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PI3K delta (PI3Kδ) inhibitors are used to treat lymphomas but safety concerns and limited target selectivity curbed their clinical usefulness. PI3Kδ inhibition in solid tumors has recently emerged as a potential novel anticancer therapy through the modulation of T-cell responses and direct antitumor activity. Here we report the exploration of IOA-244/MSC2360844, a first-in-class non–ATP-competitive PI3Kδ inhibitor, for the treatment of solid tumors. We confirm IOA-244’s selectivity as tested against a large set of kinases, enzymes, and receptors. IOA-244 inhibits the in vitro growth of lymphoma cells and its activity correlates with the expression levels of PIK3CD, suggesting cancer cell–intrinsic effects of IOA-244. Importantly, IOA-244 inhibits regulatory T cell proliferation while having limited antiproliferative effects on conventional CD4+ T cells and no effect on CD8+ T cells. Instead, treatment of CD8 T cells with IOA-244 during activation, favors the differentiation of memory-like, long-lived CD8, known to have increased antitumor capacity. These data highlight immune-modulatory properties that can be exploited in solid tumors. In CT26 colorectal and Lewis lung carcinoma lung cancer models, IOA-244 sensitized the tumors to anti-PD-1 (programmed cell death protein 1) treatment, with similar activity in the Pan-02 pancreatic and A20 lymphoma syngeneic mouse models. IOA-244 reshaped the balance of tumor-infiltrating cells, favoring infiltration of CD8 and natural killer cells, while decreasing suppressive immune cells. IOA-244 presented no detectable safety concerns in animal studies and is currently in clinical phase Ib/II investigation in solid and hematologic tumors. Significance: IOA-244 is a first-in-class non–ATP-competitive, PI3Kδ inhibitor with direct antitumor in vitro activity correlated with PI3Kδ expression. The ability to modulate T cells, in vivo antitumor activity in various models with limited toxicity in animal studies provides the rationale for the ongoing trials in patients with solid tumors and hematologic cancers.

Target engagement kinase tree

Tumor experiments showing monotherapy testing of IOA-244

IC50 curve of the ATP competitive assay and pie chart of the Kinativ assay

Table summarizing the IC50 values derived from previous published experiments performed with IOA-244

Correlation analysis of PI3Kd levels and the response to IOA-244 and Idelalisib

Table showing the raw data of the AUC value of figure 2A

PI3K delta (PI3Kδ) inhibitors are used to treat lymphomas but safety concerns and limited target selectivity curbed their clinical usefulness. PI3Kδ inhibition in solid tumors has recently emerged as a potential novel anticancer therapy through the modulation of T-cell responses and direct antitumor activity. Here we report the exploration of IOA-244/MSC2360844, a first-in-class non–ATP-competitive PI3Kδ inhibitor, for the treatment of solid tumors. We confirm IOA-244’s selectivity as tested against a large set of kinases, enzymes, and receptors. IOA-244 inhibits the in vitro growth of lymphoma cells and its activity correlates with the expression levels of PIK3CD, suggesting cancer cell–intrinsic effects of IOA-244. Importantly, IOA-244 inhibits regulatory T cell proliferation while having limited antiproliferative effects on conventional CD4+ T cells and no effect on CD8+ T cells. Instead, treatment of CD8 T cells with IOA-244 during activation, favors the differentiation of memory-like, long-lived CD8, known to have increased antitumor capacity. These data highlight immune-modulatory properties that can be exploited in solid tumors. In CT26 colorectal and Lewis lung carcinoma lung cancer models, IOA-244 sensitized the tumors to anti-PD-1 (programmed cell death protein 1) treatment, with similar activity in the Pan-02 pancreatic and A20 lymphoma syngeneic mouse models. IOA-244 reshaped the balance of tumor-infiltrating cells, favoring infiltration of CD8 and natural killer cells, while decreasing suppressive immune cells. IOA-244 presented no detectable safety concerns in animal studies and is currently in clinical phase Ib/II investigation in solid and hematologic tumors.Significance:IOA-244 is a first-in-class non–ATP-competitive, PI3Kδ inhibitor with direct antitumor in vitro activity correlated with PI3Kδ expression. The ability to modulate T cells, in vivo antitumor activity in various models with limited toxicity in animal studies provides the rationale for the ongoing trials in patients with solid tumors and hematologic cancers.

Table showing values of microsomal stability of IOA-244

Table showing the raw data of the AUC value of figure 2A

Correlation analysis of PI3Kd levels and the response to IOA-244 and Idelalisib

Phosphoinositide 3-kinase delta (PI3Kd) inhibitors are used to treat lymphomas but safety concerns and limited target selectivity curbed their clinical usefulness. PI3Kd inhibition in solid tumors has recently emerged as a potential novel anti-cancer therapy through the modulation of T cell responses and direct anti-tumor activity. Here we report the exploration of IOA-244/MSC2360844, a first-in-class non-ATP-competitive PI3Kd inhibitor, for the treatment of solid tumors. We confirm IOA-244’s selectivity as tested against a large set of kinases, enzymes, and receptors. IOA-244 inhibits the in vitro growth of lymphoma cells and its activity correlates with the expression levels of PIK3CD, suggesting cancer cell-intrinsic effects of IOA-244. Importantly, IOA-244 inhibits Treg proliferation while having limited anti-proliferative effects on conventional CD4+ T cells and no effect on CD8+ T cells. Instead, treatment of CD8 T cells with IOA-244 during activation, favors the differentiation of memory-like, long-lived CD8, known to have increased antitumor capacity. These data highlight immune-modulatory properties that can be exploited in solid tumors. In CT26 colorectal and LLC lung cancer models, IOA-244 sensitized the tumors to anti-PD-1 (Programmed cell death protein 1) treatment, with similar activity in the Pan-02 pancreatic and A20 lymphoma syngeneic mouse models. IOA-244 reshaped the balance of tumor-infiltrating cells, favoring infiltration of CD8 and NK cells, while decreasing suppressive immune cells. IOA-244 presented no detectable safety concerns in animal studies and is currently in clinical Phase Ib/II investigation in solid and hematological tumors.

Table showing values of microsomal stability of IOA-244

IC50 curve of the ATP competitive assay and pie chart of the Kinativ assay

Table summarizing the IC50 values derived from previous published experiments performed with IOA-244

Tumor experiments showing monotherapy testing of IOA-244

Target engagement kinase tree

The DNA damage response (DDR) kinases ATR, Chk1, and Wee1 play vital roles in the response to replication stress and in maintaining cancer genomic stability. Inhibitors of these kinases are currently under clinical investigation. Mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) are aggressive lymphomas whose clinical outcome is still largely unsatisfactory. These cell lymphoma subtypes are highly dependent on both Chk1 and Wee1 for survival. We investigated the activity of the ATR inhibitor AZD6738 as single agent and in combination with either Chk1 (AZD6738) or Wee1 (AZD1775) inhibitors in several preclinical models of MCL and DLBCL. This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, and validation experiments on in vivo models. Cellular and molecular mechanisms of the observed synergistic effect as well as pharmacodynamic analysis of in vivo samples were studied. AZD6738 exerted a strong synergistic cytotoxic effect in combination with both AZD7762 and AZD1775 in the 2 lymphoma subtypes regardless of their TP53, MYC, and ATM mutational status. These DDR inhibitor combinations, similarly to the Chk1/Wee1 inhibitor combination, caused a marked S-phase delay, with an increase in cyclin-dependent kinases (CDK) activity, increased DNA damage, and decreases in Wee1, MYC, and RRM2 protein levels. The synergistic in vitro activity translated to striking in vivo antitumor activity. DDR–DDR inhibitor combinations could potentially offer promising novel therapeutic strategies for patients with B-cell lymphoma.

GSEA of transcripts upregulated in cells with low sensitivity (non-responders) to AZD6738.

Primers used for real-time PCR on cDNA

GSEA of transcripts upregulated in cells with high sensitivity (responders) to AZD6738.

Supplementary Figure 1. Correlation analysis among the different drug IC50s; Supplementary Figure 2. AZD6738 IC50 in all the 36 B-cell lymphoma cells and their molecular characteristics; Supplementary Figure 3: Cell cycle and DNA damage protein levels in the different cell lines under study; Supplementary Figure 4: Correlation between drug IC50s and biomarkers of response; Supplementary Figure 5: Cell cycle perturbation induced by single and combined treatments with the inhibitors; Supplementary Figure 6: Biparametric analysis of pS10-Histone H3 and DNA in OCILY-7 cells untreated or 8, 24 and 48 h after treatment with the drug either singly or combined. Caspase-3 activity assay 8, 24 and 48h after treatment with the three drugs either singly or combined Real time PCR showing Myc, Wee1 and RRM2 expression levels after 24 and 48h of treatment with the drugs either singly or combined; Supplementary Figure 7: Body weights of mice transplanted with OCILY-7 xonografts treated or not with the single agents and with the combination. Tumor growth curves in OCILY-7 xenograft transplanted mice untreated or treated at a late stage; Supplementary Figure 8: Antitumor activity and pharmacodimamic markers of single and combined agents in JEKO1 xenografts

Supplementary Material and Methods

Baseline gene expression analysis of B cell lymphoma cell lines with higher (IC50 < 200 nM) or lower sensitive (IC50 > 400 nM) to PQR309.

Cell lines, growth medium and source. All media were supplemented with fetal bovine serum (20%), Penicillin-Streptomycin 5% (Sigma) and L-glutamine (1%).

PQR309 activity, combinations of PQR309 with additional drugs, BCL2, MYC and TP53 status in lymphoma cell lines.

YK-4-279 and TK-216 combinations. For each combination, the table shows the median Combination Index (CI) obtained after exposing cell lines to increasing concentration of two compounds at 8 x 8 concentrations of each compound (removing concentrations that give 10% or less of proliferation already with the single agent), as previously performed (3).

Purpose:Transcription factors are commonly deregulated in cancer, and they have been widely considered as difficult to target due to their nonenzymatic mechanism of action. Altered expression levels of members of the ETS-transcription factors are often observed in many different tumors, including lymphomas. Here, we characterized two small molecules, YK-4-279 and its clinical derivative, TK-216, targeting ETS factors via blocking the protein–protein interaction with RNA helicases, for their antilymphoma activity.Experimental Design:The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination; validation experiments on in vivo models; and transcriptome and coimmunoprecipitation experiments.Results:YK-4-279 and TK-216 demonstrated an antitumor activity across several lymphoma cell lines, which we validated in vivo. We observed synergistic activity when YK-4-279 and TK-216 were combined with the BCL2 inhibitor venetoclax and with the immunomodulatory drug lenalidomide. YK-4-279 and TK-216 interfere with protein interactions of ETS family members SPIB, in activated B-cell–like type diffuse large B-cell lymphomas, and SPI1, in germinal center B-cell–type diffuse large B-cell lymphomas.Conclusions:The ETS inhibitor YK-4-279 and its clinical derivative TK-216 represent a new class of agents with in vitro and in vivo antitumor activity in lymphomas. Although their detailed mechanism of action needs to be fully defined, in DLBCL they might act by targeting subtype-specific essential transcription factors.

Gene expression data after TK-216 treatment (8h) in U2932. A) Supervised analysis of transcriptome after treatment B) Gene-sets significantly enriched after treatment. C) IRF4/SPIB and lenalidomide related gene-sets significantly enriched after treatment.

Anti-tumor activity of YK-4-279 and TK-216 in lymphomas after 72 h of exposure. Genetic features for DLBCL cells (1,2) are also shown.

Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacologic inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clinical evaluation, in preclinical lymphoma models.Experimental Design: This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation experiments on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors.Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was associated with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib.Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compound that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120–9. ©2017 AACR.

Baseline gene expression analysis of B cell lymphoma cell lines sensitive to PQR309 and idelalisib (dual sensitive) or to PQR309 only (discordant).

Phosphopeptide changes induced by PQR309 in B cell lymphoma cell lines revealed using mass spectrometry analysis.

