Left unmitigated, high-dose ionizing radiation causes DNA damage resulting in cell death and manifestation of the acute radiation syndrome (ARS). Activation of the lysophosphatidic acid receptor subtype 2 (LPAR2) is one mechanism by which radiation damage can be mitigated. Our laboratory has previously developed 5-chloro-2-(N-(4-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)butyl)sulfamoyl)benzoic acid, designated as Radioprotectin-1 (RP-1), which is a potent LPAR2-specific agonist and radiation mitigator. We previously reported that an aqueous (AQ) formulation of RP-1 provides significant survival advantage in a murine gastrointestinal ARS (GI-ARS) model when given in a three-day regimen of twice-daily subcutaneous injections administered starting 24 h after irradiation. To improve the dosing formulation suitable for the field treatment of mass casualties, an extended-release dosing regimen was developed utilizing a water-in-oil-in-water (W/O/W) multilayered microemulsion (ME) formulation. The ME significantly extended plasma half-life, mean residence time, and exposure time while slowing plasma clearance compared to the AQ formulation in C57BL/6J mice as well as in non-human primates (NHP). This formulation provides a significant survival advantage in a GI-ARS model with only two subcutaneous injections at 24 and 72 h postirradiation. Additionally, RP-1 ME treatment protects intestinal crypts in irradiated mice, resulting in an increase in both total and actively regenerating crypts at day 5 postirradiation. Finally, LPAR2 activation by RP-1 leads to prolonged and sustained activation of the pro-survival kinases ERK1/2 and Akt up to 16 h postirradiation and reduces caspase-mediated apoptosis in irradiated mouse embryonic fibroblasts, providing a mechanism for RP-1 radiation mitigation.

Supplementary Figure 3. (A) ATX and LPA receptor profiling in B16F10 cells transduced with GFP lentivirus using quantitative real-time PCR. Data are expressed as mean {plus minus} SD of an experiment performed twice in quadruplicates. (B) Representative images of lungs harvested at day 21 from mice that were injected with PBS (left panel, control) and mice that were injected with 1 x 105 GFP-expressing B16F10 cells (right panel). Arrow denotes a single fluorescent tumor nodule on lung surface. Scale bar = 200 microns.

Supplementary Table 1. Primer sequences used in quantitative Real-Time PCR analysis.

Supplemental Figure Legends. Supplemental Figure Legends

Supplementary Figure 5. ATX and LPA receptor profiling in isolated rat alveolar type I-like (A) or type II (B) epithelial cells. Note that expression levels in type II alveolar cells are approximately 10-fold lower than in type I-like cells. Data presented are representative of an experiment performed three times in quadruplicates and are expressed as mean {plus minus} SD.

Introduction:The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5).Methods:Adult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels.Results:Ionizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation.Conclusion:These data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

Supplementary Figure 4. Effect of BMP22 on the basal invasion of B16F10 cells across a matrigel layer. Data are representative of an experiment performed twice in quadruplicates and are expressed as mean {plus minus} SD. *Denotes p value < 0.05 using one-way ANOVA followed by a Bonferroni post-test.

Supplementary Figure 1. Time course of ATX activity measured in the conditioned media (CM) of (A) MM1 rat hepatocarcinoma, (B) HUVEC, (C) RLMVEC, and (D) isolated murine mesothelial cells. Control represents media that were not exposed to cells. Data are expressed as mean {plus minus} SD of an experiment performed three times in triplicates.

Supplementary Figure 2. LPA1-5 receptor profiling in whole lung tissue isolated from WT, LPA1-, LPA2- and LPA5KO mice, respectively using quantitative real-time PCR. Data are expressed as mean {plus minus} SEM (n = 3 mice).

