Preclinical Models Of T-cell Lymphoma Research Articles

Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL).Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand).Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays.Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability.Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo.By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro.Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds.Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. DisclosuresSims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.

Combined oral 5-azacytidine and romidepsin are highly effective in patients with PTCL: a multicenter phase 2 study

AbstractPeripheral T-cell lymphomas (PTCLs) are uniquely vulnerable to epigenetic modifiers. We demonstrated in vitro synergism between histone deacetylase inhibitors and DNA methyltransferase inhibitors in preclinical models of T-cell lymphoma. In a phase 1 trial, we found oral 5-azacytidine and romidepsin to be safe and effective, with lineage-selective activity among patients with relapsed/refractory (R/R) PTCL. Patients who were treatment naïve or who had R/R PTCL received azacytidine 300 mg once per day on days 1 to 14, and romidepsin 14 mg/m2 on days 8, 15, and 22 every 35 days. The primary objective was overall response rate (ORR). Targeted next-generation sequencing was performed on tumor samples to correlate mutational profiles and response. Among 25 enrolled patients, the ORR and complete response rates were 61% and 48%, respectively. However, patients with T-follicular helper cell (tTFH) phenotype exhibited higher ORR (80%) and complete remission rate (67%). The most frequent grade 3 to 4 adverse events were thrombocytopenia (48%), neutropenia (40%), lymphopenia (32%), and anemia (16%). At a median follow-up of 13.5 months, the median progression-free survival, duration of response, and overall survival were 8.0 months, 20.3 months, and not reached, respectively. The median progression-free survival and overall survival were 8.0 months and 20.6 months, respectively, in patients with R/R disease. Patients with tTFH enjoyed a particularly long median survival (median not reached). Responders harbored a higher average number of mutations in genes involved in DNA methylation and histone deacetylation. Combined azacytidine and romidepsin are highly active in PTCL patients and could serve as a platform for novel regimens in this disease. This trial was registered at www.clinicaltrials.gov as #NCT01998035.

Tyrosine kinase inhibition to improve anthracycline-based chemotherapy efficacy in T-cell lymphoma

BackgroundAnthracycline-containing regimens, namely cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) or CHOEP (CHOP + etoposide), represent the current standard of care for patients with newly diagnosed peripheral T-cell lymphomas (PTCLs), although responses are unsatisfactory. In this study, we investigated the pathways able to mitigate the sensitivity to CHOP-based regimens in preclinical models of T-cell lymphoma (TCL) to select agents for the development of CHOP-based drug combinations.MethodsWe performed gene expression profiling of malignant T-cell lines exposed to CHOEP; flow cytometry was employed to study the effects of drug combinations on cell viability, cell cycle distribution, apoptosis and mitochondrial membrane depolarisation. Western blot analyses were performed to study cell signalling downstream of the T-cell receptor and apoptosis. The in vivo effect of the drug combination was tested in xenograft models.ResultsWe highlighted a modulation of tyrosine kinases belonging to the T-cell receptor pathway upon chemotherapy that provided the rationale for combining the tyrosine kinase inhibitor dasatinib with CHOEP. Dasatinib improves CHOEP activity and reduces viability in vitro. Furthermore, combination treatment results in tumour growth inhibition in in vivo xenograft mouse models.ConclusionsOur data provide the rationale for clinical testing of the dasatinib–CHOEP combination in patients with T-cell lymphoma.

Activity of the PI3K-δ,γ inhibitor duvelisib in a phase 1 trial and preclinical models of T-cell lymphoma

AbstractDuvelisib (IPI-145) is an oral inhibitor of phosphatidylinositol 3-kinase (PI3K)–δ/γ isoforms currently in clinical development. PI3K-δ/γ inhibition may directly inhibit malignant T-cell growth, making duvelisib a promising candidate for patients with peripheral (PTCL) or cutaneous (CTCL) T-cell lymphoma. Inhibition of either isoform may also contribute to clinical responses by modulating nonmalignant immune cells. We investigated these dual effects in a TCL cohort from a phase 1, open-label study of duvelisib in patients with relapsed or refractory PTCL (n = 16) and CTCL (n = 19), along with in vitro and in vivo models of TCL. The overall response rates in patients with PTCL and CTCL were 50.0% and 31.6%, respectively (P = .32). There were 3 complete responses, all among patients with PTCL. Activity was seen across a wide spectrum of subtypes. The most frequently observed grade 3 and 4 adverse events were transaminase increases (40% alanine aminotransferase, 17% aspartate aminotransferase), maculopapular rash (17%), and neutropenia (17%). Responders and nonresponders had markedly different changes in serum cytokine profiles induced by duvelisib. In vitro, duvelisib potently killed 3 of 4 TCL lines with constitutive phospho-AKT (pAKT) vs 0 of 7 lines lacking pAKT (P = .024) and exceeded cell killing by the PI3K-δ–specific inhibitor idelalisib. Administration of duvelisib to mice engrafted with a PTCL patient-derived xenograft resulted in a shift among tumor-associated macrophages from the immunosuppressive M2-like phenotype to the inflammatory M1-like phenotype. In summary, duvelisib demonstrated promising clinical activity and an acceptable safety profile in relapsed/refractory TCL, as well as preclinical evidence of both tumor cell–autonomous and immune-mediated effects. This trial was registered at www.clinicaltrials.gov as #NCT01476657.

The Investigational Aurora A Kinase Inhibitor Alisertib Exhibits Broad Activity in Preclinical Models of T-Cell Lymphoma and Is Highly Synergistic with Romidepsin

Aurora A kinase (AAK), a serine-threonine protein kinase, regulates mitotic entry, spindle formation, and cytokinesis. Alisertib is a selective AAK investigational inhibitor with demonstrated clinical activity in acute myeloid leukemia, peripheral T-cell lymphoma (PTCL), DLBCL and other hematologic malignancies. Herein we demonstrate the potent cytotoxicity and apoptotic effects of alisertib in a panel of T-cell-derived lymphoma cell-lines (TCL) (CTCL, HTLV+, T-ALL) and B-cell lymphoma cell-lines (DLBCL-ABC, DLBCL-GCB, MCL) alone and in combination with romidepsin.Single agent concentration and time effect relationships were generated for 8 TCL, 4 DLBCL (2 ABC, 2 GCB) and 4 MCL cell-lines. The mean IC50 of alisertib in TCL was 350 nM (range 100-1000nM) and in B-cell lymphoma lines (DLBCL, MCL) was 200 nM (range 20-300 nM) at 48 hours, measured by growth inhibition. In all cell lines evaluated, there was a consistent 2-log fold decrease in IC50 values at 72 hours, demonstrating the exquisite impact of time on effect. Combination studies evaluating synergy were performed testing schedule, concentration, and time effect relationships. A combinatorial experiment to identify potential synergistic interactions found no synergy with pralatrexate or proteasome inhibitors, but revealed potent synergy with romidepsin. Interestingly, simultaneous exposure of combined alisertib and romidepsin at their IC10, IC20, and IC30 demonstrated marked synergy only in TCL, but not B-cell lymphomas. The best synergy was observed at 72 hours with synergy coefficients ranging from 0.2 to 0.7. This synergistic interaction was restricted to the TCL cell-lines, with no benefit demonstrated in DLBCL or MCL cell lines. (Table 1)We further evaluated this synergistic interaction through a time lapse experiment (0-72hr) by live cell imaging and found that the combined alisertib and romidepsin treatment in H9, a CTCL cell line, led to failure of cytokinesis leading to apoptosis. Evidence for apoptosis was confirmed for alisertib in combination with romidepsin in the TCL cell-line, H9 and HH after 72 hours of exposure through increased Puma, Caspase 3 and PARP cleavage, and decreased BCL-xL and BCL2 expression; suggesting that cell death was apoptotic. AnnexinV/propridium iodide via FACS analysis confirmed induction of apoptosis. In addition, cell cycle analysis was performed following 24 hour of treatment of romidepsin and alisertib as a single agent and in combination. Alisertib produced a G2/M arrest while the combination of both single agents induced polyploidy (up to 8N). This finding may also complement the increase in apoptosis in the combination treatment versus the single agents. Furthermore, preliminary results from an in-vivoxenograft demonstrate a synergistic interaction amongst the combined treatment of alisertib and romidepsin when compared to control cohort and both single agent cohorts.These data support the observation that alisertib produces broad single-agent activity in models of TCL and demonstrates marked synergy with romidepsin. Interestingly this effect is restricted to TCL. Mechanistic studies to better understand this synergistic interaction are ongoing. It is hypothesized that the combined alisertib and romidepsin treatment synergistically inhibit the activation of stat-3, leading to apoptosis and inhibition of cellular proliferation through BCL-xL and c-MYC.Abstract 4493. Table 1:Synergy Coefficients of Alisertib in Combination with Romidepsin at 72 Hours.[1]Drug CombinationDND41(T-ALL)J.CAM 1.6(T-ALL)HH(CTCL)H9(CTCL)C5MJ(HTLV+)Romidepsin [IC10-20] + Alisertib 50 nM0.960.811.051.11.53Romidepsin [IC10-20] + Alisertib 100nM0.510.560.680.660.58Romidepsin [IC10-20] + Alisertib 1000 nM0.400.200.400.460.70[1] 1=additive; <1=synergistic; >1= subadditive DisclosuresAmengual:Acetylon Pharmaceuticals: Research Funding. O’Connor:Millennium Pharmaceuticals: Consultancy; Celgene : Consultancy.

