Search

Background Most Crohn’s disease [CD] patients require surgery. Ileitis recurs after most ileocolectomies and is a critical determinant for outcomes. The impacts of ileocolectomy-induced bile acid [BA] perturbations on intestinal microbiota and inflammation are unknown. We characterized the relationships between ileocolectomy, stool BAs, microbiota and intestinal inflammation in inflammatory bowel disease [IBD]. Methods Validated IBD clinical and endoscopic assessments were prospectively collected. Stool primary and secondary BA concentrations were compared based on ileocolectomy and ileitis status. Primary BA thresholds for ileitis were evaluated. Metagenomic sequencing was use to profile microbial composition and function. Relationships between ileocolectomy, BAs and microbiota were assessed. Results In 166 patients, elevated primary and secondary BAs existed with ileocolectomy. With ileitis, only primary BAs [795 vs 398 nmol/g, p = 0.009] were higher compared to without ileitis. The optimal primary BA threshold [≥228 nmol/g] identified ileitis on multivariable analysis [odds ratio = 2.3, p = 0.04]. Microbial diversity, Faecalibacterium prausnitzii and O-acetylhomoserine aminocarboxypropyltransferase [MetY] were decreased with elevated primary BAs. Amongst ileocolectomy patients, only those with elevated primary BAs had diversity, F. prausnitzii and MetY reductions. Those with both ileocolectomy and intermediate [p = 0.002] or high [≥228 nmol/g, p = 9.1e-11]] primary BA concentrations had reduced F. prausnitzii compared to without ileocolectomy. Those with ileocolectomy and low [ Conclusions Elevated primary BAs were associated with ileitis, and reduced microbial diversity, F. prausnitzii abundance and enzymatic abundance of MetY [acetate and l-methionine-producing enzyme expressed by F. prausnitzii], and were the only factors associated with these findings after ileocolectomy.

Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrently mutated driver genes. Here we describe a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant PIK3CA, among the most frequently genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this strategy, we developed a panel of TCRs that recognize an endogenously processed neopeptide encompassing a common PIK3CA hotspot mutation restricted by the prevalent human leukocyte antigen (HLA)-A*03:01 allele. Mechanistically, immunogenicity to this public NeoAg arises from enhanced neopeptide/HLA complex stability caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a comparatively ‘featureless’ surface dominated by the peptide’s backbone. To bind this epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface facilitated by an unusually long CDR3β loop. In patients with diverse malignancies, we observed NeoAg clonal conservation and spontaneous immunogenicity to the neoepitope. Finally, adoptive transfer of TCR-engineered T cells led to tumor regression in vivo in mice bearing PIK3CA-mutant tumors but not wild-type PIK3CA tumors. Together, these findings establish the immunogenicity and therapeutic potential of a mutant PIK3CA-derived public NeoAg.

Isocitrate dehydrogenase (IDH)-wild-type glioblastoma (GBM) is the most common and aggressive primary brain tumor which carries a very poor overall prognosis and is universally fatal. Understanding the transcriptional regulation of the proliferation of GBM tumor cells is critical for developing novel and effective treatments. In this study, we investigate the role of the transcription factor TCF12 in the regulation of GBM proliferation using human and murine GBM cell lines and an in vivo GBM xenograft model. Our study shows that TCF12 deficiency severely impairs proliferation of tumor cells in vitro by disrupting/blocking the G1 to S phase transition. We also discover that TCF12 loss significantly improves animal survival and that TCF12-deficient tumors grow much slower in vivo. Overexpression of TCF12, on the other hand, leads to an increase in the proliferation of tumor cells in vitro and more aggressive tumor progression in vivo. Interestingly, loss of TCF12 leads to upregulation of signature genes of the oligodendrocytic lineage in GBM stem cells, suggesting a role for TCF12 in inhibiting differentiation along the oligodendrocytic lineage. Transcriptomic data also reveals that loss of TCF12 leads to dysregulation of the expression of key genes in the cell cycle. Our work demonstrates critical roles of TCF12 in GBM tumor progression.

The immune system coordinates the response to cardiac injury and is known to control regenerative and fibrotic scar outcomes in the heart and subsequent chronic low-grade inflammation associated with heart failure. Here we profiled the inflammatory response to heart injury using single cell transcriptomics to compare and contrast two experimental models with disparate outcomes. We used adult mice, which like humans lack the ability to fully recover and zebrafish which spontaneously regenerate after heart injury. The extracardiac reaction to cardiomyocyte necrosis was also interrogated to assess the specific peripheral tissue and immune cell reaction to chronic stress. Cardiac macrophages are known to play a critical role in determining tissue homeostasis by healing versus scarring. We identified distinct transcriptional clusters of monocytes/macrophages in each species and found analogous pairs in zebrafish and mice. However, the reaction to myocardial injury was largely disparate between mice and zebrafish. The dichotomous response to heart damage between the mammalian and zebrafish monocytes/macrophages may underlie the impaired regenerative process in mice, representing a future therapeutic target.

