Apoptosis In Cutaneous T-cell Lymphoma Research Articles

XPO1 blockade with KPT-330 promotes apoptosis in cutaneous T-cell lymphoma by activating the p53–p21 and p27 pathways

Dysregulated nuclear-cytoplasmic trafficking has been shown to play a role in oncogenesis in several types of solid tumors and hematological malignancies. Exportin 1 (XPO1) is responsible for the nuclear export of several proteins and RNA species, mainly tumor suppressors. KPT-330, a small molecule inhibitor of XPO1, is approved for treating relapsed multiple myeloma and diffuse large B-cell lymphoma. Cutaneous T-cell lymphoma (CTCL) is an extranodal non-Hodgkin lymphoma with an adverse prognosis and limited treatment options in advanced stages. The effect of therapeutically targeting XPO1 with KPT-330 in CTCL has not been established. We report that XPO1 expression is upregulated in CTCL cells. KPT-330 reduces cell proliferation, induces G1 cell cycle arrest and apoptosis. RNA-sequencing was used to explore the underlying mechanisms. Genes associated with the cell cycle and the p53 pathway were significantly enriched with KPT-330 treatment. KPT-330 suppressed XPO1 expression, upregulated p53, p21WAF1/Cip1, and p27Kip1 and their nuclear localization, and downregulated anti-apoptotic protein (Survivin). The in vivo efficacy of KPT-330 was investigated using a bioluminescent xenograft mouse model of CTCL. KPT-330 blocked tumor growth and prolonged survival (p < 0.0002) compared to controls. These findings support investigating the use of KPT-330 and next-generation XPO1 inhibitors in CTCL.

P-153 - Inhibition of XPO1 with Selinexor (KPT-330) triggers apoptosis in cutaneous T-cell lymphoma (CTCL) by activating the p53-p21 and p27 pathways

P-153 - Inhibition of XPO1 with Selinexor (KPT-330) triggers apoptosis in cutaneous T-cell lymphoma (CTCL) by activating the p53-p21 and p27 pathways

Epigenetic Regulation of Apoptosis in Cutaneous T-Cell Lymphoma: Implications for Therapy with Methotrexate, Jak Inhibitors, and Resveratrol

Previously, we showed that deficiencies in apoptotic factors occur in cutaneous T-cell lymphoma (CTCL) and can be enhanced through epigenetic mechanisms (Stutz et al., 2012; Wu and Wood, 2011; Wu et al., 2009). Now, we have further explored the epigenetic regulation of apoptosis in CTCL with special attention to the DNA methyltransferase (DNMT)/signal transducer and activator of transcription (STAT) 3 promoter methylation complex responsible for silencing several tumor suppressor genes, including those involved in apoptosis.

160 Mechanisms governing promoter methylation and apoptosis in cutaneous T-cell lymphoma: implications for therapy with methotrexate and JAK-inhibitors

Cutaneous T-cell lymphoma (CTCL) is characterized by resistance to apoptosis involving dysregulation of the FAS ligand (FASL) - FAS death receptor pathway. CTCL cells also exhibit constitutively activated STAT proteins, which are known to upregulate and bind DNA methyltransferases (DNMTs). We hypothesized that, like many other tumor suppressor genes, FAS and FASL expression is at least partially regulated by promoter methylation. Earlier, we showed that methotrexate (MTX), a known folate antagonist, acts as a DNA methylation inhibitor via depleting the methyl donor, S-adenosylmethionine (SAM), in CTCL cells. Pyrosequencing analysis of FAS and FASL promoters showed that MTX inhibited methylation of certain CpG sites in these genes, which resulted in increased FAS/FASL expression and subsequent apoptosis via activated caspase 8. We found that shRNA mediated genetic knockdown of DNMT -1 and -3A also resulted in upregulation of FASL mRNA and protein in CTCL cell lines. However, the reduction in FASL promoter methylation induced by MTX was generally superior to that induced by shRNA knockdown and was not improved by a combination of MTX plus knockdown. Because STAT proteins help regulate DNMTs, we also evaluated the effect of multiple JAK inhibitors (fedratinib, tofacitinib, and ruxolitinib) in CTCL cells and Sezary patient’s blood. Among these, fedratinib treatment was most effective in decreasing viability, spheroid formation, and inducing apoptosis. These responses were accompanied by decreased phosphorylation of STAT3 at tyrosine-705. Taken together, our data demonstrate that enhanced apoptosis of CTCL can be induced by MTX, DNMT knockdown and JAK inhibitors. This is consistent with a mechanistic cascade in which activated STATs enhance DNMT activities that in turn suppress the expression of apoptotic factors and promote CTCL survival. Our findings suggest a therapeutic role for MTX and JAK inhibitors in the management of CTCL.

