John DeSisto

Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor

Longitudinal tracking is a powerful approach to understand the biology of single cells. In cancer therapy, outcome is determined at the molecular and cellular scale, yet relationships between cellular response and cell fate are often unknown. The selective inhibitor of nuclear export, selinexor, is in development for the treatment of various cancers. Selinexor covalently binds exportin-1, causing nuclear sequestration of cargo proteins, including key regulators of the cell cycle and apoptosis. The cell cycle effects of selinexor and the relationships between cell cycle effects and cell fates, has not been described for individual cells. Using fluorescent cell cycle indicators we report the majority of cell death after selinexor treatment occurs from a protracted G1-phase and early S-phase. G1- or early S-phase treated cells show the strongest response and either die or arrest, while those treated in late S- or G2-phase progress to mitosis and divide. Importantly, the progeny of cell divisions also die or arrest, mostly in the next G1-phase. Cells that survive selinexor are negative for multiple proliferation biomarkers, indicating a penetrant, arrested state. Selinexor acts quickly, shows strong cell cycle selectivity, and is highly effective at arresting cell growth and inducing death in cancer-derived cells.

Preclinical analysis of MTOR complex 1/2 inhibition in diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is an incurable childhood brain tumor. The mechanistic target of rapamycin (MTOR), a key oncogene, functions as two distinct signaling complexes, MTORC1 and MTORC2. We set out to determine the preclinical efficacy and mechanism of action of MTOR inhibitors in DIPG. We evaluated the MTORC1 inhibitor everolimus and the MTORC1/2 inhibitor AZD2014 in three patient-derived DIPG cell lines using cell culture models. We created dose-response curves for both compounds. We measured phenotypic effects on cell self-renewal, apoptosis, cell cycle, differentiation, senescence, and autophagy. We assessed the effects of each compound on the AKT pathway. Finally, we measured the efficacy of AZD2014 in combination with radiation therapy (RT) and a panel of FDA-approved chemotherapy drugs. While everolimus showed minimal antitumor efficacy, AZD2014 revealed IC50 levels of 410–552 nM and IC90 levels of 1.30–8.86 µM in the three cell lines. AZD2014 demonstrated increased inhibition of cell self-renewal compared to everolimus. AZD2014 decreased expression of phospho-AKT, while no such effect was noted with everolimus. Direct AKT inhibition showed similar efficacy to AZD2014, and induction of constitutive AKT activity rescued DIPG cells from the effects of AZD2014. AZD2014 exhibited synergistic relationships with both RT and various chemotherapy agents across classes, including the multikinase inhibitor ponatinib. MTORC1/2 inhibition shows antitumor activity in cell culture models of DIPG due to the effect of MTORC2 inhibition on AKT. This strategy should be further assessed for potential incorporation into combinatorial approaches to the treatment of DIPG.

NF-κB upregulation through epigenetic silencing of LDOC1 drives tumor biology and specific immunophenotype in Group A ependymoma

Background Inflammation has been identified as a hallmark of high-risk Group A (GpA) ependymoma (EPN). Chronic interleukin (IL)-6 secretion from GpA tumors drives an immune suppressive phenotype by polarizing infiltrating monocytes. This study determines the mechanism by which IL-6 is dysregulated in GpA EPN. Methods Twenty pediatric GpA and 21 pediatric Group B (GpB) EPN had gene set enrichment analysis for MSigDB Hallmark gene sets performed. Protein and RNA from patients and cell lines were used to validate transcriptomic findings. GpA cell lines 811 and 928 were used for in vitro experiments performed in this study. Results The nuclear factor-kappaB (NF-κB) pathway is a master regulator of IL-6 and a signaling pathway enriched in GpA compared with GpB EPN. Knockdown of NF-κB led to significant downregulation of IL-6 in 811 and 928. NF-κB activation was independent of tumor necrosis factor alpha (TNF-α) stimulation in both cell lines, suggesting that NF-κB hyperactivation is mediated through an alternative mechanism. Leucine zipper downregulated in cancer 1 (LDOC1) is a known transcriptional repressor of NF-κB. In many cancers, LDOC1 promoter is methylated, which inhibits gene transcription. We found decreased LDOC1 gene expression in GpA compared with GpB EPN, and in other pediatric brain tumors. EPN cells treated with 5AZA-DC, demethylated LDOC1 regulatory regions, upregulated LDOC1 expression, and concomitantly decreased IL-6 secretion. Stable knockdown of LDOC1 in EPN cell lines resulted in a significant increase in gene transcription of v-rel avian reticuloendotheliosis viral oncogene homolog A, which correlated to an increase in NF-κB target genes. Conclusion These results suggest that epigenetic silencing of LDOC1 in GpA EPN regulates tumor biology and drives inflammatory immune phenotype.

Abstract 1946: Synergistic effects of the XPO1 inhibitor selinexor with proteasome inhibitors in pediatric high-grade glioma and diffuse intrinsic pontine glioma

Background: Pediatric high-grade gliomas (HGG) and diffuse intrinsic pontine gliomas (DIPG) account for the majority of pediatric brain tumor deaths and respond poorly to chemotherapy. Selinexor, a nuclear export inhibitor, is effective against HGG and DIPG in in vitro and in vivo models, but resistance to treatment develops. We previously identified the NF-κB pathway as a likely mediator of selinexor’s activity in these tumors. NF-κB transcriptional activity is regulated by an inhibitor, IKB-α, whose levels are in turn regulated by ubiquitination and proteasomal degradation. IKB-α is a client of exportin-1 (XPO1); its nuclear levels are increased by selinexor treatment, leading to inhibition of NF-κB. We subsequently identified proteasome inhibitors as potentially synergistic with selinexor in HGG and DIPG through a screen of all FDA-approved chemotherapy agents. Proteasome inhibition has also been shown to synergize with selinexor treatment in multiple myeloma and osteosarcoma. Methods: We treated HGG cell lines (BT245 and GBM1) and DIPG cell lines (DIPG4, DIPG7 and SF7761) for five days with selinexor in combination with each of three proteasome inhibitors, bortezomib, carfilzomib and marizomib, and assayed cell viability at the conclusion of treatment. In each experiment, cells were treated with selinexor, a proteasome inhibitor, and a combination of the two drugs at several constant ratios. IC50 values were computed for each drug acting alone, and the combination index (CI) of the two drugs acting together was computed using the Chou-Talalay method. We also treated SF7761 cells with a combination of radiation (8Gy), selinexor, and a proteasome inhibitor. Results: The proteasome inhibitors had widely varying IC50 values in the cell lines treated, ranging from 1nM to 5µM. The CI for the combination of selinexor and each proteasome inhibitor was consistently less than 1 (indicating a synergistic relationship) in the cell lines tested. We found that radiation and proteasome inhibition had an antagonistic relationship (CI>1), radiation and selinexor a synergistic relationship (CI Conclusions: Selinexor and proteasome inhibitors show promise as a combination therapy for HGG and DIPG. We are conducting in vivo experiments to further explore this combination for subsequent clinical trial use. Citation Format: John DeSisto, Patrick Flannery, Trinayan Kashyap, Rakeb Lemma, Shelby Mestnik, Andrew Kung, Rajeev Vibhakar, Yosef Landesman, Adam Green. Synergistic effects of the XPO1 inhibitor selinexor with proteasome inhibitors in pediatric high-grade glioma and diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1946. doi:10.1158/1538-7445.AM2017-1946