Maralixibat in progressive familial intrahepatic cholestasis (MARCH-PFIC): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) is a group of autosomal recessive disorders, the most prevalent being BSEP deficiency, resulting in disrupted bile formation, cholestasis, and pruritus. Building on a previous phase 2 study, we aimed to evaluate the efficacy and safety of maralixibat-an ileal bile acid transporter inhibitor-in participants with all types of PFIC. METHODS: MARCH-PFIC was a multicentre, randomised, double-blind, placebo-controlled, phase 3 study conducted in 29 community and hospital centres across 16 countries in Europe, the Americas, and Asia. We recruited participants aged 1-17 years with PFIC with persistent pruritus (>6 months; average of ≥1·5 on morning Itch-Reported Outcome [Observer; ItchRO(Obs)] during the last 4 weeks of screening) and biochemical abnormalities or pathological evidence of progressive liver disease, or both. We defined three analysis cohorts. The BSEP (or primary) cohort included only those with biallelic, non-truncated BSEP deficiency without low or fluctuating serum bile acids or previous biliary surgery. The all-PFIC cohort combined the BSEP cohort with participants with biallelic FIC1, MDR3, TJP2, or MYO5B deficiencies without previous surgery but regardless of bile acids. The full cohort had no exclusions. Participants were randomly assigned (1:1) to receive oral maralixibat (starting dose 142·5 µg/kg, then escalated to 570 µg/kg) or placebo twice daily for 26 weeks. The primary endpoint was the mean change in average morning ItchRO(Obs) severity score between baseline and weeks 15-26 in the BSEP cohort. The key secondary efficacy endpoint was the mean change in total serum bile acids between baseline and the average of weeks 18, 22, and 26 in the BSEP cohort. Efficacy analyses were done in the intention-to-treat population (all those randomly assigned) and safety analyses were done in all participants who received at least one dose of study drug. This completed trial is registered with ClinicalTrials.gov, NCT03905330, and EudraCT, 2019-001211-22. FINDINGS: Between July 9, 2019, and March 4, 2022, 125 patients were screened, of whom 93 were randomly assigned to maralixibat (n=47; 14 in the BSEP cohort and 33 in the all-PFIC cohort) or placebo (n=46; 17 in the BSEP cohort and 31 in the all-PFIC cohort), received at least one dose of study drug, and were included in the intention-to-treat and safety populations. The median age was 3·0 years (IQR 2·0-7·0) and 51 (55%) of 93 participants were female and 42 (45%) were male. In the BSEP cohort, least-squares mean change from baseline in morning ItchRO(Obs) was -1·7 (95% CI -2·3 to -1·2) with maralixibat versus -0·6 (-1·1 to -0·1) with placebo, with a significant between-group difference of -1·1 (95% CI -1·8 to -0·3; p=0·0063). Least-squares mean change from baseline in total serum bile acids was -176 µmol/L (95% CI -257 to -94) for maralixibat versus 11 µmol/L (-58 to 80) for placebo, also representing a significant difference of -187 µmol/L (95% CI -293 to -80; p=0·0013). The most common adverse event was diarrhoea (27 [57%] of 47 patients on maralixibat vs nine [20%] of 46 patients on placebo; all mild or moderate and mostly transient). There were five (11%) participants with serious treatment-emergent adverse events in the maralixibat group versus three (7%) in the placebo group. No treatment-related deaths occurred. INTERPRETATION: Maralixibat improved pruritus and predictors of native liver survival in PFIC (eg, serum bile acids). Maralixibat represents a non-surgical, pharmacological option to interrupt the enterohepatic circulation and improve the standard of care in patients with PFIC. FUNDING: Mirum Pharmaceuticals.

Loss of Anti-Tumor Efficacy by Polyamine Blocking Therapy in GCN2 Null Mice.

