Search

Supplementary Table S10. p value of NOTCH2 target genes and members of its transcriptional complex that are inversely correlated to BCL6 in primary GC B-cells.

Supplementary Figure S1. BCL6 displays a specific genomic localization pattern in FL. Supplementary Figure S2. Inverse correlation between BCL6 and NOTCH2 complex genes in primary GC B-cells. Supplementary Figure S3. GC reaction is impaired in Notch2 knock-in mice. Supplementary Figure S4. GC reaction is impaired in Notch2 knock-in mice. Supplementary Figure S5. BCL6 represses NOTCH2 complex genes and Notch activity. Supplementary Figure S6. FL cells are dependent on BCL6 in a NOTCH2-dependent manner. Supplementary Figure S7. Evaluation of the in vivo potency and specificity of anti-Notch2 blockade. Supplementary Figure S8. RI-BPI suppresses FL tumors in vivo and ex vivo. Supplementary Table S5. Primers used for RT-PCR.

Supplementary Table 1, Table 2 and Table 3

Supplementary Figures 1-8 Supplementary Tables 1-2 Supplementary Materials and Methods

HSP90 is critical for maintenance of the cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here we described the role of this HSP90 form, referred to as oncogenic HSP90, in the regulation of cytosolic metabolic pathways in proliferating B-cell lymphoma cells. Oncogenic HSP90 assisted in the organization of metabolic enzymes into non-membrane–bound functional compartments. Under experimental conditions that conserved cellular proteostasis, oncogenic HSP90 coordinated and sustained multiple metabolic pathways required for energy production and maintenance of cellular biomass as well as for secretion of extracellular metabolites. Conversely, inhibition of oncogenic HSP90, in absence of apparent client protein degradation, decreased the efficiency of MYC-driven metabolic reprogramming. This study reveals that oncogenic HSP90 supports metabolism in B-cell lymphoma cells and patients with diffuse large B-cell lymphoma, providing a novel mechanism of activity for HSP90 inhibitors. Significance: The oncogenic form of HSP90 organizes and maintains functional multienzymatic metabolic hubs in cancer cells, suggesting the potential of repurposing oncogenic HSP90 selective inhibitors to disrupt metabolism in lymphoma cells.

Mutations in BRAF and RAS are often oncogenic and lead to a constitutively active MAPK pathway that promotes aberrant cell proliferation and tumor growth. Currently approved BRAF inhibitors are selective against monomeric BRAF V600 mutants. These drugs are largely inactive against non-V600 dimeric BRAF mutants and have poor brain penetration. Although there is an FDA-approved KRAS G12C mutant-selective inhibitor, there are no approved inhibitors for cancer patients who harbor other (non-G12C) KRAS and NRAS mutations which promote tumor growth likely through constitutively active RAF dimers. There remains a high unmet clinical need for a highly CNS penetrant oral RAF inhibitor that targets a broad spectrum of BRAF mutations and constitutively active RAF dimers without paradoxical activation of the MAPK signaling pathway. BDTX-4933 is a potent, reversible, CNS penetrant RAF MasterKey inhibitor designed to target a large family of oncogenic BRAF mutations including BRAF monomers and RAF dimers. The compound inhibits not only all classes (I, II, and III) of BRAF mutations but also targets constitutively active RAF dimers promoted by upstream oncogenic MAPK pathway alterations, such as RAS mutations. In a panel of cancer cell lines that endogenously express BRAF or RAS mutations, BDTX-4933 demonstrates inhibition of the MAPK pathway signaling without paradoxical activation, resulting in potent inhibition of cellular proliferation. BDTX-4933 shows target engagement, inhibiting ERK phosphorylation, in tumor models in vivo, achieving strong anti-tumor efficacy and tumor regression across tumor models driven by either BRAF or RAS mutations. Furthermore, BDTX-4933 exhibits high CNS exposure leading to dose-dependent tumor growth inhibition, and survival benefit in mice implanted intracranially with xenograft BRAF mutant tumors. BDTX-4933 has a best-in-class profile to treat cancer patients harboring BRAF mutations or RAF dimer-promoting upstream genetic alterations. IND-enabling studies for BDTX-4933 are on-going. Citation Format: Yoon-Chi Han, Pui-Yee Ng, Luisa Shin Ogawa, Shao Ning Yang, Miao Chen, Noboru Ishiyama, Tai-An Lin, Elizabeth Buck. Preclinical characterization of a brain penetrant RAF inhibitor, BDTX-4933, targeting oncogenic BRAF Class I/II/III and RAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3415.

Several lines of evidence link the canonical oncogene BCL6 to stress response. Here we demonstrate that BCL6 evolved in vertebrates as a component of the HSF1-driven stress response, which has been co-opted by the immune system to support germinal center formation and may have been decisive in the convergent evolution of humoral immunity in jawless and jawed vertebrates. We find that the highly conserved BTB corepressor binding site of BCL6 mediates stress adaptation across vertebrates. We demonstrate that pan-cancer cells hijack this stress tolerance mechanism to aberrantly express BCL6. Targeting the BCL6 BTB domain in cancer cells induces apoptosis and increases susceptibility to repeated doses of cytotoxic therapy. The chemosensitization effect upon BCL6 BTB inhibition is dependent on the derepression of TOX, implicating modulation of DNA repair as a downstream mechanism. Collectively, these data suggest a form of adaptive nononcogene addiction rooted in the natural selection of BCL6 during vertebrate evolution. Significance: We demonstrate that HSF1 drives BCL6 expression to enable stress tolerance in vertebrates. We identify an HSF1–BCL6–TOX stress axis that is required by cancer cells to tolerate exposure to cytotoxic agents and points toward BCL6-targeted therapy as a way to more effectively kill a wide variety of solid tumors. This article is highlighted in the In This Issue feature, p. 565

Description, Arginine enhances the effect of radiation in patients with brain metastasis by metabolic suppression of cancer cells., Selected patients with brain metastases (BM) are candidates for radiotherapy. A lactatogenic metabolism, common in BM, has been associated with radioresistance. We demonstrated that BM express nitric oxide (NO) synthase 2 and that administration of its substrate l-arginine decreases tumor lactate in BM patients. In a placebo-controlled trial, we showed that administration of l-arginine before each fraction enhanced the effect of radiation, improving the control of BM. Studies in preclinical models demonstrated that l-arginine radiosensitization is a NO-mediated mechanism secondary to the metabolic adaptation induced in cancer cells. We showed that the decrease in tumor lactate was a consequence of reduced glycolysis that also impacted ATP and NAD+ levels. These effects were associated with NO-dependent inhibition of GAPDH and hyperactivation of PARP upon nitrosative DNA damage. These metabolic changes ultimately impaired the repair of DNA damage induced by radiation in cancer cells while greatly sparing tumor-infiltrating lymphocytes.

HSP90 is critical for maintenance of the cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here, we described a novel role of HSP90 in the cytosolic compartmentalization of metabolic pathways in proliferating cancer cells. We found that HSP90 assists in the organization of metabolic enzymes into non-membrane-bound functional compartments termed metabosomes. Under experimental conditions that conserved the cellular proteostasis, we demonstrated that the compartmentalizing activity of HSP90 is critical to sustain the coordinated synthesis of multiple metabolites required for energy production, maintenance of the cellular biomass and secretion of immunometabolites. Conversely, inhibition of the nucleating capacity of HSP90 modified the topology of cytosolic metabosomes before protein degradation was apparent decreasing the efficiency of MYC-driven metabolic pathways. Inhibition of HSP90 decreases cancer metabolism in B-cell lymphoma cells and patients providing a novel mechanism of activity for this class of drugs.

The canonical BRAF V600E (Class I) mutation is a potent oncogene uniquely active as a RAS-independent monomer, successfully targeted by several FDA-approved inhibitors. However, these first-generation BRAF inhibitors are not active against non-canonical BRAF oncogenic mutations, including BRAF-fusions, that drive RAS-independent (Class II) or RAS-dependent (Class III) dimers. As such, developing inhibitors directed against dimeric BRAF oncogenic mutations that avoid paradoxical activation is a major unmet clinical need. We applied proprietary Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics to identify and validate a group of previously uncharacterized non-canonical oncogenic Class II and Class III BRAF mutation clusters. We further demonstrate that this ensemble of novel and previously validated non-canonical oncogenic BRAF mutants can form the basis of a differentiated drug discovery program aimed at identifying small molecules that potently and selectively target this family of dimeric BRAF mutations. Herein, we describe a small molecule inhibitor, BDTX BRAF-A, with potent anti-proliferative activity directed against tumor cells expressing a wide spectrum of non-canonical Class II/III mutations. This broad activity (“MasterKey” profile) of BDTX BRAF-A is further demonstrated in cell lines that harbor endogenous oncogenic dimer-inducing BRAF mutations and in various solid tumor patient-derived xenograft (PDX) models ex vivo. Importantly, BDTX BRAF-A did not induce paradoxical RAF activation characteristic of Class I BRAF inhibitors. Finally, BDTX BRAF-A achieves robust anti-tumor efficacy and target engagement of dimeric BRAF oncogenes in mouse models. These data support the continued development of rationally designed molecules targeting a broad range of non-canonical BRAF dimer-promoting mutations to extend the prospect of precision medicine in patients. Citation Format: Yoon-Chi Han, Pui Yee Ng, Ryan Schulz, Shao Ning Yang, Alana Lelo, Luisa Shin, Matthew O'Connor, Ivan Jewett, Noboru Ishiyama, Darlene Romashko, Shalabh Thakur, Andrei Salomatov, Sherri Smith, Elizabeth Buck, Christopher Roberts, Matthew Lucas, Tai-An Lin. Pre-clinical evaluation of next-generation inhibitor targeting a wide spectrum of oncogenic BRAF dimers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P229.

Background: Targeting FGFR genetic alterations using small molecule inhibitors is a validated therapeutic strategy for urothelial carcinoma and cholangiocarcinoma. However, the current FDA-approved pan-FGFR inhibitors, erdafitinib and pemigatinib, are subject to FGFR1-mediated dose-limiting toxicities (e.g., hyperphosphatemia). These treatments necessitate a high rate of dose reductions, interruptions, and discontinuations, thereby potentially limiting efficacy. In addition, drug-resistant mutations (e.g., gatekeeper) in FGFR2 and FGFR3 genes rapidly emerge in patients treated with these drugs. Our research goals are to reveal the full spectrum of oncogenic FGFR2 and FGFR3 mutations that drive tumor growth and to discover an inhibitor that selectively targets these mutations together with FGFR2 and FGFR3 gene fusion and drug-resistance mutations, while minimizing FGFR1 activity and associated toxicities. We hypothesize that this will deliver an FGFR precision medicine with enhanced anti-tumor activity, an improved drug resistance profile, and broader mutational coverage. Methods: Applying the Mutation-Allostery-Pharmacology (MAP) platform technology developed by Black Diamond Therapeutics, we defined a spectrum of 34 allosteric FGFR2/3 oncogenic mutations, including over 28 previously uncharacterized mutations that we now show to be oncogenic. The MAP platform allowed us to further classify those mutations into functional clusters or families of mutations that can be targeted using a single compound. While located throughout the extracellular and kinase domains, we demonstrated how these functional clusters activate FGFR2 or FGFR3. Among the mechanisms identified, a functional cluster of mutations is activated due to disulfide-bond mediated dimerization. Results: Herein, we report the discovery of a series of orally available, selective FGFR2/3 inhibitors that 1) shows antiproliferative potency across all 34 mutations; 2) spares FGFR1-wild-type; 3) is active against gatekeeper mutations and 4) shows favorable selectivity versus a subset of closely related kinases in the human kinome. In addition to being potent against FGFR2 and FGFR3 primary mutations, we demonstrated that our FGFR1 sparing inhibitors retain potency against the most prevalent FGFR2 resistant mutations. When dosed orally, one example was well tolerated and exhibited dose-dependent PK/PD and anti-tumor efficacy and regression in several FGFR2 and FGFR3 driven xenograft models in mice. In addition, when dosed at efficacious doses, no FGFR1-mediated hyperphosphatemia was observed in these animals instead of the animals that were treated with pan FGFR inhibitors. Conclusion: Our data support the development of rationally designed selective inhibitors targeting a spectrum of FGFR2/3 mutations while sparing dose limiting FGFR1 activity. Citation Format: Etienne Dardenne, Fernando Padilla, Sara Rasmussen, Shao Ning Yang, Ahmet Mentes, Luisa Shin Ogawa, Anthony Trombino, Darlene Romashko, Maria Chevtsova, Shalabh Thakur, Elisabeth Buck, Christopher Roberts, Matthew Lucas, Tai-An Lin. Discovery and characterization of selective, FGFR1 sparing, inhibitors of FGFR2/3 oncogenic mutations for the treatment of cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P246.

Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.

// Michela Boi 1, 2, * , Maria Todaro 1, 2, * , Valentina Vurchio 1 , Shao Ning Yang 3 , John Moon 3 , Ivo Kwee 4, 5, 6 , Andrea Rinaldi 4 , Heng Pan 7, 8 , Ramona Crescenzo 1, 2 , Mangeng Cheng 9 , Leandro Cerchietti 3 , Olivier Elemento 7, 8 , Maria E. Riveiro 10 , Esteban Cvitkovic 10, 11 , Francesco Bertoni 4, 12 , Giorgio Inghirami 1, 2, 13 , The AIRC 5xMille Consortium ‘Genetics-Driven Targeted Management of Lymphoid Malignancies’ 1 Department of Molecular Biotechnology and Health Science and Center for Experimental Research and Medical Studies (CeRMS), University of Torino, Torino, Italy 2 Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA 3 Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medical College, New York, NY, USA 4 Lymphoma and Genomics Research Program, IOR Institute of Oncology Research, Bellinzona, Switzerland 5 Dalle Molle Institute for Artificial Intelligence (IDSIA), Manno, Switzerland 6 Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland 7 Institute for Computational Biomedicine, Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA 8 Institute for Precision Medicine, Weill Cornell Medical College, New York, NY, USA 9 In Vitro Pharmacology, Merck Research Laboratory, Boston, MA, USA 10 Oncology Therapeutic Development, Clichy, France 11 Oncoethix SA (Now Oncoethix GmbH, A Wholly Owned Subsidiary of Merck Sharp & Dohme Corp.), Lucerne, Switzerland 12 IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland 13 Department of Pathology, and NYU Cancer Center, New York University School of Medicine, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Giorgio Inghirami, email: ggi9001@med.cornell.edu Francesco Bertoni, email: frbertoni@mac.com Keywords: anaplastic large cell lymphoma, BRD inhibitor, OTX015/MK-8628, tyrosine kinase inhibitor, gene expression profiling Received: July 07, 2016 Accepted: September 19, 2016 Published: October 25, 2016 ABSTRACT Anaplastic large cell lymphomas (ALCL) represent a peripheral T-cell lymphoma subgroup, stratified based on the presence or absence of anaplastic lymphoma kinase (ALK) chimeras. Although ALK-positive ALCLs have a more favorable outcome than ALK-negative ALCL, refractory and/or relapsed forms are common and novel treatments are needed. Here we investigated the therapeutic potential of a novel bromodomain inhibitor, OTX015/MK-8628 in ALK-positive ALCLs. The effects of OTX015 on a panel of ALK+ ALCL cell lines was evaluated in terms of proliferation, cell cycle and downstream signaling, including gene expression profiling analyses. Synergy was tested with combination targeted therapies. Bromodomain inhibition with OTX015 led primarily to ALCL cell cycle arrest in a dose-dependent manner, along with downregulation of MYC and its downstream regulated genes. MYC overexpression did not compensate this OTX015-mediated phenotype. Transcriptomic analysis of OTX015-treated ALCL cells identified a gene signature common to various hematologic malignancies treated with bromodomain inhibitors, notably large cell lymphoma. OTX015-modulated genes included transcription factors ( E2F2 , NFKBIZ, FOS , JUNB, ID1, HOXA5 and HOXC6 ) , members of multiple signaling pathways ( ITK , PRKCH, and MKNK2 ), and histones (clusters 1-3). Combination of OTX015 with the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib led to cell cycle arrest then cell death, and combination with suboptimal doses of the ALK inhibitor CEP28122 caused cell cycle arrest. When OTX015 was associated with GANT61, a selective GLI1/2 inhibitor, C1156Y-resistant ALK ALCL growth was impaired. These findings support OTX015 clinical trials in refractory ALCL in combination with inhibitors of interleukin-2-inducible kinase or SHH/GLI1.

// Thibault Dupont 1, * , ShaoNing Yang 1, * , Jayeshkumar Patel 1 , Katerina Hatzi 1 , Alka Malik 1 , Wayne Tam 3 , Peter Martin 1 , John Leonard 1 , Ari Melnick 1, 2 , Leandro Cerchietti 1 1 Hematology and Oncology Division, Weill Cornell Medical College, New York, NY, USA 2 Pharmacology Department, Weill Cornell Medical College, New York, NY, USA 3 Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Ari Melnick, e-mail: amm2014@med.cornell.edu Leandro Cerchietti, e-mail: Lec2010@med.cornell.edu Keywords: lymphoma, BCL6, BCL2, targeting, resistance Received: July 22, 2015 Accepted: November 21, 2015 Published: December 09, 2015 ABSTRACT The BCL6 oncogene plays a crucial role in sustaining diffuse large B-cell lymphomas (DLBCL) through transcriptional repression of key checkpoint genes. BCL6-targeted therapy kills lymphoma cells by releasing these checkpoints. However BCL6 also directly represses several DLBCL oncogenes such as BCL2 and BCL-XL that promote lymphoma survival. Herein we show that DLBCL cells that survive BCL6-targeted therapy induce a phenomenon of “oncogene-addiction switching” by reactivating BCL2-family dependent anti-apoptotic pathways. Thus, most DLBCL cells require concomitant inhibition of BCL6 and BCL2-family members for effective lymphoma killing. Moreover, in DLBCL cells initially resistant to BH3 mimetic drugs, BCL6 inhibition induces a newly developed reliance on anti-apoptotic BCL2-family members for survival that translates in acquired susceptibility to BH3 mimetic drugs ABT-737 and obatoclax. In germinal center B cell-like (GCB)-DLBCL cells, the proteasome inhibitor bortezomib and the NEDD inhibitor MLN4924 post-transcriptionally activated the BH3-only sensitizer NOXA thus counteracting the oncogenic switch to BCL2 induced by BCL6-targeting. Hence our study indicates that BCL6 inhibition induces an on-target feedback mechanism based on the activation of anti-apoptotic BH3 members. This oncogene-addition switching mechanism was harnessed to develop rational combinatorial therapies for GCB-DLBCL.

Fil: Cayrol, María Florencia. Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Departamento de Neuroinmunomodulación y Oncología Molecular; Argentina Fil: Díaz Flaqué, María Celeste. Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Departamento de Neuroinmunomodulación y Oncología Molecular; Argentina Fil: Tharu, Fernando. Weill Cornell Medical College. Department of Medicine. Division of Hematology and Oncology; Estados Unidos Fil: Sterle, Helena Andrea. Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Departamento de Neuroinmunomodulación y Oncología Molecular; Argentina Fil: Bolontrade, Marcela. Fundación Instituto Leloir. División de Hematología y Oncología; Argentina Fil: Amorós, Mariana. Fundación Instituto Leloir. División de Hematología y Oncología; Argentina Fil: Isse, Blanca. Universidad Nacional de Tucumán. Departamento de Bioquímica de la Nutrición; Argentina Fil: Isse, Blanca. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica Dr Bernabé Bloj; Argentina Fil: Farías, Ricardo Norberto. Universidad Nacional de Tucumán. Departamento de Bioquímica de la Nutrición; Argentina Fil: Farías, Ricardo Norberto. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica Dr Bernabé Bloj; Argentina Fil: Haelee, Ahn. Weill Cornell Medical College. Department of Medicine. Division of Hematology and Oncology; Estados Unidos Fil: Tian, Ye F. Cornell University. Sibley School of Mechanical and Aerospace Engineering; Estados Unidos Fil: Tabbò, Fabrizio. Cornell University. Weill Cornell Medical College. Department of Pathology and Laboratory Medicine; Estados Unidos Fil: Singh, Ankur. Cornell University. Sibley School of Mechanical and Aerospace Engineering; Estados Unidos; Argentina Fil: Inghirami, Giorgio. Cornell University. Weill Cornell Medical College. Department of Pathology and Laboratory Medicine; Estados Unidos Fil: Cerchietti, Leandro. Weill Cornell Medical College. Department of Medicine. Division of Hematology and Oncology; Estados Unidos Fil: Cremaschi. Graciela A. Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Departamento de Neuroinmunomodulación y Oncología Molecular; Argentina Fil: Cremaschi, Graciela A. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Laboratorio de Radioisótopos; Argentina Abstract: The interaction of lymphoid tumor cells with components of the extracellular matrix via integrin avb3 allows tumor survival and growth. This integrin was demonstrated to be the membrane receptor for thyroid hormones (THs) in several tissues. We found that THs, acting as soluble integrin avb3 ligands, activated growth-related signaling pathways in T-cell lymphomas (TCLs). Specifically, TH-activated avb3 integrin signaling promoted TCL proliferation and angiogenesis, in part, via the upregulation of vascular endothelial growth factor (VEGF). Consequently, genetic or pharmacologic inhibition of integrin avb3 decreased VEGF production and induced TCL cell death in vitro and in human xenograft models. In sum, weshowthat integrin avb3 transduces prosurvival signals into TCL nuclei, suggesting a novel mechanism for the endocrine modulation of TCL pathophysiology. Targeting this mechanism could constitute an effective and potentially low-toxicity chemotherapy-free treatment of TCL patients.

Although the BCL6 transcriptional repressor is frequently expressed in human follicular lymphomas (FL), its biological role in this disease remains unknown. Herein, we comprehensively identify the set of gene promoters directly targeted by BCL6 in primary human FLs. We noted that BCL6 binds and represses NOTCH2 and NOTCH pathway genes. Moreover, BCL6 and NOTCH2 pathway gene expression is inversely correlated in FL. Notably, BCL6 upregulation is associated with repression of NOTCH2 and its target genes in primary human and murine germinal center (GC) cells. Repression of NOTCH2 is an essential function of BCL6 in FL and GC B cells because inducible expression of Notch2 abrogated GC formation in mice and killed FL cells. Indeed, BCL6-targeting compounds or gene silencing leads to the induction of NOTCH2 activity and compromises survival of FL cells, whereas NOTCH2 depletion or pathway antagonists rescue FL cells from such effects. Moreover, BCL6 inhibitors induced NOTCH2 expression and suppressed growth of human FL xenografts in vivo and primary human FL specimens ex vivo. These studies suggest that established FLs are thus dependent on BCL6 through its suppression of NOTCH2. Significance: We show that human FLs are dependent on BCL6, and primary human FLs can be killed using specific BCL6 inhibitors. Integrative genomics and functional studies of BCL6 in primary FL cells point toward a novel mechanism whereby BCL6 repression of NOTCH2 drives the survival and growth of FL cells as well as GC B cells, which are the FL cell of origin. Cancer Discov; 7(5); 506–21. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 443

Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL biology represents an urgent need. Here we report that PTCL are sensitive to transcription-targeting drugs, and, in particular, to THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7). The STAT-signalling pathway is highly vulnerable to THZ1 even in PTCL cells that carry the activating STAT3 mutation Y640F. In mutant cells, CDK7 inhibition decreases STAT3 chromatin binding and expression of highly transcribed target genes like MYC, PIM1, MCL1, CD30, IL2RA, CDC25A and IL4R. In surviving cells, THZ1 decreases the expression of STAT-regulated anti-apoptotic BH3 family members MCL1 and BCL-XL sensitizing PTCL cells to BH3 mimetic drugs. Accordingly, the combination of THZ1 and the BH3 mimetic obatoclax improves lymphoma growth control in a primary PTCL ex vivo culture and in two STAT3-mutant PTCL xenografts, delineating a potential targeted agent-based therapeutic option for these patients., T-cell lymphomas are aggressive diseases associated with poor outcome. Here, the authors show that the THZ1, a CDK7 inhibitor, suppresses STAT transcriptional activity leading to apoptosis and sensitization to BCL2 inhibitors in T-cell lymphomas.

The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression affecting most notably genes of the NFkB and MAP kinase signaling pathways. GC B cells were predominantly hypomethylated compared with naive B cells and AICDA binding sites were highly overrepresented among hypomethylated loci. GC B cells also exhibited greater DNA methylation heterogeneity than naive B cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.

SummaryThe BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.

We report that Heat shock protein 90 (Hsp90) inhibitors selectively kill Diffuse Large B-cell Lymphomas (DLBCL) that are biologically dependent on the Bcl6 transcriptional repressor. Endogenous Hsp90 was found to interact with Bcl6 in DLBCL cells and could stabilize both Bcl6 mRNA and protein. Hsp90 formed a complex with Bcl6 at its target promoters and Hsp90 inhibitors de-repressed Bcl6 target genes. A stable mutant of Bcl6 rescued DLBCL cells from Hsp90 inhibitor induced apoptosis. Bcl6 and Hsp90 were almost invariantly co-expressed in the nuclei of primary DLBCL cells, suggesting that their interaction is relevant in this disease. We examined the pharmacokinetics, toxicity and efficacy of PU-H71, a recently developed purine derived Hsp90 inhibitor. PU-H71 preferentially accumulated in lymphomas compared to normal tissues and selectively suppressed Bcl6-dependent DLBCLs in vivo, inducing reactivation of key Bcl6 target genes and apoptosis. PU-H71 also induced cell death in primary human DLBCL specimens.

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.

Diffuse large B cell lymphomas (DLBCLs) arise from proliferating B cells transiting different stages of the germinal center reaction. In activated B cell DLBCLs (ABC-DLBCLs), a class of DLBCLs that respond poorly to current therapies, chromosomal translocations and amplification lead to constitutive expression of the B cell lymphoma 6 (BCL6) oncogene. The role of BCL6 in maintaining these lymphomas has not been investigated. Here, we designed small-molecule inhibitors that display higher affinity for BCL6 than its endogenous corepressor ligands to evaluate their therapeutic efficacy for targeting ABC-DLBCL. We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor called FX1 that has 10-fold greater potency than endogenous corepressors and binds an essential region of the BCL6 lateral groove. FX1 disrupted formation of the BCL6 repression complex, reactivated BCL6 target genes, and mimicked the phenotype of mice engineered to express BCL6 with corepressor binding site mutations. Low doses of FX1 induced regression of established tumors in mice bearing DLBCL xenografts. Furthermore, FX1 suppressed ABC-DLBCL cells in vitro and in vivo, as well as primary human ABC-DLBCL specimens ex vivo. These findings indicate that ABC-DLBCL is a BCL6-dependent disease that can be targeted by rationally designed inhibitors that exceed the binding affinity of natural BCL6 ligands.

Non-Hodgkin lymphomas are a heterogeneous group of lymphoproliferative disorders of B and T cell origin that are treated with chemotherapy drugs with variable success rate that has virtually not changed over decades. Although new classes of chemotherapy-free epigenetic and metabolic drugs have emerged, durable responses to these conventional and new therapies are achieved in a fraction of cancer patients, with many individuals experiencing resistance to the drugs. The paucity in our understanding of what regulates the drug resistance phenotype and establishing a predictive indicator is, in great part, due to the lack of adequate ex vivo lymphoma models to accurately study the effect of microenvironmental cues in which malignant B and T cell lymphoma cells arise and reside. Unlike many other tumors, lymphomas have been neglected from biomaterials-based microenvironment engineering standpoint. In this study, we demonstrate that B and T cell lymphomas have different pro-survival integrin signaling requirements (αvβ3 and α4β1) and the presence of supporting follicular dendritic cells are critical for enhanced proliferation in three-dimensional (3D) microenvironments. We engineered adaptable 3D tumor organoids presenting adhesive peptides with distinct integrin specificities to B and T cell lymphoma cells that resulted in enhanced proliferation, clustering, and drug resistance to the chemotherapeutics and a new class of histone deacetylase inhibitor (HDACi), Panobinostat. In Diffuse Large B cell Lymphomas, the 3D microenvironment upregulated the expression level of B cell receptor (BCR), which supported the survival of B cell lymphomas through a tyrosine kinase Syk in the upstream BCR pathway. Our integrin specific ligand functionalized 3D organoids mimic a lymphoid neoplasm-like heterogeneous microenvironment that could, in the long term, change the understanding of the initiation and progression of hematological tumors, allow primary biospecimen analysis, provide prognostic values, and importantly, allow a faster and more rational screening and translation of therapeutic regimens.

The chaperone HSP90 is used by B-cell lymphomas to support the stability of proteins involved in oncogenic processes such as signaling and anti-apoptosis. While HSP90 inhibitors decrease the levels of these client proteins favoring cell death they also prompt cellular counter-regulatory mechanisms that diminish the efficacy of these drugs. Improving the clinical activity of HSP90 inhibitors will depend on understanding the complexity of HSP90 functions. Here we show that HSP90 facilitates the function of MYC by improving the efficiency of metabolic pathways through the orchestration of enzymatic networks, and that HSP90 inhibition impairs the metabolic fitness of DLBCLs without client protein degradation. Moreover, drugs inducing sub-lethal metabolic stress in DLBCL cells cause apoptosis upon HSP90 inhibition. To identify metabolic enzymes actively chaperoned by HSP90 we integrated the information from proteomics and metabolomics in DLBCL cell lines. Proteomics was performed from the cytoplasmic fraction of OCI-Ly1 and OCI-Ly7 cells chemically precipitated with PU-H71, an HSP90 inhibitor that selectively binds to HSP90 contained in active multi-chaperone complexes.STRING network analysis of the metabolic client proteins identified several hubs highly enriched for enzymes involved in metabolism of nucleotides (e.g. IMPDH2, CTPS1, CAD), carbohydrates (e.g. G6PD, HK2) and proteins (e.g. MTHFR, ASNS). Functionality of the network was assessed by metabolomics from OCI-Ly1 cells treated with PU-H71 500 nM for 6 h (sub-lethal). This dose and timing assured HSP90 inhibition but no client protein degradation. The proteomics and metabolomics mapping into KEGG pathways showed a significant overlap, indicating that HSP90 preferentially interacts with proteins representing regulatory hubs to coordinate their committed activity and thus secure the flow of the pathway. We quantified the effect of HSP90 on the activity of metabolic networks by measuring glycolysis (by lactate production and medium acidification) and mitochondrial respiration (by oxygen consumption) in OCI-LY1 and OCI-LY7 cell lines upon PU-H71. We found that inhibition of HSP90 decreased glycolysis by 20-25% and respiration by 25% (p To understand the mechanistic relevance of these findings to lymphomagenesis, we analyzed the HSP90 metabolic proteome for common features and found it was significantly enriched (chi-square p Disclosures Yang: Regeneron Pharmaceuticals: Employment. Cerchietti: Lymphoma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment; Celgene: Research Funding.

Rationally designed combinations of targeted therapies for refractory cancers, such as activated B cell–like diffuse large B cell lymphoma (ABC DLBCL), are likely required to achieve potent, durable responses. Here, we used a pharmacoproteomics approach to map the interactome of a tumor-enriched isoform of HSP90 (teHSP90). Specifically, we chemically precipitated teHSP90-client complexes from DLBCL cell lines with the small molecule PU-H71 and found that components of the proximal B cell receptor (BCR) signalosome were enriched within teHSP90 complexes. Functional assays revealed that teHSP90 facilitates BCR signaling dynamics by enabling phosphorylation of key BCR signalosome components, including the kinases SYK and BTK. Consequently, treatment of BCR-dependent ABC DLBCL cells with PU-H71 attenuated BCR signaling, calcium flux, and NF-κB signaling, ultimately leading to growth arrest. Combined exposure of ABC DLBCL cell lines to PU-H71 and ibrutinib, a BCR pathway inhibitor, more potently suppressed BCR signaling than either drug alone. Correspondingly, PU-H71 combined with ibrutinib induced synergistic killing of lymphoma cell lines, primary human lymphoma specimens ex vivo, and lymphoma xenografts in vivo, without notable toxicity. Together, our results demonstrate that a pharmacoproteome-driven rational combination therapy has potential to provide more potent BCR-directed therapy for ABC DLCBL patients.

