Purpose Glioblastoma (GBM) is a deadly brain tumor marked by dysregulated signaling and aberrant cell cycle control. Molecular analyses have identified that the CDK4/6-Rb-E2F axis is dysregulated in about 80% of GBMs. Single-agent CDK4/6 inhibitors have failed to provide durable responses in GBM, suggesting a need to combine them with other agents. We investigate the efficacy of the combination of CDK4/6 inhibition and mTOR inhibition against GBM. Experimental Design Preclinical in vitro and in vivo assays using primary GBM cell lines were performed. Results We show that the CDK4/6 inhibitor palbociclib suppresses the activity of downstream mediators of the mTOR pathway, leading to rebound mTOR activation that can be blocked by the mTOR inhibitor everolimus. We further show that mTOR inhibition with everolimus leads to activation of the Ras mediator Erk that is reversible with palbociclib. The combined treatment strongly disrupts GBM metabolism, resulting in significant apoptosis. Further increasing the utility of the combination for brain cancers, everolimus significantly increases the brain concentration of palbociclib. Conclusions Our findings demonstrate that the combination of CDK4/6 and mTOR inhibition has therapeutic potential against GBM and suggest it should be evaluated in a clinical trial.
The cullin-based CRL4-CDT2 ubiquitin ligase is emerging as a master regulator of cell proliferation. CRL4-CDT2 prevents re-initiation of DNA replication during the same cell cycle “rereplication” through targeted degradation of CDT1, SET8 and p21 during S-phase of the cell cycle. We show that CDT2 is overexpressed in cutaneous melanoma and predicts poor overall and disease-free survival. CDT2 ablation inhibited a panel of melanoma cell lines through the induction of SET8- and p21-dependent DNA rereplication and senescence. Pevonedistat (MLN4924), a specific inhibitor of the NEDD8 activating enzyme (NAE), inhibits the activity of cullin E3 ligases, thereby stabilizing a vast number of cullin substrates and resulting in cancer cell inhibition in vitro and tumor suppression in nude mice. We demonstrate that pevonedistat is effective at inhibiting the proliferation of melanoma cell lines in vitro through the induction of rereplication-dependent permanent growth arrest as well as through a transient, non-rereplication-dependent mechanism. CRISPR/Cas9-mediated heterozygous deletion of CDKN1A (encoding p21) or SET8 in melanoma cells demonstrated that the rereplication-mediated cytotoxicity of pevonedistat is mediated through preventing the degradation of p21 and SET8 and is essential for melanoma suppression in nude mice. By contrast, pevonedistat-induced transient growth suppression was independent of p21 or SET8, and insufficient to inhibit tumor growth in vivo. Pevonedistat additionally synergized with the BRAF kinase inhibitor PLX4720 to inhibit BRAF melanoma, and suppressed PLX4720-resistant melanoma cells. These findings demonstrate that the CRL4-CDT2-SET8/p21 degradation axis is the primary target of inhibition by pevonedistat in melanoma and suggest that a broad patient population may benefit from pevonedistat therapy.Research in ContextThe identification of new molecular targets and effective inhibitors is of utmost significance for the clinical management of melanoma. This study identifies CDT2, a substrate receptor for the CRL4 ubiquitin ligase, as a prognostic marker and therapeutic target in melanoma. CDT2 is required for melanoma cell proliferation and inhibition of CRL4CDT2 by pevonedistat suppresses melanoma in vitro and in vivo through the induction of DNA rereplication and senescence through the stabilization of the CRL4CDT2 substrates p21 and SET8. Pevonedistat also synergizes with vemurafenib in vivo and suppresses vemurafenib-resistant melanoma cells. These findings show a significant promise for targeting CRL4CDT2 therapeutically.
The cullin RING E3 ubiquitin ligase 4 (CRL4) with its substrate receptor CDT2 (CRL4-CDT2) is emerging as a critical regulator of DNA replication through targeting CDT1, SET8, and p21 for ubiquitin-dependent proteolysis. The aberrant increased stability of these proteins in cells with inactivated CRL4-CDT2 results in DNA rereplication, which is deleterious to cells due to the accumulation of replication intermediates and stalled replication forks. Here, we demonstrate that CDT2 is overexpressed in head and neck squamous cell carcinoma (HNSCC), and its depletion by siRNA inhibits the proliferation of human papilloma virusnegative (HPV-ve) HNSCC cells primarily through the induction of rereplication. Treatment of HNSCC with the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924), which inhibits all cullin-based ligases, induces significant rereplication and inhibits HNSCC cell proliferation in culture and HNSCC xenografts in mice. Pevonedistat additionally sensitizes HNSCC cells to ionizing radiation (IR) and enhances IR-induced suppression of xenografts in mice. Induction of rereplication via CDT2 depletion, or via the stabilization or activation of CDT1, also radiosensitizes HNSCC cells. Collectively, these results demonstrate that induction of rereplication represents a novel approach to treating radioresistant HNSCC tumors and suggest that pevonedistat may be considered as an adjuvant for IR-based treatments.
Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through smallmolecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresis-tance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers.Significance: Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.
Purpose To test the hypothesis that small molecule targeting of nucleophosmin (NPM1) represents a rational approach for radiosensitization. Methods and Materials Wild type and NPM1-deficient mouse embryo fibroblasts (MEFs) were used to determine if radiosensitization produced by the small molecule YTR107 was NPM1 dependent. The stress response to ionizing radiation was assessed by quantifying pNPM1, γH2AX, and Rad51 foci, neutral comet tail moment, and colony formation. NPM1 levels in a human-derived NSCLC tissue microarray (TMA) were determined by immunohistochemistry. YTR107-mediated radiosensitization was assessed in NSCLC cell lines and xenografts. Results Use of NPM1-null MEFs demonstrated that NPM1 is critical for DNA double strand break (DSB) repair, that loss of NPM1 increases radiation sensitivity, and that YTR107-mediated radiosensitization is NPM1 dependent. YTR107 was shown to inhibit NPM1 oligomerization and impair formation of pNPM1 irradiation-induced foci (IRIF) that co-localize with γH2AX foci. Analysis of the TMA demonstrated that NPM1 is over expressed in subsets of NSCLC. YTR107 inhibited DNA DSB repair and radiosensitized NSCLC lines and xenografts. Conclusions These data demonstrate that YTR107-mediated targeting of NPM1 impairs DNA DSB repair, an event that increases radiation sensitivity.
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