The TP53 gene has been widely studied for its roles in cell cycle control, maintaining genome stability, activating repair mechanisms upon DNA damage, and initiating apoptosis should repair mechanisms fail. Thus, it is not surprising that mutations of p53 are the most common genetic alterations found in human cancer. Emerging evidence indicates that dysregulation of lipid metabolism by p53 can have a profound impact not only on cancer cells but also cells of the tumor microenvironment (TME). In particular, intermediates of the sphingolipid and lysophospholipid pathways regulate many cellular responses common to p53 such as cell survival, migration, DNA damage repair and apoptosis. The majority of these cellular events become dysregulated in cancer as well as cell senescence. In this review, we will provide an account on the seminal contributions of Prof. Lina Obeid, who deciphered the crosstalk between p53 and the sphingolipid pathway particularly in modulating DNA damage repair and apoptosis in non-transformed as well as transformed cells. We will also provide insights on the integrative role of p53 with the lysophosphatidic acid (LPA) signaling pathway in cancer progression and TME regulation.

The tumor microenvironment (TME) may be best conceptualized as an ecosystem comprised of cancer cells interacting with a multitude of stromal components such as the extracellular matrix (ECM), blood and lymphatic networks, fibroblasts, adipocytes, and cells of the immune system. At the center of this crosstalk between cancer cells and their TME is the bioactive lipid lysophosphatidic acid (LPA). High levels of LPA and the enzyme generating it, termed autotaxin (ATX), are present in many cancers. It is also well documented that LPA drives tumor progression by promoting angiogenesis, proliferation, survival, invasion and metastasis. One of the hallmarks of cancer is the ability to modulate and escape immune detection and eradication. Despite the profound role of LPA in regulating immune functions and inflammation, its role in the context of tumor immunity has not received much attention until recently where emerging studies highlight that this signaling axis may be a means that cancer cells adopt to evade immune detection and eradication. The present review aims to look at the immunomodulatory actions of LPA in baseline immunity to provide a broad understanding of the subject with a special emphasis on LPA and cancer immunity, highlighting the latest progress in this area of research.

The lipid mediator lysophosphatidic acid (LPA) in biological fluids is primarily produced by cleavage of lysophospholipids by the lysophospholipase D enzyme Autotaxin (ATX). LPA has been identified and abundantly detected in the culture medium of various cancer cell types, tumor effusates, and ascites fluid of cancer patients. Our current understanding of the physiological role of LPA established its role in fundamental biological responses that include cell proliferation, metabolism, neuronal differentiation, angiogenesis, cell migration, hematopoiesis, inflammation, immunity, wound healing, regulation of cell excitability, and the promotion of cell survival by protecting against apoptotic death. These essential biological responses elicited by LPA are seemingly hijacked by cancer cells in many ways; transcriptional upregulation of ATX leading to increased LPA levels, enhanced expression of multiple LPA GPCR subtypes, and the downregulation of its metabolic breakdown. Recent studies have shown that overexpression of ATX and LPA GPCR can lead to malignant transformation, enhanced proliferation of cancer stem cells, increased invasion and metastasis, reprogramming of the tumor microenvironment and the metastatic niche, and development of resistance to chemo-, immuno-, and radiation-therapy of cancer. The fundamental role of LPA in cancer progression and the therapeutic inhibition of the ATX-LPA axis, although highly appealing, remains unexploited as drug development to these targets has not reached into the clinic yet. The purpose of this brief review is to highlight some unique signaling mechanisms engaged by the ATX-LPA axis and emphasize the therapeutic potential that lies in blocking the molecular targets of the LPA system.

CD19-targeted CAR T cells show sustained CD19 + B cell depletion and profound therapeutic benefit in ameliorating disease in mouse models of lupus.

