Search

Purpose: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. Experimental Design: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. Results: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. Conclusions: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

Effective treatments are urgently needed for the incurable paediatric brainstem tumour Diffuse Midline Glioma (DMG). Most DMGs contain Histone H3 mutations (H3K27M), which produce extensive epigenetic dysregulation by inhibiting EZH2-mediated trimethylation of H3K27 (H3K27me3). Global depletion of the repressive H3K27me3 modification results in aberrantly open chromatin and is central to DMG tumorigenesis. Thus, targeting the epigenome is a promising avenue of treatment for DMG. We found that targeting the histone chaperone complex Facilitates Chromatin Transcription (FACT) with the curaxin drug CBL0137 to have potent pre-clinical efficacy against DMG, leading to a paediatric Phase I/II clinical trial for CBL0137 which includes a DMG/DIPG cohort (NCT04870944). In this project we aim to elucidate CBL0137’s molecular mechanism in DMG. FACT is critical for maintaining chromatin homeostasis during DNA replication, transcription, and repair. We therefore hypothesised that FACT maintains the aberrant epigenetic landscape resulting from H3K27M. Consistently, we found CBL0137 to be more cytotoxic against H3K27M-mutant, compared to H3-WT or isogenic DMG cells lacking the mutation. Furthermore, FACT directly interacts with H3K27M, and FACT inhibition increases both EZH2 catalytic activity and global H3K27me3 levels. We are now using ChIP-seq to discover the genome-wide distribution of FACT and H3K27M, and will interrogate the consequence of CBL0137 treatment on the epigenome and transcriptome. Preliminary results suggest that FACT is located at certain genes co-occupied by H3K27M, the active histone mark H3K27ac, and the BET protein BRD4. This implies that FACT is involved in maintaining open chromatin and perhaps transcription at these regions. Combining CBL0137 with other epigenetic drugs, such as BET inhibitors, could therefore represent a rational therapeutic opportunity for DMG. This work will ultimately inform mechanism-based targeted therapies to combine with CBL0137 to improve its efficacy and uncover valuable new insights into DMG epigenetics and pathobiology.

Introduction: Standard of care therapies for children with acute myeloid leukemia (AML) cause potent off-target toxicity to healthy cells, highlighting the need to develop new therapeutic approaches that are safe and specific for leukemia cells. Long non-coding RNAs (lncRNAs) are an emerging and highly attractive therapeutic target in the treatment of cancer due to their oncogenic functions and selective expression in cancer cells. However, lncRNAs have historically been considered ‘undruggable’ targets because they do not encode for a protein product. Therefore, the discovery of novel lncRNA targets and the development of efficient methods to silence them is critical for developing new therapies for AML. In this study, we describe the development of an siRNA-loaded lipid nanoparticle (LNP) for the therapeutic silencing of the novel oncogenic lncRNA LINC01257. Materials and Methods: Transcriptomic analysis of children with AML from both the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative and AAML1031 study was performed to identify AML specific lncRNAs. The subsequently identified lncRNA LINC01257 was knocked down in AML Kasumi-1 cells via electroporation of siRNA targeting LINC01257 (si-LINC01257). Changes in proliferation and cell viability were assessed via cell count and Annexin V/7AAD staining 48 hours post-transfection. LNPs incorporating non-targeting si-SCR or si-LINC01257 were synthesized through NxGen non-turbulent microfluidics. LNPs were formulated reproducing the FDA-approved Onpattro® siRNA delivery vector, D-Lin-MC3-DMA:DSPC:cholesterol:PEG-DMG. LNP-si-LINC01257 uptake was measured by confocal microscopy and flow cytometry. Kasumi-1 cells and healthy peripheral blood mononuclear cells (PBMCs) were treated with 250nM LNP-si-SCR or LNP-si-LINC01257 for 48 hours. Changes in proliferation and cell viability were assessed with cell counts and Annexin V/7AAD staining 48 hours post-treatment. Results: LINC01257 was significantly overexpressed in patients carrying the t(8;21) translocation compared to healthy controls ( P = 85% siRNA encapsulation rate. LNP-siRNAs were efficiently taken up by Kasumi-1 cells (>95% of cells) and LNP-si-LINC01257 treatment successfully ablated LINC01257 expression, which was accompanied by a 55% reduction in total cell count following 48 hours of treatment (P= 0.02). In contrast, healthy PBMCs, which do not express LINC01257, were unaffected by LNP-si-LINC01257 treatment despite comparable levels of LNP-siRNA uptake. Conclusion: Our data demonstrate the effective use of LNP-based RNA interference modalities for the silencing of cancer-driving lncRNAs and highlight their potential as a viable therapeutic and non-toxic approach in the management of AML. Citation Format: Patrick Connerty, Ernest Moles, Charles deBock, Nisitha Jayatilleke, Jenny L. Smith, Soheil Meshinchi, Chelsea Mayoh, Maria Kavallaris, Richard B. Lock. Development of siRNA-loaded lipid nanoparticles targeting long non-coding RNA LINC01257 as a novel and safe therapeutic approach for paediatric acute myeloid leukaemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P143.