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Ultra-high dose rate ("FLASH") radiotherapy (>40-60 Gy/s) is a promising new radiation modality currently in human clinical trials. Previous studies showed that FLASH proton radiotherapy (FR) improves toxicity of normal tissues compared to standard proton radiotherapy (SR) without compromising anti-tumor effects. Understanding this normal tissue sparing effect may offer insight into how toxicities from cancer therapy can be improved. Here, we show that compared to SR, FR resulted in improved acute weight recovery and survival in mice after whole-abdomen irradiation. Improved morbidity and mortality after FR were associated with greater proliferation of damage-induced epithelial progenitor cells followed by improved tissue regeneration. FR led to the accelerated differentiation of revival stem cells (revSCs), a rare damage-induced stem cell required for intestinal regeneration, and to qualitative and quantitative changes in activity of signaling pathways important for revSC differentiation and epithelial regeneration. Specifically, FR resulted in greater infiltration of macrophages producing TGF-β, a cytokine important for revSC induction, that was coupled to augmented TGF-β signaling in revSCs. In pericryptal fibroblasts, FR resulted in greater type I IFN (IFN-I) signaling, which directly stimulates production of FGF growth factors supporting revSC proliferation. Accordingly, the ability of FR to improve epithelial regeneration and morbidity was dependent on IFN-I signaling. In the context of SR, however, IFN-I had a detrimental effect and promoted toxicity. Thus, a tissue-level signaling network coordinated by differences in IFN-I signaling and involving stromal cells, immune cells, and revSCs underlies the ability of FLASH to improve normal tissue toxicity without compromising anti-tumor efficacy.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells., (© 2024. The Author(s).)

Inflammation of the gastrointestinal tract is a prevalent pathology in diseases such as inflammatory bowel disease (IBD). Currently, there are no therapies to prevent IBD, and available therapies to treat IBD are often sub-optimal. Thus, an unmet need exists to better understand the molecular mechanisms underlying intestinal tissue responses to damage and regeneration. The recent development of single-cell RNA (sc-RNA) sequencing-based techniques offers a unique opportunity to shed light on novel signaling pathways and cellular states that govern tissue adaptation or maladaptation across a broad spectrum of diseases. These approaches require the isolation of high-quality cells from tissues for downstream transcriptomic analyses. In the context of intestinal biology, there is a lack of protocols that ensure the isolation of epithelial and non-epithelial compartments simultaneously with high-quality yield. Here, we report two protocols for the isolation of epithelial and stromal cells from mouse and human colon tissues under inflammatory conditions. Specifically, we tested the feasibility of the protocols in a mouse model of dextran sodium sulfate (DSS)-induced colitis and in human biopsies from Crohn's patients. We performed sc-RNA sequencing analysis and demonstrated that the protocol preserves most of the epithelial and stromal cell types found in the colon. Moreover, the protocol is suitable for immunofluorescence staining of surface markers for epithelial, stromal, and immune cell lineages for flow cytometry analyses. This optimized protocol will provide a new resource for scientists to study complex tissues such as the colon in the context of tissue damage and regeneration. Key features • This protocol allows the isolation of epithelial and stromal cells from colon tissues. • The protocol has been optimized for tissues under inflammatory conditions with compromised cell viability. • This protocol is suitable for experimental mouse models of colon inflammation and human biopsies., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)

The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancy (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 was shown to regulate inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to epigenetically repress two inflammatory signaling pathways in neutrophils: Toll-like receptor 4 (TLR4) and type I interferon (IFN) signaling. RUNX1 loss in GMPs augments neutrophils' inflammatory response to the TLR4 ligand lipopolysaccharide through increased expression of the TLR4 coreceptor CD14. RUNX1 binds Cd14 and other genes encoding proteins in the TLR4 and type I IFN signaling pathways whose chromatin accessibility increases when RUNX1 is deleted. Transcription factor footprints for the effectors of type I IFN signaling-the signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRFs)-were enriched in chromatin that gained accessibility in both GMPs and neutrophils when RUNX1 was lost. STAT1::STAT2 and IRF motifs were also enriched in the chromatin of retrotransposons that were derepressed in RUNX1-deficient GMPs and neutrophils. We conclude that a major direct effect of RUNX1 loss in GMPs is the derepression of type I IFN and TLR4 signaling, resulting in a state of fixed maladaptive innate immunity., (© 2023 Zezulin et al.; Published by Cold Spring Harbor Laboratory Press.)

