Reversal of cancer gene expression identifies repurposed drugs for diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is an aggressive incurable brainstem tumor that targets young children. Complete resection is not possible, and chemotherapy and radiotherapy are currently only palliative. This study aimed to identify potential therapeutic agents using a computational pipeline to perform an in silico screen for novel drugs. We then tested the identified drugs against a panel of patient-derived DIPG cell lines. Using a systematic computational approach with publicly available databases of gene signature in DIPG patients and cancer cell lines treated with a library of clinically available drugs, we identified drug hits with the ability to reverse a DIPG gene signature to one that matches normal tissue background. The biological and molecular effects of drug treatment was analyzed by cell viability assay and RNA sequence. In vivo DIPG mouse model survival studies were also conducted. As a result, two of three identified drugs showed potency against the DIPG cell lines Triptolide and mycophenolate mofetil (MMF) demonstrated significant inhibition of cell viability in DIPG cell lines. Guanosine rescued reduced cell viability induced by MMF. In vivo, MMF treatment significantly inhibited tumor growth in subcutaneous xenograft mice models. In conclusion, we identified clinically available drugs with the ability to reverse DIPG gene signatures and anti-DIPG activity in vitro and in vivo. This novel approach can repurpose drugs and significantly decrease the cost and time normally required in drug discovery

Polo-Like Kinase 4 (PLK4) Is Overexpressed in Central Nervous System Neuroblastoma (CNS-NB)

Neuroblastoma (NB) is the most common extracranial solid tumor in pediatrics, with rare occurrences of primary and metastatic tumors in the central nervous system (CNS). We previously reported the overexpression of the polo-like kinase 4 (PLK4) in embryonal brain tumors. PLK4 has also been found to be overexpressed in a variety of peripheral adult tumors and recently in peripheral NB. Here, we investigated PLK4 expression in NBs of the CNS (CNS-NB) and validated our findings by performing a multi-platform transcriptomic meta-analysis using publicly available data. We evaluated the PLK4 expression by quantitative real-time PCR (qRT-PCR) on the CNS-NB samples and compared the relative expression levels among other embryonal and non-embryonal brain tumors. The relative PLK4 expression levels of the NB samples were found to be significantly higher than the non-embryonal brain tumors (p-value < 0.0001 in both our samples and in public databases). Here, we expand upon our previous work that detected PLK4 overexpression in pediatric embryonal tumors to include CNS-NB. As we previously reported, inhibiting PLK4 in embryonal tumors led to decreased tumor cell proliferation, survival, invasion and migration in vitro and tumor growth in vivo, and therefore PLK4 may be a potential new therapeutic approach to CNS-NB

Evaluation of Protein Kinase Inhibitors with PLK4 Cross-Over Potential in a Pre-Clinical Model of Cancer

Polo-like kinase 4 (PLK4) is a cell cycle-regulated protein kinase (PK) recruited at the centrosome in dividing cells. Its overexpression triggers centrosome amplification, which is associated with genetic instability and carcinogenesis. In previous work, we established that PLK4 is overexpressed in pediatric embryonal brain tumors (EBT). We also demonstrated that PLK4 inhibition exerted a cytostatic effect in EBT cells. Here, we examined an array of PK inhibitors (CFI-400945, CFI-400437, centrinone, centrinone-B, R-1530, axitinib, KW-2449, and alisertib) for their potential crossover to PLK4 by comparative structural docking and activity inhibition in multiple established embryonal tumor cell lines (MON, BT-12, BT-16, DAOY, D283). Our analyses demonstrated that: (1) CFI-400437 had the greatest impact overall, but similar to CFI-400945, it is not optimal for brain exposure. Also, their phenotypic anti-cancer impact may, in part, be a consequence of the inhibition of Aurora kinases (AURKs). (2) Centrinone and centrinone B are the most selective PLK4 inhibitors but they are the least likely to penetrate the brain. (3) KW-2449, R-1530 and axitinib are the ones predicted to have moderate-to-good brain penetration. In conclusion, a new selective PLK4 inhibitor with favorable physiochemical properties for optimal brain exposure can be beneficial for the treatment of EBT

Plasmodium falciparum gametocyte development 1 (Pfgdv1) and gametocytogenesis early gene identification and commitment to sexual development.

Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual growth. Comparison of Pfge gene transcriptional profiles in patient samples divided the genes into two groups differing in their expression in mature circulating gametocytes and providing candidates to evaluate gametocyte induction and maturation separately in vivo. The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria

Subcellular localization of PfGDV1.

<p>Parasites transformed with GFP- or HA-tagged PfGDV1 were stained with DAPI (DNA stain) and the indicated anti-sera, and then examined by fluorescence microscopy (Zeiss Axiovert 200, 1000× magnification). Images are shown of the DAPI stain (DNA), GFP-tagged PfGDV1 epifluorescence (GDV1), and antibodies specific for HA (αHA), Pfs16 (αPfs16), PfGE3 (αPfGE3), PfMCM2 (αMCM2), and PfSir2 (αSir2). The corresponding merged and bright field (BF) images are included on the right. PfGDV1 expression is indicated with an arrow; locations of parasites in the BF image are indicated with a P for parasite or S for schizont. A) A schizont (S) (<i>Upper</i>) expressing GDV1 and a doubly infected erythrocyte (<i>Lower</i>) with one parasite (P1) expressing HA-tagged PfGDV1 (αHA) and another negative (P2) for anti-HA antibodies. B) Co-staining of parasites expressing GDV1 with early gametocytogenesis markers. A doubly infected erythrocyte (<i>Upper</i>) with one parasite (P1) positive for GDV1 and αPfs16 and the other (P2) negative for both. An erythrocyte (<i>Lower</i>) infected with a parasite (P) positive for GDV1 and αPfGE3. C) Co-localization of PfGDV1 with nuclear proteins. A doubly infected erythrocyte (<i>Upper</i>) with one parasite in the plane of the image (P1) and the other below (P2). Both P1 and P2 are positive for GDV1 and αMCM2. A schizont (S) (<i>Lower</i>) expressing GDV1 stained with αSir2.</p

Identification of <i>Plasmodium falciparum</i> gametocyte development 1 gene (<i>Pfgdv1</i>).

<p>A) Schematic of chromosome 9 (1–1,541,723 bp) showing the segment deleted (arrows) in the 3D7.G_def clone. For orientation, the putative centromere is indicated by an O and the 1,500,000 bp position is marked (1500 k). Colored boxes indicate the location of annotated genes: blue, genes transcribed toward the telomere; red, transcribed toward the centromere; horizontal stripes, <i>Pfgdv1;</i> diagonal stripes, <i>var</i> and <i>rifins</i>. The 70-mer oligonucleotide that identified the chromosome 9 deletion in 3D7.G_def parasites is indicated by an asterisk (*). The numbered gray bars below the line indicate the positions of the eight PCR products used to map the chromosome 9 deletion. B) Amplification products from chromosome 9 using Indochina, 3D7.G_def, FCR3.G_def, or HB3.G_def gDNA as a template. Peak gametocytemias attained in two independent experiments are indicated on the right of the ethidium bromide-stained agarose gel of the PCR products generated using the eight primer pairs (1–8). C) Southern blot of <i>Bsa</i>BI-digested gDNA from 3D7.G+ (+), 3D7.G_def (d), 3D7.G_def +<i>Pfgdv1</i> (a), 3D7.G_def +HA.<i>Pfgdv1</i> (b) and the parental 3D7 parasites (WT). Digested gDNA was probed with <i>Pfgdv1</i> (bp 1423–1800) or <i>Pfg27</i> (bp 1–654). D–E) Gametocyte production in WT (black square), 3D7.G_def (black triangle), complemented line a, 3D7.G_def +<i>Pfgdv1</i> (Panel D, black inverted triangle) and line b, 3D7.G_def +HA.<i>Pfgdv1</i> (Panel E, unfilled inverted triangle). Cultures set up at 0.1% asexual parasitemia on day 0 were followed for gametocyte production by Giemsa-stained smears for the next 16 days. Mean gametocytemia and standard deviation of two (D) or three (E) independent experiments are shown (<i>P</i><0.003 by linear regression analysis). F) Giemsa-stained smear of parasitized erythrocytes purified on a 16% Nycodenz cushion from day 16 gametocyte cultures of WT, 3D7.G_def +HA.<i>Pfgdv1</i> (HA.<i>Pfgdv1</i>), and 3D7.G_def (G_def) lines. G) Northern blots of RNA harvested from WT 3D7 (w), 3D7.G_def (d), 3D7.G_def<i>+Pfgdv1</i> (a), and 3D7.G_def +HA.<i>Pfgdv1</i> (b) complemented lines were hybridized with probes corresponding to <i>Pfgdv1</i> (<i>gdv1</i>), <i>Pfge</i> genes (<i>ge1–3, 5–11</i>), and merozoite surface protein-1 (<i>msp1</i>) as an asexual parasite control. Autoradiographs are shown with the corresponding ethidium bromide-stained gel. H) Expression of gametocyte specific antigen Pfs48/45. Methanol-fixed WT, 3D7.G_def+<i>Pfgdv1</i> (<i>a</i>) and 3D7.G_def+HA.<i>Pfgdv1</i> (b) mature gametocyte cultures were incubated with Pfs48/45 mAb IIC5B10 (1∶250 dilution) and labeled secondary antibodies (1∶500 dilution). I) The average gametocytemia of 4 independent cultures of WT 3D7 (WT), WT 3D7 transformed with a <i>Pfgdv1</i> episomal expression plasmid (WT+HA.<i>Pfgdv1</i>) and the G_def (G_def) line is graphed. The error bars represent the SEM and a significant difference from WT and G_def is indicated by an asterisk (p<0.05 ANOVA followed by Tukey multiple comparison test).</p

Model for continuous gametocytogenesis.

<p>During each round of the asexual cycle, a proportion of the schizonts (S_c, light blue) produced are committed to producing merozoites (M_c, light blue) that will differentiate into gametocytes (G_c, G_I–V) after invading a RBC. The expression profile for <i>Pfgdv1</i> is indicated by a line under the corresponding stage.</p