Acquired Resistance to EZH2 Inhibitor GSK343 Promotes the Differentiation of Human DLBCL Cell Lines toward an ABC-Like Phenotype.

Diffuse large B-cell lymphoma (DLBCL) accounts for 40% of non-Hodgkin lymphoma, and 30% to 40% of patients will succumb to relapsed/refractory disease (rrDLBCL). Patients with rrDLBCL generally have low long-term survival rates due to a lack of efficient salvage therapies. Small-molecule inhibitors targeting the histone methyltransferase EZH2 represent an emerging group of novel therapeutics that show promising clinical efficacy in patients with rrDLBCL. The mechanisms that control acquired resistance to this class of targeted therapies, however, remain poorly understood. Here, we develop a model of resistance to the EZH2 inhibitor (EZH2i) GSK343 and use RNA-seq data and in vitro investigation to show that GCB (germinal center B-cell)-DLBCL cell lines with acquired drug resistance differentiate toward an ABC (activated B-cell)-DLBCL phenotype. We further observe that the development of resistance to GSK343 is sufficient to induce cross-resistance to other EZH2i. Notably, we identify the immune receptor SLAMF7 as upregulated in EZH2i-resistant cells, using chromatin immunoprecipitation profiling to uncover the changes in chromatin landscape remodeling that permit this altered gene expression. Collectively, our data reveal a previously unreported response to the development of EZH2i resistance in DLBCL, while providing strong rationale for pursuing investigation of dual-targeting of EZH2 and SLAMF7 in rrDLBCL.

Two-year survival with nivolumab in previously treated advanced non-small-cell lung cancer: A real-world pooled analysis of patients from France, Germany, and Canada.

OBJECTIVES: Immune checkpoint inhibitors have become the standard of care for metastatic non-small-cell lung cancer (NSCLC) progressing during or after platinum-based chemotherapy. Real-world clinical practice tends to represent more diverse patient characteristics than randomized clinical trials. We sought to evaluate overall survival (OS) outcomes in the total study population and in key subsets of patients who received nivolumab for previously treated advanced NSCLC in real-world settings in France, Germany, or Canada. MATERIALS AND METHODS: Data were pooled from two prospective observational cohort studies, EVIDENS and ENLARGE, and a retrospective registry in Canada. Patients included in this analysis were aged ≥18 years, had stage IIIB/IV NSCLC, and received nivolumab after at least one prior line of systemic therapy. OS was estimated in the pooled population and in various subgroups using the Kaplan-Meier method. Timing of data collection varied across cohorts (2015-2019). RESULTS: Of the 2585 patients included in this analyses, 1235 (47.8 %) were treated in France, 881 (34.1 %) in Germany, and 469 (18.1 %) in Canada. Median OS for the total study population was 11.3 months (95 % CI: 10.5-12.2); this was similar across France, Germany, and Canada. The OS rate was 49 % at 1 year and 28 % at 2 years for the total study population. In univariable Cox analyses, the presence of epidermal growth factor receptor mutations in nonsquamous disease, liver, or bone metastases were associated with significantly shorter OS, whereas tumor programmed death ligand 1 expression and Eastern Cooperative Oncology Group performance status 0-1 were associated with significantly prolonged OS. Similar OS was noted across subgroups of age and prior lines of therapy. CONCLUSION: OS rates in patients receiving nivolumab for previously treated advanced NSCLC in real-world clinical practice closely mirrored those in phase 3 studies, suggesting similar effectiveness of nivolumab in clinical trials and clinical practice.

The eukaryotic translation initiation factor eIF4E harnesses hyaluronan production to drive its malignant activity.

The microenvironment provides a functional substratum supporting tumour growth. Hyaluronan (HA) is a major component of this structure. While the role of HA in malignancy is well-defined, the mechanisms driving its biosynthesis in cancer are poorly understood. We show that the eukaryotic translation initiation factor eIF4E, an oncoprotein, drives HA biosynthesis. eIF4E stimulates production of enzymes that synthesize the building blocks of HA, UDP-Glucuronic acid and UDP-N-Acetyl-Glucosamine, as well as hyaluronic acid synthase which forms the disaccharide chain. Strikingly, eIF4E inhibition alone repressed HA levels as effectively as directly targeting HA with hyaluronidase. Unusually, HA was retained on the surface of high-eIF4E cells, rather than being extruded into the extracellular space. Surface-associated HA was required for eIF4E's oncogenic activities suggesting that eIF4E potentiates an oncogenic HA program. These studies provide unique insights into the mechanisms driving HA production and demonstrate that an oncoprotein can co-opt HA biosynthesis to drive malignancy.

Peroxisomes protect lymphoma cells from HDAC inhibitor-mediated apoptosis.

Peroxisomes are a critical rheostat of reactive oxygen species (ROS), yet their role in drug sensitivity and resistance remains unexplored. Gene expression analysis of clinical lymphoma samples suggests that peroxisomes are involved in mediating drug resistance to the histone deacetylase inhibitor (HDACi) Vorinostat (Vor), which promotes ROS-mediated apoptosis. Vor augments peroxisome numbers in cultured lymphoma cells, concomitant with increased levels of peroxisomal proteins PEX3, PEX11B, and PMP70. Genetic inhibition of peroxisomes, using PEX3 knockdown, reveals that peroxisomes protect lymphoma cells against Vor-mediated cell death. Conversely, Vor-resistant cells were tolerant to elevated ROS levels and possess upregulated levels of (1) catalase, a peroxisomal antioxidant, and (2) plasmalogens, ether glycerophospholipids that represent peroxisome function and serve as antioxidants. Catalase knockdown induces apoptosis in Vor-resistant cells and potentiates ROS-mediated apoptosis in Vor-sensitive cells. These findings highlight the role of peroxisomes in resistance to therapeutic intervention in cancer, and provide a novel modality to circumvent drug resistance.

Genetic and pharmacologic inhibition of eIF4E reduces breast cancer cell migration, invasion, and metastasis.

The translation initiation factor eIF4E is an oncogene that is commonly overexpressed in primary breast cancers and metastases. In this article, we report that a pharmacologic inhibitor of eIF4E function, ribavirin, safely and potently suppresses breast tumor formation. Ribavirin administration blocked the growth of primary breast tumors in several murine models and reduced the development of lung metastases in an invasive model. Mechanistically, eIF4E silencing or blockade reduced the invasiveness and metastatic capability of breast cancer cells in a manner associated with decreased activity of matrix metalloproteinase (MMP)-3 and MMP-9. Furthermore, eIF4E silencing or ribavirin treatment suppressed features of epithelial-to-mesenchymal transition, a process crucial for metastasis. Our findings offer a preclinical rationale to explore broadening the clinical evaluation of ribavirin, currently being tested in patients with eIF4E-overexpressing leukemia, as a strategy to treat solid tumors such as metastatic breast cancer.

The role of eIF4E in response and acquired resistance to vemurafenib in melanoma.

In eukaryotic cells, the rate-limiting component for cap-dependent mRNA translation is the translation initiation factor eIF4E. eIF4E is overexpressed in a variety of human malignancies, but whether it has a role in melanoma remains obscure. We hypothesized that eIF4E promotes melanoma cell proliferation and facilitates the development of acquired resistance to the BRAF inhibitor vemurafenib. We show that eIF4E is overexpressed in a panel of melanoma cell lines, compared with immortalized melanocytes. Knockdown of eIF4E significantly repressed the proliferation of a subset of melanoma cell lines. Moreover, in BRAF(V600E) melanoma cell lines, vemurafenib inhibits 4E-BP1 phosphorylation, thus promoting its binding to eIF4E. Cap-binding and polysome profiling analysis confirmed that vemurafenib stabilizes the eIF4E-4E-BP1 association and blocks mRNA translation, respectively. Conversely, in cells with acquired resistance to vemurafenib, there is an increased dependence on eIF4E for survival; 4E-BP1 is highly phosphorylated and thus eIF4E-4E-BP1 associations are impeded. Moreover, increasing eIF4E activity by silencing 4E-BP1/2 renders vemurafenib-responsive cells more resistant to BRAF inhibition. In conclusion, these data suggest that therapeutically targeting eIF4E may be a viable means of inhibiting melanoma cell proliferation and overcoming vemurafenib resistance.

Eukaryotic translation initiation factor 4E as a novel therapeutic target in hematological malignancies and beyond.