Although aberrant DNA methylation patterning is a hallmark of cancer, the relevance of targeting DNA methyltransferases (DNMT) remains unclear for most tumors. In diffuse large B-cell lymphoma (DLBCL) we observed that chemoresistance is associated with aberrant DNA methylation programming. Prolonged exposure to low-dose DNMT inhibitors (DNMTI) reprogrammed chemoresistant cells to become doxorubicin sensitive without major toxicity in vivo. Nine genes were recurrently hypermethylated in chemoresistant DLBCL. Of these, SMAD1 was a critical contributor, and reactivation was required for chemosensitization. A phase I clinical study was conducted evaluating azacitidine priming followed by standard chemoimmunotherapy in high-risk patients newly diagnosed with DLBCL. The combination was well tolerated and yielded a high rate of complete remission. Pre- and post-azacitidine treatment biopsies confirmed SMAD1 demethylation and chemosensitization, delineating a personalized strategy for the clinical use of DNMTIs. Significance: The problem of chemoresistant DLBCL remains the most urgent challenge in the clinical management of patients with this disease. We describe a mechanism-based approach toward the rational translation of DNMTIs for the treatment of high-risk DLBCL. Cancer Discov; 3(9); 1002–19. ©2013 AACR. See related commentary by Steinhardt and Gartenhaus, p. 968 This article is highlighted in the In This Issue feature, p. 953

Pericytes and vascular smooth muscle cells (VSMCs), which are recruited to developing blood vessels by platelet-derived growth factor BB, support endothelial cell survival and vascular stability. Here, we report that imatinib, a tyrosine kinase inhibitor of platelet-derived growth factor receptor β (PDGFRβ), impaired growth of lymphoma in both human xenograft and murine allograft models. Lymphoma cells themselves neither expressed PDGFRβ nor were growth inhibited by imatinib. Tumor growth inhibition was associated with decreased microvascular density and increased vascular leakage. In vivo, imatinib induced apoptosis of tumor-associated PDGFRβ(+) pericytes and loss of perivascular integrity. In vitro, imatinib inhibited PDGFRβ(+) VSMC proliferation and PDGF-BB signaling, whereas small interfering RNA knockdown of PDGFRβ in pericytes protected them against imatinib-mediated growth inhibition. Fluorescence-activated cell sorter analysis of tumor tissue revealed depletion of pericytes, endothelial cells, and their progenitors following imatinib treatment. Compared with imatinib, treatment with an anti-PDGFRβ monoclonal antibody partially inhibited lymphoma growth. Last, microarray analysis (Gene Expression Omnibus database accession number GSE30752) of PDGFRβ(+) VSMCs following imatinib treatment showed down-regulation of genes implicated in vascular cell proliferation, survival, and assembly, including those representing multiple pathways downstream of PDGFRβ. Taken together, these data indicate that PDGFRβ(+) pericytes may represent a novel, nonendothelial, antiangiogenic target for lymphoma therapy.

Rationale: The BCL6 oncogene is constitutively activated by chromosomal translocations and amplification in ABC-DLBCLs, a class of DLBCLs that respond poorly to current therapies. Yet the role of BCL6 in maintaining these lymphomas has not been investigated. BCL6 mediates its effects by recruiting corepressors to an extended groove motif. Development of effective BCL6 inhibitors requires compounds exceeding the binding affinity of these corepressors. Objectives: To design small molecule inhibitors with superior potency vs. endogenous BCL6 ligands for unmet putative therapeutic needs such as targeting ABC-DLBCL. Findings: We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor with 10-fold greater potency than endogenous corepressors. The compound, called FX1, binds in such a way as to occupy an essential region of the BCL6 lateral groove. FX1 disrupts BCL6 repression complex formation, reactivates BCL6 target genes, and mimics the phenotype of mice engineered to express BCL6 with lateral groove mutations. This compound eradicated established DLBCLs xenografts at low doses. Most strikingly, FX1 suppressed ABC-DLBCL cells as well as primary human ABC-DLBCL specimens ex vivo. Conclusions: ABC-DLBCL is a BCL6 dependent disease that can be targeted by novel inhibitors able to exceed the binding affinity of natural BCL6 ligands. Citation Format: Mariano Gonzalo Cardenas, Wenbo Yu, Wendy Beguelin, Teater R. Matthew, Huimin Geng, Rebecca Goldstein, Erin Oswald, Katerina Hatzi, Shao-Ning Yang, Joanna Cohen, Rita Shaknovich, Kenno Vanommeslaeghe, Huimin Cheng, Dongdong Liang, Hyoje Cho, Joshua Abbott, Wayne Tam, John P. Leonard, Leandro Cerchietti, Tomasz Cierpicki, Fengtian Xue, Alexander D. MacKerell, Jr., Ari Melnick. Therapeutic targeting of GCB- and ABC-DLBCLS by rationally designed BCL6 inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A11.

SummaryThe EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

Summary MALT1 cleavage activity is linked to the pathogenesis of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), a chemoresistant form of DLBCL. We developed a MALT1 activity assay and identified chemically diverse MALT1 inhibitors. A selected lead compound, MI-2, featured direct binding to MALT1 and suppression of its protease function. MI-2 concentrated within human ABC-DLBCL cells and irreversibly inhibited cleavage of MALT1 substrates. This was accompanied by NF-κB reporter activity suppression, c-REL nuclear localization inhibition, and NF-κB target gene downregulation. Most notably, MI-2 was nontoxic to mice, and displayed selective activity against ABC-DLBCL cell lines in vitro and xenotransplanted ABC-DLBCL tumors in vivo. The compound was also effective against primary human non-germinal center B cell-like DLBCLs ex vivo.

ALK positive diffuse large B-cell lymphomas (DLBCL) are a distinct lymphoma subtype associated with a poor outcome. Most of them feature a t(2;17) encoding a clathrin (CLTC)-ALK fusion protein. The contribution of deregulated ALK-activity in the pathogenesis and maintenance of these DLBCLs is not yet known. We established and characterized the first CLTC-ALK positive DLBCL cell line (LM1). LM1 formed tumors in NOD-SCID mice. The selective ALK inhibitor NVP-TAE684 inhibited growth of LM1 cells in vitro at nanomolar concentrations. NVP-TAE684 repressed ALK-activated signalling pathways and induced apoptosis of LM1 DLBCL cells. Inhibition of ALK-activity resulted in sustained tumor regression in the xenotransplant tumor model. These data indicate a role of CLTC-ALK in the maintenance of the malignant phenotype thereby providing a rationale therapeutic target for these otherwise refractory tumors.

B cell lymphoma 6 (BCL6), which encodes a transcriptional repressor, is a critical oncogene in diffuse large B cell lymphomas (DLBCLs). Although a retro-inverted BCL6 peptide inhibitor (RI-BPI) was recently shown to potently kill DLBCL cells, the underlying mechanisms remain unclear. Here, we show that RI-BPI induces a particular gene expression signature in human DLBCL cell lines that included genes associated with the actions of histone deacetylase (HDAC) and Hsp90 inhibitors. BCL6 directly repressed the expression of p300 lysine acetyltransferase (EP300) and its cofactor HLA-B-associated transcript 3 (BAT3). RI-BPI induced expression of p300 and BAT3, resulting in acetylation of p300 targets including p53 and Hsp90. Induction of p300 and BAT3 was required for the antilymphoma effects of RI-BPI, since specific blockade of either protein rescued human DLBCL cell lines from the BCL6 inhibitor. Consistent with this, combination of RI-BPI with either an HDAC inhibitor (HDI) or an Hsp90 inhibitor potently suppressed or even eradicated established human DLBCL xenografts in mice. Furthermore, HDAC and Hsp90 inhibitors independently enhanced RI-BPI killing of primary human DLBCL cells in vitro. We also show that p300-inactivating mutations occur naturally in human DLBCL patients and may confer resistance to BCL6 inhibitors. Thus, BCL6 repression of EP300 provides a basis for rational targeted combinatorial therapy for patients with DLBCL.

The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCLs). BCL6 lymphomagenic activity is dependent on its ability to recruit corepressor proteins to a unique binding site on its N-terminal BTB domain. A recombinant peptide fragment of the SMRT (silencing mediator for retinoid and thyroid hormone receptor) corepressor that blocks this site can inhibit BCL6 biologic functions. Shortening and conversion of this peptide to D-amino acid and retro configuration as well as the addition of a fusogenic motif yielded a far more potent and stable BCL6 inhibitor that still retained the specificity of the original SMRT fragment. Like the L-peptide, retroinverso BCL6 peptide inhibitor (RI-BPI) selectively killed BCR rather than OxPhos-type DLBCL cells. The RI-BPI could recapitulate the failure to form germinal centers seen in BCL6 null mice yet was nontoxic and nonimmunogenic even when administered for up to 52 weeks. RI-BPI showed superior duration of tissue penetration and could accordingly powerfully suppress the growth of human DLBCLs xenografts in a dose-dependent manner. Finally, RI-BPI could kill primary human DLBCL cells but had no effect on normal lymphoid tissue or other tumors.