GCN2 is one of the main sensors of amino acid starvation stress, and its activation in the stressful tumor microenvironment plays a crucial role in tumor survival and progression. We hypothesized that elevated polyamine biosynthesis and subsequent depletion of precursor arginine activates GCN2, thus rewiring metabolism to support tumor cell survival and drive myeloid immunosuppressive function. We sought to determine if the anti-tumor efficacy of a polyamine blocking therapy (PBT) may be mediated by its effect on GCN2. Unlike wild-type mice, PBT treatment in GCN2 knockout mice bearing syngeneic B16.F10 or EG7 tumors resulted in no tumor growth inhibition and no changes in the profile of infiltrating tumor immune cells. Studies with murine bone marrow cell cultures showed that increased polyamine metabolism and subsequent arginine depletion and GCN2 activation played an essential role in the generation and cytoprotective autophagy of myeloid derived suppressor cells (MDSCs) as well as the M2 polarization and survival of macrophages, all of which were inhibited by PBT. In all, our data suggest that polyamine-dependent GCN2 signaling in stromal cells promotes tumor growth and the development of the immunosuppressive tumor microenvironment, and that the PBT anti-tumor effect is mediated, at least in part, by targeting GCN2.

Challenges and physiological implications of sarcopenia in children and youth in health and disease.

PURPOSE OF REVIEW: Highlight the controversies and challenges associated with a sarcopenia diagnosis in infants and children and the potential physiological mechanisms contributing to this disorder. RECENT FINDINGS: Sarcopenia has been recently identified in infants and children with chronic diseases such as liver, cardiac, gastrointestinal, cancer and organ transplant recipients. However, there is no consensus regarding the definition of pediatric sarcopenia. Different sarcopenic phenotypes (sarcopenia and sarcopenic obesity) have been identified in healthy children and children with chronic disease. Both conditions have been associated with adverse clinical outcomes (e.g. delayed growth, increased hospitalization) in children and youth with chronic disease. The etiology of pediatric sarcopenia is likely multifactorial associated with malnutrition, physical inactivity and altered metabolic environments influencing skeletal muscle mass accumulation and function. Gaps in the literature include the lack of standard tools that should be used for the evaluation of skeletal muscular fitness and body composition in sarcopenia, particularly in infants and young children (<4years). SUMMARY: Longitudinal evaluation of sarcopenia expression and the underlying physiological and lifestyle factors contributing to pediatric sarcopenia are important to understand to ensure effective rehabilitation strategies can be developed and to avoid the adverse clinical consequences in children.

Difluoromethylornithine (DFMO) Enhances the Cytotoxicity of PARP Inhibition in Ovarian Cancer Cells.

Ovarian cancer accounts for 3% of the total cancers in women, yet it is the fifth leading cause of cancer deaths among women. The BRCA1/2 germline and somatic mutations confer a deficiency of the homologous recombination (HR) repair pathway. Inhibitors of poly (ADP-ribose) polymerase (PARP), another important component of DNA damage repair, are somewhat effective in BRCA1/2 mutant tumors. However, ovarian cancers often reacquire functional BRCA and develop resistance to PARP inhibitors. Polyamines have been reported to facilitate the DNA damage repair functions of PARP. Given the elevated levels of polyamines in tumors, we hypothesized that treatment with the polyamine synthesis inhibitor, α-difluoromethylornithine (DFMO), may enhance ovarian tumor sensitivity to the PARP inhibitor, rucaparib. In HR-competent ovarian cancer cell lines with varying sensitivities to rucaparib, we show that co-treatment with DFMO increases the sensitivity of ovarian cancer cells to rucaparib. Immunofluorescence assays demonstrated that, in the presence of hydrogen peroxide-induced DNA damage, DFMO strongly inhibits PARylation, increases DNA damage accumulation, and reduces cell viability in both HR-competent and deficient cell lines. In vitro viability assays show that DFMO and rucaparib cotreatment significantly enhances the cytotoxicity of the chemotherapeutic agent, cisplatin. These results suggest that DFMO may be a useful adjunct chemotherapeutic to improve the anti-tumor efficacy of PARP inhibitors in treating ovarian cancer.

Immunomodulatory role of thrombin in cancer progression.

Coagulation proteases and the generation of thrombin are increased in tumors. In addition, chemotherapeutic agents commonly used to treat malignant cancers can exacerbate cancer-associated thromboses. Thrombin can modify tumor cell behavior directly through the activation of protease-activated receptors (PAR) or indirectly by generating fibrin matrices. In addition to its role in generating fibrin to promote hemostasis, thrombin acts directly on multiple effector cells of the immune system impacting both acute and chronic inflammatory processes. Thrombin-mediated release of interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 leads to the accumulation of multiple tumor-infiltrating immunosuppressive cell populations including myeloid derived suppresser cells, M2-like macrophages, and T regulatory cells. Ablation of PAR-1 from the tumor microenvironment, but not the tumor, has been shown to dramatically reduce tumor growth and metastasis in multiple tumor models. Thrombin-activated platelets release immunosuppressive cytokines including transforming growth factor-ß that can inhibit natural killer cell activity, helping tumor cells to evade host immunosurveillance. Taken together, there is strong evidence that thrombin influences cancer progression via multiple mechanisms, including the tumor immune response, with thrombin emerging as a target for novel therapeutic strategies for cancer.

The Transition to Microsurgical Technique for Hepatic Artery Reconstruction in Pediatric Liver Transplantation.

BACKGROUND: Hepatic artery thrombosis represents a potentially fatal complication following liver transplantation. Rates of hepatic artery thrombosis are significantly higher in children, with mortality reported up to 80 percent. Microsurgical anastomosis has been shown to decrease the rate of hepatic artery thrombosis and now represents the standard of care at the authors' institution. In this article, the authors present the largest study of its type directly comparing rates of hepatic artery thrombosis with and without microsurgical reconstruction of the hepatic artery. METHODS: All pediatric patients who underwent primary orthotopic liver transplantation between 1989 and 2018 were included. Patients were divided into two cohorts: standard anastomosis with loupes, and microsurgical anastomosis under the operating microscope. The authors' primary outcome was the rate of hepatic artery thrombosis. Secondary outcomes were graft survival, patient survival, retransplantation rate, requirement for intraoperative blood products, and length of stay. RESULTS: Two hundred thirty-one children met criteria for inclusion. One hundred eighty cases were performed with loupe magnification and 51 cases were performed under the microscope. The hepatic artery thrombosis rate was lower, but not significantly so (p = 0.114), in the microsurgical group [n = 1 (2.0 percent)] compared with the standard cohort [n = 15 (8.3 percent)]. Survival analysis revealed a significant increase in graft survival with microsurgical anastomosis (p = 0.020), but not patient survival (p = 0.196). The retransplantation rate was significantly lower with microsurgical anastomosis (p = 0.021). CONCLUSIONS: Microsurgical anastomosis was associated with a clinically important decrease in hepatic artery thrombosis compared with standard loupe anastomosis. The graft survival rate was significantly higher in the microsurgical cohort, with a reduced retransplantation rate at 1 year. On this basis, the authors recommend microsurgical hepatic artery anastomosis in cases of pediatric liver transplantation. . CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Harnessing the polyamine transport system to treat BRAF inhibitor-resistant melanoma.

BRAF mutations are present in over half of all melanoma tumors. Although BRAF inhibitors significantly improve survival of patients with metastatic melanoma, recurrences occur within several months. We previously reported that BRAF mutant melanoma cells are more sensitive to a novel arylmethyl-polyamine (AP) compound that exploits their increased polyamine uptake compared to that of BRAF wildtype cells. Using an animal model of BRAF inhibitor-resistant melanoma, we show that co-treatment with the BRAF inhibitor, PLX4720, and AP significantly delays the recurrence of PLX4720-resistant melanoma tumors and decreases tumor-promoting macrophages. Development of BRAF inhibitor-resistance enriches for metastatic cancer stem cells (CSC) and increases tumor-promoting macrophages. In vitro studies demonstrated that CD304+, CXCR4+ spheroid cultures of BRAF mutant melanoma cells are resistant to PLX4720 but are more sensitive to AP compared to monolayer cultures of the same cells. AP significantly inhibited YUMM1.7 melanoma cell invasiveness across a Matrigel-coated filter using the CXCR4 ligand, SDF-1α, as the chemoattractant. AP also blocked the chemotactic effect of SDF-1α on CXCR4+ macrophages and inhibited M2 polarization of macrophages. In melanoma-macrophage co-cultures, AP prevented the PLX4720-induced release of pro-tumorigenic growth factors, such as VEGF, from macrophages and prevented the macrophage rescue of BRAF mutant melanoma cells treated with PLX4720. Our study offers a novel therapy (AP) to treat chemo-resistant melanoma. AP is unique because it targets the polyamine transport system in BRAF inhibitor-resistant CSCs and also blocks CXCR4 signaling in invasive melanoma cells and pro-tumorigenic macrophages.