Interferon-gamma (IFN-γ) has pleiotropic effects on cancer immune checkpoint blockade (ICB), including roles in ICB resistance. We analyzed gene expression in ICB-sensitive versus ICB-resistant tumor cells and identified a strong association between interferon-mediated resistance and expression of Ripk1, a regulator of tumor necrosis factor (TNF) superfamily receptors. Genetic interaction screening revealed that in cancer cells, RIPK1 diverted TNF signaling through NF-κB and away from its role in cell death. This promoted an immunosuppressive chemokine program by cancer cells, enhanced cancer cell survival, and decreased infiltration of T and NK cells expressing TNF superfamily ligands. Deletion of RIPK1 in cancer cells compromised chemokine secretion, decreased ARG1 + suppressive myeloid cells linked to ICB failure in mice and humans, and improved ICB response driven by CASP8-killing and dependent on T and NK cells. RIPK1-mediated resistance required its ubiquitin scaffolding but not kinase function. Thus, cancer cells co-opt RIPK1 to promote cell-intrinsic and cell-extrinsic resistance to immunotherapy., Competing Interests: Declaration of interests A.J.M. has received research funding from Merck. He is a scientific advisor for Takeda, H3Biomedicine, Xilio, and Related Sciences. A.J.M. is an inventor on patents related to the IFN pathway and an inventor on a filed patent related to modified CAR T cells. A.J.M. is a scientific founder for Dispatch Biotherapeutics., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Poor tumor infiltration, development of exhaustion, and antigen insufficiency are common mechanisms that limit chimeric antigen receptor (CAR)-T cell efficacy. Delivery of pattern recognition receptor agonists is one strategy to improve immune function; however, targeting these agonists to immune cells is challenging, and off-target signaling in cancer cells can be detrimental. Here, we engineer CAR-T cells to deliver RN7SL1, an endogenous RNA that activates RIG-I/MDA5 signaling. RN7SL1 promotes expansion and effector-memory differentiation of CAR-T cells. Moreover, RN7SL1 is deployed in extracellular vesicles and selectively transferred to immune cells. Unlike other RNA agonists, transferred RN7SL1 restricts myeloid-derived suppressor cell (MDSC) development, decreases TGFB in myeloid cells, and fosters dendritic cell (DC) subsets with costimulatory features. Consequently, endogenous effector-memory and tumor-specific T cells also expand, allowing rejection of solid tumors with CAR antigen loss. Supported by improved endogenous immunity, CAR-T cells can now co-deploy peptide antigens with RN7SL1 to enhance efficacy, even when heterogenous CAR antigen tumors lack adequate neoantigens., Competing Interests: Declaration of interests A.J.M. has received research funding from Merck. He is a scientific advisor for Takeda, H3Biomedicine, Related Sciences, and Xilio. A.J.M. is an inventor on patents related to the IFN pathway. A.J.M., L.R.J., and C.H.J. are inventors on a filed patent related to modified CAR-T cells. C.H.J. reports research funding from Novartis, and he is a scientific founder of Tmunity Therapeutics. A.J.M., C.H.J., and L.R.J. are scientific founders for Project 5 Therapeutics. C.H.J. also works under a research collaboration involving the University of Pennsylvania and the Novartis Institute of Biomedical Research and is an inventor of intellectual property licensed by the University of Pennsylvania to Novartis. C.H.J. is on the board of directors for AC Immune and is a scientific advisor for BluesphereBio, Cabaletta, Carisma, Cartography, Cellares, Celldex, DeCART, Decheng, Poseida, Verismo, WIRB Copernicus, and Ziopharm., (Copyright © 2021. Published by Elsevier Inc.)

In studies of electron and proton radiotherapy, ultrahigh dose rates of FLASH radiotherapy appear to produce fewer toxicities than standard dose rates while maintaining local tumor control. FLASH-proton radiotherapy (F-PRT) brings the spatial advantages of PRT to FLASH dose rates (>40 Gy/second), making it important to understand if and how F-PRT spares normal tissues while providing antitumor efficacy that is equivalent to standard-proton radiotherapy (S-PRT). Here we studied PRT damage to skin and mesenchymal tissues of muscle and bone and found that F-PRT of the C57BL/6 murine hind leg produced fewer severe toxicities leading to death or requiring euthanasia than S-PRT of the same dose. RNA-seq analyses of murine skin and bone revealed pathways upregulated by S-PRT yet unaltered by F-PRT, such as apoptosis signaling and keratinocyte differentiation in skin, as well as osteoclast differentiation and chondrocyte development in bone. Corroborating these findings, F-PRT reduced skin injury, stem cell depletion, and inflammation, mitigated late effects including lymphedema, and decreased histopathologically detected myofiber atrophy, bone resorption, hair follicle atrophy, and epidermal hyperplasia. F-PRT was equipotent to S-PRT in control of two murine sarcoma models, including at an orthotopic intramuscular site, thereby establishing its relevance to mesenchymal cancers. Finally, S-PRT produced greater increases in TGFβ1 in murine skin and the skin of canines enrolled in a phase I study of F-PRT versus S-PRT. Collectively, these data provide novel insights into F-PRT-mediated tissue sparing and support its ongoing investigation in applications that would benefit from this sparing of skin and mesenchymal tissues. SIGNIFICANCE: These findings will spur investigation of FLASH radiotherapy in sarcoma and additional cancers where mesenchymal tissues are at risk, including head and neck cancer, breast cancer, and pelvic malignancies., (©2021 The Authors; Published by the American Association for Cancer Research.)

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)., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The DNA-dependent pattern recognition receptor, cGAS (cyclic GMP-AMP synthase), mediates communication between the DNA damage and the immune responses. Mitotic chromosome missegregation stimulates cGAS activity; however, it is unclear whether progression through mitosis is required for cancercell-intrinsic activation of anti-tumor immune responses. Moreover, it is unknown whether cell cycle checkpoint disruption can restore responses in cancer cells that are recalcitrant to DNAdamage-induced inflammation. Here, we demonstrate that prolonged cell cycle arrest at the G 2 -mitosis boundary from either excessive DNA damage or CDK1 inhibition prevents inflammatory-stimulated gene expression and immune-mediated destruction of distal tumors. Remarkably, DNAdamage-induced inflammatory signaling is restored in a RIG-I-dependent manner upon concomitant disruption of p53 and the G 2 checkpoint. These findings link aberrant cell progression and p53 loss to an expanded spectrum of damage-associated molecular pattern recognition and have implications for the design of rational approaches to augment anti-tumor immune responses., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Interferon-gamma (IFNG) augments immune function yet promotes T cell exhaustion through PDL1. How these opposing effects are integrated to impact immune checkpoint blockade (ICB) is unclear. We show that while inhibiting tumor IFNG signaling decreases interferon-stimulated genes (ISGs) in cancer cells, it increases ISGs in immune cells by enhancing IFNG produced by exhausted T cells (T EX ). In tumors with favorable antigenicity, these T EX mediate rejection. In tumors with neoantigen or MHC-I loss, T EX instead utilize IFNG to drive maturation of innate immune cells, including a PD1 + TRAIL + ILC1 population. By disabling an inhibitory circuit impacting PD1 and TRAIL, blocking tumor IFNG signaling promotes innate immune killing. Thus, interferon signaling in cancer cells and immune cells oppose each other to establish a regulatory relationship that limits both adaptive and innate immune killing. In melanoma and lung cancer patients, perturbation of this relationship is associated with ICB response independent of tumor mutational burden., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host's response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14-15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual's recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4-5, 2019.

Tumor-derived extracellular vesicles (TEV) "educate" healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: We conducted a phase I trial evaluating pembrolizumab+hypofractionated radiotherapy (HFRT) for patients with metastatic cancers., Methods: There were two strata (12 patients each): (i) NSCLC/melanoma progressing on prior anti-PD-1 therapy, (ii) other cancer types; anti-PD-1-naive. Patients received 6 cycles of pembrolizumab, starting 1 week before HFRT. Patients had ≥2 lesions; only one was irradiated (8 Gy × 3 for first half; 17 Gy × 1 for second half in each stratum) and the other(s) followed for response., Results: Of the 24 patients, 20 (83%) had treatment-related adverse events (AEs) (all grade 1 or 2). There were eight grade 3 AEs, none treatment related. There were no dose-limiting toxicities or grade 4/5 AEs. Stratum 1: two patients (of 12) with progression on prior PD-1 blockade experienced prolonged responses (9.2 and 28.1 months). Stratum 2: one patient experienced a complete response and two had prolonged stable disease (7.4 and 7.0 months). Immune profiling demonstrated that anti-PD-1 therapy and radiation induced a consistent increase in the proliferation marker Ki67 in PD-1-expressing CD8 T cells., Conclusions: HFRT was well tolerated with pembrolizumab, and in some patients with metastatic NSCLC or melanoma, it reinvigorated a systemic response despite previous progression on anti-PD-1 therapy., Clinical Trial Registration: NCT02303990 ( www.clinicaltrials.gov ).

Immunotherapy in pancreatic ductal adenocarcinoma (PDA) remains a difficult clinical problem despite success in other disease types with immune checkpoint blockade (ICB) and chimeric antigen receptor T-cell therapy. Mechanisms driving immunosuppression and poor T-cell infiltration in PDA are incompletely understood. Here, we use genetically engineered mouse models of PDA that recapitulate hallmarks of human disease to demonstrate that CD40 pathway activation is required for clinical response to radiotherapy and ICB with αCTLA-4 and αPD-1. The combination of an agonist αCD40 antibody, radiotherapy, and dual ICB eradicated irradiated and unirradiated (i.e., abscopal) tumors, generating long-term immunity. Response required T cells and also short-lived myeloid cells and was dependent on the long noncoding RNA myeloid regulator Morrbid Using unbiased random forest machine learning, we built unique, contextual signatures for each therapeutic component, revealing that (i) radiotherapy triggers an early proinflammatory stimulus, ablating existing intratumoral T cells and upregulating MHC class I and CD86 on antigen-presenting cells, (ii) αCD40 causes a systemic and intratumoral reorganization of the myeloid compartment, and (iii) ICB increases intratumoral T-cell infiltration and improves the CD8 T-cell:regulatory T-cell ratio. Thus, αCD40 and radiotherapy nonredundantly augment antitumor immunity in PDA, which is otherwise refractory to ICB, providing a clear rationale for clinical evaluation. Significance: Radiotherapy and αCD40 disrupt key links between innate and adaptive immunity, ameliorating resistance to immune checkpoint blockade in pancreatic cancer via multiple cellular mechanisms. Cancer Res; 78(15); 4282-91. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The success of immune checkpoint blockade in patients with a wide variety of malignancies has changed the treatment paradigm in oncology. However, combination therapies with immune checkpoint blockade will be needed to overcome resistance and broaden the clinical utility of immunotherapy. Here we discuss a framework for rationally designing combination therapy strategies based on enhancing major discriminatory functions of the immune system that are corrupted by cancer-namely, antigenicity, adjuvanticity, and homeostatic feedback inhibition. We review recent advances on how conventional genotoxic cancer therapies, molecularly targeted therapies, epigenetic agents, and immune checkpoint inhibitors can restore these discriminatory functions. Potential barriers that can impede response despite combination therapy are also discussed., (Copyright © 2018. Published by Elsevier Inc.)

Inflammatory gene expression following genotoxic cancer therapy is well documented, yet the events underlying its induction remain poorly understood. Inflammatory cytokines modify the tumour microenvironment by recruiting immune cells and are critical for both local and systemic (abscopal) tumour responses to radiotherapy. A poorly understood feature of these responses is the delayed onset (days), in contrast to the acute DNA-damage responses that occur in minutes to hours. Such dichotomous kinetics implicate additional rate-limiting steps that are essential for DNA-damage-induced inflammation. Here we show that cell cycle progression through mitosis following double-stranded DNA breaks leads to the formation of micronuclei, which precede activation of inflammatory signalling and are a repository for the pattern-recognition receptor cyclic GMP-AMP synthase (cGAS). Inhibiting progression through mitosis or loss of pattern recognition by stimulator of interferon genes (STING)-cGAS impaired interferon signalling. Moreover, STING loss prevented the regression of abscopal tumours in the context of ionizing radiation and immune checkpoint blockade in vivo. These findings implicate temporal modulation of the cell cycle as an important consideration in the context of therapeutic strategies that combine genotoxic agents with immune checkpoint blockade.

While maintaining the integrity of the genome and sustaining bioenergetics are both fundamental functions of the cell, potential crosstalk between metabolic and DNA repair pathways is poorly understood. Since histone acetylation plays important roles in DNA repair and is sensitive to the availability of acetyl coenzyme A (acetyl-CoA), we investigated a role for metabolic regulation of histone acetylation during the DNA damage response. In this study, we report that nuclear ATP-citrate lyase (ACLY) is phosphorylated at S455 downstream of ataxia telangiectasia mutated (ATM) and AKT following DNA damage. ACLY facilitates histone acetylation at double-strand break (DSB) sites, impairing 53BP1 localization and enabling BRCA1 recruitment and DNA repair by homologous recombination. ACLY phosphorylation and nuclear localization are necessary for its role in promoting BRCA1 recruitment. Upon PARP inhibition, ACLY silencing promotes genomic instability and cell death. Thus, the spatial and temporal control of acetyl-CoA production by ACLY participates in the mechanism of DNA repair pathway choice., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cancer is a disease driven by evolutionary selection on somatic genetic and epigenetic alterations. Here, we propose Canopy, a method for inferring the evolutionary phylogeny of a tumor using both somatic copy number alterations and single-nucleotide alterations from one or more samples derived from a single patient. Canopy is applied to bulk sequencing datasets of both longitudinal and spatial experimental designs and to a transplantable metastasis model derived from human cancer cell line MDA-MB-231. Canopy successfully identifies cell populations and infers phylogenies that are in concordance with existing knowledge and ground truth. Through simulations, we explore the effects of key parameters on deconvolution accuracy and compare against existing methods. Canopy is an open-source R package available at https://cran.r-project.org/web/packages/Canopy/., Competing Interests: The authors declare no conflict of interest.

Improving efficacy of immune checkpoint blockade for cancer can be facilitated by combining these agents with each other and/or with other conventional or targeted therapies. Interferon and innate immune signaling pathways in immune and tumor cells have emerged as intriguing determinants of response and resistance, often in complex and seemingly paradoxical ways., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The immunogenic effects of chemotherapy rely on effective activation of dendritic cells to present antigen to tumor-specific T cells. However, the signals that govern how dendritic cells seek out dying cancer cells to initiate this process are poorly understood. A recent study by Vacchelli et al. provides important insight., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Background/objectives: The molecular chaperone αB-crystallin is expressed in estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 "triple-negative" breast carcinomas and promotes brain and lung metastasis. We examined αB-crystallin expression in primary breast carcinomas with metastatic data to evaluate its association with prognosis and site-specific metastases., Methods: αB-crystallin gene ( CRYAB ) expression was examined using publically available global-gene expression data (n=855 breast tumors) with first site of distant metastasis information ("855Met"). αB-crystallin protein expression was determined by immunohistochemistry using the clinically annotated tissue microarray (n=3987 breast tumors) from British Columbia Cancer Agency (BCCA). Kaplan-Meier and multivariable Cox regression analyses were used to evaluate the prognostic value of αB-crystallin. Multivariable logistic regression analysis was used to evaluate risks of αB-crystallin and other markers for site of metastasis., Results: In the 855Met dataset, αB-crystallin gene ( CRYAB) expression was an independent predictor of brain as the first distant site of relapse (HR = 1.2, (95% CI 1.0-1.4), P = 0.021). In the BCCA series, αB-crystallin protein expression was an independent prognostic marker of poor breast cancer specific survival (HR = 1.3, (95% CI 1.1-1.6), P = 0.014). Among patients with metastases, αB-crystallin was the strongest independent predictor of brain metastasis (OR = 2.99 (95% CI 1.83-4.89), P < 0.0001) and the only independent predictor of brain as the first site of distant metastasis (OR = 3.15 (95% CI1.43-6.95), P = 0.005). αB-crystallin was also associated with worse survival (3.0 versus 4.7 months, P = 0.007)., Conclusions: αB-crystallin is a promising biomarker to identify breast cancer patients at high risk for early relapse in the brain, independent of ER and HER2 status., Competing Interests: Competing interests CMP and TON are equity stock holders, and Board of Director Member of BioClassifier LLC. CMP, TON and MCUC are also listed as inventors on a patent application on the PAM50 assay. The other authors declare they have no competing interests.

Glioblastomas are aggressive adult brain tumors, characterized by inadequately organized vasculature and consequent nutrient and oxygen (O2)-depleted areas. Adaptation to low nutrients and hypoxia supports glioblastoma cell survival, progression and therapeutic resistance. However, specific mechanisms promoting cellular survival under nutrient and O2 deprivation remain incompletely understood. Here, we show that miR-124 expression is negatively correlated with a hypoxic gene signature in glioblastoma patient samples, suggesting that low miR-124 levels contribute to pro-survival adaptive pathways in this disease. As miR-124 expression is repressed in various cancer types (including glioblastoma), we quantified miR-124 abundance in normoxic and hypoxic regions in glioblastoma patient tissue, and investigated whether ectopic miR-124 expression compromises cell survival during tumor ischemia. Our results indicate that miR-124 levels are further diminished in hypoxic/ischemic regions within individual glioblastoma patient samples, compared with regions replete in O2 and nutrients. Importantly, we also show that increased miR-124 expression affects the ability of tumor cells to survive under O2 and/or nutrient deprivation. Moreover, miR-124 re-expression increases cell death in vivo and enhances the survival of mice bearing intracranial xenograft tumors. miR-124 exerts this phenotype in part by directly regulating TEAD1, MAPK14/p38α and SERP1, factors involved in cell proliferation and survival under stress. Simultaneous suppression of these miR-124 targets results in similar levels of cell death as caused by miR-124 restoration. Importantly, we further demonstrate that SERP1 reintroduction reverses the hypoxic cell death elicited by miR-124, indicating the importance of SERP1 in promoting tumor cell survival. In support of our experimental data, we observed a significant correlation between high SERP1 levels and poor patient outcome in glioblastoma patients. Collectively, among the many pro-tumorigeneic properties of miR-124 repression in glioblastoma, we delineated a novel role in promoting tumor cell survival under stressful microenvironments, thereby supporting tumor progression.

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.

Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.

Glioblastoma multiforme (GBM) and the mesenchymal GBM subtype in particular are highly malignant tumors that frequently exhibit regions of severe hypoxia and necrosis. Because these features correlate with poor prognosis, we investigated microRNAs whose expression might regulate hypoxic GBM cell survival and growth. We determined that the expression of microRNA-218 (miR-218) is decreased significantly in highly necrotic mesenchymal GBM, and orthotopic tumor studies revealed that reduced miR-218 levels confer GBM resistance to chemotherapy. Importantly, miR-218 targets multiple components of receptor tyrosine kinase (RTK) signaling pathways, and miR-218 repression increases the abundance and activity of multiple RTK effectors. This elevated RTK signaling also promotes the activation of hypoxia-inducible factor (HIF), most notably HIF2α. We further show that RTK-mediated HIF2α regulation is JNK dependent, via jun proto-oncogene. Collectively, our results identify an miR-218-RTK-HIF2α signaling axis that promotes GBM cell survival and tumor angiogenesis, particularly in necrotic mesenchymal tumors.

The c-Myc oncoprotein regulates >15% of the human transcriptome and a limited number of microRNAs (miRNAs). Here, we establish that in a human B-lymphoid cell line, Myc-repressed, but not Myc-stimulated, genes are significantly enriched for predicted binding sites of Myc-regulated miRNAs, primarily those comprising the Myc-activated miR-17~92 cluster. Notably, gene set enrichment analysis demonstrates that miR-17∼92 is a major regulator of B-cell receptor (BCR) pathway components. Many of them are immunoreceptor tyrosine inhibitory motif (ITIM)-containing proteins, and ITIM proteins CD22 and FCGR2B were found to be direct targets of miR-17∼92. Consistent with the propensity of ITIM proteins to recruit phosphatases, either MYC or miR-17~92 expression was necessary to sustain phosphorylation of spleen tyrosine kinase (SYK) and the B-cell linker protein (BLNK) upon ligation of the BCR. Further downstream, stimulation of the BCR response by miR-17-92 resulted in the enhanced calcium flux and elevated levels of Myc itself. Notably, inhibition of the miR-17~92 cluster in diffuse large B-cell lymphoma (DLBCL) cell lines diminished the BCR response as measured by SYK and BLNK phosphorylation. Conversely, human DLBCLs of the BCR subtype express higher Myc and mir17hg transcript levels than other subtypes. Hence, the Myc-miR-17-92-BCR axis, frequently affected by genomic rearrangements, constitutes a novel lymphomagenic feed-forward loop.

Increasing evidence suggests that stem-like cells mediate cancer therapy resistance and metastasis. Breast tumour-initiating stem cells (T-ISC) are known to be enriched in CD44(+) CD24(neg/low) cells. Here, we identify two T-ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44(+) CD24(low+) subpopulation generates CD44(+) CD24(neg) progeny with reduced sphere formation and tumourigenicity. CD44(+) CD24(low+) populations contain subsets of ALDH1(+) and ESA(+) cells, yield more frequent spheres and/or T-ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA-MB-231 model, metastatic potential. CD44(+) CD24(low+) but not CD44(+) CD24(neg) express activated Notch1 intracellular domain (N1-ICD) and Notch target genes. We show N1-ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44(+) CD24(low+) cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44(+) CD24(low+) cells, but CD44(+) CD24(neg) were resistant. While GSI hold promise for targeting T-ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T-ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti-cancer drug responsiveness., (© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

The miR-17∼92 cluster is thought to be an oncogene, yet its expression is low in glioblastoma multiforme (GBM) cell lines. This could allow unfettered expression of miR-17∼92 target genes such as connective tissue growth factor (CTGF; or CCN2), which is known to contribute to GBM pathogenesis. Indeed, microRNA-18a (but not other miR-17∼92 members) has a functional site in the CTGF 3' UTR, and its forced reexpression sharply reduces CTGF protein and mRNA levels. Interestingly, it also reduces the levels of CTGF primary transcript. The unexpected effects of miR-18a on CTGF transcription are mediated in part by direct targeting of Smad3 and ensuing weakening of TGFβ signaling. Having defined the TGFβ signature in GBM cells, we demonstrate a significant anti-correlation between miR-18 and TGFβ signaling in primary GBM samples from The Cancer Genome Atlas. Most importantly, high levels of miR-18 combined with low levels of the TGFβ metagene correlate with prolonged patient survival. Thus, low expression of the miR-17∼92 cluster, and specifically miR-18a, could significantly contribute to GBM pathogenesis.

Cancer lethality is mainly caused by metastasis. Therefore, understanding the nature of the genes involved in this process has become a priority. Given the heterogeneity of mutations in cancer cells, considerable focus has been directed toward characterizing metastasis genes in the context of relevant signaling pathways rather than treating genes as independent and equal entities. One signaling cascade implicated in the regulation of cell growth, invasion and metastasis is the MAP kinase pathway. Raf kinase inhibitory protein (RKIP) functions as an inhibitor of the MAP kinase pathway and is a metastasis suppressor in different cancer models. By utilizing statistical analysis of clinical data integrated with experimental validation, we recently identified components of the RKIP signaling pathway relevant to breast cancer metastasis. Using the RKIP pathway as an example, we show how prior biological knowledge can be efficiently combined with genome-wide patient data to identify gene regulatory mechanisms that control metastasis.

Tumour metastasis suppressors are inhibitors of metastasis but their mechanisms of action are generally not understood. We previously showed that the suppressor Raf kinase inhibitory protein (RKIP) inhibits breast tumour metastasis in part via let-7. Here, we demonstrate an integrated approach combining statistical analysis of breast tumour gene expression data and experimental validation to extend the signalling pathway for RKIP. We show that RKIP inhibits let-7 targets (HMGA2, BACH1) that in turn upregulate bone metastasis genes (MMP1, OPN, CXCR4). Our results reveal BACH1 as a novel let-7-regulated transcription factor that induces matrix metalloproteinase1 (MMP1) expression and promotes metastasis. An RKIP pathway metastasis signature (designated RPMS) derived from the complete signalling cascade predicts high metastatic risk better than the individual genes. These results highlight a powerful approach for identifying signalling pathways downstream of a key metastasis suppressor and indicate that analysis of genes in the context of their signalling environment is critical for understanding their predictive and therapeutic potential.

The molecular basis for breast cancer metastasis to the brain is largely unknown. Brain relapse typically occurs years after the removal of a breast tumour, suggesting that disseminated cancer cells must acquire specialized functions to take over this organ. Here we show that breast cancer metastasis to the brain involves mediators of extravasation through non-fenestrated capillaries, complemented by specific enhancers of blood-brain barrier crossing and brain colonization. We isolated cells that preferentially infiltrate the brain from patients with advanced disease. Gene expression analysis of these cells and of clinical samples, coupled with functional analysis, identified the cyclooxygenase COX2 (also known as PTGS2), the epidermal growth factor receptor (EGFR) ligand HBEGF, and the alpha2,6-sialyltransferase ST6GALNAC5 as mediators of cancer cell passage through the blood-brain barrier. EGFR ligands and COX2 were previously linked to breast cancer infiltration of the lungs, but not the bones or liver, suggesting a sharing of these mediators in cerebral and pulmonary metastases. In contrast, ST6GALNAC5 specifically mediates brain metastasis. Normally restricted to the brain, the expression of ST6GALNAC5 in breast cancer cells enhances their adhesion to brain endothelial cells and their passage through the blood-brain barrier. This co-option of a brain sialyltransferase highlights the role of cell-surface glycosylation in organ-specific metastatic interactions.

Raf kinase inhibitory protein (RKIP) negatively regulates the MAP kinase (MAPK), G protein-coupled receptor kinase-2, and NF-kappaB signalling cascades. RKIP has been implicated as a metastasis suppressor for prostate cancer, but the mechanism is not known. Here, we show that RKIP inhibits invasion by metastatic breast cancer cells and represses breast tumour cell intravasation and bone metastasis in an orthotopic murine model. The mechanism involves inhibition of MAPK, leading to decreased transcription of LIN28 by Myc. Suppression of LIN28 enables enhanced let-7 processing in breast cancer cells. Elevated let-7 expression inhibits HMGA2, a chromatin remodelling protein that activates pro-invasive and pro-metastatic genes, including Snail. LIN28 depletion and let-7 expression suppress bone metastasis, and LIN28 restores bone metastasis in mice bearing RKIP-expressing breast tumour cells. These results indicate that RKIP suppresses invasion and metastasis in part through a signalling cascade involving MAPK, Myc, LIN28, let-7, and downstream let-7 targets. RKIP regulation of two pluripotent stem cell genes, Myc and LIN28, highlights the importance of RKIP as a key metastasis suppressor and potential therapeutic agent.

Individualization of cancer management requires prognostic markers and therapy-predictive markers. Prognostic markers assess risk of disease progression independent of therapy, whereas therapy-predictive markers identify patients whose disease is sensitive or resistant to treatment. We show that an experimentally derived IFN-related DNA damage resistance signature (IRDS) is associated with resistance to chemotherapy and/or radiation across different cancer cell lines. The IRDS genes STAT1, ISG15, and IFIT1 all mediate experimental resistance. Clinical analyses reveal that IRDS(+) and IRDS(-) states exist among common human cancers. In breast cancer, a seven-gene-pair classifier predicts for efficacy of adjuvant chemotherapy and for local-regional control after radiation. By providing information on treatment sensitivity or resistance, the IRDS improves outcome prediction when combined with standard markers, risk groups, or other genomic classifiers.

The breast cancer susceptibility gene BRCA1 encodes a large protein thought to contribute to a variety of cellular processes, although the critical determinants of BRCA1-deficient tumorigenesis remain unclear. Given that BRCA1 is required for cell proliferation, suppressor mutations are believed to modify BRCA1 phenotypes and contribute to the etiology of BRCA1-deficient tumors. Here, we show that overexpression of the homologous recombinase RAD51 in a DT40 BRCA1Delta/Delta mutant rescues defects in proliferation, DNA damage survival, and homologous recombination (HR). In addition, epistasis analysis with BRCA1 and the DNA end-joining factor KU70 indicates that these factors operate independently of one another to repair double-strand breaks. Consistent with this genetic finding, cell synchronization studies show that the ability of BRCA1 to promote radioresistance is restricted to the late S and G2 phases of the cell cycle, as predicted for genes whose function is specific to homology-mediated repair rather than nonhomologous end-joining. Notably, retrospective analyses of microarray expression data reveal elevated expression of RAD51 and two of its late-acting cofactors, RAD54 and RAD51AP1, in BRCA1-deficient versus sporadic breast tumors. Taken together, our results indicate that up-regulation of HR provides a permissive genetic context for cells lacking BRCA1 function by circumventing its requirement in RAD51 subnuclear assembly. Furthermore, the data support a model in which enhanced HR activity contributes to the etiology of BRCA1-deficient tumors.

Elsewhere, we reported that multiple serial in vivo passage of a squamous cell carcinoma cells (SCC61) concurrent with ionizing radiation (IR) treatment resulted in the selection of radioresistant tumor (nu61) that overexpresses the signal transducer and activator of transcription 1 (Stat1)/IFN-dependent pathway. Here, we report that (a) the Stat1 pathway is induced by IR, (b) constitutive overexpression of Stat1 is linked with failure to transmit a cytotoxic signal by radiation or IFNs, (c) selection of parental cell line SCC61 against IFN-alpha and IFN-gamma leads to the same IR- and IFN-resistant phenotype as was obtained by IR selection, and (d) suppression of Stat1 by short hairpin RNA renders the IR-resistant nu61 cells radiosensitive to IR. We propose a model that transient induction of Stat1 by IFN, IR, or other stress signals activates cytotoxic genes and cytotoxic response. Constitutive overexpression of Stat1 on the other hand leads to the suppression of the cytotoxic response and induces prosurvival genes that, at high levels of Stat1, render the cells resistant to IR or other inducers of cell death.

The association between large tumor size and metastatic risk in a majority of clinical cancers has led to questions as to whether these observations are causally related or whether one is simply a marker for the other. This is partly due to an uncertainty about how metastasis-promoting gene expression changes can arise in primary tumors. We investigated this question through the analysis of a previously defined "lung metastasis gene-expression signature" (LMS) that mediates experimental breast cancer metastasis selectively to the lung and is expressed by primary human breast cancer with a high risk for developing lung metastasis. Experimentally, we demonstrate that the LMS promotes primary tumor growth that enriches for LMS(+) cells, and it allows for intravasation after reaching a critical tumor size. Clinically, this corresponds to LMS(+) tumors being larger at diagnosis compared with LMS(-) tumors and to a marked rise in the incidence of metastasis after LMS(+) tumors reach 2 cm. Patients with LMS-expressing primary tumors selectively fail in the lung compared with the bone or other visceral sites and have a worse overall survival. The mechanistic linkage between metastasis gene expression, accelerated tumor growth, and likelihood of metastatic recurrence provided by the LMS may help to explain observations of prognostic gene signatures in primary cancer and how tumor growth can both lead to metastasis and be a marker for cells destined to metastasize.

DNA damage results in transcriptional induction of p53 target genes, including the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) (CDKN1A) and the proapoptotic Bcl-2 family member p53 up-regulated modulator of apoptosis (PUMA). Depending on the cellular context, p21(Cip1) and PUMA mediate cell cycle arrest and apoptosis, respectively. By imposing cell cycle arrest at the expense of apoptosis, p21(Cip1) can sharply reduce the effectiveness of DNA-damaging anticancer agents in colorectal cancer cells. We investigated the link between cell cycle progression and the onset of apoptosis in DNA-damaged cells by analyzing the activation of the apoptotic cascade in p21(Cip1)-deficient HCT116 colorectal cancer cells. DNA damage induced a similar level of p53 activation and PUMA induction in p21(Cip1)-deficient cells compared with wild-type isogenic counterparts. p21(Cip1) did not act as a direct blocker of PUMA. However, only p21(Cip1)-deficient cells showed extensive cytochrome c release, mitochondrial membrane depolarization, and caspase activation. An increase in caspase activation occurred as these cells reached M-phase and incurred polyploidy. When ectopically expressed in p21(Cip1)-deficient HCT116 cells, p21(Cip1), its family member p27(Kip1), and the structurally unrelated CDK inhibitor p16(Ink4a) were similarly effective at causing cell cycle arrest and inhibiting DNA damage-induced apoptotic events such as cytochrome c release, mitochondrial membrane depolarization, and activation of the caspase cascade. These observations suggest that by blocking dysregulated cell cycle progression, CDK inhibitors can influence the sensitivity of the mitochondria to proapoptotic signals in DNA damage-induced cancer cells.

By means of in vivo selection, transcriptomic analysis, functional verification and clinical validation, here we identify a set of genes that marks and mediates breast cancer metastasis to the lungs. Some of these genes serve dual functions, providing growth advantages both in the primary tumour and in the lung microenvironment. Others contribute to aggressive growth selectively in the lung. Many encode extracellular proteins and are of previously unknown relevance to cancer metastasis.

We used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in immunodeficient mice. Individual cells from a population established in culture from the pleural effusion of a breast cancer patient showed distinct patterns of organ-specific metastasis. Single-cell progenies derived from this population exhibited markedly different abilities to metastasize to the bone, lung, or adrenal medulla, which suggests that metastases to different organs have different requirements. Transcriptomic profiling revealed that these different single-cell progenies similarly express a previously described "poor-prognosis" gene expression signature. Unsupervised classification using the transcriptomic data set supported the hypothesis that organ-specific metastasis by breast cancer cells is controlled by metastasis-specific genes that are separate from a general poor-prognosis gene expression signature. Furthermore, by using a gene expression signature associated with the ability of these cells to metastasize to bone, we were able to distinguish primary breast carcinomas that preferentially metastasized to bone from those that preferentially metastasized elsewhere. These results suggest that the bone-specific metastatic phenotypes and gene expression signature identified in a mouse model may be clinically relevant.

Metastasis is a multistep and multifunctional biological cascade that is the final and most life-threatening stage of cancer progression. Understanding the biological underpinnings of this complex process is of extreme clinical relevance and requires unbiased and comprehensive biological scrutiny. In recent years, we have utilized a xenograft model of breast cancer metastasis to discover genes that mediate organ-specific patterns of metastatic colonization. Examination of transcriptomic data from cohorts of primary breast cancers revealed a subset of site-specific metastasis genes that are selected for early in tumor progression. High expression of these genes predicts the propensity for lung metastasis independently of several classic markers of poor prognosis. These genes fulfill dual functions-enhanced primary tumorigenicity and augmented organ-specific metastatic activity. Other metastasis genes fulfill functions specialized for the microenvironment of the metastatic site and are consequently not selected for in primary tumors. These findings improve our understanding of metastatic progression, facilitate the interpretation of primary tumor gene expression data, and open several important possibilities for future clinical application.

A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Mutants were constructed which did not bind to Bax but retained anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain with the corresponding amino acids from Bax. The resulting chimera showed a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retained the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was introduced that rendered the chimera incapable of heterodimerization, the resulting protein failed to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial properties that were inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel.

Bcl-X(L), a member of the Bcl-2 family, can inhibit many forms of programed cell death. The three-dimensional structure of Bcl-X(L) identified a 60 amino acid loop lacking defined structure. Although amino acid sequence within this region is not conserved among Bcl-2 family members, structural modeling suggested that Bcl-2 also contains a large unstructured region. Compared with the full-length protein, loop deletion mutants of Bcl-X(L) and Bcl-2 displayed an enhanced ability to inhibit apoptosis. Despite enhanced function, the deletion mutants did not have significant alterations in the ability to bind pro-apoptotic proteins such as Bax. The loop deletion mutant of Bcl-2 also displayed a qualitative difference in its ability to inhibit apoptosis. Full-length Bcl-2 was unable to prevent anti-IgM-induced cell death of the immature B cell line WEHI-231. In contrast, the Bcl-2 deletion mutant protected WEHI-231 cells from death. Substantial differences were observed in the ability of WEHI-231 cells to phosphorylate the deletion mutant of Bcl-2 compared with full-length Bcl-2. Bcl-2 phosphorylation was found to be dependent on the presence of an intact loop domain. These results suggest that the loop domain in Bcl-X(L) and Bcl-2 can suppress the anti-apoptotic function of these genes and may be a target for regulatory post-translational modifications.

Heterodimerization between members of the Bcl-2 family of proteins is a key event in the regulation of programmed cell death. The molecular basis for heterodimer formation was investigated by determination of the solution structure of a complex between the survival protein Bcl-xL and the death-promoting region of the Bcl-2-related protein Bak. The structure and binding affinities of mutant Bak peptides indicate that the Bak peptide adopts an amphipathic alpha helix that interacts with Bcl-xL through hydrophobic and electrostatic interactions. Mutations in full-length Bak that disrupt either type of interaction inhibit the ability of Bak to heterodimerize with Bcl-xL.

Bcl-2-related proteins are critical regulators of cell survival that are localized to the outer mitochondrial, outer nuclear and endoplasmic reticulum membranes. Despite their physiological importance, the biochemical function of Bcl-2-related proteins has remained elusive. The three-dimensional structure of Bcl-xL, an inhibitor of apoptosis, was recently shown to be similar to the structures of the pore-forming domains of bacterial toxins. A key feature of these pore-forming domains is the ability to form ion channels in biological membranes. Here we demonstrate that Bcl-xL shares this functional feature. Like the bacterial toxins, Bcl-xL can insert into either synthetic lipid vesicles or planar lipid bilayers and form an ion-conducting channel. This channel is pH-sensitive and becomes cation-selective at physiological pH. The ion-conducting channel(s) formed by Bcl-xL display multiple conductance states that have identical ion selectivity. Together, these data suggest that Bcl-xL may maintain cell survival by regulating the permeability of the intracellular membranes to which it is distributed.

During somatic cell division, faithful chromosomal segregation must follow DNA replication to prevent aneuploidy or polyploidy. Damage to the mitotic spindle is one potential mechanism that interferes with chromosomal segregation. The accumulation of aneuploid or polyploid cells resulting from a disrupted mitotic spindle is presumably prevented by cell cycle checkpoint controls. In the course of studying cells that overexpress the apoptosis-inhibiting protein Bcl-xL, we found that these cells have an increased rate of spontaneous tetraploidization, suggesting that apoptosis may play an important role in eliminating cells that fail to complete mitosis properly. When cells expressing Bcl-xL are treated with mitotic spindle inhibitors, a significant percentage reinitiate DNA replication and become polyploid. Nevertheless, the majority of cells expressing Bcl-xL undergo a prolonged p53-dependent cell cycle arrest following mitotic spindle damage. Unexpectedly, p53 expression is not induced in mitosis, nor does it influence M-phase arrest. Instead, cells with mitotic spindle damage only transiently arrest in M phase, and despite failing to complete mitosis, appear to proceed to G1. During this subsequent growth factor-dependent phase, p53 is induced and mediates cell cycle arrest. In cells that do not overexpress Bcl-xL, elimination of the p53-dependent growth arrest with a dominant negative mutant also results in polyploidy after mitotic spindle damage, but under these conditions most cells die by apoptosis. Expression of Bcl-xL and abrogation of p53 cooperate to allow rapid and progressive polyploidization following mitotic spindle damage. Our results suggest that suppression of apoptosis by bcl-2-related genes and loss of p53 function can act cooperatively to contribute to genetic instability.