Treatment Of Cutaneous T-cell Lymphoma Research Articles

Bortezomib inhibits expression of TGF-β1, IL-10, and CXCR4, resulting in decreased survival and migration of cutaneous T cell lymphoma cells.

Increased expression of the immunosuppressive cytokines, TGF-β1 and IL-10, is a hallmark of the advanced stages of cutaneous T cell lymphoma (CTCL), where it has been associated with suppressed immunity, increased susceptibility to infections, and diminished antitumor responses. Yet, little is known about the transcriptional regulation of TGF-β1 and IL-10 in CTCL, and about their function in regulating the CTCL cell responses. In this article, we show that TGF-β1 and IL-10 expression in CTCL cells is regulated by NF-κB and suppressed by bortezomib (BZ), which has shown promising results in the treatment of CTCL. However, although the TGF-β1 expression is IκBα dependent and is regulated by the canonical pathway, the IL-10 expression is IκBα independent, and its inhibition by BZ is associated with increased promoter recruitment of p52 that characterizes the noncanonical pathway. TGF-β1 suppression decreases CTCL cell viability and increases apoptosis, and adding exogenous TGF-β1 increases viability of BZ-treated CTCL cells, indicating TGF-β1 prosurvival function in CTCL cells. In addition, TGF-β1 suppression increases expression of the proinflammatory cytokines IL-8 and IL-17 in CTCL cells, suggesting that TGF-β1 also regulates the IL-8 and IL-17 expression. Importantly, our results demonstrate that BZ inhibits expression of the chemokine receptor CXCR4 in CTCL cells, resulting in their decreased migration, and that the CTCL cell migration is mediated by TGF-β1. These findings provide the first insights into the BZ-regulated TGF-β1 and IL-10 expression in CTCL cells, and indicate that TGF-β1 has a key role in regulating CTCL survival, inflammatory gene expression, and migration.

Recent advances in primary cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of skin-homing T-cell neoplasms, which represent approximately 75% of all primary cutaneous lymphomas. Currently available drug therapies, when effective, simply control disease and the only option for curing CTCL is stem cell transplant. In the last year, there has been an incredible effort made to improve the understanding and treatment of CTCL. Recent findings indicate that epigenetic aberrations are integral to active disease. Furthermore, multiple tumor-derived immunological factors have also been shown to inhibit viability, proliferation, and cytokine production of nonmalignant T cells. Several novel targeted therapies show great potential, most promising being antibody drug conjugates targeting surface markers such as CD30 in some CTCL subtypes. Additional attractive targets involve the global modulation of epigenetic markers such as demethylation agents or HDAC inhibitors, either as single agents or in combination therapies. This is a concise review of recent advances in the field of CTCL with special focus on research articles over the preceding year.

Treating cutaneous T-cell lymphoma with highly irregular surfaces with photon irradiation using rice as tissue compensator.

Purpose: Cutaneous T-cell lymphoma (CTCL) is known to have an excellent response to radiotherapy, an important treatment modality for this disease. In patients with extremity and digit involvement, the irregular surface and depth variations create difficulty in delivering a homogenous dose using electrons. We sought to evaluate photon irradiation with rice packing as tissue equivalence and determine clinical tolerance and response.Materials and methods: Three consecutive CTCL patients with extensive lower extremity involvement including the digits were treated using external beam photon therapy with rice packing for tissue compensation. The entire foot was treated to 30–40 Gy in 2–3 Gy per fraction using 6 MV photons prescribed to the mid-plane of an indexed box filled with rice in which the foot was placed. Treatment tolerance and response were monitored with clinical evaluation.Results: All patients tolerated the treatment without treatment breaks. Toxicities included grade 3 erythema and desquamation with resolution within 4 weeks. No late toxicities were observed. All patients had a partial response by 4 weeks after therapy with two patients achieving a complete response. Patients reported improved functionality after treatment. No local recurrence has been observed.Conclusion: Tissue compensation with rice packing offers a convenient, inexpensive, and reproducible method for the treatment of CTCL with highly irregular surfaces.

Successful implementation of a rural extracorporeal photopheresis program for the treatment of cutaneous T-cell lymphoma and chronic graft-versus-host disease in a rural hospital.

Extracorporeal photopheresis (ECP) is currently standard therapy for cutaneous T-cell lymphoma (CTCL) and Graft-versus-host disease (GVHD). Of the many challenges associated with outpatient ECP treatments, commuter travel to capable facilities can fragment and compromise the patient care. In 2008, our hospital implemented an ECP program providing patients to a treatment center over 120 minutes away. This study was undertaken to describe our experience with the establishment of a regional ECP program. A retrospective review using a standardized template was performed of patients treated from May 2008 to 2012. The response to treatment was analyzed after a minimum of eight procedures. A partial response to treatment in individuals with CTCL, was more than 50% skin improvement, and GVHD, a reduction in steroid dose by 50%, liver function test improvement or documented improvement in skin findings. Of the 34 patients treated, 11 were for CTCL and 23 for GVHD. 95.8% of the 1,071 planned procedures were successfully. The average procedure time was 186 min for the UVAR-XTSTM and 93 min for the CELLEXTM. Patients travelled a median of 65.7 miles (range 4-133 miles). The median duration of therapy was 6 months (range 2-23) for CTCL and 5 months (range 1-27) for GVHD. A clinical benefit was observed in 7 of 11 (63.6%) patients with CTCL and in 15 of 23 (65.2%) with GVHD. Our regional ECP program was a viable option in improving access to care for patients requiring treatment for CTCL and chronic GVHD.

ChemInform Abstract: Novel Aromatic and Antiaromatic Systems

AbstractReview: 48 refs.

The utility of bexarotene in mycosis fungoides and Sézary syndrome.

Cutaneous T-cell lymphoma (CTCL) is an umbrella term that encompasses a group of neoplasms that have atypical T-lymphocytes in the skin. Mycosis fungoides (MF) is the most common type of CTCL and Sézary syndrome (SS) is the leukemic form. Treatment for CTCL is dependent on the stage of disease and response to previous therapy. Therapy is divided into skin-directed treatment, which tends to be first line for early-stage disease, and systemic therapy, which is reserved for refractory CTCL. Bexarotene is a rexinoid and was licensed in Europe in 2002 for use in patients with advanced disease that have been refractory to a previous systemic treatment. We review the use of bexarotene as monotherapy and in combination with other treatments.

Identification of p38β as a Therapeutic Target for the Treatment of Sézary Syndrome

Cutaneous T-Cell Lymphomas (CTCL) represent a group of hematopoietic malignancies that home to the skin and have no known molecular basis for disease pathogenesis. Sézary syndrome (SS) is the leukemic variant of CTCL. Currently, CTCL is incurable, highlighting the need for new therapeutic modalities. We have previously observed that combined smallmolecule inhibition of protein kinase C (PKC) β and glycogen synthase kinase 3 (GSK3) causes synergistic apoptosis in CTCL cell lines and patient cells. Through microarray analysis of a SS cell line, we surveyed global gene expression following combined PKCβ-GSK3 treatment to elucidate therapeutic targets responsible for cell death. Clinically relevant targets were defined as genes differentially expressed in SS patients that were modulated by combination-drug treatment of SS cells. Gene set enrichment analysis uncovered candidate genes enriched for an immune cell signature, specifically the T-cell receptor and MAPK signaling pathways. Further analysis identified p38 as a potential therapeutic target that is over-expressed in SS patients and decreased by synergistic-inhibitor treatment. This target was verified through small-molecule inhibition of p38 leading to cell death in both SS cell lines and patient cells. These data establish p38 as a SS biomarker and potential therapeutic target for the treatment of CTCL.

Deacetylation of chromatin and gene expression regulation: a new target for epigenetic therapy.

Besides the genetic information thath is encoded by DNA, heritable information can also be passed on without relying on changes in the nucleotide sequence of DNA, a phenomenon known as epigenetics. Gene expression in eukaryotes is partly regulated by epigenetic mechanisms both at the DNA and histone protein levels. Chromatin structure can be influenced by various modifications, including the reversible posttranslational processes of acetylation and deacetylation of DNA-binding proteins. Histone acetyl transferase (HAT) is referred to as the writer of this process, whereas histone deacetylase (HDAC) is the eraser of this lysine modification. Dysregulation of gene expression and changes in the HDAC expression profile have been associated with carcinogenesis, and HDAC inhibitors are already approved for the treatment of cutaneous T-cell lymphoma and peripheral T-cell lymphoma. These inhibitors are able to influence epigenetic processes by targeting HDAC activity, increasing nuclear histone acetylation status, and contributing to chromatin remodeling, thereby affecting gene expression. In addition, HDACs also act on a plethora of cytosolic proteins with many cellular functions, including angiogenesis, immune responses, and autophagy. In this review, we will give an overview of histone deacetylase and how it can regulate gene expression at the chromatin level.

Bexarotene and Hypopituitarism

ABSTRACT Objective: Bexarotene is currently approved for treatment of cutaneous T-cell lymphoma and is also under investigation for other cancers such as lung, breast, and thyroid cancer. We recently reported a patient with mycosis fungoides treated with bexarotene. The treatment induced hypopituitarism characterized by hypogonadism, hypothyroidism, and hypoadrenalism, which had not been previously reported. Methods: Here we report a second case with the same features and emphasize the possible risk of adrenal insufficiency during bexarotene treatment. Results: A 59 year-old male patient was diagnosed with anaplastic lymphoma. He was treated with 6 cycles of chemotherapy and interferon alfa 2b in the first 2 years of his treatment. Later, bexarotene treatment was initiated because lymphoma progression was observed. The patient developed hypopituitarism characterized by hypogonadism, hypothyroidism, and hypoadrenalism 2 months after bexarotene therapy. Conclusions: Clinicians should have a high index of su...

Denileukin Diftitox (Ontak) as Maintenance Therapy for Peripheral T-Cell Lymphomas: Three Cases with Sustained Remission.

Peripheral T-cell lymphomas (PTCL) are rare but markedly aggressive forms of non-Hodgkin's lymphoma (NHL). They carry a poor prognosis, with current therapeutic approach being generally ineffective. The most employed first-line treatment is CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), which still results in high rates of relapses. Denileukin diftitox is a fusion protein combining the cytotoxic portion of the diphtheria toxin and the receptor-binding domain of the interleukin-2 (IL-2) molecule, thereby targeting cells expressing the IL-2 receptor, including both T-cell and B-cell lymphomas. It has been approved for the treatment of cutaneous T-cell lymphomas, and it has documented activity in PTCL both as a single agent and as part of combination therapy. This report documents three cases of PTCL where denileukin diftitox has been used as long-term maintenance therapy after complete remission was achieved. While the overall survival rate of patients with advanced stage, refractory PTCL is generally poor (with median overall survival of 5.5 months), the three patients described in this report are all experiencing an ongoing complete remission for more than four years.

Extracorporeal photopheresis as a therapy for autoimmune diseases.

Systemic autoimmune diseases (AID) have multiorgan, heterogeneous clinical presentations and are characterized by dysregulation of the immune system, immunodeficiency, irreversible organ damage and increased morbidity and mortality. Preventing or decreasing flares of AID correlate with durable disease control, significant reduction of inflammation and prevention of disability or therapy-related toxicity. There is an urgent need for better treatment of severe, therapy-refractory AID. Extracorporeal photopheresis (ECP) is a cell-based immunomodulatory treatment which has been extensively used in variety of autoimmune disorders for the last two decades. ECP treatment is FDA approved for the treatment of cutaneous T-cell lymphoma (CTCL) with particularly promising results seen in graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HCT). Prolonged therapy is safe, well tolerated and allows reduction of systemic immunosuppression in therapy-refractory patients. Both clinical and experimental evidence suggest that ECP mechanism of action is characterized by apoptosis and phagocytosis of activated cells by antigen-presenting cells (APC), secretion of anti-inflammatory cytokines and stimulation of regulatory T cells (Tregs). The focus of this paper is to review the current evidence of ECP use in the treatment of AID. Here, we summarize the experience of nine major AID from 65 published reports. The key findings demonstrate substantial evidence of ECP feasibility, safety and in some AID also promising efficacy. However, the role of ECP in AID therapy is not established as most published studies are retrospective with limited number of patients and the trials are small or poorly standardized. The available data support future investigations of ECP as a therapeutic modality for the treatment of AID in well-designed prospective clinical studies. J

Retinoids Selectively Induce Gut-Homing Properties in Cutaneous T Cell Lymphoma (CTCL) Cells

Abstract Introduction: Cutaneous T cell lymphoma (CTCL) is a heterogeneous group of malignancies characterized by accumulation of malignant T cells within skin. Retinoids, metabolic derivatives and synthetic analogs of vitamin A, naturally imprint gut homing in T lymphocytes by inducing integrin alpha4beta7 and CCR9 expression which effectively targets the immune cells away from the skin niche. Although successfully used in CTCL therapy for decades, the therapeutic mechanism of action of retinoids in resolving CTCL remains largely speculative. How the natural role of retinoids in immune cell trafficking relates or applies to therapeutic effectiveness in CTCL has not been addressed and merits investigation. Here we provide the first evidence that the natural role of retinioids in governing immune cell localization to the gut accounts for their therapeutic benefit in CTCL. Methods: Changes in cell surface integrin levels of T cell lineages, 2 CTCL cells lines (MJ and HuT78) and 3 non-CTCL cell lines (Jurkat, Molt4 and CCRFCEM), exposed to retinoid was determined by flow cytometry. Static cell adhesion assays to various immune trafficking ligands were performed to establish if retinoids bias integrin function that would occlude skin localization and occupancy. Function blocking or activating monoclonal antibodies against specific integrin subunits were employed to delineate the specific integrin heterodimers required for retinoid-prompted adhesion. Static adhesion results were verified in soluble binding assays. TransWell migration assays were utilized to address changes in cell migration that occurred upon retinoid exposure. Results: Retinoids induced integrin beta7 subunit expression on CTCL cell surfaces and enhanced beta7-mediated cell adhesion in multiple CTCL cell lines, but not in non-CTCL lineages. The retinoid-induced adhesion observed was selective as adhesion increased to the gut-specific ligand MAdCAM-1, but not to other trafficking ligands such as VCAM-1 or fibronectin. Interestingly, retinoid-induced CTCL cell adhesion to MAdCAM-1 was potently attenuated by 1,25-dihydroxyvitamin D3, a metabolic vitamin derivative involved in inducing skin-homing immune properties. Of clinical relevance, this work entailed the use of Bexarotene, an RXR agonist and the only FDA-approved retinoid for CTCL therapy. Since immune cell trafficking involves the progressive involvement of a variety of protein ligands and receptors including chemokine receptors, we extended our study to address the migratory properties of CTCL cells. Bexarotene significantly increased the migration of CTCL cells towards the gut chemokine CCL25/TECK as compared to vehicle treated cells. Conclusions: Retinoids imprint gut homing properties selectively within CTCL cells by inducing beta 7 integrin expression and function, which may account, in part, for the therapeutic benefit of retinoids in CTCL. Retinoids, through their natural role in establishing and maintaining mucosal immunity, plausibly occlude further trafficking or terminate the residency of the malignant T cells within the skin niche. This action may predispose and heighten the susceptibility of malignant cells to additional therapies (e.g. UV light) commonly used in combination with Bexarotene. Our data establish that the natural role of retinoids in immune cell localization embody an unexplored therapeutic mechanism in CTCL treatment. Disclosures No relevant conflicts of interest to declare.

Phase II Investigator-Initiated Study of Brentuximab Vedotin in Mycosis Fungoides or Sezary Syndrome: Final Results Show Significant Clinical Activity and Suggest Correlation with CD30 Expression

Background: Brentuximab vedotin (BV) is an anti-CD30 chimeric antibody conjugated by a protease-cleavable linker to the microtubule-disrupting agent, MMAE. While CD30 expression of malignant cells in HL and ALCL is uniform, in mycosis fungoides/Sezary syndrome (MF/SS), the CD30 expression is more variable. We reported previously (ASH 2012) that BV has clinical activity in MF/SS across all CD30 expression levels. In this final report, we update the clinical data and present new biomarker and correlative tissue analyses.Methods: Patients (pts) with MF/SS stages IB-IV, ECOG 0-2, with at least 1 prior systemic therapy were enrolled in this investigator-initiated, phase II, single-arm study. CD30 expression levels in the skin (% of total mononuclear cell infiltrate) were evaluated by routine immunohistochemistry (IHC) using the BerH2 antibody. At least 2 skin biopsies were obtained per pt and the max CD30 level was used for CD30 designation. All pts (CD30, 0-100%) were treated with up to 8 cycles of BV (1.8 mg/kg) administered every 3 weeks; optional extension of up to 8 additional cycles was allowed in responders with ongoing clinical improvement. Primary endpoint was overall response rate (ORR) by consensus global response criteria. CD30 expression and clinical response data were confirmed by independent review. Tumor microenvironment was assessed utilizing IHC for CD8, CD20, CD163, FoxP3, and PD-1. Multispectral image analysis was utilized to evaluate CD30 antigen co-expression.Results: 32 pts were enrolled and all received at least one dose of BV (Table); all included in safety data, 2 excluded in efficacy analyses (no response assessment). Median age was 62 (20-87). 28/32 (88%) had advanced disease, majority with adverse prognostic features (90% large cell transformation or folliculotropism). ORR was 70% (21/30) with responses in all clinical stages. Median best mSWAT reduction (response measure in the skin) was 73% (range, 100% to -54%) with 1 CR, 7 near CR in skin (>90% reduction) (Fig 1). Median time to response was 6.6 weeks (range 3.0-27.0). Kaplan-Meyer estimate shows 79% of responses ongoing and 54% progression-free at 12 months. Related AEs observed were mostly grade 1-2 and limited to AEs previously reported with BV. By IHC, median max CD30 expression level of all pts was 13% (range 0-100%; 6 had <5%), where the median was higher in responders (CR/PR), 15%, vs. non-responders (SD/PD), 3.0%, p=0.037 (Fig 1). The correlation with CD30 level was greatest in IIB/skin tumor subset (p=0.0072). Those with CD30 level <5% had a lower likelihood of clinical response (17% vs 83%; p=0.0046). Multispectral image analysis demonstrated quantifiable CD30 staining in 19/20 (95%) samples that were interpreted as negligible CD30 by routine IHC. Of other tissue biomarkers evaluated, CD163+ cells (macrophages) were most abundant with a median of 40% (range 5-80%) of total infiltrate. To distinguish expression of CD30 by neoplastic CD4+ vs. tumor-infiltrating cells, double staining for CD30 and CD8 or CD163 was assessed. We observed significant co-expression of CD30 by CD163+ macrophages (median 9.7% of total cells assessed, range 1.0-40.9%) or CD8+ cytotoxic T cells (median 1.7%, range 0.2-14.8%) (Fig 2). Those with lower CD163 relative to CD30 stain (CD163/CD30 ratio) by routine IHC were associated with clinical response (p=0.033). This may simply reflect the influence of CD30 level; however, in light of the co-localization data, it might also suggest that infiltrating macrophages that co-express CD30 may play a role as a microenvironment factor in clinical response to BV.Conclusions: Our final analysis confirms significant clinical activity of BV in MF/SS with expected AE profile. Although clinical response was observed at all levels of CD30 expression, the likelihood of response was lower in those with negligible or very low (<5%) level. We utilized multispectral imaging to enhance detection of CD30 and also show co-expression of CD30 by tumor-infiltrating cells. These findings suggest that further studies of biomarkers are warranted and may help optimize management strategies with BV.TableCharacteristicAll Ptsn=32Evaluablen=30ORRn (%)n (%)CRPRSDPDClinical StageAll pts IB IIB IV/SS32 (100) 4 (13) 18 (56) 10 (31)1 0 0 120 3 14 34 1 2 15 0 2 321/30 (70) 3/4 (75) 14/18 (78) 4/8 (50)Transformed or folliculotropic MF29 (91)1193520/28 (71)# prior systemic tx<3 >=315 (47) 17(53)0 18 122 24 18/14 (57) 13/16 (81) [Display omitted] [Display omitted] DisclosuresKim:Actelion: Consultancy; Celgene: Advisory, Advisory Other; Eisai: Research Funding, SAC/Advisory, SAC/Advisory Other; Galderma: Advisory, Advisory Other; Millennium/Takeda: Research Funding, SAC/Advisory Other; Kyowa-Hakko-Kirin: Research Funding, SAC/Advisory Other; Oncosec: Advisory Other; Seattle Genetics: Advisory, Advisory Other, Research Funding; SHAPE: Consultancy, Research Funding. Off Label Use: Treatment of cutaneous T-cell lymphoma, also recently added to NCCN NHL practice guidelines in CTCL. Wood:Millennium: IDMC Other. Advani:Seattle Genetics: Research Funding. Horwitz:Millennium: Consultancy, Research Funding; Infinity: Research Funding; Kiowa-Kirin: Research Funding; Seattle Genetics: Consultancy, Research Funding; Spectrum: Research Funding; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding.

MK2a Substrate-Selective p38 Inhibitor CMPD1 Selectively Synergizes with HDAC Inhibitors in CD34+ Cells from Myeloid Malignancies

Background: Histone deacetylase (HDAC) inhibitors (HDIs) vorinostat (SAHA) and romidepsin are approved for the treatment of cutaneous T-cell lymphoma, while belinostat has recently been approved for the treatment of peripheral T-cell lymphoma. SAHA exhibits clinical activity in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), although insufficient to justify single-agent therapy for these indications. The precise clinical mechanism of SAHA and other HDIs remains incompletely characterized, impeding the design and implementation of rational HDI-based therapeutic combinations for myeloid malignancies. To circumvent this lack of mechanistic understanding, we conducted RNA-interference (RNAi) modifier screens of the kinome and phosphatome to identify signaling pathways that modulate SAHA (HDI) anti-leukemic activity in AML cell lines TF-1, HEL and THP-1 for rapid translation of findings from ex vivo validation to clinical application.Results: Multiple screen hits (both kinases and phosphatases) converge on distinct points of the p38-SAPK/JNK signaling pathways, suggesting involvement of these stress-activated pathways in SAHA anti-leukemic activity. However, the most well characterized p38α/β inhibitors SB202190 and LY2228820 do not augment SAHA anti-leukemic activity at any dose across the broad dose ranges tested. Similarly, the JNK inhibitor SP600125 does not modulate SAHA activity at doses that inhibit JNK. However, the putative MK2a substrate-selective p38 inhibitor CMPD1 (Boehringer-Ingelheim; Davidson, W., et. al., Biochemistry 2004 Sep 21;43(37):11658-71) selectively potentiates the activity of SAHA in all AML cell lines tested (N=8), as well as in ex vivo cultures of primary myeloid malignancies (N=14), including polycythemia vera, chronic myelomonocytic leukemia, MDS/myeloproliferative neoplasm overlap syndrome, MDS transformed to AML, and de novo AML. The observed dose-dependent synergy occurs at nanomolar doses consistent with the reported apparent inhibitory constant for CMPD1 inhibition of p38-dependent phosphorylation of MK2a. Further, SAHA + CMPD1 synergy is significantly greater in CD34+-selected as compared to CD34+-depleted cell populations ex vivo. Furthermore, CMPD1 is similarly synergistic with the HDI panobinostat in vitro, but interacts antagonistically with cytarabine and additively with 5-azacitidine. This suggests that CMPD1 interacts specifically with SAHA and panobinostat in AML. The synergistic interaction between CMPD1 and SAHA is independent of ROS induction, as pretreatment with N-acetylcysteine does not abrogate synergy, despite reducing single-agent SAHA activity. While CMPD1 is reported to be an MK2a substrate-selective p38 inhibitor, CMPD1 does not affect the regulation of several canonical MK2 targets at doses that are potently synergistic in AML cell lines, including the phosphorylation of HSP27 or AATF. To begin to probe the mechanism of CMPD1 and SAHA synergy more extensively, mRNA expression was measured by next-generation sequencing after SAHA and CMPD1 treatment alone and in combination in the AML cell line TF-1. Sequencing data is currently being analyzed and findings will be presented.Conclusion: RNAi knockdown of several p38-SAPK/JNK pathway components modulates SAHA activity in malignant myeloid cells; however, common pharmacological sensitizing targets remain elusive. We hypothesize that MK2 and/or a yet unrecognized pathway modulated by CMPD1 can be targeted for HDI combination therapy in malignant myeloid cells. Current and ongoing results from this study will provide validated targets for rational HDI combination in myeloid malignancies. DisclosuresOff Label Use: LDE225/Sonidegib as investigational agent. Mesa:Incyte, CTI, NS pharma, Gilead, Celgene: Research Funding.

Bortezomib and Methotrexate Interfere with the DNA Repair Signaling Transduction Pathways and Induce Apoptosis in Cutaneous T-Cell Lymphoma

Introduction:Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas, derived from skin-homing mature T-cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are the commonest types and together comprise 54% of all CTCL. MF evolves from patches to infiltrated plaques and eventually tumors. SS is a lymphoma-leukemia syndrome characterized by erythroderma and the presence of a malignant T-cell clone in the peripheral blood and the skin. At present, no curative treatment for CTCL is available. Therefore current CTCL research efforts are focused on elucidating the molecular mechanisms of the disease’s pathogenesis and on identifying new pharmacological targets. Several drugs have shown potentially significant activity either alone or in combination with conventional agents. Their effectiveness and their mechanisms of action comprise a current research challenge for the improvement of CTCL therapy. The aim of this study was to investigate the possible alterations in the gene expression profile (focusing on DNA Damage Signaling and DNA Repair pathways) and cell death in CTCL cell lines after treatment with two chemotherapeutic agents, Bortezomib and Methotrexate.Methods:Three CTCL cell lines were used. MyLa, (MF), SeAx and Hut-78 (both SS). Cells were cultured in RPMI 1640 and were treated with either Bortezomib (10nmol/L) or Methotrexate (10μM) for 24h. Apoptosis was determined by flow cytometry using the Annexin V/PI method. Gene expression profiling following PCR arrays analysis was performed after total RNA extraction and purification from untreated and drug-treated cells. All RNA samples’ amplification, labeling and hybridization to RT2 Profiler PCR Arrays (DNA Damage Signaling and DNA Repair PCR array) (QIAGEN) were performed according to the manufacturer’s instructions. All data were analyzed using the appropriate RT2 Profiler PCR Array data analysis tool.Results:Hut-78, Seax and Myla cells responded with statistically significant enhanced apoptosis when treated for 24h with bortezomib, compared to untreated cells, while Methotrexate led to a rather moderate increase of apoptosis in Hut-78 and Seax cells and did not affect the apoptosis of Myla cells. Microarrays analysis after bortezomib treatment revealed a great effect in the expression profile of genes involved in almost all DNA repair pathways tested, in all three cell lines, with Hut-78 being affected the most. Specifically, in all cell lines, there was a significant down-regulation of a large number of genes involved in the Double Strand Breaks DNA Repair mechanism, (i.e. BRCA1, BRCA2, RAD50, RAD51, RAD51C, XRCC2, XRCC3, XRCC4, XRCC5 and XRCC6) as well as of genes involved in the Mismatch Repair pathway (i.e. MLH1, MLH3, MSH2, MSH5, MSH6) and the Nucleotide Excision Repair mechanism (i.e. DDB2, LIG1 and RAD23A), compared to untreated cells. On the contrary, bortezomib had a small effect on Base Excision repair mechanism, mostly downregulating the expression of XRCC1 gene in Hut-78 and Myla cells. Methotrexate treatment also led to a significant down-regulation of genes involved in the DSB (RAD50, XRCC4, XRCC6), MMR (MSH4) and NER (CDK7, RAD23A) repair mechanisms in Hut-78 cells but had a rather much more moderate effect on the expression profile of Seax and Myla cells, where fewer genes were affected.Conclusions:Our data clearly demonstrate a differential effect of bortezomib and methotrexate in terms of apoptosis induction on CTCL cells with bortezomib inducing apoptosis of both MF and SS derived cell lines and methotrexate being rather inactive on SS derived cells. We showed that both drugs, but mostly bortezomib significantly down-regulate a large number of genes involved in the DSB, MMR and NER mechanisms, suggesting a possible mechanism, among probably others, for the enhanced sensitivity to apoptosis of SS and MF cell lines after treatment. Bortezomib’s significant effect could be easily understood, since it is a well known proteasome inhibitor and has been previously related to inhibition of NF-kB and accumulation of pro-apoptotic proteins, while it has also been reported that cancer cells are more sensitive to proteasome inhibition than normal cells. Although these results need to be further confirmed, they appear very encouraging for understanding the mechanisms of action of these drugs in CTCL with the view to ameliorate their use in clinical practice. DisclosuresNo relevant conflicts of interest to declare.