Phosphoprotein changes induced by PQR309 in B cell lymphoma cell lines revealed using Reverse Phase Protein Array (RPPA) analysis.

Supplementary figures, table legends, Table S1, S9 Figure S1. In vitro antitumor activity of the dual PI3K/mTOR inhibitor apitolisib and its correlation with PQR309. Figure S2. Antitumor In vivo activity of PQR309 in the treatment of RI-1 and SU-DHL-6 xenograft model. Figure S3. Apoptosis is induced by co-treatment of PQR309 with venetoclax or panobinostat in primary cells and in the DLBCL SU-DHL-6 cell line. Figure S4. Specific baseline gene expression signatures are associated with higher or lower sensitivity to PQR309. Figure S5. In vitro antitumor activity of the PI3K� inhibitor idelalisib and its correlation with PQR309. Figure S6. PQR309 induced decrease in phosphorylation of AKT-Ser473 and p70S6K-Thr389 in DLBCL cell lines. Figure S7. PQR309 reduces AKT-Ser473 phosphorylation in lymphoma cell lines. Figure S8. Transcriptional expression signature of ABC DLBCL cell lines induced by PQR309. Figure S9. ABC and GCB DLBCL PQR309-treated signatures were highly overlapping. Figure S10. Transcript expression levels of PQR309-treated samples. Figure S11. Changes in protein phosphorylation and RNA expression differently contribute to PQR309 affected biologic pathways in ABC DLBCL. Figure S12. PQR309 can largely regulate the same genes affected by the BTK inhibitor ibrutinib, the PI3K� idelalisib, the dual PI3K�/� inhibitor duvelisib (A), the dual PI3K�/� inhibitor AZD8835 or the AKT inhibitor AZD5363 (B). Figure S13. BCR pathway signature is similarly affected after ibrutinib, idelalisib and duvelisib treatments in ABC DLBCL cell lines. Figure S14. The dual PI3K/mTOR inhibitor PQR309 and the PIM inhibitor AZD1208 synergize in DLBCL cell lines. Supplementary Table 1. List of phosphoresidues investigated by Carna Bioscience to perform RPPA analysis. Supplementary Table 9. Transcripts differentially expressed in ABC DLBCL cell lines treated with ibrutinib (A), idelalisib (B) or duvelisib (C) versus DMSO.

Gene expression data after YK-4-279 or TK-216 treatment (18h) in U2932, TMD8, OCILY10 and SUDHL2. A) Supervised analysis of transcriptome after treatment. B) Gene-sets significantly enriched after treatment.

Phosphoproteins changes induced by PQR309 in B cell lymphoma cell lines revealed using Pathscan Akt Signaling Antibody Array Kit.

Gene expression changes in DLBCL cell lines after exposure to PQR309.

Introduction: CXCR4 is expressed in different B cell lymphoid neoplasms, and its levels are upregulated by BCR/PI3K blockade. High expression has been associated with resistance to BCR/PI3K inhibitors and gene mutations with reduced sensitivity to BTK inhibitors. We assessed whether its pharmacological inhibition with the potent CXCR4 inhibitor SPX5551 (POL5551) increases the antitumor activity of anti-lymphoma agents in lymphoma models, including some with secondary resistance to PI3K/BTK inhibitors. Methods: Antiproliferative activity was measured in mantle cell lymphoma (MCL, n=10), chronic lymphocytic leukemia (CLL,2), and marginal zone lymphoma (MZL, 2 + 4 derivatives with secondary resistance to PI3K and BTK inhibitors) cell lines exposed (72h) to increasing doses of compounds as single and in combination. Drugs included ibrutinib, copanlisib, idelalisib. The beneficial effect of the combinations versus the single agents was considered both as synergism (Chou-Talalay combination index), and as potency and efficacy (MuSyC algorithm). Mechanisms of action were studied by RNA-Seq, cell cycle and apoptosis assays, and immunofluorescence. Results: Combination of SPX5551 with conventional or targeted therapies was beneficial, either additive or synergistic, across all tested lymphomas. The combination of SPX5551 and ibrutinib was superior to single treatments in MCL, MZL or CLL, with stronger benefit in MZL and MCL than CLL. SPX5551/copanlisib showed synergism in both MCL and MZL, and SPX5551 addition overcame resistance in MZL with secondary resistance to idelalisib, copanlisib or ibrutinib. These results were confirmed using the clinically available drug candidate balixafortide (SPX6326, POL6326).RNA-Seq in the MCL REC1 model showed a much bigger impact of the SPX5551+ibrutinib on transcriptome than single agents: 2100 transcripts differentially expressed in the combination group vs DMSO (P Conclusions: Results suggest that a combination with the CXCR4 inhibitor SPX5551 adds beneficial effect to BCR inhibitors and might overcome resistance to PI3K/BTK inhibitors. Adding SPX551 to the treatment of patients with B cell lymphoma warrants further exploration. Citation Format: Laura Barnabei, Alberto J. Arribas, Luciano Cascione, Francesca Guidetti, Filippo Spriano, Chiara Tarantelli, Andrea Rinaldi, Anastasios Stathis, Davide Rossi, Emanuele Zucca, Johann Zimmermann, Francesco Bertoni. Pharmacological inhibition of CXCR4 increases the anti-tumor activity of conventional and targeted therapies in B cell lymphoma models [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 492.

Background: Marginal zone lymphoma (MZL) is an indolent yet incurable B cell malignancy. Two BTK inhibitors, ibrutinib and zanubrutinib, are FDA approved for relapsed/refractory MZL patients. PI3K inhibitors have also shown clinical activity. The identification of the mechanisms of resistance can provide useful information to optimize the use of the agents. We previously reported an IL6 driven MZL model of PI3K inhibitors resistance developed by prolonged exposure to the PI3Kδ inhibitor idelalisib (Arribas, Haematologica 2022). Here, we present the detailed characterization of a second model with resistance to both BTK and PI3K inhibitors. Methods: MTT assay. RNA-Seq, whole exome sequencing, miRNA and methylation profiling. FACS and ELISA analyses. Results: Resistant cells, developed by continuous exposure of the cell line Karpas1718 to idelalisib, showed resistance to various inhibitors of BTK (ibrutinib, zanubrutinib, acalabrutinib and pirtobrutinib) and PI3K (idelalisib, duvelisib, copanlisib and umbralisib). No mutations affecting BTK, PLCG2 or CXCR4 were identified in resistant cells, which had higher expression of genes involved in ERBB signaling (HBEGF, NRG2, ERBB4), cell proliferation (PBK, MKI67, TCL1A) and DNA recombination (RAG1, RAG2) than parental cells. We confirmed cell surface ERBB4 up-regulation, and the cytoplasmatic expression and secretion of its ligand HBEGF in resistant cells, which led to increased levels of p-AKT and p-ERK. The miRNAs miR-29c and let-7c, known negative regulators of the HBEGF-ERBB axis, were fully methylated and down-regulated in resistant compared to parental cells. ERBB4 genetic silencing improved sensitivity to PI3Kδ inhibitor, and exposure to let-7c or miR-29c mimics decreased secreted HBEGF and recovered sensitivity to PI3K inhibitors in resistant cells. Addition of recombinant HBEGF (rHBEGF) induced resistance to BTK and to PI3K inhibitors in parental cells and in other lymphoma models including mantle cell lymphomas and diffuse large B cell lymphomas (DLBCL). The rHBEGF induced resistance was reverted adding the ERBB inhibitor lapatinib. To extend our findings to the clinical context, using two MZL and one DLBCL expression datasets, we showed HBEGF and ERBB4 expression in clinical specimens. Finally, HBEGF levels appeared elevated in the serum of CLL patients with primary or acquired resistance to PI3Kδ or to BTK inhibitors, compared to patients responding to the drugs and paired for similar clinical features. Conclusions: We characterized a novel B cell lymphoma model of secondary resistance to BTK and PI3K inhibitors. Our results indicate that epigenetic plasticity led to the activation of HBEGF-ERBB signaling sustaining resistance to BTK/PI3K inhibitors, which can be overcome using epigenetic agents and ERBB inhibitors. These therapeutics approaches could be tested in novel clinical trials. AJA, SN: equally contributed. Citation Format: Alberto J. Arribas, Sara Napoli, Luciano Cascione, Laura Barnabei, Giulio Sartori, Eleonora Cannas, Eugenio Gaudio, Chiara Tarantelli, Afua A. Mensah, Filippo Spriano, Antonella Zucchetto, Francesca M. Rossi, Andrea Rinaldi, Manuel Castro de Moura, Anastasios Stathis, Georg Stussi, Valter Gattei, Jennifer R. Brown, Manel Esteller, Emanuele Zucca, Davide Rossi, Francesco Bertoni. ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms [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 394.

Purpose: Transcription factors are commonly deregulated in cancer, and they have been widely considered as difficult to target due to their nonenzymatic mechanism of action. Altered expression levels of members of the ETS-transcription factors are often observed in many different tumors, including lymphomas. Here, we characterized two small molecules, YK-4-279 and its clinical derivative, TK-216, targeting ETS factors via blocking the protein–protein interaction with RNA helicases, for their antilymphoma activity. Experimental Design: The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination; validation experiments on in vivo models; and transcriptome and coimmunoprecipitation experiments. Results: YK-4-279 and TK-216 demonstrated an antitumor activity across several lymphoma cell lines, which we validated in vivo. We observed synergistic activity when YK-4-279 and TK-216 were combined with the BCL2 inhibitor venetoclax and with the immunomodulatory drug lenalidomide. YK-4-279 and TK-216 interfere with protein interactions of ETS family members SPIB, in activated B-cell–like type diffuse large B-cell lymphomas, and SPI1, in germinal center B-cell–type diffuse large B-cell lymphomas. Conclusions: The ETS inhibitor YK-4-279 and its clinical derivative TK-216 represent a new class of agents with in vitro and in vivo antitumor activity in lymphomas. Although their detailed mechanism of action needs to be fully defined, in DLBCL they might act by targeting subtype-specific essential transcription factors.

Background: The high variability in clinical responses observed in cancer patients highlights the need of a tailored therapeutic approach. A possible modality is to assess drugs sensitivity directly in the patients, by introducing small drug delivering devices in tumor sites for a very short period and then looking at the local anti-tumor effect. Here, we present the design of an innovative drug eluting device and its test with the BTK inhibitor ibrutinib as an example of small molecules. Methods: Mathematical models considered factors related to drug (MW), physical properties, desired concentrations in surrounding tissue, polymers and tissue physical features to identify the optimal polymers and the drug loading for the desired release profile over 24h. In vitro proliferation was measured with the MTT assay, in vivo experiments done in NOD-SCID mice (license TI05/2021), and immunohistochemistry on FFPE xenograft sections stained for Ki67 and cleaved caspase 3 (CASP-3). Results: Device was designed as an arrow-shaped cylinder, with flat end and flatter sections to be filled with the drug-eluting polymers. Prototypes were built in nylon6,6, a biocompatible but stable polymer. Ibrutinib was incorporated in low MW polyester poly-ε-caprolactone (PCL) as biopolymer by solvent casting. Polymeric coating onto devices was done with a dedicated automatic device.Devices loaded with biopolymer and different concentrations of ibrutinib or “empty” biopolymers were first in vitro tested using diffuse large B cell lymphoma (DLBCL) cell lines. Over 72h, devices with drug inhibited proliferation of the ibrutinib-sensitive TMD8 and OCI-Ly10 cell lines, but not of the ibrutinib-resistant SU-DHL-2 and U2932. No effect was seen with devices with ibrutinib-free biopolymers. Devices, empty or loaded with ibrutinib (5μg), were then inserted in xenografts of ibrutinib-sensitive cell line OCI-Ly10 and ibrutinib resistant U2932. After 24h, mice were sacrificed and xenografts analyzed. By Ki67 and CASP-3 a reduced cell proliferation and an increased apoptosis in the region surrounding the device was observed in the ibrutinib-sensitive xenografts, conversely nor reduced cell proliferation nor apoptosis induction were identified in the ibrutinib-resistant xenografts. Conclusions: We have created a prototype of a device that can locally release drugs allowing the evaluation of anti-tumor activity, optimizing cures tailored to single patient. The system can be further developed to include multiple drugs, including e.g. antibodies. Citation Format: Giuseppe Perale, Eugenio Gaudio, Tommaso Casalini, Luca Aresu, Anna Rita De Corso, Filippo Spriano, Chiara Tarantelli, Anastasios Stathis, Andrea Castrovinci, Francesco Bertoni. A novel implantable device to in vivo assess anti-cancer agents [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 1879.

Background. Novel therapeutic targets are needed to improve the outcome of individuals with cancer. EG-011 is a small molecule with in vitro and in vivo anti-tumor activity in lymphoma and acute leukemias, but, at least so far, no activity in solid tumor models (Gaudio et al AACR 2019). Since no information was available on its target, we have now performed experiments showing that EG-011 targets WASp. Methods. Target identification: kinase screens with DiscoverX KINOMEscan, ProQinase Wildtype-Profiler; thermal proteomic profiling. Target validation: pyrene actin polymerization assay. In vitro drug treatment of cell lines followed for changes in actin filaments (F-actin) by confocal imaging with Alexa Fluor 488 Phalloidin. Results. Extensive kinome screens excluded kinases as targets of EG-011. We then applied thermal proteomic profiling to identify new protein targets interacting with EG-011. Over 3,300 proteins from the soluble proteome from the EG-011 sensitive mantle cell lymphoma cell line REC1 were analyzed and 48 possible protein targets were initially identified. Among the proteins undergoing a thermal shift, WASp was among the most highly destabilized by EG-011. Due to the pattern of expression of WASp, compatible with the anti-tumor activity observed only in hematological cancers (Gaudio et al AACR 2019), we performed further experiments to confirm the possibility that EG-011 targets WASp. One of the main functions of WASp is the regulation of actin filaments formation. Pyrene actin polymerization assays demonstrated that EG-011 activated the auto-inhibited form of WASP with strong actin polymerization. Further confirmation was obtained using confocal imaging of cell lines exposed to DMSO or EG-011 (500 nM, 5 μM) and stained for F-actin. An increase in actine polymerization was seen in EG-011 sensitive (VL51) and not in resistant (Z138) cell lines at 4, 8 and 24h with both concentrations. Conclusions. These data demonstrate that EG-011 is the “first-in-class” activator of the auto-inhibited form of WASp with selective anti-tumor activity in lymphomas. Citation Format: Filippo Spriano, Laura Barnabei, Ana Maria Carrasco Del Amor, Meagan R. Tomasso, Chiara Tarantelli, Eugenia Riveiro, Natalina Pazzi, Shae B. Padrick, Susana Cristobal, Franco Cavalli, Eugenio Gaudio, Francesco Bertoni. EG-011 is a first-in-class Wiskott-Aldrich syndrome protein (WASp) activator with anti-tumor activity [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 1817.

Background: Avanbulin (BAL27862) is a microtubule-targeting agent (MTA) that promotes tumor cell death by modulating the spindle assembly checkpoint. Preclinical studies in glioblastoma (GBM) have shown that avanbulin is also active in human cancer cells resistant to other MTAs used in the clinic. Lisavanbulin (BAL101553) is a water-soluble, oral, lysine prodrug of avanbulin, currently in phase 1/2 for patients with recurrent GBM and, in combination with radiotherapy, for newly diagnosed GBM patients. Preclinical studies and clinical signals have also suggested an association between lisavanbulin activity and expression of microtubule plus-end binding protein (EB1). MTAs are active agents for lymphoma patients, as exemplified by the inclusion of vincristine in the R-CHOP regimen, standard treatment for diffuse large B cell lymphoma (DLBCL). Thus, our aim was to evaluate avanbulin, the active moiety of lisavanbulin, for its potential anti-lymphoma activity. Methods: Lymphoma cell lines derived from activated B cell like (ABC) and germinal center B cell type (GCB) DLBCL were exposed to increasing doses of avanbulin; cell proliferation was measured with MTT. Apoptosis induction was performed with annexin V staining on cells incubated in a live cell imaging system (Incucyte) treated with 5, 10, 20 or 40nM avanbulin. Images were taken every 4h for 72h. Cell cycle analysis was performed after 24, 48 and 72h of drug exposure at 20nM. Results: 26 DLBCL cell lines, treated for 72h with increasing avanbulin doses, showed high anti-proliferative activity of the compound, with a median IC50 = 11nM. No differences in terms of IC50 were observed comparing ABC (n=8) to GCB (n=18) DLBCLs, nor based on MYD88, TP53 and BCL2 status. Cell lines with MYC translocation were less sensitive than the ones without translocation. All lymphoma cell lines exhibited high EB1 RNA expression, with no correlation with sensitivity to avanbulin.Live cell imaging demonstrated a strong apoptosis induction in 15 out of 17 cell lines tested at 20 and 40nM. Among the 15 cell lines undergoing apoptosis, 8 cell lines already showed an apoptosis induction within 24h of drug exposure, 6 lines between 24 and 48h and 1 line after 48h exposure. These data confirmed the high activity of avanbulin already seen with the MTT assay.Cell cycle experiments performed at 24, 48 and 72h confirmed the strong cytotoxic effect of avanbulin in 4 DLBCL cell lines, including 2 ABC (OCILY3 and TMD8) and 2 GCB (SUDHL5 and SUDHL16) lines. Sub-G0 accumulation was already present at 24h in all 4 cell lines tested, with an increase over time. Especially at 24h, where we also observed a G2-M arrest in 3 out of 4 cell lines. Conclusions: Our data demonstrate the high cytotoxic anti-lymphoma activity of avanbulin, suggesting a potential activity of its prodrug lisavanbulin in lymphoma patients. Citation Format: Filippo Spriano, Luca Aresu, Luciano Cascione, Alberto J. Arribas, Nicole Forster-Gross, Felix Bachmann, Heidi Lane, Francesco Bertoni. Avanbulin, the active moiety of the tumor checkpoint controller lisavanbulin (BAL101553), has anti-lymphoma activity [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 2575.

Background. Marginal zone lymphoma (MZL) is a heterogeneous B-cell malignancy for which no standard treatment exists. B-cell receptor (BCR) signaling dysregulation is a hallmark of MZL and can mimic antigen dependent BCR activation. However, single treatment with BCR signaling inhibitors shows limited complete response rate and relapsing patients. IRAK4 is a protein kinase downstream the Toll-like receptor signaling. CA-4948 is the first-in-class inhibitor of IRAK4 and it is in phase 1/2 studies for patients with relapsed/refractory clinical studies with favorable activity in lymphomas and myeloid neoplasms. Here, we assessed IRAK4 inhibition with CA-4948 against a panel of MZL cell lines in which we have developed secondary resistance to inhibitors of PI3K (idelalisib, copanlisib) or BTK (ibrutinib). Methods. Models with secondary resistance were developed by continuous exposing cell lines derived from splenic MZL (Karpas 1718 and VL51) to high doses of drugs for long period of times. Resistant and parental lines were exposed to increasing doses of CA-4948, alone or in combination with compounds they are resistant to. Cell viability was tested by MTT assay (72h treatment). Synergy of combinations was evaluated according to the Chou-Talalay combination index (CI), as well as potency and efficacy, estimated according to the MuSyC algorithm. Results. The parental Karpas1718, bearing a MYD88 L265P mutation, had an IC50 of 3.29 µM for CA-4948 as single agent, while the parental VL51 and all resistant models presented IC50 values in the range of 19-35 µM. IRAK4 inhibition with CA-4948 had strong benefit when combined to downstream BCR inhibitors. In resistant cells, CA-4948 was strongly synergistic with idelalisib, ibrutinib and, at lesser extent, with copanlisib. CA-4948 in combination with ibrutinib was strongly synergistic especially in the VL51 ibrutinib resistant model compared to the parental one. CA-4948 (from 1 to 5 µM) recovered sensitivity to ibrutinib at IC50 values close to the parental condition. Similarly, CA-4948 in combination with idelalisib was also synergistic and increased sensitivity to idelalisib in idelalisib resistant VL51. Less benefit was seen adding CA-4948 to copanlisib: the combination was synergistic in VL51 parental cell, but with only weak efficacy. In the Karpas1718, CA-4948 was synergistic either in idelalisib resistant or in parental, with improved benefit in the latter, in which synergistic effect was observed when combined with both idelalisib and ibrutinib. The synergistic activity of CA-4948 was mainly due to improved efficacy rather than potency of the combinatorial partners. Conclusions. Our results in MZL cell lines show that the IRAK4 inhibitor CA-4948 is synergistic with small molecules targeting BCR signaling, especially with idelalisib and ibrutinib. Data also suggest that concentrations of CA-4948 comparable to those achieved in patients might overcome or reduce secondary resistance to the tested tyrosine kinase inhibitors. Citation Format: Francesca Guidetti, Alberto J. Arribas, Filippo Spriano, Laura Barnabei, Reinhard von Roemeling, Elizabeth Martinez, Emanuele Zucca, Francesco Bertoni. Pharmacological inhibition of IRAK4 with CA-4948 is beneficial in marginal zone lymphoma models with secondary resistance to PI3K and BTK inhibitors [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 P073.

Background: PI3Kδ is expressed in B cells and has a central role in the B-cell receptor signaling. Copanlisib is a highly selective PI3Kδ and PI3Kα inhibitor, and it is currently under clinical development in indolent lymphomas including marginal zone lymphoma (MZL). Copanlisib is Food and Drug Administration (FDA) approved for the treatment of patients with relapsed or refractory follicular lymphoma. Nevertheless, a subset of patients can eventually relapse due to acquired resistance. A better understanding of resistance mechanisms could help to design improved therapies; hence, we generated MZL cell lines resistant to copanlisib. Materials and Methods: Cells were kept on copanlisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 3 weeks of drug-free culture. Multidrug resistance phenotype was ruled out by confirming sensitivity to vincristine. Cells underwent transcriptome profiling (RNA-Seq) and immunophenotypic analysis. Results: The RES models were obtained from VL51 cell line with over 50-fold times higher IC50s than PAR counterparts. Of note, the copanlisib-resistant lines showed decreased sensitivity to other PI3K inhibitors such as duvelisib (50-fold) and idelalisib (5-fold) and to the BTK inhibitor ibrutinib (15-fold), suggesting that the mechanism observed here might drive resistance to other downstream B-cell receptor inhibitors. Gene expression profiles of RES showed the upregulation of cytokine signaling (IL1A, IL1B, CXCR4), NFkB (LTA, TNF), MAPK (RASGRP4, RASGRP2), and JAK-STAT (STAT3, JAK3) signaling pathways and negative regulators of apoptosis (CD44, JUN). Conversely, repressed genes in RES were involved in cell adhesion (ITGA4, ITGB1), antigen presentation (HLAs), and IFN response (PARP12, GBP6). Consistent with the overexpression of antiapoptotic signaling genes, RES cells exhibited also resistance to the BCL2-inhibitor venetoclax, either as a single as in combination with copanlisib. Flow cytometry confirmed the CXCR4 upregulation and the downregulation of CD49d (ITGA4), paired with reduced CD20 and CD81 surface expression. In accordance, addition of a CXCR4 inhibitor overcame resistance to copanlisib. Conclusions: We created a model of secondary resistance to the PI3K inhibitor copanlisib, derived from an MZL cell line. This model will help in clarifying mechanisms of resistance to the drug and to evaluate alternative therapeutic approaches. Indeed, we already identified novel potential targets, such as IL1 and CXCR4, that might be exploited in overcoming resistance to copanlisib and are worthy of further investigation. Citation Format: Alberto J. Arribas, Sara Napoli, Luciano Cascione, Eugenio Gaudio, Roberta Bordone-Pittau, Marilia Barreca, Giulio Sartori, Chiara Tarantelli, Filippo Spriano, Andrea Rinaldi, Anastasios Stathis, Georg Stussi, Davide Rossi, Emanuele Zucca, Francesco Bertoni. Mechanisms of resistance to the PI3K inhibitor copanlisib in marginal zone lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-46.

Background: Pracinostat (SB939) is a class I, II and IV histone deacetylase inhibitor (HDACi), currently in phase 3 in combination with azacitidine for acute myeloid leukemia (NCT03151408). We have reported its strong preclinical activity in lymphomas as a single agent and in combination with several targeted anti-lymphoma agents (AACR 2018, AACR 2019). Here we report the anti-proliferative activities of pracinostat and its primary metabolite SB991 as single agents and in combination with the demethylating agent 5-azacitidine in acute myeloid leukemia (AML) cell lines. Methods. To assess anti-proliferative activity cells were exposed to increasing doses of pracinostat, SB991 (Helsinn) and vorinostat (LC Laboratories) alone or in combination (pracinostat and SB991) with increasing doses of 5-azacitidine (Selleckchem) for 72h, followed by the MTT assay. The Chou-Talalay index was used to determine synergism. Combination indexes (CI) defined the following: additive effect (CI 0.9-1.1), synergism (CI 0.3-0.9) and antagonism (CI > 1.1).For apoptosis and cell cycle analyses cells were treated with 250 nM pracinostat for 72 hours. Results. HL-60 and MV-4-11 cells responded well to pracinostat with IC50 values of 362 nM and 93 nM, respectively. IC50 values obtained for SB991, the main metabolite of pracinostat, were 233 nM and 85 nM for HL60 and MV-4-11, respectively. These IC50 values compared well with those obtained for vorinostat, which equaled 710 nM for HL-60 and 205 nM for MV-4-11. Combination of pracinostat with 5-azacitidine showed synergism in both AML cell lines (CI = 0.51 for HL-60 and CI = 0.69 for MV-4-11). Combination of SB991 with 5-azacitidine achieved synergism in MV-4-11 cells (CI = 0.54) but was not beneficial in HL-60 cells (CI = 1.23). Pracinostat moderately induced apoptosis in HL-60 cells while MV-4-11 cells underwent massive apoptosis. Cell cycle analysis revealed marked G2/M arrest plus moderate accumulation of cells in the sub-G1 phase for HL-60 cells, which was in agreement with the moderate induction of apoptosis measured by Annexin V staining. Also in agreement with the apoptosis analysis, MV-4-11 cells treated with pracinostat all accumulated in the sub-G1 phase. Conclusions. Pracinostat and its main metabolite SB991 exhibited robust anti-proliferative activity as single agents in HL-60 and MV-4-11, comparing favourably with the FDA approved HDACi vorinostat. Pracinostat mediated its anti-proliferative action by inducing apoptosis and G2/M cell cycle arrest. Combination of pracinostat with 5-azacitidine enhanced its activity in AML cells. These results provide pre-clinical rationale for the ongoing phase 3 trials of pracinostat in combination with azacitidine in AML. Citation Format: Afua Adjeiwaa Mensah, Filippo Spriano, Eugenio Gaudio, Annalisa Bonifacio, Emanuela Lovati, Claudio Pietra, Francesco Bertoni. The histone deacetylase inhibitor pracinostat and its metabolite SB991 are active as single agents and in combination with 5-azacitidine in acute myeloid leukemia cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B009. doi:10.1158/1535-7163.TARG-19-B009

Introduction. Targeting the DNA repairing pathways represents an intriguing new approach for cancer treatment. BAY 1895344 is a potent, highly selective and orally available ATR inhibitor (Luecking et al, AACR 2017) in its early clinical development in patients with advanced solid tumors and lymphomas (NCT03188965). We performed evaluated its antitumor activity in a large panel of lymphoma cell lines. Methods. IC50s and caspase 3/7 activation were obtained in cell lines derived from human B (n=50) or T cell (n=9), murine (n=2) and canine (n=1) lymphomas exposed to increasing doses of BAY 1895344 for 72h. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation vs controls. Transcriptome data (Illumina HumanHT 12 Expression BeadChips, HTG Biomarker Panel) were analyzed with GSEA (statistical significance: absolute NES >1.5, FDR < 0.01). In vivo efficacy was evaluated in xenograft studies of human lymphoma in mice. Results. BAY 1895344 showed anti-tumor activity with a median IC50 of 60 nM (95%C.I.; 3-500 nM) across the 62 cell lines, comprising mainly germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL, n.=17), activated B-cell DLBCL (8), mantle cell lymphoma (MCL, 10), marginal zone lymphoma (6), T cell lymphomas (9). BAY 1895344 was mostly cytotoxic with apoptosis induction in 38/62 (61%) of the cell lines. Sensitivity was not affected by lymphoma histology or DLBCL cell of origin, TP53/BCL2/MYC status or ATM/ATR loss. A xenograft experiment (MCL Rec-1 cell line) demonstrated complete tumor remission with BAY 1895344 (50 mg/kg, twice daily, 3 days on and 4 days off). Comparing the gene expression profile of 4 less (IC50>200nM) vs 3 very sensitive (IC50 Conclusion. A strong anti-tumor activity, associated with specific gene expression signatures, was seen with BAY 1895344 in lymphoma models, providing further support for the on-going phase I study. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Filippo Spriano, Luciano Cascione, Alberto Arribas, Emanuele Zucca, Anastasios Stathis, Antje Margret Wengner, Francesco Bertoni. The ATR inhibitor BAY 1895344 shows strong preclinical activity in lymphomas and appears associated with specific gene expression signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 274.

