Pediatric high-grade gliomas (pHGGs) are the leading cause of cancer-related deaths in children in the USA. Sixteen percent of hemispheric pediatric and young adult HGGs encode Gly34Arg/Val ...substitutions in the histone H3.3 (H3.3-G34R/V). The mechanisms by which H3.3-G34R/V drive malignancy and therapeutic resistance in pHGGs remain unknown. Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we demonstrate that this mutation led to the downregulation of DNA repair pathways. This resulted in enhanced susceptibility to DNA damage and inhibition of the DNA damage response (DDR). We demonstrate that genetic instability resulting from improper DNA repair in G34R-mutant pHGG led to the accumulation of extrachromosomal DNA, which activated the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway, inducing the release of immune-stimulatory cytokines. We treated H3.3-G34R pHGG-bearing mice with a combination of radiotherapy (RT) and DNA damage response inhibitors (DDRi) (i.e., the blood-brain barrier-permeable PARP inhibitor pamiparib and the cell-cycle checkpoint CHK1/2 inhibitor AZD7762), and these combinations resulted in long-term survival for approximately 50% of the mice. Moreover, the addition of a STING agonist (diABZl) enhanced the therapeutic efficacy of these treatments. Long-term survivors developed immunological memory, preventing pHGG growth upon rechallenge. These results demonstrate that DDRi and STING agonists in combination with RT induced immune-mediated therapeutic efficacy in G34-mutant pHGG.
In this work, we designed, characterized, and investigated the performance of hydrolyzed galactomannan (hGM)-based amphiphilic nanoparticles for selective intratumoral accumulation in pediatric ...patient-derived sarcomas. To create a self-assembly amphiphilic copolymer, the side chain of hGM was hydrophobized with poly(methyl methacrylate) (PMMA) by utilizing a graft free radical polymerization reaction. Different hGM and MMA weight feeding ratios were used to adjust the critical aggregation concentration and the size and size distribution of the nanoparticles. The ability to actively target glucose transporter-1 (GLUT-1) was studied by fluorescence confocal microscopy and imaging flow cytometry in vitro on Rh30 (rhabdomyosarcoma) and patient-derived Ewing sarcoma (HSJD-ES-001) cell lines with different expression levels of GLUT-1. Results confirmed that the nanoparticles are internalized by ∼100% of the cells at 37 °C. Furthermore, we investigated the biodistribution of the nanoparticles in pediatric patient-derived models of two deadly musculoskeletal tumors, rhabdomyosarcoma and Ewing sarcoma. Outstandingly, the intratumoral accumulation of the nanoparticles correlated very well with the expression level of GLUT1 gene in each patient-derived tumor (P = 0.0141; Pearson’s correlation test). Finally, we demonstrated the encapsulation capacity of these nanoparticles by loading 7.5% (w/w) of the hydrophobic first-generation tyrosine kinase inhibitor imatinib. These findings point out the potential of this new type of nanoparticle to target GLUT-1-expressing tumors and selectively deliver anticancer agents.
Diffuse intrinsic pontine glioma (DIPG), or high-grade brainstem glioma (BSG), is one of the major causes of brain tumor-related deaths in children. Its prognosis has remained poor despite numerous ...efforts to improve survival. Panobinostat, a histone deacetylase inhibitor, is a targeted agent that has recently shown pre-clinical efficacy and entered a phase I clinical trial for the treatment of children with recurrent or progressive DIPG.
A collaborative pre-clinical study was conducted using both a genetic BSG mouse model driven by PDGF-B signaling, p53 loss, and ectopic H3.3-K27M or H3.3-WT expression and an H3.3-K27M orthotopic DIPG xenograft model to confirm and extend previously published findings regarding the efficacy of panobinostat in vitro and in vivo.
In vitro, panobinostat potently inhibited cell proliferation, viability, and clonogenicity and induced apoptosis of human and murine DIPG cells. In vivo analyses of tissue after short-term systemic administration of panobinostat to genetically engineered tumor-bearing mice indicated that the drug reached brainstem tumor tissue to a greater extent than normal brain tissue, reduced proliferation of tumor cells and increased levels of H3 acetylation, demonstrating target inhibition. Extended consecutive daily treatment of both genetic and orthotopic xenograft models with 10 or 20 mg/kg panobinostat consistently led to significant toxicity. Reduced, well-tolerated doses of panobinostat, however, did not prolong overall survival compared to vehicle-treated mice.
