Microwave transitions and ground state rotational constants are reported for five newly synthesized deuterium isotopologues of cis-1,3,5-hexatriene (cHTE). These rotational constants along with those of the parent and the three (13)C species are used with vibration-rotation constants calculated from an MP2/cc-pVTZ model to derive an equilibrium structure. That structure is improved by the mixed estimation method. In this method, internal coordinates from good-quality quantum chemical calculations (with appropriate uncertainties) are fit simultaneously with moments of inertia of the full set of isotopologues. The new structure of cHTE is confirmed to be planar and is stabilized by an interaction between the hydrogen atoms H2 and H5, which form a bond and participate in a six-membered ring. cHTE shows larger structural effects of π-electron delocalization than does butadiene with the effects being magnified in the center of the molecule. Thus, strong structural evidence now exists for an increase in π-electron delocalization as the polyene chain lengthens.

Molecular alterations in the histone methyltransferase EZH2 and the antiapoptotic protein Bcl-2 frequently co-occur in diffuse large B-cell lymphoma (DLBCL). Because DLBCL tumors with these characteristics are likely dependent on both oncogenes, dual targeting of EZH2 and Bcl-2 is a rational therapeutic approach. We hypothesized that EZH2 and Bcl-2 inhibition would be synergistic in DLBCL. To test this, we evaluated the EZH2 inhibitor tazemetostat and the Bcl-2 inhibitor venetoclax in DLBCL cells, 3-dimensional lymphoma organoids, and patient-derived xenografts (PDXs). We found that tazemetostat and venetoclax are synergistic in DLBCL cells and 3-dimensional lymphoma organoids that harbor an EZH2 mutation and an IGH/BCL2 translocation but not in wild-type cells. Tazemetostat treatment results in upregulation of proapoptotic Bcl-2 family members and priming of mitochondria to BH3-mediated apoptosis, which may sensitize cells to venetoclax. The combination of tazemetostat and venetoclax was also synergistic in vivo. In DLBCL PDXs, short-course combination therapy resulted in complete remissions that were durable over time and associated with superior overall survival compared with either drug alone.

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors that act on the mevalonate pathway and inhibit synthesis of cholesterol, geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP). In preclinical studies, these agents have been shown to inhibit proliferation, trigger apoptosis and promote cell differentiation of leukemia. Proposed mechanisms include cholesterol deprivation and inhibition of isoprenylation of important signaling molecules. Case reports and early clinical studies suggest a therapeutic potential for statins in acute myeloid leukemia (AML). In the other leukemias there are limited clinical data, but in vitro studies provide a strong rationale for future studies involving statins. The effects of statins on the immune system may lend these agents to a role in allogeneic stem cell transplant. While many of the studies are early, statins have the future potential to be integrated into conventional chemotherapy regimens with limited side effects.

The 2-d1 and 3-d1 isotopologues of trans-hexatriene have been synthesized, and their high-resolution (0.0015 cm-1) IR spectra have been recorded. For each of the isotopologues the rotational structure in four C-type bands for out-of-plane vibrational modes has been analyzed, and the ground state combination differences (GSCDs) have been pooled. Ground state rotational constants have been fitted to the GSCDs. For the 2-d species, A0, B0, and C0 values of 0.7837254(5), 0.0442806(3), and 0.0419299(2) cm-1 were fitted to 2450 GSCDs. For the 3-d species, A0, B0, and C0 values of 0.7952226(8), 0.0446149(7), and 0.0422661(4) cm-1 were fitted to 2234 GSCDs. For the eleven out-of-plane modes of the two isotopologues, predictions of wavenumbers corrected for anharmonicity and harmonic intensities have been computed and compared with experiment where possible.

