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PET, image-guided HDAC inhibition of pediatric diffuse midline glioma improves survival in murine models

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Science Advances  24 Jul 2020:
Vol. 6, no. 30, eabb4105
DOI: 10.1126/sciadv.abb4105
  • Fig. 1 PETobinostat is bioactive against DIPG in vitro.

    (A) Cell survival studies where PETobinostat was incubated with cells for either 2, 3, or 4 days reveal how PETobinostat has high cytotoxicity against an array of DIPG (DIPG XVII, DIPG XXV, SF8628, SF9427, and mBSG) and adult glioma (U251 and 4701) cells lines; reactivity against brainstem astrocytes (control) is diminished. (B) Western blotting demonstrating dose-dependent recovery of H3Ac following PETobinostat treatment in DIPG IV and DIPG XVII. (C) Dynamic studies in DIPG IV and SF8628 demonstrate how a minimum of 6-hour exposure to PETobinostat is needed to affect cytotoxicity. Cytotoxicity increases as exposure to the drug increases. (D) Western blotting illustrates how a minimum of 6-hour exposure is necessary to reestablish H3Ac in DIPG IV and DIPG XVII cell lines.

  • Fig. 2 PETobinostat is effective against DIPG in vivo.

    (A) Flank model of DIPG was generated by implanting luciferase-tagged SF8628 cells; luciferin signal was used to confirm tumor presence. (B) Caliper-tumor volume was monitored during and after treatment; panobinostat (blue) and PETobinostat (red) slowed down tumor growth when compared to vehicle (green). n.s., not significant. (C) Western blotting performed on the last day of treatment (red arrow) revealed recovery of H3Ac in panobinostat- or PETobinostat-treated animals, but not in vehicle-treated ones. Photo credit: Umberto Tosi, Weill Cornell Medicine.

  • Fig. 3 PETobinostat delivery route comparison.

    (A to E) PET images taken either at the beginning of the scan (t = 0) or 2 hours thereafter of mice injected with PETobinostat either via intraperitoneally (IP) (A), intravenously (IV) (B), intravenously following mannitol administration (C), or [18F]fluoride ion given intraperitoneally (D), or CED of PETobinostat (E). Only CED shows significant PETobinostat cranial accumulation. (F) Quantification of the PET signal showing significant PETobinostat cranium accumulation following CED but not for other methods of administration. Less PETobinostat was observed in the gut. No difference was seen in the spine. (G) Postmortem scintillation biodistribution showing PETobinostat accumulation only in the brain with CED delivery. PETobinostat accumulation in other organs (stomach, intestine, spleen, and lung) varies with route of administration as expected. CPM, counts per minute.

  • Fig. 4 PETobinostat post-CED ehavior.

    (A to D) PETobinostat was administered via CED to naïve mice at a rate of 0.167 μl/min (1 hour) (A and B) or 1.67 μl/min (6 min) (C and D); mice were then monitored via PET for up to 6 hours (images obtained at each hour-mark for the first 3 hours are shown). The CED site is shown by the white arrow (A and C). Relative PETobinostat concentration as it clears from the cranium following CED are shown (B and D); each line represents each mouse in the cohort. (E) PET-extrapolated data (points) were fit to a single exponential decay equation for each delivery rate (P < 0.0001). (F) After CED, tissue was harvested and processed by LC-MS (black). Data show compound integrity and PETobinostat clearance consistent with PET data (red). (G) Dynamic PET scanning showing the clearance of PETobinostat from the skull following co-infusion with Gd-DTPA. (H) Repeated MRI showing Gd-DTPA clearance from the injection site. (I) Data were fit to a linear equation, which show significantly more rapid clearance of PETobinostat (P < 0.0001).

  • Fig. 5 PETobinostat in tumor in vivo kinetics.

    (A) Tumor-bearing mice were scanned with T1-weighted MRI, which confirmed tumor presence (arrow). (B) Following CED of PETobinostat (100 μCi, 10 μl, 100 μM at a rate of 1.67 μl/min), mice were imaged with dynamic PET/CT, later overlaid with MRI, which allowed the determination of PETobinostat concentration at time 0 and thereafter. (C) Significant mouse-to-mouse variability is observed when data points are plotted against time (one color per mouse). (D) PET-extrapolated data were plotted on a one-phase decay equation, which yielded a half-life of 70.3 min. The dotted red line approximates a 1 μM concentration, assumed to be therapeutic.

  • Fig. 6 PETobinostat is efficacious in a DIPG mouse models.

    (A and B) Implantation of luciferase-tagged SF8628 cells resulted in the creation of intracranial tumors with pontine features like human DIPG (A) and high mitotic activity (B). (C and D) H&E staining was used to assess tumor cellularity by counting nuclei in high-magnification fields (C); tumors treated via CED had lower cellularity (D). HPF, high-power field. (E and F) Ki67 staining was used to assess tumor mitotic activity (E); tumors treated via CED had fewer dividing cells (F). (G and H) Cc3 staining was used to assess apoptotic cells; black arrows show Cc3+ cells (G); CED-treated tumors had an increase in apoptotic cells, consistently with panobinostat’s and PETobinostat’s mechanism of action (H). (I and J) Staining for H3Ac (I) revealed a recovery of acetylation following treatment (J). (K) Log-rank survival analysis of tumor-bearing RCAS mice. Systemic treatments (either vehicle or panobinostat intraperitoneally), 4× CED of vehicle, or a single CED of panobinostat results in a much shorter survival than 4× CED treatments with either panobinostat or PETobinostat.

Supplementary Materials

  • Supplementary Materials

    PET, image-guided HDAC inhibition of pediatric diffuse midline glioma improves survival in murine models

    Umberto Tosi, Harikrishna Kommidi, Oluwaseyi Adeuyan, Hua Guo, Uday Bhanu Maachani, Nandi Chen, Taojunfeng Su, Guoan Zhang, David J. Pisapia, Nadia Dahmane, Richard Ting, Mark M. Souweidane

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