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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

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

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.

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

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

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.

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.

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.

PurposeThe DNA damage response (DDR) is the cellular process devoted to the preservation of an intact genome. The DDR is often deregulated in lymphoma cells due to high levels of DNA damage, tumor suppressor inactivation, increased replication stress observed after oncogene activation, or high amounts of reactive oxygen species. The ataxia telangiectasia and Rad3-related (ATR) kinase is a crucial factor of DDR in the response to DNA single strand breaks. ATR inhibitors are a class of agents that have shown considerable clinical potential in this context.Experimental DesignWe characterized the activity of the ATR inhibitor elimusertib (BAY 1895344) in a panel of lymphoma cell lines. Furthermore, we evaluated the activity of elimusertib in combination with the clinically approved PI3K inhibitor copanlisib inin vitroandin vivolymphoma models.ResultsElimusertib exhibitedin vitroactivity across a variety of lymphoma subtypes which was associated with expression of genes related to replication stress. Elimusertib also demonstrated wide-spread anti-tumor activity that was stronger compared to ceralasertib, another ATR inhibitor, in several tumor models. This activity was present in both DDR-proficient and DDR-deficient lymphoma models. Furthermore, elimusertib had synergistic antitumor activity in combination with copanlisib.ConclusionsPotent antitumor activity of elimusertib was demonstrated in several lymphoma models which is associated with high expression of gene transcripts coding for proteins that are involved in DDR and cell cycle regulation. Combination of ATR and PI3K inhibition by treatment with elimusertib and copanlisib had synergistic efficacy providing a potential new treatment option for lymphoma patients.Translational relevanceThe DNA damage response (DDR) is often deregulated in lymphoma cells. Here, we characterized the activity of elimusertib, an inhibitor of the ataxia telangiectasia and Rad3-related kinase (ATR) that is involved in the DDR. Elimusertib was demonstrated to exhibit anti-lymphoma activity across several lymphoma cell lines and tumor models. Copanlisib is an inhibitor of PI3K kinase family which has also shown activity in various lymphomas. Combined treatment with elimusertib and copanlisib resulted in a synergistic antitumor effect in lymphoma models. The combination of elimusertib and copanlisib could potentially constitute a new chemotherapy-free treatment option for lymphomas.

Inhibitors of phosphatidylinositol 3-kinase (PI3K) and Bruton tyrosine kinase (BTK) represent a recognized option for the treatment of patients affected by indolent B cell lymphomas. However, small molecules as single agents show limited success in their ability in inducing complete responses, with only partial remission achieved in most patients, suggesting the need for combination therapies. IRAK4 is a protein kinase downstream of the Toll-like receptor signaling (TLR), a driver pathway of secondary tumor° resistance in both hematological and solid tumor malignancies. Activation of IRAK4 upon TLRs and IL-1 receptor (IL-1R) stimulation and through the adaptor protein MYD88 initiates a signaling cascade that induces cytokine and survival factor expression mediated by the transcription factor NF-κB. MYD88-L265P encoding mutations occur in diffuse large B-cell lymphomas, in lymphoplasmacytic lymphomas and in few marginal zone lymphomas (MZL). The IRAK4 inhibitor emavusertib (CA-4948) has shown early safety and clinical activity in lymphoma and leukemia patients. In this preclinical study, we assessed emavusertib effectiveness in MZL, both as single agent and in combination with targeted agents, with a particular focus on its capability to overcome resistance to BTK and PI3K inhibitors. We showed that the presence of MYD88 L265P mutation in bona fide MZL cell lines confers sensitivity to the IRAK4 inhibitor emavusertib as single agent. Emavusertib-based combinations improved the sensitivity of MZL cells to BTK and PI3K inhibitors, including cells with a secondary resistance to these agents. Emavusertib exerted its activity via inhibition of NF-κB signaling and induction of apoptosis. Considering the early safety data from clinical trials, our study identifies the IRAK4 inhibitor emavusertib as a novel compound to be explored in trials for patients with MYD88-mutated indolent B cell lymphomas as single agent and as combination partner with BTK or PI3K inhibitors in unselected populations of patients.