423 Gentian violet induces pro-apoptotic and anti-proliferative effects in cutaneous T-cell lymphoma (CTCL): Preclinical studies

Previously, we showed that triggering the extrinsic apoptotic pathway by up-regulating activation-induced cell death (AICD) is a very effective way to kill CTCL cells in-vitro and ex-vivo (J Invest Dermatol. 2015; 135: 861-8). To identify novel small molecules that induce extrinsic apoptosis in CTCL, we used CTCL line HH in an ELISA assay to detect cleaved caspase 8 induced by 1710 unique compounds in the NIH Clinical Collection, Selleck Kinase Inhibitor Library and Prestwick Chemical Library. As assessed by Annexin V/PI flow cytometry of HH, the non-prescription topical antimicrobial remedy, gentian violet (GV), induced more total apoptosis than mechlorethamine (FDA-approved for topical therapy of CTCL). Furthermore, GV induced roughly 4-6X greater apoptosis in CTCL lines (MyLa, SeAx, Hut78, HH, SZ4) than in normal keratinocytes, suggesting a favorable topical toxicity profile. In addition to increasing caspase 8, GV also upregulated death receptors DR4, DR5 and ligands TRAIL, FASL. This is consistent with induction of extrinsic apoptosis via the TRAIL and FAS pathways. Increased phosphorylation of phospholipase C-gamma 1, Ca++ influx and ROS were also detected in CTCL lines, indicating the mechanism of FASL upregulation involves key elements of the AICD pathway. Methotrexate significantly increased GV apoptosis in Sezary blood cells and all CTCL lines tested except FAS-null SeAx, suggesting the role of FAS in this process. In ex-vivo studies, 1μM GV induced up to 90% CTCL apoptosis in Sezary blood cells. GV also reduced expression of anti-apoptotic MCL-1 and pro-proliferative NFkB components while increasing IkB levels that promote NFkB catabolism. Furthermore, GV inhibited cell proliferation and induced cell cycle arrest. Although purple at neutral pH, GV can be rendered colorless by altering its pH. These preclinical findings broaden our knowledge of the anti-neoplastic effects of GV and provide a rationale for clinical studies of its utility as a novel inexpensive topical therapy for CTCL.

Decitabine Priming Enhances Mucin 1 Inhibition Mediated Disruption of Redox Homeostasis in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous neoplasm and patients with relapsed/refractory disease exhibit resistance to standard therapies. We have previously demonstrated that the Mucin 1 C-terminal subunit (MUC1-C) plays a critical role in protection from oxidative stress in CTCL cells. Targeting of MUC1-C with a pharmacologic inhibitor, GO-203, was associated with apoptosis in CTCL. However, disease responses were incomplete underscoring the need for combinatorial strategies that could exploit the vulnerability of CTCL cells to oxidative signals. Cell lines, primary samples, and xenograft models of CTCL were used to assess synergy of GO-203 with decitabine, a hypomethylating agent. Present studies demonstrate that exposure of CTCL cells to decitabine in combination with GO-203, increased the generation of reactive oxygen species (ROS) levels and decreased levels of scavenger molecules, NADP, NADPH, glutathione, and TIGAR, critical to intracellular redox homeostasis. Dual exposure to GO-203 and decitabine resulted in marked downregulation of DNA methyl transferases demonstrating significant synergy of these agents in inducing global and gene specific hypomethylation. Accordingly, treatment with decitabine and GO-203 upregulated the ROS generating enzymes, NADPH oxidase 4 and dual oxidase 2 potentially due to their effect on epigenomic regulation of these proteins. In concert with these findings, exposure to decitabine and GO-203 resulted in heightened apoptotic death in CTCL cell lines, patient-derived primary samples and in a murine xenograft model. These findings indicate that decitabine intensifies MUC1-C inhibition induced redox imbalance and provides a novel combination of targeted and epigenetic agents for patients with CTCL. Mol Cancer Ther; 16(10); 2304-14. ©2017 AACR.