Facilitating axon regeneration in the injured central nervous system remains a challenging task. RAF-MAP2K signaling plays a key role in axon elongation during nervous system development. Here, we show that conditional expression of a constitutively kinase-activated BRAF in mature corticospinal neurons elicited the expression of a set of transcription factors previously implicated in the regeneration of zebrafish retinal ganglion cell axons and promoted regeneration and sprouting of corticospinal tract (CST) axons after spinal cord injury in mice. Newly sprouting axon collaterals formed synaptic connections with spinal interneurons, resulting in improved recovery of motor function. Noninvasive suprathreshold high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) activated the BRAF canonical downstream effectors MAP2K1/2 and modulated the expression of a set of regeneration-related transcription factors in a pattern consistent with that induced by BRAF activation. HF-rTMS enabled CST axon regeneration and sprouting, which was abolished in MAP2K1/2 conditional null mice. These data collectively demonstrate a central role of MAP2K signaling in augmenting the growth capacity of mature corticospinal neurons and suggest that HF-rTMS might have potential for treating spinal cord injury by modulating MAP2K signaling.

The epicardium, a mesothelial cell tissue that encompasses vertebrate hearts, supports heart regeneration after injury through paracrine effects and as a source of multipotent progenitors. However, the progenitor state in the adult epicardium has yet to be defined. Through single-cell RNA-sequencing of isolated epicardial cells from uninjured and regenerating adult zebrafish hearts, we defined the epithelial and mesenchymal subsets of the epicardium. We further identified a transiently activated epicardial progenitor cell (aEPC) subpopulation marked byptx3aandcol12a1bexpression. Upon cardiac injury, aEPCs emerge from the epithelial epicardium, migrate to enclose the wound, undergo epithelial-mesenchymal transition (EMT), and differentiate into mural cells andpdgfra+hapln1a+mesenchymal epicardial cells. These EMT and differentiation processes are regulated by the Tgfβ pathway. Conditional ablation of aEPCs blocked heart regeneration through reduced Nrg1 expression and mesenchymal cell number. Our findings identify a transient progenitor population of the adult epicardium that is indispensable for heart regeneration and highlight it as a potential target for enhancing cardiac repair.

Tumor-specific CD8 T cells (TST) found in patients with cancer are unable to halt cancer progression. TST are dysfunctional and cannot produce effector cytokines or kill target cells1. TST dysfunction, also known as exhaustion, has been thought to be driven by chronic T cell receptor (TCR)/antigen stimulation over days to weeks, encoded by exhaustion/dysfunction-associated epigenetic and transcriptional programs1,2. However, we know little about (i) the interplay between CD8 T cell function and epigenetics during the initial hours after activation in both functional (acute infection) or dysfunctional contexts (tumors) or (ii) the kinetics of CD8 T cell effector or dysfunction differentiation and relationship to cell division. Nevertheless, is it widely thought that T cell effector differentiation requires cell division3. Here we tracked differentiation of naive antigen-specific CD8 T cells by cell division within the first hours (0-60 hours) after antigen activation in tumor-bearing hosts, comparing to T cells undergoing functional effector differentiation during acute infection (E). Surprisingly, while TST and E exhibited the same rapid activation and cell division kinetics, TST failed to acquire effector function and implemented exhaustion-associated chromatin features. Notably, epigenetic encoding of TST dysfunction began within 6 hours of antigen encounter, even prior to cell division. These findings not only challenge the paradigm that chronic T cell receptor stimulation drives dysfunction/exhaustion in tumors, but also refute for CD8 T cells the prevailing notion that cell division is required to initiate epigenetic remodeling and differentiation3-7. Interestingly, while T cell dysfunctional fate decisions are made within hours of antigen encounter, continued TCR stimulation and epigenetic remodeling progressively stabilize, or “imprint” the dysfunctional state. Our study defines for the first time the regulation and kinetics driving the rapid divergence of T cell fate choice prior to cell division in the context of tumors versus infection.

Facilitating axon regeneration in the injured central nervous system remains a challenging task. RAF–MEK signaling plays an important role in axon elongation during nervous system development. Here we show that activation of B-RAF in mature corticospinal neurons elicited the expression of a discrete set of transcription factors previously implicated in the regeneration of zebrafish optic nerve axons. Genetic activation of B-RAF–MEK signaling promoted robust regeneration and sprouting of corticospinal tract axons after injury. Newly sprouting axon collaterals formed synaptic connections with spinal interneurons, correlating with the recovery of skilled motor function. Seeking a non-invasive way to stimulate axon regeneration, we found that suprathreshold high-frequency repetitive transcranial magnetic stimulation activates the B-RAF canonical effectors MEK1/2 and requires MEK1/2 activity to promote corticospinal axon regeneration and sprouting after injury. These data demonstrate a central role of neuron-intrinsic RAF–MEK signaling in enhancing the growth capacity of mature corticospinal neurons and propose HF-rTMS as a potential therapy for spinal cord injury.One Sentence SummaryGenetic or HF-rTMS-mediated activation of B-RAF– MEK signaling promotes CST axon sprouting and functional regeneration after a spinal cord injury.

Contains fulltext : 244608.pdf (Publisher’s version ) (Closed access) Aberrant inflammation in the CNS has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a new approach to derive microglia from human pluripotent stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular cross-talk along the neuroinflammatory axis in vitro. We used the tri-culture system to model neuroinflammation in Alzheimer's disease with hPSCs harboring the APP(SWE)+/+ mutation and their isogenic control. We found that complement C3, a protein that is increased under inflammatory conditions and implicated in synaptic loss, is potentiated in tri-culture and further enhanced in APP(SWE)+/+ tri-cultures due to microglia initiating reciprocal signaling with astrocytes to produce excess C3. Our study defines the major cellular players contributing to increased C3 in Alzheimer's disease and presents a broadly applicable platform to study neuroinflammation in human disease.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) ‘resets’ these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices—which do not have the constraints of bioprinted scaffolds—the ‘reset’ vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call ‘Organ-On-VascularNet’. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting., The transient reactivation of ETV2 in adult human endothelial cells reprograms these cells to become adaptable vasculogenic endothelia that in three-dimensional matrices self-assemble into vascular networks that can transport blood and physiologically arborize organoids and decellularized tissues.

Efforts to cure HIV have focused on reactivating latent proviruses to enable elimination by CD8+ cytotoxic T-cells. Clinical studies of latency reversing agents (LRA) in individuals treated with antiretroviral therapy (ART) have shown increases in HIV transcription, but without reductions in virologic measures, or evidence that HIV-specific CD8+ T-cells were productively engaged. Here, we show that the SARS-CoV-2 mRNA vaccine BNT162b2 activates the RIG-I/TLR – TNF – NFκb axis, resulting in transcription of HIV proviruses with minimal perturbations of T-cell activation and host transcription. T-cells specific for the early gene-product HIV-Nef uniquely increased in frequency and acquired effector functon(granzyme-B) in ART-treated individuals following SARS-CoV-2 mRNA vaccination. These parameters of CD8+ T-cell induction correlated strongly with significant decreases in cell-associated HIV mRNA, suggesting killing or suppression of cells transcribing HIV. These results are, to our knowledge, the first observation of an intervention-induced reduction in a measure of HIV persistence, accompanied by precise immune correlates, in ART-suppressed individuals. However, we did not observe significant depletions of intact proviruses, underscoring challenges to achieving (or measuring) HIV reservoir reductions. Overall, our results support prioritizing the measurement of granzyme-B-producing Nef-specific responses in latency reversal studies and add impetus to developing HIV-targeted mRNA therapeutic vaccines that leverage built-in LRA activity.

The purpose of this study is to identify key regulatory pathways that potentially drive abnormal gene expression program in relapsed Acute Myeloid Leukemia (AML) patients, by integrative computational analyses on multi-omics molecular profiles. Relapsed AML remains a clinical challenge. Epigenetic heterogeneity may contribute to transcriptional dysregulation and disease progression in AML. However, what specific transcriptional programs and potential regulatory mechanisms contribute to disease relapse are not yet well understood. To characterize the global transcriptional landscapes in relapsed AML, we integrated genomics data from two cohorts of matched diagnosis and relapse patient specimens. We identified 5,416 differentially expressed genes (DEGs) between diagnosis and relapse in Cohort I. Unsupervised clustering yielded three distinct DEG groups: group A, B and C genes that were predominantly (88%) down-regulated, divergently regulated, or predominantly (65%) up-regulated, respectively, upon relapse. The expression pattern of all DEGs separated the patients into two clusters, most robustly by Group B genes. Interestingly, the majority of DEGs did not associate with changes in gene promoter methylation. Similar patterns were observed in Cohort II. We used Binding Analysis for Regulation of Transcription (BART) to identify transcriptional regulators (TRs) that potentially regulated the DEGs not associated with DNA methylation changes, and assessed the differential expression of identified TRs during disease progression. PU.1 was identified as a potential TR for Group A genes and was down-regulated upon relapse. GATA1 and TAL1 were identified as regulating Group B genes and were up-regulated in patient cluster1 and down-regulated in cluster2, consistent with the expression pattern of Group B genes. RBBP5 was a top predicted TR for Group C genes and was up-regulated upon relapse. We next validated the potential functionality of those predicted factors. In NSG mice transplanted with a human AML specimen, TAL1 and GATA1 were downregulated in AML cells collected four weeks after chemotherapy treatment, and were inferred as TRs for the down-regulated genes, similar to the patient data. PU.1 was inferred as regulating the up-regulated genes. Furthermore, we found that the level of differential expression of TAL1, GATA1, and PU.1 in each patient specimen associated with the correlation of DEG profiles between the patient specimen and TR perturbation in human-derived hematopoietic cell lines. Our results support the possibility that in some AML patients, TRs with roles in hematopoiesis and leukemia might contribute to disease relapse. Further mechanistic studies deciphering the molecular and phenotypic events facilitated by these TRs will yield significant insight into disease biology and possible therapeutic targeting approaches in relapsed AML. Citation Format: Zhenjia Wang, Yaseswini Neelamraju, Cem Meydan, Nicholas Dunham, Jorge Gandara, Tak Lee, Subhash Prajapati, Franck Rapaport, Caroline Sheridan, Paul Zumbo, Michael Becker, Lars Bullinger, Martin Carroll, Richard D’Andrea, Richard Dillon, Ross Levine, Christopher E. Mason, Ari Melnick, Donna Neuberg, Stefan Bekiranov, Chongzhi Zang, Francine E. Garrett-Bakelman. Gene expression profiles reveal distinct regulatory activities of transcription factors GATA1 and TAL1 upon AML relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3155.

