ALBERT

Description: The genetic background of follicular lymphomas (FLs) diagnosed in advanced clinical stages III/IV, and which are frequently characterized by t(14;18), has been substantially unraveled. Molecular features, as exemplified in the clinicogenetic risk model m7FLIPI, are important tools in risk stratification. In contrast, little information is available concerning localized-stage FL (clinical stages I/II), which accounts for ∼20% of newly diagnosed FL in which the detection rate of t(14;18) is only ∼50%. To investigate the genetic background of localized-stage FL, patient cohorts with advanced-stage FL or localized-stage FL, uniformly treated within phase 3 trials of the German Low-Grade Lymphoma Study Group, were comparatively analyzed. Targeted gene expression (GE) profiling of 184 genes using nCounter technology was performed in 110 localized-stage and 556 advanced-stage FL patients. By penalized Cox regression, a prognostic GE signature could not be identified in patients with advanced-stage FL, consistent with results from global tests and univariate regression. In contrast, it was possible to define robust GE signatures discriminating localized-stage and advanced-stage FL (area under the curve, 0.98) by penalized logistic regression. Of note, 3% of samples harboring an “advanced-stage signature” in the localized-stage cohort exhibited inferior failure-free survival (hazard ratio [HR], 7.1; P = .0003). Likewise, in the advanced-stage cohort, 7% of samples with a “localized-stage signature” had prolonged failure-free survival (HR, 2.3; P = .017) and overall survival (HR, 3.4; P = .072). These data support the concept of a biological difference between localized-stage and advanced-stage FL that might contribute to the superior outcome of localized FL.

Description: Introduction:Considering the increasing numbers of lymphoma patients (pts) surviving long-term, late effects of current treatment strategies such as secondary (sec) malignancies gain increasing importance. Many treatment regimens used in pts with lymphoma (R-CHOEP, DA-EPOCH-R, BEACOPP) include etoposide, a cytotoxic agent reported to increase sec leukemias. In order to further investigate the role of etoposide in inducing sec malignancies in pts with aggressive lymphoma we analyzed the R-MegaCHOEP trial (Schmitz et al., Lancet Oncology 2012) where young, high-risk pts with aggressive B-cell lymphomas had received R-CHOEP or R-MegaCHOEP, a regimen containing very high doses of etoposide (4g/m2). We compared rates of secondary tumors to incidences found in young patients treated with R-CHOP only. Methods:We analyzed 1536 pts aged 18-60 years with aggressive B-cell lymphoma treated in the prospective phase 3 trials FLYER (NCT00278421; n=588, median observation time (OT)=66 months), UNFOLDER (NCT00278408; n=695, median OT=72 months) and MegaCHOEP (NCT00129090; n=253, median OT=112 months) to compare the cumulative incidences of sec neoplasms. We performed a competing risk analysis for time from randomization to occurrence of sec malignancy (myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) or other) according to first-line therapy with R-CHOP (n=1283) vs. R-CHOEP (n=127) vs. R-MegaCHOEP (n=126). We used a cause-specific hazard model adjusted for gender, age (〉50 vs. 50 years showed a trend for increasing sec AML. We found very similar incidences of sec solid tumors (not MDS/AML) affecting 4% of pts treated with R-CHOP or R-MegaCHOEP and 6% of pts following R-CHOEP regimen. The only factor significantly increasing the risk of sec solid tumors was age 〉50 years (HR 2.6, 95%CI (1.5; 4.3), p

Description: Despite the development of novel targeted drugs, the molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) still poses a major therapeutic challenge. DLBCL can be classified into at least two major subtypes, i.e. germinal center B-cell-like (GCB) and the aggressive activated B-cell-like (ABC) DLBCL, each characterized by specific gene expression profiles and mutation patterns. Here we demonstrate a broad anti-tumor effect of dimethyl fumarate (DMF) on both DLBCL subtypes, which is mediated by the induction of ferroptosis, a form of cell death driven by the peroxidation of phospholipids. Due to high expression of arachidonate 5-lipoxygenase in concert with low glutathione and glutathione peroxidase 4 levels, DMF induces lipid peroxidation and thus ferroptosis particularly in GCB DLBCL. In ABC DLBCL cells, which are addicted to NF-κB and STAT3 survival signaling, DMF treatment efficiently inhibits the activity of the IKK complex and JAK kinases. Interestingly, the BCL-2 specific BH3 mimetic ABT-199 and an inhibitor of ferroptosis suppressor protein 1 synergize with DMF in inducing cell death in DLBCL. Collectively, our findings identify the clinically approved drug DMF as a promising novel therapeutic option in the treatment of both GCB and ABC DLBCL.

Description: Cancer genome sequencing studies have identified frequent driver mutations that alter the function of transcription factors (TFs). However, most TFs, are classically "undruggable". The discovery that IMiDs [immunomodulatory drugs e.g.Lenalidomide (LEN) and Pomalidomide (POM)] exert powerful antimyeloma effects by triggering the proteasomal degradation of the critical TFsIKZF1 and IKZF3has led togreat interest in the discovery of"degraders"in other cancers. Existing "down" assays to identify destabilizers (e.g.measuring the loss of the protein of interest [POI]) often have(1) poor signal/noise ratios and narrow dynamic ranges and (2) generate uninterestinghits e.g.drugs thatinhibit transcription or translation. We present a novel positive-selection "up" assay, compatible with high throughput screening, for identifying drugs or sgRNAs that destabilize a POI. We have used this platform to identify novel destabilizers of IKZF1. Our assay is based on a previously published "suicide gene", a variant of the nucleotide salvage gene deoxycytidine kinase (DCK). This variant (DCK*) has mutations that increase its specificity for the synthetic substrate 2-Bromovinyldeoxyuridine (BVdU). Cells that express DCK* are killed by BVdU. We made mammalian expression vectors co-expressing GFP and DCK*, IKZF1, or a DCK*-IKZF1 fusion protein as a single bicistronic mRNA and stably introduced each into 293T cells. In the absence of POM, cells expressing DCK* or DCK*-IKZF1 are killed by BVdU . However, in the presence of an IKZF1 destabilizer, e.g. POM, the DCK*-IKZF1 protein is degraded, making the cells resistant to BVdU. Cells expressing DCK* alone are unaffected by POM and serve as a specificity control for the assay (Panel A). We did a pilot chemical "up" screen with a ~ 2000 bioactive compound library (that included LEN and POM) using 293T cells expressing DCK*-IKZF1. In parallel, we conducted a "down" screen, using 293T cells co-expressing an IKZF1-Firefly Luciferase (Fluc) fusion protein and Renilla luciferase (Rluc) from a single bicistronic mRNA. Compounds that decreased the ratio of Fluc/Rluc activity were scored as hits. As expected, LEN and POM scored in both assays,but there was considerably more noise in the down assay. Lastly, we used the DCK*-IKZF1 cells to screen a library of uncharacterized IMiD derivatives. The screen correctly identified a novel IMiD derivative (MI-2-61) and a known next-generation IMiD (Avadomide) with greater potency against IKZF1 than POM. To identify novel degraders of IKZF1, we used the DCK*-IKZF1 cells and DCK* control cells to screena metabolic inhibitor/anticancer library of ~600 compounds. We identified Spautin-1 as a compound that rescues DCK*-IKZF1 cells, but not DCK* control cells from BVdU toxicity. Spautin-1 downregulates exogenous IKZF1 in 293T cells (Panels B and C) and endogenous IKZF1 in KMS11 myeloma cells. Spautin-1 reportedly suppresses autophagy through inhibition of USP10 and USP13. However, siRNA mediated knockdown of USP10 and USP10 neither altered IKZF1 protein levels, nor blocked downregulation of IKZF1 by Spautin-1. Moreover, Spautin-1 downregulated IKZF1 in 293FT cells in which autophagy was disabled by CRISPR/Cas9-mediated disruption of genes crucial to the autophagy pathway suggesting that the downregulation of IKZF1 by Spautin-1 occurs via a novel mechanism. Unlike IMiDs, the downregulation of IKZF1 by Spautin-1 does not require the CRBN E3 ligase. However, it is blocked by inhibitors of the E1 ubiquitin activating enzyme or the proteasome, but not by neddylation inhibitors required for Cullin-dependent E3 ligases. These data suggest that Spautin-1 triggers the proteasomal degradation of IKZF1 using a non-Cullen E3 ligase. The downregulation of exogenous IKZF1 by Spautin-1 requires the N-terminus of IKZF1, but not the zinc finger domain (ZF2) targeted by IMiDs. Preliminary structure-activity relationship (SAR) studies identified both active and inactive Spautin-1 derivatives, suggesting that downregulation of IKZF1 by Spautin-1 reflects a specific protein binding event and that Spautin-1's potency and specificity can be optimized further. We are undertaking studies to identify the mechanism by which Spautin-1 degrades IKZF1 in the hope that it may represent a novel druggable pathway to therapeutically degrade IKZF1, and validate the use of this platform to discover degraders of "undruggable" targets in other cancers. Figure Disclosures Koduri: Cedilla Therapeutics: Consultancy. Paulk:Novartis: Current Employment. Harris:ONO Pharmaceutical: Consultancy. Buhrlage:Adenoid Cystic Carcinoma Foundation: Other: Scientific Advisory Board. Kaelin:Cedilla Therapeutics: Other: Scientific Founder; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; Fibrogen: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Tango Therapeutics: Other: Founder; Tracon Therapeutics: Membership on an entity's Board of Directors or advisory committees.