Review of Cardiotoxicity in Pediatric Cancer Patients: During and after Therapy

With the improvement in survival from childhood cancer, late effects of therapy are becoming more apparent. Cardiac disease, one of these late effects, has a significant impact on the life of survivors of childhood cancers. Most survivors are followed by primary care doctors and adult subspecialists after they have graduated from pediatric centers. Since much of the cardiac toxicity of therapy occurs years off of therapy, it is important for these physicians to be aware of how to monitor survivors for the development of cardiac toxicities. In this paper we will discuss the incidence of cardiac disease during treatment and in survivors, what treatment modalities contribute to its development and modalities utilized to screen for cardiac disease. Recommendations for posttherapy monitoring will be emphasized

ETV6 germline mutations cause HDAC3/NCOR2 mislocalization and upregulation of interferon response genes

ETV6 is an ETS family transcription factor that plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that led to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation was also reflected in patient-derived platelet transcripts and drove aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction

Uncovering Cardioprotective Strategies For Anthracycline Therapy By Analysis Of Redox and Apoptotic Effects

Abstract Anthracyclines are among the most powerful drugs used for the treatment of leukemia, however their use has been associated with cardiotoxicity. Reactive oxygen species (ROS) are generated in both cancer and normal cells after anthracycline exposure and have been implicated in both early and late onset cardiotoxicity. Counteracting this ROS generation are intracellular antioxidants such as the ubiquitous antioxidant glutathione (GSH), levels of which are depleted upon anthracycline exposure. Basal expression of GSH pathway components and other antioxidants vary greatly between different cell types. Due to this differential expression of cellular antioxidants in cardiomyocytes versus leukemia cells, we posit that anthracyclines exert distinct effects on oxidative stress and consequent apoptosis induction in leukemia cells and nontransformed hematopoietic cells (PBMC) relative to cardiomyocytes. As a result, we expect potentially varied mechanisms of cell death induction in these cell lines after anthracycline treatment. To test this hypothesis, the acute leukemia cell lines Jurkat and ML-1 and the cardiomyocyte line H9C2 were used. Dose responses with the anthracyclines, doxorubicin and daunorubicin, were carried out and trypan blue exclusion and propidium iodide staining followed by flow cytometry were used to assess viability and DNA fragmentation respectively. Cardiomyocytes had a 25-150 fold higher IC50 value than the acute leukemia cell lines, indicating selectivity. To assess whether apoptosis was induced by anthracyclines, caspase 3 activity was measured and found to be increased at 24 hours in Jurkat cells which preceded decreases in viability, supporting an apoptotic mechanism of cell death. GSH levels also decreased markedly after 24 hours of treatment with anthracyclines in this cell line, however, a pan-caspase inhibitor did not block GSH depletion, indicating that these events occur independent of each other. To evaluate whether antioxidants conferred protection against loss of viability in all cell types, cells were pretreated for at least 30 minutes with antioxidants and then treated with doxorubicin and daunorubicin for 24 hours. Antioxidants used were N-acetylcysteine (NAC, a GSH precursor and amino acid source), GSH ethyl ester (cell permeable form of GSH), tiron (free radical scavenger) and trolox (a water soluble form of vitamin E). GSH ethylester did not prevent cytotoxicity of anthracyclines in acute leukemia lines or cardiomyocytes. Therefore boosting GSH levels in leukemia cells does not reverse cytotoxicity. Trolox, however, did block anthracycline induced cell death in ML-1 cells, suggesting that vitamin E supplementation would counteract leukemia cell specific effects of anthracyclines on AML cells. Tiron protected PBMC from doxorubicin cytotoxicity but did not protect leukemia cells or cardiomyocytes, hinting at a protective strategy for normal non-leukemia blood cells. Interestingly, NAC did not interfere with the cytotoxic effects of anthracyclines on acute leukemia cells or PBMC, but protected H9C2 cells from daunorubicin cytotoxicity. Taken together, these data reveal differential protective effects of antioxidants in cardiomyocytes and PBMCs relative to ALL and AML cells. Our work indicates that NAC can protect cardiomyocytes without interfering with anthracycline cytotoxicity in acute leukemia cells. In humans, one randomized control trial tested the addition of NAC to doxorubicin therapy, detecting no evidence of cardioprotective activity by chronic administration of NAC. However, the schedule used for administration of NAC in that study may not have been optimal, and biomarkers for oxidative stress reduction by NAC were not incorporated into the trial. Previously, other antioxidants have been used with very limited clinical success and possible contributing factors include inadequate sample size, choice of agent, dose used, duration of intervention and the lack of biomarker endpoints. Designing a cardioprotective and antioxidant strategy with attention to these factors may prove to be efficacious in protecting cardiac cells without interfering with the antitumoral effect of anthracyclines. To this end, our data suggests that trolox and vitamin E analogues should not be used in acute leukemia as they may interfere with the cytotoxic action of anthracyclines but NAC or cysteine may be used as cardioprotectants. Disclosures: No relevant conflicts of interest to declare

Comparative oncology approach to drug repurposing in osteosarcoma.