T cell receptor (TCR) signal strength is a key determinant of T cell responses. We developed a cancer mouse model in which tumor-specific CD8 T cells (TST cells) encounter tumor antigens with varying TCR signal strength. High-signal-strength interactions caused TST cells to up-regulate inhibitory receptors (IRs), lose effector function, and establish a dysfunction-associated molecular program. TST cells undergoing low-signal-strength interactions also up-regulated IRs, including PD1, but retained a cell-intrinsic functional state. Surprisingly, neither high- nor low-signal-strength interactions led to tumor control in vivo, revealing two distinct mechanisms by which PD1hi TST cells permit tumor escape; high signal strength drives dysfunction, while low signal strength results in functional inertness, where the signal strength is too low to mediate effective cancer cell killing by functional TST cells. CRISPR-Cas9–mediated fine-tuning of signal strength to an intermediate range improved anti-tumor activity in vivo. Our study defines the role of TCR signal strength in TST cell function, with important implications for T cell–based cancer immunotherapies.

SUMMARYInflammation is essential to the disruption of tissue homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define the dynamics and recall of an epigenetic memory of inflammation that impacts cell fate decisions in a context-dependent manner.

Tissue homeostasis depends on responses to environmental insults to restore cellular phenotype, microenvironment composition, and tissue architecture. Inflammation is essential to the disruption of homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define a capacity for an environmental insult to control future cell-fate decisions in a context-dependent manner. The ability of epigenetic memory to potentiate tumor initiation both broadens the relationship between inflammation and cancer and raises the possibility that inducing epigenetic ‘amnesia’ of an inflammatory insult could be leveraged as a novel cancer prevention strategy. Citation Format: David J. Falvo, Adrien Grimont, Paul Zumbo, Julie L. Yang, Alexa Osterhoudt, Grace Pan, Andre F. Rendeiro, John Erby Wilkinson, Friederike Dundar, Olivier Elemento, Rhonda K. Yantiss, Doron Betel, Richard Koche, Rohit Chandwani. An epigenetic memory of inflammation controls context-dependent lineage plasticity and KRAS-driven tumorigenesis in the pancreas. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR008.

The most prominent KRAS variants (G12D, G12V, G12R) that together represent around 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are so far undruggable. While the role of several Kras mediators have been characterized in the initiation of PDAC, very little is known about the hierarchy of Kras effectors in the maintenance of the tumor. In recent years, strategies targeting the effectors downstream of mutant KRAS have offered scope for combined inhibition of EGFR and CRAF or MEK/RAF. However, these studies, unfortunately, have been limited by either narrow interrogation of downstream effectors in mouse models, or by the use of two-dimensional cell culture systems that may not recapitulate dependencies of the tumor in situ, respectively. To systematically interrogate the potential molecular dependencies in pancreatic tumor maintenance across several combinations of driver mutations, we have deployed in vitro and in vivo approaches in which we have selectively targeted key mediators of known KRAS-dependent pathways. These include Craf, Braf, PI3K, RalA, RalB, and Rac1, for which we have used inducible GFP-coupled shRNAs in 3D mouse and human tumor organoids harboring KrasG12D and p53R172H mutation. Using competition, cell cycle, and volumetric assays, we have uncovered that Rac1, Kras, and Craf are essential to the growth of PDAC organoids, whereas Braf, PI3K, RalA and RalB are dispensable. Interestingly, Rac1 depletion led to the strongest phenotype among the Kras mediators with a reduction of macropinocytosis, cell migration and colony formation in vitro. In an orthotopic pancreatic injection model, we observed that Rac1 inhibition in vivo led to diminished primary tumor growth, improved survival, and a reduction of metastatic incidence and outgrowth. In parallel, we performed RNA-sequencing on Kras-, Craf-, Rac1-, RalA- and Renilla-depleted organoids and found that Rac1 depletion rewires tumor cells to acquire a more PanIN-like phenotype, highlighting the importance of these proteins for the maintenance of PDAC cells. We also identify in Rac1- depleted cells evidence of deregulation of reactive oxygen species (ROS) and induction of a senescence-associated secretory phenotype (SASP) compared to control organoids. With a cytokine and chemokine array, we confirmed the increase of SASP chemokines (Csf3, Cxcl1, Cxcl2 and Cxcl5) and also detected bona fide senescence via SA-βgal staining. Finally, using several Rac1 inhibitors, we recapitulate the importance of Rac1 in PDAC growth. These data suggest that among the pleiotropic signaling downstream of mutant Kras, Rac1 is a critical node in PDAC maintenance that promotes tumor cell proliferation and senescence escape. Our findings point towards future efforts to couple Rac1 inhibition to define therapeutic synergies with immunotherapy and/or radiation. All together, we anticipate these findings can inform the subsequent development of novel therapies to address these vulnerabilities. Citation Format: Adrien Grimont, David J. Falvo, Paul Zumbo, Grace Pan, John Nguyen, Rhonda K. Yantiss, Doron Betel, Laura Martin, Steven D. Leach, Rohit Chandwani. Rac1 is essential for the maintenance of established KrasG12D-driven pancreatic ductal adenocarcinoma through senescence escape [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B055.

Inflammation is essential to the disruption of tissue homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. A longstanding question has been whether a history of past injuries instructs subsequent homeostatic responses to future stimuli – despite being distantly separated in time. Thus, we employ here Mist1-CreERT2; LSL-tdTomato lineage-tracing mice to investigate the long-term effects of a transient inflammatory episode on pancreatic tissue homeostasis. We delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and reveal that the pancreatic acinar cell compartment durably retains specific inflammation-induced epigenetic changes even 18-weeks after exposure to the original inflammatory stimulus. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. Motif analysis demonstrates the enrichment of AP-1/Fra1 motifs at these persistently accessible memory regions—a transcriptional effector activated downstream of the Ras/MAPK pathway. This epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that pancreatic acinar cells exhibit rapid malignant transformation upon re-challenge with oncogenic stress via inflammatory memory recall, with robust reactivation of genes associated with differentially accessible memory regions. Together, our findings define the dynamics and recall of an epigenetic memory of inflammation that impacts cell fate decisions in a context-dependent manner. Citation Format: David J. Falvo, Adrien Grimont, Paul Zumbo, Julie L. Yang, Alexa Osterhoudt, Grace Pan, Andre F. Rendeiro, John E. Wilkinson, Friederike Dündar, Olivier Elemento, Rhonda K. Yantiss, Doron Betel, Richard Koch, Rohit Chandwani. An epigenetic memory of inflammation controls context-dependent lineage plasticity in the pancreas [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR009.

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1–6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, ‘non-exhausted’ immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4(+) T cells, indicating increased tension between proapoptotic and prosurvival family members—and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-X(L) due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4(+) T cells with HIV resulted in increased BCL-X(L) protein expression, and treatment with two selective BCL-X(L) antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4(+) T cells. In a primary cell model of latency, both BCL-X(L) antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4(+) T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple “kick and kill” modalities—relative to latency models. We also interpret our results as encouraging further exploration of BCL-X(L) antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in “reservoirs” of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a “prosurvival” factor, BCL-X(L), that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-X(L) antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce “natural” reservoirs in ex vivo CD4(+) T cells—adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-X(L) antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.

Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrent mutations in driver genes that are restricted by prevalent HLA alleles. Here, we report on a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant (Mut)PIK3CA, among the most common genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this method, we developed a library of TCRs that recognize an endogenously processed neoepitope containing a commonPIK3CAhotspot mutation that is restricted by HLA-A*03:01. Mechanistically, immunogenicity to this public NeoAg arises primarily from enhanced stability of the neopeptide/HLA complex caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a relatively “featureless” surface dominated by the peptide’s backbone. To overcome the challenge of binding such an epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface aided by an unusually long β-chain complementarity-determining region 3 (CDR3β) loop. In a pan-cancer cohort of patients with diverse malignancies that express thePIK3CApublic NeoAg, we observed spontaneous immunogenicity, NeoAg clonal conservation, and in a limited number of cases, evidence of targeted immune escape. Together, these results establish the immunogenic potential of MutPIK3CA, creating a framework for off-the-shelf immunotherapies targeting this public NeoAg.

Tumor-specific CD8 T cells (TST) found in patients with cancer are dysfunctional and unable to halt cancer progression. TST dysfunction, also known as exhaustion, is encoded by specific epigenetic and transcriptional programs and thought to be induced by chronic antigen stimulation. Using a genetic liver cancer mouse model, we followed the fate of naïve TST in tumor-bearing hosts. Strikingly, we found that within hours after encountering tumor antigen, TST lost effector function, expressed high levels of inhibitory receptors, and displayed chromatin peak accessibility changes previously characterized in late-stage dysfunction. This rapid onset challenges the paradigm that chronic antigen/T cell receptor stimulation is required to induce dysfunction/exhaustion. Moreover, dysfunction-associated epigenetic features were established even prior to cell division, opposing the prevailing notion that cell divisions are required for epigenetic remodeling and T cell differentiation. To determine whether these early dysfunction hallmarks were “imprinted”, we reisolated TST from malignant lesions and transferred them into antigen-free hosts. While loss of effector function was imprinted within hours of tumor exposure, other dysfunctional hallmarks, including PD1 expression, required several days of tumor exposure and additional chromatin remodeling to become stably encoded. Our study reveals the rapid induction and epigenetic remodeling which underlies TST commitment to the dysfunctional fate, highlighting the need to target early activation-induced pathways to improve anti-cancer T cell immunity. Supported by T32GM007347-43

HIV persists, despite antiviral immune responses and effective antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T-cells, indicating increased tension between pro-apoptotic and pro-survival family members – as well as raising the possibility that the inhibition of pro-survival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to further study BCL2L1 (encoding the protein BCL-XL), due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T-cells with either a clinical isolate, a CCR5-tropic strain, or a CXCR4-tropic strain of HIV resulted in increased BCL-XL protein expression; and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to disproportionate cell death compared to uninfected CD4+ T-cells. In a primary cell model of latency, both BCL-XL antagonists drove significant reductions in total HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1, or in combination with the highly potent latency reversing agent combination PMA + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T-cells from ART-suppressed donors. Our results add to growing evidence that bonafide reservoir-harboring cells are resistant to multiple “kick and kill” modalities - relative to latency models - and uncover BCL-XL antagonists as a facile approach to probing mechanistic underpinnings. We also interpret our results as encouraging of further exploration of BCL-XL antagonists for cure, where combination approaches may unlock the ability to eliminate ex vivo reservoirs.

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, remains the second leading cause of cancer-related death. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP-531. We report potent anti-tumor effects of unacylated ghrelin, dependent on cells being cultured in 3D in a biologically-relevant extracellular matrix. The mechanism of unacylated ghrelin-mediated growth inhibition involves activation of Gαi and suppression of MAPK signaling. AZP-531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

Breast implant-associated lymphoma (BIA-ALCL) has recently been recognized as an independent peripheral T-cell lymphoma (PTCL) entity. In this study, we generated the first BIA-ALCL patient-derived tumor xenograft (PDTX) model (IL89) and a matching continuous cell line (IL89_CL#3488) to discover potential vulnerabilities and druggable targets. We characterized IL89 and IL89_CL#3488, both phenotypically and genotypically, and demonstrated that they closely resemble the matching human primary lymphoma. The tumor content underwent significant enrichment along passages, as confirmed by the increased variant allele frequency (VAF) of mutations. Known aberrations (JAK1 and KMT2C) were identified, together with novel hits, including PDGFB, PDGFRA, and SETBP1. A deep sequencing approach allowed the detection of mutations below the Whole Exome Sequencing (WES) sensitivity threshold, including JAK1G1097D, in the primary sample. RNA sequencing confirmed the expression of a signature of differentially expressed genes in BIA-ALCL. Next, we tested IL89&rsquo, s sensitivity to the JAK inhibitor ruxolitinib and observed a potent anti-tumor effect, both in vitro and in vivo. We also implemented a high-throughput drug screening approach to identify compounds associated with increased responses in the presence of ruxolitinib. In conclusion, these new IL89 BIA-ALCL models closely recapitulate the primary correspondent lymphoma and represent an informative platform for dissecting the molecular features of BIA-ALCL and performing pre-clinical drug discovery studies, fostering the development of new precision medicine approaches.

Background: “Public” neoantigens represent immunogenic epitopes encompassing hotspot mutations in driver oncogenes that are also restricted by common HLA alleles. In contrast with patient-specific “private” neoantigens, public neoantigens are conceptually attractive because they are tumor-specific, clonally conserved, and shared across patients. Whether PIK3CA, the most common driver oncogene in breast and endometrial cancer, can yield public neoepitopes that may be exploited for cancer immunotherapy is unknown. Methods: We have developed a high-throughput, single-cell functional assay for the discovery and retrieval of TCR a/b gene sequences that confer specific recognition of endogenously processed and presented public neoantigens and not the corresponding wild type (WT) sequence. In this approach, donor-derived T cells are sensitized with autologous antigen presenting cells (APCs) electroporated with RNA encoding PIK3CA hotspot mutations. Expanded T cells are subsequently divided into paired daughter wells for short-term co-culture with APCs electroporated with minigenes containing either mutant or WT PIK3CA sequences. Acutely re-stimulated T cells from paired wells are subject to single-cell a/b TCR VDJ and RNA sequencing. TCR a/b gene sequences associated with selective upregulation of TCR signaling transcripts to mutant but not WT PIK3CA stimulation are subsequently cloned into retroviral vectors to confirm reactivity. Results: Using this method, we have retrieved multiple TCRs that confer specific recognition of a public neoepitope derived from a PIK3CA hotspot mutation (PIK3CAH1047L). These TCRs are restricted by HLA-A*03:01, an allele present in 20.5% of the North American population. Immune-precipitation/tandem mass spectrometry analysis determined that the endogenously processed and presented public neoepitope is a 9 amino acid sequence containing a HisàLeu substitution at position 2. To understand the mechanistic basis for the immunogenicity of this public neoepitope, we generated x-ray crystallography structures of mutant and WT epitopes bound to HLA-A*03:01 at ~2Å resolution. These studies revealed significant topologic overlap in the bound peptides. By contrast, the thermal and kinetic stability of the mutant peptide/HLA-A*03:01 complex was significantly enhanced relative to the WT complex, as measured by differential scanning fluorimetry and fluorescence anisotropy assays. Peripheral blood T cells genetically engineered with PIK3CA public neoantigen-specific TCRs cytolytically cleared target cells in a HLA/mutation-specific manner, leaving HLA-mismatched or WT target cells unperturbed. Conclusions: These findings reveal for the first time the existence of an endogenously processed and presented public neoantigen derived from a PIK3CA hotspot mutation. These results open the possibility of targeting this common driver oncogene using adoptively transferred and genetically redirected T cells. Citation Format: Smita Chandran, Jiaqi Ma, Martin G. Klatt, Friederike Dündar, Paul Zumbo, Matthew R. Femia, Doron Betel, David A. Scheinberg, Brian M. Baker, Christopher A. Klebanoff. T cell receptor gene therapy for a public neoantigen derived from mutated PIK3CA, a dominant driver oncogene in breast and endometrial cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN01-03. doi:10.1158/1535-7163.TARG-19-CN01-03

Introduction: Peripheral T-cell lymphomas (PTCLs) include heterogeneous entities of rare and aggressive neoplasms. The improved understanding of the biological/molecular mechanisms driving T-cell transformation and tumor maintenance has powerfully propelled new therapeutic programs. However, despite this progress, PTCLs remain an unmet medical need. Recurrent aberrations and the deregulated activation of distinct signaling pathways have been mapped and linked to selective subtypes. The JAK/STAT signaling pathway's deregulated activation plays a pathogenetic role in PTCL, including ALCL subtypes. STATs regulate the differentiation/phenotype, survival and cell-growth, metabolism, and drug resistance of T-cell lymphomas as well as host immunosuppressive microenvironments. Although many drugs' discovery programs were launched, a plethora of compounds has failed. Methods: We have discovered heterobifunctional molecules by an iterative medicinal chemistry SAR campaign that potently and selectively degrade STAT3 in a proteasome-dependent manner. Conventional PTCL cell lines and Patient Derived Tumor Xenograft (PDTX) and/or derived cell lines (PDTX-CL), carrying either WT- or mutated-STAT3, were exposed to increasing amounts (50nM⇒5µM) of STAT3-degraders. Proteins and mRNA transcripts (2⇒144hrs) were assessed by deep-proteomics and paired-end RNA sequencing, combined with WB/flow cytometry and qRT-PCR. Cell-titer-glo, cell titer blue, Annexin-V and S-cell cycle analyses were used as readouts. Chromatin accessibility, STAT3 DNA binding, 3D chromosomal architecture reorganization and 5-hmC profiling were assessed by ATACseq, CHIPseq and Hi-C and H3K27ac Hi-CHIP and mass-spectrometry. Drug testing/discovery combinations (96-well-plate) were performed using a semi-automated flow-cytometry. A battery of PTCL PDTX models were tested in pre-clinical trials. Results: Treatment of ALK+ ALCL (SU-DHL1) led to the rapid (~6hrs.) and profound down-regulation of STAT3 followed by the loss of canonical STAT3-regulated proteins (SOCS3, MYC, Granzyme B, GAS1, and IL2RA), without appreciable changes for other STAT family members (STAT1, STAT5b). In vitro, cytoplasmic, nuclear, and mitochondrial STAT3 downregulation was maintained up to 144 hrs. Loss of STAT3 in ALK+/- ALCL and BIA-ALCL cells was associated with major transcriptional changes (7116-10615 and 15114 DEGs in ALK- and ALK+ ALCL, respectively), underscoring public/shared as well as private time-dependent signatures. Main down-regulated pathways included JAK-STAT, MAPK, NF-kB, PI3K, TGFb, and TNFa. Comparison of STAT3 shRNA (ALK+ ALCL) and STAT3 degrader (ALK-/ALK+ ALCL) signatures demonstrated a substantial and concordant gene modulation (24hrs) among all models with the highest overlaps between ALK+ ALCL (Figure 3). To identify direct STAT3 gene targets, we analyzed CHIPseq peaks and predicted bindings sites, demonstrating that canonical genes, i.e., SOCS3, Granzyme B, GAS1, IL2RA, STAT3, and CD30, were significantly downregulated. Conversely, CD58, CD274, and MCH-I/II were upregulated at late time points. By mapping the STAT3 binding sites in ALK+ and ALK- ALCL, we have identified 1077 and 2763 STAT3 peaks within promoter/5'-/3'- and distant intergenic regions, corresponding to both coding and non-coding genes. Therapeutically, in vitro treatments led to cell cycle arrest and profound growth inhibition, and over time increased cell death of PTCL cells, including ALCL. Accordingly, growth inhibition of ALCL xenograft and PDTX tumors was also achieved (Figure 2). To identify drugs that could synergize withSTAT3-degrader activity, we tested a compound library (40) targeting pro-tumorigenic PTCL pathways as well as FDA-approved compounds. Ongoing studies are in progress. Conclusion: We have discovered selective STAT3 degraders which control PTCL growth. STAT3 degraders are powerful tools to define the STAT3 pathogenetic mechanisms and dissect genes/pathways to be targeted for T-cell lymphoma eradication. These data provide additional rationale for testing STAT3 degraders in the clinic for the treatment of aggressive malignancies including PTCL/ALCL. Figure 1 Figure 1. Disclosures Yang: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: Owkin: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Johnson and Johnson: Research Funding; Eli Lilly: Research Funding; Janssen: Research Funding; Champions Oncology: Consultancy; Freenome: Consultancy, Other: Current equity holder in a privately-held company; One Three Biotech: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding. Horwitz: Affimed: Research Funding; Aileron: Research Funding; ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; Celgene: Research Funding; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding. DeSavi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, it remains the second leading cause of cancer-related death in the US. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. One major area of research relates to its reported effects to increase insulin sensitivity in diabetics, this being the basis for the development of unacylated ghrelin analog, AZP531 or livoletide, now in clinical trials for the treatment of Prader-Willi Syndrome. The mechanism of action of unacylated ghrelin is largely uncharacterized, in any system, because it does not bind to or activate the cognate ghrelin receptor, GHSR1a. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP531. We report potent effects of unacylated ghrelin, at picomolar doses, on the growth of breast cancer cells, dependent on 3D culture and activation of Gαi. Suppression of MAPK signaling by unacylated ghrelin leads to cell cycle arrest and apoptosis. AZP531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

Precision medicine has generated diagnoses for many patients with challenging undiagnosed disorders. Some individuals remain without a diagnosis despite comprehensive testing, and this impedes their treatment. This report addresses the role of personalized medicine in identifying effective therapy for an undiagnosed disease. A 22-year-old woman presented with chronic severe recurrent trismus, facial pain, progressive multicentric inflammatory and fibrotic masses, and high C-reactive protein. Sites of disease included the pterygomaxillary region, masseter muscles, mandible, lung, pericardium, intrabdominal cavity, and retroperitoneum. A diagnosis was not established after an extensive assessment, including multiple biopsies. The patient was subsequently evaluated under the Undiagnosed Diseases Program at the National Institutes of Health. Large scale genotyping, proteomic studies, and in vitro and gene expression analyses of fibroblasts obtained from a major disease locus were performed. Germline genetic testing did not identify strong candidate genes; proteomic studies of the patient's serum and bronchoalveolar lavage fluid and gene expression analyses of her cells were consistent with dysregulation of the tumor necrosis factor-alpha pathway. The patient's cultured fibroblasts were incubated with selected drugs, and cell proliferation was inhibited by hydroxychloroquine. Treatment of the patient with hydroxychloroquine conferred prolonged beneficial clinical effects, including stabilization of trismus and reduction of corticosteroid dose, C-reactive protein, and size of masses. This case represents an example of precision medicine applied to discover effective treatments for individuals with enigmatic undiagnosed disorders.

Radiation therapy (RT) used at immunogenic doses (8GyX3) leads to cytosolic accumulation of DNA that binds to the DNA sensor cGAS and activates the cGAMP synthesis, resulting in STING activation and downstream interferon type I (IFN-I) production by cancer cells [1-3]. RT-induced IFN-I is critical for effective anti-tumor immune responses in combination with immune-checkpoint blockade therapy [1]. We have recently demonstrated that tumor-derived exosomes (TEX) secreted by irradiated (8GyX3) TSA murine carcinoma cells (RT-TEX) contain more DNA compared to TEX produced by untreated cells (UT-TEX). Interestingly, only the DNA carried by RT-TEX is capable of STING-dependent activation of recipient dendritic cells (DCs) resulting in CD40, CD80 and CD86 upregulation and IFN-I production. Mice vaccination with RT-TEX, but not UT-TEX, elicits tumor-specific CD8+ T-cell responses that protect mice from tumor development [4]. Here, we tested the hypothesis that not only quantitative but also qualitative differences between the DNA cargo of RT-TEX and UT-TEX may explain its differential ability to activate DCs. To this end, internal DNA purified from TEX and from the cytosolic fraction of the parent TSA cells was characterized for length using Agilent Bioanalyzer. Furthermore, DNA TEX was analyzed by whole-genome sequencing (WGS) and by whole-genome bisulfite sequencing for methylation status. In addition, the percentage of global methylation of DNA of the TSA cells was quantified by 5-methyl cytosine DNA Elisa kit. All experiments were performed in biological triplicates. DNA size analysis revealed an enrichment of DNA fragments with size between 60 and 250 bp in RT-TEX compared to UT-TEX, and in the cytosolic fraction of irradiated compared to untreated TSA cells. The WGS showed that the entire genome was represented in DNA contained within TEX, regardless of the treatment of the parent cells. More than 99% of TEX DNA was of nuclear origin, but mitochondrial DNA was increased in RT-TEX (6.2 fold change, p=0.006). Interestingly, the RT treatment increases the DNA abundance of several regions (median p=0.006). Interestingly, we found reduced levels of methylation in exosomal and total DNA purified from irradiated compared to untreated TSA cells. Our data support the hypothesis that there are potentially important qualitative differences in the DNA cargo of TEX derived from irradiated compared to untreated cancer cells that reflect molecular changes occurring in parent cells. For instance, the enrichment in DNA fragments with size between 60-250 bp in RT-TEX is especially intriguing in light of the recent report that cGAS is optimally activated by this DNA length range [5]. The impact of these qualitative differences in the cargo DNA on activation of the IFN-I pathway in innate immune cells that uptake TEX is under investigation. Identification of DNA signature associated with TEX ability to activate the cGAS/STING pathway could provide a blood-based biomarker for the immunogenic tumor response to radiotherapy. Citation Format: Sheila Spada, Paul Zumbo, Doron Betel, Tuo Zhang, Nils-Petter Rudqvist, Sandra Demaria. Characteristics of the interferon-stimulatory DNA cargo of exosomes produced by irradiated breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2263.

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)–JUN family genes as key barriers of DE differentiation. The JNK–JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK–JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine. CRISPR screens identify JNK–JUN family genes as repressors of definitive endoderm differentiation in human pluripotent stem cells. JUN co-occupies stem cell enhancers with OCT4, NANOG, SMAD2 and SMAD3 and inhibits the exit from pluripotency.

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic research. However, use of FFPE samples in genomic studies has been limited by technical challenges resulting from nucleic acid degradation. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues preserved in formalin for different amounts of time using 2 DNA microarray protocols and 2 whole-transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other methods by having the highest correlations of differentially expressed genes (DEGs), and best overlap of pathways, between FRO and FFPE groups. The effect of sample time in formalin (18 h or 3 weeks) on gene expression profiles indicated that test article treatment, not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18 h and 3 week FFPE samples compared with FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of time in paraffin on genomic profiles. Ribo-depletion RNA-seq analysis of 8-, 19-, and 26-year-old control blocks resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, untranslated region, intronic, and ribosomal fractions of the transcriptome. Overall, our results indicate that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples.

We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the US Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific-filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed for all examined platforms, including qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings.