Juvenile social play behavior is a shared trait across a wide variety of mammalian species. When play is characterized by the frequency or duration of physical contact, males usually display more play relative to females. The endocannabinoid system contributes to the development of the sex difference in social play behavior in rats. Treating newborn pups with a nonspecific endocannabinoid agonist, WIN55,212-2, masculinizes subsequent juvenile rough-and-tumble play behavior by females. Here we use specific drugs to target signaling through either the CB1 or CB2 endocannabinoid receptor (CB1R or CB2R) to determine which modulates the development of sex differences in play. Our data reveal that signaling through both CB1R and CB2R must be altered neonatally to modify development of neural circuitry regulating sex differences in play. Neonatal co-agonism of CB1R and CB2R masculinized play by females, whereas co-antagonism of these receptors feminized rates of male play. Because of a known role for the medial amygdala in the sexual differentiation of play, we reconstructed Golgi-impregnated neurons in the juvenile medial amygdala and used factor analysis to identify morphological parameters that were sexually differentiated and responsive to dual agonism of CB1R and CB2R during the early postnatal period. Our results suggest that sex differences in the medial amygdala are modulated by the endocannabinoid system during early development. Sex differences in play behavior are loosely correlated with differences in neuronal morphology.

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 in epithelial tumorigenesis, but how a temporally remote inflammatory episode impacts tumor development is unknown. Herein we employ lineage-traced mouse models to unveil the presence of an epigenetic memory of inflammation. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to the same molecular baseline. In vivo, the memory is associated with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis when instead subjected to an oncogenic Kras mutation. We find that memory is a cell-intrinsic property, primarily encoded in chromatin, that features persistent derepression of metaplastic genes and is recalled with oncogenic stress. Together, our findings define a capacity for an environmental insult to potentiate future tumor initiation, broadening the relationship between inflammation and cancer.SIGNIFICANCEInflammation is a key characteristic of epithelial cancers. Here, we demonstrate that resolution of pancreatitis in vivo is accompanied by memory, featuring persistent epigenetic and transcriptional alterations that are recalled to increase the susceptibility to oncogenic stress later in life.

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.

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.

Pancreatic cancer initiation features abundant rewiring of the normal acinar cell,. but little is known about the chromatin specification of pancreatic cell types and the epigenetic dysregulation of normal acinar cells in tumor initiation. To address these questions, we employed a lineage-traced autochthonous mouse model to examine systematically perturbed acinar cells. We coupled the spatiotemporal control of inflammation (via caerulein injections) with inducible oncogene activation (mutant Kras) in the adult mouse along with bulk RNA-seq and ATAC-seq to sorted acinar and acinar-derived cells. In addition, we generated Ptf1a-TdTomato mice to reliably sort pancreatic progenitors at e10.5 and e15.5. We observe that Kras activation alone does not disturb acinar cell chromatin nor the histologic appearance of the pancreas. By contrast, caerulein alters chromatin significantly in metaplasia and even in regeneration, with putative enhancers derepressed despite normal histology. In the context of Kras activation and caerulein administration, we find a broad and stable reorganization of chromatin, reflecting cooperativity between oncogenic stress and an inflammatory insult. We also find that in PanIN, the chromatin state bears few, if any, ductal, progenitor, or islet features and instead reflects a largely novel cell fate. To understand the dependencies of these findings on an inflammatory insult and mutant Kras, we leveraged temporal resolution of pancreatitis and the iKras system to define the reversibility of this epigenetic rewiring. Notably, neither resolution of inflammation nor withdrawal of mutant Kras expression are sufficient to revert an acinar cell to its initial chromatin state. Analysis of the acinar-derived cells undergoing the transition to PanIN reveals the induction of specific proliferative and progenitor master transcription factors and activation of associated transcriptional programs. In these data we also observe a specific unveiling of the AP-1 isoform Fra-1 (Fosl1) transcript, protein, and binding sites in chromatin. To address if Fra-1-associated alterations to chromatin are bona fide requirements for tumorigenesis, we coupled conditional Fra-1 knockout alleles with the iKras system, finding nearly complete ablation of PanIN in the absence of Fra-1. Together, our findings suggest that (1) loss of acinar cell identity is resistant to oncogenic stress and is susceptible to inflammation; (2) the acquired acinar cell fate reflects neither ‘pure’ metaplasia nor transdifferentiation nor dedifferentiation events, and (3) acinar cell regeneration is incomplete. In contrast to recent studies, we demonstrate that pancreatic tumorigenesis does not re-establish a progenitor cell fate, but hijacks the AP-1 transcription factors for tumor-specific genomic locations, with Fra-1 emerging as a dependency in tumorigenesis. Our data thus highlight the complexity of cell fate decisions in the preneoplastic pancreas and reveal key regulators of acinar cell identity. Citation Format: David Falvo, Jason Pitarresi, Alexa Osterhoudt, Adrien Grimont, Ben Stanger, Steven D. Leach, Anil K. Rustgi, Rohit Chandwani. Chromatin dynamics in vivo define coordinate functions of inflammation and mutant Kras in pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-005.

Cellular decision-making is highly dependent on cues present in the environment. As such, biologic systems have evolved efficient ways to respond to salient and often transient stimuli by encoding and transducing relevant extracellular information to chromatin. Additionally, accumulating evidence suggests that cells are capable of remembering previously experienced stimuli, allowing them to adjust their behaviors appropriately when confronted in the future with a similar or different stimulus. However, the ability of cells to respond to a transient stimulus and to encode a persistent record of that experience following its resolution has yet to be explored comprehensively in a preneoplastic context. Here we determine the lasting effect in the pancreas of an acinar-to-ductal metaplasia (ADM) event, to elucidate the impact of prior inflammation on tissue function and subsequent tumorigenesis. In a lineage-traced model of acinar cell dedifferentiation, we demonstrate that following ADM, histologically normal acinar cells exhibit broad gains in chromatin accessibility that persist well after exposure to the initial ADM-inducing stimulus. We show that the effect on chromatin is not reflected in the transcriptional state of acinar cells. Moreover, we employ extensive flow-cytometry based analysis of immune cell and niche acinar populations to show there is no long-term cell-extrinsic population, nor a significant expansion of either the Dclk1+ or Nk1r+ acinar populations. Finally, we employ single-cell RNA-seq analyses to define the alterations to epithelial cells and the associated microenvironment long after regeneration following an episode of pancreatitis. These data suggest that inflammation-exposed acinar cells are capable of establishing an altered, persistently accessible chromatin state that lasts long after the initial lineage destabilizing event. Currently we are defining if, following resolution of pancreatitis, activation of mutant Kras will be sufficient to induce premalignant transformation within the acinar compartment that manifests as pancreatic intraepithelial neoplasia (PanIN)—the most common precursor lesion to pancreatic ductal adenocarcinoma (PDAC). Understanding how this postinflammatory chromatin state synergizes with oncogenic stress will further explain why inflammation is a hallmark risk factor for cancer development. Together, our studies will characterize the establishment, maintenance, and maladaptive role of epigenetic memory in the development of pancreatic cancer. Citation Format: David J. Falvo, Rohit Chandwani. The establishment, maintenance, and maladaptive role of epigenetic memory in mediating pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A12.

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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.