Epigenetically Enhanced Photodynamic Therapy (ePDT) is Superior to Conventional Photodynamic Therapy for Inducing Apoptosis in Cutaneous T-Cell Lymphoma.

Conventional photodynamic therapy with aminolevulinate (ALA-PDT) selectively induces apoptosis in diseased cells and is highly effective for treating actinic keratoses. However, similar results are achieved only in a subset of patients with cutaneous T-cell lymphoma (CTCL). Our previous work shows that the apoptotic resistance of CTCL correlates with low expression of death receptors like Fas cell surface death receptor (FAS), and that methotrexate upregulates FAS by inhibiting the methylation of its promoter, acting as an epigenetic derepressor that restores the susceptibility of FAS-low CTCL to caspase-8-mediated apoptosis. Here, we demonstrate that methotrexate increases the response of CTCL to ALA-PDT, a concept we refer to as epigenetically enhanced PDT (ePDT). Multiple CTCL cell lines were subjected to conventional PDT versus ePDT. Apoptotic biomarkers were analyzed in situ with multispectral imaging analysis of immunostained cells, a method that is quantitative and 5× more sensitive than standard immunohistology for antigen detection. Compared to conventional PDT or methotrexate alone, ePDT led to significantly greater cell death in all CTCL cell lines tested by inducing greater activation of caspase-8-mediated extrinsic apoptosis. Upregulation of FAS and/or tumor necrosis factor-related apoptosis-inducing ligand pathway components was observed in different CTCL cell lines. These findings provide a rationale for clinical trials of ePDT for CTCL.

Ubiquitin-specific protease 2 decreases p53-dependent apoptosis in cutaneous T-cell lymphoma.

Treatment of advanced cutaneous T-cell lymphomas (CTCL) is challenging because they are resistant to conventional chemotherapy. USP2 has been shown to promote resistance to chemotherapeutic agents in several cancer models.We show here USP2 is expressed in quiescent and activated T-cells and its expression is 50% lower in CTCL cell lines (MyLa2000, SeAx and Hut-78) than in normal T-cells. USP2 is expressed in neoplastic cells in early, plaque-stage mycosis fungoides (MF) and is downregulated in advanced tumor stages. Upon treatment with psoralen with UVA (PUVA) or a p53 activator, nutlin3a, USP2 expression is significantly increased in MyLa2000 (p53wt/wt), but not in SeAx (p53mut) or Hut-78 (p53−/−). USP2 knockdown decreases MyLa2000 cell viability after PUVA by 50% but not Hut-78, suggesting that the function of USP2 in CTCL cells is p53-dependent. Furthermore, USP2 knockdown results in a decreased Mdm2 expression and upregulation of p53. Taken together, our findings suggest that USP2 stabilizes Mdm2 which antagonizes pro-apoptotic activity of p53 and possibly contributes to therapeutic resistance in CTCL.

282 Small molecule screening identifies novel compounds that induce significant apoptosis in cutaneous T-cell lymphoma (CTCL)

282 Small molecule screening identifies novel compounds that induce significant apoptosis in cutaneous T-cell lymphoma (CTCL)

ONC201 Induces Apoptosis in Cutaneous T-Cell Lymphoma Cells through a Mechanism That Involves the Integrated Stress Response and Inactivation of Jak/Stat Signaling

