Autophagy inhibition improves the effectiveness and overcomes RAF pathway inhibition (RAFi) resistance across multiple CNS tumors and molecularly distinct resistance mechanisms. Mechanistic links between autophagy and apoptotic cell death may explain this ability to improve RAFi response and reverse resistance. RAFi sensitive (MAF 794, AM38) and resistant (MAF 794R, MAF 905-3, AM38R, B76) BRAFV600E CNS tumor cell lines were analyzed at baseline, following RAFi (vemurafenib), autophagy inhibition (chloroquine or shRNAs), and combination therapy. Growth assays and caspase activation were monitored by Incucyte Zoom. qRT-PCR evaluated key pro-apoptotic BH3-only members of the BCL-2 family. Broad BH-3 profiling was completed using the Letai JC-1 Plate-Based protocol. Western blot analysis assessed protein levels. Combination pharmacologic treatment caused alterations in key pro-apoptotic BH3-only proteins including an increase in BNIP3L and PUMA. Genetically inhibiting autophagy with shRNAs for ATG5 and ATG7 (proteins required for formation of the autophagosome) produced similar results with increases in both protein and mRNA levels of BNIP3L and PUMA following RAFi treatment. This suggested autophagy-mediated regulation of BH3 proteins functions to determine cellular apoptotic threshold. Caspase activation demonstrated increased effectiveness of combined RAFi and autophagy inhibition overcoming the apoptotic threshold compared to single drug treatment. BH3 profiling demonstrated a dependence on BCL-2 to inhibit apoptosis. BH3 mimetics competitively bind to pro-survival BCL-2 family members, blocking their protective effects and pushing tumor cells towards apoptosis. Autophagy inhibition can also improve treatment response by overcoming the apoptotic threshold in RAFi resistant cells and magnifying the apoptotic response in sensitive cells. BH3 profiling reveals CNS BRAFV600E are BCL-2 dependent cells, unprimed for apoptosis, which may be good candidates for additional treatment with BH3 mimetics such as venetoclax. This presents an attractive treatment for MAPK activated CNS tumors by enhancing apoptotic cell death by targeting the MAPK pathway, autophagy and BH3.
Atypical teratoid/rhabdoid tumors (AT/RT) are CNS tumors with a 5-year survival of ~35%. AT/RT is characterized by loss-of-function mutations in the SMARCB1 component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) complex. Based on preliminary CRISPR-Cas9 gene essentiality screen results identifying AT/RT vulnerabilities, we hypothesized that interaction between CDK7 and the SWI/SNF complex via SMARCB1 provides a potential target to improve clinical survival of patients. CDK7 expression was identified by microarray in AT/RT, medulloblastoma, glioblastoma and normal brain. Established cell lines (BT12, BT16, CHLA06), patient derived lines (MAF-737, MAF-1298, MAF-1337), normal human astrocytes (NHA) and NIH3T3 mouse embryonic fibroblast cells were utilized for in vitro response to CDK7 inhibition. Murine cerebellar xenografts of MAF-737 were utilized to evaluate genetic and pharmacologic response to CDK7 inhibition. The NCI Approved Oncology Drugs (AOD-9) Panel was evaluated with an IC25 dose of CDK7 inhibitor THZ2 to identify potential synergistic combinations. CDK7 is up-regulated in AT/RT compared to other brain tumors or normal brain. In vitro, AT/RT cells are highly susceptible to CDK7 pharmacologic inhibition with nM IC50 levels. AT/RT cells with shRNA against CDK7 implanted in vivo show significantly reduced growth. Evaluation of in vivo tumors treated with THZ2 demonstrate decreased Ki-67 and reduced pRBP1 demonstrating effective inhibition of the target as well as a decrease in cell proliferation. Combination therapy of THZ2 with the AOD-9 Panel found significant synergy with antimetabolite therapies, specifically pemetrexed, pralatrexate, and methotrexate. There was no synergy with other standard chemotherapy. Our findings demonstrate that CDK7 is highly expressed in AT/RT and necessary for proliferation of AT/RT cells, suggesting it as a potential therapeutic target. Antimetabolites, which are currently used in several AT/RT protocols, synergized with CDK7 inhibition offers a potential future combination therapy for patients.
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