NAC) to blockage of ROS and p53-defective H1299 NSCLC cell line. Pretreatment of melanoma cells with NAC significantly decreased the DNA damage, p53 up-regulation and cytotoxic effect of MTA. MTA was able to induce p53 expression leading to up-regulation of p53-dependent genes Mcl-1 and PIDD, followed by a postranscriptional regulation of Mcl-1 improving apoptosis. CONCLUSIONS: We found that MTA induced DNA damage and mitochondrial-mediated apoptosis in human melanoma cells in vitro and that the associated apoptosis was both caspase-dependent and -independent and p53-mediated. Our data suggest that MTA may be of therapeutic relevance for the future treatment of human malignant melanoma.

Targeting oncogene and non-oncogene addiction to inflame the tumour microenvironment.

Immune checkpoint inhibitors (ICIs) have revolutionized the clinical management of multiple tumours. However, only a few patients respond to ICIs, which has generated considerable interest in the identification of resistance mechanisms. One such mechanism reflects the ability of various oncogenic pathways, as well as stress response pathways required for the survival of transformed cells (a situation commonly referred to as 'non-oncogene addiction'), to support tumour progression not only by providing malignant cells with survival and/or proliferation advantages, but also by establishing immunologically 'cold' tumour microenvironments (TMEs). Thus, both oncogene and non-oncogene addiction stand out as promising targets to robustly inflame the TME and potentially enable superior responses to ICIs.

Cytofluorometric assessment of cell cycle progression in irradiated cells.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects upon the accumulation of unrepaired damage to macromolecules, notably DNA. The ability of ionizing radiation to prevent malignant cells from replicating and to cause their demise is indeed an integral component of the anticancer activity of RT. Neoplastic cells are generally more sensitive to the cytostatic and cytotoxic effects of RT than their healthy counterparts as they exhibit increased proliferative rate and limited capacity for DNA repair. This provides a rather comfortable therapeutic window for clinical RT usage, especially with the development of novel, technologically superior RT modalities that minimize the exposure of normal tissues. Thus, while accumulating evidence indicates that cancer control by RT also involves the activation of tumor-targeting immune responses, assessing cell cycle progression in irradiated cells remains a central approach for investigating radiosensitivity in preclinical tumor models. Here, we detail a simple, flow cytometry-assisted method to simultaneously assess cell cycle distribution and active DNA replication in cultured estrogen receptor (ER)+ breast cancer MCF7 cells. With minimal variations, the same technique can be straightforwardly implemented to a large panel of human and mouse cancer cell lines.

Quantification of cytosolic DNA species by immunofluorescence microscopy and automated image analysis.

When employed according to specific doses and fractionation schedules, radiation therapy (RT) elicits potent tumor-targeting immune responses that rely on the secretion of type I interferon (IFN) by irradiated cancer cells. Most often, this is initiated by the ability of RT to promote the cytosolic accumulation of double-stranded DNA (dsDNA) molecules, which are detected by cyclic GMP-AMP synthase (CGAS) to engage the stimulator of interferon response cGAMP interactor 1 (STING1)-dependent transactivation of type I IFN-coding genes via interferon regulatory factor 3 (IRF3). Here, we describe a simple protocol for the quantification of cytosolic dsDNA species by immunofluorescence microscopy coupled to automated image analysis, as enabled by precise sample processing conditions that permeabilize plasma-but not nuclear or inner mitochondrial-membranes. As compared to subcellular fractionation-based techniques, this approach is compatible with assessments in individual cells aimed at gauging inter-cellular heterogeneity, as well as subcellular tests including co-localization studies.

RT-PCR-assisted quantification of type I IFN responses in irradiated cancer cells.

It is now clear that radiation therapy (RT) can be delivered in doses and according to fractionation schedules that actively elicit immunostimulatory effects. While such effects are often sufficient to drive potent anticancer immunity culminating with systemic disease eradication, the immunostimulatory activity of RT stands out as a promising combinatorial partner for bona fide immunotherapeutics including immune checkpoint inhibitors (ICIs). Accumulating preclinical and clinical evidence indicates that the secretion of type I interferon (IFN) by irradiated cancer cells is a sine qua non for RT to initiate ICI-actionable tumor-targeting immune responses. Here, we detail a simple protocol to quantitatively assess type I IFN responses in irradiated mouse hormone receptor (HR)+ TS/A cells by RT-PCR. With minimal variations, the same technique can be straightforwardly adapted to quantify type I IFN-associated transcriptional responses in a variety of human and mouse cancer cells maintained in vitro.

Cytofluorometric assessment of acute cell death responses driven by radiation therapy.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects on malignant cells, largely reflecting the ability of ionizing radiation to cause direct and indirect damage to macromolecules including DNA and lipids. While low-dose RT generally causes limited cytotoxicity in an acute manner (as it imposes insufficient cellular damage to compromise homeostasis, or instead induces the delayed demise of cells that fail to complete mitosis successfully), high RT doses can mediate an acute wave of cell death that begins to manifest shortly (24-72h) after irradiation. Here, we provide two straightforward techniques to assess the acute cytotoxic effects of RT by the flow cytometry-assisted quantification of plasma membrane permeabilization (PMP, a late-stage manifestation of cell death) and either mitochondrial outer membrane permeabilization (MOMP) or phosphatidylserine (PS) externalization (two early-stage signs of cell death) in mouse mammary adenocarcinoma TS/A cells. With minor variations, the same protocols can be straightforwardly adapted to measure acute cell death responses as elicited by RT in a large panel of human and mouse cancer cells lines of different histological derivation.

PDIA3 epitope-driven immune autoreactivity contributes to hepatic damage in type 2 diabetes.

A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs, including the liver, ultimately driving nonalcoholic steatohepatitis. In this setting, environment-driven lipotoxicity and glucotoxicity induce liver damage, which promotes dendritic cell activation and generates a major histocompatibility complex class II (MHC-II) immunopeptidome enriched with peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and the stress responses. Here, we demonstrated that lipotoxicity and glucotoxicity, as driven by a high-fat and high-fructose (HFHF) diet, promoted MHC-II presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3), which is involved in immunogenic cell death. Increased MHC-II presentation of PDIA3 peptides was associated with antigen-specific proliferation of hepatic CD4+ immune infiltrates and isotype switch of anti-PDIA3 antibodies from IgM to IgG3, indicative of cellular and humoral PDIA3 autoreactivity. Passive transfer of PDIA3-specific T cells or PDIA3-specific antibodies also exacerbated hepatocyte death, as determined by increased hepatic transaminases detected in the sera of mice subjected to an HFHF but not control diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions, including autoimmune hepatitis, primary biliary cholangitis, and type 2 diabetes. Together, our data indicated that metabolic insults caused by an HFHF diet elicited liver damage and promoted pathogenic immune autoreactivity driven by T and B cell PDIA3 epitopes.

Ketosis versus carbotoxicity - metabolism determines the outcome of cancer immunotherapy.

Carbohydrate-rich diets have been consistently associated with detrimental effects for human health, including diabetes and obesity. Moreover, high glucose levels appear to mediate immunosuppressive effects in preclinical tumor models. Recent data from Ferrere and colleagues point to the intriguing possibility that carbotoxicity may originate from the abolition of ketosis.

Possible mechanisms of cancer prevention by nicotinamide.

Nicotinamide (NAM) is a precursor of vitamin B3 commonly sold over the counter as a nutritional supplement with anti-aging properties. Accumulating preclinical evidence indicates that NAM also mediates oncopreventive effects against a variety of neoplasms. Supporting the translational relevance of dietary NAM supplementation, results from a Phase 3 randomized clinical trial have demonstrated that oral NAM was safe and efficiently reduced the incidence of new non-melanoma skin cancers and actinic keratosis amongst high-risk individuals. However, the molecular and cellular mechanisms that underlie this ability of NAM to delay carcinogenesis remain to be clarified, as discussed in this short review. LINKED ARTICLES: This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc.

Targeting Serine in Cancer: Is Two Better Than One?

Several recent preclinical studies have demonstrated that simultaneously blocking exogenous and endogenous sources of serine in malignant cells mediates superior anticancer effects as compared with limiting either source alone. Here, we critically summarize key developments in targeting serine to treat cancer and discuss persisting challenges for implementing such a therapeutic approach in patients.