Treatment Of Cutaneous T-cell Lymphoma Research Articles

Abstract 651: Ex vivo and in vivo characterization of IPH4102, a humanized anti-KIR3DL2 antibody for the treatment of cutaneous T-cell lymphomas

Abstract KIR3DL2 belongs to the killer immunoglobulin (Ig)-like receptors (KIRs) family and is composed of three extracellular Ig-like domains. KIR3DL2 is naturally expressed on some NK cells and minor subpopulations of CD8+ and CD4+ T cells. Physiologically, KIRD3L2 is an inhibitory receptor for human leukocyte antigen (HLA) class I molécules regulating NK cell activation. Interestingly, KIR3DL2 is also expressed on several subtypes of T lymphomas/leukemias, and in particular on Advanced cutaneous T cell lymphomas (CTCL), making it a unique therapeutic target in cancer. From a wide collection of anti-KIR3DL2 monoclonal antibodies (mAbs) with distinct properties related to several epitopes, we have selected IPH4102 as the lead therapeutic candidate. IPH4102 is a humanized IgG1 that selectively kills KIR3DL2-positive cells in vitro, mainly through antibody-dependent cell cytotoxicity (ADCC). IPH4102 also improves survival of mice engrafted with KIR3DL2-positive tumors. More importantly, IPH4102 exerts remarkable anti-tumor activity against primary KIR3DL2-positive tumor cells ex vivo: using blood taken from Sézary Syndrome patients as experimental model, we demonstrated the efficient killing of primary leukemic cells by autologous NK cells engaged by IPH4102. IPH4102 IND-enabling studies are ongoing. In parallel, other anti-KIR3DL2 mAbs were also developed as unique and sensitive tools for the detection by immunohistochemistry of KIR3DL2 on tumor biopsies. Owing to its efficacy profile and to the highly restricted expression pattern of its target KIR3DL2, IPH4102 stands as a very promising therapeutic option for the treatment of advanced cutaneous T cell lymphomas. Citation Format: Anne Marie-Cardine, Nicolas Viaud, Arnaud Dujardin, Rachel Joly, Laurent Gauthier, Cecile Bonnafous, Mathieu Blery, Carine Paturel, Armand Bensussan, Martine Bagot, Helene Sicard. Ex vivo and in vivo characterization of IPH4102, a humanized anti-KIR3DL2 antibody for the treatment of cutaneous T-cell lymphomas . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 651. doi:10.1158/1538-7445.AM2014-651

Abstract 5112: Histone deacetylase and proteasome inhibitors synergistically induce apoptosis in colon cancer, multiple myeloma and CTCL cells through induction of the immediate early genes ATF3 and JUN

Abstract Background/Aim: Histone deacetylase inhibitors (HDACi) are approved for the treatment of cutaneous T-cell lymphoma. We have previously demonstrated that HDACi-mediated apoptosis is linked to the induction of a defined transcriptional response involving up-regulation of the immediate-early (IE) genes FOS, JUN, ATF3, EGR1 and EGR3, in cell lines derived from multiple tumour types. To identify other drugs which induce a similar transcriptional response we compared the HDACi-induced transcriptional response with that induced by >1000 bioactive molecules using the Broad Institute's Connectivity Map database. The proteasome inhibitor, MG-262, was identified as inducing the most comparable transcriptional response to HDACi. Notably, combination treatment with HDAC and proteasome inhibitors has been shown to synergistically induce apoptosis in vitro, and a phase III trial of this combination demonstrated activity in patients multiple myeloma. The aim of this study was to determine whether these effects are mediated through the additive induction of IE genes. Methods: The effect of the HDACi SAHA and the clinically approved proteasome inhibitor Bortezomib, alone, and in combination, on apoptosis induction was determined in colorectal, multiple myeloma and CTCL cell lines PI staining and FACS analysis. Quantitative RT-PCR was performed to determine gene expression changes upon drug treatment. MAPK pathway activation and histone hyper-acetylation was determined by western blot analysis. Results: Single agent treatment of multiple cancer cells with SAHA or Bortezomib induced expression of the IE genes FOS, JUN and ATF3. In both cases IE gene induction was sustained over 24h. Proteasome inhibitor-induced IE gene expression and apoptosis was selectively dependent on activation of the JNK and p38 stress response pathways, while HDACi-mediated effects were selectively dependent upon the Sp1 and Sp3 transcription factors, indicating these agents induce IE gene expression and apoptosis via independent mechanisms. Co-treatment with Bortezomib and SAHA resulted in synergistic induction of IE gene expression and apoptosis in cell lines derived from multiple tumour types. Importantly, apoptosis induction by the combination was partially inhibited by siRNA-mediated downregulation of two IE genes, ATF3 and JUN. Conclusions: This study provides insight into the mechanistic basis by which combination treatment with HDAC and proteasome inhibitors synergistically induces apoptosis in tumour cells. Citation Format: Janson WT Tse, Anderly C. Chueh, Ian Y. Luk, Fiona Chionh, Yvonne Yeung, Georgia A. Corner, Dominic CH Ng, Hoanh Tran, Amardeep S. Dhillon, John M. Mariadason. Histone deacetylase and proteasome inhibitors synergistically induce apoptosis in colon cancer, multiple myeloma and CTCL cells through induction of the immediate early genes ATF3 and JUN. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5112. doi:10.1158/1538-7445.AM2014-5112

Abstract 5533: RNAi-mediated depletion of histone deacetylases highlights the potential for isoform-specific inhibitors in B-cell lymphoma and acute myeloid leukemia

Abstract Introduction: Histone deacetylase (HDAC) inhibitors are a novel class of drugs with demonstrated activity in hematological malignancies. Vorinostat and romidepsin are FDA approved for treatment of cutaneous T-cell lymphoma and inhibit HDACs1, 2, 3 and 6 or HDAC1, 2 and 3, respectively. It is unknown which HDACs are most important for the survival of hematological tumors, however a targeted approach using HDAC-selective inhibitors could improve efficacy and reduce toxicity in the clinic. Here we utilised RNAi technology and an HDAC-specific inhibitor to investigate whether depletion/inhibition of individual or multiple HDACs could phenocopy the effects of pan-HDACi in B-cell lymphoma and acute myeloid leukemia (AML). Methods: HDACs were individually knocked down in murine Eμ-Myc (HDACs1, 2, 3, 6) and AML (MLL-AF9+NrasG12D; HDACs 1-11) cells using constitutive (pLMS/pLMN) or Tetracycline-inducible (pTRMPVIN) vectors in vitro and in vivo. Eµ-Myc cells were treated with HDAC3-specific inhibitor RGFP966. Cell proliferation, apoptosis, cell cycle, protein expression and gene knockdown were assessed by FACS, western blot and qRT-PCR. Global gene expression was assessed using RNAseq technology. Results: Loss of HDACs1, 2 or 6 had no long term effects on cell growth, while Eμ-Myc and AML cells depleted of HDAC3 were reproducibly lost from culture in vitro and in vivo. This phenotype was not prevented by Bcl-2 over-expression, caspase inhibition or knockout of p21 in Eμ-Myc but appeared dependent on Trp53 expression, including specific mutants of Trp53. HDAC3 knockdown altered the transcription of <0.05% genes in Eμ-Myc cells. Importantly, HDAC3-specific inhibitor RGFP966 reduced the growth rate of Eμ-Myc cells at low micromolar concentrations (0.5-1µM) while inducing apoptosis above 2µM, also partially dependent on Trp53 status. Conclusions: Our results have revealed exquisite sensitivities of murine B cell lymphoma and AML cells to depletion of HDAC3 in vitro and in vivo. This strongly suggests that HDAC3-specific inhibitors could prove useful for the treatment of various hematological malignancies. Further work investigating the molecular events underpinning the loss of proliferation induced by HDAC3 knockdown/inhibition and the effects of depleting multiple HDACs are currently underway. Ultimately, we aim to use this technology to discover efficacious HDACi with the best toxicity profile for the treatment of hematological malignancies. Citation Format: Geoffrey M. Matthews, Leonie Cluse, Eric Wang, Mareike Roth, Chris Vakoc, Wenyan Miao, James Rusche, Johannes Zuber, Ricky W. Johnstone. RNAi-mediated depletion of histone deacetylases highlights the potential for isoform-specific inhibitors in B-cell lymphoma and acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5533. doi:10.1158/1538-7445.AM2014-5533

Treatment options for primary cutaneous T-cell lymphoma

Primary cutaneous T-cell lymphoma (CTCL) represents a group of diseases characterized by the abnormal proliferation of lymphocytes derived from the skin,with mycosis fungoides (MF) as the most common type.The choice of treatment for CTCL is mainly based on the tumor-node-metastasis-blood (TNMB) classification and staging system for cutaneous lymphomas proposed by the International Society for Cutaneous Lymphomas (ISCL)-World Health Organization-European Organization for Research and Treatment of Cancer (EORTC) in 2007.Other factors to be considered should include the time to onset of action and duration of sustained remission,as well as tolerability,side effects,convenience and cost of treatment.Supportive and local therapies are recommended for early CTCL,while systemic chemotherapy,with or without immunomodulatory therapy,is recommended for advanced CTCL.Some specific cytokines,monoclonal antibodies,and fusion toxins have been tried to treat CTCL. Key words: Lymphoma, T-cell, cutaneous; Therapeutic uses

Treatment of cutaneous T-cell lymphoma with oral alitretinoin.

Cutaneous T-cell lymphoma (CTCL) is a potentially life-limiting malignant disease. Treatment strategies in CTCL aim at disease control and remission with the lowest possible side-effects. Recent reports suggest that the new vitamin A derivative alitretinoin might be a well-tolerated treatment option. We analysed the files of 11 CTCL patients with mycosis fungoides (n = 10) or Sézary syndrome (n = 1), who were treated with oral alitretinoin alone or in combination with standard treatment based on individual off-label treatment decisions. Patients had been monitored every 4-8 weeks with skin examination and laboratory analyses. Ten of 11 patients (90.9%) showed a marked improvement of their CTCL skin lesions and no progress of the disease during treatment with alitretinoin, one patient showed no response to the treatment (9.1%). Four of the responding patients (40.0%) had a complete response and 6 (60.0%) had a partial response. Average time to response was 2.5 months. Duration of treatment varied depending on whether patients had reached complete or partial remission. In general, alitretinoin was well tolerated. One of 11 patients developed high non-fasting average serum cholesterol (>300 mg/dL) and 1/11 a mean non-fasting triglyceride value >500 mg/dL. In 3/11 patients, thyroid-stimulating hormone declined without clinical symptoms during treatment, with one of the patients also showing a decreased thyroxin level. In our group of CTCL patients we noticed a low rate of side-effects and an overall good clinical response to treatment with alitretinoin. Further studies are required to substantiate this early clinical observation.

Molecular mechanisms of endometrial stromal sarcoma and undifferentiated endometrial sarcoma as premises for new therapeutic strategies

Endometrial stromal sarcoma (ESS) and undifferentiated endometrial sarcoma (UES) are very rare gynecologic malignancies. Due to the rarity and heterogeneity of these tumors, little is known about their epidemiology, pathogenesis, and molecular pathology. Our previous studies have described deregulation of histone deacetylases expression in ESS/UES samples. Some of these enzymes can be inhibited by substances which are already approved for treatment of cutaneous T-cell lymphoma. On the basis of published data, they may also provide a therapeutic option for ESS/UES patients. Our review focuses on molecular mechanisms of ESS/UES. It describes various aspects with special emphasis on alteration of histone deacetylation and its possible relevance for novel therapies.

Treatment of cutaneous T-cell lymphoma: enter mechlorethamine gel

Treatment of cutaneous T-cell lymphoma: enter mechlorethamine gel

NEW AND EMERGING HDAC INHIBITORS FOR THE TREATMENT OF DISEASES

Histone deacetylases (HDACs) are critical in regulating gene expression and transcription. They also play a fundamental role in regulating cellular activities such as cell proliferation, survival and differentiation. Inhibition of histone deacetylases has generated many fascinating results including a new strategy in human cancer therapy. Suberoylanilide hydroxamic acid (SAHA) and romidepsin are the two drugs approved by US FDA for the treatment of cutaneous T-cell lymphoma. The HDAC inhibitors (HDACIs) like trichostatin A and SAHA are also emerging as new promising drugs for various conditions like rheumatoid arthritis, colitis, systemic lupus erythematosus and CNS disorders. This review, along with chemical classification of HDACIs, emphasizes on the therapeutic potential of various HDACIs against different diseases.

