Numerous Tumor Cell Lines Research Articles

Reprogramming tumor microenvironment with precise photothermal therapy by calreticulin nanobody-engineered probiotics

Targeted therapies have revolutionized traditional cancer treatments by precisely targeting tumor cells, enhancing efficacy and safety. Despite this advancement, the proportion of cancer patients eligible for such therapies remains low due to the absence of suitable targets. Here, we investigate whether the translocation of the immunogenic cell death (ICD) marker calreticulin (CALR) from the endoplasmic reticulum (ER) to the cell surface following ICD induction can serve as a target for targeted therapies. To target CALR, a nanobody Nb215 identified from a naïve VHH phage library with high binding affinity to both human and mouse CALR was employed to engineer probiotic EcN 1917. Our results demonstrated that CALR nanobody-modified EcN-215 coupled with the photothermal dye indocyanine green (ICG) was able to exert NIR-II imaging-guide photothermal therapy (PTT). Moreover, PTT with EcN-215/ICG can reshape the tumor microenvironment by enhancing the infiltration of CD45+CD3+ T cells and CD11b+F4/80+ macrophages. Furthermore, the antitumor activity of CALR-targeted EcN-215/ICG is synergistically enhanced by blocking CD47-SIRPα axis. Collectively, our study provides a proof of concept for CALR-targeted therapy. Given that CALR translocation can be induced by various anticancer therapies across numerous tumor cell lines, CALR-targeted therapies hold promise as a novel approach for treating multiple types of cancers.

Antitumor Effects and the Potential Mechanism of 10-HDA against SU-DHL-2 Cells.

10-hydroxy-2-decenoic acid (10-HDA), which is a unique bioactive fatty acid of royal jelly synthesized by nurse bees for larvae and adult queen bees, is recognized for its dual utility in medicinal and nutritional applications. Previous research has indicated that 10-HDA exerts antitumor effects on numerous tumor cell lines, including colon cancer cells, A549 human lung cancer cells, and human hepatoma cells. The present study extends this inquiry to lymphoma, specifically evaluating the impact of 10-HDA on the SU-DHL-2 cell line. Our findings revealed dose-dependent suppression of SU-DHL-2 cell survival, with an IC50 of 496.8 μg/mL at a density of 3 × 106 cells/well after 24 h. For normal liver LO2 cells and human fibroblasts (HSFs), the IC50 values were approximately 1000 μg/mL and over 1000 μg/mL, respectively. The results of label-free proteomics revealed 147 upregulated and 347 downregulated differentially expressed proteins that were significantly enriched in the complement and coagulation cascades pathway (adjusted p-value = 0.012), including the differentially expressed proteins prothrombin, plasminogen, plasminogen, carboxypeptidase B2, fibrinogen beta chain, fibrinogen gamma chain, and coagulation factor V. The top three hub proteins, ribosomal protein L5, tumor protein p53, and ribosomal protein L24, were identified via protein-protein interaction (PPI) analysis. This result showed that the complement and coagulation cascade pathways might play a key role in the antitumor process of 10-HDA, suggesting a potential therapeutic avenue for lymphoma treatment. However, the specificity of the effect of 10-HDA on SU-DHL-2 cells warrants further investigation.

Example of a simple and scalable manufacturing process for immunomodulatory alpha neutrophils, a specifically designed immuno-cell therapy with direct and indirect anti-cancer activity for solid tumour indications.

e14512 Background: Recent findings have demonstrated neutrophils play a key role in tumour control and in facilitating successful responses to cancer immunotherapy. At LIfT Biosciences we have developed the first-in-class, allogeneic, off-the-shelf, stem cell-derived immuno-cell therapy with anti-cancer neutrophil properties. Immunomodulatory Alpha Neutrophils (IMANs) exert their anti-cancer activity via multiple direct and indirect mechanisms, ensuring direct antigen-independent tumour cell killing, as well as impacting anti-cancer function of other direct acting cells such as NK and gamma delta T cells, and further immunomodulation of the environment to boost adaptive immune responses ensuring maximal T cell activity can be achieved. IMANs have the potential to provide major clinical benefits in the treatment of multiple solid tumours. Methods: We present a patent-pending, GMP-compliant manufacturing process starting with CD34+ cells isolated from mobilized peripheral blood of healthy donors. Extensive characterisation of starting material in combination with in-process and raw material controls results in a simple, consistent, scalable manufacturing process using known and established manufacturing platforms. IMANs are granulopoietic myeloid progenitors derived from the expansion of CD34+ cells followed by differentiation in proprietary media compositions, composed of critical priming agents which are known to promote anti-cancer neutrophil phenotype and function. Neutrophil functional heterogeneity is becoming increasingly appreciated and we have designed our manufacturing process to produce activated, committed, persistent IMANs with multimodal mechanisms of action. IMANs are composed of distinct neutrophil progenitor populations. IMANs are cytotoxic towards numerous solid tumour cell lines, expressing tumour-ablating Neutrophil elastase. Furthermore, IMANs exert immunomodulatory properties through cytokine and chemokine secretion, and expression of ligands for T and NK cell co-stimulatory receptors. Results: The manufacturing process has been optimised with vast expansion capacity, showing up to 1000-fold increase from starting cell populations and efficient cryopreservation with exceptional post-thaw recovery. Conclusions: Cryopreserved IMANs offer a cost-effective and globally accessible cell therapy with the potential for long term clinical benefit.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Abstract 3567: Comprehensive preclinical studies of BT-001: An oncolytic vaccinia virus armed with Treg-depleting @CTLA4 and GM-CSF

Abstract Background Immune checkpoint blockade (ICB) is a clinically proven concept to treat cancer. Still, a majority of cancer patients including those with poorly immune infiltrated “cold” tumors are resistant to currently available ICB therapies. Oncolytic viruses are natural immunomodulators able to turn “cold” tumors “hot” and, for some of them, to deliver therapeutic payloads intratumorally. Payloads of choice are those with tolerability and/or pharmacokinetic/biodistribution issues, such as @CTLA4 antibodies and GM-CSF respectively. Here, Transgene and BioInvent present a preclinical characterization of a highly efficacious and potentially safe strategy to target CTLA-4 and GM-CSF using vectorization in an oncolytic vaccinia virus. Methods A novel human IgG1 CTLA-4 antibody (4-E03) was identified using function-first screening for mAbs and targets associated with checkpoint blocking and superior Treg depleting activity. A tumor-selective oncolytic Vaccinia vector was then engineered to encode this novel αCTLA-4 as full-length antibody (i.e. distinct transgenes for light and heavy chains) and GM-CSF (VVGM-αhCTLA4, BT-001). Viruses encoding matching mouse surrogate payloads were additionally generated, enabling proof-of-concept studies in syngeneic immune competent mouse tumor models. Results Our studies demonstrate that two chains cloning approach allowed to express @CTLA4 at a high level in numerous infected human tumor cell lines without affecting replication, oncolytic activity or genetic stability of the virus. Intratumoral (i.t.) administration of VVGM-αCTLA4 achieved tumor-restricted CTLA-4 receptor saturation at least for 10 days, Treg-depletion antigen cross-presentation, stronger systemic expansion of tumor-specific CD8+ T cells and antitumor immunity compared with systemic αCTLA-4 antibody or unarmed virus therapies. Remarkably, in a clinically relevant mouse model resistant to systemic immune checkpoint blockade, i.t. VVGM-αCTLA4 synergized with αPD-1 to reject “cold” tumors. Conclusion Our findings demonstrate in vivo proof-of-concept for tumor delivery by Vaccinia virus of GM-CSF and strongly Treg-depleting, immune checkpoint blocking, vectorized αCTLA-4. This vector-based delivery is a highly effective and safe strategy to target CTLA-4 which overcomes current limitations of approved αCTLA-4 regimens. A clinical trial evaluating i.t. VVGM-αhCTLA4 (BT-001) alone and in combination with αPD-1 in metastatic or advanced solid tumors has been initiated. Citation Format: Jean-Baptiste Marchand, Monika Semmrich, Christelle Ziller, Matilda Rehn, Laetitia Fend, Petra Holmkvist, Nathalie Silvestre, Carolin Svensson, Kleinpeter Patricia, Jules Deforges, Fred Junghus, Linda Mårtensson, Johann Foloppe, Ingrid Teige, Björn Frendéus, Eric Quéméneur. Comprehensive preclinical studies of BT-001: An oncolytic vaccinia virus armed with Treg-depleting @CTLA4 and GM-CSF [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 3567.

Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis.

