Search

Data integration to align cells across batches has become a cornerstone of most single cell data analysis pipelines, critically affecting downstream analyses. Yet, when the batches are expected to biologically differ, how much signal is erased during integration? Currently, there are no guidelines for when the biological differences between samples are separable from batch effects, and thus, data integration usually involve a lot of guesswork: Cells across batches should be aligned to be “appropriately” mixed, while preserving “main cell type clusters”. We show evidence that current paradigms for single cell data integration are unnecessarily aggressive, removing biologically meaningful variation. To remedy this, we present a novel statistical model and computationally scalable algorithm, CellANOVA, to recover biological signal that is lost during single cell data integration. CellANOVA utilizes a “pool-of-controls” design concept, applicable across diverse settings, to separate unwanted variation from biological variation of interest. When applied with existing integration methods, CellANOVA allows the recovery of subtle biological signals and corrects, to a large extent, the data distortion introduced by integration. Further, CellANOVA explicitly estimates cell- and gene-specific batch effect terms which can be used to identify the cell types and pathways exhibiting the largest batch variations, providing clarity as to which biological signals can be recovered.

NLR family, apoptosis inhibitory proteins (NAIPs) detect bacterial flagellin and structurally related components of bacterial type III secretion systems (T3SS), and recruit NLR family, CARD domain containing protein 4 (NLRC4) and caspase-1 into an inflammasome complex that induces pyroptosis. NAIP/NLRC4 inflammasome assembly is initiated by the binding of a single NAIP to its cognate ligand, but a subset of bacterial flagellins or T3SS structural proteins are thought to evade NAIP/NLRC4 inflammasome sensing by not binding to their cognate NAIPs. Unlike other inflammasome components such as NLRP3, AIM2, or some NAIPs, NLRC4 is constitutively present in resting macrophages, and not thought to be regulated by inflammatory signals. Here, we demonstrate that Toll-like receptor (TLR) stimulation upregulates NLRC4 transcription and protein expression in murine macrophages, which licenses NAIP detection of evasive ligands. TLR-induced NLRC4 upregulation and NAIP detection of evasive ligands required p38 MAPK signaling. In contrast, TLR priming in human macrophages did not upregulate NLRC4 expression, and human macrophages remained unable to detect NAIP-evasive ligands even following priming. Critically, ectopic expression of either murine or human NLRC4 was sufficient to induce pyroptosis in response to immunoevasive NAIP ligands, indicating that increased levels of NLRC4 enable the NAIP/NLRC4 inflammasome to detect these normally evasive ligands. Altogether, our data reveal that TLR priming tunes the threshold for NAIP/NLRC4 inflammasome activation and enables inflammasome responses against immunoevasive or suboptimal NAIP ligands.Significance StatementCytosolic receptors in the neuronal apoptosis inhibitor protein (NAIP) family detect bacterial flagellin and components of the type III secretion system (T3SS). NAIP binding to its cognate ligand engages the adaptor molecule NLRC4 to form NAIP/NLRC4 inflammasomes culminating in inflammatory cell death. However, some bacterial pathogens evade NAIP/NLRC4 inflammasome detection, thus bypassing a crucial barrier of the immune system. Here, we find that, in murine macrophages, TLR-dependent p38 MAPK signaling increases NLRC4 expression, thereby lowering the threshold for NAIP/NLRC4 inflammasome activation in response to immunoevasive NAIP ligands. Human macrophages were unable to undergo priming-induced upregulation of NLRC4 and could not detect immunoevasive NAIP ligands. These findings provide a new insight into species-specific regulation of the NAIP/NLRC4 inflammasome.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced GI injury. Through single-cell RNA-sequencing of the irradiated mouse intestine, we find that p53 target genes are specifically enriched in stem cells of the regenerating epithelium, including revival stem cells that promote animal survival after GI damage. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce revival stem cells. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells that is controlled by an Mdm2-mediated negative feedback loop. These results suggest that p53 suppresses severe radiation-indued GI injury by promoting intestinal epithelial cell reprogramming.One-Sentence SummaryAfter severe radiation injury to the intestine, transient p53 activity induces revival stem cells to promote regeneration.

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.

