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Abstract Diffuse large B-cell lymphomas (DLBCL) are broadly dependent on anaplerotic metabolism regulated by mitochondrial SIRT3. Herein we find that translational upregulation of ATF4 is coupled with anaplerotic metabolism in DLBCLs due to nutrient deprivation caused by SIRT3 driving rapid flux of glutamine into the tricarboxylic acid (TCA) cycle. SIRT3 depletion led to ATF4 downregulation and cell death, which was rescued by ectopic ATF4 expression. Mechanistically, ATF4 translation is inhibited in SIRT3-deficient cells due to the increased pools of amino acids derived from compensatory autophagy and decreased glutamine consumption by the TCA cycle. Absence of ATF4 further aggravates this state through downregulation of its target genes, including genes for amino acid biosynthesis and import. Collectively, we identify a SIRT3–ATF4 axis required to maintain survival of DLBCL cells by enabling them to optimize amino acid uptake and utilization. Targeting ATF4 translation can potentiate the cytotoxic effect of SIRT3 inhibitor to DLBCL cells. Significance: We discovered the link between SIRT3 and ATF4 in DLBCL cells, which connected lymphoma amino acid metabolism with ATF4 translation via metabolic stress signals. SIRT3–ATF4 axis is required in DLBCL cells regardless of subtype, which indicates a common metabolic vulnerability in DLBCLs and can serve as a therapeutic target. This article is highlighted in the In This Issue feature, p. 1

Locus control region (LCR) functions define cellular identity and play critical roles in diseases such as cancer, although the hierarchy of structural components and associated factors that drive functionality are incompletely understood. Here we show that OCA-B, a B cell-specific coactivator essential for germinal center (GC) formation, forms a ternary complex with the lymphoid-enriched OCT2 and GC-specific MEF2B transcription factors, and that this complex occupies and activates an LCR that regulates the BCL6 proto-oncogene and is uniquely required by normal and malignant GC B-cells. Mechanistically, through OCA-B-MED1 interactions, this complex is required for Mediator association with the BCL6 promoter. Densely tiled CRISPRi screening indicates that only LCR segments heavily bound by this ternary complex are essential for its function. Our results demonstrate how an intimately linked complex of lineage- and stage-specific factors converges on specific and highly essential enhancer elements to drive the function of a cell-type defining LCR.

MALT1 inhibitors are promising therapeutic agents for B-cell lymphomas that are dependent on constitutive or aberrant signaling pathways. However, a potential limitation for signal transduction–targeted therapies is the occurrence of feedback mechanisms that enable escape from the full impact of such drugs. Here, we used a functional genomics screen in activated B-cell–like (ABC) diffuse large B-cell lymphoma (DLBCL) cells treated with a small molecule irreversible inhibitor of MALT1 to identify genes that might confer resistance or enhance the activity of MALT1 inhibition (MALT1i). We find that loss of B-cell receptor (BCR)- and phosphatidylinositol 3-kinase (PI3K)-activating proteins enhanced sensitivity, whereas loss of negative regulators of these pathways (eg, TRAF2, TNFAIP3) promoted resistance. These findings were validated by knockdown of individual genes and a combinatorial drug screen focused on BCR and PI3K pathway–targeting drugs. Among these, the most potent combinatorial effect was observed with PI3Kδ inhibitors against ABC-DLBCLs in vitro and in vivo, but that led to an adaptive increase in phosphorylated S6 and eventual disease progression. Along these lines, MALT1i promoted increased MTORC1 activity and phosphorylation of S6K1-T389 and S6-S235/6, an effect that was only partially blocked by PI3Kδ inhibition in vitro and in vivo. In contrast, simultaneous inhibition of MALT1 and MTORC1 prevented S6 phosphorylation, yielded potent activity against DLBCL cell lines and primary patient specimens, and resulted in more profound tumor regression and significantly improved survival of ABC-DLBCLs in vivo compared with PI3K inhibitors. These findings provide a basis for maximal therapeutic impact of MALT1 inhibitors in the clinic, by disrupting feedback mechanisms that might otherwise limit their efficacy.

Somatic missense mutations of BTG1 are exclusive to germinal center (GC)-derived B cell lymphomas (~12% of DLBCLs) and are most prevalent in ABC-DLBCL (p=0.0184 vs GCB-DLBCL), particularly in the MCD/cluster 5 subtype, which features extranodal dissemination and unfavorable outcome. However, the relevance, mechanism of action and biological contribution of BTG1 mutations have not been studied. Using a rigorous genomic covariate analysis, we identified BTG1 mutations as a top genetic driver in DLBCL. Furthermore, molecular dynamics simulations indicated that BTG1 recurrent mutations, including the most frequent Q36H, disrupted the protein structure, with likely deleterious functional consequences. To investigate the effect of BTG1 mutation in GC B cells, we generated a conditional Btg1Q36H knock in mouse crossed to the B cell specific Cd19Cre line. Surprisingly, there was no apparent phenotype in GC B cells or other B cell populations. However, placing Btg1 Q36H and WT GC B cells in competition within the same mouse through adoptive transfer revealed a dramatic competitive advantage of Btg1 Q36H cells, virtually taking over the GC reaction. To gain further insight into this striking fitness advantage, we performed RNAseq in Btg1 Q36H GCs, which showed marked enrichment for genes induced in positively selected GC B cells, including MYC targets and biosynthetic pathways. The same genes were also enriched in BTG1 mutant DLBCL patients in 2 independent cohorts. Furthermore, Btg1 Q36H GC B cells displayed greater RNA content and cell size, reflecting increased fitness. Positive selection normally triggers a brief Myc pulse in GC B cells. We therefore crossed our Btg1Q36H mice to the MycGFPprotein fusion reporter and observed higher proportion of Myc GFP+ cells in Btg1 Q36H GCs. For mechanistic studies, we generated isogenic BTG1 Q36H or BTG1 WT human DLBCL cell lines. BTG1 Q36H cells exhibited enrichment for the same positively selected GC B and MYC target genes, as well as greater RNA content and cell size. BTG1 family members were suggested to interact with RNA. Performing RNA-immunoprecipitation, we discovered that ~800 transcripts associated with BTG1 WT, but not BTG1 Q36H. Notably, these corresponded to the same positively selected GC B and MYC target genes, including MYC itself. BTG1 was shown to regulate mRNA stability in other cell types. However, BTG1 Q36H did not alter MYC mRNA stability and instead facilitated MYC protein synthesis, thus disrupting a novel GC context-specific checkpoint mechanism, whereby BTG1 normally attenuates spurious MYC translation to tightly restrict fitness potential. In GC B cells, Myc induction coincides with S phase entry, but G2/M progression requires re-entry into the proliferative dark zone. To characterize GC dynamics in vivo, we performed targeted single cell RNAseq in competing Btg1 Q36H and WT GC B cells and noted earlier and higher proportion of positively selected Btg1 Q36H GC B cells having committed to G2/M and the proliferative program. We confirmed faster S phase completion in competing Btg1 Q36H GC B cells by in vivo EdU/BrdU labelling and greater re-entry into the proliferative dark zone by in vivo antigen delivery to synchronize GC B cells at the time of positive selection. Given that MCD-DLBCLs express high levels of BCL2, we crossed our Btg1Q36H mice to the VavP-Bcl2 model. As compared to VavP-Bcl2, VavP-Bcl2+Btg1 Q36H mice displayed shorter survival (p=0.0005), earlier onset of lymphoma, dysplastic B cell infiltration into non lymphoid organs and they contained highly mutated, selected and clonal tumor B cells. Moribund VavP-Bcl2+Btg1 Q36H mice uniquely featured sheets of large, immunoblastic lymphoma cells, characteristic of ABC-DLBCLs. Most notably, examining ABC-DLBCLs from 5 independent cohorts showed inferior clinical outcome for BTG1 mutant patients (p=0.0011) and independent association of BTG1 mutation with inferior overall survival by multivariable Cox regression (p=0.0190). Collectively, we find that BTG1 mutations mediate lymphomagenesis through an entirely novel mechanism of action that recapitulates the embryonic MYC-dependent "super-competitive" phenotype originally described in Drosophila imaginal disc cells. In the GC, "super-competition" is provided by BTG1 mutation via a subtle acceleration of MYC induction and GC dynamics, conferring dramatic fitness and the potential to transform into aggressive lymphomas. Disclosures Hoehn: Prellis Biologics: Consultancy. Elemento: Janssen: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Owkin: Consultancy, Other: Current equity holder; Champions Oncology: Consultancy; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Johnson and Johnson: Research Funding. Scott: NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling--licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. ; AstraZeneca: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Research Funding; Rich/Genentech: Research Funding. Melnick: Constellation: Consultancy; Epizyme: Consultancy; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceuticals: Research Funding; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees.

ABC-DLBCLs are among the most aggressive DLBCLs. Genome sequencing studies identified BCL10 gain-of-function mutations in DLBCL mostly within the ABC-DLBCLs. ABC-DLBCLs were recently split into further subtypes according to their genomic characteristics, among which BCL10 somatic mutations were most common in BN2/Cluster 1 cases. These are likely transformed marginal zone lymphomas and were recently suggested to be sensitive to ibrutinib treatment. Pooling publicly available data we noted that most BCL10 mutations are missense or nonsense SNVs affecting its CARD domain, MALT1 binding domain, and the C-terminal S/T rich domain. We generated a large panel of mutant BCL10 constructs including the top hotspot missense mutation R58Q (CARD domain) and nonsense truncation mutations such as E140X. We found that almost all mutants induced aberrantly strong induction of NF-κB activity in lymphoma cells as compared to WT BCL10, indicating that they induce gain of function. BCL10 forms a high order complex with CARD11 and MALT1 (CBM signalosome) downstream of BCR signaling. Normally, CARD11 activation induces polymerization of BCL10 which induces MALT1 activity and downstream NF-κB signaling. To investigate the impact of BCL10 mutants on CBM complex formation we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 species. Both BCL10R58Q and BCL10E140X manifested faster and even spontaneous polarization compared to BCL10WT. BCL10R58Q formed thicker and more heavily bundled filaments (~20 nm) that provide greater surface area to dock signaling proteins, whereas filaments formed by BCL10E140X had the normal ~10 nm structure. Even though the BCL10E140X deletes the canonical MALT1 binding site, the mutant filament still featured robust MALT1 recruitment. Cryo-EM studies revealed that BCL10R58Q mutant gains new interactions within the filament structure that could explain the observed stabilization and bundling effects. Notably, cryo-EM structure of the BCL10E140X mutant in complex with MALT1 showed that it retains its interaction with MALT1 in the filament form despite its predicted lack of interaction with MALT1 in the monomeric form due to the C-terminal deletion. To gain further functional insight we performed mass spectrometry to identify proteins interacting with WT, BCL10R58Q and BCL10E140X. BCL10R58Q featured gain of many novel protein interactors including NF-κB2 and TAB1 etc. consistent with bundled filament formation enabling more signaling protein recruitment. However, the BCL10E140X interactome was quite different and most notably featured loss of binding to negative regulators of non-canonical NF-κB. NF-κB2 (p100/p52) level was indeed elevated in the presence of this mutant. In addition, both BCL10 mutant classes showed aberrant activation of canonical and non-canonical NF-κB activation (IkBa/p65 and p52) through distinct mechanisms. As a functional readout of BCL10 function, we generated a MALT1 GloSensor reporter DLBCL lines to detect MALT1 protease activity. Indeed, both classes of mutations showed potent induction of MALT1 protease activity (GloSensor), enhanced cleavage of canonical MALT1 target proteins (Western Blot), expression of canonical NF-κB target genes (QPCR) such as IL6 and IL10. In striking contrast to BCL10WT, and consistent with our structural data showing spontaneous polymerization of BCL10 mutants, we found that CARD11 knockdown did not impair MALT1 activation, NF-κB signaling, or cell growth in ABC-DLBCL lines expressing both BCL10 mutants. This CARD11 independence was concerning, since it suggests that BCL10 mutant lymphomas might be resistant to drugs targeting upstream components of the BCR signaling pathway such as ibrutinib. Indeed, expression of BCL10R58Q and BCL10E140X (but not BCL10WT) in various ABC-DLBCL cell lines abrogated the ability of ibrutinib to inhibit MALT1 (GloSensor), NF-κB activity (Reporter), cell growth (Growth inhibition) as well as proliferation. Collectively, we find that BCL10 mutations induce aberrant canonical and non-canonical NF-κB activity through novel and structurally distinct biochemical mechanisms that are at least partially dependent on MALT1. BCL10 mutation should be considered as a biomarker for ibrutinib resistance in ABC-DLBCL, so that alternative targeted therapies can be prioritized for these patients. Disclosures Fontan: Johnson & Johnson: Current Employment. Melnick:Epizyme: Consultancy; Jubilant: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding.

Somatic missense mutations in histone 1 genes occur in ~30% of follicular lymphomas and DLBCL and 85% of Hodgkin's lymphomas, with significant mutual co-occurrence among these alleles, most frequently involving H1C and H1E. We crossed constitutive H1C+/-H1E+/- mice with VavP-Bcl2 transgenic mice and observed significant acceleration of lymphomagenesis (p=0.0001). Lymphoma H1 mutations affect the H1 globular domain or C-terminus. We found that the globular domain mutants fail to insert into chromatin whereas C-ter mutants fail to compact chromatin as shown by atomic force microscopy, in vitro assembled nucleosome arrays, and FRET assays in live cells. Hence both types of mutation confer loss of function. Constitutive H1C/E knockout mice are healthy and have no overt phenotype. However, immunization with T-cell dependent antigen caused significant GC hyperplasia (p=0.013) and disruption of polarity due to expansion in the number of centrocytes. Notably, H1C/EDKO GC B-cells readily outcompeted WT GC B-cells in mixed chimera experiments indicating that they have superior fitness (p=0.0086). To understand the mechanism through which this occurs we performed RNA-seq in H1C/EDKO GC B-cells which revealed an aberrant gene expression signature composed almost entirely of transcriptional activation (n=721 upregulated and n=61 downregulated q=0.05, LFC=log(1.5)). Strikingly, these same genes are upregulated during induced pluripotency (iPS cell) reprogramming, and are normally silenced during early development by the PRC2 complex (p This prompted us to extensively characterize the epigenome of purified H1C/EDKO vs WT GC B-cells using High-throughput chromosome conformation capture (Hi-C), ATAC-seq, and ChIP-seq for multiple histone marks. From the topological standpoint the genome is distributed in two compartments: Compartment A, consisting of unpacked chromatin available for gene regulatory processes, and compartment B, which is highly packed, silenced and inaccessible. Beyond this organization, sets of genes are organized into boundary delimited domains called TADs that share regulatory information. Remarkably, the primary effect of H1 loss of function was the shifting of approximately 256 TADs from compartment B to compartment A (there was no A to B shift). These TADs manifested highly significant gain of chromatin accessibility by ATACs-seq and featured reciprocal loss of H3K27me3 and gain of H3K36me2. These "B to A" TADs yielded increased looping connectivity and contained the primitive stem cell genes that were upregulated. Indeed, B to A shifting enabled critical stem cell enhancers to interact with and activate various stem cell genes as shown by v4C (e.g. KLF5, PRDM5, MEIS1, etc). Most remarkably, the 3D architecture of H1C/H1EDKO GC B-cells was similar to that of intermediate stages of iPS cell reprogramming. Consistent with this finding, H1C/EDKO cells exhibited 4-fold greater reprogramming to the iPS state than WT cells after OKSM transduction, and these iPS cells exhibited reduced H3K27me3, stronger pluripotency potential, and impaired differentiation. This effect was rescued by transduction of WT H1, but not globular or C terminal domain mutant H1. Strikingly, the H1C/EDKO primitive stem cell gene expression signature was highly significantly enriched (NES 1.24, FDR Collectively, we find that H1 isoforms are bona fide lymphoma tumor suppressors. We speculate that H1 mutations are limited to GC derived lymphomas due to the stoichiometric need for H1 proteins in these rapidly dividing cells. To the best of our knowledge these are the first data to implicate disruption of topological packing order of chromatin as a cancer driver mechanism, as well as the first data to provide a mechanism whereby mature B-cells can acquire cancer stem cell-like characteristics. Disclosures Melnick: Janssen: Research Funding; Epizyme: Consultancy; Constellation: Consultancy.

MALT1 inhibition is a promising strategy against B-cell receptor (BCR)-dependent lymphomas including ABC DLBCL, CLL and MCL. MALT1 is downstream of the most frequently mutated genes in the BCR and Toll-like receptor (TLR) pathways. MALT1 inhibitors are active in Ibrutinib-resistant BTK and PLCγ2 mutant CLL or CARD11 mutant ABC DLBCL. Therefore, MALT1 inhibitors, which recently began first in man clinical testing, have the potential to cover a larger patient population than drugs against more upstream targets in the BCR pathway including BTK inhibitors. However, activity of inhibitors targeting signaling mediators can be limited by feedback mechanisms counteracting and/or bypassing the need for a specific pathway. MALT1 is central to NF-κB activation downstream of the BCR. Therefore, activation of alternative pathways leading to full or partial activation of the BCR program or other pro-survival pathways might enable cell survival and set off MALT1i resistance. In order to map the landscape of resistance/sensitivity to MALT1 inhibition in ABC DLBCL and assist design of combinatorial regimens, we carried out a loss-of-function screen in HBL-1 (MALT1i sensitive) with or without MI-2 (small molecule irreversible inhibitor of MALT1) to identify genes capable of modulating response to MALT1 inhibition. Our analyses showed that loss of BCR and PI3K activators enhanced sensitivity, while loss of negative regulators of these pathways promoted MALT1i resistance. These findings were validated by knockdown of individual genes with two independent hairpins against activators CD79B, CARD11, BTK or the negative regulator TNFAIP3. Next, we carried out a combinatorial drug screen anchored in MALT1 inhibition by MI-2 or C3 (irreversible substrate-mimetic MALT1 inhibitor) and focused on inhibitors against signaling hubs in the BCR/PI3K pathways in 4 MALT1i sensitive cell lines. This combinatorial screen confirmed that concurrent inhibition of MALT1 and other BCR and PI3K pathways' proteins is additive (0.9>CI (combination index) In depth analysis of proliferation and cell death by CFSE dilution and Annexin V staining revealed that both MALT1/PI3K-i and MALT1/MTORC1-i combinations significantly enhanced growth inhibition and apoptosis in TMD8 and HBL-1 compared to individual agents. Results with MI-2 and C3 were comparable. Short exposure to MI-2 or C3 increased MTORC1 activity as assessed by S6K-Thr389 and S6-Ser235/6 phosphorylation in TMD8 and HBL-1 indicating that MALT1 protease activity modulates MTORC1 activation. MTOR activation is tumorigenic and can mediate chemotherapy resistance. Increased p-S6 following MALT1 inhibition (FC=1.4-2) was blocked by Idelalisib (PI3Ki). However, only Rapamycin, an MTORC1 inhibitor, reduced p-S6 levels relative to vehicle, FC=-5 alone or in combination. In vivo, MALT1/PI3K-i (MI-2/Idelalisib) significantly delayed tumor progression compared to single treatments (p MALT1/MTORC1-i regimens were also highly synergistic in 2 patient-derived xenografted (PDX) ABC DLBCL ex vivo. PDXs were cultured in gelatin/silicate nanoparticle hydrogel 3-D organoids and co-cultured with CD40L expressing cells. Compound pairs were assayed for synergy using 4x4 matrices and growth inhibition evaluated by flow cytometry. Synergy ZIP δ-score ranged 7-14 in 2 specimens for 2 MALT1i. Combined, these results suggest that: 1) MTORC1 activation constitutes a survival feedback mechanism activated after MALT1i treatment that might be leveraged by tumoral cells to evade MALT1 inhibition and, 2) that simultaneous targeting of MTORC1 could improve response and prevent resistance to MALT1 inhibitors. Disclosures Melnick: Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy.

Introduction: Among the tumor immune escape mechanisms described to date, alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial role in the development of diffuse large B-cell lymphoma (DLBCL). Although the frequency of loss of MHC expression differs between ABC- and GCB-DLBCL cell of origin (COO) subtypes, distinct genetic alterations and molecular features that affect MHC expression and the composition of immune cells in the tumor microenvironment remain ill-defined. Here, we aimed to uncover the biologic and genomic basis underlying acquired loss of MHC expression. Method: We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL and uniformly treated with R-CHOP in British Columbia. We performed targeted resequencing, SNP6.0 array and RNAseq for genetic analyses. Immunohistochemical (IHC) staining of MHC-I and -II was performed on tissue microarrays (n=332). COO was assigned by the Lymph2Cx assay in 323 cases (183 GCB, 104 ABC and 36 unclassifiable). Immune cell composition was assessed by IHC, flow cytometry and gene expression profiling (GEP)-based deconvolution of cellular signatures. To experimentally confirm decreased MHC expression induced by EZH2 mutation, we measured surface MHC-I and -II expression on tumor B cells using EZH2Y641/BCL2 mouse model which was previously established (Beguelin et al, Cancer Cell 2013). We also treated human DLBCL cells harboring EZH2 mutation and wild type using EZH2 inhibitor (EPZ-6438), and evaluated their surface MHC-I and -II expression. Results: Loss of MHC-I and -II expression was observed in 43% and 28% of DLBCL cases, respectively. MHC-II loss of expression was significantly associated with the reduction of tumor-infiltrating lymphocytes (TILs), especially CD4 positive T-cells (FOXP3+ cells, PD-1+ cells, and CD4+ naïve and memory T-cells), and cytolytic activity (GZMB and PRF1 mRNA expression) in GCB-DLBCL (all; p We next performed GEP using RNAseq separately in each COO subtype. Interestingly, only four genes (HLA-DMA, DRA, DPA1 and CD74) were differentially expressed according to MHC-II expression (FDR Correlative genetic analysis revealed that, as expected, mutations of CIITA and RFXAP were detected more frequently in MHC-II-negative GCB-DLBCL (p=0.01 and 0.003, respectively). Strikingly, CD83 mutations, which elevate and stabilize MHC-II expression in centrocytes of the light zone (LZ), were significantly enriched in MHC-II positive GCB-DLBCL (p= 0.008), suggesting that these mutations affecting the antigen presentation machinery are selectively acquired in GCB-DLBCL tumors to further reduce and increase the surface MHC-II expression. Genetic analysis also highlighted that EZH2 mutations were most significantly enriched in MHC-II-negative as well as MHC-I-negative GCB-DLBCL cases (both, p Conclusion: Our findings provide important implications for understanding the cancer biology underlying acquired loss of MHC expression. The restoration of MHC expression by EZH2 inhibitors suggests a novel approach of epigenetically enhancing tumor recognition and eradication in combination with immune therapies. Disclosures Sehn: Abbvie: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Connors:Roche Canada: Research Funding; Takeda: Research Funding; Merck: Research Funding; F Hoffmann-La Roche: Research Funding; Cephalon: Research Funding; Seattle Genetics: Honoraria, Research Funding; Amgen: Research Funding; Bayer Healthcare: Research Funding; Bristol Myers-Squibb: Research Funding; Lilly: Research Funding; NanoString Technologies: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding; Genentech: Research Funding. Gascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Roche: Research Funding; Janssen: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Juno Therapeutics: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding.

Understanding the molecular basis of therapy-resistant DLBCL is a critical unmet need. We explored whether the family of Sirtuin proteins might contribute to such effects. Analysis of four independent clinically annotated patient cohorts revealed that higher SIRT3 expression was linked to inferior outcome (p=4.7e-5). This was not the case for any other of the sirtuins. SIRT3 mRNA and protein expression were also much higher in DLBCL patients as compared to normal germinal center (GC) B-cells. Among the seven sirtuins, only SIRT3 depletion universally suppressed proliferation, induced cell cycle arrest, suppressed colony formation, and induced apoptosis in a large panel of DLBCL cell lines regardless of cell of origin, OxPhos status, or somatic mutation profiles. Constitutive Sirt3-/- mice manifested completely normal GC formation after T-cell dependent antigen immunization. However SIRT3 depleted human DLBCL cells manifested inferior engraftment and tumor formation in mice (p=0.023 for hairpin#1, p=0.045 for hairpin#2). Sirt3 inducible knockdown caused strong regression of established DLBCL xenografts. We examined whether SIRT3 was important in lymphoma initiation by crossing VavP-Bcl2 mice with Sirt3-/- animals. As compared to VavP-Bcl2 controls, the VavP-Bcl2/Sirt3-/- mice manifested significantly longer overall survival (P=0.0035), and greatly reduced tumor burden and systemic lymphoma infiltration of organs. SIRT3 is exclusively localized to mitochondria and hence its actions are likely metabolic. We therefore performed metabolomic profiling in SIRT 3 depleted DLBCL cell lines. This analysis revealed profound suppression of the TCA (tricarboxylic acid) cycle, with reduced TCA metabolites such as citrate, alpha-ketoglutarate, succinate, fumarate, malate, etc. SIRT3 depletion caused significant reduction in acetyl-CoA pools as measured by solid phase extraction and LC-MS, indicating that SIRT3 is required to maintain the production of key metabolic intermediates from the TCA cycle. To define the nature of the TCA defect we performed metabolic tracing studies using 13C-labeled glutamine and glucose. The results revealed that SIRT3 drives the TCA cycle through glutaminolysis. We showed that SIRT3 mediates this effect by directly deacetylating and hence hyper-activating the enzymatic activity of mitochondrial glutamine dehydrogenase (GDH). Indeed GDH overexpression could fully rescue the collapse of the TCA, cell proliferation arrest and apoptosis induced by SIRT3 depletion. SIRT3 knockdown was also rescued by feeding cells DMKG (which mimics alpha-ketoglutarate) and hence bypasses the need for SIRT3 mediated glutaminolysis. Because SIRT3 depletion caused metabolic collapse, DLBCL cells manifested potent induction of autophagy, as shown by ratios of LC3II/LC3I in DLBCL cells and using a mCherry-EGFP-LC3 reporter to measure autophagic flux. This autophagy effect was rescued by feeding cells with DMKG or by overexpressing GDH, which uncouple the TCA cycle from SIRT3 dependency. Notably the ratio of LC3II/LCI and perturbed autophagy flux was also Increased in lymphoma cells from VavP-Bcl2;sirt3-/- vs. VavP-Bcl2;sirt3+/+ mice. These data nominate SIRT3 as a putative therapeutic target. Therefore we designed a nanomolar-potency SIRT3 selective small molecule inhibitor including a mitochondrial-targeting motif that concentrates drug in the mitochondrial matrix. This compound (called YC8-02), phenocopied all the effects of SIRT3 depletion including proliferation arrest, apoptosis, TCA collapse by metabolomics study, hyperacetylation of mitochondrial proteins, suppression of GDH activity, and induction of autophagy. Yet YC8-02 had no effect on normal B-cells. Moreover, YC8-02 treatment of chemotherapy resistant DLBCL cell lines restored their sensitivity to clinically relevant doxorubicin concentrations. In summary, SIRT3 is a novel metabolic oncoprotein widely required for DLBCL cells to satisfy their metabolic needs by enhancing the activity of the TCA cycle through glutaminolysis. SIRT3 is a crucial new therapeutic vulnerability especially impactful for the most resistant DLBCLs regardless of their somatic mutations. YC8-02 and its newer derivatives are a promising and entirely new mechanism-based approach to help these patients. Disclosures Cerchietti: Leukemia and Lymphoma Society: Research Funding; Lymphoma Research Foundation: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment; Celgene: Research Funding.

The apical junctional complex (AJC), composed of tight and adherens junctions, maintains epithelial barrier function. Since cigarette smoking and chronic obstructive pulmonary disease (COPD), the major smoking-induced disease, are associated with increased lung epithelial permeability, we hypothesized that smoking alters the transcriptional program regulating airway epithelial AJC integrity. Transcriptome analysis revealed global down-regulation of physiological AJC gene expression in the airway epithelium of healthy smokers (n = 59) compared to nonsmokers (n = 53) in association with changes in canonical epithelial differentiation pathways such as PTEN signaling accompanied by induction of cancer-related AJC components. The overall expression of AJC-related genes was further decreased in COPD smokers (n = 23). Exposure of airway epithelial cells to cigarette smoke extract in vitro resulted in down-regulation of several AJC genes paralleled by decreased transepithelial resistance. Thus, cigarette smoking induces transcriptional reprogramming of airway epithelial AJC architecture from its physiological pattern necessary for barrier function toward a disease-associated molecular phenotype.