DLCBL has recently been classified into genetically defined subtypes based on groupings of particular genetic lesions (Chapuy et al, Nat Med 2018; Schmitz et al, NEJM 2018). One predominant cluster, C3 or EZB, is defined by mutations in the chromatin modifying genes EZH2, KMT2D, and CREBBP as well as alterations in BCL2 including mutations and/or translocation of BCL2 to the IgH enhancer. Since tumors in this cluster are likely dependent on both EZH2 and BCL2, and these oncogenes carry out their effects through distinct mechanisms and pathways, targeting both of these oncogenes is a rational therapeutic approach. We hypothesized that EZH2 inhibition and BCL2 inhibition would be synergistic in DLBCL with characteristics of the C3/EZB cluster. To test this, we evaluated the EZH2 inhibitor tazemetostat and the BCL2 inhibitor venetoclax in DLBCL cells, 3D lymphoma organoids, and patient derived xenografts. To assess the effect of combination therapy with tazemetostat and venetoclax, we administered each drug alone and the combination in a panel of DLBCL cell lines, including cells with and without EZH2 mutation and BCL2 translocation. In DLBCL cells with both a BCL2 translocation and EZH2 mutation, the combination resulted in increased killing compared to either drug alone (Figure 1, SUDHL6 (p<0.005), WSU-DLCL2 (p<0.005), and OCI-LY1 (p<0.005)). In contrast, in cells with WT EZH2 and no BLC2 translocation, the effect of the combination was not different than either drug alone. To evaluate for synergy, cells were exposed to increasing doses of each drug alone and the combination. The combined response was evaluated using the Chou-Talalay method. Synergy between tazemetostat and venetoclax was observed in SUDLH-6 (CI value 0.03), WSU-DLCL2 (CI 0.26) and OCI-LY1 (CI 0.06) but not in Farage and LY7 both of which have WT EZH2 and no BCL2 translocation. Since cell lines in suspension do not reflect lymph node architecture, we developed a 3D lymphoma "organoid" culture system that consists of extracellular matrix, lymphoma cells, and stromal cells (Tian et al, Biomaterials 2015; Beguelin et al, Nat Commun 2017). GCB-DLBCLs express integrin αvβ3 that may bind to RGD peptides or vitronectin in tumor extracellular matrix. Based on this, we developed matrix metalloproteinase (MMP)-degradable 3D lymphoma hydrogels functionalized with RGD peptides. To generate organoids, we homogeneously encapsulated DLBCL cells (40,000/gel) in 10 µL hydrogel droplets fabricated in individual wells of a 96-well plate. We established two organoid systems to evaluate response to tazemetostat/venetoclax combination therapy: 1) OCI-LY1 organoids; 2) patient derived xenograft (PDX) organoids. The PDX organoids were generated from a patient tumor after propagation in NSG mice. The PDX tumor harbors both a BCL2 translocation and EZH2 mutation. Organoid viability was evaluated using immunofluorescence for calcein AM (live cells) and ethidium homodimer (dead cells) as well as flow cytometry. In both OCI-Ly1 organoids and PDX organoids, tazemetostat and venetoclax had minimal activity as single agents, however the combination resulted in significant cell killing (Figure 2). To investigate potential mechanisms of synergy, we evaluated RNA-seq profiles of a panel of DLBCL cell lines (n=26) treated with vehicle vs. EZH2 inhibitor (Brach et al, Mol Cancer Ther 2017). Preliminary data suggest that EZH2 inhibition induces expression of pro-apoptotic proteins genes including BCL2L11 (Wilcoxon p=0.01), BAD (p=0.02), BMF (p<0.01), BCL2L13 (p=0.02), and BCL2L14 (p<0.01). BCL2 inhibition with venetoclax may be further enhancing pro-apoptotic signals and lymphoma cell death, especially in C3/EZB DLBCL tumors with dependence on BCL2. In summary, using novel model systems, we have demonstrated that BCL2 inhibition combined with EZH2 inhibition results in synergistic anti-tumor effect that is anticipated to be especially effective as precision therapy for the newly identified cluster 3/EZB DLBCL subtype. A clinical trial of this combination is currently in development. Disclosures Melnick: Janssen: Research Funding; Constellation: Consultancy; Epizyme: Consultancy. Roth:Janssen: Consultancy; ADC Therapuetics: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees.