Ben P. Martin

Activation of HIV Transcription with Short-Course Vorinostat in HIV-Infected Patients on Suppressive Antiretroviral Therapy

Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. Trial Registration ClinicalTrials.gov NCT01365065

Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist

Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.

HDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses

The identification of recurrent somatic mutations in genes encoding epigenetic enzymes has provided a strong rationale for the development of compounds that target the epigenome for the treatment of cancer. This notion is supported by biochemical studies demonstrating aberrant recruitment of epigenetic enzymes such as histone deacetylases (HDACs) and histone methyltransferases to promoter regions through association with oncogenic fusion proteins such as PML-RARα and AML1-ETO. HDAC inhibitors (HDACi) are potent inducers of tumor cell apoptosis; however, it remains unclear why tumor cells are more sensitive to HDACi-induced cell death than normal cells. Herein, we assessed the biological and molecular responses of isogenic normal and transformed cells to the FDA-approved HDACi vorinostat and romidepsin. Both HDACi selectively killed cells of diverse tissue origin that had been transformed through the serial introduction of different oncogenes. Time-course microarray expression profiling revealed that normal and transformed cells transcriptionally responded to vorinostat treatment. Over 4200 genes responded differently to vorinostat in normal and transformed cells and gene ontology and pathway analyses identified a tumor-cell-selective pro-apoptotic gene-expression signature that consisted of BCL2 family genes. In particular, HDACi induced tumor-cell-selective upregulation of the pro-apoptotic gene BMF and downregulation of the pro-survival gene BCL2A1 encoding BFL-1. Maintenance of BFL-1 levels in transformed cells through forced expression conferred vorinostat resistance, indicating that specific and selective engagement of the intrinsic apoptotic pathway underlies the tumor-cell-selective apoptotic activities of these agents. The ability of HDACi to affect the growth and survival of tumor cells whilst leaving normal cells relatively unharmed is fundamental to their successful clinical application. This study provides new insight into the transcriptional effects of HDACi in human donor-matched normal and transformed cells, and implicates specific molecules and pathways in the tumor-selective cytotoxic activity of these compounds.

NKT cell adjuvant-based tumor vaccine for treatment of myc oncogene-driven mouse B-cell lymphoma.

Immunomodulators are effective in controlling hematologic malignancy by initiating or reactivating host antitumor immunity to otherwise poorly immunogenic and immune suppressive cancers. We aimed to boost antitumor immunity in B-cell lymphoma by developing a tumor cell vaccine incorporating α-galactosylceramide (α-GalCer) that targets the immune adjuvant properties of NKT cells. In the Eμ-myc transgenic mouse model, single therapeutic vaccination of irradiated, α-GalCer-loaded autologous tumor cells was sufficient to significantly inhibit growth of established tumors and prolong survival. Vaccine-induced antilymphoma immunity required NKT cells, NK cells, and CD8 T cells, and early IL-12-dependent production of IFN-γ. CD4 T cells, gamma/delta T cells, and IL-18 were not critical. Vaccine treatment induced a large systemic spike of IFN-γ and transient peripheral expansion of both NKT cells and NK cells, the major sources of IFN-γ. Furthermore, this vaccine approach was assessed in several other hematopoietic tumor models and was also therapeutically effective against AML-ETO9a acute myeloid leukemia. Replacing α-GalCer with β-mannosylceramide resulted in prolonged protection against Eμ-myc lymphoma. Overall, our results demonstrate a potent immune adjuvant effect of NKT cell ligands in therapeutic anticancer vaccination against oncogene-driven lymphomas, and this work supports clinical investigation of NKT cell-based immunotherapy in patients with hematologic malignancies.

CDK9 inhibition by dinaciclib potently suppresses Mcl-1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo

MYC dysregulation confers a poor prognosis to diffuse large B-cell lymphoma (DLBCL), and effective therapeutic strategies are lacking in relapsed/refractory DLBCL, Burkitt lymphoma and intermediate forms.1, 2 As a master transcriptional regulator, MYC recruits transcription complexes containing RNA polymerase II (Pol II) to facilitate effective transcriptional elongation of MYC gene targets.3 Pol II is fully activated by phosphorylation of a critical serine residue at position 2 within heptapeptide repeats in the carboxy-terminal domain (CTD), a function performed by the positive transcription elongation factor b (P-TEFb; comprising CDK9 and cyclin T1).4 It has been shown that MYC binds and recruits P-TEFb to its targets as a means to activate Pol II.3, 5, 6 More recently, CDK9-mediated transcriptional elongation was reported as essential for tumor maintenance in a genetically defined MYC-driven model of hepatocellular carcinoma.7 Thus, CDK9 dependence may represent a druggable vulnerability in lymphomas with dysregulated MYC expression.