SAHA triggered MET activation contributes to SAHA tolerance in solid cancer cells

Although SAHA is approved for the treatment of cutaneous T-cell lymphoma by the U.S. Food and Drug Administration, clinical trials using SAHA as a monotherapy or in combination with other chemotherapeutic agents in solid tumors have not met with success, and the mechanisms of tolerance remain unknown. In this study, using the prostate cancer cell line PC3 and the non-small lung cancer cell line A549, which have limited sensitivity to SAHA, we found that SAHA triggered MET and AKT phosphorylation at clinical concentrations. siRNA silencing of MET enhanced SAHA induced apoptosis in PC3 and A549 cells. However, MET protein expression and HGF secretion were not affected by SAHA, suggesting that the SAHA-induced MET activation was not due to MET over-expression or HGF paracrine secretion. However, mRNA and protein expression of the laminin receptor integrin α5β1 was up-regulated by SAHA prior to MET activation. Silencing of integrin α5β1 abolished SAHA-triggered MET phosphorylation, suggesting the involvement of integrin α5β1 in MET activation. Further, the combination of SAHA and XL184 resulted in a synergistic induction of cancer cell apoptosis and a synergistic inhibition of tumor growth. These data indicate that SAHA triggered MET activation in an HGF independent manner. This effect is partially involved in the resistance to SAHA in solid cancers, warranting further clinical investigation into combining SAHA with MET inhibitors in solid cancer treatment.

Suberoylanilide Hydroxamic Acid (SAHA)-Induced Dynamics of a Human Histone Deacetylase Protein Interaction Network

Histone deacetylases (HDACs) are targets for cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma. To obtain a better mechanistic understanding of the Sin3/HDAC complex in cancer, we extended its protein-protein interaction network and identified a mutually exclusive pair within the complex. We then assessed the effects of SAHA on the disruption of the complex network through six homologous baits. SAHA perturbs multiple protein interactions and therefore compromises the composition of large parts of the Sin3/HDAC network. A comparison of the effect of SAHA treatment on gene expression in breast cancer cells to a knockdown of the ING2 subunit indicated that a portion of the anticancer effects of SAHA may be attributed to the disruption of ING2's association with the complex. Our dynamic protein interaction network resource provides novel insights into the molecular mechanism of SAHA action and demonstrates the potential for drugs to rewire networks.

Novel aromatic and antiaromatic systems.

We contributed to the field of non-benzenoid aromatic compounds by creating the cyclopropenyl cation and various of its derivatives, including cyclopropenone; it was the first aromatic system with other than six pi electrons in a single ring, and the simplest aromatic system. The pioneering work of Tetsuo Nozoe in tropolone chemistry was celebrated with the founding of ISNA, the International Symposium on Non-Benzenoid Aromatic Compounds, where I described our work in the field. It fit the prediction that aromaticity would be found in systems with 4n + 2 pi electrons, where n is an integer. I was also concerned with the properties of monocyclic systems with 4n cyclically conjugated pi electrons. They were expected not to be aromatic, but the interesting question was whether they were actually antiaromatic, especially destabilized by the cyclic conjugation in such 4n species as the cyclopropenyl anion, cyclobutadiene, and cyclopentadienyl cation. The evidence supports antiaromaticity in these cases. We also examined compounds where 4n cyclic pi systems were fused with aromatic systems, and most interestingly systems in which two 4n pi systems were fused. In these cases the periphery of the molecules had 4n + 2 pi electrons, for aromaticity, but the components were antiaromatic. Recently we have studied electrical conductivities in aromatic molecules such as thiophene and saw that aromaticity added resistance to the systems, so non-aromatic compounds are better conductors and antiaromatic compounds are predicted to be the best of all.

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 &amp;gt;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