Abstract B40: Mouse clinical trials: integrating PDX models of sarcoma subtypes with genomics to replicate patient responses to cancer therapeutics

Abstract Objective: Sarcomas are clinically and genetically heterogeneous tumors that are often difficult to treat. Patient-derived xenograft (PDX or TumorGraft) models have been shown to accurately reflect the characteristics of patient tumors and may be useful tools for developing personalized treatment strategies and deployment in mouse clinical trials assessing novel therapies. We evaluated the accuracy of PDX models in reproducing clinical responses to standard and experimental drugs used for sarcoma treatment. Methods: Fresh tumor tissue (comprising 172 distinct explants) was collected by surgery or biopsy from 150 patients with sarcoma and implanted into immunodeficient mice. Tumors successfully engrafting were screened using next-generation sequencing technology to identify key genomic alterations with therapeutic implications. PDX sensitivity to standard of care and experimental agents was evaluated and tumor growth inhibition/regression values and clinical RECIST outcomes determined. Drug screening results were correlated with individual patient outcomes. Results: Of the 172 implanted tumors, 145 have completed the implantation process, with 86 (59%) successfully establishing a PDX model. Engraftment rate depended on sarcoma subtype and specimen origin (surgical explant versus biopsy). Next generation sequencing of models from major sarcoma subtypes (Ewing sarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, and rhabdomyosarcoma) highlighted alterations in 454 genes, including those informing treatment selection such as PIK3CA, MET, and CDK4. A total of 26 PDX models from 25 patients across the major sarcoma subtypes were screened in 148 drug tests employing 64 FDA-approved drugs/combinations such as ifosfamide, and gemcitabine/docetaxel, and 26 experimental therapies in clinical trial. In 13/13 (100%) cases with available data, a significant correlation between patient clinical response and PDX model outcome was noted (p=0.0004; Fisher's exact test). Conclusions: Given the close match between patient clinical responses and PDX model outcomes, these results validate the concept of mouse clinical trials for determining the efficacy of novel therapies in sarcoma prior to broad application in expensive human trials. Moreover, the retention of alterations in key genes influencing therapeutic decision-making suggests a use for PDX models in functionally validating genomic hypotheses in a pre-clinical setting. Citation Format: Amanda Katz, Raphael E. Pollock, Leonard H. Wexler, Carlos Rodriguez-Galindo, Jonathan C. Trent, Robert Maki, Jennifer Jaskowiak, Lindsay Ryland, Daniel Ciznadija, Angela Davies, Keren Paz. Mouse clinical trials: integrating PDX models of sarcoma subtypes with genomics to replicate patient responses to cancer therapeutics. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B40.</jats:p

C6-ceramide nanoliposomes target the Warburg effect in chronic lymphocytic leukemia.

Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the "Warburg effect", we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect

Earning and learning: Role congruence, state/trait factors and adjustment to university life

Background. Undertaking term-time employment is increasingly commonplace for university students. Much research suggests that combining 'earning and learning' may be detrimental to university life, generating role conflicts, increasing stress and reducing academic success, participation and overall adjustment to university. Potential positive effects of term-time employment on well-being are often neglected. Aims. This study adopted a balanced perspective, investigating the relationship between role congruence in academic, social and career domains and adjustment, with state and trait psychological factors as mediators/moderators. Methods. A questionnaire measuring perceived role congruence, adjustment to university life and psychological state and trait characteristics was mailed to all undergraduates at a Scottish university during term-time. Sample. Results for a subsample of 625 university students currently in term-time employment were included in this paper. Results. Findings suggested that students generally perceived employment and university roles to be in balance, and there was no difference in adjustment for students whether currently in term-time employment or not. However, psychological factors, particularly positive affectivity and stress were important mediators of the relationship between role congruence and adjustment. Conclusions. Since promoting role congruence may enhance students' adjustment and well-being, the study has implications for universities, student welfare organizations and employers offering term-time employment

Nanoliposomal C6-ceramide selectively induces cell death in CLL cells.

<p>JVM3 cells were treated with varying doses of ghost or C6-ceramide or dihydro-C6-ceramide nanoliposomes for 24 hours then A). MTT assay, B). Trypan blue staining was performed. ANOVA statistical test was used to determine dose dependency between various C6-ceramide treatment groups. <i>P</i> < .0001. C). JVM3 cells were treated with different doses of ghost or C6-ceramide nanoliposomes for 24 hours, then cells were stained with annexin V and 7AAD for apoptosis assay. D). Percentage of apoptotic cells was determined via TUNEL analysis after 24 hours. E). PBMC isolated from either CLL patients (n=3) or normal donors (n=3) were treated with 25 µM ghost or C6-ceramide nanoliposome for 2, 16 and 24 hours, then MTT assay was performed. F). Percentage of apoptotic cells was determined in PBMC from normal donors (n=3) via annexin V/7AAD staining. Cells were treated with different doses of ghost or C6-ceramide nanoliposome for 24 hours or treated with 25 µM ghost or C6-ceramide nanoliposome for 2 and 24 hours. * <i>P</i> < 0.05. </p

C6-ceramide targets the glycolytic pathway.

<p>JVM3 cells were treated with varying doses of ghost or C6-ceramide nanoliposomes for 24 hours, then A) lactate production was analyzed, B) ATP production was analyzed; * <i>P</i> < 0.05. The graphs depict average results from three independent experiments. C). GAPDH was effectively knocked down in JVM3 cells via a lentiviral shRNA approach (inset). ATP production was then assessed after 24 hours of treatment with 50µM of ghost or 25µM of C6-ceramide nanoliposomes; * <i>P</i> < 0.05; ** <i>P</i> < 0.005. D). Glucose uptake was assessed after JVM3 cells were treated with ghost and C6-ceramide nanoliposomes for 24 hours. Cytochalasin B was used as a positive control. E). JVM3 cells were treated with ghost or C6-ceramide nanoliposomes for 24 hours, then Western Blot analysis was performed for GLUT1. </p

Nanoliposomal C6-ceramide displays anti-leukemic effect in a CLL animal model.

<p>A). Two weeks after ten million JVM3 cells were inoculated in the right flank of female Balb/c Nu/nu mice, animals were treated with 40 mg/kg ghost (n=8) or C6-ceramide (n=8) nanoliposomes via tail vein injection. Treatment regimen was every other day over a three week period of time. Tumor size was assessed every other day; * <i>P</i> < 0.05; ** <i>P</i> < 0.005. B). Leukemic mice following an identical dose regimen were sacrificed on day 8 (n=5), day 14 (n=6) and day 17 (n=6) of C6-ceramide treatment and on day 17 for ghost treatment. Immunoblot analysis for GAPDH protein expression was performed on tumor tissues. A representative blot from day 17 is shown (Figure 6B inset); * <i>P</i> < 0.05. </p

Pharmacological and molecular confirmation that nanoliposomal C6-ceramide targets the glycolytic pathway at the level of GAPDH.

<p>A). JVM3 cells were pre-treated for 2 hours with 10mM pyruvate, then treated with ghost or C6-ceramide nanoliposomes for 24 hours. MTT cell viability assay was performed. B). ATP production in these cells was also determined; ** <i>P</i> < 0.005. C). JVM3 cells were transduced with lentiviral particles overexpressing GAPDH or viral particles expressing RFP (control). Post transduction, Western blotting analysis was done to determine levels of GAPDH in experimental and control cells (insert). Cells were treated with ghost or C6-ceramide nanoliposomes for 24 hours. Cell viability was determined using MTT assay and alamarBlue assay; * <i>P</i> < 0.005.</p

C6-ceramide nanoliposomes target GAPDH in CLL.

<p>A). JVM3 cells were treated with (i) 25µM of ghost or C6-ceramide nanoliposomes for varying times, (ii) varying doses of ghost or C6-ceramide nanoliposomes for 24 hours, then Western Blot analysis was performed for GAPDH. Densitometry analysis from replicate experiments (n=4) was performed via ImageJ software. In Figure A(ii) the representative blot demonstrates a decrease in GAPDH at 12.5µM C6-ceramide, a result not observed in the other two distinct replicate experiments. * <i>P</i> < 0.05; ** <i>P <0 .005</i>. B). CLL patient cells (n=3, analyzed individually) were treated with different doses of ghost or C6-ceramide nanoliposomes for 24 hours, then Western Blot analysis was performed for GAPDH. * <i>P</i> < 0.05. The blot represents the effect of C6-ceramide nanoliposomes on one patient sample, however, the graph is an average of the effect on all three patient cells. C). JVM3 cells were treated with different doses of ghost and C6-ceramide nanoliposomes for 24 hours, then qRT-PCR analysis was performed for expression of GAPDH mRNA. Expression was normalized to 18S. * <i>P</i> < 0.05. D). PBMC from normal donors (n=3) were treated with 25 µM ghost or C6-ceramide nanoliposome for 24 hours and then Western Blot analysis was performed for GAPDH. E). Basal protein expression of GAPDH was determined via Western Blot analysis on PBMC isolated from normal donors (n=8) or from CLL patients with either a lower WBC count (n=9) or higher WBC count (n=5). Patients with WBC counts >50,000 cells/µL were identified as patients with high WBC count. * <i>P</i> < 0.05. F). CLL patient cells with either a lower WBC count (n=4) or higher WBC count (n=3) were treated with 25µM ghost or C6-ceramide nanoliposomes for 2, 16 and 24 hours then an MTT assay was performed * <i>P</i> < 0.05. </p