Supplemental Methods: Mouse strains and flow cytometry antibodies Supplemental Figure 1: Individual tumor kinetics of the unirradiated tumor in the PDA model. Tumor kinetics (mean and individual) of the irradiated tumor and survival in the B16-F10 melanoma model. Supplemental Figure 2: Individual tumor kinetics of the unirradiated tumor in antibody depleted mice (Cd4, CD8 and Ly6c) and knockout mice (Morrbid, Batf3, IFN gamma, CD40.) Mean tumor kinetics of the unirradiated tumor and survival in uMT and B220 knockout mice. Supplemental Figure 3: Mean tumor kinetics of the unirradiated tumor and survival in antibody depleted mice (IFNAR) and knockout mice (MyD88, TLR4, IFNAR, Sting.) Supplemental Figure 4: Flow cytometry plots for the top RF variables for the effect of RT Supplemental Figure 5: As for Supplemental figure 4, but for CD40 Supplemental Figure 6: As for supplemental Figure 4, but for the peak response Supplemental Figure 7: Heat map of the RF variables that predict response in irradiated and unirradiated tumors at 24 and 72 hours. Supplemental Figure 8: Histological samples of CD8 T cell infiltrate and SMA expression. Supplemental Figure 9: Graphical representation

Supplemental Methods

Table S2. Genes that are upregulated in the S-PRT-treated bone revealed by RNASeq analysis

Figure S1 A) Gene Ontology (GO) enrichment analysis of the differentially expressed (upregulated) genes in the F-PRT-treated mouse skin. (B) Gene Ontology (GO) enrichment analysis of the differentially expressed (downregulated) genes in the S-PRT-treated mouse leg skin. (C) Gene Ontology (GO) enrichment analysis of the differentially expressed (downregulated) genes in the F-PRT-treated mouse skin. N=4 in all analyses.

Supplementary Figure 2 from RAD51 Up-regulation Bypasses BRCA1 Function and Is a Common Feature of BRCA1-Deficient Breast Tumors

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 BRCA1Δ/Δ 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. [Cancer Res 2007;67(20):9658–65]

Supplementary Figure Legends 1-3 from RAD51 Up-regulation Bypasses BRCA1 Function and Is a Common Feature of BRCA1-Deficient Breast Tumors

Supplementary Figure 1 from RAD51 Up-regulation Bypasses BRCA1 Function and Is a Common Feature of BRCA1-Deficient Breast Tumors

Supplementary Figure 3 from RAD51 Up-regulation Bypasses BRCA1 Function and Is a Common Feature of BRCA1-Deficient Breast Tumors

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.

Supplemental Materials & Methods

Evasion of antitumor immunity and resistance to therapies in solid tumors are aided by an immunosuppressive tumor microenvironment (TME). We found that TME factors, such as regulatory T cells and adenosine, downregulated type I interferon receptor IFNAR1 on CD8

The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancies (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 regulates inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to restrict the inflammatory response of neutrophils to toll-like receptor 4 (TLR4) signaling. Loss of RUNX1 in GMPs increased the TLR4 coreceptor CD14 on neutrophils, which contributed to neutrophils’ increased inflammatory cytokine production in response to the TLR4 ligand lipopolysaccharide. RUNX1 loss increased the chromatin accessibility of retrotransposons in GMPs and neutrophils and induced a type I interferon signature characterized by enriched footprints for signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRF) in opened chromatin, and increased expression of interferon-stimulated genes. The overproduction of inflammatory cytokines by neutrophils was reversed by inhibitors of type I IFN signaling. We conclude that RUNX1 restrains the chromatin accessibility of retrotransposons in GMPs and neutrophils, and that loss of RUNX1 increases proinflammatory cytokine production by elevating tonic type I interferon signaling.

Persistent inflammation including type-one interferon (IFN-I) can cause immunosuppression. We show that delayed administration of the JAK1 inhibitor itacitinib after anti-PD1 improves immune function and anti-tumor response in mice, and results in high response rates (67%) in a phase-2 clinical trial for metastatic non-small cell lung cancer with tumor PDL1≥50%. In contrast to patients with low inflammation who responded to anti-PD1, patients with elevated inflammation had poor immune and tumor responses to anti-PD1 that improved after adding itacitinib. Itacitinib promoted features of CD8 T cell plasticity and therapeutic responses of exhausted and effector-memory clonotypes. Patients with persistent IFN-I signaling refractory to itacitinib showed progressive CD8 T cell terminal differentiation and progressive disease. Thus, JAK inhibition may improve anti-PD1 efficacy by pivoting T cell differentiation dynamics.

