Treatment with a Cholecystokinin Receptor Antagonist, Proglumide, Improves Efficacy of Immune Checkpoint Antibodies in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated deaths worldwide. Treatment with immune checkpoint antibodies has shown promise in advanced HCC, but the response is only 15-20%. We discovered a potential target for the treatment of HCC, the cholecystokinin-B receptor (CCK-BR). This receptor is overexpressed in murine and human HCC and not in normal liver tissue. Mice bearing syngeneic RIL-175 HCC tumors were treated with phosphate buffer saline (PBS; control), proglumide (a CCK-receptor antagonist), an antibody to programmed cell death protein 1 (PD-1Ab), or the combination of proglumide and the PD-1Ab. In vitro, RNA was extracted from untreated or proglumide-treated murine Dt81Hepa1-6 HCC cells and analyzed for expression of fibrosis-associated genes. RNA was also extracted from human HepG2 HCC cells or HepG2 cells treated with proglumide and subjected to RNA sequencing. Results showed that proglumide decreased fibrosis in the tumor microenvironment and increased the number of intratumoral CD8+ T cells in RIL-175 tumors. When proglumide was given in combination with the PD-1Ab, there was a further significant increase in intratumoral CD8+ T cells, improved survival, and alterations in genes regulating tumoral fibrosis and epithelial-to-mesenchymal transition. RNAseq results from human HepG2 HCC cells treated with proglumide showed significant changes in differentially expressed genes involved in tumorigenesis, fibrosis, and the tumor microenvironment. The use of the CCK receptor antagonist may improve efficacy of immune checkpoint antibodies and survival in those with advanced HCC.

Target-Specific Nanoparticle Polyplex Down-Regulates Mutant Kras to Prevent Pancreatic Carcinogenesis and Halt Tumor Progression.

Survival from pancreatic cancer is poor because most cancers are diagnosed in the late stages and there are no therapies to prevent the progression of precancerous pancreatic intraepithelial neoplasms (PanINs). Inhibiting mutant KRASG12D, the primary driver mutation in most human pancreatic cancers, has been challenging. The cholecystokinin-B receptor (CCK-BR) is absent in the normal pancreas but becomes expressed in high grade PanIN lesions and is over-expressed in pancreatic cancer making it a prime target for therapy. We developed a biodegradable nanoparticle polyplex (NP) that binds selectively to the CCK-BR on PanINs and pancreatic cancer to deliver gene therapy. PanIN progression was halted and the pancreas extracellular matrix rendered less carcinogenic in P48-Cre/LSL-KrasG12D/+ mice treated with the CCK-BR targeted NP loaded with siRNA to mutant Kras. The targeted NP also slowed proliferation, decreased metastases and improved survival in mice bearing large orthotopic pancreatic tumors. Safety and toxicity studies were performed in immune competent mice after short or long-term exposure and showed no off-target toxicity by histological or biochemical evaluation. Precision therapy with target-specific NPs provides a novel approach to slow progression of advanced pancreatic cancer and also prevents the development of pancreatic cancer in high-risk subjects without toxicity to other tissues.

AIB1 is a novel target of the high-risk HPV E6 protein and a biomarker of cervical cancer progression.

The high-risk human papillomaviruses (HPV-16, -18) are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7 proteins that are uniformly retained and expressed in cervical cancers and required for maintenance of the tumorigenic phenotype. The E6 and E7 proteins were first identified as targeting the p53 and pRB tumor suppressor pathways, respectively, in host cells, thereby leading to disruption of cell cycle controls. In addition to p53 degradation, a number of other functions and critical targets for E6 have been described, including telomerase, Myc, PDZ-containing proteins, Akt, Wnt, mTORC1, as well as others. In this study, we identified Amplified in Breast Cancer 1 (AIB1) as a new E6 target. We first found that E6 and hTERT altered similar profiling of gene expression in human foreskin keratinocytes (HFK), independent of telomerase activity. Importantly, AIB1 was a common transcriptional target of both E6 and hTERT. We then verified that high-risk E6 but not low-risk E6 expression led to increases in AIB1 transcript levels by real-time RT-PCR, suggesting that AIB1 upregulation may play an important role in cancer development. Western blots demonstrated that AIB1 expression increased in HPV-16 E6 and E7 expressing (E6E7) immortalized foreskin and cervical keratinocytes, and in three of four common cervical cancer cell lines as well. Then, we evaluated the expression of AIB1 in human cervical lesions and invasive carcinoma using immunohistochemical staining. Strikingly, AIB1 showed positivity in the nucleus of cells in the immediate suprabasal epithelium, while nuclei of the basal epithelium were negative, as evident in the Cervical Intraepithelial Neoplasia 1 (CIN1) samples. As the pathological grading of cervical lesions increased from CIN1, CIN2, CIN3 carcinoma in situ and invasive carcinoma, AIB1 staining increased progressively, suggesting that AIB1 may serve as a novel histological biomarker for cervical cancer development. For cases of invasive cervical carcinoma, AIB1 staining was specific to cancerous lesions. Increased expression of AIB1 was also observed in transgenic mouse cervical neoplasia and cancer models induced by E6E7 and estrogen. Knockdown of AIB1 expression in E6E7 immortalized human cervical cells significantly abolished cell proliferation. Taken together, these data support AIB1 as a novel target of HPV E6 and a biomarker of cervical cancer progression.

Predominant contribution of the dose received from constituent heavy-ions in the induction of gastrointestinal tumorigenesis after simulated space radiation exposure.

Gastrointestinal (GI) cancer risk among astronauts after encountering galactic cosmic radiation (GCR) is predicted to exceed safe permissible limits in long duration deep-space missions. Current predictions are based on relative biological effectiveness (RBE) values derived from in-vivo studies using single-ion beams, while GCR is essentially a mixed radiation field composed of protons (H), helium (He), and heavy ions. Therefore, a sequentially delivered proton (H) → Helium (He) → Oxygen (O) → Silicon (Si) beam was designed to simulate simplified-mixed-field GCR (Smf-GCR), and Apc1638N/+ mice were total-body irradiated to sham or γ (157Cs) or Smf-GCR followed by assessment of GI-tumorigenesis at 150 days post-exposure. Further, GI-tumor data from equivalent doses of heavy-ions (i.e., 0.05 Gy of O and Si) in 0.5 Gy of Smf-GCR were compared to understand the contributions of heavy-ions in GI-tumorigenesis. The Smf-GCR-induced tumor and carcinoma count were significantly greater than γ-rays, and male preponderance for GI-tumorigenesis was consistent with our earlier findings. Comparison of tumor data from Smf-GCR and equivalent doses of heavy ions revealed an association between higher GI-tumorigenesis where dose received from heavy-ions contributed to > 95% of the total GI-tumorigenic effect observed after Smf-GCR. This study provides the first experimental evidence that cancer risk after GCR exposure could largely depend on doses received from constituent heavy-ions.

Proglumide Reverses Nonalcoholic Steatohepatitis by Interaction with the Farnesoid X Receptor and Altering the Microbiome.

Nonalcoholic steatohepatitis (NASH) is associated with obesity, metabolic syndrome, and dysbiosis of the gut microbiome. Cholecystokinin (CCK) is released by saturated fats and plays an important role in bile acid secretion. CCK receptors are expressed on cholangiocytes, and CCK-B receptor expression increases in the livers of mice with NASH. The farnesoid X receptor (FXR) is involved in bile acid transport and is a target for novel therapeutics for NASH. The aim of this study was to examine the role of proglumide, a CCK receptor inhibitor, in a murine model of NASH and its interaction at FXR. Mice were fed a choline deficient ethionine (CDE) diet to induce NASH. Some CDE-fed mice received proglumide-treated drinking water. Blood was collected and liver tissues were examined histologically. Proglumide's interaction at FXR was evaluated by computer modeling, a luciferase reporter assay, and tissue FXR expression. Stool microbiome was analyzed by RNA-Sequencing. CDE-fed mice developed NASH and the effect was prevented by proglumide. Computer modeling demonstrated specific binding of proglumide to FXR. Proglumide binding in the reporter assay was consistent with a partial agonist at the FXR with a mean binding affinity of 215 nM. FXR expression was significantly decreased in livers of CDE-fed mice compared to control livers, and proglumide restored FXR expression to normal levels. Proglumide therapy altered the microbiome signature by increasing beneficial and decreasing harmful bacteria. These data highlight the potential novel mechanisms by which proglumide therapy may improve NASH through interaction with the FXR and consequent alteration of the gut microbiome.

CD69+ resident memory T cells are associated with graft-versus-host disease in intestinal transplantation.

Graft-versus-host disease (GvHD) is a common, morbid complication after intestinal transplantation (ITx) with poorly understood pathophysiology. Resident memory T cells (TRM ) are a recently described CD69+ memory T cell subset localizing to peripheral tissue. We observed that T effector memory cells (TEM ) in the blood increase during GvHD and hypothesized that they derive from donor graft CD69+TRM migrating into host blood and tissue. To probe this hypothesis, graft and blood lymphocytes from 10 ITx patients with overt GvHD and 34 without were longitudinally analyzed using flow cytometry. As hypothesized, CD4+ and CD8+CD69+TRM were significantly increased in blood and grafts of GvHD patients, alongside higher cytokine and activation marker expression. The majority of CD69+TRM were donor derived as determined by multiplex immunostaining. Notably, CD8/PD-1 was significantly elevated in blood prior to transplantation in patients who later had GvHD, and percentages of HLA-DR, CD57, PD-1, and naïve T cells differed significantly between GvHD patients who died vs. those who survived. Overall, we demonstrate that (1) there were significant increases in TEM at the time of GvHD, possibly of donor derivation; (2) donor TRM in the graft are a possible source; and (3) potential biomarkers for the development and prognosis of GvHD exist.

Rejection of intestinal allotransplants is driven by memory T helper type 17 immunity and responds to infliximab.

Intestinal transplantation (ITx) can be life-saving for patients with advanced intestinal failure experiencing complications of parenteral nutrition. New surgical techniques and conventional immunosuppression have enabled some success, but outcomes post-ITx remain disappointing. Refractory cellular immune responses, immunosuppression-linked infections, and posttransplant malignancies have precluded widespread ITx application. To shed light on the dynamics of ITx allograft rejection and treatment resistance, peripheral blood samples and intestinal allograft biopsies from 51 ITx patients with severe rejection, alongside 37 stable controls, were analyzed using immunohistochemistry, polychromatic flow cytometry, and reverse transcription-PCR. Our findings inform both immunomonitoring and treatment. In terms of immunomonitoring, we found that while ITx rejection is associated with proinflammatory and activated effector memory T cells in the blood, evidence of treatment efficacy can only be found in the allograft itself, meaning that blood-based monitoring may be insufficient. In terms of treatment, we found that the prominence of intra-graft memory TNF-α and IL-17 double-positive T helper type 17 (Th17) cells is a leading feature of refractory rejection. Anti-TNF-α therapies appear to provide novel and safer treatment strategies for refractory ITx rejection; with responses in 14 of 14 patients. Clinical protocols targeting TNF-α, IL-17, and Th17 warrant further testing.

Innate and adaptive immunity associated with resolution of acute woodchuck hepatitis virus infection in adult woodchucks.

Viral and/or host factors that are directly responsible for the acute versus chronic outcome of hepatitis B virus (HBV) infection have not been identified yet. Information on immune response during the early stages of HBV infection in humans is mainly derived from blood samples of patients with acute hepatitis B (AHB), which are usually obtained after the onset of clinical symptoms. Features of intrahepatic immune response in these patients are less studied due to the difficulty of obtaining multiple liver biopsies. Woodchuck hepatitis virus (WHV) infection in woodchucks is a model for HBV infection in humans. In the present study, five adult woodchucks were experimentally infected with WHV and then followed for 18 weeks. Blood and liver tissues were frequently collected for assaying markers of WHV replication and innate and adaptive immune responses. Liver tissues were further analyzed for pathological changes and stained for important immune cell subsets and cytokines. The increase and subsequent decline of viral replication markers in serum and liver, the elicitation of antibodies against viral proteins, and the induction of virus-specific T-cell responses indicated eventual resolution of acute WHV infection in all animals. Intrahepatic innate immune makers stayed unchanged immediately after the infection, but increased markedly during resolution, as determined by changes in transcript levels. The presence of interferon-gamma and expression of natural killer (NK) cell markers suggested that a non-cytolytic response mechanism is involved in the initial viral control in liver. This was followed by the expression of T-cell markers and cytolytic effector molecules, indicating the induction of a cytolytic response mechanism. Parallel increases in regulatory T-cell markers suggested that this cell subset participates in the overall immune cell infiltration in liver and/or has a role in regulating AHB induced by the cytolytic response mechanism. Since the transcript levels of immune cell markers in blood, when detectable, were lower than in liver, and the kinetics, except for NK-cells and interferon-gamma, did not correlate well with their intrahepatic expression, this further indicated enrichment of immune cells within liver. Conclusion: The coordinated interplay of innate and adaptive immunity mediates viral clearance in the woodchuck animal model of HBV infection. The initial presence of NK-cell associated interferon-gamma response points to an important role of this cytokine in HBV resolution.

De novo hepatocellular carcinoma 18 years after liver and small bowel transplantation in a one-year-old pediatric patient.

De novo HCC following transplantation in a child is a rare occurrence. Even within the adult liver transplantation population, there are a limited number of published cases. In this report, we present a case of de novo HCC found in a child, post-multivisceral transplantation. A 19-year-old boy, at the age of one, received liver and small bowel transplantation due to short gut syndrome secondary to midgut volvulus and total parenteral nutrition-associated liver disease. Eighteen years later, he was found to have a large mass involving the right hepatic dome consistent with HCC. To the best of our knowledge, this is the second reported case after gut transplantation and the third case post-liver transplantation in the pediatric population.

LSD1 activates a lethal prostate cancer gene network independently of its demethylase function.

Medical castration that interferes with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, clinical progression is universal, and tumors with AR-independent resistance mechanisms appear to be increasing in frequency. Consequently, there is an urgent need to develop new treatments targeting molecular pathways enriched in lethal prostate cancer. Lysine-specific demethylase 1 (LSD1) is a histone demethylase and an important regulator of gene expression. Here, we show that LSD1 promotes the survival of prostate cancer cells, including those that are castration-resistant, independently of its demethylase function and of the AR. Importantly, this effect is explained in part by activation of a lethal prostate cancer gene network in collaboration with LSD1's binding protein, ZNF217. Finally, that a small-molecule LSD1 inhibitor-SP-2509-blocks important demethylase-independent functions and suppresses castration-resistant prostate cancer cell viability demonstrates the potential of LSD1 inhibition in this disease.