BACKGROUND:Osteosarcoma is an orphan disease for which little improvement in survival has been made since the late 1980s. New drug discovery for orphan diseases is limited by the cost and time it takes to develop new drugs. Repurposing already approved FDA-drugs can help overcome this limitation. Another limitation of cancer drug discovery is the lack of preclinical models that accurately recapitulate what occurs in humans. For OS using dogs as a model can minimize this limitation as OS in canines develops spontaneously, is locally invasive and metastasizes to the lungs as it does in humans. METHODS:In our present work we used high-throughput screens to identify drugs from a library of 2,286 FDA-approved drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. The identified lead compound was then tested for synergy with 7 other drugs that have demonstrated activity against OS. These results were confirmed with in vitro assays and an in vivo murine model of OS. RESULTS:We identified 13 drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. Auranofin was selected for further in vitro combination drug screens. Auranofin showed synergistic effects with vorinostat and rapamycin on OS viability and apoptosis induction. Auranofin demonstrated single-agent growth inhibition in both human and canine OS xenografts, and cooperative growth inhibition was observed in combination with rapamycin or vorinostat. There was a significant decrease in Ki67-positive cells and an increase in cleaved caspase-3 levels in tumor tissues treated with a combination of auranofin and vorinostat or rapamycin. CONCLUSIONS:Auranofin, alone or in combination with rapamycin or vorinostat, may be useful new treatment strategies for OS. Future studies may evaluate the efficacy of auranofin in dogs with OS as a prelude to human clinical evaluation

Cost-Effectiveness of the International Late Effects of Childhood Cancer Guideline Harmonization Group Screening Guidelines to Prevent Heart Failure in Survivors of Childhood Cancer

PURPOSE: Survivors of childhood cancer treated with anthracyclines and/or chest-directed radiation are at increased risk for heart failure (HF). The International Late Effects of Childhood Cancer Guideline Harmonization Group (IGHG) recommends risk-based screening echocardiograms, but evidence supporting its frequency and cost-effectiveness is limited. PATIENTS AND METHODS: Using the Childhood Cancer Survivor Study and St Jude Lifetime Cohort, we developed a microsimulation model of the clinical course of HF. We estimated long-term health outcomes and economic impact of screening according to IGHG-defined risk groups (low [doxorubicin-equivalent anthracycline dose of 1-99 mg/m and/or radiotherapy \u3c 15 Gy], moderate [100 to \u3c 250 mg/m or 15 to \u3c 35 Gy], or high [≥ 250 mg/m or ≥ 35 Gy or both ≥ 100 mg/m and ≥ 15 Gy]). We compared 1-, 2-, 5-, and 10-year interval-based screening with no screening. Screening performance and treatment effectiveness were estimated based on published studies. Costs and quality-of-life weights were based on national averages and published reports. Outcomes included lifetime HF risk, quality-adjusted life-years (QALYs), lifetime costs, and incremental cost-effectiveness ratios (ICERs). Strategies with ICERs \u3c $100,000 per QALY gained were considered cost-effective. RESULTS: Among the IGHG risk groups, cumulative lifetime risks of HF without screening were 36.7$175,000 per QALY gained for all strategies for low-risk survivors, representing approximately 40% of those for whom screening is currently recommended. CONCLUSION: Our findings suggest that refinement of recommended screening strategies for IGHG high- and low-risk survivors is needed, including careful reconsideration of discontinuing asymptomatic left ventricular dysfunction and HF screening in low-risk survivors

FDA-approved compounds that show inhibition of osteosarcoma cell proliferation.

<p>FDA-approved compounds that show inhibition of osteosarcoma cell proliferation.</p

Auranofin shows synergistic effects with vorinostat and rapamycin on the viability of MG-63 cells.

<p>MG-63 cells were treated with various concentrations of auranofin along with vorinostat (<b>A</b>) or rapamycin (<b>B</b>) for 48 hours, and the cytotoxicity was determined. Representative Bliss independence plots are shown on the left. Summaries of all combination indices (numbers) calculated by Chou-Talalay plots using data obtained from varying combination pairs of auranofin and vorinostat or rapamycin (right).</p

Auranofin, in combination with vorinostat or rapamycin, significantly reduces tumor growth of KHOS/NP and Abrams cells in mice.

<p><b>A-D.</b> Tumor formation assays in nude mice subcutaneously injected with KHOS/NP (1X10⁶ (<b>A</b>, <b>B</b>) or Abrams (1.5X10⁶ (<b>C</b>, <b>D</b>)) OS cells. When tumors reached 3 mm in diameter, mice were intraperitoneally injected with DMSO or auranofin (AF), along with vorinostat (VST (<b>A</b>, <b>C</b>), or rapamycin (RPM (<b>B</b>, <b>D</b>). Tumor sizes were three-dimensionally measured twice a week. Graphs showing sizes of tumors formed in mice (top). Note that the results of DMSO and auranofin alone in (<b>C)</b> were also used in (<b>D)</b>, since experiments in (<b>C)</b> and (<b>D)</b> were performed at the same time. Error bars: means ± S.D. (n = 5 animals for each group in (<b>A</b> and <b>B)</b>; n = 4 animals each group in (<b>C</b> and <b>D</b>)). **, P < 0.01; Student’s <i>t</i> test. NS: Not significant. Representative images of tumors formed in mice at day 21 (bottom). (<b>E)</b> Representative images of immunohistochemistry for Ki67 and cleaved caspase-3 using KHOS/NP tumors treated with DMSO or indicated drugs (magnification, 40X). Scale bars, 200 μm.</p

Primary screening of FDA-approved library identifies auranofin as a potential drug for OS therapy.

<p><b>A.</b> A diagram of the primary screening to select auranofin. <b>B.</b> Auranofin chemical structure (top) and concentration-response curves of auranofin cytotoxic effects on human (MG-63 and KHOS/NP) and canine (Abrams and D17) OS cells (bottom). Graph also includes IC₅₀ values for each cell line. Error bars: means ± S.D. from 3-independent experiments.</p