Purpose Recent studies suggest that ultrahigh-dose-rate, “FLASH,” electron radiation therapy (RT) decreases normal tissue damage while maintaining tumor response compared with conventional dose rate RT. Here, we describe a novel RT apparatus that delivers FLASH proton RT (PRT) using double scattered protons with computed tomography guidance and provide the first report of proton FLASH RT-mediated normal tissue radioprotection. Methods and Materials Absolute dose was measured at multiple depths in solid water and validated against an absolute integral charge measurement using a Faraday cup. Real-time dose rate was obtained using a NaI detector to measure prompt gamma rays. The effect of FLASH versus standard dose rate PRT on tumors and normal tissues was measured using pancreatic flank tumors (MH641905) derived from the KPC autochthonous PanCa model in syngeneic C57BL/6J mice with analysis of fibrosis and stem cell repopulation in small intestine after abdominal irradiation. Results The double scattering and collimation apparatus was dosimetrically validated with dose rates of 78 ± 9 Gy per second and 0.9 ± 0.08 Gy per second for the FLASH and standard PRT. Whole abdominal FLASH PRT at 15 Gy significantly reduced the loss of proliferating cells in intestinal crypts compared with standard PRT. Studies with local intestinal irradiation at 18 Gy revealed a reduction to near baseline levels of intestinal fibrosis for FLASH-PRT compared with standard PRT. Despite this difference, FLASH-PRT did not demonstrate tumor radioprotection in MH641905 pancreatic cancer flank tumors after 12 or 18 Gy irradiation. Conclusions We have designed and dosimetrically validated a FLASH-PRT system with accurate control of beam flux on a millisecond time scale and online monitoring of the integral and dose delivery time structure. Using this system, we found that FLASH-PRT decreases acute cell loss and late fibrosis after whole-abdomen and focal intestinal RT, whereas tumor growth inhibition is preserved between the 2 modalities.

Although scRNA-seq is now ubiquitously adopted in studies of intratumor heterogeneity, detection of somatic mutations and inference of clonal membership from scRNA-seq is currently unreliable. We propose DENDRO, an analysis method for scRNA-seq data that clusters single cells into genetically distinct subclones and reconstructs the phylogenetic tree relating the subclones. DENDRO utilizes transcribed point mutations and accounts for technical noise and expression stochasticity. We benchmark DENDRO and demonstrate its application on simulation data and real data from three cancer types. In particular, on a mouse melanoma model in response to immunotherapy, DENDRO delineates the role of neoantigens in treatment response.

TPS626 Background: Systemic therapy with atezolizumab and bevacizumab (atezo/bev) has improved outcomes for advanced HCC, but results in objective responses in fewer than 30% of patients. Stereotactic body radiotherapy (SBRT) is currently used for small HCC tumors that do not require systemic therapy but has also been shown in a number of clinical trials of other solid tumors to enhance the anti-cancer immune response. Of particular interest, our prior experience with SBRT in a 17Gy fraction has demonstrated the ability to restore sensitivity to immunotherapy in advanced solid tumors, even those previously refractory to immunotherapy. We hypothesize that repeated high dose fractions of radiation will act as an immune booster and will improve on outcomes of patients with advanced HCC. Since the combination of SBRT and atezo/bev has not yet been tested prospectively in patients with HCC, and because bevacizumab is a known radiosensitizer, we are conducting a phase I trial to evaluate the safety of repeated SBRT doses. Methods: This is a single-site phase I clinical trial utilizing a Rolling 6 design to determine the safety of 1, 2, or 3 doses of SBRT fractions in combination with atezo/bev. Up to 18 total patients will be enrolled (n=6 per cohort). Patients must be naïve to systemic therapy with Child-Pugh A or B liver function, at least one lesion amenable to radiation, and a measurable lesion that will not receive radiation. Patients with uncontrolled ascites or hepatic encephalopathy are excluded. Atezo/bev is administered at the standard doses every 21 days. SBRT will start 1 week after the first infusion of this combination. Patients will receive 1, 2, or 3 17Gy fractions of SBRT at 4-week intervals (cohorts 2 and 3 only). Patients will undergo serial collections of circulating cell-free DNA (ccfDNA), methylated DNA, and peripheral blood mononuclear cells to investigate the application of these markers as a predictor of response. Following completion of SBRT, patients will continue with atezo/bev until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint is the proportion of patients experiencing dose-limiting toxicities (grade 3 or higher radiation-related toxicities graded by the Common Terminology Criteria for Adverse Events, Version 5). Secondary endpoints are overall survival, progression-free survival, objective response rate, duration of response, and toxicity rates. Enrollment began 9/2022 and complete accrual is expected by June 2024. NCT05488522. Clinical trial information: NCT05488522 .

Sweat out the fat with TSLP! Thymic stromal lymphopoietin (TSLP) is a cytokine that can promote immune responses that characterize allergic diseases. Choa et al . found that mice engineered to produce elevated TSLP displayed selective white adipose tissue loss that protected them from obesity, insulin resistance, and steatohepatitis (see the Perspective by Schneider). Protection was not mediated by eosinophils, regulatory T cells, or innate lymphoid cells. Rather, TLSP induced the migration of conventional T cells to sebaceous glands in the skin. Once there, these T cells promoted white adipose tissue loss by the hypersecretion of sebum, a lipid-rich substance that augments the skin’s barrier function. This mechanism, which likely evolved to enhance cutaneous antimicrobial defenses, could be possibly targeted in future treatments for obesity. —STS

Although cancer immunotherapy with PD-(L)1 blockade is now routine treatment for patients with lung cancer, remarkably little is known about acquired resistance. We examined 1,201 patients with NSCLC treated with PD-(L)1 blockade to clinically characterize acquired resistance, finding it to be common (occurring in more than 60% of initial responders), with persistent but diminishing risk over time, and with distinct metastatic and survival patterns compared to primary resistance. To examine the molecular phenotype and potential mechanisms of acquired resistance, we performed whole transcriptome and exome tumor profiling in a subset of NSCLC patients (n=29) with acquired resistance. Systematic immunogenomic analysis revealed that tumors with acquired resistance generally had enriched signals of inflammation (including IFNγ signaling and inferred CD8+ T cells) and could be separated into IFNγ upregulated and stable subsets. IFNγ upregulated tumors had putative routes of resistance with signatures of dysfunctional interferon signaling and mutations in antigen presentation genes. Transcriptomic profiling of cancer cells from a murine model of acquired resistance to PD-(L)1 blockade also showed evidence of dysfunctional interferon signaling and acquired insensitivity to in vitro interferon gamma treatment. In summary, we characterized clinical and molecular features of acquired resistance to PD-(L)1 blockade in NSCLC and found evidence of ongoing but dysfunctional IFN response. The persistently inflamed, rather than excluded or deserted, tumor microenvironment of acquired resistance informs therapeutic strategies to effectively reprogram and reverse acquired resistance.

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.

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)

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

Summary 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.

SummaryThe DNA dependent pattern recognition receptor, cGAS mediates communication between genotoxic stress and the immune system. Mitotic chromosome missegregation is an established stimulator of cGAS activity, however, it is unclear if progression through mitosis is required for cancer cell intrinsic activation of immune mediated anti-tumor responses. Moreover, it is unknown if disruption of cell cycle checkpoints can restore responses in cancer cells that are recalcitrant to DNA damage induced inflammation. Here we demonstrate that prolonged cell cycle arrest at the G2-mitosis boundary from either CDK1 inhibition or excessive DNA damage prevents inflammatory stimulated gene expression and immune mediated destruction of distal tumors. Remarkably, DNA damage induced inflammatory signaling is restored in a cGAS-and RIG-I-dependent manner upon concomitant disruption of p53 and the G2 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 antitumor immune responses.

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

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.

BACKGROUND Nearly 1 in 5 Americans will develop skin cancer, and as a result, survivors of skin cancer compose one of the largest groups of cancer survivors. Survivorship care plans (SCPs) are an important tool for improving patient outcomes and provide critical information to both survivors and health care professionals. Recent efforts have been made to expand SCP utilization; however, which patients currently receive SCPs is poorly understood. METHODS This study used 596 individuals with a diagnosis of melanoma (n = 391) or nonmelanoma skin cancer (n = 205) who had used an Internet-based SCP tool from May 2010 to December 2016 to model the patient and provider characteristics that determine SCP utilization. RESULTS Survivors were predominantly white (95.3%) and female (56.5%). Survivors who received a treatment summary were more likely to also receive an SCP. University and nonuniversity cancer centers used SCPs at a higher rate than other care settings. Survivors whose care was managed by a team rather than just an individual physician were also more likely to receive an SCP. Survivors older than 70 years at diagnosis were almost twice as likely to receive a plan as survivors who were diagnosed at a younger age. CONCLUSIONS With a convenience sample of skin cancer survivors, it is possible to model factors that predict the receipt of SCPs. Important variables include the diagnosis age, treatment setting, physician type, and treatment-summary utilization. A closer examination of these variables identified several disparities in care-plan use and, therefore, opportunities to improve the distribution of SCPs. Further validation in additional cohorts of survivors is necessary to confirm these conclusions. Cancer 2017. © 2017 American Cancer Society.