Lymphoma cells have frequent deregulation of their epigenome. The Bromodomain (BRD) and Extra-Terminal domain (BET) proteins are key regulators of the transcription process. The acetyltransferases cyclic AMP response element binding protein (CREB)-binding protein (CBP) and the E1A interacting protein of 300 kDa (EP300) are highly homologous BRD-containing transcriptional co-activators and are often mutated in diffuse large B cell lymphoma (DLBCL). Targeting the individual classes of proteins is a new therapeutic approach, with BET inhibitors having both preclinical and early clinical anti-lymphoma activity. NEO2734 and NEO1132 are novel chemically distinct dual inhibitors of both BET and CREBBP/EP300 proteins with different affinity for their targets. Here, we present initial data exploring their anti-tumor activity in DLBCL models. Lymphoma cell lines were exposed to increasing doses of compounds for 72h followed by MTT assay. Twenty-seven DLBCL cell lines were exposed to NEO2734 and NEO1132. NEO2734 showed anti-tumor activity with a median IC50 of 157 nM (95% C.I., 135-214). While, NEO1132 showed a lower activity compared to NEO2734, with a median IC50 of 400nM (95% C.I., 316-622). For both compounds, cell lines derived from activated B-cell-like (ABC) DLBCL (n.=8) were more sensitive than those derived from germinal center B-cell (GCB) DLBCL (n.=19) (P=0.009, P=0.03 respectively). No differences were observed based on double hit MYC/BCL2 (yes, n.=6; no, n.=14), MYC (translocation: yes, n=8; no, n.=13), BCL2 (translocation: yes, n=12; no, n.=6), TP53 (inactive: yes, n=14; no, n.=6), CREBBP (mutated, n.=10; wild type, n=16), or EP300 (mutated, n.=5; wild type, n.=20) gene status. All the cell lines were also exposed to a BET inhibitor (birabresib, OTX015) (Boi et al, Clinical Cancer Res 2015) and to a CREBBP/EP300 inhibitor (CBP30) (Hammitzsch et al, PNAS 2015). The median IC50 values of the two molecules were 237 nM (95% C.I., 171-344) and 5.5 μM (95% C.I., 4.2-8.3 μM) respectively. The four compounds presented a similar pattern of anti-proliferative activity across all the cell lines (NEO2734 vs NEO1132 R2=0.98, P < 0.001; NEO2734 vs birabresib: R2=0.84, P < 0.001; NEO2734 vs CBP30, R2 = 0.73, P < 0.001; birabresib vs CBP30, R2 = 0.73, P < 0.001) but with different degrees of IC50. NEO2734 was significantly more potent than birabresib (P=0.0182), CBP30 (P Citation Format: Filippo Spriano, Eugenio Gaudio, Chiara Tarantelli, Gaetanina Golino, Luciano Cascione, Emanuele Zucca, Anastasios Stathis, Francis Giles, Francesco Bertoni. NEO1132 and NEO2734, novel dual bromodomain inhibitors of both BET and CREBBP/EP300, compared to single BET or CREBB/EP300 inhibitors in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3829.

Background: Pracinostat (SB939) is a class I, II and IV histone deacetylase inhibitor (HDACi), in phase 3 in combination with azacitidine for acute myeloid leukemia (NCT03151408). We reported a strong preclinical activity as a single agent in lymphomas (Mensah et al, AACR 2018). Interestingly, anti-tumor activity differed between metabolically-defined subtypes of diffuse large B-cell lymphoma (DLBCL): lower in OxPhos than in B cell receptor (BCR) cell lines. Thus, we now report RNA-Seq on OxPhos and BCR DLBCL cell lines treated with pracinostat. We also present results of a combination screening with 9 anti-lymphoma drugs for future clinical development of this HDACi. Methods: For RNA-Seq, OxPhos (Toledo, Pfeiffer, WSU-DLCL2) and BCR (SU-DHL-4, SU-DHL-6, OCI-LY-1) cell lines were treated with pracinostat or DMSO for 6 hours (h) or 14 days (d). Absolute fold change > 2 with adjusted P Combinations (72 h) with 5-azacytidine, ibrutinib, lenalidomide, bendamustine, everolimus, rituximab, idelalisib, bortezomib and copanlisib were evaluated on germinal center B-cell (GCB: SU-DHL-6, VAL) and activated B cell-like (ABC: OCI-LY-10, TMD8) DLBCL using the Chou-Talalay combination index (CI). Results: At transcriptome level, pracinostat determined a time-dependent modulation of proliferation and cell cycle genes with differences between OxPhos and BCR cell lines. The more sensitive BCR showed a higher number of modulated genes at both timepoints than the less sensitive OxPhos DLBCL. Moreover, while the upregulated transcripts increased with time in both metabolic clusters [doubled in BCR (1201 vs 591) and almost tripled in OxPhos (330 vs 120)], the opposite was true for downregulated genes in BCR (358 vs 600) but not in less sensitive OxPhos (186 vs 187). Combinations of pracinostat with ibrutinib [0.37 (0.01 - 0.73)] or lenalidomide [0.29 (0.11 - 0.46)] were synergistic in ABC. Idelalisib [0.67 (0.46 - 0.92)] and everolimus [0.78 (0.57 - 1.11)] were both effective in combination with pracinostat in all ABC and 1 GCB, while rituximab was synergistic in 1 ABC and all GCB [0.61 (0.32 - >1.1)]. Copanlisib [0.87 (0.45 - 0.94)] and 5-azacytidine [0.96 (0.64 - 1.14)] were additive with pracinostat in 3/4 cell lines and were synergistic in the remaining. Combination with bortezomib was beneficial in 3 cell lines [0.94 (0.5 - 1.53)] and bendamustine combined effectively with pracinostat in 2 of the 4 cell lines [1.1 (0.89 - 1.3)]. Conclusions: Modulation of the DLBCL transcriptome by pracinostat occurs already at 6 h, suggestive of a rapid effect of pracinostat on chromatin architecture, but it differs between OxPhos and BCR DLBCL. In terms of combinations, pracinostat addition to other targeted agents was largely beneficial. Citation Format: Afua A. Mensah, Filippo Spriano, Eugenio Gaudio, Chiara Chiara Tarantelli, Luciano Cascione, Andrea Rinaldi, Emanuela Lovati, Emanuele Zucca, Anastasios Stathis, Claudio Pietra, Francesco Bertoni. The histone deacetylase inhibitor pracinostat modulates the transcriptome of diffuse large B-cell lymphoma cells and is active in combination with several targeted agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4735.