The role of p21 waf1/cip1 and p27 Kip1 in HDACi-mediated tumor cell death and cell cycle arrest in the Eμ-myc model of B-cell lymphoma

Following the establishment of histone deacetylases (HDACs) as promising therapeutic targets for the reversal of aberrant epigenetic states associated with cancer, the development of HDAC inhibitors (HDACi) and their underlying mechanisms of action has been a significant area of scientific interest. HDACi induce diverse biological responses including the inhibition of cell proliferation by blocking progression through the G1 or G2/M phases of the cell cycle. As a putative tumor-suppressor protein, p21waf1/cip1 influences cell proliferation by inhibiting the activity of cyclin–cyclin-dependent kinase (CDK) complexes at the G1/S and G2/M cell cycle checkpoints. HDACi transcriptionally activate CDKN1A, and it has been proposed that induction of p21waf1/cip1 can determine if a cell undergoes apoptosis or cell cycle arrest following HDACi treatment. In the Eμ-myc transgenic mouse model of B-cell lymphoma, knockout of cdkn1a had no effect on disease latency, indicating that p21waf1/cip1 did not function as a tumor suppressor in this system. Although HDACi robustly induced expression of p21waf1/cip1 in wild-type Eμ-myc lymphomas, deletion of cdkn1a did not sensitize the lymphoma cells to HDACi-induced apoptosis and HDACi-induced cell cycle arrest still occurred. However, knockdown of cdkn1b in cdkn1a knockout lymphomas resulted in defective vorinostat-mediated arrest at G1/S indicating an essential role of p27Kip1 in mediating this biological response to vorinostat. These data demonstrate that induction of cdkn1a does not regulate HDACi-mediated tumor cell apoptosis and refute the notion that p21waf1/cip1 is an obligate mediator of HDACi-induced cell cycle arrest.

Antitumor activities and on-target toxicities mediated by a TRAIL receptor agonist following cotreatment with panobinostat†

The recent development of novel targeted anticancer therapeutics such as histone deacetylase inhibitors (HDACi) and activators of the TRAIL pathway provide opportunities for the introduction of new treatment regimens in oncology. HDACi and recombinant TRAIL or agonistic anti‐TRAIL receptor antibodies have been shown to induce synergistic tumor cell apoptosis and some therapeutic activity in vivo. Herein, we have used syngeneic preclinical models of human solid cancers to demonstrate that the HDACi panobinostat can sensitize tumor cells to apoptosis mediated by the anti‐mouse TRAIL receptor antibody MD5‐1. We demonstrate that the combination of panobinostat and MD5‐1 can eradicate tumors grown subcutaneously and orthotopically in immunocompetent mice, while single agent treatment has minimal effect. However, escalation of the dose of panobinostat to enhance antitumor activity resulted in on‐target MD5‐1‐mediated gastrointestinal toxicities that were fatal to the treated mice. Studies performed in mice with knockout of the TRAIL receptor showed that these mice could tolerate doses of the panobinostat/MD5‐1 combination that were lethal in wild type mice resulting in superior tumor clearance. Given that clinical studies using HDACi and activators of the TRAIL pathway have been initiated, our preclinical data highlight the potential toxicities that could limit the use of such a treatment regimen. Our studies also demonstrate the power of using syngeneic in vivo tumor models as physiologically relevant preclinical systems to test the antitumor effects and identify potential side effects of novel anticancer regimens.

