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Velcrin compounds are a class of small molecules that induce complex formation between PDE3A and SLFN12, killing cancer cells that express elevated levels of these two proteins by a mechanism independent of PDE3A enzymatic inhibition. Instead, PDE3A binding stimulates the RNase activity of SLFN12, resulting in cleavage of the specific SLFN12 substrate, tRNA-Leu-TAA. Cleavage of tRNA-Leu-TAA in turn causes ribosomal pausing, inhibition of protein synthesis, and cancer cell death. Unlike traditional targeted therapies that leverage dependencies created in cancer cells by genomic alterations, velcrins instead kill cancer cells by a gain-of-function mechanism dependent on the RNase activity of SLFN12. In a collaboration between the Broad Institute and Bayer Pharmaceuticals, we developed the first velcrin, BAY 2666605, to enter Phase I clinical trials. BAY 2666605 is active in cell line and patient-derived xenografts of several tumor types, specifically where elevated levels of the two biomarkers, PDE3A and SLFN12, are expressed. Biomarker-positive tumors are especially enriched among melanomas, and we have consistently observed tumor regression in biomarker-positive melanoma tumor models in vivo. BAY 2666605 furthermore shows drug-like properties, excellent brain penetration, increased stimulation of SLFN12 RNase activity, and reduced inhibition of PDE3A enzymatic activity compared with most other velcrins and approved PDE3A inhibitors. BAY 2666605 has recently entered a First-in-Human study (NCT04809805) in patients with advanced solid tumors that co-express PDE3A and SLFN12, including melanoma, ovarian cancer, and sarcoma. Citation Format: Stefan Gradl, Sooncheol Lee, Martin Lange, Xiaoyun Wu, Silvia Goldoni, Timothy Lewis, Charlotte Kopitz, Colin Garvie, Philip Lienau, Stephanie Hoyt, Henrik Seidel, Stephan Kaulfuss, Manuel Ellermann, Luc de Waal, Adrian Tersteegen, Sven Golfier, Detlev Suelzle, Christa Hegele-Hartung, James Carr, Frederick Brookfield, Michael Bruening, Melanie Berthold, Thibaud Jourdan, Monica Schenone, Galen Gao, Joseph McGaunn, Antje Wengner, Elisa Aquilanti, Franziska Siegel, Marine Garrido, Annette Walter, Isabelle Genvresse, Andrew Cherniack, Stuart Schreiber, Knut Eis, Ashley Eheim, Matthew Meyerson, Heidi Greulich. BAY 2666605: The first PDE3A-SLFN12 complex inducer for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND04.

SUMMARYRecent data suggests that colon tumors contain a subpopulation of therapy resistant quiescent cancer stem cells (qCSCs) that are the source of relapse following treatment. Here, using colon cancer patient-derived organoids (PDOs) and xenograft (PDX) models, we identify a rare population of long-term label-retaining (PKH26Positive) qCSCs that can re-enter the cell cycle to generate new tumors. RNA-sequencing analyses demonstrated that these cells are enriched for stem cell associated gene sets such as Wnt and hedgehog signaling, epithelial-to-mesenchymal transition (EMT), embryonic development, tissue development and p53 pathway but have downregulated expression of genes associated with cell cycle, transcription, biosynthesis and metabolism. Furthermore, qCSCs are enriched for p53 interacting negative regulators of cell cycle, includingAKAP12, CD82, CDKN1A, FHL2, GPX3, KIAA0247, LCN2, TFF2, UNC5BandZMAT3, that we show are indicators of poor prognosis and may be targeted for qCSC abolition. Interestingly, CD82, KIAA0247 and UNC5B proteins localize to the cell surface and may therefore be potential markers for the prospective isolation of qCSCs. These data support the temporal inhibition of p53 signaling for the elimination of qCSCs and prevention of relapse in colorectal cancer.

SUMMARYRecent data support a hierarchical model of colon cancer driven by a population of cancer stem cells (CSCs). Greater understanding of the mechanisms that regulate CSCs may therefore lead to more effective treatments. Serial limiting dilution xenotransplantation assays of colon cancer patient-derived tumors demonstrated ALDHPositivecells to be enriched for tumorigenic self-renewing CSCs. In order to identify CSC modulators, we performed RNA-sequencing analysis of ALDHPositiveCSCs from a panel of colon cancer patient-derived organoids (PDOs) and xenografts (PDXs). These studies demonstrated CSCs to be enriched for embryonic and neural development gene sets. Functional analyses of genes differentially expressed in both ALDHPositivePDO and PDX CSCs demonstrated the neural crest stem cell (NCSC) regulator and wound response geneEGR2to be required for CSC tumorigenicity and to control expression of homeobox superfamily embryonic master transcriptional regulatorHOXgenes and the embryonic and neural stem cell regulatorSOX2. In addition, we identifyEGR2,HOXA2,HOXA4,HOXA5,HOXA7,HOXB2,HOXB3and the tumor suppressorATOH1as new prognostic biomarkers in colorectal cancer.

Summary Recent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer., Graphical Abstract, Highlights • Colon tumors contain therapy-resistant quiescent cancer stem cells (qCSCs) • qCSC gene expression mirrors that of quiescent stem cells of the regenerating gut • qCSCs are enriched for p53 signaling genes • qCSC elimination may be achieved by inhibiting downstream targets of p53 signaling, Cancer stem cells ; Quiescence; Organoids; Colon cancer; p53 signaling; Transcriptomics

Recent evidence demonstrates that colon cancer stem cells (CSCs) can generate neurons that synapse with tumor innervating fibers required for tumorigenesis and disease progression. Greater understanding of the mechanisms that regulate CSC driven tumor neurogenesis may therefore lead to more effective treatments. RNA-sequencing analyses of ALDH

Streptococcus equiis the causative agent of strangles, the most frequently diagnosed infectious disease of horses worldwide. The disease is characterized by abscessation and swelling of the lymph nodes of the head and neck, which can literally strangle the horse to death.S. equiproduces four recently acquired phage-associated bacterial superantigens (sAgs; SeeH, SeeI, SeeL, and SeeM) that share homology with the mitogenic toxins ofStreptococcus pyogenes. The aim of this study was to characterize the contribution of each of theseS. equisAgs to mitogenic activityin vitroand quantify the sAg-neutralizing capacity of sera from naturally infected horses in order to better understand their role in pathogenicity. Each of the sAgs was successfully cloned, and soluble proteins were produced inEscherichia coli. SeeI, SeeL, and SeeM induced a dose-dependent proliferative response in equine CD4 T lymphocytes and synthesis of gamma interferon (IFN-γ). SeeH did not stimulate equine peripheral blood mononuclear cells (PBMC) but induced proliferation of asinine PBMC. Allelic replacement mutants ofS. equistrain 4047 with sequential deletion of the superantigen genes were generated. Deletion ofseeI, seeL, andseeMcompletely abrogated the mitogenic activity and synthesis of IFN-γ, in equine PBMC, of the strain 4047 culture supernatant. Sera from naturally infected convalescent horses had only limited sAg-neutralizing activities. We propose thatS. equisAgs play an important role inS. equipathogenicity by stimulating an overzealous and inappropriate Th1 response that may interfere with the development of an effective immune response.

Introduction Copanlisib (BAY 80 6946) is a highly selective pan class I PI3K-i with predominant inhibitory activity against PI3Kδ and PI3Kα, in clinical development as single agent and in combination for lymphoma patients. To address single agent antitumor activity in different lymphomas and to understand the molecular basis of resistance mechanisms for rational combination, we performed a screening of copanlisib as single agent and in combination with 15 other anticancer agents in 17 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL). Methods MCL (Jeko1, Rec1, JVM2, Granta519, Maver1, Mino1, SP-49, SP-53, UPN1, Z138), MZL (Karpas1718, VL51, SSK41, ESKOL, HAIR-M, HC-1) and CLL (MEC1) cell lines were exposed to increasing doses of copanlisib alone and in combination with other compounds using the fixed ratio set-up. Tested compounds included approved and experimental inhibitors of key regulatory pathways. Synergy was assessed via Chou-Talalay combination index (CI). Gene expression profiling (GEP) was done using Illumina Human HT12Expression BeadChips and GSEA (FDR Results Copanlisib showed antitumor activity in most cell lines (median IC50=22nM; 95%C.I.: 15-98). The other most active drugs were bortezomib (5nM; 5-7), romidepsin (34nM; 2-94), roniciclib (23nM; 18-29), panobinostat (161nM; 11-1263), MI2 (490nM; 224-1000). The remaining had median IC50s >500nM. Copanlisib-containing combinations often gave synergy/additive effects: copanlisib with venetoclax was beneficial in 16/17; with MI2 in 15; with palbociclib or ibrutinib in 14; with BAY 1125976 or panobinostat in 13; with lenalidomide or BAY 1238097 in 12; with rituximab in 11; with romidepsin in 10; with roniciclib in 9; with bortezomib in 8; with BAY 1143572 in 7; with bendamustine in 6; with ruxolitinib in 2. Combinations with venetoclax and with palbociclib were the most promising, achieving CI values Conclusion Copanlisib was active in MCL, MZL and CLL models. Combinations with BCL2-i venetoclax and CDK4/CDK6-i palbociclib were the most synergistic. Specific GEP features might predict lymphomas that could benefit from these regimens. Citation Format: Eugenio Gaudio, Ivo Kwee, Filippo Spriano, Chiara Tarantelli, Andrea Rinaldi, Thibaud Jourdan, Melanie Berthold, Alberto Arriibas, Anastasion Stathis, Davide Rossi, Ningshu Liu, Martin Lange, Oliver Politz, Emanuele Zucca, Francesco Bertoni. The phosphatidylinositol-3-kinase (PI3K) inhibitor (i) copanlisib is active in preclinical models of B-cell lymphomas as single agent and in combination with conventional and targeted agents including venetoclax and palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 154. doi:10.1158/1538-7445.AM2017-154

Introduction. Copanlisib is a pan class-I PI3K inhibitor (i) with preferred activity towards PI3Kα and PI3Kδ that is currently in clinical development as single agent and in combination for patients with non-Hodgkin lymphomas. Here, we performed a screening of copanlisib as single agent and in combination with 15 other anti-cancer agents on a panel of 17 lymphoma cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL). Methods. Cell lines derived from MCL (Jeko1, Rec1, JVM2, Granta519, Maver1, Mino1, SP-49, SP-53, UPN1, Z138), MZL (Karpas1718, VL51, SSK41, ESKOL, HAIR-M, HC-1) and CLL (MEC1) were exposed to increasing doses of copanlisib alone and in combination with increasing doses of other compounds using the fixed ratio set-up. Tested compounds were approved as well as experimental inhibitors of key regulatory pathways: e.g. anti-CD20 monoclonal antibody rituximab, BTK-i ibrutinib, CDK-i roniciclib, DNA damage agent bendamustine, HDAC-i panobinostat, HDAC1/2-i romidepsin, immunomodulatory lenalidomide, JAK1/2-i ruxolitinib, MALT-i MI2, proteasome-i bortezomib. Synergy was assessed with Chou-Talalay combination index (CI): synergism ( 1.1). Gene expression profiling (GEP) was done using the Illumina-HumanHT-12 Expression-BeadChips and GSEA (FDR Results. Copanlisib showed anti-tumor activity in the vast majority of cell lines with a median IC50 of 22nM (95%C.I.; 15-98) across all the cell lines, 36nM (6-175) in MZL, 22nM (95%C.I., 3-103) in MCL, 23nM in the CLL MEC1. Among all cell lines, the most active other compounds were bortezomib (IC50 5nM; 95%C.I., 5-7), romidepsin (34nM, 2-94), roniciclib (23nM, 18-29), panobinostat (161nM, 11-1263), MI2 (490nM; 224-1000) with remaining having median IC50s >500nM. Among these the only showing higher activity in one histotype was panobinostat, more active in MCL than MZL (IC50 17 vs 557nM, P 0.04). As pattern of activity across the cell lines, copanlisib was positively correlated with ibrutinib (R 0.58, P 0.001) and negatively with ruxolitinib (R -0.55, P 0.021). When copanlisib-containing combinations were tested in the 17 cell lines, different ones frequently appeared synergistic or additive: for example copanlisib plus MI2, 88% (15); plus ibrutinib, 82% (14); plus AKT-i or panobinostat, 76% (13); plus lenalidomide or BET-i, 71% (12); plus rituximab, 65% (11); plus romidepsin, 59% (10); plus roniciclib, 53% (9); plus bortezomib, 47% (8); plus bendamustine, 35% (6); plus ruxolitinib, 12% (2). For combinations with the most promising effect, baseline GEP identified gene-sets associated with different sensitivity to these combinations. High expression of genes involved in interferon signaling, oxidative phosphorylation, fatty acid metabolism, apoptosis, PI3K/AKT/mTOR and IL6/JAK/STAT signaling and low expression of genes involved in cell cycle were associated with synergism to copanlisib combinations with several compounds. Interestingly, largely the opposite was observed for the combination with others, with better synergy in cells with high expression of E2F/MYC targets and genes involved in cell cycle and low expression of transcripts involved in interferon PI3K/AKT/mTOR and IL6/JAK/STAT signaling. Conclusion. Copanlisib was active in cell lines derived from MCL, MZL and CLL. Combinations of copanlisib with a variety of additional anti-cancer compounds were synergistic and/or additive when combined in different cell lines. The combinations of the synergy profiles in the cell lines tested and the specific GEP features might predict lymphomas that could benefit from these regimens. Disclosures Jourdan: Bayer Pharma AG: Employment. Berthold:Bayer Pharma AG: Employment. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Lange:Bayer Pharma AG: Employment. Politz:Bayer Pharma AG: Employment.