Abstract 1693: The combination of romidepsin and bendamustin is synergistically cytotoxic and reverses the malignant phenotype in preclinical models of T-cell lymphoma

Abstract Background: Peripheral T-cell lymphomas (PTCLs) represent approximately 10-15% of all non-Hodgkin lymphomas (NHL) in the Western world, and their incidence is increasing. Cases of PTCL tend to have an aggressive clinical course, with poor patient responses to conventional chemotherapy and poor long-term survival. So far, treatment approaches have mirrored diffuse large B-cell lymphoma (DLBCL) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) and CHOP-like chemotherapy are commonly used despite suboptimal results. Histone deacetylase inhibitors (HDACs) have been approved for the treatment of relapsed or refractory PTCLs given their single-agent activity in these diseases. To continue to improve responses in patients with PTCL, it is important to assess the utility of romidepsin as frontline therapy and as a component of combination therapies. Aim: Interactions between romidepsin (R) and bendamustine (B), a potent cytotoxic alkylating drug active against a panel of other lymphoproliferative disorders, was investigated in in preclinical models of T-cell Lymphoma. Experimental Design: Assays for cytotoxicity on 5 different T-cell lymphoma and leukemia cell lines (Jurkat, HD-MAR2, Karpas, Sup-T1, HH), mathematical analysis for synergism (Chou-Talalay equation), flow cytometry, and the Itk-Syk transgenic mouse model (K. Pechloff, 2010) were used to explore the in vitro and in vivo activities of R and B alone and in combination in T-cell lymphoid malignancies. Results: In vitro, romidepsin and bendamustine exhibited concentration- and time-dependent cytotoxicity against all the 5 different T-cell lymphoma and leukemia cell lines. Romidepsin showed synergism when combined with bendamustine in all cell lines studied. Romidepsin also induced potent apoptosis and caspase activation when combined with bendamustine across the panel. The impact of schedule on the activity of the combination was determined by assessing cell viability after treatment with B and R as follows: (1) simultaneous exposure; (2) B pretreatment followed by exposure to R; and (3) R pretreatment followed by exposure to B. In a new mouse model of PTCL in which a status of permanent T cell activation mediated by the Itk-Syk transcript induces highly malignant PTCLs with 100% penetrance that resemble the human disease, the combination of romidepsin and bendamustine enhanced efficacy compared with either drug alone. Conclusions: Collectively, these data strongly support the potential therapeutic role of romidepsin in combination with bendamustine for PTCLs and might constitute the basis for future phase I-II clinical trials. Citation Format: Cristiana Carniti, Silvia Gimondi, Antonio Vendramin, Sara Rizzitano, Paolo Corradini. The combination of romidepsin and bendamustin is synergistically cytotoxic and reverses the malignant phenotype in preclinical models of T-cell lymphoma. [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 1693. doi:10.1158/1538-7445.AM2014-1693

