Mulcahy Levy, J. | Search bioRxiv

bioRxiv2024

The assessment of therapeutic autophagy inhibition in NF1 mutated tumor cells

MAPK pathway activation is found across central nervous system tumors. Neurofibromin 1 (NF1) protein is a GTPase-activating protein and tumor suppressor that negatively regulates RAS protein activity. A NF1 loss of function mutation results in MAPK pathway upregulation and is associated with pediatric gliomas. We previously demonstrated autophagy inhibition improves tumor response to BRAF inhibition. Here we investigate the role of autophagy in NF1 cells in the presence or absence of MEK inhibition (MEKi). NF1 wild type (WT) and NF1 knockout (KO) (HSC1{lambda}, immortalized human Schwann) cells were evaluated. NF1 loss was confirmed, and cell growth was monitored by Incucyte. MAPK/ERK pathway upregulation and autophagy were evaluated via Western blot analysis and RAS pull-down. Effects of MEKi, autophagy inhibition (pharmacologic and genetic), and combination therapy were evaluated via short- and long-term growth assays. NF1 KO cells exhibited MAPK pathway upregulation and increased growth. Additionally, NF KO cells demonstrated increased autophagic activity. Loss of NF1 resulted in increased sensitivity to MEKi and autophagy inhibition alone and in combination. We also demonstrate that the autophagy inhibitor chloroquine (CQ) can improve MEKi sensitivity in a patient harboring a somatic mutation of NF1. This combination is currently being evaluated in clinical trial (NCT04201457).

cancer biology↗

bioRxiv2019

Comprehensive molecular characterization of pediatric treatment-induced high-grade glioma: A distinct entity despite disparate etiologies with defining molecular characteristics and potential therapeutic targets

Treatment-induced high-grade gliomas (TIHGGs) are an incurable late complication of cranial radiation therapy or combined radiation/chemotherapy used to treat pediatric cancer. We assembled a cohort of 33 TIHGGs from multiple institutions. The primary antecedent malignancies were medulloblastoma, acute lymphoblastic leukemia, astrocytoma, and ependymoma. We performed methylation profiling, RNA-seq, and genomic sequencing (whole-genome or whole-exome) on TIHGG samples. Methylation profiling revealed that TIHGGs cluster primarily with the pediatric receptor tyrosine kinase I subtype (26/31 samples). Common TIHGG copy-number alterations include Chromosome (Ch.) 1p loss/1q gain, Ch. 4 loss, Ch. 6q loss, and Ch. 13 and Ch. 14 loss; focal alterations include PDGFRA and CDK4 gain and loss of CDKN2A and BCOR. Relative to de novo pediatric high-grade glioma (pHGG), BCOR loss (p=0.004) and CDKN2A loss (p=0.005) were significantly increased. Transcriptomic analysis identified two distinct TIHGG subgroups, one with a lesser mutation burden (0.12 mut/Mb), Ch. 1p loss/1q gain (5/6 samples), and stem cell characteristics, and one with a greater mutation burden (1.08 mut/Mb, p<0.0002), depletion of DNA repair pathways, and inflammatory characteristics. We observed increased chromothripsis in TIHGG versus pHGG (67% vs. 31%, p=0.036), which was associated with extrachromosomal circular DNA-mediated amplification of PDGFRA and CDK4. In vitro drug screening in one primary, patient-derived TIHGG cell line from each expression subgroup identified microtubule inhibitors/stabilizers, DNA-damaging agents, MEK inhibition, and, in the inflammatory subgroup, proteasome inhibitors as potentially effective therapies. This study provides a comprehensive molecular profile of TIHGG, including mechanistic insights to TIHGG oncogenesis, and identifies potentially effective therapeutic modalities for further investigation.

cancer biology↗