Class I HDACs Affect DNA Replication, Repair, and Chromatin Structure: Implications for Cancer Therapy.

The contribution of epigenetic alterations to cancer development and progression is becoming increasingly clear, prompting the development of epigenetic therapies. Histone deacetylase inhibitors (HDIs) represent one of the first classes of such therapy. Two HDIs, Vorinostat and Romidepsin, are broad-spectrum inhibitors that target multiple histone deacetylases (HDACs) and are FDA approved for the treatment of cutaneous T-cell lymphoma. However, the mechanism of action and the basis for the cancer-selective effects of these inhibitors are still unclear. While the anti-tumor effects of HDIs have traditionally been attributed to their ability to modify gene expression after the accumulation of histone acetylation, recent studies have identified the effects of HDACs on DNA replication, DNA repair, and genome stability. In addition, the HDIs available in the clinic target multiple HDACs, making it difficult to assign either their anti-tumor effects or their associated toxicities to the inhibition of a single protein. However, recent studies in mouse models provide insights into the tissue-specific functions of individual HDACs and their involvement in mediating the effects of HDI therapy. Here, we describe how altered replication contributes to the efficacy of HDAC-targeted therapies as well as discuss what knowledge mouse models have provided to our understanding of the specific functions of class I HDACs, their potential involvement in tumorigenesis, and how their disruption may contribute to toxicities associated with HDI treatment. Impairment of DNA replication by HDIs has important therapeutic implications. Future studies should assess how best to exploit these findings for therapeutic gain.

Mechanisms of Histone Deacetylase Inhibitor-Regulated Gene Expression in Cancer Cells

Class I and II histone deacetylase inhibitors (HDACis) are approved for the treatment of cutaneous T-cell lymphoma and are undergoing clinical trials as single agents, and in combination, for other hematological and solid tumors. Understanding their mechanisms of action is essential for their more effective clinical use, and broadening their clinical potential. HDACi induce extensive transcriptional changes in tumor cells by activating and repressing similar numbers of genes. These transcriptional changes mediate, at least in part, HDACi-mediated growth inhibition, apoptosis, and differentiation. Here, we highlight two fundamental mechanisms by which HDACi regulate gene expression—histone and transcription factor acetylation. We also review the transcriptional responses invoked by HDACi, and compare these effects within and across tumor types. The mechanistic basis for how HDACi activate, and in particular repress gene expression, is not well understood. In addition, whether subsets of genes are reproducibly regulated by these agents both within and across tumor types has not been systematically addressed. A detailed understanding of the transcriptional changes elicited by HDACi in various tumor types, and the mechanistic basis for these effects, may provide insights into the specificity of these drugs for transformed cells and specific tumor types. Understanding the mechanisms by which HDACi regulate gene expression and an appreciation of their transcriptional targets could facilitate the ongoing clinical development of these emerging therapeutics. In particular, this knowledge could inform the design of rational drug combinations involving HDACi, and facilitate the identification of mechanism-based biomarkers of response.

The structural basis of an NADP+-independent dithiol oxidase in FK228 biosynthesis

The disulfide bond is unusual in natural products and critical for thermal stability, cell permeability and bioactivity. DepH from Chromobacterium violaceum No. 968 is an FAD-dependent enzyme responsible for catalyzing the disulfide bond formation of FK228, an anticancer prodrug approved for the treatment of cutaneous T-cell lymphoma. Here we report the crystal structures of DepH and DepH complexed with a substrate analogue S,S'-dimethyl FK228 at 1.82 Å and 2.00 Å, respectively. Structural and biochemical analyses revealed that DepH, in contrast to the well characterized low molecular weight thioredoxin reductases (LMW TrxRs), is an NADP+-independent dithiol oxidase. DepH not only lacks a conserved GGGDXAXE motif necessary for NADP+ binding in the canonical LMW TrxRs, but also contains a 11-residue sequence which physically impedes the binding of NADP+. These observations explain the difference between NADP+-independent small molecule dithiol oxidases and NADP+-dependent thioredoxin reductases and provide insights for understanding the catalytic mechanism of dithiol oxidases involved in natural product biosynthesis.

Quantification of vorinostat and its main metabolites in plasma and intracellular vorinostat in PBMCs by liquid chromatography coupled to tandem mass spectrometry and its relation to histone deacetylase activity in human blood

Vorinostat (suberoylanilide hydroxamic acid) is the first approved histone deacetylase (HDAC) inhibitor for the treatment of cutaneous T-cell lymphoma after progressive disease following two systemic therapies. Intracellular access of vorinostat is essential to exert its epigenetic effects. Therefore, we studied the relationship between vorinostat extracellular (plasma) and intracellular (peripheral blood mononuclear cells, PBMCs) concentration and assessed its concentration-effect relationship by HDAC activity testing. Assays were developed and validated for the low nanomolar quantification of vorinostat and two inactive metabolites in human plasma and PBMCs. For the vorinostat extraction from plasma and PBMCs solid-phase extraction and liquid-liquid extraction methods were applied. Extraction recoveries ranged from 88.6% to 114.4% for all analytes and extraction methods. Extracts were chromatographed on a Phenomenex Luna column isocratically (plasma) or by gradient (PBMCs) consisting of acidic ammonium acetate, acetonitrile, and methanol. The analytes were quantified using deuterated internal standards and positive electrospray tandem mass spectrometry (multiple reaction monitoring) with lower limits of quantification of 11.0 ng/mL (plasma) and 0.1 ng/3 × 10(6) cells (PBMCs). The calibrated ranges were linear for vorinostat in plasma 11.0-1100 (11,000) ng/mL (metabolites) and PBMCs 0.1-10.0 ng/3 × 10(6) cells with correlation coefficients >0.99, an overall accuracy varying between -6.7% and +3.8% in plasma, -8.1% and -1.5% in PBMCs, and an overall precision ranging from 3.2% to 6.1% in plasma and 0.8% to 4.0% in PBMCs (SD batch-to-batch). The application to blood samples from healthy volunteers incubated with vorinostat revealed accumulation of vorinostat in PBMCs, effective intracellular HDAC inhibition at therapeutic vorinostat concentrations and a direct vorinostat concentration dependency to HDAC inhibition.

Histone deacetylase inhibitors

The histone deacetylase inhibitors (HDACi) are a group of small molecules that target histone deacetylases (HDACs) by inhibiting their activity. HDACi have a long history of use in neurology and psychiatry as antiepileptics and mood stabilizers. More recently, they have been investigated as possible treatments for cancer. HDACi have undergone rapid clinical development in recent years, on the basis of their preclinical in-vitro and in-vivo antitumor activity in hematological malignancies and solid tumors. Many HDACi have entered phase I-III clinical trials. Among the HDACi, vorinostat and romidepsin are currently the most extensively studied. In 2006 and 2009, respectively, they received approval by the United States Food and Drug Administration for treatment of cutaneous T-cell lymphoma and romidepsin for the treatment of peripheral T-cell lymphoma. Other HDACi, such as panobinostat and valproic acid, also demonstrated activity as therapeutic anticancer agents. In this article we give an overview of the clinical studies of HDACi in solid tumors. We start with a short description of the working mechanism of HDACi in general.

Mutagenesis Studies of the 14 Å Internal Cavity of Histone Deacetylase 1: Insights toward the Acetate-Escape Hypothesis and Selective Inhibitor Design

Histone deacetylase (HDAC) proteins are promising targets for cancer treatment, as shown by the approval of two HDAC inhibitors for the treatment of cutaneous T-cell lymphoma. HDAC1 in particular has been linked to cell growth and cell cycle regulation and is therefore an attractive target for anticancer drugs. The HDAC1 active site contains a hydrophobic 11 Å active-site channel, with a 14 Å internal cavity at the bottom of the active site. Several computational and biochemical studies have proposed an acetate-escape hypothesis where the acetate byproduct of the deacetylation reaction escapes via the 14 Å internal cavity. Selective HDAC inhibitors that bind to the 14 Å cavity have also been created. To understand the influence of amino acids lining the HDAC1 14 Å cavity in acetate escape and inhibitor binding, we used mutagenesis coupled with acetate competition assays. The results indicate that amino acids lining the 14 Å cavity are critical for catalytic activity and acetate competition, confirming the role of the cavity in acetate escape. In addition, these mutagenesis studies will aid in HDAC1-inhibitor design that exploits the 14 Å cavity.

COX and VEGFR-2 inhibitors as potentiating agents of new HDAC inhibitors as epigenetic modulators in cancer

Recently, among anticancer agents, of growing interest are the kinases inhibitors. Many of them are under clinical trials while some have been already approved by FDA as angiogenesis blockers (Vascular endothelial growth factor receptor 2 (VEGFR- 2) inhibitors), such as Sunitinib and Vandetanib. HDACI (histone deacetylase inhibitors) are another newest drug group recently approved for the treatment of Cutaneous T-cell lymphoma (CTCL) (Wagner et al., 2010) and several others HDACIs are in pre-clinical and clinical stages. Interestingly, many scientific reports indicate the synergistic effects of the HDACI and VEGFR-2 inhibitors combination. Lately, has been proposed and showed that cycle oxygenases (COXs) inhibitors could reduce the adverse side effects upon HDACI administration. Furthermore, the anticancer effectiveness of COX-2 inhibitors in the treatment of gastric cancer animal models has been demonstrated (Thiel et al., 2011). Taking consideration of these observations we aim to study a new antiproliferative therapy with the combined use of three potentially synergic drug classes: HDACI, inhibitors of VEGFR-2 and COX-2. The antitproliferative evaluation of the more promising molecules will be evaluated both in vitro, in colangiocarcinoma and hepatocarcinoma cell lines trough MTT proliferation assay and Caspase3 Elisa kit, and in vivo models. Along with individual compound assays, synergistic effects will be also determined. The main findings derived from individual compound assay studies will be integrated and used to set-up the best combination of compounds for a multi-target approach that we expect to be highly effective against cancer.

Guidelines on the use of extracorporeal photopheresis

BackgroundAfter the first investigational study on the use of extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma was published in 1983 with its subsequent recognition by the FDA for its refractory forms, the technology has shown significant promise in the treatment of other severe and refractory conditions in a multi-disciplinary setting. Among the major studied conditions are graft versus host disease after allogeneic bone marrow transplantation, systemic sclerosis, solid organ transplant rejection and inflammatory bowel disease.Materials and methodsIn order to provide recognized expert practical guidelines for the use of this technology for all indications the European Dermatology Forum (EDF) proceeded to address these questions in the hands of the recognized experts within and outside the field of dermatology. This was done using the recognized and approved guidelines of EDF for this task.Results and conclusionThese guidelines provide at present the most comprehensive available expert recommendations for the use of extracorporeal photopheresis based on the available published literature and expert consensus opinion.

Towards selective inhibition of histone deacetylase isoforms: what has been achieved, where we are and what will be next.

Histone deacetylases (HDACs) are widely studied targets for the treatment of cancer and other diseases. Up to now, over twenty HDAC inhibitors have entered clinical studies and two of them have already reached the market, namely the hydroxamic acid derivative SAHA (vorinostat, Zolinza) and the cyclic depsipeptide FK228 (romidepsin, Istodax) that have been approved for the treatment of cutaneous T-cell lymphoma (CTCL). A common aspect of the first HDAC inhibitors is the absence of any particular selectivity towards specific isozymes. Some of molecules resulted to be “pan”-HDAC inhibitors, while others are class I selective. In the meantime, the knowledge of HDAC biology has continuously progressed. Key advances in the structural biology of various isozymes, reliable molecular homology models as well as suitable biological assays have provided new tools for drug discovery activities. This Minireview aims at surveying these recent developments as well as the design, synthesis and biological characterization of isoform-selective derivatives.

Anticancer activity of SAHA, a potent histone deacetylase inhibitor, in NCI-H460 human large-cell lung carcinoma cells in vitro and in vivo

Suberoylanilide hydroxamic acid (SAHA), a potent pan-histone deacetylase (HDAC) inhibitor, has been clinically approved for the treatment of cutaneous T-cell lymphoma (CTCL). SAHA has also been shown to exert a variety of anticancer activities in many other types of tumors, however, few studies have been reported in large-cell lung carcinoma (LCC). Our study aimed to investigate the potential antitumor effects of SAHA on LCC cells. Here, we report that SAHA was able to inhibit the proliferation of the LCC cell line NCI-H460 in a dose- and time-dependent manner, induced cell apoptosis and G2/M cell cycle arrest, decreased AKT and ERK phosphorylation, inhibited the expression of pro-angiogenic factors (VEGF, HIF-1α) in vitro, and suppressed tumor progression in an NCI-H460 cell nude mouse xenograft model in vivo. These results indicate that SAHA can exert its strong antitumor effects in LCC patient.

STAT3 Serine Phosphorylation and HDAC Inhibition In CTCL

Phosphorylation of signal transducer and activator of transcription 3 (STAT3) is essential for cell survival, proliferation and differentiation. STAT3 phosphorylation results from signaling by cytokines and growth factors, and constitutive STAT3 activity is characteristic of a number of human malignancies, including Cutaneous T Cell Lymphoma (CTCL). Furthermore, we now know that STAT3 is also required for the initiation and maintenance of the Th17 differentiation program. Th17 cells are a subset of CD4 T helper cells that have been implicated in chronic inflammatory conditions like rheumatoid arthritis and psoriasis. Mycosis fungoides (MF) and the leukemic variant of this disease, Sezary syndrome (SS), are the most frequently encountered forms of CTCL and in both of these diseases, the cell of origin – as far as the type of Teffector cell involved, has not been defined. Recent results from our laboratory and that of our colleagues have lead us to believe that Th17 cells may either be the cells of origin in CTCL or may act as critical mediators of chronic inflammation that creates a favorable environment for tumor growth in the context of this malignancy.In an effort to elucidate the role of STAT3 as a transforming factor in T cell malignancies, we generated a mouse model wherein T cell specific expression of a hyper-active STAT3 mutant protein (STAT3C) leads to the development of a lymphoproliferative disease that is highly reminiscent of CTCL. We are now taking advantage of this unique mouse model, patient biospecimens and carefully characterized CTCL cell lines to dissect the role of STAT3 signaling cascade in the malignant transformation and maintenance of CTCL.Most recently, our attention has been focused on understanding the mechanism of action of epigenetic therapy in the form of histone deacetylase inhibitors (HDACi), which is highly effective in the treatment of CTCL. We hypothesize that HDAC inhibitors affect the STAT3 mediated Th17 differentiation and thus have clinical efficacy in this disease. In addition to the regulation of chromatin accessibility through the regulation of histone modifications, HDACi have also been implicated in a less conventional mode of protein regulation directly influencing STAT3 serine phosphorylation. To dissect the action of HDACi on malignant cells, we took advantage of CTCL cell lines and cultured these with and without Romidepsin, which is an effective HDAC inhibitor used in clinic. MyLa2059 and PB2B are MF cell lines with skin only phenotype whereas SeAx and SeZ4 are SS cell lines. The cells were cultured for 48 hours with no Romidepsin, 5nm and 50 nm Romidepsin. After 48 hours, cells were fixed and permeabilized using BD fix-perm protocol. Cells were then stained to assess Serine 727 STAT3 and Tyrosine Y705 STAT3 phosphorylation and analyzed using flowcytometry. We found that Romidepsin affected serine phosphorylation exclusively in CTCL cell lines (Figure 1). This leads us to believe that STAT3 serine phosphorylation might play an important role in lymphomagenesis and can act as a potential therapeutic target. [Display omitted] The role of serine phosphorylation in the context of STAT3 signaling is hotly debated and we are now attempting to characterize the role of Serine STAT3 phosphorylation in the context of CTCL. We are also hoping to recapitulate these observations in patients' biospecimens collected before and after treatment with HDAC inhibitors. We will also study the role of serine phosphorylation in STAT3 activity in carcinogenesis using our mouse model with phenotypic and pathological characteristics similar to CTCL. We hope that these studies will advance our knowledge about the role that Stat 3 signaling plays in MF/SS malignant transformation and cancer progression and help us develop target specific treatment options for the clinical practice. Disclosures:Hymes:Celgene: Consultancy.

RP6530, a Dual PI3Kδ/γ Inhibitor, Attenutates AKT Phosphorylation and Induces Apoptosis In Primary Cutaneous T Cell Lymphoma (CTCL) Cells

Background Mycosis fungoides (MF) is the most common form of CTCL. The peripheral blood of MF patients is variably involved with malignant skin-homing T cells, with Sézary syndrome representing an advanced form characterized by erythroderma and frank leukemic expansion of the cells. The Class 1A phosphoinositide-3-kinase (PI3K) signaling cascade is involved in control of a variety of cellular responses including cell-cycle and anti-apoptotic pathways. Inappropriate activation / phosphorylation of the downstream effector, Akt, via dysregulated PI3K signaling has been detected in a variety of cancers including CTCL, and may contribute to proliferation and survival of malignant cells. While the α and β isoforms of PI3K are ubiquitous in their distribution, expression of δ and γ is majorly associated with immune cells including T-lymphocytes. Selective pharmacologic targeting of PI3K δ/γ may therefore have therapeutic potential. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor. RP6530 demonstrates high potency against PI3Kδ (IC50=25 nM) and γ (IC50=33 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency has been confirmed in target-specific assays, namely anti-FcεR1-(EC50=38 nM) or fMLP (EC50=39 nM) induced CD63 expression in human whole blood basophils, LPS-induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS-induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). In this study, we examined the effects of RP6530 on Akt phosphorylation in CTCL cell lines HH and Sez4 and in purified malignant T cells from six CTCL patients exhibiting Sézary Syndrome. Methods Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Malignant T cells were isolated by negative selection using an antibody plus magnetic bead kit (Miltenyi Biotec) designed for CD4+ T cell isolation supplemented with patient-specific anti-CD7 and/or CD26 antibodies. Purity of isolated cells as tested by flow cytometry was >96%. Purified malignant T cells were cultured in RPMI+1% BSA overnight then treated ± inhibitor for 1.5 hr, with serum plus a cytokine mix (IL2+7+15) added for the final 30 minutes to maximize Akt phosphorylation. The ratio of phosphorylated, pSer473-Akt to total Akt was then measured by phospho-flow cytometry. Results RP6530 caused dose-dependent inhibition of Akt Ser473 phosphorylation, with significant inhibition detectable even at the lowest concentration tested (10 nM, P = 0.035) and reaching 39.29% at 1000 nM. Inhibition observed was significantly greater (P = 0.0133) than that seen with the potent PI3K control inhibitor LY294002 (27.50%) at the same concentration. Non-linear regression analysis (Prism 5.0) of the mean pSer473-Akt:total-Akt ratio indicated the IC50 for RP6530 (198.2 nM) at ∼4-fold lower than for LY (939.7 nM). In addition, apoptosis induction as measured by Annexin V binding, was tested in purified malignant T cells from four Sezary Syndrome patients. Following a 48hr incubation with RP6530, a dose-dependent increase in late apoptotic, Annexin V+ Propidium Iodide (PI)+, cells was seen. The compound was effective even at lower concentrations, with 60.15% more Annexin V+ PI+ cells seen in cultures treated with 100 nM inhibitor than untreated cultures (P= 0.027), and with a 120.43% increase seen at the highest concentration tested (10 μM). Conclusion Results from in vitro studies suggest the therapeutic potential of RP6530 in the treatment of CTCL with peripheral blood involvement. Findings provide a rationale for future clinical trials in T-cell malignancies. Disclosures: Girardi: Rhizen Pharmaceuticals S.A.: Research Funding. Vakkalanka:Rhizen Pharmaceuticals, S.A.: Employment, Equity Ownership; Incozen Therapeutics Pvt. Ltd.: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics Pvt. Ltd.: Employment. Bertoni:Rhizen Pharmaceuticals SA: Research Funding.