The Combination Of Hypomethylating Agents and Histone Deacetylase Inhibitors(HDACi) Are Synergistically Cytotoxic and Reverse The Malignant Phenotype In Preclinical Models Of T-Cell Lymphoma

Both HDAC inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) are known to influence global expression patterns in hematologic malignancies. Little is known about the combination of these two drug classes in lymphoid malignancies. HDACIs have marked single agent activity in the T- cell lymphomas (TCL), although the mechanism of action is not well defined. DNMTIs affect cytosine methylation of genomic DNA and have activity mainly restricted to the myeloid derived hematologic malignancies. The single agent efficacy and synergistic interaction of a panel of HDACIs (panobinostat, belinostat, romidepsin and vorinostat) and DNMTIs (decitabine (DEC), 5-azacytadine (5-AZA)) was evaluated in models of TCL. The molecular basis for the synergistic effect of HDACIs and DNMTIs was evaluated by gene expression profiling (GEP) and CpG methylation CTCL.Single agent concentration and time effect relationships were generated for 2 CTCL (HH, H9) and 2 T-ALL (P12, PF382) cell lines. Romidepsin and belinostat were the most potent HDACIs with the mean 48 hour IC50 of 8.8 nM (range 1.7-2.7 nM) and 85 nM (range 36-136 nM), respectively. Cell viability was not affected by treatment with DEC or 5-AZA at 24 and 48 hours at concentrations as high as 20 μM. Reduction in viability was first demonstrated after 72 hours of exposure to DEC, with the mean IC50 of 14.8 μM (range 0.4 μM- >20uM). Simultaneous exposure of combinations of DEC plus romidepsin or DEC plus belinostat at their IC10, IC20, and IC50 produced marked synergy in all TCL derived cell lines. Simultaneous exposure of DEC plus romidepsin demonstrated the deepest synergy at 72 hours with synergy coefficients in the range of 0.3. Cells treated with the combination of DEC plus romidepsin also demonstrated significant induction of apoptosis as evaluated by annexinV/propridium iodide via FACS analysis and an increase in acetylated histone 3 by immunoblot.The in vivo activity of the combination of DEC plus belinostat was investigated in a xenograft model of CTCL using HH, the most resistant TCL derived cell line. Mice were treated with DEC 1.5 mg/kg (day 29, 33, 35, 37, 39, 41, 43) and/or belinostat 100 mg/kg (day 29-day 47). The combination mouse cohort demonstrated statistically significant tumor growth delay compared to DEC alone (p=0.002) and belinostat alone (p=0.001).The interaction of DEC and romidepsin was analyzed by GEP and methylation array. Interestingly, the baseline malignant phenotype seen in the CTCL cell-lines was reversed. A significant down-regulation of genes involved in biosynthetic pathways including protein and lipid synthesis, and a significant up-regulation of genes responsible for cell cycle arrest were seen. The vast majority (114/138; 92%) of genes modulated by the single agents were similarly modulated by the combination. However, the latter induced a further significant change in the transcriptome, affecting an additional 390 genes. Similarly, methylation array data was analyzed following treatment of these drugs alone and in combination. DEC induced de-methylation of 190 different gene regions corresponding to 175 genes and an additional 335 loci. Interestingly, when combined with romidepsin the number of demethylated gene regions decreased to 85 corresponding to 79 genes, 78 of which were common with DEC and 148 additional loci. The comparison of gene expression and methylation demonstrated a significant inverse relationship (R2 = 0.657) with genes found to be differentially expressed in GEP and methylation analysis. (Figure 1) [Display omitted] These data support the observation that DNMTIs in combination with HDACIs produces significant synergistic activity in models of TCL. Further evaluation of the mechanism of action with DNMTIs in combination with HDACIs is ongoing, and a clinical trial of the combination is now open. Disclosures:O'Connor:Celgene Pharmaceuticals: Consultancy; Spectrum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Hypomethylating Agents in T-cell lymphoma. Amengual:Acetylon Pharmacueticals, INC: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Combination of Histone Deacetylase Inhibitors and Hypomethylating Agents Exhibits Marked Synergy In Preclinical Models of T-Cell Lymphoma