Background: ONC201 (also called TIC10) is an orally active, first-in-class small molecule that is in phase II clinical trials for advanced cancers based on its ability to activate apoptosis in tumor cells, but not normal cells, in a p53-independent manner. Recent studies have implicated the integrated stress response as an early stage mechanism of ONC201 that may lead to the previously observed downstream anti-cancer effects. This study was conducted to evaluate the anti-tumor activity of ONC201 on Cutaneous T-Cell Lymphoma (CTCL) cell lines and Sézary cells.Methods: We treated 8 CTCL cell lines (H9, HH, Hut78, Mac2A, MJ, MyLA, Pb2b and SeAx), peripheral blood mononuclear cells (PBMC) from 5 Sézary syndrome (SS) patients and 6 healthy donors with ONC201. MTS viability Assay was used to assess the anti-proliferation effect. Apoptosis was assessed using Annexin V FITC/PI staining and sub-G1 analysis by flow cytometry. Protein expression by western blotting was analyzed in 3 CTCL cell lines (HH, Hut78, MJ) and in PBMCs from 2 SS patients treated for 72 hours with ONC201including Activating Transcription Factor 4 (ATF4), interferon regulatory factor 7 (IRF7/pIRF7), Signal transducer and activator of transcription 3 (STAT3), Janus Kinase 3 (JAK3/pJAK3) and NF-κB family members (p65, RelB, c-Rel and p105).Results: MTS viability assay showed pronounced cell growth inhibition, significantly increasing in a time-dependent manner in H9, HH, Hut78, Mac2A, MJ, MyLa, Pb2b, and SeAx lines after a 96-hour incubation within a very narrow efficacy threshold ranging from 1.25 to 10µM (n = 8, p < 0.05). To determine whether growth inhibition of ONC201 is due to cell-cycle arrest and/or to apoptosis in CTCL cell lines, sub-G1 analysis by flow cytometry was measured in 3 CTCL cell lines (HH, Hut78 and MJ). The percentages of HH, Hut78 and MJ cells with sub-G1 population increased, suggesting that cells are undergoing apoptosis (n = 3, p < 0.05). Induction of apoptosis was further confirmed by Annexin V FITC/PI staining, revealing dose and time dependent apoptosis induction in all 8 cell lines (Figure 1). To confirm cell line results in refractory ex vivo samples, we tested the pro-apoptotic effects of ONC201 on PBMCs from 5 SS patients who had high circulating CD4+CD26- malignant T-cells compared with PBMCs from 6 healthy donors. ONC201 induced significant levels of apoptosis in PBMCs from SS patients, but not in normal PBMCs (Figure 1, p < 0.001). Western blot analysis revealed that ONC201 increased the expression of ATF4, a hallmark of the integrated stress response and a negative regulator of IRF7. ONC201 decreased expression of IRF7/pIRF7 and down-regulated JAK3/pJAK3 and STAT3 expression in CTCL cell lines and PBMCs from SS patients. ONC201 also decreased the protein expression of NF-κB family members ( p65, RelB, c-Rel and p105) that can cause resistance to apoptosis in CTCL cells.Conclusions: ONC201 appears to be active as a single oral agent. It may impact key signaling pathways in CTCL preclinical models by inhibiting proliferation and inducing apoptosis through a mechanism that involves the integrated stress response, leading to inactivation of JAK/STAT signaling and down-regulation of the NFκB pathway. [Display omitted] DisclosuresDuvic:Eisai: Research Funding; MiRagen Therapeutics: Consultancy; Cell Medica Ltd: Consultancy; Tetralogics SHAPE: Research Funding; Soligenics: Research Funding; Array Biopharma: Consultancy; Innate Pharma: Research Funding; Allos (spectrum): Research Funding; Rhizen Pharma: Research Funding; Spatz Foundation: Research Funding; Oncoceutics: Research Funding; Huya Bioscience Int'l: Consultancy; Therakos: Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Most drugs for ctcl are non approved. This drug is a small molecule and is not approved for any cancer. Tarapore:Oncoceutics, Inc: Employment, Equity Ownership. Allen:Oncoceutics, Inc: Employment, Equity Ownership.

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.

Ellipticine induces apoptosis in T-cell lymphoma via oxidative DNA damage

The tumor suppressor p53 is often mutated in human cancers. Restoring its antitumor activity has been shown to be a promising therapeutic approach for cancer treatment. Here we analyzed the activity and mechanism of a p53 reactivator, ellipticine, in a cellular model of cutaneous T-cell lymphoma (CTCL), a disease that is progressive, chemoresistant and refractory to treatment. We tested the effect of ellipticine in three cell lines with different p53 status: MyLa2000 (p53wt/wt), SeAx (G245Sp53) and Hut-78 (R196Stopp53). Ellipticine caused apoptosis in MyLa2000 and SeAx and restored the transcriptional activity of G245Sp53 in SeAx. However, p53 siRNA knockdown experiments revealed that p53 was not required for ellipticine-induced apoptosis in CTCL. The lipophilic antioxidant α-tocopherol inhibited ellipticine-dependent apoptosis and we linked the apoptotic response to the oxidative DNA damage. Our results provide evidence that ellipticine-induced apoptosis is exerted through DNA damage and does not require p53 activation in T-cell lymphoma.