The eukaryotic translation initiation factor 4E (eIF4E) is an oncogene elevated in a large number of cancers. The oncogenic potential of eIF4E arises from its critical roles in the cytoplasm in the mRNA translation and in the nucleus in the mRNA export of specific subset of transcripts. These transcripts can be regulated at the cytoplasmic, nuclear export or at both levels. We therefore analyzed eIF4E expression in 105 cases of DLBCLs by IHC, and found that is expressed in almost all the tumors (88%) and for the majority of cases (72%), eIF4E was expressed in both cellular compartments. Since elevated eIF4E activity leads to increased protein production that favors a subset of transcripts including prominent DLBCL oncogenes such as MYC and BCL2, we analyzed eIF4E expression in “double-hit” and “triple hit” DLBCLs. All the pts samples and DLBCL cell lines harboring two or more oncogenic mutations expressed eIF4E at high levels, suggesting a potential mechanistic association. Cells engineered to overexpress wild-type eIF4E, or mutants active in translation but not export (S53A) or mutants active in export but not translation (W73A), confirmed that eIF4E regulates the nuclear export and translation of MYC and BCL2, and discovered that this mechanism regulates additional DLBCL oncogenes such as BCL6. EIF4E immunoprecipitation from triple-hit DLBCL nuclear fractions followed by mRNA amplification by QPCR (RIP-QPCR) determined that MYC, BCL2 and BCL6 were direct eIF4E mRNA export targets. Polysomal profiling demonstrated that these oncogenes are also preferentially translated by eIF4E cytosolic activity. Accordingly, treatment of triple-hit DLBCL cell lines with the eIF4E competitive inhibitor ribavirin significantly increased the nuclear entrapment of BCL6, MYC and BCL2 transcripts, decreased their proportion of heavy polysomes and lead to a decrease in their protein abundance. In an extended panel of six double- and triple-hit DLBCL cell lines, ribavirin induced cell killing in all of them at clinically achievable concentrations. To assess the anti-lymphoma effect of ribavirin in a more clinically relevant context, we established a patient derived xenograft (PDX) in NSG mice. The specimen was isolated from a naïve treatment patient harboring a triple-hit ABC-type DLBCL featuring BCL6 translocation (3q27), BCL2 translocation and MYC amplification. The patient presented a treatment-refractory disease. We then expanded PDX into 10 NSG mice and when tumors were palpable, mice were randomized to receive vehicle or ribavirin for 10 days. We found a significant reduction in tumor growth in PDX. We also found that ribavirin induced mRNA nuclear entrapment of BCL2 and BCL6 and decrease protein levels of BCL2, BCL6 and MYC by day 10. We demonstrated that Hsp90 activity is required to maintain eIF4E-containing heavy polysomes in these oncogene transcripts. Accordingly, Hsp90 inhibition with PU-H71 rapidly promotes ribosomal disassembly leading to increased 40S, 60S and monosomes and decreased heavy polysomes. EIF4E target transcripts such as MYC, BCL6 and BCL2 were more affected, consequently decreasing the protein levels of these transcripts. The combination of Hsp90 and eIF4E inhibition reduced more efficiently the protein levels of these transcripts. To determine whether this molecular effect translates into an improved anti-lymphoma effect, we xenografted two triple-hit DLBCL cell lines in SCID mice and we treated them with vehicle control, ribavirin, PU-H71 or the combination for 10 days. We found that the combination of ribavirin with PU-H71 suppressed lymphoma growth more profoundly in both models. This effect was associated with a decrease in proliferation rate (measured by Ki67 staining) and reduced BCL6 expression. There were no macroscopic or microscopic evidence of toxicity in these mice. In summary, our data provides a novel and potentially non-toxic mechanistically-based approach to target B-cell lymphomas harboring multiple oncogene activation. Given the prevalence of simultaneous chromosomal aberrations in other hematological malignancies we envision a wider role for this strategy. Citation Format: Biljana Kraljacic-Culkjovic, Tharu Fernando, Rebecca Goldstein, Charles Mctavish, Jayeshkumar Patel, Shaoning Yang, Fabrizio Tabbo, Ari Melnick, Giorgio Inghirami, Katherine LB Borden, Leandro Cerchietti. EIF4E deregulation drives simultaneous expression of B-cell lymphoma oncogenes. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B12.

Mutation and constitutive expression of MYC and BCL2 and/or BCL6 (a.k.a. double and triple-hit lymphomas) defines subsets of diffuse large B-cell lymphoma (DLBCL) pts with particularly poor outcome. Almost 60% of pts with BCL2 and MYC translocations die within six months of diagnosis due to chemorefractory disease, a prognosis that cannot be overcome with intense chemotherapy. A further hindrance to patient survival is that these double and triple-hit lymphomas are frequently found in the elderly who have limited tolerability to aggressive chemotherapeutic regimens. To identify chemoresistant double and triple-hit DLBCL, we screened 38 DLBCL cell lines for mutations in BCL2, MYC and BCL6 by FISH and response to chemotherapy administration in vitro. We found 3 triple-hit and 2 double-hit DLBCL cell lines. Previous work in our laboratory showed that the nuclear export protein, XPO1, is a critical regulator of MYC, BCL2 and BCL6 mRNA transport in DLBCL. We therefore analyzed XPO1 amplification (by PCR) and expression (by immunoblot) in these cell lines and found that all 5 cells expressed XPO1 at higher levels than centroblasts and that, in at least 2 of them, this was result of gene amplification. Inhibition of XPO1 with selinexor, a Selective Inhibitor or Nuclear Export (SINE) compound, increased nuclear localization of MYC, BCL2 and BCL6 transcripts and consequently decreased their protein expression. Moreover, selinexor administration induced chemosensitization in doxorubicin-resistant DLBCL cells through decreasing DNA damage repair mechanisms as demonstrated by comet assays. To determine the potential extent of DLBCL patients that could benefit from such a treatment, we first analyzed 110 DLBCL patient samples by immunohistochemistry and found that XPO1 expression was increased in 85 cases compared to normal tonsils. Within the cohort analyzed, 6 patients had double or triple hit lymphoma (by FISH) and all overexpressed XPO1. Since this population has higher incidence of chemorefractory disease, we decided to develop a pre-clinical model of this disease using primary patient samples. We developed two patient-derived xenograft (PDX) models representing a double and a triple-hit lymphoma. In both cases XPO1 was also amplified. We compared the molecular and pathological characteristics of 12 generations of PDXs with the original pt sample, and found that protein expression and mutations of these genes were stable. We therefore administered selinexor alone, CHOP alone (cyclophosphamide, vincristine, doxorubicin and dexamethasone) and the combination of selinexor with CHOP to these PDXs. We found that selinexor significantly decreased tumor growth, compared to vehicle or CHOP alone. Moreover, combinatorial treatment indicated that selinexor was able to revert the chemorefractoriness of these DLBCLs, without inducing toxicity by clinical chemistry or pathologic examination. Analysis of selinexor vs. vehicle treated PDXs indicated a decrease in protein levels of MYC, BCL6, BCL2 as well as DNA damage and checkpoint regulators such as CHK1. In summary, selinexor has potent anti-proliferative effects in double/triple-hit DLBCL and can be safely combined with CHOP to enhance cancer cell death. These data present a new therapeutic approach for pts with double/triple hit lymphomas, and provide rational support for the study of selinexor/CHOP combination in clinical trials. Citation Format: Rossella Marullo, ShaoNing Yang, Tami Rashal, Yosef Landesman, Robert Carlson, Sharon Shacham, Leandro C. Cerchietti. XPO1 is a rational target for double and triple-hit aggressive B-cell lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-062. doi:10.1158/1538-7445.AM2015-LB-062

Peripheral T-cell lymphomas (PTCL) are clinically aggressive diseases with poor response to available (largely B-cell lymphoma–tailored) chemotherapy regimens and dismal survival. To identify active drugs for PTCL patients, we performed a cell-based progressive screen from a library of 105 anti-neoplastic drugs in clinical use. Primary screening was done in the PTCL-NOS cell line (OCI-Ly12), using three drug concentrations. We identified 3 active drug groups within the clinical-range limit: HDAC inhibitors (HDI) (romidepsin), proteasome inhibitors (bortezomib, carfilzomib) and transcription inhibitors (dactinomycin). Secondary screening was conducted for the active drug groups with 6 drug concentrations, in an extended panel of 9 TCL cell lines. We also expanded the drugs in each group by adding vorinostat, panobinostat and valproic acid for HDI and SNS032 (CDK9>2>>7 inhibitor) and THZ1 (CDK7>12 inhibitor) for transcription inhibitors. We demonstrated that the most active drugs (within serum achievable concentrations) were bortezomib, carfilzomib, romidepsin, dactinomycin and THZ1. Since proteasome inhibitors and romidepsin are being currently tested in PTCL clinical trials, we focused on the transcriptional inhibitors. Actinomycin inhibits the transcription initiation complex and prevents RNA elongation by RNA-POL2; given this broad mechanism of action, it is associated with serious side effects that limit its clinical use either alone or in combination. On the other hand, a recently discovered CDK7>12 inhibitor, THZ1, showed minimal side effects in vivo (Kwiatkowski N, et al. Nature 2014 Jun22), and high potency in our secondary screening yielding IC50s in TCL of ~200nM. We therefore used THZ1 to investigate the functional relevance of CDK7/12 targeting in PTCL. CDK7 is a critical component of both CDK-activating kinase (CAK) and transcription factor II human (TFIIH) complexes that phosphorylates cell cycle CDKs and C-terminal domain (CTD) of RNA-POL2, respectively. CDK7 inhibition decreases the phosphorylation of RNA-POL2 at Ser5 and Ser2, leading to transcriptional inhibition of susceptible loci. CDK12 in complex with CCNK displays a CTD kinase activity that is required for RNA splicing. To better understand the sensitivity of TCL to THZ1, we first determined the expression of all CAK components (CDK7, CCNH, and MAT1A) and CDK12/CCNK in 9 TCL cell lines that were found overexpressed compared to normal T cells from tonsils. Treatment of OCI-Ly12 and OCI-Ly13.2 (ALCL-ALK negative) cell lines with THZ1 for up to 48 h induced dose- and time-dependent decrease of phospho-POL2-ser5 and phospho-POL2-ser2, followed by PARP cleavage, caspase 7/3 activation and apoptosis. Genes with super-enhancers were found to be more susceptible to THZ1, and we also found these enhancers in genes associated with PTCL prognosis such as MCL1, JAK1 and MYC. Accordingly, THZ1 decreased mRNA and protein levels of MCL1, JAK1 and MYC as early as 3 hours after treatment. This was followed by decreasing levels of BCL2, BCL-XL, JUND and NFkB, and increased expression of pro-apoptotic proteins such as BAX at later time points. The decrease in JAK1 abundance led up to 70% reduction of phospho-STAT3 activity (as determined by immunoblotting and EMSA-like assays). Moreover, induction of STAT3 phosphorylation by IL-7/IL-15 treatment partially rescued the effect of THZ1, suggesting that JAK1 is a relevant target of CDK7 in PTCLs. The THZ1-dependent decrease in anti-apoptotic BCL2-family members prompted us to determine whether CDK7/12 inhibition can re-sensitize TCL to these drugs. We found either synergistic or re-sensitization effects on combination of THZ1 with BH3-family inhibitors, ABT-737 and obatoclax, in both OCI-LY12 and OCI-Ly13.2 cells. In sum, we identified a mechanism by which CDK7/12 inhibition, with the irreversible clinical candidate compound THZ1, simultaneously inhibits prominent PTCL survival pathways (JAK/STAT3, MYC and BCL2) causing apoptosis and re-sensitization to BCL2-family inhibitors. Disclosures Off Label Use: THZ1 (CDK7 and CDK12 inhibitor) for inducing apoptosis and sensitization to BCL2 inhibitors in peripheral T cell lymphomas.. Kwiatkowski:Syros Pharmaceuticals: One of the inventors Patents & Royalties. Zhang:Syros Pharmaceuticals: One of the inventors Patents & Royalties. Gray:Syros Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Reprogramming of tumor cell metabolism contributes to disease aggressiveness and chemoresistance, but how this process is regulated on the molecular levels is unclear. Hsp90 regulates diverse cellular processes through its interaction with co-chaperones and client proteins. Although basally expressed, Hsp90 is almost universally overexpressed in malignant cells due to increased levels of internal (oxidants, genomic instability, unfolded proteins) and external (hypoxia, nutrients limitation, drugs) stressors. Indeed, Hsp90 is starting to be acknowledged also for its role as an integrator of nutrient sensing, tumor bioenergetics and metabolic stress response (Chae et al, Cancer Cell, 2012). Here we report that active cytosolic Hsp90 binds and stabilizes enzymes regulating central metabolic processes in DLBCL, and provides an actionable target for this disease. Despite its overexpression, not all DLBCL are equally responsive to Hsp90 inhibitors. Using a panel of 10 DLBCL cell lines we found that those with higher glycolytic activity were more likely to respond to the Hsp90 inhibitors 17-DMAG and PU-H71. Global metabolomics profiling (GC-MS and UPLC-MS) of Hsp90-dependent (OCI-Ly7, OCI-Ly1, SU-DHL6, OCI-Ly10) vs. Hsp90-independent (Karpas422) cells identified “purine metabolism” (p=0.000027) and “pyrimidine metabolism” (p=0.0017) as upregulated, whereas “TCA cycle” was downregulated (p=0.000027). We validated these findings by short-term (6 h) exposure of OCI-Ly1 and OCI-Ly7 cells with non-cytotoxic doses of PU-H71 and found a highly reduced oxygen consumption rate (OCR) as well as glycolytic rate (ECAR). This was accompanied by a sharp decrease in glucose consumption and lactate production and by only a slight reduction in glutamine consumption. Unexpectedly, the drop in glycolysis and respiration in OCI-Ly1 and OCI-Ly7 did not lead to a significant decrease either in ATP or reducing power, suggesting that actually both the production and the utilization of ATP and redox equivalents are shut down when Hsp90 activity (an ATP-hydrolyzing enzyme) is inhibited. To identify the molecular basis for these differences, we characterized the active Hsp90-associated proteome in DLBCL by using chemical affinity purification followed by MS in cytosolic fractions of two Hsp90-dependent DLBCL cell lines (OCI-Ly1 and OCI-Ly7) in duplicates. This method pulls down only active Hsp90 multichaperone complexes (Moulick et al, Nat Chem Biol, 2011). Pathway enrichment analysis identified “cellular metabolism” as a significant process actively chaperoned by Hsp90 (ratio of enrichment 4.35, p=8.22E-15). Specifically, “nucleotides” and “carbohydrates” metabolism were the two most represented pathways, suggesting their critical role in Hsp90-dependent DLBCL cells. To functionally validate these findings, we integrated the proteomics analysis with serum metabolomics obtained from 10 OCI-Ly7-xenografted mice treated with PU-H71 vs vehicle (75 mg/m2 for 24 h (n=5)). Bioinformatic analysis revealed that PU-H71-treated mice had significant changes in 122 metabolites including lower levels of xanthine, hypoxanthine, adenosine and inosine, suggesting an alteration of the purines metabolism. IMPDH1/2 is the enzyme catalyzing the first committed and rate-limiting step of de novo guanine nucleotide biosynthesis. To determine whether IMPDH stability depends on Hsp90 activity, we treated a panel of 6 DLBCL cell lines (including OCI-Ly7) with their GI50 for PU-H71 or 17-DMAG for up to 24 h and found a time-dependent decrease in IMPDH2 protein levels. Finally, SCID mice were xenografted with OCI-Ly1 and SU-DHL6 and treated with vehicle, PU-H71, mycophenolate mofetil (MMF, the pro-drug of the IMPDH inhibitor mycophenolic acid) and the combination of PU-H71 and MMF. We found that the combination of drugs exhibited a greater antitumor effect than each drug alone (p=0.002 for SU-DHL6 and P Altogether, our work supports a role for Hsp90 as a necessary component in a subset of DLBCLs to build-up the metabolic features that allow these tumor cells to meet the requirements for their unrestrained growth and proliferation. Disclosures No relevant conflicts of interest to declare.