INTRODUCTION: The eukaryotic translation initiation factor 4E (eIF4E) is a key regulator of protein synthesis, and an oncogene. Its expression and activity are frequently elevated in cancer, and have been shown to correlate with poor prognosis. Efforts to target eIF4E have thus yielded much interest, with some clinical success. AREAS COVERED: We provide an overview of eIF4E function and regulation, and its role in hematological malignancies and solid tumors. Activation of eIF4E via upstream signaling pathways that are frequently deregulated in cancer and the role of eIF4E phosphorylation are discussed. We present an updated review of different approaches to target eIF4E function in the lab and in the clinic. EXPERT OPINION: The prospect of effectively targeting eIF4E in cancer is very attractive, because eIF4E is a common downstream node on which multiple oncogenic signaling pathways converge. However, efforts to do so have yielded limited clinical success so far. While active-site inhibitors of mammalian target of rapamycin show some promise, and inhibitors of eIF4E phosphorylation may emerge as clinical candidates, the only drug to date that has demonstrated antitumor activity associated with eIF4E inhibition in patients is ribavirin. Further studies will certainly aid the design of better compounds and rational combination therapies.

Expression of leukemia-associated fusion proteins increases sensitivity to histone deacetylase inhibitor-induced DNA damage and apoptosis.

Histone deacetylase inhibitors (HDI) show activity in a broad range of hematologic and solid malignancies, yet the percentage of patients in any given malignancy who experience a meaningful clinical response remains small. In this study, we sought to investigate HDI efficacy in acute myeloid leukemia (AML) cells expressing leukemia-associated fusion proteins (LAFP). HDIs have been shown to induce apoptosis, in part, through accumulation of DNA damage and inhibition of DNA repair. LAFPs have been correlated with a DNA repair-deficient phenotype, which may make them more sensitive to HDI-induced DNA damage. We found that expression of the LAFPs PLZF-RARα, PML-RARα, and RUNX1-ETO (AML1-ETO) increased sensitivity to DNA damage and apoptosis induced by the HDI vorinostat. The increase in apoptosis correlated with an enhanced downregulation of the prosurvival protein BCL2. Vorinostat also induced expression of the cell-cycle regulators p19(INK4D) and p21(WAF1) and triggered a G2-M cell cycle arrest to a greater extent in LAFP-expressing cells. The combination of LAFP and vorinostat further led to a greater downregulation of several base excision repair (BER) enzymes. These BER genes represent biomarker candidates for response to HDI-induced DNA damage. Notably, repair of vorinostat-induced DNA double-strand breaks was found to be impaired in PLZF-RARα-expressing cells, suggesting a mechanism by which LAFP expression and HDI treatment cooperate to cause an accumulation of damaged DNA. These data support the continued study of HDI-based treatment regimens in LAFP-positive AMLs.

Vorinostat induces reactive oxygen species and DNA damage in acute myeloid leukemia cells.

Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.

Ribavirin treatment effects on breast cancers overexpressing eIF4E, a biomarker with prognostic specificity for luminal B-type breast cancer.

PURPOSE: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of specific, growth-stimulatory mRNAs and is frequently overexpressed in cancer. EXPERIMENTAL DESIGN: Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node-negative breast cancers were analyzed for eIF4E expression levels and correlation with distant metastasis-free survival (DMFS), overall and within each intrinsic breast cancer subtype. RESULTS: At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating with reduced mRNA export and protein expression of important eIF4E targets. This effect was suppressed by knockdown of eIF4E. Although eIF4E expression is elevated in all breast cancer cell lines, variability in ribavirin responsiveness was observed, indicating that other factors contribute to an eIF4E-dependent phenotype. Assessment of the prognostic value of high eIF4E mRNA in patient tumors found that significant discrimination between good and poor outcome groups was observed only in luminal B cases, suggesting that a specific molecular profile may predict response to eIF4E-targeted therapy. CONCLUSIONS: Inhibition of eIF4E is a potential breast cancer therapeutic strategy that may be especially promising against specific molecular subtypes and in metastatic as well as primary tumors.

Inhibition of DNA methyltransferase activates tumor necrosis factor alpha-induced monocytic differentiation in acute myeloid leukemia cells.

Transcriptional silencing via promoter methylation of genes important for cell growth and differentiation plays a key role in myeloid leukemogenesis. We find that clinically achievable levels of 5-aza-2'-deoxycytidine (5-AZA-dC), a potent inhibitor of DNA methylation, can modify chromatin and restore the ability of tumor necrosis factor alpha (TNFalpha) to induce monocytic differentiation of the acute myeloid leukemia cells NB4 and U937. Although 5-AZA-dC cannot fully induce differentiation, we show that 5-AZA-dC acts directly on TNFalpha-responsive promoters to facilitate TNFalpha-induced transcriptional pathways leading to differentiation. 5-AZA-dC regulates the expression of Dif-2, a TNFalpha target gene, by deacetylating chromatin domains in a methylation-dependent manner. Chromatin immunoprecipitation analyses of the Dif-2 promoter show histone hyperacetylation and a recruitment of the nuclear factor-kappaB transcription factor in response to 5-AZA-dC. Furthermore, 5-AZA-dC plus TNFalpha enhances the level of phosphorylated RNA Pol II at the Dif-2 promoter via synergistic recruitment of TFIIH. We conclude that nonspecific changes in chromatin can allow a specific transcriptional inducer to overcome blocks in leukemic cell differentiation. Our results support the concept of low doses of 5-AZA-dC acting in combination with other agents to target epigenetic changes that drive malignant growth in leukemic cells. [Cancer Res 2009;69(1):55-64].

Rexinoids modulate steroid and xenobiotic receptor activity by increasing its protein turnover in a calpain-dependent manner.

The steroid and xenobiotic receptor SXR (human pregnane X receptor) is a nuclear receptor that plays a key role in the body's detoxification response by regulating genes involved in drug metabolism and transport. SXR ligands include a wide range of compounds, which induce transcription of SXR target genes via activation of a heterodimeric transcription factor consisting of SXR and the related nuclear receptor retinoid X receptor (RXR). We investigated the effect of RXR-selective ligands, rexinoids, on SXR/RXR activity. In agreement with previous reports, we found that rexinoids are weak activators of SXR, but we also found that they can antagonize SXR activation by the potent SXR agonist rifampicin. This antagonism included suppression of rifampicin-induced expression of SXR target genes, as well as reduced binding of SXR/RXR to SXR response elements both in vivo and in vitro. Interestingly, two rexinoids, bexarotene (LGD1069/Targretin) and LG100268, caused a rapid and sustained decrease in the protein levels of both SXR and RXR. The decrease in SXR level was due to an enhanced rate of protein degradation and was dependent on calpain activity, as opposed to rexinoid-induced RXR degradation, which is mediated via the proteasome. Thus, we have demonstrated a novel, rexinoid-modulated mechanism regulating SXR protein stability, which may explain why rexinoids are only weak activators of SXR/RXR-mediated transcription, despite reports that they bind to SXR with high affinity. We suggest that the ability of rexinoids to induce degradation of both SXR and RXR, in combination with competition for binding to SXR, can also explain why rexinoids antagonize the activation of SXR by drugs like rifampicin.

Retinoic acid modulates chromatin to potentiate tumor necrosis factor alpha signaling on the DIF2 promoter.

Transcriptional activation by nuclear hormone receptors is well characterized, but their cooperation with other signaling pathways to activate transcription remains poorly understood. Tumor necrosis factor alpha (TNFalpha) and all-trans retinoic acid (RA) induce monocytic differentiation of acute promyelocytic leukemia (APL) cells in a synergistic manner. We used the promoter of DIF2, a gene involved in monocytic differentiation, to model the mechanism underlying the cooperative induction of target genes by RA and TNFalpha. We show a functional RA response element in the DIF2 promoter, which is constitutively bound by PML/RARalpha in APL cells. RA stimulates release of corepressors and recruitment of chromatin modifying proteins and additional transcription factors to the promoter, but these changes cause only a modest induction of DIF2 mRNA. Co-stimulation with RA plus TNFalpha facilitates binding of NF-kappaB to the promoter, which is crucial for full induction of transcription. Furthermore, RA plus TNFalpha greatly enhanced the level of RNA Pol II phosphorylation on the DIF2 promoter, via synergistic recruitment of TFIIH. We propose that RA mediates remodeling of chromatin to facilitate binding of transcription factors, which cooperate to enhance Pol II phosphorylation, providing a mechanism whereby nuclear receptors interact with other signaling pathways on the level of transcription.

ERbeta sensitizes breast cancer cells to retinoic acid: evidence of transcriptional crosstalk.

