Comparison of pharmacological inhibitors of lysine-specific demethylase 1 in glioblastoma stem cells reveals inhibitor-specific efficacy profiles.

Introduction: Improved therapies for glioblastoma (GBM) are desperately needed and require preclinical evaluation in models that capture tumor heterogeneity and intrinsic resistance seen in patients. Epigenetic alterations have been well documented in GBM and lysine-specific demethylase 1 (LSD1/KDM1A) is amongst the chromatin modifiers implicated in stem cell maintenance, growth and differentiation. Pharmacological inhibition of LSD1 is clinically relevant, with numerous compounds in various phases of preclinical and clinical development, but an evaluation and comparison of LSD1 inhibitors in patient-derived GBM models is lacking. Methods: To assess concordance between knockdown of LSD1 and inhibition of LSD1 using a prototype inhibitor in GBM, we performed RNA-seq to identify genes and biological processes associated with inhibition. Efficacy of various LSD1 inhibitors was assessed in nine patient-derived glioblastoma stem cell (GSC) lines and an orthotopic xenograft mouse model. Results: LSD1 inhibitors had cytotoxic and selective effects regardless of GSC radiosensitivity or molecular subtype. In vivo, LSD1 inhibition via GSK-LSD1 led to a delayed reduction in tumor burden; however, tumor regrowth occurred. Comparison of GBM lines by RNA-seq was used to identify genes that may predict resistance to LSD1 inhibitors. We identified five genes that correlate with resistance to LSD1 inhibition in treatment resistant GSCs, in GSK-LSD1 treated mice, and in GBM patients with low LSD1 expression. Conclusion: Collectively, the growth inhibitory effects of LSD1 inhibition across a panel of GSC models and identification of genes that may predict resistance has potential to guide future combination therapies.

Small molecule targeting of the STAT5/6 Src homology 2 (SH2) domains to inhibit allergic airway disease.

Asthma is a chronic inflammatory disease of the lungs and airways and one of the most burdensome of all chronic maladies. Previous studies have established that expression of experimental and human asthma requires the IL-4/IL-13/IL-4 receptor α (IL-4Rα) signaling pathway, which activates the transcription factor STAT6. However, no small molecules targeting this important pathway are currently in clinical development. To this end, using a preclinical asthma model, we sought to develop and test a small-molecule inhibitor of the Src homology 2 domains in mouse and human STAT6. We previously developed multiple peptidomimetic compounds on the basis of blocking the docking site of STAT6 to IL-4Rα and phosphorylation of Tyr641 in STAT6. Here, we expanded the scope of our initial in vitro structure-activity relationship studies to include central and C-terminal analogs of these peptides to develop a lead compound, PM-43I. Conducting initial dose range, toxicity, and pharmacokinetic experiments with PM-43I, we found that it potently inhibits both STAT5- and STAT6-dependent allergic airway disease in mice. Moreover, PM-43I reversed preexisting allergic airway disease in mice with a minimum ED50 of 0.25 µg/kg. Of note, PM-43I was efficiently cleared through the kidneys with no long-term toxicity. We conclude that PM-43I represents the first of a class of small molecules that may be suitable for further clinical development against asthma.

Combinatorial effects of histone deacetylase inhibitors (HDACi), vorinostat and entinostat, and adaphostin are characterized by distinct redox alterations.

PURPOSE: Amongst the epigenetically targeted therapies, targeting of the histone deacetylases (HDACs) has yielded numerous drugs for clinical use in hematological malignancies, but none as yet for acute lymphocytic leukemia (ALL). Single agent activity of HDAC inhibitors (HDACi) has been elusive in ALL, and has prompted study of combinatorial strategies. Because several HDACi raise levels of intracellular oxidative stress, we evaluated combinations of two structurally distinct HDACi with the redox active compound adaphostin in ALL. METHODS: The HDACi vorinostat and entinostat were tested in combination with adaphostin in human ALL cell lines. DNA fragmentation, caspase activation, mitochondrial disruption and levels of  intracellular peroxides, superoxide and glutathione were measured in cells treated with the HDACi/adaphostin combinations. Antioxidant blockade of cell death induction and gene expression profiling of cells treated with vorinostat/adaphostin versus entinostat/adaphostin combinations were evaluated. RESULTS: Both combinations synergistically induced apoptotic DNA fragmentation, which was preceded by an increase in superoxide levels, a reduction in mitochondrial membrane potential, and an increase in caspase-9 activation. The antioxidant N-acetylcysteine (NAC) blocked superoxide generation and prevented reduction of mitochondrial membrane potential. NAC decreased DNA fragmentation and caspase activity in cells treated with adaphostin and vorinostat, but not in those treated with adaphostin and entinostat. Gene expression arrays revealed differential regulation of several redox genes prior to cell death induction. CONCLUSIONS: A redox modulatory agent, adaphostin, enhances efficacy of two HDACi, vorinostat or entinostat, but via different mechanisms indicating a point of divergence in the mechanisms of synergy between the two distinct HDACi and adaphostin.

Preclinical activity of combined HDAC and KDM1A inhibition in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive form of brain cancer. Our previous studies demonstrated that combined inhibition of HDAC and KDM1A increases apoptotic cell death in vitro. However, whether this combination also increases death of the glioma stem cell (GSC) population or has an effect in vivo is yet to be determined. Therefore, we evaluated the translational potential of combined HDAC and KDM1A inhibition on patient-derived GSCs and xenograft GBM mouse models. We also investigated the changes in transcriptional programing induced by the combination in an effort to understand the induced molecular mechanisms of GBM cell death. METHODS: Patient-derived GSCs were treated with the combination of vorinostat, a pan-HDAC inhibitor, and tranylcypromine, a KDM1A inhibitor, and viability was measured. To characterize transcriptional profiles associated with cell death, we used RNA-Seq and validated gene changes by RT-qPCR and protein expression via Western blot. Apoptosis was measured using DNA fragmentation assays. Orthotopic xenograft studies were conducted to evaluate the effects of the combination on tumorigenesis and to validate gene changes in vivo. RESULTS: The combination of vorinostat and tranylcypromine reduced GSC viability and displayed efficacy in the U87 xenograft model. Additionally, the combination led to changes in apoptosis-related genes, particularly TP53 and TP73 in vitro and in vivo. CONCLUSIONS: These data support targeting HDACs and KDM1A in combination as a strategy for GBM and identifies TP53 and TP73 as being altered in response to treatment.

Expression and activity of Fyn mediate proliferation and blastic features of chronic myelogenous leukemia.

The BCR-ABL1 oncogene is a tyrosine kinase that activates many signaling pathways, resulting in the induction of chronic myeloid leukemia (CML). Kinase inhibitors, such as imatinib, have been developed for the treatment of CML; however, the terminal, blast crisis phase of the disease remains a clinical challenge. Blast crisis CML is difficult to treat due to resistance to tyrosine kinase inhibitors, increased genomic instability and acquired secondary mutations. Our recent studies uncovered a role for Fyn in promoting BCR-ABL1 mediated cell growth and sensitivity to imatinib. Here we demonstrate that Fyn contributes to BCR-ABL1 induced genomic instability, a feature of blast crisis CML. Bone marrow cells and mouse embryonic fibroblasts derived from Fyn knockout mice transduced with BCR-ABL1 display slowed growth and clonogenic potential as compared to Fyn wild-type BCR-ABL1 expressing counterparts. K562 cells overexpressing constitutively active Fyn kinase were larger in size and displayed an accumulation of genomic abnormalities such as chromosomal aberrations and polyploidy. Importantly, loss of Fyn protected mouse embryonic fibroblast cells from increased number of chromosomal aberrations and fragments induced by BCR-ABL1. Together, these results reveal a novel role for Fyn in regulating events required for genomic maintenance and suggest that Fyn kinase activity plays a role in the progression of CML to blast crisis.

Inhibition of the NADPH oxidase regulates heme oxygenase 1 expression in chronic myeloid leukemia.