Proteasome inhibition as a novel mechanism of the proapoptotic activity of γ-secretase inhibitor I in cutaneous T-cell lymphoma

We have previously discovered that Notch1 is expressed on malignant T cells in cutaneous T-cell lymphoma (CTCL), and is required for survival of CTCL cell lines. Notch can be inhibited by γ-secretase inhibitors (GSIs), which differ widely in their ability to induce apoptosis in CTCL. To investigate whether GSI-I, in addition to inhibiting Notch, induces apoptosis in CTCL by proteasome inhibition, as GSI-I is very potent and has structural similarity to the proteasome inhibitor MG-132. Cell lines derived from CTCL (MyLa, SeAx, JK, Mac1 and Mac2a) were treated with GSI-I and two other proteasome inhibitors (MG-132 and bortezomib). The effects on cell viability, apoptosis and proteasome activity were measured, as was the impact on the prosurvival, nuclear factor κB (NF-κB) pathway. In CTCL, GSI-I had proteasome-blocking activity with a potency comparable to the proteasome inhibitors MG-132 and bortezomib. Proteasome inhibition was the main mechanism responsible for GSI-I-induced cell death, as tiron, a compound known to reverse the effect of MG-132, restored proteasome activity and largely abrogated the cytotoxic effect of GSI-I. Although inactivation of NF-κB is an important mechanism of action for proteasome inhibitors, we demonstrated an apparent activation of NF-κB. Furthermore, we showed that while the tumour suppressor protein p53 was induced during proteasome inhibition, it was dispensable for CTCL apoptosis, as both SeAx cells, which harbour p53 mutations that attenuate the apoptotic capacity, and HuT-78 cells, which have a deleted p53 gene, demonstrated potent apoptotic response. GSI-I represents an interesting drug with a dual mechanism of action comprising inhibition of both Notch and the proteasome.

MiR-122 regulates p53/Akt signalling and the chemotherapy-induced apoptosis in cutaneous T-cell lymphoma.

Advanced cutaneous T-cell lymphoma (CTCL) is resistant to chemotherapy and presents a major area of medical need. In view of the known role of microRNAs (miRNAs) in the regulation of cellular signalling, we aimed to identify the functionally important miRNA species, which regulate apoptosis in CTCL. Using a recently established model in which apoptosis of CTCL cell lines is induced by Notch-1 inhibition by γ-secretase inhibitors (GSIs), we found that miR-122 was significantly increased in the apoptotic cells. miR-122 up-regulation was not specific for GSI-1 but was also seen during apoptosis induced by chemotherapies including doxorubicin and proteasome blockers (bortezomib, MG132). miR-122 was not expressed in quiescent T-cells, but was detectable in CTCL: in lesional skin in mycosis fungoides and in Sézary cells purified from peripheral blood. In situ hybridization results showed that miR-122 was expressed in the malignant T-cell infiltrate and increased in the advanced stage mycosis fungoides. Surprisingly, miR-122 overexpression decreased the sensitivity to the chemotherapy-induced apoptosis via a signaling circuit involving the activation of Akt and inhibition of p53. We have also shown that induction of miR-122 occurred via p53 and that p53 post-transcriptionally up-regulated miR-122. miR-122 is thus an amplifier of the antiapoptotic Akt/p53 circuit and it is conceivable that a pharmacological intervention in this pathway may provide basis for novel therapies for CTCL.

Reduction of Fas/CD95 Promoter Methylation, Upregulation of Fas Protein, and Enhancement of Sensitivity to Apoptosis in Cutaneous T-Cell Lymphoma

To explore the relationships among (Fas) promoter methylation, Fas expression, and apoptotic sensitivity in cutaneous T-cell lymphoma (CTCL). Laboratory investigation. Dermatology research unit of a university medical center. Five CTCL lines and Sézary syndrome blood. Treatment of cells with 5-azacytidine (aza), methotrexate, and interferon alfa-2b. Fas promoter methylation, Fas expression, and sensitivity to Fas-mediated apoptosis. Fas promoter methylation correlates inversely with the level of Fas transcript, protein, and apoptotic sensitivity in CTCL. Increased DNA methylation also correlates with decreased NFkB (nuclear factor kappa-light chain enhancer of activated B cells) binding to the Fas promoter. All of these relationships were reversed by the DNA-demethylating agent, 5-aza. We found that methotrexate also functions as a DNA-demethylating agent by depleting methyl donors and, together with interferon alfa-2b, upregulates Fas and enhances sensitivity to Fas-mediated apoptosis. These findings help explain the previously reported impressive responses of patients with advanced CTCL to combination therapy with methotrexate and interferon alfa. They also provide a new rationale for the treatment of CTCL with methotrexate and its use in combination with other agents.

Low FAS/CD95 Expression by CTCL Correlates with Reduced Sensitivity to Apoptosis that Can Be Restored by FAS Upregulation

FAS expression was generally low in 30 of 31 cutaneous T-cell lymphoma (CTCL) cases (mycosis fungoides/Sezary syndrome, SS) as well as in 5 of 6 large plaque parapsoriasis cases (a CTCL precursor). To investigate this phenomenon, we explored FAS transcript levels, cell-surface FAS protein expression and susceptibility to FAS-mediated apoptosis in four CTCL lines (MyLa, HH, SZ4, and SeAx), freshly isolated leukemic cells from a patient with SS, an acute lymphoblastic leukemia T-cell line (Jurkat), and JFL (a FAS-low variant of Jurkat). Results confirmed low FAS expression by the leukemic SS cells, HH, SZ4, SeAx, and JFL relative to normal peripheral blood mononuclear leukocytes and the other cell lines. There was a direct correlation among FAS transcript level, FAS protein level, and FAS-mediated apoptotic sensitivity in the CTCL samples. When the FAS-deficient cell lines were transfected with a wild-type FAS construct, FAS expression and sensitivity to FAS-mediated apoptosis were restored. In aggregate, these findings provide evidence that like normal T cells, CTCL cells exhibit a mechanistic connection between transcriptional regulation of FAS and sensitivity to FAS-mediated apoptosis, point to the development of FAS deficiency as one molecular mechanism responsible for acquired resistance to apoptosis in CTCL, and indicate that upregulation of FAS expression can restore sensitivity to apoptosis.

Bortezomib‐induced apoptosis in mature T‐cell lymphoma cells partially depends on upregulation of Noxa and functional repression of Mcl‐1

Bortezomib, a proteasome inhibitor that was originally developed as an inhibitor of nuclear factor-κB pathways, is currently used for the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL). The mechanisms of action of this antitumor agent have been studied by several investigators. Here, we explore the underlying mechanisms of bortezomib-induced apoptosis in cutaneous T-cell lymphoma (CTCL) and adult T-cell leukemia/lymphoma (ATLL) at the level of mitochondrial membrane injury. In all cell lines including (KMS-12-PE [MM], HUT78 [CTCL], ATN1 [ATLL], and MT4 [ATLL]), antiapoptotic factors such as c-Flip and XIAP were downregulated after exposure to bortezomib, probably via inhibition of nuclear factor-κB signaling. In addition, among the members of the BH3-only family, upregulation of Noxa was consistently seen at both the transcriptional and protein levels in a p53-independent manner after exposure to bortezomib. Repression of Noxa by small interfering RNA partially rescued CTCL and ATLL cells from bortezomib-induced apoptosis. Immunoprecipitation assays indicated time-dependent binding of Noxa and Mcl-1 in all cell types, suggesting that functional repression of Mcl-1 led to the loss of mitochondrial outer membrane potential. Similar results were also obtained in primary tumor cells from patients with ATLL. Taken together, we conclude that bortezomib-induced apoptosis in ATLL and CTCL cells at least partly depends on the upregulation of Noxa and functional repression of Mcl-1, as is also the case in MM and malignant melanoma.

