Pan-histone Deacetylase Inhibitor Panobinostat Research Articles

Highly Specific Blood-Brain Barrier Transmigrating Single-Domain Antibodies Selected by an In Vivo Phage Display Screening.

A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.

Abstract 49: Brain penetrant HDAC inhibitor RG2833 induces DIPG cell death and synergizes with lomustine

Abstract Diffuse intrinsic pontine glioma (DIPG) is a universally fatal pediatric brain tumor. The pan-histone deacetylase (HDAC) inhibitor panobinostat showed great activity against DIPG in pre-clinical models, but has poor blood brain barrier penetration and demonstrated significant toxicity in clinical trials. RG2833 (RGFP109) is a selective HDAC1/3 inhibitor with established brain penetration. In clinical trials, the Cmax(plasma) of RG2833 was 32uM. RG2833 demonstrated cytotoxicity against temozolomide-resistant glioblastoma and downregulated the NFĸB pathway. Because this pathway is overexpressed in DIPG and may play a role in DIPG cell growth and survival, we hypothesized that RG2833 would kill DIPG cells. Treatment of DIPG cell lines with RG2833 as a single agent inhibits cell growth in the 5 to 10μM range (MTS assay for HSJD007 p=0.0004 10μM vs DMSO, JHH-DIPG1 p=0.001 10μM vs DMSO, SF-7761 p=0.04 10μM vs DMSO, SU-DIPG13 p=0.01 10μM vs DMSO by t-test). RG2833 induces apoptosis by 48 hours as measured by Western blot for cPARP and cleaved caspase 3 immunofluorescence (HSJD007 p<0.003 8μM vs DMSO, JHH-DIPG1 p=0.0026 10μM vs DMSO by t-test). RG2833 also slows cell proliferation as measured by Western blot for phospho-Rb and immunofluorescence for BrdU (HSJD007 p=0.008 8μM vs DMSO, JHH-DIPG1 p=0.0002 10μM vs DMSO by t-test). Western blot confirmed a dose-dependent increase in histone 3 acetylation with RG2833 treatment. We detected increased acetylated p65 and decreased expression of the NFĸB regulated pro-survival genes BCL2, BCL-xL, and XIAP with RG2833 treatment in vitro. Treatment of DIPG flank tumor mouse models with RG2833 alone for 5 days suppressed tumor growth (p< 0.005 by t-test). Combination treatment with RG2833 and lomustine was synergistic against DIPG cell lines in vitro (ZIP synergy scores by SynergyFinder for JHH-DIPG1 17.8 and HSJD007 17.7). We will next assess combination treatment in vitro and in vivo for apoptotic effects. This data indicates that selective HDAC inhibitor RG2833 may be a promising therapeutic candidate for DIPG. Citation Format: Katherine K. Barnett, Orlandi Novak, Charles Eberhart, Eric Raabe. Brain penetrant HDAC inhibitor RG2833 induces DIPG cell death and synergizes with lomustine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 49.

Abstract 1917: The HDAC inhibitor panobinostat elicits preferential cytotoxic and antiproliferative effects in IDH mutant glioblastoma

Abstract Glioblastoma (GBM) is an incurable form of brain cancer with a median survival of ~15 months. Identification of a CpG Island Methylator Phenotype (CIMP) subtype of GBM (G-CIMP) represents a significant clinical discovery as these patients have an enhanced survival, with a median survival of 3 years. G-CIMP is characterized by a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2) which results in production of the oncometabolite 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent DNA demethylases. This mutation occurs early in gliomagenesis and further results in aberrant DNA methylation and widespread transcriptional repression. Histone deacetylase (HDAC) enzymes are localized to methylated chromatin via methyl-binding domain (MBD) proteins, so we hypothesized that IDH mutant GBM exhibits enhanced reliance on HDAC activity, which is functionally significant to their cell proliferation and survival. Using a panel of 6 patient-derived cell lines grown in defined growth media, we show that G-CIMP is significantly more sensitive in vitro to the clinically approved pan histone deacetylase (HDAC) inhibitor panobinostat, with IDH mutant GBM cells exhibiting ~4 and ~10 fold lower IC/EC50 values in flow cytometric cell viability and apoptosis assays compared to IDH wild type (WT) GBM cells, respectively. For IDH mutant GBM, the average IC50 value was 13.5 nM for cell viability assays and the average EC50 value was 4.8 nM for apoptosis assays. Induction of cleaved caspase 3, a marker of apoptosis, was observed only in IDH mutant GBM cells when exposed to 10 nM panobinostat over 5 days. Analysis of proliferation via BrdU incorporation assays show that treatment with 10 nM panobinastat for 48 hours results in a 90% reduction in proliferation of IDH mutant GBM compared to only 25% reduction in IDH WT GBM cells. Molecular analysis via western blot shows that various acetylated chromatin marks, i.e. H3K14/18/27Ac, are preferentially upregulated in our IDH mutant GBM cell lines when exposed to 10 nM panobinostat for 48 hours. This was also supported by ELISA data showing that IDH mutant GBM cells have ~2 fold increase in histone H3 acetylation compared to IDH WT cells in response to 10 nM and 50 nM panobinostat. These data led us to perform a HDAC activity assay with nuclear extracts from our IDH WT and mutant GBM cells, but this assay showed no significant difference in basal HDAC activity. Overall, our results support a hypothesis where IDH mutant GBMs are more sensitive to panobinostat via preferential uptake of the drug. These data ultimately suggest that G-CIMP tumor cells display a profound sensitivity to the cytotoxic and antiproliferative effects mediated by exposure to the HDAC inhibitor panobinostat. These studies provide a strong foundation for future preclinical work evaluating the use of HDAC inhibitors as a personalized cancer therapy to treat G-CIMP. Citation Format: Thomas K. Sears, Kevin D. Woolard. The HDAC inhibitor panobinostat elicits preferential cytotoxic and antiproliferative effects in IDH mutant glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1917.