AbstractAbstract 3937CHOP and CHOP-like chemotherapy programs remain the most commonly used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite often sub-optimal results. Histone deacetylase inhibitors (HDACIs) are epigenetic agents known to be active in T-cell lymphoma. Recently romidepsin (R) was approved for patients with relapsed or refractory CTCL. Both R and belinostat (B) are being investigated in patients with relapsed or refractory PTCL. We have previously shown that hypomethylating agents as decitabine (D) produce synergistic interactions with HDACIs in B-cell lymphomas. We investigated the in vitro and in vivo activity of D, R and B alone or in combination 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 (GSI) (P12, PF-382). For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition on a Biotek Synergy HT. The IC50s for D, B and R were calculated using the Calcusyn software (Biosoft). Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR) based on the GraphPad software (RRR<1 are defining synergism). Apoptosis was assessed by staining with Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Whole cell lysate proteins were extracted and quantified according to Bradford assay. After electrophoresis on a gradient 4–20% SDS-PAGE gels the proteins were transferred to nitrocellulose membrane. After blocking and incubation with the primary and the secondary antibodies, the chemiluminescent agent was added and the x-ray films were exposed to the membranes. The IC50s for belinostat alone at 24, 48 and 72 hours were generally in the nanomolar range: H9: 108.1nM – 35.7nM – 29.1nM; HH: 240.1nM - 67.6nM – 39.01nM; P12: 386.9nM – 99.9nM – 99.8nM; PF 382: 267.1nM – 135nM – 118.3nM. The IC50s for romidepsin alone at 24, 48 and 72 hours were generally in the low nanomolar range: H9: 5nM – 2.1nM – 2.2nM; HH: 14nM – 2.6nM - 2.5nM; P12: 6.2nM – 2.4nM – 2.1nM; PF382: 6.1nM – 1.7nM – 1.5nM. The IC50s for D alone at 72 and 96 hours were in the micromolar range: H9: 7.4uM – 3.7uM; HH: > 20 uM. In the cytotoxicity assays, the combination of D and B or R at 72 hours showed synergism in all the cell lines studied. The most representative RRRs are showed in table 1.Table 1D0.5 uM1uMB (nM)RRRH9500.70.7700.60.61000.40.5PF 3821500.80.70.5 uM1 uMR (nM)RRRH90.50.90.910.80.820.30.3PF 38210.80.71.50.40.420.10.1When H9, HH, P12 and PF382 cell lines were treated with D and B or R for 72 hours, all the combination groups showed significantly more apoptosis than the single drug exposures and controls. Table 2 displays the range of apoptosis induction for B, R and D or for them used in combination and the RRR value after the analysis for the most significant data.Table 2BDB + DRRR(% Apoptotic + Dead Cells)H9100nM (22.9%)500nM (17.9%)51.5%0.7HH100nM (42.9%)1uM (46.9%)61.3%0.8P 12150nM (16%)1uM (42.7%)80.1%0.4PF 382100nM (8.3%)1uM (27.9%)40.1%0.8RDR + DH92nM (22.2%)500nM (17.9%)63.6%0.5HH2nM (80%)1uM (46.9%)89.7%0.6P 122nM (9.9%)10uM (58.7%)98%0.03PF 3822nM (54.5%)500nM (17.9%)88.7%0.2Increased acetylation of H3 was observed when H9 cells were treated with R alone and synergistically increased after exposing cells to the combination of D + B and D + R. The expression of phosphorylated Stat3 was decreased after exposure of H9 cells to the combination of D and R. Additional interrogation of the effects of this epigenetic therapy on the JAK-STAT signaling pathway are now underway. An in vivo xenograft study in six to eight weeks old female SCID beige mice injected subcutaneously with 2 × 107 HH cells has also begun and will be reported. Mice were separated into different cohorts and treated with intraperitoneal injections of D or B or their combination according to the following schedules: D alone at 1.5 mg/kg on days 1, 5; B alone at 35 mg/Kg/day for 7 days. Collectively, the data suggest that the combination of a hypomethylating agent like D and a HDACI (B and R) are synergistic in in vitro models of human T-cell lymphoma, and may lead to a new platform for the treatment of these diseases. Disclosures:O'Connor:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.