Introduction: Anal cancer is one of few malignancies for which the incidence continues to rise in the US. It is highly associated with high risk (HR) HPV infection with HPV 16 and 18 accounting for the vast majority of cases. Understanding the molecular mechanisms underlying anal cancer progression would be valuable for the purposes of developing novel clinical management strategies. However, there remains a paucity of experimental models allowing for the study of HPV-associated anal cancer development. Herein, we describe an in vitro raft tissue culture system model to study anal squamous neoplastic progression via anal intraepithelial neoplasa (AIN) induced by HPV infection.Methods: Primary human anal keratinocyte (HAK) cells were transfected with either HPV16 or 18 DNA and grown in a raft tissue culture system. The progression of dysplastic changes was sequentially examined at ~5 passage intervals by histologic review including hematoxylin-eosin (H&E) staining, immunohistochemistry (p16, Ki67, cytokeratins CK5/6, CK8/18 and CK10) and in situ hybridization (ISH) with an HPV16/18 RNA probe cocktail. The degree of dysplasia was determined by the degree of disorganization of the epithelial architecture, presence of mitotic/dysplastic cells, p16 positivity, diffuse Ki67 staining, increased number of basal-parabasal type cells (CK5/6 and CK10 markers) and lack of epithelial differentiation.Results: Advanced dysplastic changes (corresponding to AIN2-3 lesions) were already apparent at the earliest of initial passages of transfected HAK cells (passage 8 for HPV16 and 7 for HPV 18). Later passages suggested development of AIN3/squamous cell carcinoma in situ in both lines with HPV16 HAK cells showing progressively increased labeling of CK8/18 and HPV18 HAK cells demonstrating increasing cell keratinization and epithelial disintegration. In both cell lines, HPV was present in both episomal and integrated forms throughout all stages of progression as suggested by ISH signal patterns.Conclusions: A raft tissue culture model system has been developed to study HPV-induced dysplastic progression in human anal keratinocytes. Importantly, this system is of great interest as an in vitro tool to characterize the sequential molecular alterations associated with the development of anal cancer which in turn, may have implications for novel screening, prevention and treatment strategies.Citation Format: Irina V. Getun, Abidemi Ajidahun, Janice Milici, Sreejata Chatterjee, Jorge Solares, Abul Elahi, Leah E. Hendrick, Evan S. Glazer, Louisa Balazs, Craig Meyers, David Shibata. A study of HPV induced anal cancer progression in a raft tissue culture system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3786.

Alcohol consumption has been shown to cause dysbiosis, but the mechanism involved in it is unknown. Recurrent colitis is known to induce expression of α-defensins in the colon, but the effect of alcohol consumption on it is not known. We investigated the effect of ethanol on α-defensin expression in the small intestine and colitis-induced expression in colon in mice. Furthermore, we evaluated the effect of human defensin-5 (HD5) on ethanol and colitis-induced gut barrier dysfunction and mucosal damage. Recurrent colitis was induced by feeding dextran sulfate sodium (DSS), 3 cycles of 5-days each with 15 days intervals, followed by 30-days remission. Ethanol was fed during the intervals and recovery in a liquid diet with or without HD5. Expression of α-defensins, tight junction (TJ) integrity and cytokine/chemokine expression were analyzed. Chronic ethanol feeding reduced α-defensin expression in the small intestine and colitis-induced defensin expression in the colon. HD5 attenuated the growth of enterotoxigenic Bacteriodes fragilis and E. coli, but had no effect on non-toxigenic Bacteriodes fragilis or probiotics, the Lactobacilli. Ethanol and colitis elevated Enterobacteriaceae, Firmicutes and Firmicutes to Bacteriodetes ratio in colonic mucosa. HD5 feeding attenuated ethanol and colitis-induced dysbiosis, disruption of intestinal epithelial TJ, mucosal inflammation, expression of pro-inflammatory cytokines and chemokines in the small intestine and colon, and endotoxemia. These results demonstrate that ethanol suppresses intestinal α-defensin expression, leading to dysbiosis, barrier dysfunction, inflammation and endotoxemia. HD5 feeding attenuates intestinal injury caused by ethanol and colitis, indicating that defensin expression is a potential target for treatment of alcoholic tissue injury and colitis.