Diffuse large B-cell lymphomas (DLBCLs) arise from germinal center (GC) B-cells. Normal GC B-cells clonally expand and undergo somatic hypermutation of their immunoglobulin loci to produce high-affinity antibodies. Induction of BCL6, a transcription factor that represses genes involved in DNA damage sensing and checkpoint activation, is essential for GC B-cells to tolerate replicative and genotoxic stress without inducing cell cycle arrest. We previously showed that BCL6 forms a complex with tumor enriched HSP90 (TE-HSP90) to repress target genes in DLBCL cells. Based on these facts, we hypothesized that BCL6 is a component of a conserved stress response program, required for GC formation and maintenance of established lymphoma cells. Along these lines, we report that treatment of mice with TE-HSP90-selective inhibitor PU-H71 completely abrogates GC formation after immunization by T-cell dependent antigen. Stress responses are coordinated by the transcription factor heat shock factor 1 (HSF1), which is activated by phosphorylation. Immunofluorescence of human tonsillar sections revealed HSF1-pSer326-positive cells within BCL6+ GC B-cells. We found that HSF1-/- mice manifest a 40% decrease in GC B-cells after immunization and significantly (p=0.0073) decreased titers of high-affinity immunoglobulin compared to WT mice, indicating defective affinity maturation. Mixed chimera experiments revealed that the GC defect is exclusively due to malfunction of GC B-cells and not other cell types. Because of this GC B-cell defect, we reasoned that HSF1 might induce BCL6 expression. We identified three conserved heat shock elements (HSEs) in the BCL6 promoter. Quantitative ChIP analysis demonstrated HSF1 binding to these HSEs in human GC B-cells. Heat shock induced the BCL6 promoter in reporter assays and resulted in an increase in BCL6 nascent transcripts and protein. However this induction did not occur in HSF1-/-B-cells or after HSF1 knockdown. These data suggest that BCL6 is a stress response gene downstream of HSF1. To determine whether BCL6 is involved in mediating a stress tolerant phenotype, we performed serial stress response assays (using heat shock) in B220+ splenocytes from BCL6+/+ or BCL6-/- mice. Whereas BCL6+/+ cells were able acquire stress tolerance if preconditioned with an initial heat shock, BCL6-/- splenocytes failed to adapt to stress and died. To understand the mechanistic basis of this finding, we generated knockin mice with point mutations that disrupt the repressor activity of the BCL6 BTB (BCL6BTB) or the BCL6 RD2 (BCL6RD2) domain. Interestingly while splenocytes from BCL6RD2 mutant mice displayed normal stress tolerance responses, BCL6BTB mutant B-cells were completely deficient similar to BCL6-/-B-cells. Likewise the BCL6 BTB domain inhibitor RI-BPI also abrogated the BCL6 stress tolerance function. DLBCLs are dependent on many of the same molecular mechanisms as normal GC B-cells (e.g. BCL6). Indeed the lentiviral transduction of HSF1 shRNAs in DLBCL cell lines reduced BCL6 protein levels by more than 50% resulting in a 70%-90% loss in viability. HSF1 is known to help tumor cells survive exposure to chemotherapy drugs, and the BCL6 BTB domain is required for stress tolerance. Thus we hypothesized that BCL6 BTB domain targeted therapy (RI-BPI) would synergistically kill DLBCL when combined with chemotherapy. We treated 7 DLBCL cell lines with RI-BPI in combination with doxorubicin, vincristine, dexamethasone, mechlorethamine (in place of cyclophosphamide), and their combination CHOP. Almost all combinations resulted in an additive or synergistic effect on DLBCL growth inhibition. Moreover the combination of RI-BPI and doxorubicin in a DBLCL xenograft model was more potent and significantly (p Collectively we demonstrate that BCL6 is an HSF1-dependent stress tolerance factor and mediates this effect via its BTB domain. This phenomenon occurs during normal GC formation and in lymphoma cells. Thus targeting the BTB domain of BCL6 pharmacologically in combination with other chemotherapy is a viable strategy to eradicate lymphomas. Disclosures No relevant conflicts of interest to declare.

Malignant T cell proliferation, survival and drug resistant are dependent on a combination of external stimuli delivered by the microenvironment. Previous studies have shown that the transmembrane receptor integrin αVβ3 plays a critical role mediating the interaction of T cell lymphoma (TCL) cells with external signals. Integrin αVβ3 ligands include extracellular matrix-associated signaling proteins and soluble factors such as thyroid hormones (TH). We have also shown that THs stimulate the proliferation of TCLs through complimentary intracellular pathways involving the αVβ3 integrin. We therefore hypothesized that targeting integrin αVβ3 could represent a novel strategy to treat TCL patients. To determine survival pathways induced by TH on αVβ3 integrins, we evaluated the transcriptional changes (RNA-seq) induced by physiological concentrations of cell impermeable T3/T4 coupled to agarose (TH-AG) vs. control in the TCL cell line CUTLL1. We identified 123 up- and 5 down-regulated transcripts (p< 0.01), belonging to “angiogenesis” (e.g. VEFGB), “lymphocyte proliferation/differentiation” and “DNA replication/transcription” (e.g. DBP, IL4, EDF1, DOK2) pathways. Target-gene promoter analysis suggested that TH acting on αVβ3 integrin activated NFkB (that was later confirmed by EMSA-like assays). We then focused on the angiogenesis pathway since (i) VEGF expression and angiogenesis correlate with survival and prognosis in TCL patients, and (ii) we found a positive correlation between integrin αVβ3 and VEGFA and VEGFB expression in 169 cases of TCLs. We analyzed T3/T4-αVβ3-dependent increase of VEGFB and VEGFA in an extended panel of cell lines (n=5) representing the spectrum of immature and peripheral TCL. Similarly to CUTLL1, treatment with TH-AG increased VEGFB and VEGFA mRNA levels in Jurkat (TCL/L), HuT-78 (CTCL), OCI-Ly12 (PTCL-NOS) and Karpas299 (ALCL-ALK+) cells. Increase in VEGF production was completely abrogated by knocking-down either αV or β3components with specific si-RNAs (vs. siRNA control) in CUTLL1, HuT-78 and OCI-Ly12 cells. Moreover, exposing HMEC1 endothelial cells to conditioned medium from CUTLL1 cells treated with TH-AG vs control increased they proliferation and migration (to 481 ± 118 cells from 206 ± 82 cells, respectively, p= 0.01). Importantly, this higher migration was completely abrogated when conditioned medium was obtained from CUTLL1 cells knocked-down for either αV or β3 and treated with TH-AG. To determine whether targeting αVβ3integrin could be of therapeutic benefit for TCL, we developed TCL xenografts in SCID mice using CUTLL1 cells transfected with si-control, si-αV and si-β3, and monitored tumor growth and angiogenesis. We found that CUTLL1 transfected with si-αV and si-β3 developed significant smaller tumors than si-control. Also, tumors from integrin knocked-down cells, showed decreased tumor vascularization (by CD31) and VEGF expression. To determine the translational impact of this strategy, we assessed the effect of cilengitide, a selective αVβ3 integrin inhibitor in phase 3 for glioma, in pre-clinical models of PTCL-NOS and ALCL-ALK+. For PTCL-NOS we xenografted OCI-Ly12 cells in NOD/SCID mice (n=12) and for ALCL-ALK+ we developed a patient-derived tumorgraft (PDT) in NSG mice (n=8). Cilentide treatment for 10 days (vs. vehicle), at human equivalent dose, induced tumor remission in both models (p Disclosures No relevant conflicts of interest to declare.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease. Combination of gene expression and metabolic profiling of DLBCLs uncovered two biologically relevant entities: BCR-DLBCL (∼60% of DLBCL pts) characterized by a glycolytic metabolism and B-cell receptor (BCR) signaling activation; and OXPHOS-DLBCL characterized by enhanced mitochondrial energy transduction and increased glutathione levels. To identify small molecules that can serve as biological probes and potential therapeutics for these subgroups, we conducted a phenotypic screening in BCR-DLBCLs vs. OXPHOS-DLBCLs. We first tested cell growth inhibition capacity of 84,000 drug-like small molecules in one BCR (OCI-Ly7) vs. OXPHOS (Toledo) cell lines for 48 h at 20 µM. We identified 3,684 compounds that inhibited >90% the growth of either or both cell lines. In a secondary screening we selected compounds that showed a dose-response effect lower than 10 µM. We identified 31 BCR-selective, 8 OXPHOS-selective and 5 non-selective compounds. We then determined potency and phenotypic-selectivity of active compounds in an expanded panel of BCR-glycolytic (OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly10, SU-DHL6) vs. OXPHOS (OCI-Ly4, Karpas422, Toledo, WSU-DLCL2) cell lines. Five compounds were validated as BCR-glycolytic specific and 3 as bi-specific and their GI50 values (50% growth inhibition) were determined. We further characterized the most selective (>5-fold) and potent (low µM) BCR-glycolytic specific small molecules, termed LI-2 and LI-5. These compounds showed GI50 between 500 nM - 3 µM in BCR-glycolytic vs 9-20 µM in OXPHOS DLBCLs. LI-2 and LI-5 selectively induced 60-80% caspase-dependent apoptosis (p≤0.05) after 24 h at 2.5 µM in BCR-glycolytic but not in OXPHOS. To determine phenotypic changes upon treatment with LI-2 and LI-5, we performed RNA-sequencing on BCR-glycolytic DLBCL OCI-Ly7 and SUDHL-6 cells. LI-2 signature included 91 up- and 76 down-regulated transcripts (FC>1.2, FDR adjusted p Citation Format: Mariano G. Cardenas, Matt R. Teater, Nieves Calvo-Vidal, Shao Ning Yang, J F. Glickman, Ari Melnick, Leandro Cerchietti. Small molecule phenotypic targeting of aggressive B-cell lymphomas. [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 4232. doi:10.1158/1538-7445.AM2014-4232

T-cell non-Hodgkin lymphoma (T-NHL) responds poorly to standard chemotherapeutic regimens like CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone). In order to identify better therapeutic approaches we conducted a viability screening using a library of known anti-neoplastic agents. We performed a first screening in the PTCL-NOS cell line OCI-Ly12 using 101 approved oncology drugs obtained from the National Cancer Institute in 3 concentrations (10, 1, 0.1 μM). We measured cell viability after 48 h of exposure and calculated 50% growth inhibitory concentrations (GI50s). We found 30 drugs with GI50s< 10 μM that were further tested at 0.1 μM in 3 additional cell lines: anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL): Karpas299, ALK-negative ALCL: OCI-Ly13.2, and cutaneous T-cell lymphoma (CTCL): HuT78. In this secondary screening, we found 3 drugs that killed > 70% of cells in all 4 cell lines: bortezomib (proteasome inhibitor), romidepsin (histone deacetylase -HDAC- inhibitor) and dactinomycin (RNA polymerase -RNA-Pol- inhibitor). We then tested these 3 compounds with additional HDAC inhibitors (panobinostat, vorinostat, trichostatin A), at 6 concentrations, in an extended panel of 7 T-NHL cell lines (tertiary screening). We found that only romidepsin and dactinomycin had GI50s lower than their respective maximum plasma concentration (Cmax) in humans; we therefore focused further mechanistic studies in these two compounds. Romidepsin GI50 ranged from 10 nM (in Karpas299) to 200 nM (in Mac2A and SU-DHL1), which were lower than its Cmax (1850 nM). Administering the drug one or two times over the 48 h exposure yielded equivalent anti-lymphoma effect. To further characterize this effect and determine whether the biological change was transient or permanent, we treated cells with romidepsin once with their GI50 dose and measured cell proliferation, apoptosis and extent of histone 3 lysine 27 acetylation (H3K27Ac) by immunoblotting at 4, 8, 12 h, then daily for 7 days. We found that the main cellular effect of romidepsin was inhibition of cell proliferation rather than inducing cell death. This effect was associated with increasing H3K27Ac as early as 8 h, peaking at 24 h (∼30 fold to control), maintaining for 3 days with subsequently slow declining until day 7 (∼15 fold to control). Since romidepsin response rate in relapsed/refractory PTCL patients is ∼30%, we wished to determine whether it could be more active when combined with chemotherapy. We found that in all T-NHL cell lines, concomitant administration with doxorubicin or vincristine yielded synergistic killing effect by isobologram analysis. This data suggested that romidepsin could be a potent chemosensitizer in PTCLs. Dactinomycin is a weak reversible RNA-Pol elongation inhibitor that affects all 3 RNA-Pols, which confers broad anti-neoplastic activity, but also significant toxicity. To determine whether more selective RNA-Pol2 inhibitors are active in PTCL, we first analyzed the expression of cyclin-dependent kinase (CDK) complexes, CDK7/CCNH and CDK9/CCNT, responsible for RNA-Pol2 activation through phosphorylation, in T-NHL cell lines. We found overexpression of CDK7 by 3.06 fold (± 0.3) and CCNH by 2.1 fold (± 0.2) in all 7 PTCLs cell lines compared to tonsilar normal T cells. This prompted us to investigate the anti-proliferative effect of a recently described CDK7 inhibitor (CDK7i). CDK7i showed anti-proliferative effect in all 7 T-NHL cell lines tested with GI50 ranging from 0.2 μM (OCI-Ly13.2) to 2.3 μM (Mac2A), which were 5-10 times lower than the CDK7i chemical control. We found that this anti-proliferative effect correlated with a primary decrease in phospho-Ser5 RNA-Pol2 followed by a decrease in phospho-Ser2 RNA-Pol2 ultimately causing transcriptional inhibition of active loci. This effect led to a decrease in BCL2 and MYC mRNA levels in treated cells, which suggested that CDK7i could represents the first of a novel class of selective transcriptional inhibitors for PTCL treatment. In sum, we identified romidepsin as the most active drug for T-NHL and described a potential chemosensitizing effect. We also uncovered a potential novel target, CDK7, for the treatment of this disease. Disclosures: No relevant conflicts of interest to declare.

The EZH2 histone methyltransferase is the enzymatic core of the Polycomb repressor 2 (PRC2 complex), is highly upregulated in germinal center (GC) B cells and is targeted by gain-of-function somatic mutations that enhance its ability to trimethylate histone 3 lysine 27 in diffuse large B cell lymphomas (DLBCLs) and follicular lymphomas (FLs). We explored the significance and mechanism of action of EZH2 in normal GC development and lymphomagenesis. We observed that EZH2-conditional knockout mice and mice exposed to the novel EZH2-specific inhibitor GSK503 both completely failed to form GCs or high affinity antibodies. Using ChIP-seq, sequential QChIP, RNA-seq and functional assays we demonstrated that EZH2 mediates the GC phenotype through de novo formation of bivalently marked chromatin domains (characterized by overlapping H3K27me3 repressive mark with the H3K4me3 activation mark) at the promoters of target genes involved in cell cycle regulation (e.g. CDKN1A) and in GC exit and terminal differentiation program (e.g. IRF4 and PRDM1). Notably, mutant EZH2 caused hyper-repression of these bivalent genes through increased H3K27me3, which we showed is causal to the mutant EZH2 phenotype. Mice engineered to conditionally express lymphoma-associated EZH2Y641F exhibited aberrant suppression of bivalent gene expression leading to increased proliferation, blockade of terminal differentiation, and massive GC hyperplasia. Transcriptional profiles of human DLBCL patients revealed that those with mutant EZH2 display a unique signature consisting of silencing of GC bivalent genes, suggesting that mutant EZH2 contributes to human lymphomagenesis through paralysis of bivalent chromatin domains. This scenario is reminiscent of the role of the transcriptional repressor BCL6, which is also required for GC formation. BCL6 also represses CDKN1A, IRF4 and PRDM1 and is required to maintain the proliferation and survival of DLBCL cells. Notably BCL6 represses its targets by associating with BCoR, which forms a variant of Polycomb repressor 1 (PRC1) complex. We hypothesized that EZH2 and BCL6 cooperate to mediate the GC B-cell phenotype and when aberrantly active may cooperate to form GC-derived B-cell lymphomas. Using ChIP-seq studies we found that the target promoters of BCL6-BCoR complex (but not promoters with BCL6 complexes lacking BCoR) significantly overlap with EZH2 bivalent promoter genes in primary human GC B cells and lymphoma cells (Hypergeometric test, p=1.5x10-26). Treatment of DLBCL cells with EZH2 or BCL6 inhibitors or siRNA partially derepressed these genes indicating that both factors cooperate and are required to mediate full repression of these crucial loci. To determine whether EZH2 and BCL6 cooperate to generate GC-derived lymphomas, we transduced bone marrow of IµHABCL6 mice (which mimic BCL6 translocations in DLBCL) with retrovirus encoding mutant EZH2Y641F or GFP alone, and transplanted them into lethally irradiated recipients. Only EZH2Y641F/BCL6 mice showed an accelerated lethal phenotype (log-rank test, p=0.007), with reduced median survival (EZH2Y641F: 309 days, empty vector: 453 days). Serial bone marrow transplantation resulted in even further increased lethality (log-rank test, p=0.004; median survival EZH2Y641F: 127 days, empty vector: 169 days). Given the oncogenic cooperation between BCL6 and EZH2, we hypothesized that rational combinatorial therapy with BCL6 and EZH2 inhibitors might synergistically kill DLBCLs. Indeed, by combining the EZH2 inhibitor GSK343 and the RI-BPI, a drug that inhibits BCL6 by abrogating its interaction with BCoR, we observed a potent synergistic effect on the inhibition of DLBCL cell lines proliferation. The combination of these two inhibitors in mice bearing DLBCL xenografts accordingly suppressed tumor growth more effectively than either agent alone. Finally, the combination also yielded further killing of primary human DLBCL cells growth in a co-culture system that we developed for testing primary human specimens. In summary we identified the first epigenetic mechanism of lymphomagenesis involving aberrant repression of GC-specific bivalent domains by EZH2 (PRC2) in cooperation with BCL6-BCoR (PRC1) complexes, as well as a rational epigenetic-based and molecular targeted therapeutic approach with the potential to eradicate lymphomas without harming normal tissues. Disclosures: Creasy: GlaxoSmithKline: Employment.