Editor's Note: Role of CAAT/Enhancer Binding Protein Homologous Protein in Panobinostat-mediated Potentiation of Bortezomib-induced Lethal Endoplasmic Reticulum Stress in Mantle Cell Lymphoma Cells.

Purpose: Bortezomib induces unfolded protein response (UPR) and endoplasmic reticulum stress, as well as exhibits clinical activity in patients with relapsed and refractory mantle cell lymphoma (MCL). Here, we determined the molecular basis of the improved in vitro and in vivo activity of the combination of the pan-histone deacetylase inhibitor panobinostat and bortezomib against human, cultured, and primary MCL cells. Experimental Design: Immunoblot analyses, reverse transcription-PCR, and immunofluorescent and electron microscopy were used to determine the effects of panobinostat on bortezomib-induced aggresome formation and endoplasmic reticulum stress in MCL cells. Results: Treatment with panobinostat induced heat shock protein 90 acetylation; depleted the levels of heat shock protein 90 client proteins, cyclin-dependent kinase 4, c-RAF, and AKT; and abrogated bortezomib-induced aggresome formation in MCL cells. Panobinostat also induced lethal UPR, associated with induction of CAAT/enhancer binding protein homologous protein (CHOP). Conversely, knockdown of CHOP attenuated panobinostat-induced cell death of MCL cells. Compared with each agent alone, cotreatment with panobinostat increased bortezomib-induced expression of CHOP and NOXA, as well as increased bortezomib-induced UPR and apoptosis of cultured and primary MCL cells. Cotreatment with panobinostat also increased bortezomib-mediated in vivo tumor growth inhibition and improved survival of mice bearing human Z138C MCL cell xenograft. Conclusion: These findings suggest that increased UPR and induction of CHOP are involved in enhanced anti-MCL activity of the combination of panobinostat and bortezomib. Clin Cancer Res; 16(19); 4742–54. ©2010 AACR.

The mTOR inhibitor everolimus overcomes CXCR4-mediated resistance to histone deacetylase inhibitor panobinostat through inhibition of p21 and mitotic regulators

Although having promising anti-myeloma properties, the pan-histone deacetylase inhibitor (HDACi) panobinostat lacks therapeutic activity as a single agent. The aim of the current study was to elucidate the mechanisms underlying multiple myeloma (MM) resistance to panobinostat monotherapy and to define strategies to overcome it. Sensitivity of MM cell lines and primary CD138+ cells from MM patients to panobinostat correlated with reduced expression of the chemokine receptor CXCR4, whereas overexpression of CXCR4 in MM cell lines increased their resistance to panobinostat. Decreased sensitivity to HDACi was associated with reversible G0/G1 cell growth arrest while response was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators revealed the pro-survival mTOR pathway to be regulated by CXCR4 overexpression. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance to HDACi and induced synergistic cell death. The combination of panobinostat/everolimus resulted in sustained DNA damage and irreversible suppression of proliferation accompanied by robust apoptosis. Gene expression analysis revealed distinct genetic profiles of single versus combined agent exposure. Whereas panobinostat increased the expression of the cell cycle inhibitor p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated the expression of DNA repair genes and mitotic checkpoint regulators. Importantly, the combination of panobinostat with everolimus effectively targeted CXCR4-expressing resistant MM cells in vivo in the BM niche. In summary, our results uncover the mechanism responsible for the strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel potential therapeutic approach to treat MM.

The distribution, clearance, and brainstem toxicity of panobinostat administered by convection-enhanced delivery.

OBJECTIVE The pan-histone deacetylase inhibitor panobinostat has preclinical efficacy against diffuse intrinsic pontine glioma (DIPG), and the oral formulation has entered a Phase I clinical trial. However, panobinostat does not cross the blood-brain barrier in humans. Convection-enhanced delivery (CED) is a novel neurosurgical drug delivery technique that bypasses the blood-brain barrier and is of considerable clinical interest in the treatment of DIPG. METHODS The authors investigated the toxicity, distribution, and clearance of a water-soluble formulation of panobinostat (MTX110) in a small- and large-animal model of CED. Juvenile male Wistar rats (n = 24) received panobinostat administered to the pons by CED at increasing concentrations and findings were compared to those in animals that received vehicle alone (n = 12). Clinical observation continued for 2 weeks. Animals were sacrificed at 72 hours or 2 weeks following treatment, and the brains were subjected to neuropathological analysis. A further 8 animals received panobinostat by CED to the striatum and were sacrificed 0, 2, 6, or 24 hours after infusion, and their brains explanted and snap-frozen. Tissue-drug concentration was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Large-animal toxicity was investigated using a clinically relevant MRI-guided translational porcine model of CED in which a drug delivery system designed for humans was used. Panobinostat was administered at 30 μM to the ventral pons of 2 juvenile Large White-Landrace cross pigs. The animals were subjected to clinical and neuropathological analysis, and findings were compared to those obtained in controls after either 1 or 2 weeks. Drug distribution was determined by LC-MS/MS in porcine white and gray matter immediately after CED. RESULTS There were no clinical or neuropathological signs of toxicity up to an infused concentration of 30 μM in both small- and large-animal models. The half-life of panobinostat in rat brain after CED was 2.9 hours, and the drug was observed to be distributed in porcine white and gray matter with a volume infusion/distribution ratio of 2 and 3, respectively. CONCLUSIONS CED of water-soluble panobinostat, up to a concentration of 30 μM, was not toxic and was distributed effectively in normal brain. CED of panobinostat warrants clinical investigation in patients with DIPG.

Refining Radiation for the Next Century.