We have previously demonstrated that the small molecule octadecenyl thiophosphate (OTP), a synthetic mimic of the growth factor-like mediator lysophosphatidic acid (LPA), showed radioprotective activity in a mouse model of total-body irradiation (TBI) when given orally or intraperitoneally 30 min before exposure to 9 Gy γ radiation. In the current study, we evaluated the effects of OTP, delivered subcutaneously, for radioprotection or radiomitigation from -24 h before to up to +72 h postirradiation using a mouse TBI model with therapeutic doses at around 1 mg/kg. OTP was injected at 10 mg/kg without observable toxic side effects in mice, providing a comfortable safety margin. Treatment of C57BL/6 mice with a single dose of OTP over the time period from -12 h before to +26 h after a lethal dose of TBI reduced mortality by 50%. When administered at +48 h to +72 h postirradiation (LD50/30 to LD100/30), OTP reduced mortality by ≥34%. OTP administered at +24 h postirradiation significantly elevated peripheral white blood cell and platelet counts, increased crypt survival in the jejunum, enhanced intestinal glucose absorption and reduced endotoxin seepage into the blood. In the 6.4-8.6 Gy TBI range using LD50/10 as the end point, OTP yielded a dose modification factor of 1.2. The current data indicate that OTP is a potent radioprotector and radiomitigator ameliorating the mortality and tissue injury of acute hematopoietic as well as acute gastrointestinal radiation syndrome.

In this study we characterized the effects of radiation injury on the expression and function of the autotaxin (ATX)–LPA2 GPCR axis. In IEC-6 crypt cells and jejunum enteroids quantitative RT-PCR showed a time- and dose-dependent upregulation of lpa2 in response to γ-irradiation that was abolished by mutation of the NF-κB site in the lpa2 promoter or by inhibition of ATM/ATR kinases with CGK-733, suggesting that lpa2 is a DNA damage response gene upregulated by ATM via NF-κB. The resolution kinetics of the DNA damage marker γ-H2AX in LPA-treated IEC-6 cells exposed to γ-irradiation was accelerated compared to vehicle, whereas pharmacological inhibition of LPA2 delayed the resolution of γ-H2AX. In LPA2-reconstituted MEF cells lacking LPA1&3 the levels of γ-H2AX decreased rapidly, whereas in Vector MEF were high and remained sustained. Inhibition of ERK1&2 or PI3K/AKT signaling axis by pertussis toxin or the C311A/C314A/L351A mutation in the C-terminus of LPA2 abrogated the effect of LPA on DNA repair. LPA2 transcripts in Lin−Sca-1+c-Kit+ enriched for bone marrow stem cells were 27- and 5-fold higher than in common myeloid or lymphoid progenitors, respectively. Furthermore, after irradiation higher residual γ-H2AX levels were detected in the bone marrow or jejunum of irradiated LPA2-KO mice compared to WT mice. We found that γ-irradiation increases plasma ATX activity and LPA level that is in part due to the previously established radiation-induced upregulation of TNFα. These findings identify ATX and LPA2 as radiation-regulated genes that appear to play a physiological role in DNA repair.

Pharmacological mitigation of injuries caused by high-dose ionizing radiation is an unsolved medical problem. A specific nonlipid agonist of the type 2 G protein coupled receptor for lysophosphatidic acid (LPA2) 2-[4-(1,3-dioxo-1H,3H-benzoisoquinolin-2-yl)butylsulfamoyl]benzoic acid (DBIBB) when administered with a postirradiation delay of up to 72 hr reduced mortality of C57BL/6 mice but not LPA2 knockout mice. DBIBB mitigated the gastrointestinal radiation syndrome, increased intestinal crypt survival and enterocyte proliferation, and reduced apoptosis. DBIBB enhanced DNA repair by augmenting the resolution of γ-H2AX foci, increased clonogenic survival of irradiated IEC-6 cells, attenuated the radiation-induced death of human CD34(+) hematopoietic progenitors and enhanced the survival of the granulocyte/macrophage lineage. DBIBB also increased the survival of mice suffering from the hematopoietic acute radiation syndrome after total-body irradiation. DBIBB represents a drug candidate capable of mitigating acute radiation syndrome caused by high-dose γ-radiation to the hematopoietic and gastrointestinal system.

Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression.Thepresentstudysoughttounderstandthemechanisticrole of LPA G protein–coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found