CTCL Cell Research Articles

Role of STAT3 and Th17 Cells in Cutaneous T Cell Lymphoma

AbstractAbstract 66 BackgroundCutaneous T cell Lymphoma (CTCL) is a heterogeneous group of non-Hodgkin's lymphomas characterized by skin-homing malignant T cells. The etiology of CTCL remains enigmatic, but constitutive Signal Transducer and Activator of Transcription 3 (STAT3) activity is characteristic of this malignancy. CTCL cell lines undergo growth arrest and apoptosis following inhibition of STAT3 signaling, suggesting that STAT3 may be a pro-survival factor in these cells. STAT3 phosphorylation is also required for the initiation and maintenance of the Th17 differentiation program, a recently identified subset of CD4+ T helper cells, implicated in a number of chronic inflammatory conditions including rheumatoid arthritis and psoriasis. Furthermore, we know that Histone Deacetylase inhibitors (HDACi) namely Vorinostat and Romidepsin are very active agents for treatment of CTCL but their mechanism of action is still to be elucidated. Our goal is to probe the contribution of Th17 mediated inflammation in CTCL and to examine whether HDAC inhibitors work by modulating Th17 inflammation in the context of this malignancy. MethodsUsing conditional gene targeting approach we have generated a transgenic 100% penetrant mouse model of CTCL wherein expression of a hyper-active STAT3 mutant protein (STAT3C) selectively in T lymphocytes results in skin pathology highly reminiscent of this human malignancy. To have a better understanding of the contribution of STAT3 in CD4 T cell differentiation program and CTCL, we used lymphocytes and splenocytes from 8 weeks old STAT3Cstopfl/fl (without CD4Cre, thus no pathology) and control YFPstopfl/fl (Yellow Fluorescent Protein floxed) mice (Figure 1a). We isolated naïve CD4 cells using Dynabeads and treated these CD4 cells with a transducible Cre enzyme (Tat-Cre) to delete the upstream stop cassette ex-vivo. These cells were then plated with stimulating α CD3/CD28 antibodies in various cytokine cocktails as shown in Figure 1b. These cells were harvested after 84 hours and stimulated with PMA/Inomycin for 4 hours for flowcytometric analysis. We are now using this in-vitro approach as well as our mouse model and primary tissue samples from CTCL patients to explore the impact of HDAC inhibitors on Th17/Treg balance. ResultsUpon Tat-Cre treatment we were able to analyze GFP+ (and therefore STAT3C expressing) vs. GFP- (control) T cells in the same differentiation conditions. Hyperactive STAT3C mutant protein favored TH17 differentiation as GFP Positive cells had significantly augmented IL17A production (Figure 1b). On the other hand, STAT3C expressing GFP positive cells in Th0 and Th1 differentiation conditions had less IFN-γ production compared to the GFP negative cells and control YFP fractions. Even the GFP negative fraction of STAT3C had less IFN-γ and more IL17A production than the control YFP fractions due to the cytokine milieu created by STAT3C expressing cells present in the same well. Conclusions:These results highlight an intriguing possibility that STAT3 dependent Th17 cells play a role in CTCL pathogenesis. Using our newly developed mouse model and CTCL patients' blood and skin samples (IRB approved), we are trying to distinguish whether Th17 cells are instigators of chronic inflammation that contributes to the malignant transformation of T cells or that Th17 cells may actually be the cells of origin in this malignancy. Our ongoing experiments also include the effects of HDACi on this ex-vivo CD4 T cell differentiation. In addition, we are studying the hallmarks of Th17 differentiation namely IL-17A, IL-22 and pSTAT3 positivity in the skin biopsy and blood samples of CTCL patients before and after treatment with HDACi. These experiments will help us determine the impact of HDAC inhibition on STAT3 activity and Th17 differentiation and have a better understanding of their mechanism of action in CTCL. Our hope is that our study will guide us to comprehend the contribution of chronic inflammation in carcinogenesis and potentially identify novel treatment targets and strategies in the field of hematological malignancies. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A proteomic study of early signaling events regulating the Fas-FasL Death Inducing Signaling Complex (163.20)

Abstract Clearance of activated T cells by the Fas-FasL pathway is a critical mechanism of peripheral CD4 T cell tolerance. This process is preferentially higher in the effector subset of memory CD4+ T cells due to efficient recruitment and formation of the Fas death inducing signaling complex (DISC). In order to elucidate mechanisms of this heightened apoptosis sensitivity, we tested for differential DISC interacting proteins in Fas sensitive and Fas resistant CTCL cell lines using 2D-DIGE and Mass Spectrometry. We found several candidate proteins and invitro studies were done to confirm Fas receptor interactions through overexpression and Flourescence Energy Transfer (FRET) studies. One of these targets, Drebrin, is an actin binding protein previously found to play a role in T cell synapse formation by facilitating CXCR4 recruitment. Overexpression in 293 indicates that Drebrin interacts with Fas receptor. Further, endogenous drebrin preassociates with Fas receptor and remains associated with the Fas DISC even after Fas ligation only in Type I SKW cell line. Interestingly, Fas DISC associated Drebrin gets cleaved after Fas treatment and invitro caspase cleavage studies indicate that drebrin is a specific downstream target of caspase-8. Our studies propose Drebrin as a novel Fas interacting protein. Investigations are currently underway to study the mechanisms by which Drebrin modifies the Fas-DISC dynamics and the significance of the caspase-cleaved Drebrin in this process.

Abstract A20: Differential rates of proliferation and apoptosis observed in human cutaneous T-cell lymphoma cell lines, in response to treatment with 9-cis UAB30, a novel rexinoid analog

Abstract Cutaneous T-cell lymphoma (CTCL) designates a heterogeneous group of malignancies, characterized by the infiltration and detection of transformed T-cell lymphocytes in the skin. Although there is no cure for CTCL, a wide range of treatment options have been developed for use in the clinical setting. Bexarotene, a retinoid X receptor (RXR) agonist or rexinoid, is a commonly used treatment modality for CTCL. However, documented increases in the levels of triglycerides of CTCL patients, resulting from treatment with bexarotene, point to concerns over the long-term use and efficacy of this anticancer agent. We have recently synthesized a novel rexinoid, 9-cis UAB30, which does not elevate triglycerides in vivo and have begun evaluating its use as a potential chemotherapeutic agent for the treatment of CTCL. In this study, we compared the efficacy of 9-cis UAB30 to that of bexarotene in reducing cell proliferation and inducing apoptosis, in the three human CTCL cell lines HH, MJ, and Hut78. A standard XTT assay was used to determine the rate of cell proliferation of CTCL cells. In all three lines, 9-cis UAB30 was equally and sometimes more effective than bexarotene at suppressing cell proliferation. Using an annexin V binding assay, we next analyzed the rate of apoptosis in these human CTCL lines. Interestingly, we observed the MJ cell line to be resistant to apoptosis, when treated with either compound. The HH cell line exhibited a similar increase in apoptosis, whether treated with 9-cis UAB30 or bexarotene. Finally, the Hut78 cell line showed a greater sensitivity to apoptosis, when treated with 9-cis UAB30 as compared to bexarotene. Taken together, these preliminary results indicate that 9-cis UAB30 appears to be equally if not more effective than bexarotene at suppressing cell proliferation and appears to exert differential effects on the rate of apoptosis that may be cell-line specific. These differential responses of human CTCL cells have implications for the development of next-generation, rexinoid-based therapies and reveal that 9-cis UAB30 may be a better alternative for the treatment of CTCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A20.

Constitutive activation of STAT3 in Sézary syndrome is independent of SHP-1

Constitutive and persistent activation of STAT3 has been implicated in the pathogenesis of many malignancies. Studies of CTCL cell lines have previously suggested that aberrant activation of STAT3 is mediated via silencing of the negative regulator SHP-1 by promoter methylation. In this study of ex vivo tumour cell populations from 18 Sézary syndrome (SS) patients, constitutive phosphorylation of STAT3, JAK1 and JAK2 was present in all patients, but was absent in comparative CD4+ T-cells from healthy controls. Furthermore, no loss or significant difference in SHP-1 expression was observed between patients and healthy control samples. Methylation-specific PCR analysis of the SHP-1 CpG island in 47 SS patients and 11 healthy controls did not detect any evidence of methylation. Moreover, small interfering RNA knockdown of SHP-1 had no effect on phosphorylation of STAT3. In contrast, treatment of SS tumour cells with the pan-JAK inhibitor pyridone 6 led to downregulation of phosphorylated STAT3 (pSTAT3), its target genes and induction of apoptosis. No evidence for common JAK1/JAK2-activating mutations was found. These data demonstrate that constitutive activation of STAT3 in SS is not due to the loss of SHP-1, but is mediated by constitutive aberrant activation of JAK family members.

MicroRNA profiling reveals that miR-21, miR486 and miR-214 are upregulated and involved in cell survival in Sézary syndrome

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma and its pathogenesis is still unknown. Diagnosis/prognosis may strongly ameliorate the management of SS individuals. Here, we profiled the expression of 470 microRNAs (miRNAs) in a cohort of 22 SS patients, and we identified 45 miRNAs differentially expressed between SS and controls. Using predictive analysis, a list of 19 miRNAs, including miR-21, miR-214, miR-486, miR-18a, miR-342, miR-31 and let-7 members were also found. Moreover, we defined a signature of 14 miRNAs including again miR-21, potentially able to discriminate patients with unfavorable and favorable outcome. We validated our data for miR-21, miR-214 and miR-486 by qRT-PCR, including an additional set of array-independent SS cases. In addition, we also provide an in vitro evidence for a contribution of miR-214, miR-486 and miR-21 to apoptotic resistance of CTCL cell line.

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

AbstractCutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non–mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRASG13D mutation; one Sézary syndrome and one CD30+ CTCL harbored a NRASQ61K amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRASQ61K mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRASQ61K mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

STAT3 knockdown by siRNA induces apoptosis in human cutaneous T-cell lymphoma line Hut78 via downregulation of Bcl-xL

Cutaneous T-cell lymphomas (CTCLs) are non-Hodgkin’s lymphomas resulting from clonal expansion and localization of malignant T-lymphocytes to the skin. CTCL cells have defective apoptosis. Signal transducers and activators of transcription (STAT) are a family of transcription factors known to play important roles in the development and progression of several human cancers by promoting cell proliferation and protecting against apoptosis. In this study, we investigated the specific role of STAT3, a major component of the STAT family, in growth and survival of human CTCL cell line Hut78. Western immunoblot analysis showed elevated expression of STAT3 and phospho-STAT3(Y705) in human CTCL cells as compared to freshly isolated peripheral blood lymphocytes (PBLs). Specific knockdown of STAT3 expression in Hut78 cells by RNA interference induced morphological and biochemical changes indicating apoptotic cell death. Moreover, STAT3 inhibition downregulated the expression of Bcl2 family of anti-apoptotic gene Bcl-xL. These observations suggest that STAT3 is required for the survival of CTCL cells and strongly indicate that targeting STAT3 using siRNA techniques may serve a novel therapeutic strategy for the treatment of CTCL.

Development of Immunogenic Tumor-Loaded Dendritic Cells Through Physical Perturbation and Apoptotic Cell Loading

To improve understanding of the forces that drive monocytes to transition into dendritic cells (), we developed an experimental system that converts monocytes to DC by passage of leukocytes through a 400 μm silica bead column. The results demonstrate that overnight culture of column-treated monocytes causes a phenotypic conversion that is characteristically displayed by immature DC. These phenotypic changes were enhanced when the DC were loaded with apoptotic cells, leading to increased expression of the DC maturation-associated markers CD83, CD80 and the chemokine receptor CCR7. The DC demonstrated potent induction of allogeneic T cell proliferation and the capacity to activate autologous CD8+ T cells. The CD8 T cells expressed augmented levels of perforin, IFN-γ and TNF-α and mediated CTCL cell apoptosis. These studies demonstrate that physical contact with silica beads combined with loading of apoptotic tumor cells induces synchronized, rapid conversion of human monocytes to DC, which can efficiently stimulate CD8+ T cells. These results may aid in the development of more efficient DC vaccines that can be loaded with the universe of antigens available in apoptotic tumor cells in a rapid, clinically practical fashion.

Oncolytic measles virus in cutaneous T-cell lymphomas mounts antitumor immune responses in vivo and targets interferon-resistant tumor cells

AbstractSome cutaneous T-cell lymphomas, (CTCLs) clonal T cells are deficient in interferon signaling, making them promising targets for viral oncolysis. We evaluated cytopathic effects of measles virus (MV) in CTCL. CTCL cell lines and infiltrating lymphocytes in CTCL expressed MV receptors CD150 and CD46. In a phase 1 dose escalation trial a total of 16 injections of live MV, Edmonston-Zagreb vaccine strain, were given intratumorally to 5 patients with CTCL. Patients had antimeasles-serum antibodies and were pretreated with interferon-α to prevent uncontrolled virus spread. The well-tolerated treatment with MV resulted in clinical responses. Evaluation of biopsies, before and at 11 days after injection, by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated local viral activity with positive staining for MV nucleoprotein (NP), an increase of the interferon γ (IFN-γ)/CD4 and IFN-γ/CD8 mRNA ratios and a reduced CD4/CD8 ratio. All patients demonstrated an increased antimeasles antibody titer after therapy. The data demonstrate that CTCLs are promising targets for an MV-based oncolytic therapy.

Syndrome de sézary

The Sezary cell is a mature lymphocyte with a convoluted, cerebriform nucleus on cytomorphologic and ultrastructural samples. It is a major diagnostic feature of Sezary syndrome, clinically defined by the rapid onset of a pruriginous erythroderma with diffused adenopathies. Sezary cells can also reveal blood involvement by tumor cells in patients with mycosis fungoides. In a suggestive clinical context, peripheral blood smear examination is essential, whatever the results of the total blood count, to identify a significant percentage of characteristic cells. Most Sezary cells have a phenotype of mature memory T-lymphocytes, CD2 +, CD3 +, CD4 +, CD5 +, CD8 −, CD45RO +, CD45RA −, TCR α/β +, but rarely they can also exhibit a CD8 +CD4 − or CD4 +CD8 + phenotype, and/or a lack of expression of CD2, CD3, CD4, or CD5 antigens. Patients with a Sezary syndrome often have an increase in CD4 + lymphocytes. The CD4 +CD7 − population is also increased, but tumor cells have been identified both in the CD4 +CD7 + and in the CD4 +CD7 − populations. Sezary cells are found in the CD4 +CD26 − population, but there are yet no specific phenotypic markers of Sezary cells. IL-7 is a growth factor for Sezary cells, and can induce the establishment of long-term tumor T-cell lines. We recently identified three other surface antigens on Sezary cells. The Natural Killer (NK) receptor CD158k/KIR3DL2/p140, normally expressed by NK and CD8 + T-cells was detected on the surface of CTCL cell lines as well as on freshly isolated CD4 + peripheral blood lymphocytes from SS patients. SC5 is a newly described activation-related intracellular inhibitory receptor expressed on the surface of a minor PBL subset. We found that SC5 expression was significantly increased in SS cells and correlated to p140 expression, ILT-2/CD85j is another inhibitory receptor expressed by tumor T-lymphocytes. Both SC5 and ILT-2 molecules are functional on CTCL cells, as their triggering in vitro leads to the recruitement of SHP-1 and to the specific inhibition of CTCL malignant cell proliferation induced by CD3/TCR stimulation. The expression of these antigens and the cytokine-induced upregulation of bcl-2 could partly explain the survival and the inhibition of the apoptosis of Sezary cells. Their identification on circulating SS tumor cells might be of importance both for the understanding of CTCL pathophysiology and for the treatment of SS patients.

AD2, a human molecule involved in the interaction of T cells with epidermal keratinocytes and thymic epithelial cells.

Interactions between T cells and epithelial cells of the thymus are essential for normal T cell development, and interactions between T cells and skin epidermal keratinocytes occur in the context of inflammatory skin diseases and cutaneous T cell malignancies. On the basis of observations that the T cell ALL cell line, HSB, bound to IFN-gamma activated epidermal keratinocytes (41 +/- 5% of EK with three or more HSB cells bound), whereas the CTCL cell line H9 bound poorly (8 +/- 3%), we have raised mAb 13H12 that identified a 36 kDa molecule, termed AD2, that was highly expressed on HSB but not on H9 T cells. mAb 13H12 inhibited the binding of HSB T cells to IFN-gamma-treated epidermal keratinocytes (43 +/- 5% inhibition, p < 0.01), inhibited the binding of peripheral blood T cells to IFN-gamma-treated EK (62 +/- 3% inhibition, p < 0.001), and inhibited the binding of IFN-gamma-treated human thymic epithelial cells to thymocytes (39 +/- 3% inhibition, p < 0.01). Although AD2 was expressed at a low level on all T cells, AD2 was highly expressed on the CD3-CD4-CD8-, CD3-CD4low+ CD8-, and CD3-CD4+ CD8+ subsets of immature thymocytes in the thymic subcapsular and inner cortex. Taken together, these data suggest a role for the AD2 molecule in interactions of T cells with epithelial cells of skin and thymus.

Testing CTCL cell lines for the expression of the rag genes

Testing CTCL cell lines for the expression of the rag genes