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.

p27 shifts from CDK inhibitor to oncogene when phosphorylated by PI3K effector kinases. Here, we show that p27 is a cJun coregulator, whose assembly and chromatin association is governed by p27 phosphorylation. In breast and bladder cancer cells with high p27pT157pT198 or expressing a CDK-binding defective p27pT157pT198 phosphomimetic (p27CK−DD), cJun is activated and interacts with p27, and p27/cJun complexes localize to the nucleus. p27/cJun up-regulates TGFB2 to drive metastasis in vivo. Global analysis of p27 and cJun chromatin binding and gene expression shows that cJun recruitment to many target genes is p27 dependent, increased by p27 phosphorylation, and activates programs of epithelial–mesenchymal transformation and metastasis. Finally, human breast cancers with high p27pT157 differentially express p27/cJun-regulated genes of prognostic relevance, supporting the biological significance of the work.

2530 Background: Neoantigen depleted malignancies such as colorectal cancer demonstrate primary resistance to immune checkpoint blockade, and solid tumors in general have shown resistance to chimeric antigen receptor (CAR) T cell therapy. However, CAR-T cells have been shown to be capable of delivering various therapeutic molecules in a targeted fashion to the tumor microenvironment, in some cases through extracellular vesicles (EVs). In vivo studies have shown that the presentation of foreign viral peptides by solid tumors can reprogram bystander virus-specific cytotoxic T cells (CTLs) against tumor cells. In this study, we demonstrate that CAR-T cells can deliver engineered peptide antigens to solid tumors, leading to presentation on tumor cells and anti-tumor response. Methods: Second generation CAR-T cells (41BB endodomain) targeting human CD19 (19BBz) or human mesothelin (M5BBz) were generated via retroviral and lentiviral transduction respectively. CAR-T cells were engineered to co-express peptides such as SIINFEKL of ovalbumin and NLVPMVATV of CMV pp65 among others. Peptides were isolated from EVs via ultracentrifugation. For in vivo studies, C57BL/6 or NSG mice were injected on the flank with relevant tumors and treated with peptide-CAR-T cells. In vitro studies utilized flow cytometry and xCELLigence killing assays. Results: Murine 19BBz CAR-T cells expressing the SIINFEKL peptide of ovalbumin (ova-19BBz) were found to transfer SIINFEKL peptide to tumor cells via EVs in vitro and in vivo, leading to peptide presentation on MHC-I of tumor cells. This resulted in significantly delayed tumor growth in tumor bearing mice transfused with OT-I T cells to mimic an existing antigen specific T cell pool. We expanded on these findings by isolating EVs from human M5BBz CAR-T cells expressing CMV viral peptides. Peptide-CAR-T EVs were co-cultured with human ovarian cancer cells to assess presentation to Jurkat T cells. Finally, we utilized primary human T cells from CMV+ healthy donors to assess the clinical feasibility of our peptide delivery approach. Conclusions: CAR-T cells can be engineered to deliver peptides to tumor cells for presentation and subsequent targeting by antigen specific CTLs. This represents a novel strategy for the treatment of non-immunogenic tumors.

