Abstract 3025: PRMT5 inhibition reduces viability and stemness of pediatric high grade glioma
Authors: Brown Elizabeth J. , Balaguer-Lluna Leire , Cribbs Adam , Philpott Martin , Carcaboso Ángel M. , Farnie Gillian , Bullock Alex N. 
Published: July 2021

No current therapies halt the progression of Diffuse Intrinsic Pontine Glioma (DIPG), a pediatric brainstem tumor, with a median survival of only 6-9 months. DIPG cells carry characteristic missense histone H3 K27M mutations and activating ACVR1 mutations in 80% and 25% of cases respectively, and have transcriptomes similar to that of oligodendrocyte precursor cells (OPCs). We aim to find novel therapeutic targets for DIPG cells carrying these ACVR1 and H3 mutations.

We performed parallel adherent and spheroid in vitro viability screens in patient derived DIPG cells using a small molecule epigenetic library generated by the SGC (https://www.thesgc.org/chemical-probes/epigenetics). 16/51 probes significantly reduced the viability of HSJD-DIPG-007 cells (ACVR1 R206H, H3.3 K27M) grown adherently for 7 days, of which 6 compounds inhibit the protein arginine methyltransferase (PRMT) family. The 2 most effective compounds inhibit PRMT5 (GSK591 and LLY-283) reducing viability &gt;50% in adherent and spheroid screens. Dose response curves in 5 DIPG and 1 glioblastoma (GBM) cell line confirmed a potent reduction of viability in ACVR1 mutant cells, with GI50s of 13, 57 and 79nM after GSK591 inhibition in HSJD-DIPG-007, SU-DIPG-XXI and SU-DIPG-IV cells respectively. In comparison ACVR1 WT cells were at least 10x less sensitive to GSK591 with GI50s of 2.9, &gt;10, &gt;10μM in HSJD-DIPG-011, HSJD-GBM-002 and SU-DIPG-VI cells.

To provide mechanistic insight as to how PRMT5 inhibition reduced viability of DIPG cells RNA-sequencing was performed at 5 time points, over a 7 day treatment with LLY-283 (GI40). Transcriptomic analysis found PRMT5 inhibition significantly enriched neural differentiation genes (including NLGN3, MDK, APOE and DKK1) suggesting a reduction in stemness. To explore this further we performed an in vitro limiting dilution assay after 7 days pre-treatment with the LLY-283 GI80, and confirmed that PRMT5 inhibition reduced single cell colony formation. This reduction of stemness could represent the conversion of DIPG cells from their typical OPC-like state into a more differentiated, less malignant phenotype.

Following in vitro efficacy of PRMT5 inhibition, orthotopic patient derived DIPG xenografts (PDX) were treated with LLY-283 over 28 days (50mg/kg, 3 days on 4 days off). Like many single agent therapies tested in this highly aggressive DIPG PDX model, PRMT5 did not improve survival. However a reduction in stemness, as seen in vitro¸ does not always reduce primary tumor burden. Therefore proteomic characterization of neural differentiation in the primary PDX samples is underway, and investigation into secondary tumor initiation would be warranted.

Our data shows PRMT5 inhibitors are a promising new target for DIPG, specifically in ACVR1 mutant DIPG, and justifies further in vivo investigations into changes in tumor initiation capacity and exploration of rational combinations to improve survival outcome.

Citation Format: Elizabeth J. Brown, Leire Balaguer-Lluna, Adam Cribbs, Martin Philpott, Ángel M. Carcaboso, Gillian Farnie, Alex N. Bullock. PRMT5 inhibition reduces viability and stemness of pediatric high grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3025.