![][1] Wilhelm Roentgen's report more than a century ago describing X-ray imaging fostered in the application of ionizing radiation (IR) for cancer therapy. Today, about half of all the patients with cancer are treated with radiotherapy and it remains one of our most effective

PDTM-14. THE DISTRIBUTION, CLEARANCE AND TOXICITY OF PANOBINOSTAT ADMINISTERED TO JUVENILE RAT AND PIG BRAINSTEM BY CONVECTION ENHANCED DELIVERY

AbstractThe pan-histone deacetylase inhibitor panobinostat has preclinical efficacy against DIPG and the oral formulation has entered a phase one clinical trial. However, panobinostat does not cross the blood brain barrier in humans. Convection enhanced delivery (CED) is a novel neurosurgical drug delivery technique that bypasses the blood brain barrier and is of considerable clinical interest in DIPG. We investigated the toxicity, distribution and clearance of a water-soluble formulation of panobinostat in small and large animal models of CED. 30 juvenile male Wistar rats received panobinostat administered to the pons by CED at increasing concentration and were compared to animals that received vehicle alone. Clinical observation continued for two weeks. Animals were sacrificed at 72 hours or two weeks following treatment and the brains were subjected to neuropathological analysis. A further 6 animals received panobinostat by CED to the striatum and were sacrificed zero, two or six hours after infusion, their brains explanted and snap-frozen. Brainstem drug concentration was determined by LC-MS/MS. Large animal toxicity was investigated using a clinically relevant MRI guided translational porcine model of CED using a drug delivery system designed for human use. 30 μM panobinostat was administered to the ventral pons of two juvenile Large White/Landrace-cross pigs and subject to clinical and neuropathological analysis compared to control after one or two weeks. Drug distribution was determined by LC-MS/MS in porcine white and gray matter immediately after CED. There were no clinical or neuropathological signs of toxicity up to an infused concentration of 30 μM in both small and large animal models. The half-life of panobinostat in rat brain after CED was 3 hours and the drug was observed to distribute in porcine white and gray matter with a volume infusion/distribution ratio of two and three respectively. CED of water-soluble panobinostat warrants investigation in children with DIPG.

TRTH-07. THE DISTRIBUTION, CLEARANCE AND TOXICITY OF PANOBINOSTAT ADMINISTERED TO JUVENILE RAT AND PIG BRAINSTEM BY CONVECTION ENHANCED DELIVERY

The pan-histone deacetylase inhibitor panobinostat has preclinical efficacy against DIPG and the oral formulation has entered a phase one clinical trial. However, panobinostat does not cross the blood brain barrier in humans. Convection enhanced delivery (CED) is a novel neurosurgical drug delivery technique that bypasses the blood brain barrier and is of considerable clinical interest in DIPG. We investigated the toxicity, distribution and clearance of a water-soluble formulation of panobinostat in small and large animal models of CED. 30 juvenile male Wistar rats received panobinostat administered to the pons by CED at increasing concentration and were compared to animals that received vehicle alone. Clinical observation continued for two weeks. Animals were sacrificed at 72 hours or two weeks following treatment and the brains were subjected to neuropathological analysis. A further 6 animals received panobinostat by CED to the striatum and were sacrificed zero, two or six hours after infusion, their brains explanted and snap-frozen. Brainstem drug concentration was determined by LC-MS/MS. Large animal toxicity was investigated using a clinically relevant MRI guided translational porcine model of CED using a drug delivery system designed for human use. 30 μM panobinostat was administered to the ventral pons of two juvenile Large White/Landrace-cross pigs and subject to clinical and neuropathological analysis compared to control after one or two weeks. Drug distribution was determined by LC-MS/MS in porcine white and gray matter immediately after CED. There were no clinical or neuropathological signs of toxicity up to an infused concentration of 30 μM in both small and large animal models. The half-life of panobinostat in rat brain after CED was 3 hours and the drug was observed to distribute in porcine white and gray matter with a volume infusion/distribution ratio of two and three respectively. CED of water-soluble panobinostat warrants investigation in children with DIPG.

Convection enhanced delivery of panobinostat (LBH589)-loaded pluronic nano-micelles prolongs survival in the F98 rat glioma model.

BackgroundThe pan-histone deacetylase inhibitor panobinostat is a potential therapy for malignant glioma, but it is water insoluble and does not cross the blood–brain barrier when administered systemically. In this article, we describe the in vitro and in vivo efficacy of a novel water-soluble nano-micellar formulation of panobinostat designed for administration by convection enhanced delivery (CED).Materials and methodsThe in vitro efficacy of panobinostat-loaded nano-micelles against rat F98, human U87-MG and M059K glioma cells and against patient-derived glioma stem cells was measured using a cell viability assay. Nano-micelle distribution in rat brain was analyzed following acute CED using rhodamine-labeled nano-micelles, and toxicity was assayed using immunofluorescent microscopy and synaptophysin enzyme-linked immunosorbent assay. We compared the survival of the bioluminescent syngenic F98/Fischer344 rat glioblastoma model treated by acute CED of panobinostat-loaded nano-micelles with that of untreated and vehicle-only-treated controls.ResultsNano-micellar panobinostat is cytotoxic to rat and human glioma cells in vitro in a dose-dependent manner following short-time exposure to drug. Fluorescent rhodamine-labelled nano-micelles distribute with a volume of infusion/volume of distribution (Vi/Vd) ratio of four and five respectively after administration by CED. Administration was not associated with any toxicity when compared to controls. CED of panobinostat-loaded nano-micelles was associated with significantly improved survival when compared to controls (n=8 per group; log-rank test, P<0.001). One hundred percent of treated animals survived the 60-day experimental period and had tumour response on post-mortem histological examination.ConclusionCED of nano-micellar panobinostat represents a potential novel therapeutic option for malignant glioma and warrants translation into the clinic.

