Chou-Talalay Combination Index Research Articles

Mycophenolic Acid Has Intrinsic Antiviral Properties and Potentiates the Antiviral Effect of Ribavirin on Parainfluenza Virus

Respiratory tract infection with parainfluenza virus (PIV) is increasingly associated with chronic lung allograft dysfunction in lung transplant recipients (LTR). We evaluated the antiviral properties of ribavirin (RBV) and mycophenolic acid (MPA) on PIV, since MPA often is standard immunosuppression in LTR and therefore a clinically prevalent combination in LTR treated for PIV infection. Antiviral assays with RBV and MPA on PIV-3 were conducted on LLC-MK2 cells. Interactions between the compounds from these experiments were evaluated using Loewe interaction analysis and Chou-Talalay combination index. Next, primary human airway epithelial cells cultured at an air-liquid interface system (ALI-system) were infected with PIV-3 at the apical site and treated at the basal site with either RBV at 30 µM or 100 µM (day +1 until +11, i.e. a therapeutic regimen) or MPA at 0.33 µM (day -1 until +11, i.e. prophylactic start of treatment) to mimic the "real world" scenario in LTR (apical infection + systemic treatment). Apical washes were collected for 20 consecutive days and viral RNA was quantified using qPCR. Median 50% effective concentrations to inhibit viral replication (EC50) of RBV and MPA were 14 (IQR 2.4) µM and 0.26 (IQR 0.20) µM in the PIV-3/LLC-MK2 system. Additivity of the two compounds was indicated by both Loewe analysis and a combination index of 0.67 (SD 0.15). In the ALI-system RBV (30µM, = 7.3 mg/L in plasma) or MPA (0.33µM, = 3.5mg/L) alone had only a marginal effect on viral replication, but when combined a more pronounced inhibitory effect was observed during the treatment period (Fig 1A). This combined effect was even more pronounced when the RBV concentration was increased to 100µM (=24mg/L, Fig 1B). MPA at clinically relevant concentrations potentiates the antiviral activity of RBV in vitro. The balance of the immunosuppressive- and antiviral effects of MPA and the combination with RBV in LTR with PIV remains to be established.

In silico Prediction Followed By in Vitro validation Identifies the Survivin Inhibitor YM155 As a Potent Secondary Drug Against Pi-Resistant Myeloma

Drug resistance is a major cause of concern in cancer chemotherapy that not only reduces efficacy but also causes toxicity through overdosing. Multiple myeloma (MM) is the second-most common haematological malignancy in the USA that remains a challenging disease to cure due to the presence of significant complexity and heterogeneity at molecular level with high treatment cost. Proteasome inhibitor, the standard of care drug, has proven to remarkably improve myeloma patient outcomes but is associated with dose limiting toxicities and drug resistance. Therefore, new combination treatment regimens are urgently needed for the treatment of PI-resistant MM that can lower the required dose of standard-of-care drug without compromising on treatment efficacy. We have created an optimization-regularization based computational prediction method called secDrug that uses large-scale pharmacogenomics databases to identify novel secondary drugs against PI-resistance when used as single agent or in combination. We applied secDrug to PIs-resistant cell lines (>100) in the Genomics of Drug Sensitivity in Cancer (GDSC) database and predicted the top drugs that can be best combined with PIs to overcome PI-resistance in MM. These include: Survivin inhibitor (YM155), Nicotinamide phospho ribosyl transferase or Nampt inhibitor (FK866), PIKfyve inhibitor (YM201636), Raf inhibitor (PLX-4720), Bcl2 inhibitor (Navitoclax),HSP90 inhibitor (17-AAG), AKT inhibitor (KIN00102), HDAC inhibitors (Panobinostat, SAHA), S6K1-specific inhibitor (PF-4708671), and the neddylation inhibitor (MLN4924). To validate our prediction results, we treated human multiple myeloma cell lines (HMCLs) highly resistant to the proteasome inhibitors Bortezomib, Carfilzomob, Oprozomib and Ixazomib and >10 clonally derived acquired resistant cells with the top predicted best secondary drugs. Next, we treated three pairs of parental PI-sensitive (P) and clonally-derived PI-resistant (R) cell lines from PI-sensitive (P) HMCLs representing acquired PI-response with the top predicted drug combinations. Our preliminary in vitro data confirmed our in silico predictions of secondary drugs based on secDrug analysis. Among these, YM155 and YM155 + Ixazomib showed strikingly very high in vitro cytotoxicity against PI- resistant MM cell lines (Figure 1). The results, based on Chou-Talalay's combination index (CI) theorem, show that this combination works highly synergistically where the IC50 of ixazomib in MM cells was significantly reduced in presence of YM155. Similar results were obtained for the other top predicted combination regimens. Our findings provide a strong case for combining YM155 with ixazomib to enhance sensitivity or overcome resistance to ixazomib and thereby improve patient outcome. Further, this study provides additional support towards using secDrug for predicting secondary drugs in cancers resistant to the standard-of-care therapy. Currently, we are exploring the molecular mechanisms behind the synergism between proteasome inhibitors and the top drugs identified by our algorithm as candidates for secondary combination therapies using next-gen transcriptomics and single-cell analysis approaches. Earlier studies have shown that YM155 potentially induces selective cell death in human pluripotent stem cells. Further, the presence of cancer stem cells sub population/ MM-CSCs in drug-resistant tumors have been shown to significantly contribute to the development of PI-resistance in MM. Hence, we propose that YM155 reverses PI-resistance by reducing the stem cell load in multiple myeloma cell line model system. Therefore, our future plan is to assess the effects of YM155 on MM-cancer stem cells like subclones/ sub population/ MM-CSCs including CD19- CD138- quiescent stem cells, ALDH+ and side populations/SP) in drug-resistant tumors. Overall, our in vitro validation results corroborated with our in silico prediction of secondary drugs. Disclosures No relevant conflicts of interest to declare.

Repurposing Clofazimine As a Novel Drug for the Treatment of PI-Resistant Stem Cell-like Subclones in Myeloma

Multiple myeloma (MM) is the second-most common hematopoietic malignancy in the United States with estimated new reported cases of 32,110 and deaths of 12,960 in 2019. Despite recent improvement in therapies, including proteasome inhibitors (PIs) - the standard-of-care drug, MM still remains a difficult-to-cure disease with significant complexity and heterogeneity at the molecular level. Recent studies have shown that the presence of cell sub populations with potential cancer stem cell-like properties (MM-CSCs), including CD19- CD138- quiescent stem cells, dormant cells, ALDH+ cells and side populations/SP, in drug-resistant tumors significantly contribute to the development of PI resistance in myeloma. However, no study so far has attempted to develop drugs specifically targeting MM-CSCs involved in drug resistance. Further, the gene signature underlying these sub cellular populations are yet to be revealed. We have previously demonstrated that Clofazimine (CFZ), an anti-leprosy drug, could be successfully repurposed for the treatment of Chronic myeloid leukemia (CML) by specifically targeting quiescent stem cell population (CD34+CD38-, CFSE-bright) in drug resistant patient samples. Here, we hypothesized that we will repurpose CFZ as a novel drug for the treatment of PI-resistant MM by targeting the sub-cellular stem cell-like subclones. Therefore, in the current study we utilized our panel of >50 human myeloma cell lines (HMCLs) representing innate PI-response/resistance and >10 pairs (parental and PI-resistant lines generated using dose escalation over a period of time) of clonally derived PI-resistant HMCLs representing acquired resistance to repurpose CFZ for the management of PI-resistant MM. Cells were treated with CFZ and the PIs Bortezomib (Bz), Carfilzomib (Cz), Ixazomib (Ix) and Oprozomib (Opz) as single agents or in combination and in vitro cytotoxicity was determined using CellTiter-Glo assay. Synergy between CFZ and PIs was analyzed by Calcusyn software based on Chou-Talalay's combination index (CI) theorem. Drug-induced apoptosis, ROS generation, mitochondrial membrane potential and cell cycle arrest were evaluated by Flow cytometry. Sub-cellular stem cell-like populations were isolated using cell sorting by Flow Cytometry (FCM/FACS). Tumorigenic potential of purified MM-CSCs was determined using colony forming assay, carboxyfluorescein succinimidyl ester (CFSE) assay, Aldeflour activity and analysis of side population in resistant MM cells. We found very promising preliminary results where CFZ alone showed very potent inhibition of cell viability in HMCLs. Further, treatment with CFZ + PIs significantly improved the therapeutic index of PI administration to the cells. Similar results were observed in parental (P) vs clonally derived PI-resistant (VR) cell lines. We next evaluated if CFZ alone or in combination with PIs could erode quiescent CD138+ cells in parental-P/clonally derived PI-resistant cells-VR. Hence, we labelled RPMI-8226 (parental cells), RPMI8226-VR with CFSE and treated them with drugs for 96h. While PIs failed to reduce CFSE-bright (non-dividing) cells, CFZ alone or in combination with Ixazomib significantly reduced their number and increased CFSE-dim (dividing cell) population. Evaluation of apoptosis in these cells revealed that CFZ alone caused apoptosis in both CFSE-bright and CFSE-dim cells while combining CFZ +PI caused a more robust effect amounting to their near-obliteration. We next assessed side populations/SP in resistant cells. We found that % SP was higher in resistant cells as compared to parental cells. CFZ alone or in combination (CFZ+PI) reduced %SP in PI- resistant cell lines. Based on these results, we propose that CFZ may have strong potential to increase the therapeutic efficacy of PIs when used in combination by specifically targeting MM-CSCs sub cellular population in MM - which we are investigating further. Currently, we are: 1) validating the potency of CFZ (alone or CFZ+PI combination therapy) in primary myeloma cells/PMCs from newly diagnosed patients, followed by 2) bulk mRNA sequencing and 3) single-cell transcriptomic analysis of MM-CSCs in HMCLs and PMCs to evaluate molecular pathways involved in stem-cell based PI-resistance and to characterize PI-resistant sub-cellular stem cell populations based on gene expression signatures using our prediction analysis software SCATTome. Disclosures No relevant conflicts of interest to declare.

Radiosensitization and Micronucleus Formation are Induced by Centrosome Clustering Inhibition with Griseofulvin in Prostate Cancer Cell Lines

Ionizing radiation (IR) leads to micronucleus-dependent immunogenic signaling through cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and type I interferon. Inhibition of supernumerary centrosome clustering in tumor cells with griseofulvin can increase micronucleus formation and mitotic cell death but has not been studied in prostate cancer. We hypothesize that co-administration of IR and centrosome clustering inhibition (CCi) will radiosensitize prostate cancer cells and increase cGAS signaling, a potential avenue for enhancing the interplay between radiotherapy and immunotherapy. Murine Myc-CaP and human PC3 and 22rv1 prostate cancer cells were grown in culture using standard techniques. CCi was achieved with griseofulvin. Clonogenic survival was assessed across a range of griseofulvin (3-30 μM) and IR (1-12 Gy) doses, both as single interventions and combination treatments. Micronuclei (MN) were visualized and quantified by DAPI staining. Bipolar and multipolar spindles were identified by immunofluorescence against alpha-tubulin and pericentrin. In untreated Myc-CaP cells, MN were identified in 2.8% of cells. Griseofulvin treatment resulted in dose-dependent increases in MN, seen in 15% of cells after 30 μM treatment. IR also resulted in dose-dependent MN formation, with 4 Gy inducing MN in 24% of cells. Similar effects were observed in PC3 and 22rv1 cells, with 30 μM griseofulvin inducing MN in 5.2% and 5.8% of cells, while 9 Gy IR induced MN in 7.4% and 11.7%. After treatment 30 μM griseofulvin, multipolar spindles were identified in 31.3% of Myc-Cap, 10% of PC3, and 18.5% of 22rv1 cells, as compared to 4.6%, 4%, and 0% in untreated cells. Pretreatment of Myc-CaP cells with 30 μM griseofulvin prior to 3 Gy IR resulted in supra-additive decreases in clonogenic survival with a Chou-Talalay combination index (CI) of 0.69. In PC3 and 22rv1 cells, 30 μM griseofulvin enhanced 1 Gy IR with CI of 0.25 and 0.31, respectively. Griseofulvin and IR induce dose-dependent micronucleus formation in murine and human prostate cancer cell lines, and griseofulvin also promotes multipolar mitosis. Treatment with griseofulvin prior to IR decreases clonogenic survival across tested cell lines. These data suggest that combination treatment with griseofulvin and IR may improve local responses to radiotherapy and, through micronucleus-dependent immunogenic signaling, lead to more effective abscopal response generation in metastatic prostate cancer. Immediate future directions include investigation of cGAS/STING/interferon signaling axis and evaluation in an immune competent syngeneic mouse model with concurrent immune checkpoint inhibition.

Abstract 4735: 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 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 <0.01 were used as thresholds. 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.

Abstract 4796: EG-011 is a novel small molecule with in vitro and in vivo anti-tumor activity against lymphoma

Abstract Introduction: Despite the improvements, still too many patients die for their lymphomas and novel compounds are needed. We present a new small molecule, EG-011 (PCT/EP2018/057678), with in vitro and in vivo anti-cancer activity in lymphoma models. Methods: Lymphoma and solid tumor cell lines were exposed to a large range of concentrations of EG-011 as single agent for 72h, followed by MTT proliferation assay and IC50 calculation. Cell viability of twelve acute lymphoblastic leukemia (ALL) primary patient cells from different high-risk subgroups (VNN2+, E2A-HLF, refractory T and IKZF plus) co-culture with marrow-derived MSCs were assayed after 72h of incubation with EG-011 and controls. Apoptosis assay was measured with annexin V by FACS. Xenografts were established s.c. into the left flanks of female NOD-SCID mice; treatment (200 mg/kg, i.p. 5 days per week) started with already established tumors. Combinations were evaluated with Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1) after 72 hr treatments. Results: EG-011 presented a median IC50 of 2.25 μM in 62 lymphoma cell lines (95% C.I. 1-5μM). A higher activity was observed in a group of 21 cell lines that had a median IC50 of 250 nM (95% C.I. 40-600 nM). Among these there were 11 germinal center B cell (GCB) diffuse large B cell lymphomas (DLBCL) (sensitive n=11/21, resistant n=9/41, P < 0.05), 4 mantle cell lymphoma (MCL) (sensitive n=4/21, resistant n=6/41, P n.s.), 3 marginal zone lymphoma (sensitive n=3/21, resistant n=2/41, P n.s.). EG-011 did not show any anti-proliferative activity in a panel of 25 solid tumor cell lines (IC50s > 10 μM), Among 12 primary ALL samples, 7 were sensitive to EG-011 with IC50 values between 0.3-4.6 µM after 72h, 5 displayed IC50 higher than 20 µM. A dose-dependent increase in cell death (20-55%) was observed in lymphoma cell lines (OCI-LY-19 and REC1) (500 nM and 2 μM; 72h). No cytotoxicity was seen in PBMCs from two healthy donors after treatment at 1 and 10 μM for 24h and 48h. In an in vivo xenograft experiment with the MCL REC-1 cell line, EG-011 delayed tumor growth (Day 6, Day 7, Day 9, P < 0.05) and tumor weight. EG-011-treated tumors were 2.2-fold smaller than controls (P < 0.001). Combinations were tested in DLBCL (OCI-LY-1, OCI-LY-8, TMD8) and MCL (REC1, MINO). EG-011 was synergistic with rituximab, bendamustine, venetoclax, ibrutinib and lenalidomide in all tested cell lines. Conclusion: The selective anti-lymphoma activity, in both in vitro and in vivo models, and the observed in vitro synergisms with FDA approved targeted agents make EG-011 a novel intriguing new drug candidate deserving further preclinical studies. Citation Format: Eugenio Gaudio, Filippo Spriano, Chiara Tarantelli, Matilde Guala, Eugenia Riveiro, Gaetanina Golino, Antonio Lupia, Giosuè Costa, Roberta Rocca, Luciano Cascione, Silvia Jenni, Yi-Chien Tsai, Beat Bornhauser, Stefano Alcaro, Francesco Paduano, Francesco Trapasso, Emanuele Zucca, Anastasios Stathis, Natalina Pazzi, Franco Cavalli, Francesco Bertoni. EG-011 is a novel small molecule with in vitro and in vivo anti-tumor activity against 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 4796.

Abstract 2661: Targeting endoplasmic reticulum-resident proteins for the treatment of B cell cancer

Abstract IRE-1 splices XBP-1 mRNAs, leading to activation of the functional XBP-1 transcription factor. We showed that genetic deletion of XBP-1 in chronic lymphocytic leukemia (CLL) cells decelerated malignant progression of CLL in mice. We synthesized and characterized a specific inhibitor, B-I09, which could block the RNase activity of IRE-1 with high potency and efficacy. B-I09 clearly suppressed activation of the IRE-1/XBP-1 pathway, as evidenced by the decreased mRNA and protein levels of XBP-1 in intact cells. B-I09 specifically targeted mouse CLL cells in vivo by inducing apoptosis. Because XBP-1 deficiency could compromise the BCR signaling, a crucial survival signal for CLL, we tested whether pharmacological inhibition of XBP-1 could enhance the effect of inhibitors of the BCR signaling by combining B-I09 with ibrutinib (a Bruton’s tyrosine kinase inhibitor) to treat human CLL cells. A strong pharmacological synergism was determined using the Chou-Talalay combination index method, suggesting that B-I09 could help ibrutinib to achieve higher cytotoxicity at a lower dose, addressing ibrutinib’s toxicity issue. Our studies also led us to discover that IRE-1 interacted with STING, an ER-resident protein critical for cytoplasmic DNA sensing and interferon production. We showed that the IRE-1/XBP-1 pathway was required for the interferon-producing function of STING, and that agonists of STING selectively triggered mitochondria-mediated apoptosis in malignant B cells. Upon stimulation, STING was degraded inefficiently in malignant B cells, implying that prolonged activation of STING could lead to apoptosis. In CLL-bearing mice, injection of the STING agonist, 3'3'-cGAMP, induced apoptosis and regression of leukemia. Similarly efficacious effects were elicited by 3'3'-cGAMP injection in syngeneic or immunodeficient NSG mice grafted with malignant B cells. These data suggested that STING agonists could directly eradicate CLL and other B cell malignancies in vivo. IRE-1 and STING are thus useful ER-resident protein targets for the treatment of B cell cancer. Citation Format: Chih-Hang Anthony Tang, Juan R. Del Valle, Chih-Chi Andrew Hu. Targeting endoplasmic reticulum-resident proteins for the treatment of B cell cancer [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 2661.

COPANLISIB SYNERGIES WITH CONVENTIONAL AND TARGETED AGENTS INCLUDING VENETOCLAX IN PRECLINICAL MODELS OF B‐ AND T‐CELL LYMPHOMAS

Introduction: Copanlisib has shown activity in lymphomas, but the number of patients achieving complete remission is relatively low, a problem shared by many targeted agents. We performed a pharmacological screening to identify active combinations. Methods: We tested copanlisib as single agent and in combination with other molecules in cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and T cell lymphomas (T-NHLs). Synergism was evaluated with Chou-Talalay combination index, as previously done (Tarantelli et al, CCR 2018). Results: Copanlisib showed an in vitro dose-dependent antitumor activity in the vast majority of the models with a median IC50 of 75 nM (95% C.I., 21-160 nM). The drug was active both in B- (n = 17) and T-NHLs (n = 9), but the latter were less sensitive (285 vs 22 nM, P = 0.0002). The in vitro activity was confirmed using the human JeKo-1 MCL xenograft model. We evaluated 16 combination partners, including rituximab (B-NHL), crizotinib and brentuximab vedotin (T-NHLs), lenalidomide, bendamustine, agents targeting AKT1/2, BCL2, BET, BTK, CDKs (pan, CDK4/6, CDK9), HDAC, JAK1/2, MALT1, and the proteasome. In B-NHLs, copanlisib/venetoclax were the most beneficial (14 synergism, 2 additive), and the 4thbest in T-NHLs (5 synergism, 1 additive). The combination led to increased apoptosis, as shown by a dose-dependent increased PARP cleavage in 3 MZL, and 1 MCL cell lines and by a reduction of cell viability in 2 MCL primary cells. In MZL (SSK41) and MCL (Jeko1) 24h exposure to copanlisib determined a dose-dependent down-regulation of pAKT and anti-apoptotic proteins (BCL-XL in the MZL and of MCL1 in the MCL). These changes were maintained in cells exposed to the combination, but not after venetoclax alone. An in vivo experiment using the SSK41 MZL cell line validated the beneficial effect of this combination and also of copanlisib/lenalidomide (in vitro beneficial in 12/17 B- and 4/9 T-NHL). In T-NHLs, benefit was observed in all ALCL-ALK+ with copanlisib/crizotinib (3/4 synergisms). All but one of the non ALCL-ALK+ cell lines achieved a synergism with copanlisib/ruxolitinib. Copanlisib/brentuximab vedotin was synergistic in the non ALCL-ALK+ and Sezary Syndrome cell lines. Transcriptome analysis identified the PIM1 and PIM2 kinases and the anti-apoptotic gene MCL1 as up-regulated in the MZL cell line HAIR-M exposed to copanlisib (5nM, for 4, 8 and 12h). Thus, we tested combinations with the PIM inhibitor AZD1208 and the MCL1 inhibitor MIK665 (S63845) in 3 MCL and 3 MZL cell lines. Both combinations were synergistic in the HAIR-M and beneficial in additional 5/5 and 3/5 cell lines, respectively. Conclusions: Our study identified a series of active copanlisib-containing combinations and provided the rational for the design of the new SAKK 66/18 phase I study exploring copanlisib plus venetoclax in relapsed/refractory B-cell lymphomas. Keywords: marginal zone lymphoma (MZL); PI3K/AKT/mTOR; venetoclax. Disclosures: Lange, M: Employment Leadership Position: Bayer AG. Jourdan, T: Employment Leadership Position: Bayer AG. Berthold, M: Employment Leadership Position: Bayer AG. Rossi, D: Consultant Advisory Role: Gilead, AbbVie, Janssen, AstraZeneca; Honoraria: Gilead, AbbVie Janssen, Roche; Research Funding: Gilead, AbbVie, Janssen. Stathis, A: Research Funding: Bayer, Merck, Pfizer, Novartis, Roche; Other Remuneration: travel grant from AbbVie. Liu, N: Employment Leadership Position: Bayer AG. Zucca, E: Consultant Advisory Role: Roche, Mei Pharma, Astra Zeneca; Research Funding: Roche, Janssen; Other Remuneration: travel grants from Abbvie and Gilead; expert statements provided to Gilead. Politz, O: Employment Leadership Position: Bayer AG. Bertoni, F: Research Funding: Acerta, Bayer AG; Other Remuneration: travel grants from Astra Zeneca, PIQUR Therapeutics AG.

Combined targeting of PI3K and MEK effector pathways via CED for DIPG therapy.

BackgroundMidline gliomas like diffuse intrinsic pontine glioma (DIPG) carry poor prognosis and lack effective treatment options. Studies have implicated amplifications in the phosphatidylinositol 3-kinase (PI3K) signaling pathway in tumorigenesis; compensatory activation of parallel pathways (eg, mitogen-activated protein kinase [MEK]) may underlie the resistance to PI3K inhibition observed in the clinic.MethodsThree patient-derived cell lines (SU-DIPG-IV, SU-DIPG-XIII, and SF8628) and a mouse-derived brainstem glioma cell line were treated with PI3K (ZSTK474) and MEK (trametinib) inhibitors, alone or in combination. Synergy was analyzed using Chou-Talalay combination index (CI). These agents were also used alone or in combination in a subcutaneous SU-DIPG-XIII tumor model and in an intracranial genetic mouse model of DIPG, given via convection-enhanced delivery (CED).ResultsWe found that these agents abrogate cell proliferation in a dose-dependent manner. Combination treatments were found to be synergistic (CI < 1) across cell lines tested. They also showed significant tumor suppression when given systemically against a subcutaneous DIPG model (alone or in combination) or when given via direct intracranial injection (CED) in a intracranial DIPG mouse model (combination only, median survival 47 vs 35 days post-induction, P = .038). No significant short- or long-term neurotoxicity of ZSTK474 and trametinib delivered via CED was observed.ConclusionsOur data indicate that ZSTK474 and trametinib combinatorial treatment inhibits malignant growth of DIPG cells in vitro and in vivo, prolonging survival. These results suggest a promising new combinatorial approach using CED for DIPG therapy, which warrants further investigation.

Cyclin-dependent kinase inhibitors AZD5438 and R547 show potential for enhancing efficacy of daunorubicin-based anticancer therapy: Interaction with carbonyl-reducing enzymes and ABC transporters

Daunorubicin (DAUN) has served as an anticancer drug in chemotherapy regimens for decades and is still irreplaceable in treatment of acute leukemias. The therapeutic outcome of DAUN-based therapy is compromised by its cardiotoxicity and emergence of drug resistance. This phenomenon is often caused by pharmacokinetic mechanisms such as efflux of DAUN from cancer cells through ATP-binding cassette (ABC) transporters and its conversion to less cytostatic but more cardiotoxic daunorubicinol (DAUN-OL) by carbonyl reducing enzymes (CREs). Here we aimed to investigate, whether two cyclin-dependent kinase inhibitors, AZD5438 and R547, can interact with these pharmacokinetic mechanisms and reverse DAUN resistance. Using accumulation assays, we revealed AZD5438 as potent inhibitor of ABCC1 showing also weaker inhibitory effect to ABCB1 and ABCG2. Combination index analysis, however, shown that inhibition of ABCC1 does not significantly contribute to synergism between AZD5438 and DAUN in MDCKII-ABCC1 cells, suggesting predominant role of other mechanism. Using pure recombinant enzymes, we found both tested drugs to inhibit CREs with aldo-keto reductase 1C3 (AKR1C3). This interaction was further confirmed in transfected HCT-116 cells. Moreover, these cells were sensitized to DAUN by both compounds as Chou-Talalay combination index analysis showed synergism in AKR1C3 transfected HCT-116, but not in empty vector transfected control cell line. In conclusion, we propose AZD5438 and R547 as modulators of DAUN resistance that can prevent AKR1C3-mediated DAUN biotransformation to DAUN-OL. This interaction could be beneficially exploited to prevent failure of DAUN-based therapy as well as the undesirable cardiotoxic effect of DAUN-OL.

Abstract P6-21-07: The stapled peptide ALRN-6924, a dual inhibitor of MDMX and MDM2, and the CDK4/6 inhibitors palbociclib or abemaciclib synergistically enhance each other's in vitro and in vivo anticancer activity

Abstract Background ALRN-6924 is a cell-penetrating α-helical stapled peptide that disrupts the interaction of the p53 tumor suppressor protein and its inhibitors, MDMX and MDM2. Reactivation of p53 with ALRN-6924 in TP53-wild-type tumors triggers cell cycle arrest and apoptosis resulting in antitumor efficacy. CDK4/6 inhibitors induce apoptosis, senescence, and cell growth arrest via the interrelated Rb pathway, and co-amplification of MDM2 and CDK4 (both on chromosome 12q13) is a known oncogenic driver, suggesting that combinations of ALRN-6924 and CDK4/6i's may be synergistic. This study evaluates the antitumor efficacy and pharmacodynamics (PD) of ALRN-6924 combined with palbociclib or abemaciclib. Methods ALRN-6924 was tested in combination with palbociclib or abemaciclib in MCF-7 breast cancer cell lines and MDM2- and CDK4-co-amplified SJSA-1 sarcoma cell lines using WST-1 cell viability assays. Synergy was quantified by the Chou-Talalay combination index method. Single agents and combinations were evaluated in cell culture using assays for apoptosis (Caspase 3/7 cleavage), proliferation (BrdU), senescence (ß-Galactosidase), colony growth (Giemsa), and Western blot analysis of p53, p21, Rb, phospho-Rb, FOXM1, and phospho-FOXM1; and E2F1 mRNA. In vivo combinations were tested in athymic nude mouse MCF-7 and SJSA-1 xenograft models, with cell cycle assays (EdU) measured in tumor samples by flow cytometry. Results ALRN-6924 combinations with palbociclib or abemaciclib display synergistic in vitro anti-proliferative activity in MCF-7 and SJSA-1 cells. ALRN-6924 induces senescence in vitro as a monotherapy and in combination with CDK4/6i's. Western blot assays show that ALRN-6924/palbociclib combinations trigger sustained on-mechanism biomarker activation, vs. transient activation with single agents. Phospho-Rb and phospho-FOXM1 down-regulation, p53 and p21 up-regulation, and repression of E2F1 mRNA are sustained after wash-out in combination, but not in single agent-treated cells. MCF-7 and SJSA-1 tumor growth inhibition was improved in mice treated with ALRN-6924 combinations with either palbociclib or abemaciclib vs. single agent. EdU assays show that ALRN-6924/palbociclib combinations inhibit SJSA-1 tumor cell proliferation in vivo. Body weights and mortality data show the combination of ALRN-6924 with palbociclib 75 mg/kg/day was well tolerated; the combination with abemaciclib 100 mg/kg/day was tolerated with interruption and dose-reduction. No pharmacokinetic (PK) drug-drug interactions were noted in nude mice due to different modes of metabolism for ALRN-6924 (proteolysis) and palbociclib (CYP3A). Conclusions This study demonstrates that ALRN-6924 and CDK4/6i combinations show synergistic activity. PD biomarkers indicate on-mechanism in vitro activity that is sustained after wash-out. In vivo efficacy, biomarker, PK, and tolerability results, plus clinical evidence that the most frequent and concerning safety issues for CDK4/6i's (neutropenia, leukopenia, infections) do not overlap with ALRN-6924's reported safety profile (Meric-Bernstam et al., ASCO 2017) support the development of combination regimens for breast cancer and other malignancies. Citation Format: Annis A, Carvajal LA, Ren J-G, Sutton D, Santiago S, Narasimhan N, Guerlavais V, Aivado M. The stapled peptide ALRN-6924, a dual inhibitor of MDMX and MDM2, and the CDK4/6 inhibitors palbociclib or abemaciclib synergistically enhance each other's in vitro and in vivo anticancer activity [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-21-07.

Telmisartan induces melanoma cell apoptosis and synergizes with vemurafenib in vitro by altering cell bioenergetics.

ObjectiveDespite recent advancements in targeted therapy and immunotherapies, prognosis for metastatic melanoma patients remains extremely poor. Development of resistance to previously effective treatments presents a serious challenge and new approaches for melanoma treatment are urgently needed. The objective of this study was to examine the effects of telmisartan, an AGTR1 inhibitor and a partial agonist of PPARγ, on melanoma cells as a potential agent for repurposing in melanoma treatment.MethodsExpression of AGTR1 and PPARγ mRNA in melanoma patient tumor samples was examined in publicly available datasets and confirmed in melanoma cell lines by qRT-PCR. A panel of melanoma cell lines was tested in viability, apoptosis and metabolic assays in presence of telmisartan by flow cytometry and immunocytochemistry. A cytotoxic effect of combinations of telmisartan and targeted therapy vemurafenib was examined using the Chou-Talalay combination index method.ResultsBoth AGTR1 and PPARγ mRNA were expressed in melanoma patient tumor samples and decreased compared to the expression in the healthy skin. In vitro, we found that telmisartan decreased melanoma cell viability by inducing cell apoptosis. Increased glucose uptake, but not utilization, in the presence of telmisartan caused the fission of mitochondria and release of reactive oxygen species. Telmisartan altered the cell bioenergetics, thereby synergizing with vemurafenib in vitro, and even sensitized vemurafenib-resistant cells to the treatment. ConclusionsGiven that the effective doses of telmisartan examined in our study can be administered to patients and that telmisartan is a widely used and safe antihypertensive drug, our findings provide the scientific rationale for testing its efficacy in treatment of melanoma progression.

Abstract 1894: Identification of novel OTX015-containing combinations for lymphoma treatment

Abstract Introduction. Epigenetics represents and intriguing target of therapy for lymphomas. The epigenetic mechanisms are regulated by several players (e.g. HDAC, HAT, BET) and among them the BET proteins became of interest a few years ago with demonstrated preclinical anti-cancer activity with the first compounds and early clinical responses first in class clinical-grade BET inhibitor OTX015 (MK-8628/birabresib). Despite a widespread anti-proliferative activity, the cytotoxicity effect of BET inhibitors is limited, and, here, we aimed to identify drugs that improve OTX015 activity performing a screening with the BET inhibitor with a library 348 compounds in two lymphoma cell lines. Methods. Two cell lines derived from germinal center B cell (GCB) diffuse large B cell lymphoma (DLBCL) (OCI-LY-19 and WSU-DLCL2) were exposed to OTX015 (single dose, 100 nM) in combination with two different doses (20 and 1,000 nM) of 348 compounds. Compounds giving a 1.5-fold decreased proliferation with the combination than with the individual compounds were further evaluated in two additional lymphoma cell lines, exposed (72h) to increasing doses of OTX015 alone and in combination with increasing doses of other selected compounds. Synergy was assessed with Chou-Talalay combination index (CI): synergism (&amp;lt;0.9), additive (0.9-1.1), antagonism/no benefit (&amp;gt; 1.1). Results. OCI-LY-19 and WSU-DLCL2 were treated with OTX015 (100 nM) and 348 compounds as single agents and in combination. The combinations of OTX015 with a series of compounds achieved improved anti-tumor activity than single agents. Besides HDAC and mTOR inhibitors, in accordance with what previously reported (Boi, Gaudio, Bonetti et al, Clinical Cancer Res 2015), the ABL/SRC inhibitor dasatinib, the AKT1/2/3 inhibitor MK-2206, the JAK2 inhibitor TG101209 and the LRRK2 inhibitor LRRK2-IN appeared interesting combination partners. These screening results were validated in the DLBCL cell lines with targeted experiments, as well as in chronic lymphocytic leukemia (CLL) (OTX015 plus MK-2206) and mantle cell lymphoma (MCL) (REC1) (Dasatinib, LRRK2-IN). Experiments in additional cell lines and assessing the effects on the apoptosis are on-going and will be presented. Conclusion. A chemical screening has identified novel OTX015-containing combinations active in lymphoma cell lines that are worth of further investigations. Work supported by a San Salvatore Foundation grant. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Filippo Spriano, Alberto J. Arribas, Luciano Cascione, Emanuele Zucca, Anastasios Stathis, Francesco Bertoni. Identification of novel OTX015-containing combinations for lymphoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1894.

Abstract 955: Combined inhibition of MEK and mTOR pathways is effective in NRAS Q61K mutant small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a heterogeneous disease, consisting of a phenotypically diverse population of cells, often described as neuroendocrine (NE) and non-NE. Though previous studies suggest that aberrant RAS signaling is involved in the transition from NE to non-NE profile, the characteristics of SCLC with RAS mutations have not been fully examined due to the rarity of the occurrence and the lack of resources available to study its biology. Methods: We created a cell line (DFCI168) from the pleural effusion of the SCLC patient with a rare NRASQ61K mutation. A patient-derived xenograft (PDX) model from this patient was successfully established by subcutaneous implantation of cancer cells in NSG mice. The efficacy of MEK and mTOR inhibitors was evaluated in DFCI168 and the commercially available lung cancer cell lines with NRAS Q61 mutations (SW1271, H1299, H2087, H2347). The Chou-Talalay combination index was used to evaluate drug synergy. Retroviral NRASQ61K transduction was performed on a classical SCLC cell line Glc16 to examine whether NRAS activation leads to acquisition of non-NE features. Results: DFCI168 showed non-NE/mesenchymal features both morphologically and biologically, which is exemplified by an elongated spindle shape, mesenchymal marker (VIM) expression and silenced NE markers (SYP, ASCL1) expression. NRASQ61K transduction on Glc16 achieved the transition from NE to non-NE/mesenchymal phenotype. Although DFCI168 showed marked sensitivity to MEK inhibitors both in vitro (trametinib IC50=2nmol/L, selumetinib IC50=50nmol/L) and in vivo, the tumors regrew after withdrawal of treatment with trametinib in xenograft mice, and caused sustained activation of the mTOR pathway during MEK inhibitor treatment in vitro. The combination of MEK and mTOR inhibitors (trametinib and TORIN2) prevented mTOR activation at 100nmol/L and yielded synergistic effects (CI=0.61). The commercially available lung cancer cell lines (SW1271, H1299, H2087, H2347) also responded synergistically to the trametinib/TORIN2 combination except H1299, a cell line with a known PTEN promoter methylation. Conclusion: We demonstrate that the oncogenic NRAS mutation defines a distinct molecular subset of SCLC, and is sufficient to cause the phenotypic switch from NE to non-NE/mesenchymal profile. Such tumors are likely to benefit from the dual targeted therapy with MEK and mTOR inhibitors. Our findings highlight the future potential of targeted therapies for SCLC patients with NRAS activating mutations. Citation Format: Atsuko Ogino, Mika Lin, Prafulla C. Gokhale, Margaret K. Wilkens, Jihyun Choi, Antonio Calles, Man Xu, Marzia Capelletti, Geoffrey Oxnard, Nathanael S. Gray, Paul Kirschmeier, Pasi A. Jänne. Combined inhibition of MEK and mTOR pathways is effective in NRAS Q61K mutant small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 955.

Abstract 796: The BTK inhibitor BGB-3111 is synergistic with other anti-lymphoma targeted agents

Abstract Introduction. BGB-3111 is a novel generation irreversible BTK inhibitor (Hu et al AACR2017) under active clinical investigation for lymphoid tumors. Here, we evaluated BGB-3111 in combination with other anti-cancer agents on a panel of human lymphoma cell lines. Methods. Cell lines derived from activated B cell (ABC) diffuse large B cell lymphoma (DLBCL) (n=3), mantle cell lymphoma (MCL) (n=2) and chronic lymphocytic leukemia (CLL) (n=2) were exposed to increasing doses of BGB-3111 alone and in combination with increasing doses of other compounds (72h). Synergy was assessed with Chou-Talalay combination index (CI): synergism (&amp;lt;0.9), additive (0.9-1.1), antagonism/no benefit (&amp;gt; 1.1). Cell cycle analysis was performed after 24h of treatment. Results. As single agent BGB-3111 showed anti-tumor activity in the nanomolar range in two ABC-DLBCL (TMD8, IC50 0.4 nM; OCI-LY-10, 1.5 nM) and in one MCL (REC1, IC50 0.9 nM) cell lines, while the remaining four cell lines resulted resistant (IC50s &amp;gt; 5µM). The pattern of activity was similar to what seen with ibrutinib and other 2nd generation BTK inhibitors (Gaudio et al, ENA 2016). BGB-3111 was then valuated in combination with targeted agents. In ABC-DLBCL, synergism was achieved in 3/3 cell lines when BGB-3111 was combined with the MEK inhibitor pimasertib or with BCL2 inhibitor venetoclax. The combination with BET inhibitor OTX-015 was synergistic in 2/3 cell lines, while the combination with the XPO1 antagonist selinexor was beneficial in 2/3 (1 synergism, 1 additive). In CLL cell lines, the best combinations were BGB-3111 with OTX015 or with selinexor with 2/2 synergisms. The results of the combinations with pimasertib or venetoclax were discordant (pimasertib, 1 synergism, 1 no benefit; venetoclax, 1 synergism, 1 no benefit). Both MCL cell lines achieved synergism combining BGB-3111 with pimasertib, or selinexor, or venetoclax. The combination with OTX-015 was also beneficial, but synergism was observed in only one of the two cell lines, and additive in the remaining. The improved anti-tumor activity of the combination versus the single agents were confirmed performing cell cycle analysis in an ABC-DLBCL (OCI-LY-10) with an increased subG0 phase when BGB3111 was combined with venetoclax, pimasertib and OTX-015. Conclusion. BGB-3111 was active as single and the combination with inhibitors of key regulatory pathways in cell lines derived from ABC-DLBCL, CLL and MCL. Citation Format: Chiara Tarantelli, Lu Zhang, Elisabetta Curti, Filippo Spriano, Eugenio Gaudio, Luciano Cascione, Alberto Arribas, Emanuele Zucca, Anastasios Stathis, Davide Rossi, Francesco Bertoni. The BTK inhibitor BGB-3111 is synergistic with other anti-lymphoma targeted agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 796.

Abstract B041: PQR309-containing combinations show synergistic antilymphoma activity

Abstract Background: The PI3K/AKT/mTOR pathway represents an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor under clinical investigation for patients affected by lymphomas and solid tumors. Here, we present novel data obtained with PQR309 in combination with other targeted agents in lymphoma models. Materials and Methods: Established lymphoma cell lines derived from mantle cell lymphoma (MCL) and from diffuse large B cell lymphoma (DLBCL) of the activated B cell like (ABC) or the germinal center B cell type (GCB) were exposed to combinations of PQR309 with selected compounds for 72 hrs. Synergy was assessed with Chou-Talalay combination index (CI): synergism (&amp;lt;0.9), additive (0.9-1.1), antagonism/no benefit (&amp;gt; 1.1). Results: PQR309 was evaluated in combination with additional targeted agents. The combination with the BTK inhibitor ibrutinib was synergistic in 3/3 ABC-DLBCL (HBL1, median CI 0.26, 95%C.I. 0.22-0.29; OCI-LY-10, median CI 0.67, 95%C.I. 0.43-0.75; OCI-LY-3, median CI 0.68, 95%C.I. 0.6-0.73) and in 4/4 MCL (JEKO1, median CI 0.53, 95%C.I. 0.47-0.64; REC1, median CI 0.6, 95%C.I. 0.37-0.69; SP49, median CI 0.68, 95%C.I. 0.55-0.81; UPN1, median CI 0.69, 95%C.I. 0.64-0.75) cell lines tested. When PQR309 was assayed in combination with the BCL2 inhibitor venetoclax in MCL, 3/4 were synergistic (JEKO1, median CI 0.82, 95% CI 0.75-1.01; REC1, median CI 0.49, 95% CI 0.35-0.78; SP49, median CI 0.8, 95% CI 0.67-0.93) while one had no benefit (UPN1, CI &amp;gt;3). The combination with the novel anti-BRD4 Protac ARV-825 was synergistic in 4/8 cell lines (RI1, median CI 0.61, 95% CI 0.51-0.66; KARPAS422, median CI 0.68, 95% CI 0.56-0.81; OCI-LY-18, median CI 0.78, 95% CI 0.71-1.04; SU-DHL-6, median CI 0.71, 95% CI 0.65-0.88), additive in 2/8 (U2932, median CI 0.98, 95% CI 0.9-1.14; DOHH2, median CI 0.97, 95% CI 0.67-1.09) and with no benefit in 2/8 (OCI-LY-3, median CI 1.29, 95% CI 1.02-1.39; TMD8, median CI 1.35, 95% CI 1.33-1.4) cell lines. Finally, following the previous demonstration of an upregulation of PIM1 kinase after exposure to PQR309 and synergism combining PQR309 with the PIM inhibitor AZD1208 in a few DLBCL cell lines (Tarantelli et al, ASH 2015), we tested the efficacy of this combination in a larger panel of DLBCL cell lines. Seven out of seven DLBCL lymphoma cell lines benefited from the combination, achieving synergism to PQR309 plus AZD1208 (OCI-LY-10, median CI 0.51, 95% CI 0.47-0.57; OCI-LY-3, median CI 0.52, 95% CI 0.49-0.66; RI1, median CI 0.2, 95% CI 0.16-0.22; U2932, median CI 0.37, 95% CI 0.34-0.44; DOHH2, median CI 0.34, 95% CI 0.31-0.41; KARPAS422, median CI 0.86, 95% CI 0.8-0.92; OCI-LY-18, median CI 0.54, 95% CI 0.46-0.66). Conclusions: PQR309 showed synergistic activity in B cell lymphomas when combined with inhibitors of BTK, BCL2, BRD4, and PIM, suggesting these combinations are worthy of being further investigated. Citation Format: Chiara Tarantelli, Elena Bernasconi, Filippo Spriano, Petra Hillmann, Eugenio Gaudio, Luciano Cascione, Ivo Kwee, Doriano Fabbro, Emanuele Zucca, Vladimir Cmiljanovic, Francesco Bertoni. PQR309-containing combinations show synergistic antilymphoma activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B041.

Abstract B182: The novel tyrosine kinase inhibitors UJ26 and UJ30 are active in solid tumor and hematologic cancer cell lines

Abstract Background: Tyrosine kinase (TK) inhibitors represent the class of targeted antitumoral drugs that has been so far more successful. Here, we present two novel TK inhibitors, UJ26 and UJ30, with antitumor activity in preclinical models of solid tumors and hematologic cancers. Materials and Methods: Biochemical kinase profiling was performed (DiscoverX, CA, USA). Cell lines included in the NCI60 panel and lymphoma cell lines were exposed to increasing doses of UJ26, UJ30, and, as control, to the SRC inhibitor dasatinib for 72 hours. Cell proliferation was evaluated by using MTT assay. In vitro synergy was assessed with the Chou-Talalay combination index (CI): synergism (&amp;lt;0.9), additive (0.9-1.1), antagonism/no benefit (&amp;gt; 1.1). DLBCL RI-1 cells (15x106) were s.c. inoculated in NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice. Tumor size was measured on regular basis and drug treatments with UJ26 and UJ30 both given i.p, at the dose of 100mg/kg started when tumors reached 5 mm in diameter (100 mm3). Results: The two novel kinase inhibitors UJ26 and UJ30 presented broad specificity comparable to that of dasatinib, able to inhibit clinically relevant kinases ABL1, EGFR, FLT3, KIT, and MET. UJ26 and UJ30 presented antitumor activity in the NCI60 cell line panel against models of hematologic malignancies and solid tumors, in particular in the K562 leukemia and KM12 colon cancer cell lines (GI50 &amp;lt; 50 nM). Antiproliferative activity of UJ26 and UJ30 was then assessed in 8 B-cell lymphoma cell lines derived from mantle cell lymphoma (MCL) (Jeko1, MAVER1), chronic lymphocytic leukemia (MEC-1, PCL-12), and diffuse large B cell lymphoma (DLBCL) of the activated B cell like (ABC) (RI-1, SU-DHL-2) or of the germinal center B cell (GCB) (VAL, SU-DHL-4). Compounds were active with median IC50s of 600nM (95%C.I. 300-1065nM) and 250nM (95%C.I. 100-1049nM), respectively. Their antitumor activity appeared more potent than dasatinib (median IC50=1325nM, 95%C.I. 100-16625nM), in particular in 3 of the cell lines (MAVER1, SU-DHL-2 and MEC1). UJ26 and UJ30 were evaluated in combination with the BET inhibitor OTX015, exposing the four most sensitive lymphoma cell lines to increasing concentrations of the single compound or of both compounds for 72h. Both the UJ26/OTX015 combination, as well as the UJ30/OTX015 combination, were synergistic in all the cell lines. In vivo experiments showed that UJ26 was very toxic already after the first dose with swelling of the injection area. UJ26-treated mice started to recover after 2 weeks and were not treated any more, and there was no antitumor effect. UJ30 given once every 5 days reduced tumor volume compared to control group (D5, D11, D15; P &amp;lt; 0.05) and was well tolerated. UJ30-treated xenografts were three times smaller than both control and UJ26 groups at the end of the experiment (D15, UJ30 median tumor volume=867, 95%C.I. 600-1267.5). Body weight loss was observed after treatment with compound UJ26 whereas UJ30 showed similar weight growth as in the control group. Experiment was terminated after 2 weeks for vehicle- and UJ26-treated groups (D15) while UJ30-treated mice were sacrificed at day 20. Conclusions: The novel TK inhibitors UJ26 and UJ30 showed preclinical antitumor activity and in particular UJ30 is worth further investigations. CN, AC, FB: co-senior authors. Citation Format: Chiara Tarantelli, Eugenio Gaudio, Andrea Unzue, Pawel Sledz, Hillarie Ekeh, Elena Bernasconi, Filippo Spriano, Cristina Nevado, Amedeo Caflisch, Francesco Bertoni. The novel tyrosine kinase inhibitors UJ26 and UJ30 are active in solid tumor and hematologic cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B182.

An improved model for the analysis of combined antimicrobials: a replacement for the Chou-Talalay combination index method.

To rationalize confusion in the literature concerning the analysis of combined antimicrobials, specifically to see if the combination index (CI) method of analysis was as rigorous as claimed. Data from previous studies of the inhibition of Staphylococcus aureus by mixed antimicrobials were re-analysed using the CI method and a model which takes account of differences in the concentration exponents of individual antimicrobials. The Chou-Talalay combination index method for the analysis of combined antimicrobials was found to be valid only in the specific cases where concentration exponents were equal. In these cases, the CI method was found to be a function of the residuals of fitting the additive model to the observed data. Where concentration exponents were not equal, the CI method was invalid, whereas the additive model took these differences into account. The CI method can be replaced wholly by the additive model described. The model allows simple regression to be used to analyse whole data sets and provides simple graphical output.

Insulin-like growth factor receptor and sphingosine kinase are prognostic and therapeutic targets in breast cancer

BackgroundTargeting the type 1 insulin-like growth factor receptor (IGF1R) in breast cancer remains an ongoing clinical challenge. Oncogenic IGF1R-signaling occurs via activation of PI3K/AKT/MAPK downstream mediators which regulate cell proliferation and protein synthesis. To further understand IGF1R signaling we have investigated the involvement of the oncogenic IGF1R-related sphingosine kinase (SphK) pathway.MethodsThe prognostic (overall survival, OS) and therapeutic (anti-endocrine therapy) co-contribution of IGF1R and SphK1 were investigated using breast cancer patient samples (n = 236) for immunohistochemistry to measure total and phosphorylated IGF1R and SphK1. Kaplan-Meier and correlation analyses were performed to determine the contribution of high versus low IGF1R and/or SphK1 expression to OS in patients treated with anti-endocrine therapy. Cell viability and colony formation in vitro studies were completed using estrogen receptor (ER) positive and negative breast cancer cell-lines to determine the benefit of IGF1R inhibitor (OSI-906) and SphK inhibitor (SKI-II) co-therapy. Repeated measures and 1-way ANOVA were performed to compare drug treatments groups and the Chou-Talalay combination index (CI) was calculated to estimate drug synergism in vitro (CI < 1).ResultsHigh IGF1R and SphK1 protein co-expression in tumor tissue was associated with improved OS specifically in ER-positive disease and stratified for anti-endocrine therapy. A significant synergistic inhibition of cell viability and/or colony formation following OSI-906 and SKI-II co-treatment in vitro was evident (p < 0.05, CI < 1).ConclusionWe conclude that high IGF1R and SphK1 co-expression act together as prognostic indicators and are potentially, dual therapeutic targets for the development of a more effective IGF1R-directed combination breast cancer therapy.

Tetrandrine and arsenic trioxide synergistically inhibit proliferation of HCC1937 triple negative breast cancer cells

ObjectiveTo evaluate the effects of tetrandrine plus arsenic trioxide on HCC1937 cells, a triple negative breast cancer cell line, and to explore possible mechanisms. MethodsThe 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method was used to compare the antiproliferative effects of tetrandrine, arsenic trioxide alone and tetrandrine plus arsenic trioxide on HCC1937 cells. The median-effect principle (Chou-Talalay combination index method) was used to examine the interaction between the two drugs. Flow cytometry was used to evaluate effects of treatment with tetrandrine, arsenic trioxide or the combination of both on HCC1937 cell apoptosis. Real-time polymerase chain reaction and western blotting were performed to evaluate changes in apoptosis-related gene expression and protein levels. ResultsTetrandrine and arsenic trioxide each inhibited HCC1937 cell proliferation in a dose-dependent manner. The cell inhibition rate of HCC1937 cells treated with a combination of tetrandrine and arsenic trioxide was significantly higher than with either compound alone. The two drugs produced a synergistic effect when the inhibition rate was 20%-40%. Flow cytometry results showed that treatment with the two drugs increased the proportion of apoptotic cells. In the combination treated group, caspase-3 activation and PARP cleavage were significantly higher than in the other groups. Moreover, Bcl-2 and survivin expression were decreased, whereas that of both Bid and Bax was increased. ConclusionThese findings demonstrated that tetrandrine plus arsenic trioxide had synergistic efficacy on induction of apoptosis in HCC1937 cells.