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Cell-based immunotherapies have had remarkable success in the clinic, specifically in the treatment of hematologic malignancies. However, these strategies have had limited efficacy in patients with solid tumors. To better understand the challenges involved, the National Cancer Institute (NCI) convened an initial workshop with immuno-oncology thought leaders in December 2018 and a follow-up workshop in December 2020. The goals of the NCI workshops on cell-based immunotherapy for solid tumors were to discuss the current state of the field of cell-based immunotherapy, obtain insights into critical knowledge gaps, and identify ways in which NCI could facilitate progress. At both meetings, subjects emphasized four main types of challenges in further developing cell-based immunotherapy for patients with solid tumors: scientific, technical, clinical, and regulatory. The scientific barriers include selecting appropriate targets, ensuring adequate trafficking of cell therapy products to tumor sites, overcoming the immunosuppressive tumor microenvironment, and identifying appropriate models for these investigations. While mouse models may provide some useful data, the majority of those that are commonly used are immunodeficient and unable to fully recapitulate the immune response in patients. There is therefore a need for enhanced support of small early-phase human clinical studies, preferably with adaptive trial designs, to provide proof of concept for novel cell therapy approaches. Furthermore, the requirements for manufacturing, shipping, and distributing cell-based therapies present technical challenges and regulatory questions, which many research institutions are not equipped to address. Overall, workshop subjects identified key areas where NCI support might help the research community in driving forward innovation and clinical utility: 1) provide focused research support on topics such as tumor target selection, immune cell fitness and persistence, cell trafficking, and the immunosuppressive tumor microenvironment; 2) support the rapid translation of preclinical findings into proof of concept clinical testing, harmonize clinical trial regimens, and facilitate early trial data sharing (including negative results); 3) expand manufacturing support for cell therapies, including vectors and reagents, and provide training programs for technical staff; and 4) develop and share standard operating procedures for cell handling and analytical assays, and work with the Food and Drug Administration to harmonize product characterization specifications.

Objective To introduce a novel preclinical animal model of psoriatic arthritis (PsA) in R26Stat3Cstopfl/fl CD4Cre mice, and to investigate the role of Th17 cytokines in the disease pathogenesis. Methods We characterized a novel murine model of Th17-driven cutaneous and synovio-entheseal disease directed by T cell-specific expression of a hyperactive Stat3 allele. By crossing R26Stat3Cstopfl/fl CD4Cre mice onto an interleukin-22 (IL-22)-knockout background or treating the mice with a neutralizing antibody against IL-17, we interrogated how these Th17 cytokines could contribute to the pathogenesis of PsA. Results R26Stat3Cstopfl/fl CD4Cre mice developed acanthosis, hyperkeratosis, and parakeratosis of the skin, as well as enthesitis/tendinitis and periarticular bone erosion in different joints, accompanied by osteopenia. T cell-specific expression of a hyperactive Stat3C allele was found to drive the augmented Th17 response in these animals. Careful characterization of the mouse bone marrow revealed an increase in osteoclast progenitor (OCP) and RANKL-producing cells, which contributed to the osteopenia phenotype observed in the mutant animals. Abrogation of the Th17 cytokines IL-17 or IL-22 improved both the skin and bone phenotype in R26Stat3Cstopfl/fl CD4Cre mice, revealing a central role of Th17 cells in the regulation of OCP and RANKL expression on stromal cells. Conclusion Perturbation of the IL-23/Th17 axis instigates Th17-mediated inflammation in R26Stat3Cstopfl/fl CD4Cre mice, leading to cutaneous and synovio-entheseal inflammation and bone pathologic features highly reminiscent of human PsA. Both IL-17A and IL-22 produced by Th17 cells appear to play critical roles in promoting the cutaneous and musculoskeletal inflammation that characterizes PsA.

Cutaneous T-cell lymphoma is a heterogeneous group of lymphomas characterized by the accumulation of malignant T cells in the skin. The molecular and cellular etiology of this malignancy remains enigmatic, and what role antigenic stimulation plays in the initiation and/or progression of the disease remains to be elucidated. Deep sequencing of the tumor genome showed a highly heterogeneous landscape of genetic perturbations, and transcriptome analysis of transformed T cells further highlighted the heterogeneity of this disease. Nonetheless, using data harvested from high-throughput transcriptional profiling allowed us to develop a reliable signature of this malignancy. Focusing on a key cytokine signaling pathway previously implicated in cutaneous T-cell lymphoma pathogenesis, JAK/STAT signaling, we used conditional gene targeting to develop a fully penetrant small animal model of this disease that recapitulates many key features of mycosis fungoides, a common variant of cutaneous T-cell lymphoma. Using this mouse model, we show that T-cell receptor engagement is critical for malignant transformation of the T lymphocytes and that progression of the disease is dependent on microbiota.

Th17 cells are a proinflammatory subset of effector T cells that have been implicated in the pathogenesis of asthma. Their production of the cytokine IL-17 is known to induce local recruitment of neutrophils, but the direct impact of IL-17 on the lung epithelium is poorly understood. In this study, we describe a novel mouse model of spontaneous IL-17–driven lung inflammation that exhibits many similarities to asthma in humans. We have found that STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs. IL-17 secretion then leads to neutrophil infiltration and lung epithelial changes, in turn leading to a chronic inflammatory state with increased mucus production and decreased lung function. We used this model to investigate the effects of IL-17 activity on airway epithelium and identified CXCL5 and MIP-2 as important factors in neutrophil recruitment. The neutralization of IL-17 greatly reduces pulmonary neutrophilia, underscoring a key role for IL-17 in promoting chronic airway inflammation. These findings emphasize the role of IL-17 in mediating neutrophil-driven pulmonary inflammation and highlight a new mouse model that may be used for the development of novel therapies targeting Th17 cells in asthma and other chronic pulmonary diseases.

Fogli, L. K., M. S. Sundrud, S. Goel, S. Bajwa, K. Jensen, E. Derudder, A. Sun, M. Coffre, C. Uyttenhove, J. Van Snick, M. Schmidt-Supprian, A. Rao, G. Grunig, J. Durbin, S. S. Casola, K. Rajewsky, and S. B. Koralov. 2013. T cell–derived IL-17 mediates epithelial changes in the airway and drives

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

Mantle cell lymphoma (MCL) is a disease characterized by gross cell cycle dysregulation driven by the constitutive overexpression of cyclin D1. The identification of a “proliferation signature” in MCL, underscores the necessity of new therapeutic approaches aimed at lowering the proliferative signature of the disease, theoretically shifting the prognostic features of the disease. Romidepsin, an HDAC inhibitor (HDACi) approved for the treatment of relapsed T-cell lymphoma, is thought to induce cell cycle arrest and apoptosis. Central to the block of cell proliferation is the up-regulation of the cdk inhibitor p21Cip1/Waf1. However up-regulation of p21Cip1/Waf1 has also been shown to reduce sensitivity to romidepsin. HDACi activates p21Cip1/Waf1 expression via ATM and KU60019, a specific ATM inhibitor, has been shown to decrease the p21Cip1/Waf1 protein levels in a concentration dependent manner. We sought to explore the effect of the combination of romidepsin and KU60019 in inducing cell death in MCL. Analysis of romidepsin treated Jeko-1 cell extracts showed a marked effect on the expression of proteins involved in cell cycle regulation. Decrease expression of Emi1, a mitotic regulator required for the accumulation of the APC/C substrates was observed. Emi1 is also responsible for the stability of the E3 ubiquitin ligase Skp2 that specifically recognizes and promotes the degradation of phosphorylated cdk inhibitor p27. However, decrease in Emi1 protein levels, upon addition of romidepsin, was not followed by an increased expression of the cdk inhibitor p27. On the other end, increased expression of the cdk inhibitor p21Cip1/Waf1, was a common feature of all romidepsin treated MCL lines analyzed. Cell cycle analysis via Fluorescent Activated Cell Sorting (FACS) of romidepsin treated Jeko-1 cells showed an accumulation of romidepsin treated cells in the G2/M phase when compared to the control suggesting a p21Cip1/Waf1 induced cell cycle arrest. For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-GloTM followed by acquisition on a Biotek Synergy HT and IC50s calculated using the Calcusyn software. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR). Synergy analyses were performed using Jeko-1, Maver-1 and Z-138 cells treated with different concentrations of romidepsin corresponding to IC10-20 in combination with KU60019 at a concentration of 2.5, 5.0, 7.5 and 15 umol/L for 24, 48 and 72 hours. A synergistic cytotoxic effect was observed in all MCL cell lines when the HDACi was combined with KU60019 throughout the range of all concentrations. The RRR analysis showed a strong synergism at 48 and 72 hours in virtually all combinations of HDACi and KU60019 in all three cell lines. The results of drug:drug combination in two of the three cell lines are shown below. Protein expression analysis of Jeko-1 and Maver-1cells treated with single agents or combinations for 48 hours revealed changes in a host of proteins known to be involved in cell cycle control and apoptosis. The increased p21 protein expression upon addition of romidepsin, was not observed when the romidepsin treatment was combined with the KU60019. Increased activation of the programmed cell death proteins Caspase 8, induced by Fas, and Caspase 3 was observed upon combinations of the single agents in all three cell lines, resulting in an increased cleavage of Poly (ADP-ribose) polymerase (PARP-1). Finally, the abundance of the anti-apoptotic proteins Bcl-XL and BCL-2 showed a significant decrease after treatment with romidepsin plus increase concentrations of KU60019 when compared with their abundance in the presence of the single agents. Cell cycle analysis of Jeko-1 cells treated for 24 hours with single agents and combination suggests that the increased apoptosis is the result of inhibition of the p21Cip1/Waf1 induced G2/M cell cycle arrest by KU60019. Overall, these data demonstrated that the combination of romidepsin and KU60019 was synergistically effective in inhibiting the in vitro growth of the mantle cell lymphoma lines. Jeko-1 Maver-1 Disclosures: O'Connor: Celgene: Consultancy, Research Funding.

Abstract 66 Background Cutaneous 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. Methods Using 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. Results Upon 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. Disclosures: No relevant conflicts of interest to declare.

Abstract 2727 CHOP and CHOP-like chemotherapy remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) are presently approved for the treatment of relapsed or refractory cutaneous T- cell lymphomas (CTCL) and peripheral T-cell lymphomas (PTCL) given their marked single agent activity in these diseases. The interaction between the HDACIs (depsipeptide (R) and belinostat (B)) and a DNMT inhibitor (decitabine (D)) was investigated in vitro, in vivo and at the molecular level in different T-cell lymphoma and leukemia cell lines including CTCL (H9, HH), and T- acute lymphoblastic leukemia (T-ALL) lines resistant to gamma-secretase inhibitors (P12, PF-382). For all cytotoxicity assays, a luminescence based cell viability assay was used (CellTiter-Glo™) followed by acquisition on a Biotek Synergy HT. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR An in vivo xenograft study in 6–8 weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells was performed. Mice were separated into different cohorts and treated i.p. for 3 cycles with D or B or their combination according to the following schedules: D at 1.5 mg/kg on days 1, 3, 5; B at 40 mg/kg/day for 10 days (I cycle); D at 1.5mg/kg on days 15,17,19,21; B at 65 mg/kg/day for 10 days (II cycle); D at 1.5 mg/kg on days 29,31,33,35,37,39,41,43; B at 100mg/kg for 19 days (III cycle). Statistically significantly tumor growth inhibition was observed in the combination cohort compared to all the other cohorts (analysis on day 42, 45). We analyzed the molecular basis for this synergistic effect by evaluating gene expression patterns using the Illumina Human HT-12 v4 Expression BeadChip microarrays. These analyses revealed differentially expressed genes and modulated pathways for each of the single treatment conditions and the combination. As shown in Figure 1, a set of genes (A) is down-regulated by both drugs. Other genes (B) are up-regulated by D and the effect is maintained in the combination. Other genes (C+E) are slightly up-regulated by R, though not significantly modified by D, and more strongly up-regulated in the combination group. Similarly, genes to some extent up-regulated by D but not by R (D+F) appeared to be more significantly affected by the combination. As shown in Figure 2, the effects of the two drugs are largely different (only 39 genes modified in common by all the treatment groups). Most of the effects induced by the single agent treatment are maintained in the combination group (174 genes out of 191 for romidepsin and 211 genes out of 221 for decitabine). Interestingly, an additional 944 genes appeared to be modulated uniquely by the combination treatment strongly supporting the hypothesis of synergism also at the molecular level. Collectively, the data suggest that the combination of a DNMTI and HDACIs is synergistic in in vitro and in vivo model of T-cell lymphoma and is able to synergistically reverse the malignant signature at the molecular level. These data may constitute the basis for future phase I-II clinical trials. Disclosures: O'Connor: celgene: Consultancy, Research Funding; merck: Research Funding; Novartis: Research Funding; spectrum: Research Funding.