The mTOR Inhibitor Everolimus Overcomes CXCR4-Mediated Resistance to HDAC Inhibitor Panobinostat through Inhibition of p21 and Mitosis Regulators, Sensitizing MM Cells to DNA-Damaged Induced Apoptosis

Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Despite the initial efficacious treatment, MM patients often become refractory to common anti-MM drugs, therefore novel therapies are in need. Pan-histone deacetylase (HDAC) inhibitor panobinostat exerts multiple cytotoxic actions in MM cells in vitro, and was approved for the treatment of relapsed/refractory MM in combination with bortezomib and dexamethasone. Although having promising anti-MM properties, panobinostat lacks therapeutic activity as monotherapy. The aim of the current study was to elucidate the mechanisms underlying MM resistance to panobinostat and to define strategies to overcome it.Results: Panobinostat at the low concentrations (IC50 5-30 nM) suppressed the viability in MM cell lines (n=7) and primary CD138+ cells from MM patients (n=8) in vitro. Sensitivity to panobinostat correlated with reduced expression of chemokine receptor CXCR4, while overexpression of CXCR4 or its ligand CXCL12 in RPMI8226 and CAG MM cell lines significantly (p<0.001) increased their resistance to panobinostat, pointing to the role of the CXCR4 axis in HDACi response. Notably, similar expression levels of class I HDACs (HDAC1-3) were detected in MM cells with either low or high CXCR4. Interaction with BM stromal cells that represent the source of CXCL12 also protected MM cells from panobinostat-induced apoptosis, further strengthening a role for CXCR4 downstream pathway. Decreased sensitivity to cytotoxic effect was concomitant with reduced histone (H3K9 and H4K8) acetylation in response to panobinostat treatment. In addition, resistance to HDACi was associated with the reversible G0/G1 cell growth arrest, whereas sensitivity was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators involved in CXCR4-mediated HDACi resistance revealed the pro-survival AKT/mTOR pathway to be regulated by both CXCR4 over-expression and interaction with BMSCs. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance and induced synergistic cell death of MM cell lines and primary MM cells, but not of normal mononuclear cells (CI<0.4). This effect was concurrent with the increase in DNA double strand breaks, histone H2AX phosphorylation, loss of Dψm, cytochrome c release, caspase 3 activation and PARP cleavage. The increase in DNA damage upon combinational treatment was not secondary to the apoptotic DNA fragmentation, as it occurred similarly when apoptosis onset was blocked by caspase inhibitor z-VAD-fmk. Kinetics studies also confirmed that panobinostat-induced DNA damage preceded apoptosis induction. Strikingly, combined panobinostat/everolimus treatment resulted in sustained DNA damage and irreversible suppression of MM cell proliferation accompanied by robust apoptosis, in contrast to the modest effects induced by single agent. Gene expression analysis revealed distinct genetic profiles of single versus combined exposures. Whereas panobinostat increased the expression of cell cycle inhibitors GADD45G and p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated DNA repair genes, including RAD21, Ku70, Ku80 and DNA-PKcs. Furthermore, combined treatment markedly decreased both mRNA and protein expression of anti-apoptotic factors survivin and BCL-XL, checkpoint regulator CHK1, and G2/M-specific factors PLK1, CDK1 and cyclin B1, therefore suppressing the DNA damage repair and inhibiting mitotic progression. Given the anti-apoptotic role of p21, the synergistic lethal effect of everolimus could be attributed to its ability to suppress p21 induction by panobinostat ensuing the shift in the DNA damage response toward apoptosis.Conclusions: Collectively, our findings indicate that CXCR4/CXCL12 activity promotes the resistance of MM cells to HDACi with panobinostat through mTOR activation. Inhibition of mTOR by everolimus synergizes with panobinostat by simultaneous suppression of p21, G2/M mitotic factors and DNA repair machinery, rendering MM cells incapable of repairing accumulated DNA damage and promoting their apoptosis.Our results unravel the mechanism responsible for strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel therapeutic strategy to eradicate MM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Phase II Study of Panobinostat with Lenalidomide and Weekly Dexamethasone in Myeloma