Multiple studies indicate that ultra-high dose rate, “FLASH” Radiotherapy (F-RT), augments the therapeutic index compared to Standard RT (S-RT) by sparing normal tissue from damage while maintaining tumor responses. We previously reported the first studies using FLASH proton RT (F-PRT), demonstrating that compared to S-PRT, F-PRT spares mice from intestinal epithelium fibrosis and leads to improved overall survival at single doses from 13-16 Gy. Moreover, F-PRT spares a higher percentage of proliferating crypt cells and better maintains the villi architecture 3.5 days post-RT. Importantly, F-PRT was equipotent with S-PRT in controlling syngeneic pancreatic tumors in the same mouse strain. Here, we present new data into potential cellular mechanisms leading to the sparing effect. Using single-cell transcriptomics, we find that F-PRT leads to activation of RIG-I-mediated signaling in the immune cell compartment, which has been linked to normal tissue radioprotection. F-PRT led to higher levels of proliferation genes (Myc, mTOR) in the stem/progenitor cell compartment, consistent with higher proliferative rates. Using a model of epithelial-specific knockout of p53, we find that mice treated with 13.5 Gy S-PRT quickly succumb to acute radiation syndrome while F-PRT significantly improves overall survival. This suggests that F-PRT may preserve a higher proportion of progenitor epithelial cells, leading to reduced fibrosis and long-term toxicity. We also expanded studies to include normal skin damage and control of fibrosarcoma tumors. F-PRT comprehensively decreased toxicity relative to S-PRT, as measured by survival endpoints of mortality/compulsory euthanasia. F-PRT decreased perivascular inflammation, dermal inflammatory infiltrates, and dermal fibrosis on histopathology. Critically, the effect of F-PRT on control of syngeneic sarcomas grown in the leg was indistinguishable from that of S-PRT. Based on these results, we initiated a pilot trial for canine osteosarcoma. To date, we have treated 20 canine patients with a single pre-op fraction of S-PRT or F-PRT. Early results following RNAseq from normal and tumor bone tissue revealed that F-PRT and S-PRT differentially affect myogenesis, adipogenesis, EMT, and inflammatory response. While multiple studies with electron and proton F-RT have demonstrated its normal tissue sparing effects, the mechanism at the molecular level remains elusive. Several models have been proposed, including depletion of oxygen in the vicinity of the DNA, ROS recombination, etc. We will present data from studies of hypoxia and its impact on the FLASH effect and discuss this and alternative models for preferential radioprotection. Our results support the notion that F-PRT significantly augments in the therapeutic window of radiotherapy. Several key questions concerning the mechanism of normal tissue sparing, impact of S-RT vs. F-RT on different tissues and biophysical parameters such as fractionation and optimal dose rates will need to be addressed prior to full clinical implementation of this new modality. Citation Format: Ioannis Verginadis, Anastasia Velalopoulou, Sarah Hagan, Denisa Goia, Khayrullo Shoniyozov, Michele Kim, Eric Diffenderfer, Lei Dong, James Metz, Keith Cengel, Theresa Busch, Andy Minn, Amit Maity, Constantinos Koumenis. Preclinical studies with proton FLASH radiotherapy in mice and canines: Biological effects, biophysical considerations and potential mechanisms [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-019.

Detection of genetically distinct subclones and profiling the transcriptomic differences between them is important for studying the evolutionary dynamics of tumors, as well as for accurate prognosis and effective treatment of cancer in the clinic. For the profiling of intra-tumor transcriptional heterogeneity, single cell RNA-sequencing (scRNA-seq) is now ubiquitously adopted in ongoing and planned cancer studies. Detection of somatic DNA mutations and inference of clonal membership from scRNA-seq, however, is currently unreliable. We propose DENDRO, an analysis method for scRNA-seq data that detects genetically distinct subclones, assigns each single cell to a subclone, and reconstructs the phylogenetic tree describing the tumor’s evolutionary history. DENDRO utilizes information from single nucleotide mutations in transcribed regions and accounts for technical noise and expression stochasticity at the single cell level. The accuracy of DENDRO was benchmarked on spike-in datasets and on scRNA-seq data with known subpopulation structure. We applied DENDRO to delineate subclonal expansion in a mouse melanoma model in response to immunotherapy, highlighting the role of neoantigens in treatment response. We also applied DENDRO to primary and lymph-node metastasis samples in breast cancer, where the new approach allowed us to better understand the relationship between genetic and transcriptomic intratumor variation.

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.

Following publication of the original article [1], the author reported that an author name, Roberta Zappasodi, was missed in the authorship list. The correction has been implemented in the original article as well. The publisher apologized for any inconvenience this might has caused.

Although cancer cell intrinsic mechanisms are important in driving aggressive features of cancer, stromal cells in the tumor microenvironment also serve critical roles. In particular, heterotypic interaction between cancer cells and stromal fibroblasts can generate signals for cancer progression, therapy resistance, and inflammatory responses. Among the signals resulting from cellular cross-talk include anti-viral pathways that induce interferon-stimulated genes (ISGs). These observations suggest 1) the existence of signals in the tumor microenvironment that mimic a viral infection, 2) resulting activation of anti-viral signaling may contribute to how stromal cells promote aggressive features of cancer, and 3) mechanisms must exist to prevent this anti-viral signaling under non-pathological conditions. We have demonstrated that exosomes and their RNA produced by stromal fibroblasts represents one such “virus mimic” by engaging RNA pattern recognition receptors (PRRs) such as RIG-I. Triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous non-coding RNA normally shielded from PRR recognition by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 that is deployed in stromal exosomes. After these stromal exosomes are transferred to immune cells, the unshielded RN7SL1 can drive an inflammatory response. Upon exosome transfer to breast cancer cells, unshielded RN7SL1 activates the RIG-I to enhance tumor growth, metastasis, and therapy resistance. The significance of this “virus mimicry” and failure to shield endogenous RN7SL1 from PRR activation is corroborated by evidence from patient tumors and blood. In total, these results demonstrate how exosomes, unshielding of endogenous RNAs, and engagement of pattern recognition receptors couple stromal activation with aggressive features of breast cancer. Citation Format: Minn A. Exosome-mediated stimulation of metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES2-3.

Much of our understanding on resistance mechanisms to conventional cancer therapies such as chemotherapy and radiation has focused on cell intrinsic properties that antagonize the detrimental effects of DNA and other cellular damage. However, it is becoming clear that the immune system and/or innate immune signaling pathways can integrate with these intrinsic mechanisms to profoundly influence treatment efficacy. In this context, recent evidence indicates that interferon (IFN) signaling has an important role in this integration by influencing immune and intrinsic/non-immune determinants of therapy response. However, IFN signaling can be both immunostimulatory and immunosuppressive, and the factors determining these outcomes in different disease settings are unclear. Here I discuss the regulation and molecular events in cancer that are associated with these dichotomous functions.

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.

Nearly 1 in 5 Americans will develop skin cancer, and as a result, survivors of skin cancer compose one of the largest groups of cancer survivors. Survivorship care plans (SCPs) are an important tool for improving patient outcomes and provide critical information to both survivors and health care professionals. Recent efforts have been made to expand SCP utilization; however, which patients currently receive SCPs is poorly understood.This study used 596 individuals with a diagnosis of melanoma (n = 391) or nonmelanoma skin cancer (n = 205) who had used an Internet-based SCP tool from May 2010 to December 2016 to model the patient and provider characteristics that determine SCP utilization.Survivors were predominantly white (95.3%) and female (56.5%). Survivors who received a treatment summary were more likely to also receive an SCP. University and nonuniversity cancer centers used SCPs at a higher rate than other care settings. Survivors whose care was managed by a team rather than just an individual physician were also more likely to receive an SCP. Survivors older than 70 years at diagnosis were almost twice as likely to receive a plan as survivors who were diagnosed at a younger age.With a convenience sample of skin cancer survivors, it is possible to model factors that predict the receipt of SCPs. Important variables include the diagnosis age, treatment setting, physician type, and treatment-summary utilization. A closer examination of these variables identified several disparities in care-plan use and, therefore, opportunities to improve the distribution of SCPs. Further validation in additional cohorts of survivors is necessary to confirm these conclusions. Cancer 2018;124:183-91. © 2017 American Cancer Society.

Immune escape of malignant cells is an important hallmark of cancer, necessary for tumor formation and progression. Accordingly, in recent years, therapies that enhance the immune system have had remarkable success in treating a myriad of malignancies. Particularly successful has been immune checkpoint blockade (ICB), which is a therapy that targets T-cell inhibitory receptors, or immune checkpoints. Despite these encouraging clinical results, most patients do not respond to such agents. Therefore, determining methods to better target and enhance the therapeutic efficacy of ICB is of paramount importance. One appealing approach is to use standard anticancer therapies, such as radiation, chemotherapy, and targeted biologics, to favorably modulate the immune system and enhance the anticancer immune response. For example, although radiation therapy has classically been thought of as a local therapy, there is significant potential for combining radiation therapy with ICB to both optimize local control and to treat metastatic disease. This concept is supported by numerous preclinical studies and clinical case reports and has since led to many early and ongoing clinical trials. However, it is still unclear how to optimally combine radiation and ICB to maximize the therapeutic effect. In this review, we highlight relevant preclinical and clinical studies in the field of radiation and ICB and discuss optimal strategies for combination therapies moving forward.

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.