DLBCL features marked molecular heterogeneity. Gene overexpression due to genetic lesions or by other mechanisms activates powerful oncogenic pathways such as MYC, BCL6, BCL2 and MCL1; that are usually expressed concomitantly. Regardless the underlying mechanism, genes must first be transcribed into mRNA and then translated into proteins in the cytosol to exert their oncogenic functions. While most transcripts representing bulk mRNA are exported to the cytosol using the TAP/NXF1 complex, a specific subset of transcripts that contain a conserved sequence (4E-SE) are exported using the eIF4E/LRPPRC/XPO1 complex. EIF4E is frequently elevated in many malignances and exhibit oncogenic potential that arises from its critical roles in the nuclear export and cytosolic translation of oncogenic transcripts. EIF4E competitive inhibitors, such as ribavirin (RIB), as well as XPO1 inhibitors such as KPT-330, abrogate its pro-survival function by decreasing export and translation of target mRNAs. We hypothesized that eIF4E could have a role in the expression of oncogenic transcripts and proteins in DLBCL patients. In this case, eIF4E nuclear pore complex inhibitors would constitute a new therapeutic approach for this disease. We first analyzed the expression of eIF4E in DLBCLs by gene expression (RNA-seq and qPCR) and immunohistochemistry (IHC). Compared to centroblasts, primary DLBCL (n=69) and cell lines (n=25) showed significant overexpression of eIF4E (p BCL6, the most frequently involved oncogene in DLBCL, contains a 4E-SE sequence in its transcript making it a potential eIF4E target. To determine whether in fact BCL6 was an eIF4E target, we analyzed BCL6 transcript cytosolic/nuclear ratio (C/N) in DLBCL cells engineered to overexpress or knockdown eIF4E. eIF4E overexpression and knocking-down caused 80% increase and 40% decrease in BCL6 C/N respectively, and this was accompanied by coincident BCL6 protein changes. To further characterize the nuclear eIF4E contribution to BCL6 expression we infected DLBCL cells with control vector (GFP), eIEF4EWT (overexpression), eIF4EW73A (mutant with no translation activity) and eIF4ES53A (mutant with no export activity). Only eIEF4EWT and eIF4EW73Awere able to increase and maintain BCL6 mRNA and protein levels, suggesting that BCL6 is, at least, an export target of eIF4E. To more directly test this, we performed eIF4E-immunoprecipitation followed by RNA-seq or qPCR (for validation) in DoHH2 and SUDHL6 cells. We found that BCL6, together with other 150 transcripts including the oncogenes MYC, MCL1, BCL2, BCLXL and OCD1, was significantly and differentially bound to eIF4E (vs. IgG control) in both cell lines. Additional functional experiments validated these oncogenes transcripts as eIF4E targets in DLBCL cells. In DLBCLs with cytosolic eIF4E overexpression, BCL6 and other oncogenes with complex 5’UTRs, such as MYC, BCL2 and MCL1, could be also behave as preferential translational targets. In order to test this, we isolated nine polysomal fractions from SUDHL6 cells treated with RIB 30 μM or vehicle for up to 96 h. We found that RIB treatment significantly decreased BCL6, MYC, BCL2 and MCL1 transcripts in polysomes. Non-complex transcripts such as actin were unaffected. This translated in decreased protein levels of BCL6, MYC, BCL2 and MCL1 in treated cells. Our data therefore suggested that BCL6 is a new eIF4E target transcript and RIB decreases BCL6 transcript and subsequently protein levels by inhibiting both mRNA nuclear export and preferential translation. To assess whether this could be capitalized therapeutically, we exposed a panel of 10 DLBCL cell lines for 48 h to eIF4E nuclear pore complex inhibitors RIB and KPT-330. We found that RIB and KPT-330 have potent anti-lymphoma activity in these cells. We then tested this concept in vivo in established OCI-Ly1 xenografts that were randomized into 2 groups of 7 mice each and treated with vehicle or RIB 80 mg/kg/day. After 10 days of treatment, RIB significantly decreased tumor proliferation (p=0.025) without inducing toxicity. In sum, this study showed that BCL6 is a new eIF4E target transcript and that eIF4E nuclear pore complex inhibitors could represent a new therapeutic approach for DLBCL pts, especially for those with expression of multiple oncogenes. Disclosures: No relevant conflicts of interest to declare.

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of T-cell non-Hodgkin lymphomas (T-NHL) derived from skin-homing mature T cells. Thyroid hormones (THs) are crucial regulators of cellular differentiation, growth and metabolism. We recently found that THs stimulate the proliferation and metabolism of T cells and that this pathway is co-opted by T-NHL cells to maintain their malignant phenotype. Accordingly, a decrease in TH levels affects the proliferation of T-NHL cells. We also found that THs modulate these functions by activating both canonical nuclear (TR) and membrane receptors (mTR, that for most cells is represented by integrin αvβ3). We now hypothesized that the characterization of the relative contribution of these receptors to the malignant phenotype of T-NHLs will allow for a more precise therapeutic intervention. The study presented here was conducted to determine (i) whether THs are pro-survival factors in CTCL and (ii) the relative contribution of TR and mTR to the effect of THs in CTCLs. To test this hypothesis, we first analyzed the expression of TR and mTR in CTLC cells (HuT 78, MJ and HuT102) vs. normal T cells (from peripheral blood) by qRT-PCR and immunoblotting. We found that CTCL expressed 2 to 10 fold more THRA, ITGAV and ITGB3 mRNA and protein than their normal counterpart (p To determine the specific transcriptional programs regulated by mTR and TR, T-NHLs were treated with free T3/T4 (for total TR activity) and agarose-coupled T3/T4 (for mTR activity) for 24h and analyzed by mRNA-sequencing. Differentially regulated genes were further analyzed using Ingenuity Pathway Analysis. We found the TNFR and interleukin-2 pathways (e.g. TNFRSF19, TNFRSF9, IL2RA) among the top programs regulated by free THs through canonical nuclear TR activation whereas mTR activation upregulated genes involved in angiogenesis induction (e.g. PDGFR, VEGFA, VEFGB), lymphocyte proliferation/differentiation and DNA replication (e.g. IL4, DOK2). We independently validated these genes in HuT-78 and MJ cell lines by qRT-PCR. Taken together, these data suggest that TR and mTR regulate distinct transcriptional programs that are complimentary towards CTCL proliferation. Considering that (i) mTR are over-expressed in CTCL vs. normal T cells (in contrast to canonical nuclear TR that are broadly expressed) and (ii) mTR regulate pro-survival pathways in CTLC, we wished to determine whether mTR inhibition will be sufficient to decrease CTCL proliferation. We therefore tested the effect of mTR abrogation by using siRNA to ITGAV and ITGB3 as well as the effect of pharmacological mTR inhibitors (i.e: RGD peptide and Tetrac) in CTCL proliferation. We found that mTR inhibition with siRNA or compounds affected mTR-induced transcriptional programs. In fact, the TH-ag mediated upregulation of VEGFB, VEGFA, DOK2 and IL4 mRNA levels were totally abrogated in CTCL cells transfected with the siRNA to ITGAV and ITGB3. Moreover, almost 100% of the TH-ag-induced and 80% of the free-TH-induced CTCL proliferative effect was abrogated by ITGAV- and/or ITGB3-targeting siRNA. Taken together these data suggest that THs are important inducers of CTCL proliferation by transcriptionally regulating major survival pathways, and inhibition of mTR could constitute a selective chemotherapy-free treatment for CTCLs. Disclosures: No relevant conflicts of interest to declare.

Abstract 3706 Background: Diffuse large B cell lymphoma (DLBCL) is potentially curable with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), but many patients either do not respond to treatment or relapse after achieving remission. Since outcomes after relapse are poor, the optimal strategy to improve survival would be to improve initial therapy. Aberrant DNA methylation contributes to the phenotype of many tumors, including DLBCL. DNA methyltransferase inhibitor (MTI) drugs such as azacitidine can reactivate epigenetically silenced genes resulting in direct anti-tumor effects and/or chemosensitization. Preclinical studies show that combination therapy with MTIs and chemotherapy is synergistic in killing DLBCL xenografts while non-toxic to the host. This chemosensitization effect is mediated, in part, by TGF-beta signaling restoration due to SMAD1 demethylation and re-expression. We therefore hypothesized that the combination of the MTI azacitidine with R-CHOP would be feasible and improve outcomes in patients with DLBCL. Methods: We performed a phase I study of epigenetic priming with azacitidine in patients with newly diagnosed DLBCL receiving R-CHOP. Patients were eligible if they had not received previous therapy, had preserved organ function, and did not have active viral hepatitis. Patients were treated with subcutaneous azacitidine daily for 5 days at escalating doses, followed by standard R-CHOP administered on day 8. Cycles were repeated every 21 days for 6 cycles. Hematopoietic growth factor was administered according to ASCO guidelines. Azacitidine dose escalation was undertaken according to the continuous reassessment model using the following dose levels: dose level 1: 25 mg/m2 daily; dose level 2: 50 mg/m2 daily; dose level 3: 75 mg/m2daily. Dose limiting toxicity (DLT) was defined as grade 3 or greater non-hematologic toxicity or grade 4 hematologic toxicity lasting more than 7 days. Neutropenic fever was considered DLT only if grade 4. Tumor and blood samples for correlative studies were obtained prior to initiation of treatment and after cycle one of azacitidine, prior to initiation of R-CHOP. Results: The study has completed accrual, with 12 patients enrolled and treated: one at dose level 1, one at dose level 2, and ten at dose level 3. Eleven patients were of high or high intermediate risk by International Prognostic Index (IPI), and one was of low intermediate risk. Eight patients have completed or discontinued therapy, and 4 remain on treatment. One patient on dose level 3 experienced DLT consisting of hepatitis C reactivation after 2 cycles of study therapy; that patient completed an additional 4 cycles of treatment with CHOP alone. All patients experienced grade 3 or 4 neutropenia, none lasting more than 7 days. Four patients experienced grade 3 neutropenic fever. One patient discontinued study therapy after cycle 1 due to complications of a GI bleed presumed secondary to tumor response; he completed therapy with R-CHOP. All 7 patients who have completed post-treatment restaging have achieved complete remission and are eligible for secondary efficacy analysis. At median follow up of 10 months (range 1.5 to 25 months), one patient (who experienced DLT and completed therapy with CHOP alone) has relapsed. Correlative analysis of tumor and blood samples obtained prior to and after the first cycle of azacitidine show global hypomethylation, decreased SMAD1 promoter methylation and increased SMAD1 expression following azacitidine treatment. Paired patient tumor samples showed improved sensitivity to chemotherapy after treatment with azacitidine ex vivo. Conclusions: Epigenetic priming with azacitidine prior to standard R-CHOP is feasible and shows promising clinical outcomes in patients with previously untreated DLBCL. Correlative studies support a role for TGF-beta signaling pathway in mediating chemosensitization in these patients. Disclosures: Off Label Use: Azacitidine is approved for use in MDS. Discussion here is off label. Martin:Millennium Pharmaceuticals, Inc.: Speakers Bureau. Leonard:Celgene: Consultancy, Honoraria.