Our collaborative pre-clinical study confirms that panobinostat is an effective targeted agent against DIPG human and murine tumor cells in vitro and in short-term in vivo efficacy studies in mice but does not significantly impact survival of mice bearing H3.3-K27M-mutant tumors. We suggest this may be due to toxicity associated with systemic administration of panobinostat that necessitated dose de-escalation.
Tuberculosis (TB) is the second most deadly infectious disease. Despite potentially curative pharmacotherapies being available for over 50
years, the length of the treatment and the pill burden can ...hamper patient lifestyle. Thus, low compliance and adherence to administration schedules remain the main reasons for therapeutic failure and contribute to the development of multi-drug-resistant (MDR) strains. Pediatric patients constitute a high risk population. Most of the first-line drugs are not commercially available in pediatric form. The design of novel antibiotics attempts to overcome drug resistance, to shorten the treatment course and to reduce drug interactions with antiretroviral therapies. On the other hand, the existing anti-TB drugs are still effective. Overcoming technological drawbacks of these therapeutic agents as well as improving the effectiveness of the drug by targeting the infection reservoirs remains the central aims of Pharmaceutical Technology. In this framework, nanotechnologies appear as one of the most promising approaches for the development of more effective and compliant medicines. The present review thoroughly overviews the state-of-the-art in the development of nano-based drug delivery systems for encapsulation and release of anti-TB drugs and discusses the challenges that are faced in the development of a more effective, compliant and also affordable TB pharmacotherapy.
Pediatric high-grade gliomas are among the deadliest of childhood cancers due to limited knowledge of early driving events in their gliomagenesis and the lack of effective therapies available. In ...this study, we investigate the oncogenic role of PPM1D, a protein phosphatase often found truncated in pediatric gliomas such as DIPG, and uncover a synthetic lethal interaction between PPM1D mutations and nicotinamide phosphoribosyltransferase (NAMPT) inhibition. Specifically, we show that mutant PPM1D drives hypermethylation of CpG islands throughout the genome and promotes epigenetic silencing of nicotinic acid phosphoribosyltransferase (NAPRT), a key gene involved in NAD biosynthesis. Notably, PPM1D mutant cells are shown to be sensitive to NAMPT inhibitors in vitro and in vivo, within both engineered isogenic astrocytes and primary patient-derived model systems, suggesting the possible application of NAMPT inhibitors for the treatment of pediatric gliomas. Overall, our results reveal a promising approach for the targeting of PPM1D mutant tumors, and define a critical link between oncogenic driver mutations and NAD metabolism, which can be exploited for tumor-specific cell killing.
Worldwide, over 40 million people are infected with the Human Immunodeficiency Virus (HIV). The High Activity Antiretroviral Therapy (HAART) combines at least three antiretroviral (ARV) drugs and, ...for over a decade, has been used to extend the lifespan of the HIV-infected patients. Chronic intake of HAART is mandatory to control HIV infection. The frequent administration of several drugs in relatively high doses is a main cause of patient incompliance and a hurdle toward the fulfillment of the pharmacotherapy. High adherence to HAART does not lead to complete HIV virus elimination from the host. Intracellular and anatomical viral reservoirs are responsible for the perpetuation of the infection. Active transport mechanisms involving proteins of the ATP-binding cassette superfamily prevent the penetration of ARV drugs into the brain and may account for the limited bioavailability after oral administration. A new research that addresses from simple organoleptic or technological problems to more complex issues involving the targeting of specific tissues and organs has emerged. With the aim to reduce dosing frequency, to improve the compliance of the existing pharmacotherapy and to target viral reservoirs, the design of drug delivery systems (DDS) is becoming complementary to new drug discovery. Based on the common molecular features that characterize the different families of ARV drugs, the present review describes state-of-the-art ARV DDS and thoroughly discusses the challenges in the development of medicines with enhanced biopharmaceutical properties. In addition, a number of specific issues such as pediatric HAART, preventive pharmacotherapy and specific HIV-associated ethical issues are addressed in an integrative manner. Finally, the impact of such novel drug development on the Pharmaceutical Technology field is discussed.
Drug Delivery Systems as the cornerstone of HIV pharmacotherapy optimization with regard to patient needs, drug formulation requirements and virus targeting toward a more effective pharmacotherapy.