Liver Transplantation in Locally Unresectable, Undifferentiated Embryonal Cell Sarcoma.

BACKGROUND: Undifferentiated embryonal cell sarcoma (UESL) of the liver is the third most common malignant liver disease of childhood presenting as a rapidly enlarging intraabdominal mass. This systematic review explores the practicality of liver transplantation as a viable option in the treatment armamentarium for locally advanced undifferentiated embryonal cell sarcoma. METHODS: A systematic review of the literature was performed using Medline and Embase, from inception of databases to December 31, 2018. Keywords and MeSH headings used were embryonal sarcoma, mesenchymal sarcoma, and liver transplant. Reviews and manuscripts with incomplete data were excluded. RESULTS: Twenty-eight patients had orthotopic liver transplantation (OLT) as a curative treatment option. The median age at presentation was 8 and 27 years in the pediatric and adult population, respectively, with a similar male to female ratio. A majority of the patients presented with abdominal pain, palpable mass, and a normal alpha-feto-protein. The median tumor size was 15 cm mainly affecting the right lobe (62%) of the liver. Eighty-two percent of the patients underwent primary OLT and 5 patients had salvage OLT. One death (3.6%) was due to initial misdiagnosis and management for hepatoblastoma. Recurrence was noted in 7.1% of the population. The median follow-up was noted to be 28.5 months. The documented survival rate post-liver transplant for UESL was 96%. CONCLUSIONS: Based on available data and the very positive results therein, liver transplantation is a practical and justifiable use of a scarce resource as a treatment option for locally unresectable, undifferentiated embryonal cell sarcoma. The authors propose (accepting existence of different proposals) neoadjuvant therapy before curative resection, and if not achievable, then liver transplantation followed by adjuvant chemotherapy is an option for suitable candidates. For recurrent tumors after surgical resection, adjuvant therapy with salvage liver transplantation is an option.

Single Topic Conference on Autoimmune Liver Disease from the Canadian Association for the Study of the Liver.

Autoimmune liver disease (AILD) spans a spectrum of chronic disorders affecting the liver parenchyma and biliary system. Three main categories of AILD are autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC). This review condenses the presentation and discussions of the Single Topic Conference (STC) on AILD that was held in Ottawa, Ontario, in November 2019. We cover generalities regarding disease presentation and clinical diagnosis; mechanistic themes; treatment paradigms; clinical trials, including approaches and challenges to new therapies; and looking beyond traditional disease boundaries. Although these diseases are considered autoimmune, the etiology and role of environmental triggers are poorly understood. AILDs are progressive and chronic conditions that affect survival and quality of life. Advances have been made in PBC treatment because second-line treatments are now available (obeticholic acid, bezafibrate); however, a significant proportion still present suboptimal response. AIH treatment has remained unchanged for several decades, and data suggest that fewer than 50% of patients achieve a complete response and as many as 80% develop treatment-related side effects. B-cell depletion therapy to treat AIH is in an early stage of development and has shown promising results. An effective treatment for PSC is urgently needed. Liver transplant remains the best option for patients who develop decompensated cirrhosis or hepatocellular carcinoma within specific criteria, but recurrent AILD might occur. Continued efforts are warranted to develop networks for AILD aimed at assessing geo-epidemiological, clinical, and biochemical differences to capture the new treatment era in Canada.

Polyamine Blocking Therapy Decreases Survival of Tumor-Infiltrating Immunosuppressive Myeloid Cells and Enhances the Antitumor Efficacy of PD-1 Blockade.

Despite unprecedented advances in the treatment of cancer through the use of immune checkpoint blockade (ICB), responses are not universal and alternative strategies are needed to enhance responses to ICB. We have shown previously that a novel polyamine blocking therapy (PBT), consisting of cotreatment with α-difluoromethylornithine (DFMO) to block polyamine biosynthesis and a Trimer polyamine transport inhibitor, decreases myeloid-derived suppressor cells (MDSC) and M2-like tumor-associated macrophages (TAM). Both MDSCs and TAMs promote tumor progression, inhibit antitumor immunity, and limit the efficacy of ICB. In this study, we investigated the use of PBT to heighten therapeutic responses to PD-1 blockade in mice bearing 4T1 mammary carcinoma and B16F10 melanoma tumors. Whereas PBT inhibited primary tumor growth in both tumor models, 4T1 lung metastases were also dramatically decreased in mice treated with PBT. Reductions in MDSC and TAM subpopulations in 4T1 tumors from PBT-treated mice were accompanied by reduced cytoprotective autophagy only in tumor-infiltrating MDSC and macrophage subpopulations but not in the lung or spleen. PBT treatment blunted M2-like alternative activation of bone marrow-derived macrophages and reduced STAT3 activation in MDSC cultures while increasing the differentiation of CD80+, CD11c+ macrophages. PBT significantly enhanced the antitumor efficacy of PD-1 blockade in both 4T1 and B16F10 tumors resistant to anti-PD-1 monotherapy, increasing tumor-specific cytotoxic T cells and survival of tumor-bearing animals beyond that with PBT or PD-1 blockade alone. Our results suggest that cotreatment with DFMO and the Trimer polyamine transport inhibitor may improve the therapeutic efficacy of immunotherapies in patients with cancer with resistant tumors.

Deficits in Muscle Strength and Physical Performance Influence Physical Activity in Sarcopenic Children After Liver Transplantation.

Sarcopenia is a muscle disease characterized by reduced skeletal muscle mass (SMM), muscle strength, and physical performance. Reduced SMM has been identified in children after liver transplantation (LT), but no information related to muscle strength/physical performance or lifestyle factors contributing to sarcopenia is available. We hypothesized that sarcopenia, as determined by measures of SMM, muscle strength, and physical performance, is highly prevalent in children after LT and is related to poor diet quality (DQ) and physical inactivity. A cross-sectional study in post-LT children (n = 22) and age-matched healthy controls (n = 47) between the ages of 6 and 18 years examining body composition (dual energy X-ray absorptiometry and multiple skinfold), measures of muscle strength (handgrip, sit-to-stand, and push-ups), physical performance (6-minute walk test and stair climb test), diet (3-day food intake), and physical activity (accelerometer) was conducted. Low muscle strength/physical performance and SMM (SMM z scores ≤-1.5) were defined by values 2 standard deviations below the mean values for age- and sex-matched controls. Sarcopenia occurred in 36% of children who underwent LT, and they had significantly lower scores for muscle strength (sit-to-stand and push-up tests) and physical performance (stair climb test) than controls (P < 0.05). Deficits in physical performance in children with sarcopenia were predominantly revealed by longer stair climbing times (P = 0.03), with no differences in other muscle tests. Low SMM, muscle strength, and physical performance were associated with a lower amount of time spent in fairly and very active physical activity, but no associations with DQ were found. Sarcopenia is highly prevalent in children after LT and is related to lower moderate-to-vigorous physical activity. Development of effective rehabilitation strategies to treat sarcopenia are needed in post-LT children.

Polyamine-stimulation of arsenic-transformed keratinocytes.

Tumor promotion is strongly associated with inflammation and increased polyamine levels. Our understanding of relevant mechanisms responsible for arsenic-induced cancer remains limited. Previous studies suggest that arsenic targets and dysregulates stem cell populations that remain dormant in the skin until promoted to be recruited out of the bulge stem cell region, thus giving rise to skin tumors. In this study, we explored a possible mechanism by which increased keratinocyte polyamine biosynthesis promotes tumorsphere formation and invasiveness of arsenic-transformed HaCaT keratinocytes (As-HaCaT). Unlike parental HaCaT cells, As-HaCaT cells were tumorigenic in athymic nude mice, and the CD45negative epithelial tumor cells had enriched expression of Toll-Like Receptor 4 (TLR4), CD34 and CXCR4 as did As-HaCaT tumorsphere cultures compared to As-HaCaT monolayer cultures. Ornithine decarboxylase (ODC) overexpressing keratinocytes (Ker/ODC) release increased levels of the alarmin high mobility group box 1 (HMGB1). Ker/ODC conditioned medium (CM) stimulated As-HaCaT but not parental HaCaT tumorsphere formation, and this was inhibited by glycyrrhizin, an inhibitor of HMGB1, and by TAK242, an inhibitor of the HMGB1 receptor TLR4. Compared to parental HaCaT cells, As-HaCaT cells demonstrated greater invasiveness across a Matrigel-coated filter using either fibroblast CM or SDF-1α as chemoattractants. Addition of Ker/ODC CM or HMGB1 dramatically increased As-HaCaT invasiveness. Glycyrrhizin and TAK242 inhibited this Ker/ODC CM-stimulated invasion of As-HaCaT cells but not HaCaT cells. These results show that polyamine-dependent release of HMGB1 promotes the expansion of stem cell-like subpopulations in arsenic-transformed keratinocytes while also increasing their invasiveness, suggesting that polyamines may be a potential therapeutic target for the prevention and treatment of arsenic-initiated skin cancers.

Preclinical study of the long-range safety and anti-inflammatory effects of high-dose oral meglumine.

Meglumine is a methylamino derivative of sorbitol that is an approved drug excipient. Recent preclinical studies suggest that administration of high-dose oral meglumine can exert beneficial medicinal effects to treat diabetes, obesity, and fatty liver disease (NAFLD/nonalcoholic steatohepatitis [NASH]). Here we address gaps in knowledge about the pharmacology and toxicology of this substance administered at high concentrations to explore its medicinal potential. We observed that high-dose meglumine limited secretion of proinflammatory cytokines and cell adhesion molecules from activated human THP-1 or murine RAW264.7 monocytes. Preclinical pharmacokinetic analysis in Swiss mice confirmed that meglumine was orally available. Informed by this data, oral doses of 18 to 75 mM meglumine were administered ad libitum in the drinking water of Sprague-Dawley rats and two cohorts of C57BL/6 mice housed in different vivariums. In a 32-week study, urinary isoprostane levels trended lower in subjects consistent with the possibility of anti-inflammatory effects. In full lifespan studies, there was no detrimental effect on longevity. Heart function evaluated in C57BL/6 mice using an established noninvasive cardiac imaging system showed no detrimental effects on ejection fraction, fractional shortening, left ventricle function or volume, and cardiac output in mice up to 15-month old, with a potential positive trend in heart function noted in elderly mice consistent with earlier reported benefits on muscle stamina. Finally, in a transgenic model of inflammation-associated skin carcinogenesis, the incidence, number, and growth of skin tumors trended lower in subjects receiving meglumine. Overall, the evidence obtained illustrating the long-range safety of high-dose oral meglumine support the rationale for its evaluation as a low-cost modality to limit diabetes, hypertriglyceridemia, and NAFLD/NASH.

A Novel Polyamine-Targeted Therapy for BRAF Mutant Melanoma Tumors.

Mutant serine/threonine protein kinase B-Raf (BRAF) protein is expressed in over half of all melanoma tumors. Although BRAF inhibitors (BRAFi) elicit rapid anti-tumor responses in the majority of patients with mutant BRAF melanoma, the tumors inevitably relapse after a short time. We hypothesized that polyamines are essential for tumor survival in mutant BRAF melanomas. These tumors rely on both polyamine biosynthesis and an upregulated polyamine transport system (PTS) to maintain their high intracellular polyamine levels. We evaluated the effect of a novel arylpolyamine (AP) compound that is cytotoxic upon cellular entry via the increased PTS activity of melanoma cells with different BRAF mutational status. Mutant BRAF melanoma cells demonstrated greater PTS activity and increased sensitivity to AP compared to wild type BRAF (BRAFWT) melanoma cells. Treatment with an inhibitor of polyamine biosynthesis, α-difluoromethylornithine (DFMO), further upregulated PTS activity in mutant BRAF cells and increased their sensitivity to AP. Furthermore, viability assays of 3D spheroid cultures of mutant BRAF melanoma cells demonstrated greater resistance to the BRAFi, PLX4720, compared to 2D monolayer cultures. However, co-treatment with AP restored the sensitivity of melanoma spheroids to PLX4720. These data indicate that mutant BRAF melanoma cells are more dependent on the PTS compared to BRAFWT melanoma cells, resulting in greater sensitivity to the PTS-targeted cytotoxic AP compound.

A novel polyamine blockade therapy activates an anti-tumor immune response.

Most tumors maintain elevated levels of polyamines to support their growth and survival. This study explores the anti-tumor effect of polyamine starvation via both inhibiting polyamine biosynthesis and blocking the upregulated import of polyamines into the tumor. We demonstrate that polyamine blockade therapy (PBT) co-treatment with both DFMO and a novel polyamine transport inhibitor, Trimer PTI, significantly inhibits tumor growth more than treatment with DFMO or the Trimer PTI alone. The anti-tumor effect of PBT was lost in mice where CD4+ and CD8+ T cells were antibody depleted, implying that PBT stimulates an anti-tumor immune effect that is T-cell dependent. The PBT anti-tumor effect was accompanied by an increase in granzyme B+, IFN-γ+ CD8+ T-cells and a decrease in immunosuppressive tumor infiltrating cells including Gr-1+CD11b+ myeloid derived suppressor cells (MDSCs), CD4+CD25+ Tregs, and CD206+F4/80+ M2 macrophages. Stimulation with tumor-specific peptides elicited elevated antigen-specific IFN-γ secretion in splenocytes from PBT-treated mice, indicating that PBT treatment stimulates the activation of T-cells in a tumor-specific manner. These data show that combined treatment with both DFMO and the Trimer PTI not only deprives polyamine-addicted tumor cells of polyamines, but also relieves polyamine-mediated immunosuppression in the tumor microenvironment, thus allowing the activation of tumoricidal T-cells.

Thrombin inhibition and cisplatin block tumor progression in ovarian cancer by alleviating the immunosuppressive microenvironment.

Cancer is often associated with an increased risk of thrombotic complications which can be aggravated by treatment with chemotherapeutics such as cisplatin. Multiple lines of evidence suggest that thrombin activity promotes tumor growth and metastasis. We examined the effect of co-treatment with dabigatran etexilate, a direct thrombin inhibitor, and cisplatin using the murine ID8 ovarian cancer model. Mice receiving co-treatment with both dabigatran etexilate and low dose cisplatin had significantly smaller tumors, developed less ascites and had lower levels of circulating activated platelets and tissue factor (TF) positive microparticles than those treated with dabigatran etexilate or cisplatin alone. Co-treatment with dabigatran etexilate and cisplatin significantly decreased the number of Gr1+/CD11b+ myeloid derived suppresser cells and CD11b+/CD11c+ dendritic cells in the ascites of ID8 tumor-bearing mice. Co-treatment also significantly reduced levels of pro-tumorigenic cytokines including TGF-ß, VEGF, IL-6, IL-10, and MCP-1 in the ascites while increasing IFN-γ production by CD8+ effector T cells in the tumor ascites. These results demonstrate that co-treatment with dabigatran etexilate significantly augments the anti-tumor activity of cisplatin in ovarian tumor progression by alleviating the immunosuppressive microenvironment, suggesting that thrombin may be a potential therapeutic target for treatment of ovarian cancer.

Polyamine Antagonist Therapies Inhibit Neuroblastoma Initiation and Progression.

PURPOSE: Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma. EXPERIMENTAL DESIGN: We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second rate-limiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity. RESULTS: An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance. CONCLUSIONS: Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted. Clin Cancer Res; 22(17); 4391-404. ©2016 AACR.

Thrombin inhibition and cyclophosphamide synergistically block tumor progression and metastasis.

Cancer is often associated with an increased risk of thrombotic events which are exacerbated by treatment with chemotherapeutics such as cyclosphosphamide (CP). Evidence suggests that thrombin can stimulate tumor progression via formation of fibrin and activation of protease-activated receptors (PARs) and platelets. We examined the effect of co-treatment with CP and dabigatran etexilate, a direct inhibitor of thrombin, using the murine orthotopic 4T1 tumor model. Mice receiving co-treatment with both low dose CP and dabigatran etexilate had significantly smaller mammary tumors and fewer lung metastases than mice treated with CP or dabigratran etexilate alone. Co-treatment with dabigatran etexilate and low dose CP also significantly decreased the number of arginase(+)Gr-1(+)CD11b(+) myeloid derived suppressor cells as well as levels of TGF-ß in spleens from tumor bearing mice. 4T1 tumors express procoagulant tissue factor (TF) and spontaneously release TF(+) microparticles which are potent procoagulant factors that promote thrombin generation. Treatment with dabigatran etexilate alone prevented tumor-induced increases in circulating TF(+) microparticles and also decreased the numbers of tumor-induced activated platelets by 40%. These results show that co-treatment with dabigatran etexilate and CP synergistically inhibits growth and metastasis of mammary tumors, suggesting that oral administration of the thrombin inhibitor dabigatran etexilate may be beneficial in not only preventing thrombotic events in cancer patients but also in treating malignant tumors themselves.

Translational development of difluoromethylornithine (DFMO) for the treatment of neuroblastoma.

Neuroblastoma is a childhood tumor in which MYC oncogenes are commonly activated to drive tumor progression. Survival for children with high-risk neuroblastoma remains poor despite treatment that incorporates high-dose chemotherapy, stem cell support, surgery, radiation therapy and immunotherapy. More effective and less toxic treatments are sought and one approach under clinical development involves re-purposing the anti-protozoan drug difluoromethylornithine (DFMO; Eflornithine) as a neuroblastoma therapeutic. DFMO is an irreversible inhibitor of ornithine decarboxylase (Odc), a MYC target gene, bona fide oncogene, and the rate-limiting enzyme in polyamine synthesis. DFMO is approved for the treatment of Trypanosoma brucei gambiense encephalitis ("African sleeping sickness") since polyamines are essential for the proliferation of these protozoa. However, polyamines are also critical for mammalian cell proliferation and the finding that MYC coordinately regulates all aspects of polyamine metabolism suggests polyamines may be required to support cancer promotion by MYC. Pre-emptive blockade of polyamine synthesis is sufficient to block tumor initiation in an otherwise fully penetrant transgenic mouse model of neuroblastoma driven by MYCN, underscoring the necessity of polyamines in this process. Moreover, polyamine depletion regimens exert potent anti-tumor activity in pre-clinical models of established neuroblastoma as well, in combination with numerous chemotherapeutic agents and even in tumors with unfavorable genetic features such as MYCN, ALK or TP53 mutation. This has led to the testing of DFMO in clinical trials for children with neuroblastoma. Current trial designs include testing lower dose DFMO alone (2,000 mg/m(2)/day) starting at the completion of standard therapy, or higher doses combined with chemotherapy (up to 9,000 mg/m(2)/day) for patients with relapsed disease that has progressed. In this review we will discuss important considerations for the future design of DFMO-based clinical trials for neuroblastoma, focusing on the need to better define the principal mechanisms of anti-tumor activity for polyamine depletion regimens. Putative DFMO activities that are both cancer cell intrinsic (targeting the principal oncogenic driver, MYC) and cancer cell extrinsic (altering the tumor microenvironment to support anti-tumor immunity) will be discussed. Understanding the mechanisms of DFMO activity are critical in determining how it might be best leveraged in upcoming clinical trials. This mechanistic approach also provides a platform by which iterative pre-clinical testing using translational tumor models may complement our clinical approaches.

Value of serum cystatin C in estimating renal function in children with non-renal solid organ transplantation.

Children with non-renal solid organ transplants are surviving longer, but outcome is complicated by CKD. Accurate and frequent renal function monitoring is imperative to recognize and institute measures early to reverse, prevent, or arrest progression. This study of 59 children determined the accuracy (P30), bias, sensitivity and specificity between measured renal function by NM-GFR, and estimated GFR by three formulas: Filler (serum cystatin C), mSchwartz (serum creatinine), and CKiD (serum cystatin C, creatinine, urea, and height). Mean GFR by all formulas differed significantly from NM-GFR. Filler and mSchwartz formulas significantly increased the proportion of patients with GFR ≥ 90 mL/min/1.73 m(2) (CKD stage 1) while decreasing those with GFR 60-89 mL/min/1.73 m(2) (CKD stage 2). All formulas overestimated GFR. CKiD showed the highest P30 and lowest bias (79.7%; 6.9 mL/min/1.73 m(2) ) followed by Filler (67.7%; 19.9 mL/min/1.73 m(2) ) and Schwartz (57.6%; 26.8 mL/min/1.73 m(2) ) for all GFR values. All formulas performed best with GFR ≥ 90 mL/min/1.73 m(2) , but CKiD was the only formula to achieve 91.1% accuracy. All formulas showed high sensitivities, but low specificities at NM-GFR cutoff at 90. Thus, GFR estimated by CKiD followed by Filler formula is an adequate method to monitor renal function closely and frequently in these children.