Background:Treatment options for multiple myeloma (MM) refractory (ref) to immunomodulatory drugs (IMID) and proteasome inhibitors (PI) are urgently needed. Epigenetic agents e.g. the pan histone deacetylase inhibitor (HDACi) panobinostat (pan) to modulate the acetylation of histones and oncogenic proteins are promising. Preclinically in MM, pan is syngeristic when combined with dexamethasone (dex), lenalidomide (len), and bortezomib (btz) (Ocio EM et al. Haematologica 2010). The phase 3 PANORMA 1 study of 768 patients randomized to receive IV btz and dex with either pan or placebo revealed a 3.9 month increase in PFS and increased CR rates with pan but at the cost of an increase in grade 3/4 diarrhea from 8% to 25% (San Miguel, Lancet 2014).The safety/preliminary efficacy of pan-len-dex was assessed in a phase 1b study (Mateos et al, ASCO 2010) but was complicated by high dose dex toxicities. The maximum tolerated doses were used for this phase 2 study, however, we attenuated the schedule so that pan is given thrice weekly every other week (instead of weekly) and dex is given only weekly (table 1).Methods:Inclusion criteria were patients with rel or rel/ref MM (including IMID/PI ref), measurable disease, adequate organ function and hematologic parameters. Patients previously treated with a HDACi or currently receiving QTC prolonging medications were excluded.The primary objective in this open label, single arm phase 2 study was to evaluate the best overall response rate (ORR). Secondary objectives were to evaluate safety, duration of response (DOR), and overall and progression-free survival (PFS). Each drug was administered at the doses and schedule shown in Table 1.Results:26 evaluable patients with progressive disease (PD) at screening have been enrolled with a median age of 64 (44% > 65 yo) and a median of 2 lines of therapy over 4 years since diagnosis. High-risk molecular findings were present in 14 patients (54%), including 4 with del p53 and 10 with gain of 1q21 by FISH (4 with concurrent t(4;14)). 22 (85%) were len- ref, & 35, 54, 23% were ref to each: pomalidomide (pom), btz, & carfilzomib.Responses include 2 complete responses (CR), 4 very good partial responses (VGPR), 4 PRs, 9 minimal responses (MR), and 3 stable diseases (SD), for an ORR of 38%, CBR (>MR) of 73%, and a median DOR of 6 mos. The median PFS was 6.5 mos. In the 22 len-ref pts, there were 4 VGPRs, 2 PRs, 8 MRs & 3 SDs, with a median PFS of 5.5 mos. Responses were even seen in 10 pom-ref pts including 1 VPGR, 1 PR, and 4 MRs.Grade 3/4 toxicities (regardless of drug attribution) were primarily hematologic, with neutropenia (40%), thrombocytopenia (23%) and anemia (4%) respectively. Grade 3/4 nonhematologic AEs included infections in 5 (1 while neutropenic), 3 diarrhea (transient), 4 fatigue & 1 pulmonary embolus and 1 pt each with: neck pain, QTc prolongation & weight loss. Patients requiring dose reductions of len/pan respectively were 4/4 for ANC, 5/1 for plts, 1/1 for febrile neutropenia & 5 len for fatigue, & 1 pan for asymptomatic T wave inversions. No doses were held or reduced for GI toxicities.Preliminary results from RNA-seq of bone marrow (BM) aspirates comparing > PR responders (R) vs < PR non responders (NR) identify 261 differentially expressed transcripts (p<0.05). Network analysis revealed "Antigen Presentation/Cell mediated immunity" as a top network function with TLR3 and MHC Class II complex as focus molecules. Immunophenotyping of the tumor microenvironment showed increased CD1c+ myeloid dendritic cells in Rs and conversely, increased CD123+ plasmacytoid dendritic cells in NRs (p<0.05). BM cytokine analysis revealed higher IL-6 and alpha-interferon levels at baseline in Rs. Interestingly, protein levels of Cereblon, Ikaros and Aiolos were not significantly different in the 2 groups.Conclusions:In rel/ref MM, the completely oral pan len dex demonstrates encouraging ORR, DOR, and PFS, even in len-ref patients with high-risk molecular findings, indicating the essential role of pan in attaining responses. In notable contrast to PANORMA 1, this regimen is well tolerated with no significant GI toxicities and primarily expected hematologic toxicities. Updated results of planned 27 pts, including correlatives, will be presented at the annual meeting.Table 1Study Drug DosesStudyPan 20 mg poLen 25 mg poDex 40 mg poMateos et alDay 1,3,5,8,10,12,15,17,19Day 1-21Day 1-4, 9-12,17-20Current studyDay 1,3,5,15,17,19Day 1-21Day 1, 8, 15 DisclosuresChari:Onyx: Consultancy, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Array BioPharma: Consultancy, Research Funding; Millenium/Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Catamero:Millennium / Takeda: Other: Lecturer; Onyx: Other: Lecturer; Celgene: Honoraria, Other: Lecturer. Verina:Celgene: Other: Lecturer. Jagannath:Celgene: Honoraria; Janssen: Honoraria; Merck: Honoraria; Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

Synergistic anti-leukemic interactions between panobinostat and MK-1775 in acute myeloid leukemia ex vivo

MK-1775 is the first-in-class selective Wee1 inhibitor which has been demonstrated to synergize with CHK1 inhibitors in various malignancies. In this study, we report that the pan-histone deacetylase inhibitor (HDACI) panobinostat synergizes with MK-1775 in acute myeloid leukemia (AML), a malignancy which remains a clinical challenge and requires more effective therapies. Using both AML cell line models and primary patient samples, we demonstrated that panobinostat and MK-1775 synergistically induced proliferation arrest and cell death. We also demonstrated that panobinostat had equal anti-leukemic activities against primary AML blasts derived from patients either at initial diagnosis or at relapse. Interestingly, treatment with panobinostat alone or in combination with MK-1775 resulted in decreased Wee1 protein levels as well as downregulation of the CHK1 pathway. shRNA knockdown of CHK1 significantly sensitized AML cells to MK-1775 treatment, while knockdown of Wee1 significantly enhanced both MK-1775- and panobinostat-induced cell death. Our results demonstrate that panobinostat synergizes with MK-1775 in AML cells, at least in part through downregulation of CHK1 and/or Wee1, providing compelling evidence for the clinical development of the combination treatment in AML.

Pre-clinical activity of BET protein antagonist-based combinations against ibrutinib-sensitive or -resistant human Mantle Cell Lymphoma cells

In Mantle Cell Lymphoma (MCL) cells, activated B cell receptor (BCR) and NFkB signaling exerts pro-growth and pro-survival activity. Treatment with the Bruton’s Tyrosine Kinase (BTK) inhibitor ibrutinib induces clinical responses, but long-term durable remissions and cure remains elusive. BET (bromodomain and extra-terminal) proteins, including BRD4, bind to acetylated lysine(s) on histones, assemble transcriptional co-regulators at the enhancers and promoters and increase the transcription of MCL-relevant oncogenes, e.g., MYC, BCL-2, CDK4/6, cyclin D1. Additionally, BRD4 is essential for NFkB activity. Here, we determined that BET protein antagonists JQ1 or I-BET151, which disrupt the binding of BRD4 to acetylated histones, dose-dependently inhibit the growth and induce apoptosis of cultured (MO2058 and Mino, Z138 and Jeko1) and primary MCL (pMCL) cells. Treatment with JQ1 attenuated the mRNA and protein expression of MYC, BCL2, MCL-1 and CDK4/6, while inducing the levels of HEXIM1, p21 and p27 in MCL cells. Importantly, JQ1 treatment also attenuated the nuclear RelA and inhibited the expression of several NFkB target genes, including XIAP, IkBα, cFLIP, cIAP2, as well as BTK. Compared to ibrutinib alone, co-treatment with JQ1 and ibrutinib markedly inhibited pBTK, BTK, pPLCγ2, pAKT and nuclear RelA levels and synergistically induced apoptosis of the cultured and pMCL cells. Following tail-vein infusion and engraftment of Mino cells in the bone marrow and spleen, co-treatment with JQ1 and ibrutinib, versus each agent alone, significantly improved the survival of NOD/SCID mice. By exposing parental Mino cells to escalating levels of ibrutinib, we isolated Ibrutinib-resistant Mino/IR cells (>20 fold resistant). Compared to Mino, Mino/IR cells demonstrated higher levels of BTK, AKT, XIAP, BCL-2 and CDK6, but reduced levels of NOXA, PUMA and p21. Co-treatment with JQ1 and the pan-histone deacetylase inhibitor panobinostat (PS) or the BCL-2 antagonist venetoclax (ABT199), or palbocyclib (CDK4/6 inhibitor) synergistically induced apoptosis of Mino and cultured pMCL, as well as of Mino/IR cells. These findings highlight promising BET protein (BA) antagonist-based combinations with ibrutinib, panobinostat, venetoclax or palbociclib against MCL cells. They also identify potential BA-based combinations against ibrutinib-resistant MCL cells.