Pralatrexate (PDX) Compliments the Activity of the Proteasome Inhibitor Bortezomib (B) in in Vitro Models of Lymphoid T-Cell Malignancies

Pralatrexate (10-propargyl-10-deazaaminopterin, PDX) is a novel antifolate with greater affinity for the reduced folate carrier (RFC-1) and foly-polyglutamyl synthase (FPGS). PDX is emerging as a promising drug for the treatment of chemotherapy resistant T-cell lymphomas and leukemias. Bortezomib (B) is a modified dipeptidyl boronic acid that induces apoptosis by inhibiting the 26S proteasome in a variety of hematologic malignancies, including multiple myeloma and non-Hodgkin's lymphoma. Recently, NF- B has shown a prominent role in inducing resistance to apoptosis in cutaneous T-cell lymphoma (CTCL), which supports a potential therapeutic role for bortezomib in the treatment of these patients. We investigated the in vitro and/or in vivo activity of PDX and B alone or in combination in different T cell lymphoma and leukemia cell lines including CTCL (H9), T- acute lymphoblastic leukemia (T-ALL) lines resistant or sensitive to gamma- secretase inhibitors (GSI) (P12, PF 382 and CEM are GSI resistant while KOKPT-1, DND-41 and HPB-ALL are GSI sensitive lines). For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition on a Biotek Synergy HT. Drug : drug interactions were analyzed using the Calcusyn software (Biosoft) with a combination index (CI) < 1 showing synergism, CI=1 reflecting additivity, and a CI>1 suggesting antagonism. As an alternative, calculation of the relative risk ratios (RRR) was performed based on the GraphPad software with RRR<1 defining synergism. Apoptosis was assessed by staining with Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Caspase 8 and 9 activation was evaluated using Active Caspase 8 and 9 staining kits (Biovision) followed by FACSCalibur acquisition. The IC50s for PDX alone at 48 and 72 hours were generally in the low nanomolar range: H9: 1.1nM – 2.5nM; P12: 1.7nM – 2.4nM; CEM: 3.2nM – 4.2nM; PF 382: 5.47nM – 2.72nM; KOPT-K1: 1nM – 1.69nM; DND 41: 97.37nM - 1.21nM; HPB-ALL: 247.78nM - 0.77nM. The IC50s for bortezomib alone at 48 and 72 hours were: H9: 5.99nM – 5.27nM; P12: 4.71nM; PF 382: 2.22nM. In the cytotoxicity assays, the combination of PDX and B at 48 hours showed synergism in H9 (CI ≤ 0.38), P12 (CI≤ 0.513) and PF382 (CI≤ 0.352). When H9, P12 and PF382 cell lines were treated with B and/or PDX (both drugs in a range from 2 to 6 nM) for 48 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. H9: B alone: 26% – 53%, PDX: 22% – 58%, B+PDX: 69% – 89%, RRR for the combination ≤ 0.9; P12: B alone: 40% – 57%, PDX alone: 24% – 49%, B+PDX: 64% – 87%; RRR ≤ 0.9; PF382 B alone 72%, PDX alone 65%, B+PDX 94%; RRR ≤ 0.65. The combination of PDX and B also revealed an increase in caspase 8 and 9 activation compared to the single drugs in H9 (relative risk ≤0.81 and ≤0.74 respectively). The percentage of cells with activated caspase 8 or 9 was approximately double in the combination groups compared to the single treatments. An in vivo xenograft study in six to eight weeks old female SCID beige mice injected subcutaneously with 1 × 107 H9 cells is underway. Mice were separated into different cohorts and treated with intraperitoneal injections of PDX or B or their combination according to the following schedules: PDX alone at 15, 30 or 60 mg/kg on days 1, 4, 8 and 11; B alone at 0.5mg/Kg on days 1, 4, 8, and 11 and all the possible combination of the two drugs. Collectively, the data to data suggest that PDX and bortezomib are potentially synergistic in in vitro models of human T-cell lymphoma. Should the in vivo data confirm the in vitro observations, it is possible this combination of two T-cell active drugs will form the basis of future combination Phase 1 – 2 studies.

Bortezomib-Induced Apoptosis in Cutaneous T-Cell Lymphoma and Adult T-Cell Leukemia/Lymphoma Cells Partially Depends on up-Regulation of Noxa, Functional Repression of Mcl-1, and Down-Regulation of C-Flip and XIAP.

Bortezomib, a proteasome inhibitor, which was originally developed as an inhibitor of NF-kappaB pathways, is currently used for the treatment of multiple myeloma (MM) and mantle cell lymphoma (MCL). The mechanisms of action of this anti-tumor agent have been studied by several investigators. As bortezomib has been reported effective for a fraction of peripheral T-cell lymphomas including adult T-cell leukemia/lymphoma (ATLL) and cutaneous T-cell lymphoma (CTCL) at clinically achievable concentrations, it is expected to be applicable for these refractory T-cell malignancies. In this study, we have explored the underlying mechanisms of bortezomib-induced apoptosis in cell lines established from CTCL and ATLL together with those from MM by particularly focusing on the level of mitochondrial membrane injury. It was because the apoptosis induced by bortezomib in CTCL and ATLL cells accompanied the activation of caspase-3, -8, and -9, and the disturbance of mitochondrial membrane potential preceded the process of bortezomib-induced apoptosis when detected by DiIC1 and Annexin V staining. In 6 MM lines including KMS-12-PE, 2 CTCL lines including Hut78, and 7 ATLL lines including ATN1 and MT4, anti-apoptotic factors such as c-Flip and XIAP were down-regulated after exposure to bortezomib, probably via inhibition of NF-kappaB signaling. This reduction of c-Flip and XIAP was also confirmed in the primary tumor cells derived from the patients with ATLL after exposure to 20nM bortezomib. These findings indicate that the activation of both intrinsic and extrinsic apoptosis pathways led to activation of caspases via reduced expression of negative regulators of apoptosis. Among the members of the BH3-only family protein, up-regulation of Noxa was consistently seen at both the transcriptional and protein levels in a p53-independent and c-Myc-independent manner after exposure to bortezomib, whereas the expression of other BH3-only family proteins showed no consistent changes. Of anti-apoptotic Bcl-2 family proteins, accumulation of Mcl-1 and its cleaved form were observed, but no altered expression of other Bcl-2 family members was seen. Specific repression of Noxa by small interfering RNA partially rescued CTCL and ATLL cells from bortezomib–induced apoptosis, suggesting a crucial role of Noxa in bortezomib-induced apoptosis in these cell types as well as in MM cells. Immunoprecipitation assays indicated time-dependent binding of Noxa and Mcl-1 in all cell types, suggesting that functional repression of Mcl-1 led to the loss of mitochondrial outer membrane potential. Similar results showing transcriptional and translational up-regulation of Noxa followed by increased amount of the Mcl-1/Noxa complexes after exposure to 20nM bortezomib were also obtained in primary tumor cells derived from patients with ATLL. Taken together, we conclude that bortezomib-induced apoptosis in ATLL/CTCL cells at least partly depends on up-regulation of Noxa and functional repression of Mcl-1 in addition to the reduced amount of c-Flip and XIAP proteins, although the mechanisms leading to the transcriptional activation of Noxa after exposure to bortezomib remains to be clarified.

Blockade of Death Receptor-Mediated Pathways Early in the Signaling Cascade Coincides with Distinct Apoptosis Resistance in Cutaneous T-Cell Lymphoma Cells

Control of apoptosis via death ligands plays a basic role for lymphocyte homeostasis and lymphoma development. In this study, cutaneous T-cell lymphoma (CTCL) cell lines revealed pronounced resistance to death ligands as compared to cell lines of T-cell acute lymphoblastic leukemia (T-ALL). The proapoptotic activity of tumor necrosis factor (TNF)-alpha was blocked, sensitivity to TNF-related apoptosis-inducing ligand was significantly reduced, and 1/4 CTCL cell lines was resistant to CD95 activation. In parallel, there was no activation of effector caspase-3 and initiator caspase-8 in nonresponsive CTCL cells, whereas caspase-10 was cleaved selectively in sensitive CTCL cells. No indication for a responsibility of typical downstream regulators of apoptosis was obtained, but loss of CD95 was found in 1/4, loss of TNF-R1 in 3/4, loss of caspase-10 in 2/4, loss of Bid in 1/4, and overexpression of cellular flice inhibitory protein was found in 4/4 CTCL cell lines. This clearly indicates an inhibition of apoptosis early in the extrinsic cascade, namely at the formation of the death-inducing signaling complex. Parallels with regard to expression of apoptosis regulators were seen in peripheral blood mononuclear cells and biopsies of CTCL patients. This study may indicate defects in apoptosis in CTCL and may help to guide CTCL therapy.