Induction of autophagy does not alter the anti-tumor effects of HDAC inhibitors

Dear Editor, HDAC inhibitors (HDACis) can suppress the growth and/or survival of tumor cells and thus far two HDACis, vorinostat and romidepsin, have been approved by the FDA for the treatment of hematological malignancies.1 Vorinostat is a potent HDACi that mediates tumor cell-selective apoptosis and we and others have previously shown that the induction of apoptosis correlates with therapeutic efficacy in mouse models of hematological cancer.2, 3, 4, 5 Although apoptosis may be the preferred mode of HDACi-induced cell death, it is clear that in tumor cells with nonfunctional apoptotic cascades, HDACi are capable of inducing a caspase-independent form of cell death.5, 6, 7 We recently demonstrated that Eμ-myc lymphomas devoid of Apaf-1 displayed a delayed cell death in response to HDACi treatment, and loss of Apaf-1 failed to affect therapeutic efficacy.5 The delayed HDACi-induced cell death was concomitant with biochemical and morphological changes characteristic of autophagy.5 Herein we utilized the genetically tractable Eμ-myc mouse model to address conflicting reports in the field regarding the importance of autophagy in regulating the anti-tumor responses of HDACi.6, 7, 8 To determine whether autophagy had any role in regulating the response of Eμ-myc/Apaf-1−/− cells to HDACi, we knocked down the expression of two key autophagy proteins (Atg5 and Atg7) using constitutive short-hairpin RNAs (shRNAs). We developed two distinct shRNAs against Atg5 and one shRNA against Atg7 that efficiently silenced the expression of Atg5 and Atg7, respectively (Figure 1a). To determine if knockdown of Atg5 or Atg7 impaired HDACi-mediated autophagy, we treated Atg5- and Atg7-shRNA-expressing Eμ-myc/Apaf-1−/− lymphomas with vorinostat and determined the ratio of LC3-I/LC3-II by western blot. In Eμ-myc/Apaf-1−/− cells with decreased expression of Atg5 or Atg7, vorinostat-induced processing of LC3-I to LC3-II was greatly reduced (Figure 1a). Figure 1 Vorinostat does not require a functional autophagic pathway to induce tumor cell death in Eμ-myc/Apaf-1−/− lymphomas. (a) Eμ-myc/Apaf-1−/− lymphomas were retrovirally transduced with constructs expressing ... We next evaluated the response of Atg5- and Atg7-shRNA-expressing Eμ-myc/Apaf-1−/− lymphomas to increasing concentrations of vorinostat over time. Consistent with our previous results,5 a delay in the kinetics of cell death was observed in vorinostat-treated Eμ-myc/Apaf-1−/− compared with Eμ-myc lymphomas (Figure 1b). However, depletion of Atg5 or Atg7 did not affect vorinostat-mediated tumor cell death, indicating that inhibition of autophagy neither protected Eμ-myc/Apaf-1−/− cells from HDACi-mediated cell death nor potentiated its anticancer effect. To evaluate the response of Atg5- and Atg7-shRNA-expressing Eμ-myc/Apaf-1−/− lymphomas to vorinostat in vivo, we transplanted lymphomas into recipient mice and treated tumor-bearing mice with vorinostat. Consistent with our in vitro data, Atg5- and Atg7-shRNA-expressing lymphomas were equally responsive to vorinostat and cleared with similar kinetics as control-shRNA-expressing lymphomas (Figure 1c). Finally, we determined whether inhibition of autophagy had any effect on the therapeutic efficacy of vorinostat in Eμ-myc/Apaf-1−/− lymphomas. We observed an enhanced survival in mice bearing Atg5-shRNA Eμ-myc/Apaf-1−/− lymphomas after treatment with vorinostat (median survival vorinostat: 43.5 days, median survival vehicle: 25 days, P<0.0084), which was similar to the therapeutic response seen with control-shRNA-expressing lymphomas (median survival vorinostat: 48 days compared with vehicle: 23.5 days, P<0.0001). In conclusion, our data demonstrate that there is no evidence supporting an essential role for autophagy in regulating the response of apoptosis-deficient tumors to HDACi. Recent data suggest that in tumor cells with apoptotic defects, inhibition of autophagy may potentiate the therapeutic response mediated by HDACi.7 In contrast, we provide evidence that combining autophagy inhibitors with HDACi may not be clinically beneficial in lymphomas with apoptotic defects.

Long term, continuous exposure to panobinostat induces terminal differentiation and long term survival in the TH-MYCN neuroblastoma mouse model

Neuroblastoma is the most common extra‐cranial malignancy in childhood and accounts for ∼15% of childhood cancer deaths. Amplification of MYCN in neuroblastoma is associated with aggressive disease and predicts for poor prognosis. Novel therapeutic approaches are therefore essential to improving patient outcomes in this setting. The histone deacetylases are known to interact with N‐Myc and regulate numerous cellular processes via epigenetic modulation, including differentiation. In this study, we used the TH‐MYCN mouse model of neuroblastoma to investigate the antitumor activity of the pan‐HDAC inhibitor, panobinostat. In particular we sought to explore the impact of long term, continuous panobinostat exposure on the epigenetically driven differentiation process. Continuous treatment of tumor bearing TH‐MYCN transgenic mice with panobinostat for nine weeks led to a significant improvement in survival as compared with mice treated with panobinostat for a three‐week period. Panobinostat induced rapid tumor regression with no regrowth observed following a nine‐week treatment period. Initial tumor response was associated with apoptosis mediated via upregulation of BMF and BIM. The process of terminal differentiation of neuroblastoma into benign ganglioneuroma, with a characteristic increase in S100 expression and reduction of N‐Myc expression, occurred following prolonged exposure to the drug. RNA‐sequencing analysis of tumors from treated animals confirmed significant upregulation of gene pathways associated with apoptosis and differentiation. Together our data demonstrate the potential of panobinostat as a novel therapeutic strategy for high‐risk neuroblastoma patients.

Detection of Apoptotic Cells Using Propidium Iodide Staining

Flow cytometry assays are often used to detect apoptotic cells in in vitro cultures. Depending on the experimental model, these assays can also be useful in evaluating apoptosis in vivo. In this protocol, we describe a propidium iodide (PI) flow cytometry assay to evaluate B-cell lymphomas that have undergone apoptosis in vivo. B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with the apoptosis inducer vorinostat (a histone deacetylase inhibitor). Tumor samples collected from the lymph nodes and/or the spleen are used to prepare a single-cell suspension that is exposed to a hypotonic solution containing the fluorochrome PI. The DNA content of the cells, now labeled with PI, is analyzed by flow cytometry. Nuclear DNA content is lost during apoptosis, resulting in a hypodiploid (or sub-G1) DNA profile during flow cytometry. In contrast, healthy cells display a sharp diploid DNA profile.