BACKGROUND: Patients with chronic myelogenous leukemia (CML) in blast crisis have a poor response to tyrosine kinase inhibitors designed to inhibit the breakpoint cluster region-v-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1) oncogene. Recent work has demonstrated that heme oxygenase 1 (HO-1) expression is increased in BCR-ABL1-expressing cells and that the inhibition of HO-1 in CML leads to reduced cellular growth, suggesting that HO-1 may be a plausible target for therapy. The objective of the current study was to clarify the mechanism of HO-1 overexpression and the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a contributor to this mechanism in CML. METHODS: HO-1 expression was evaluated in bone marrow specimens from patients with CML in various stages of disease, in a transplantation-based model for CML, and in CML cell lines. Chemical and genetic inhibition of the NADPH oxidase was carried out in CML cells. RESULTS: Specimens from patients with CML in blast crisis displayed higher levels of HO-1 staining than specimens from patients with CML in chronic or accelerated phase. HO-1 up-regulation in BCR-ABL1-expressing cells was suppressed by diphenyleneiodonium (DPI), a chemical inhibitor of the NADPH oxidase. Targeting the NADPH oxidase through RNA interference (RNAi) to Ras-related C3 botulinum toxin substrate 1 (Rac1), a dominant-negative Rac1 construct or an inhibitor of Rac1 activity also blunted HO-1 protein expression. Moreover, inhibition of the NADPH oxidase by RNAi directed toward the 47-kd cytosolic subunit of Nox (p47phox) similarly abrogated HO-1 levels. CONCLUSIONS: BCR-ABL1 expression up-regulated HO-1, a survival factor for CML cells. This up-regulation was more pronounced in blast crisis CML relative to early stage disease and was mediated by the NADPH oxidase components Rac1 and p47phox. The expression of p47phox was increased in BCR-ABL1-expressing cells.

Therapeutic strategies to enhance the anticancer efficacy of histone deacetylase inhibitors.

Histone acetylation is a posttranslational modification that plays a role in regulating gene expression. More recently, other nonhistone proteins have been identified to be acetylated which can regulate their function, stability, localization, or interaction with other molecules. Modulating acetylation with histone deacetylase inhibitors (HDACi) has been validated to have anticancer effects in preclinical and clinical cancer models. This has led to development and approval of the first HDACi, vorinostat, for the treatment of cutaneous T cell lymphoma. However, to date, targeting acetylation with HDACi as a monotherapy has shown modest activity against other cancers. To improve their efficacy, HDACi have been paired with other antitumor agents. Here, we discuss several combination therapies, highlighting various epigenetic drugs, ROS-generating agents, proteasome inhibitors, and DNA-damaging compounds that together may provide a therapeutic advantage over single-agent strategies.

Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors.

Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM.

Role for the nuclear receptor-binding SET domain protein 1 (NSD1) methyltransferase in coordinating lysine 36 methylation at histone 3 with RNA polymerase II function.

The NSD (nuclear receptor-binding SET domain protein) family encodes methyltransferases that are important in multiple aspects of development and disease. Perturbations in NSD family members can lead to Sotos syndrome and Wolf-Hirschhorn syndrome as well as cancers such as acute myeloid leukemia. Previous studies have implicated NSD1 (KMT3B) in transcription and methylation of histone H3 at lysine 36 (H3-K36), but its molecular mechanism in these processes remains largely unknown. Here we describe an NSD1 regulatory network in human cells. We show that NSD1 binds near various promoter elements and regulates multiple genes that appear to have a concerted role in various processes, such as cell growth/cancer, keratin biology, and bone morphogenesis. In particular, we show that NSD1 binding is concentrated upstream of gene targets such as the bone morphogenetic protein 4 (BMP4) and zinc finger protein 36 C3H type-like 1 (ZFP36L1/TPP). NSD1 regulates the levels of the various forms of methylation at H3-K36 primarily, but not exclusively, within the promoter proximal region occupied by NSD1. At BMP4 we find that this reduces the levels of RNAP II recruited to the promoter, suggesting a role for NSD1-dependent methylation in initiation. Interestingly, we also observe that the RNAP II molecules that lie within BMP4 have inappropriate persistence of serine-5 phosphorylation and reduced levels of serine-2 phosphorylation within the C-terminal domain (CTD) of the large subunit of RNAP II. Our findings indicate that NSD1 regulates RNAP II recruitment to BMP4, and failure to do so leads to reduced gene expression and abrogated levels of H3K36Me and CTD phosphorylation.