Microtubules are major components of the cellular cytoskeleton, ubiquitously founded in all eukaryotic cells. They are involved in mitosis, cell motility, intracellular protein and organelle transport, and maintenance of cytoskeletal shape. Avanbulin (BAL27862) is a microtubule-targeted agent (MTA) that promotes tumor cell death by destabilization of microtubules. Due to its unique binding to the colchicine site of tubulin, differently from other MTAs, avanbulin has previously shown activity in solid tumor cell lines. Its prodrug, lisavanbulin (BAL101553), has shown early signs of clinical activity, especially in tumors with high EB1 expression. Here, we assessed the preclinical anti-tumor activity of avanbulin in diffuse large B cell lymphoma (DLBCL) and the pattern of expression of EB1 in DLBCL cell lines and clinical specimens.Avanbulin showed a potentin vitroanti-lymphoma activity, which was mainly cytotoxic with potent and rapid apoptosis induction. Median IC50 was around 10nM in both activated B cell like (ABC) and germinal center B cell like (GCB) DLBCL. Half of the cell lines tested showed an induction of apoptosis already in the first 24h of treatment, the other half in the first 48h. EB1 showed expression in DLBCL clinical specimens, opening the possibility for a cohort of patients that could potentially benefit from treatment with lisavanbulin.These data show the basis for further preclinical and clinical evaluation of lisavanbulin in the lymphoma field.

BTK and PI3K inhibitors are among the drugs approved for the treatment of patients with lymphoid neoplasms. Although active, their ability to lead as single agents to long-lasting complete remission is rather limited especially in the lymphoma setting. This indicates that tumor cells often develop resistance to the drugs. Here, we show that the overexpression of ERBB4 and its ligands represents a modality for B cell neoplastic cells to bypass the anti-tumor activity of BTK and PI3K inhibitors and that targeted pharmacological interventions can restore sensitivity to the small molecules.We started from a marginal zone lymphoma (MZL) cell line, Karpas-1718, kept under prolonged exposure to the PI3Kδ inhibitor idelalisib until acquisition of resistance, or with no drug. Cells underwent transcriptome, miRNA and methylation profiling, whole exome sequencing, and pharmacological screening which led to the identification of the overexpression of ERBB4 and its ligands HBEGF and NRG2 in the resistant cells. Cellular and genetic experiments demonstrated the involvement of this axis in blocking the anti-tumor activity of various BTK and PI3K inhibitors, currently used in the clinical setting. Addition of recombinant HBEGF induced resistance to BTK and PI3K inhibitors in parental cells but also in additional lymphoma models. Combination with the ERBB inhibitor lapatinib was beneficial in resistant cells and in other lymphoma models already expressing the identified resistance factors. Multi-omics analysis underlined that an epigenetic reprogramming affected the expression of the resistance-related factors, and pretreatment with demethylating agents or EZH2 inhibitors overcame the resistance. Resistance factors were shown to be expressed in clinical samples, further extending the findings of the study.In conclusions, we identified a novel ERBB4-driven mechanism of resistance to BTK and PI3K inhibitors and treatments that appear to overcome it.Key pointsA mechanism of secondary resistance to the PI3Kδ and BTK inhibitors in B cell neoplasms driven by secreted factors.Resistance can be reverted by targeting ERBB signaling.

We synthesized a new inhibitor of tubulin polymerization, the pyrrole (1-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrrol-3-yl)(3,4,5-trimethoxy-phenyl)methanone 6 (RS6077). Compound 6 inhibited the growth of multiple cancer cell lines, with IC

Hematological cancers are among the most common cancers in adults and in children. Despite significant improvements in therapies, many patients still succumb to the disease, therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family proteins regulate actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations, auto-inhibited and active conformations. Here, we describe the development of EG-011, a first-in-class small molecule activator of the WASp auto-inhibited form. EG-011 possesses in vitro and in vivo anti-tumor activity as single agent in lymphoma, leukemia and multiple myeloma, including models of secondary resistance to PI3K, BTK and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization induced by EG-011 was demonstrated with multiple techniques. Transcriptome analysis highlighted homology with drugs inducing actin polymerization.Key pointsEG-011 is a novel small molecule with anti-tumor activity in hematological cancers, including resistant lymphoma and multiple myeloma modelsEG-011 is a first-in-class small molecule activator of the auto-inhibited form of the Wiskott-Aldrich syndrome protein (WASp)

Histone deacetylase inhibitors (HDACis) are antitumor agents with distinct efficacy in hematologic tumors. Pracinostat is a pan-HDACi with promising early clinical activity. However, similar to other HDACis, its activity as a single agent is limited. Diffuse large B-cell lymphoma (DLBCL) includes distinct molecular subsets or metabolically defined subtypes that rely in different ways on the B-cell receptor signaling pathway, oxidative phosphorylation, and glycolysis for their survival. The antitumor activity of pracinostat has not been determined in lymphomas. We performed preclinical in vitro activity screening of 60 lymphoma cell lines that included 25 DLBCLs. DLBCL cells belonging to distinct metabolic subtypes were treated with HDACis for 6 hours or 14 days followed by transcriptional profiling. DLBCL xenograft models enabled assessment of the in vivo antilymphoma activity of pracinostat. Combination treatments with pracinostat plus 10 other antilymphoma agents were performed. Western blot was used to assess acetylation levels of histone and nonhistone proteins after HDACi treatment. Robust antiproliferative activity was observed across all lymphoma histotypes represented. Focusing on DLBCL, we identified a low-sensitivity subset that almost exclusively consists of the oxidative phosphorylation (OxPhos)-DLBCL metabolic subtype. OxPhos-DLBCL cells also showed poorer sensitivity to other HDACis, including vorinostat. Transcriptomic analysis revealed fewer modulated transcripts but an enrichment of antioxidant pathway genes after HDACi treatment of OxPhos-DLBCLs compared with high-sensitivity B-cell receptor (BCR)–DLBCLs. Pharmacologic inhibition of antioxidant production rescued sensitivity of OxPhos-DLBCLs to pracinostat whereas BCR-DLBCLs were unaffected. Our study provides novel insights into the antilymphoma activity of pracinostat and identifies a differential response of DLBCL metabolic subtypes to HDACis.

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.

Antibody drug conjugates represent an important class of anti-cancer drugs in both solid tumors and hematological cancers. Here, we report preclinical data on the anti-tumor activity of the first-in-class antibody drug conjugate MEN1309/OBT076 targeting CD205. The 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. CD205 was first shown frequently expressed in lymphomas, leukemias and multiple myeloma by immunohistochemistry on tissue microarrays. Anti-tumor activity of MEN1309/OBT076 as single agent was then shown across 42 B-cell lymphoma cell lines with a median IC50 of 200 pM and induction of apoptosis in 25/42 (59.5%) of the cases. The activity appeared highly correlated with its target expression. After in vivo validation as the single agent, the antibody drug conjugate synergized with the BCL2 inhibitor venetoclax, and the anti-CD20 monoclonal antibody rituximab. The first-in-class antibody drug targeting CD205, MEN1309/OBT076, demonstrated strong pre-clinical anti-tumor activity in lymphoma, warranting further investigations as a single agent and in combination.

PI3Kδ inhibitors are active in patients with lymphoid neoplasms and a first series of them have been approved for the treatment of multiple types of B-cell lymphoid tumors, including marginal zone lymphoma (MZL). The identification of the mechanisms underlying either primary or secondary resistance is fundamental to optimize the use of novel drugs. Here we present a model of secondary resistance to PI3Kδ inhibitors obtained by prolonged exposure of a splenic MZL cell line to idelalisib. The VL51 cell line was kept under continuous exposure to idelalisib. The study included detailed characterization of the model, pharmacological screens, silencing experiments, and validation experiments on multiple cell lines and on clinical specimens. VL51 developed resistance to idelalisib, copanlisib, duvelisib, and umbralisib. An integrative analysis of transcriptome and methylation data highlighted an enrichment of upregulated transcripts and low-methylated promoters in resistant cells, including IL-6/STAT3- and PDGFRA-related genes and surface CD19 expression, alongside the repression of the let-7 family of miRNA, and miR-125, miR-130, miR-193 and miR-20. The IL-6R blocking antibody tocilizumab, the STAT3 inhibitor stattic, the LIN28 inhibitor LIN1632, the PDGFR inhibitor masitinib and the anti-CD19 antibody drug conjugate loncastuximab tesirine were active compounds in the resistant cells as single agents and/or in combination with PI3Kδ inhibition. Findings were validated on additional in vitro lymphoma models and on clinical specimens. A novel model of resistance obtained from splenic MZL allowed the identification of therapeutic approaches able to improve the antitumor activity of PI3Kδ inhibitors in B-cell lymphoid tumors.

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.

Antibody-drug conjugates (ADC) are a novel way to deliver potent cytotoxic compounds to cells expressing a specific antigen. Four ADC targeting CD19, including SAR3419 (coltuximab ravtansine), have entered clinical development. Here, we present huB4-DGN462, a novel ADC based on the SAR3419 anti-CD19 antibody linked via sulfo-SPDB to the potent DNA-alkylating agent DGN462. huB4-DGN462 had improved in vitro anti-proliferative and cytotoxic activity compared to SAR3419 across multiple B-cell lymphoma and human acute lymphoblastic leukemia cell lines. In vivo experiments using lymphoma xenografts models confirmed the in vitro data. The response of B-cell lymphoma lines to huB4-DGN462 was not correlated with CD19 expression, the presence of BCL2 or MYC translocations, TP53 inactivation or lymphoma histology. In conclusion, huB4-DGN462 is an attractive candidate for clinical investigation in patients with B-cell malignancies.

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.

Driver genes with a mutually exclusive mutation pattern across tumor genomes are thought to have overlapping roles in tumorigenesis. In contrast, we show here that mutually exclusive prostate cancer driver alterations involving the ERG transcription factor and the ubiquitin ligase adaptor SPOP are synthetic sick. At the molecular level, the incompatible cancer pathways are driven by opposing functions in SPOP. ERG upregulates wild type SPOP to dampen androgen receptor (AR) signaling and sustain ERG activity through degradation of the bromodomain histone reader ZMYND11. Conversely, SPOP-mutant tumors stabilize ZMYND11 to repress ERG-function and enable oncogenic androgen receptor signaling. This dichotomy regulates the response to therapeutic interventions in the AR pathway. While mutant SPOP renders tumor cells susceptible to androgen deprivation therapies, ERG promotes sensitivity to high-dose androgen therapy and pharmacological inhibition of wild type SPOP. More generally, these results define a distinct class of antagonistic cancer drivers and a blueprint toward their therapeutic exploitation., Nature Communications, 12, ISSN:2041-1723

The marine environment is a rich source of biologically active molecules for the treatment of human diseases, especially cancer. The adaptation to unique environmental conditions led marine organisms to evolve di erent pathways than their terrestrial counterparts, thus producing unique chemicals with a broad diversity and complexity. So far, more than 36,000 compounds have been isolated from marine micro- and macro-organisms including but not limited to fungi, bacteria, microalgae, macroalgae, sponges, corals, mollusks and tunicates, with hundreds of new marine natural products (MNPs) being discovered every year.Marine-based pharmaceuticals have started to impactmodern pharmacology and different anti-cancer drugs derived frommarine compounds have been approved for clinical use, such as: cytarabine, vidarabine, nelarabine (prodrug of ara-G), fludarabine phosphate (pro-drug of ara-A), trabectedin, eribulin mesylate, brentuximab vedotin, polatuzumab vedotin, enfortumab vedotin, belantamab mafodotin, plitidepsin, and lurbinectedin. This review focuses on the bioactive molecules derived from the marine environment with anticancer activity, discussing their families, origin, structural features and therapeutic use., This publication is based upon work from COST Action CA18238 (Ocean4Biotech), supported by COST (European Cooperation in Science and Technology) programme. This work was partially supported by the Applied Molecular Biosciences Unit-UCIBIO (UID/Multi/04378/2019), financed by national funds from FCT/MCTES. Additionally, support was received from the FCT/MCTES through grant IF/00700/2014 (to SPG); the Slovenian Research Agency research core funding P1-0245 (to AR); TÜB˙ITAK grant number 216Z167 (to AE); the Ministerio de Ciencia e Innovación (grant RTA 2015-00010-C03-02) (to MC); the Agustin de Betancourt Programme (Cabildo de Tenerife, TFinnova Programme supported by MEDI and FDCAN funds) (to ARDM); the MetaboCell project of Canceropôle Provence-Alpes-Côte d’Azur and the Provence-Alpes-Côte d’Azur Region (to MM). This project (to OPT, Grant-Aid Agreements No. PBA/MB/16/01 and PDOC/19/02/01) was carried out with the support of the Marine Institute and funded under the Marine Research Programme by the Irish Government.