A phase II study of panobinostat with lenalidomide and weekly dexamethasone in myeloma.

8528 Background: Preclinical data support deacetylation of oncogenic proteins/histones with the pan histone deacetylase inhibitor (HDACi) panobinostat (pan), which renders MM cells more susceptible...

Increased expression of amyloid precursor protein promotes proliferation and migration of AML1/ETO-positive leukemia cells and be inhibited by panobinostat.

Amyloid precursor protein (APP) is ahighly conserved integral membrane protein extensively expressed in various types of cells. Previously we found that overexpression of APP in patients with AML1/ETO-positive acute myeloid leukemia (AML) associated with ahigher incidence of extramedullaryinfiltrationin and indicate apoor prognosis. In this study, we attempted to define the roles of APP in AML1/ETO-positive leukemia cells. Western blotting and qRT-PCR analysis showed that protein levels of APP are significantly higher in Kasumi-1, at(8;21)/ AML1/ETO-positive M2-type AML cell line. Stable knockdown of APP by lentivirus-based RNA interference (RNAi) dramatically impaired colony-formation and migration ability of Kasumi-1 cells, whereas APP knockdown had very little effect on cell viability, apoptosis, cell cycle and differentiation. We further explored whether the pan-histone deacetylase inhibitor panobinostat could deplete the protein levels of APP in Kasumi-1 cells. Treatment with panobinostat caused depletion of APP in Kasumi-1 cells. These findings indicate that overexpression of APP is involved in promoting proliferation and migration of AML1/ETO-positive leukemia cells and can be inhibited by panobinostat, which provide an attractive prospect for treatment of AML1/ETO-positive AML.

Pre-clinical efficacy of combined therapy with novel β-catenin antagonist BC2059 and histone deacetylase inhibitor against AML cells.

The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survival of AML stem/blast progenitor cells (BPCs). Deregulated WNT signaling inhibits degradation of β-catenin, causing increased nuclear translocation and co-factor activity of β-catenin with the transcriptional regulator TCF4/LEF1 in AML BPCs. Here, we determined the pre-clinical anti-AML activity of the anthraquinone oxime-analog BC2059 (BC), known to attenuate β-catenin levels. BC treatment disrupted the binding of β-catenin with the scaffold protein TBL1 (transducin β-like 1) and proteasomal degradation and decline in the nuclear levels of β-catenin. This was associated with reduced transcriptional activity of TCF4 and expression of its target genes, cyclin D1, c-MYC and survivin. BC treatment dose-dependently induced apoptosis of cultured and primary AML BPCs. Treatment with BC also significantly improved the median survival of immune-depleted mice engrafted with either cultured or primary AML BPCs exhibiting nuclear expression of β-catenin. Co-treatment with the pan-histone deacetylase inhibitor panobinostat and BC synergistically induced apoptosis of cultured and primary AML BPCs, including those expressing FLT3-ITD, as well as further significantly improved the survival of immune-depleted mice engrafted with primary AML BPCs. These findings underscore the promising pre-clinical activity and warrant further testing of BC against human AML, especially those expressing FLT3-ITD.

Mechanistic Role of HEXIM1 Induction in BRD4-Antagonist Mediated Growth Inhibition, Differentiation and in Vivo Lethal Activity Against Human AML Blast Progenitor Cells

The BET (bromodomain and extra terminal) protein family, including BRD4 are chromatin reader proteins that bind to acetylated lysines on histone proteins and recruit transcriptional regulatory complexes to gene promoters, thereby coupling histone acetylation to RNA polymerase II (RNAP2)-mediated transcript elongation. Preferential binding of BRD4 to clustered enhancers or ‘super enhancers’ enables BRD4, through the mediator complex, to regulate the transcription of specific oncogenes, including c-MYC, BCL2 and CDK4/6 in AML blast progenitor cells (BPCs). Through its C-terminal domain (CTD), BRD4 recruits pTEFb (positive transcription elongation factor b), the heterodimer composed of cyclin T and CDK9, which phosphorylates the heptad repeats on the CTD of RNAP2 at serine 2. The kinase activity of CDK9 in pTEFb is also required for the phosphorylation of DSIF and NELF, which abrogates their inhibitory hold on the pause-release of RNAP2 and productive transcript elongation of specific oncogenes and their target genes. pTEFb exists in two forms, the catalytically active form, which is associated with BRD4 and RNAP2, and a catalytically inactive form associated with 7SK snRNP (small nuclear ribonucleoprotein) and the inhibitory protein HEXIM1 (hexamethylene bis-acetamide or HMBA-inducible protein 1). The transition of pTEFb between the inactive and active complex is dynamic and regulated by cell growth and stress signaling. HEXIM1 binds to and sequesters pTEFb, thereby inhibiting its kinase activity for RNAP II. We recently reported (Mol Cancer Ther, 2014) that the BRD4 antagonist JQ1 and I-BET151 alone, and synergistically with the pan-histone deacetylase inhibitor panobinostat, inhibits the growth and induces apoptosis in cultured (OCI-AML3 and MOLM13) and patient-derived primary AML (pAML) BPCs. JQ1 treatment also improved the survival of the immune-depleted mice engrafted with MOLM13 or pAML BPCs. Notably, the lethal activity of JQ1 was associated with marked induction of HEXIM1. Therefore, we hypothesized that HEXIM1 induction, by sequestering pTEFb and inhibiting RNAP2, contributes to the growth inhibitory and lethal effects of JQ1. We additionally hypothesized that co-treatment with those agents that further augment HEXIM1 induction would synergize with JQ1 against AML BPCs. To validate these hypotheses, we achieved a stable lentivirus mediated knockdown (KD) of the mRNA and protein expressions of HEXIM1 by approximately 90% in OCI-AML3 and MOLM13 cells, as well as transient KD in pAML BPCs. Compared to the parental control cells, OCI-AML3/HKD and MOLM13/HKD cells showing HEXIM1 knockdown (HKD) exhibited reduced binding of HEXIM1 with cyclin T (as revealed by cyclin T pull-down and immunofluorescence microscopy), but showed augmented suspension culture growth and increased protein levels (by Western blot) of cyclin T, pRNAP2 and cMYC levels. However, in contrast to the parental control cells, JQ1 (1000 nM) treatment-mediated HEXIM1 induction and binding to cyclin T was markedly inhibited in the HKD AML BPCs, which was associated with significant inhibition of JQ1-induced pRNAP2, as well as differentiation and apoptosis of HKD BPCs (p < 0.01). Notably, whereas treatment with JQ1 alone improved the median survival of NOD/SCID mice engrafted with MOLM13 BPCs, this survival improvement was significantly impaired in the mice engrafted with MOLM13/HKD AML BPCs (p < 0.02). This indicated that HEXIM1-induction mechanistically contributes to JQ1-induced growth inhibition, differentiation and apoptosis of AML BPCs. Further, the synergistic apoptosis observed in MOLM13, OCI-AML3 and primary AML cells induced by co-treatment with JQ1 (250 to 1000 nM) and PS (5 to 10 nM) or HMBA (1 to 2 mM) was also associated with greater induction of HEXIM1. However, this synergy was impaired in the cultured and pAML BPCs with HEXIM1 knockdown. These findings confirm that HEXIM1 induction is a biomarker and plays a mechanistic role in mediating growth inhibition, differentiation and apoptosis induced by BRD4 antagonist in human AML BPCs. These findings support, and we have embarked upon, studies involving future pre-clinical development of those agents that in combination significantly augment BRD4 antagonist-induced HEXIM1 in human AML BPCs. DisclosuresNo relevant conflicts of interest to declare.

Superior Pre-Clinical Activity of BET (Bromodomain and Extra terminal) Protein Antagonist Combined with Ibrutinib, Panobinostat or Carfilzomib Against Human Mantle Cell Lymphoma (MCL) Cells

Poor-risk Mantle Cell Lymphoma (MCL) demonstrate several genetic alterations involving cyclin D1, RB1, ATM, p53, INK4a/ARF, BMI1, HDM2, CDK4, CDKN2A, NOTCH1 & 2, c-MYC, BCL2 and BIM, as well as in the Toll like receptor (TLR), B-cell receptor (BCR) and NFkB signaling genes. Activated BCR signaling is also associated with downstream pro-growth and pro-survival activity of NFκB in the poor-risk MCL. Pre-clinical and clinical studies have shown that ibrutinib, a selective, orally bioavailable, irreversible inhibitor of BTK is active against B cell neoplasms, especially MCL. Treatment with ibrutinib induces clinical responses, but long-term durable remissions and cure remains uncertain. BET (bromodomain and extra terminal) protein family members, including BRD4, bind to acetylated lysine(s) on the histone proteins, help assemble transcriptional co-regulators at the transcriptionally active target oncogene 'super’ enhancers and promoters, and regulate the expression of important MCL-relevant oncogenes, e.g., c-MYC, BCL-2, CDK4/6 and cyclin D1. Additionally, BRD4 has been shown to bind acetylated RelA and is essential for NFκB activity. Here, we determined that treatment with the prototype BET protein antagonist JQ1 (250 to 2000 nM) or I-BET151, which disrupts the binding of BRD4 to acetylated histones, dose-dependently inhibited growth and exerted lethal activity against cultured (MO2058 and Mino, Z138 and JeKo-1) and patient-derived, primary MCL (pMCL) cells. Treatment with JQ1 reduced the occupancy of BRD4 and RNA pol II (RNAP2) on the promoters of c-MYC and BCL2, as well as attenuated the mRNA (by qPCR) and protein expression (by Western blot) of c-MYC, BCL2, MCL-1 and, CDK4/6, without affecting the levels of SOX11, but induced the levels of HEXIM1, p21, p27 and cleaved PARP in MCL cells. Importantly, JQ1 treatment reduced p-BTK, BTK and p-PLCγ2 levels in MCL cells. JQ1 treatment also attenuated the nuclear RelA (by Western and confocal immunofluorescence analyses) and inhibited the expression of several NFkB target genes, including XIAP, Iκbα, cFLIP and cIAP2 expressions. Treatment with ibrutinib (2 to 10 µM) attenuated p-BTK, p-PLCγ2, p-AKT and c-MYC levels but induced p21 levels, as well as induced apoptosis of cultured (MO2058, Mino and Z138 and JeKo-1 cells) and pMCL cells. Importantly, co-treatment with JQ1 and ibrutinib markedly inhibited p-BTK, BTK, p-PLCγ2, p-AKT and nuclear RelA levels and synergistically induced apoptosis of the cultured (JeKo-1, MO2058 and Mino) and pMCL cells (CI values < 1.0 by isobologram analysis). Further, following tail-vein infusion and engraftment of Mino cells (3 million cells/mouse) in the bone marrow and spleen of NOD/SCID mice, as compared to each agent alone, co-treatment with JQ1 (50 mg/kg/day, IP) and ibrutinib (25 mg/kg/day IP) significantly improved the survival of the NOD/SCID mice (p < 0.01). We also isolated ibrutinib-resistant Mino/IR cells ( > 20 fold resistant), following a continuous exposure of the parental Mino cells in culture to escalating levels of ibrutinib, which exhibited high levels of p-BTK, p-PLCγ2, c-MYC and CDK6. Co-treatment with JQ1 and the pan-histone deacetylase inhibitor panobinostat (PS) synergistically induced apoptosis of not only Mino but also Mino/IR cells. We also isolated carfilzomib-resistant JeKo-1/CZR cells ( > 10 fold resistant). As compared to JeKo-1, JeKo-1/CZR cells demonstrated higher protein levels of proteasome subunits and increased base-line and lesser EGCG (Epigallocatechin-3-gallate)-suppressible proteasome activity. Mutations in the PSMB5 were not detected. JeKo-1/CZR cells also displayed higher levels of c-MYC, cyclin D1, MCL-1 and CDK4. Notably, co-treatment with JQ1 and PS or ibrutinib also synergistically induced apoptosis of not only JeKo-1 but also JeKo-1/CZR cells (CI values < 1.0 by isobologram analysis). Taken together, these pre-clinical findings demonstrate synergistic preclinical activity of BET protein (BA) antagonist-based combinations with ibrutinib, panobinostat or carfilzomib against human MCL cells. Collectively, they also define potential BA-based combinations to overcome resistance to ibrutinib and carfilzomib in human MCL. DisclosuresNo relevant conflicts of interest to declare.

A Phase II, Single-Center, Open-Label Study of Oral Panobinostat in Combination with Lenalidomide and Weekly Dexamethasone in Patients with Multiple Myeloma

Background: Treatment options for patients with multiple myeloma (MM) refractory (ref) to immunomodulatory drugs and proteasome inhibitors are urgently needed. A promising strategy is the use of epigenetic agents such as the pan histone deacetylase inhibitor (HDACi) panobinostat (pan) to modulate the acetylation of histones and proteins involved in oncogenesis. Pre-clinical studies with pan demonstrate synergy against MM cells when combined with dexamethasone (dex), lenalidomide (len), and bortezomib (btz) (Ocio EM et al. Haematologica 2010). Interim data from the phase 3 PANORMA 1 study of 768 patients randomized to receive IV btz and dex with either pan or placebo revealed a 3.9 month increase in PFS with pan along with an increase in CR rates. However, this was accompanied by 25% grade 3/ 4 diarrhea versus 8% in the placebo arm.The safety and preliminary efficacy of the pan-len-dex triplet regimen was assessed in a phase 1b study of relapsed (rel) or rel/ref MM patients (Mateos et al, ASCO 2010) but was complicated by high dose dex toxicities. The maximum tolerated dose of pan and dex in that study are the doses selected for this phase 2 study. However we investigated a modified schedule (table 1) of this triplet regimen. Here, pan is given thrice weekly only every other week (instead of weekly) and dex is given weekly (instead of three 4 day pulses).Patients and Methods: Inclusion criteria were patients with rel or rel/ref MM, measurable disease, adequate performance status, organ function, and hematologic parameters. Patients previously treated with a HDACI or currently receiving medications with a risk of prolonging the QTc interval were excluded.The primary objective was to evaluate the best overall response rate (ORR). Secondary objectives were to evaluate safety, response duration, and overall and progression-free survival. Each drug was administered at the doses and schedule shown in Table 1.Results: Overall, 13 evaluable patients with progressive disease (PD) at screening have been enrolled, including 9 len-refractory (2 also pomalidomide refractory) and 4 len sensitive with a median of 4 and 3 lines of prior therapy respectively (range 1–10). High-risk molecular findings were present in 9 patients, including 6 with gain of 1q21 by FISH and 3 with del p53. Three of the patients with gain of 1q21 also had t (4;14).Of the 13 patients, there have been 3 very good partial responses (VGPR), 2 partial responses (PR), 3 minimal responses (MR), 4 stable diseases (SD), and 1 PD, for an ORR of 38% and clinical benefit rate (CBR i.e. MR or greater) of 61 %. With a median follow up of 4.5 months the median progression free survival and duration of response have not been reached. Of the 9 patients who were len refractory, there were 2 VGPR, 2 MRs, 4 SD, and 1 PD for a 22% ORR and 44% CBR. Notably, 3 len refractory patients remain on treatment for 11, 16, and 16 months including 2 with gain of 1q21 that have attained VGPRs.Grade 3/4 toxicities (regardless of drug attribution) were primarily hematologic, with neutropenia, thrombocytopenia, lymphopenia, and anemia noted in 7, 4, 1, and 1 patients respectively. Grade 3/4 nonhematologic AEs included infections in 4 (with 1 one occurring while neutropenic), and 1 patient with each of the following: pulmonary embolus, neck pain, QTc prolongation, and weight loss 1. Dose modifications for neutropenia were required in 4 patients for len and in 2 patients for pan. 1 additional patient required pan dose reduction for asymptomatic T wave inversions. Nausea was noted in 2 patients and diarrhea in 3 with 2 additional patients experiencing both – however these were transient, Grade 1/2, and did not require dose modifications. Importantly, no patients discontinued therapy for toxicity.Conclusions: In rel/ref MM patients, pan in combination with len and dex demonstrates durable responses, even in len-refractory patients with high-risk molecular findings, indicating the essential role of pan in attaining a response. These results suggest that pan modulates expression of genes to restore sensitivity to len, In notable contrast to the PANORMA 1 results, this completely oral regimen is well tolerated with no Grade 3/4 GI toxicities and primarily expected hematologic toxicities. Updated results, including correlative studies, will be presented at the annual meeting.Table 1:Study Drug DosesPanobinostatLenalidomideDexamethasone20 mg po Day 1, 3, 5, 15, 17,1925 mg po Day 1-2140 mg po Day 1, 8, 15 DisclosuresChari:Array Biopharma: Membership on an entity’s Board of Directors or advisory committees; Millenium : Consultancy, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Jagannath:Celgene: Honoraria; Millennium: Honoraria; Sanofi: Honoraria.