The ability of retinoids to inhibit breast cancer cell growth correlates with estrogen receptor (ER) alpha status, as shown by the antiproliferative effects of retinoids in ERalpha-positive breast cancer cells and their use as chemopreventive agents in premenopausal women. The discovery of ERbeta, also present in breast cancer cells, has added a new level of complexity to this malignancy. To determine the retinoid response in ERbeta-expressing breast cancer cells, we used retroviral transduction of ERbeta in ER-negative MDA-MB-231 cells. Western blot and immunofluorescence confirmed expression and nuclear localization of ERbeta, whereas functionality was shown using an estrogen response element-containing reporter. A significant retinoic acid (RA)-mediated growth inhibition was observed in the transduced ERbeta-positive cells as shown by proliferation assays. Addition of estradiol, tamoxifen, or ICI 182,780 had no effect on cell growth and did not alter RA sensitivity. We observed that retinoids altered ERbeta-mediated transcriptional activity from an estrogen response element, which was confirmed by decreased expression of the pS2 gene, and from an activator protein response element. Conversely, the expression of ERbeta altered RA receptor (RAR) beta expression, resulting in greater induction of RARbeta gene expression on RA treatment, without altered expression of RARalpha. Our data provide evidence of transcriptional crosstalk between ERbeta and RAR in ERbeta-positive breast cancer cells that are growth inhibited by RA.

Enhanced retinoid-induced apoptosis of MDA-MB-231 breast cancer cells by PKC inhibitors involves activation of ERK.

Retinoids are vitamin A derivatives, which cause growth inhibition, differentiation and/or apoptosis in various cell types, including some breast cancer cells. In general, estrogen receptor (ER)-positive cells are retinoic acid (RA) sensitive, whereas ER-negative cells are resistant. In this report, we show that ER-negative MDA-MB-231 cells are strongly growth inhibited by retinoids in combination with a PKC inhibitor. While neither RA nor GF109203X (GF) has a significant growth inhibitory effect in these cells, RA+GF potently suppress proliferation. We found that RA+GF induce apoptosis, as shown by an increase in fragmented DNA, Annexin-V-positive cells and caspase-3 activation. Apoptosis was also induced by GF in combination with two synthetic retinoids. Expression of phosphorylated as well as total PKC was decreased by GF and this was potentiated by RA. In addition, treatment with GF caused a strong and sustained activation of ERK1/2 and p38-MAPK, as well as a weaker activation of JNK. Importantly, inhibition of ERK but not p38 or JNK suppressed apoptosis induced by RA+GF, indicating that activation of ERK is specifically required. In support of this novel finding, the ability of other PKC inhibitors to cause apoptosis in combination with RA correlates with ability to cause sustained activation of ERK.

The N-terminal of the estrogen receptor (ERalpha) mediates transcriptional cross-talk with the retinoic acid receptor in human breast cancer cells.

Transcriptional cross-talk exists between the estrogen receptor (ERalpha) and retinoic acid receptor (RAR) pathways in human breast cancer cells. We have previously shown that re-expression of ERalpha in ER-negative cells stimulates the transcriptional and growth inhibitory effects of all-trans-retinoic acid (tRA) by a mechanism that is independent of the ER ligands estradiol and tamoxifen. In this study, we generated cell lines stably expressing ERalpha-deletion mutants to elucidate the mechanism whereby ERalpha modulates RAR transcriptional activity. Using RT-PCR and RNAse protection assays, we observed that expression of ERalpha suppresses basal expression of the RA-responsive gene RARbeta2, while allowing it to be strongly induced by tRA. Repression of basal RARbeta2 transcription was confirmed by transient expression of the reporter plasmid betaRE-tk-CAT, containing the RARbeta2 promoter. In the ERalpha-negative cells, on the other hand, transcription was only weakly induced by RA. We further determined that this effect of ERalpha on RARbeta induction required the N-terminal AF-1-containing region, including the DNA-binding domain, but was independent of the C-terminal ligand-binding domain. Consistent with these results, the ER agonist estradiol and the AF-2 antagonist 4-hydroxytamoxifen had no significant effect on betaRARE activity. Conversely, the full ER antagonist ICI 182,780, which blocks ERalpha AF-1 activity, was able to completely relieve repression of basal betaRARE activity. The effect of ERalpha is specific for RAR-mediated transcription and does not occur on promoters containing typical response elements for the Vitamin D or thyroid hormone receptors. Moreover, the cross-talk between ERalpha and RAR does not seem to be mediated by sequestration of a number of common co-regulators, suggesting a novel mechanism whereby the N-terminal region of ERalpha modulates the transcriptional activity of RAR.