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Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor that is located in the pons and primarily affects children. Nearly 80% of DIPGs harbor mutations in histone H3 genes, wherein ...lysine 27 is substituted with methionine (H3K27M). H3K27M has been shown to inhibit polycomb repressive complex 2 (PRC2), a multiprotein complex responsible for the methylation of H3 at lysine 27 (H3K27me), by binding to its catalytic subunit EZH2. Although DIPGs with the H3K27M mutation show global loss of H3K27me3, several genes retain H3K27me3. Here we describe a mouse model of DIPG in which H3K27M potentiates tumorigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M-expressing tumors require PRC2 for proliferation. Furthermore, we demonstrate that small-molecule EZH2 inhibitors abolish tumor cell growth through a mechanism that is dependent on the induction of the tumor-suppressor protein p16
. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the DIPG mouse model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M-expressing DIPGs, and that inhibition of EZH2 is a potential therapeutic strategy for the treatment of these tumors.
H3K27-altered Diffuse Midline Glioma (DMG) is a universally fatal paediatric brainstem tumour. The prevalent driver mutation H3K27M creates a unique epigenetic landscape that may also establish ...therapeutic vulnerabilities to epigenetic inhibitors. However, while HDAC, EZH2 and BET inhibitors have proven somewhat effective in pre-clinical models, none have translated into clinical benefit due to either poor blood-brain barrier penetration, lack of efficacy or toxicity. Thus, there remains an urgent need for new DMG treatments. Here, we performed wider screening of an epigenetic inhibitor library and identified inhibitors of protein arginine methyltransferases (PRMTs) among the top hits reducing DMG cell viability. Two of the most effective inhibitors, LLY-283 and GSK591, were targeted against PRMT5 using distinct binding mechanisms and reduced the viability of a subset of DMG cells expressing wild-type TP53 and mutant ACVR1. RNA-sequencing and phenotypic analyses revealed that LLY-283 could reduce the viability, clonogenicity and invasion of DMG cells in vitro, representing three clinically important phenotypes, but failed to prolong survival in an orthotopic xenograft model. Together, these data show the challenges of DMG treatment and highlight PRMT5 inhibitors for consideration in future studies of combination treatments.
Diffuse intrinsic pontine gliomas (DIPGs) are highly infiltrative malignant glial neoplasms of the ventral pons that, due to their location within the brain, are unsuitable for surgical resection and ...consequently have a universally dismal clinical outcome. The median survival time is 9-12 months, with neither chemotherapeutic nor targeted agents showing substantial survival benefit in clinical trials in children with these tumors. We report the identification of recurrent activating mutations in the ACVR1 gene, which encodes a type I activin receptor serine/threonine kinase, in 21% of DIPG samples. Strikingly, these somatic mutations (encoding p.Arg206His, p.Arg258Gly, p.Gly328Glu, p.Gly328Val, p.Gly328Trp and p.Gly356Asp substitutions) have not been reported previously in cancer but are identical to mutations found in the germ line of individuals with the congenital childhood developmental disorder fibrodysplasia ossificans progressiva (FOP) and have been shown to constitutively activate the BMP-TGF-β signaling pathway. These mutations represent new targets for therapeutic intervention in this otherwise incurable disease.
Glioblastoma is the most lethal brain tumor and harbors glioma stem cells (GSCs) with potent tumorigenic capacity. The function of GSCs in tumor propagation is maintained by several core ...transcriptional regulators including c-Myc. c-Myc protein is tightly regulated by posttranslational modification. However, the posttranslational regulatory mechanisms for c-Myc in GSCs have not been defined. In this study, we demonstrate that the deubiquitinase USP13 stabilizes c-Myc by antagonizing FBXL14-mediated ubiquitination to maintain GSC self-renewal and tumorigenic potential. USP13 was preferentially expressed in GSCs, and its depletion potently inhibited GSC proliferation and tumor growth by promoting c-Myc ubiquitination and degradation. In contrast, overexpression of the ubiquitin E3 ligase FBXL14 induced c-Myc degradation, promoted GSC differentiation, and inhibited tumor growth. Ectopic expression of the ubiquitin-insensitive mutant T58A-c-Myc rescued the effects caused by FBXL14 overexpression or USP13 disruption. These data suggest that USP13 and FBXL14 play opposing roles in the regulation of GSCs through reversible ubiquitination of c-Myc.
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