F-box protein 5 (FBXO5), an essential subunit of the ubiquitin protein ligase complex, is increasingly recognized to exhibit important biological effects in regulating tumor occurrence and progression. The present research was intended to systematically investigate the latent roles of FBXO5 in prognosis and immunological function across cancers. Pan-cancer analyses of FBXO5 were performed based upon publicly available online databases, mainly including the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), UCSC Xena, cBioPortal, and ImmuCellAI, revealing the possible relationships between FBXO5 and prognosis, DNA methylation, tumor microenvironment (TME), infiltration of immune cells, immune-related genes, immune checkpoints, tumor mutation burden (TMB), and microsatellite instability (MSI). The results suggested that FBXO5 was expressed at a high level in numerous tumor cell lines with significant upregulation in most cancers as opposed to normal tissues. Of note, elevated expression of FBXO5 was significantly related to an unfavorable prognosis in many cancer types. Furthermore, DNA methylation and TME were confirmed to display evident correlation with the expression of FBXO5 in several malignancies. Moreover, FBXO5 expression was remarkably positively correlated with the levels of infiltrating Treg cells and Tcm cells in most tumors, but negatively correlated with tumor-infiltrating CD8+ T cells, NK/NKT cells, and Th2 cells. Meanwhile, FBXO5 was demonstrated to be co-expressed with the genes encoding immune activating and suppressive factors, chemokines, chemokine receptors, and major histocompatibility complex (MHC). Immune checkpoints, TMB, and MSI were also overtly associated with FBXO5 dysregulation among diverse kinds of cancers. Additionally, the enrichment analyses showed close relationships between FBXO5 expression and the processes related to cell cycle and immune inflammatory response. These findings provided a detailed comprehension of the oncogenic function of FBXO5. Because of its crucial roles in cancer immunity and tumorigenesis, FBXO5 may serve as a novel prognostic indicator and immunotherapeutic target for various malignancies.

Duclauxin Derivatives From Fungi and Their Biological Activities.

Duclauxin is a heptacyclic oligophenalenone dimer consisting of an isocoumarin and a dihydroisocoumarin unit. These two tricyclic moieties are joined by a cyclopentane ring to form a unique hinge or castanets-like structure. Duclauxin is effective against numerous tumor cell lines because it prevents adenosine triphosphate (ATP) synthesis by inhibiting mitochondrial respiration. There are about 36 reported natural duclauxin analogs mainly produced by 9 Penicillium and Talaromyces species (T. duclauxii, T. aculeatus, T. stipitatus, T. bacillisporus, T. verruculosus, T. macrosporus, P. herquei, P. manginii, and Talaromyces sp.). These metabolites exhibit remarkable biological activities, including antitumor, enzyme inhibition, and antimicrobial, showing tremendous potential in agricultural and medical applications. This review highlights the chemical structures and biological activities of fungal duclauxins, together with biosynthesis, absolute configuration, and mode of action for important duclauxins. Furthermore, phylogenetic analysis and correct names of Penicillium and Talaromyces species producing duclauxins are presented in this review.

Pleiotropic Pharmacological Actions of Capsazepine, a Synthetic Analogue of Capsaicin, against Various Cancers and Inflammatory Diseases.

Capsazepine is a synthetic analogue of capsaicin that can function as an antagonist of TRPV1. Capsazepine can exhibit diverse effects on cancer (prostate cancer, breast cancer, colorectal cancer, oral cancer, and osteosarcoma) growth and survival, and can be therapeutically used against other major disorders such as colitis, pancreatitis, malaria, and epilepsy. Capsazepine has been reported to exhibit pleiotropic anti-cancer effects against numerous tumor cell lines. Capsazepine can modulate Janus activated kinase (JAK)/signal transducer and activator of the transcription (STAT) pathway, intracellular Ca2+ concentration, and reactive oxygen species (ROS)-JNK-CCAAT/enhancer-binding protein homologous protein (CHOP) pathways. It can inhibit cell proliferation, metastasis, and induce apoptosis. Moreover, capsazepine can exert anti-inflammatory effects through the downregulation of lipopolysaccharide (LPS)-induced nuclear transcription factor-kappa B (NF-κB), as well as the blockage of activation of both transient receptor potential cation channel subfamily V member 1 (TRPV1) and transient receptor potential cation channel, subfamily A, and member 1 (TRPA1). This review briefly summarizes the diverse pharmacological actions of capsazepine against various cancers and inflammatory conditions.

A genome-wide RNAi screen identifies the SMC5/6 complex as a non-redundant regulator of a Topo2a-dependent G2 arrest.

The Topo2a-dependent arrest is associated with faithful segregation of sister chromatids and has been identified as dysfunctional in numerous tumour cell lines. This genome-protecting pathway is poorly understood and its characterization is of significant interest, potentially offering interventional opportunities in relation to synthetic lethal behaviours in arrest-defective tumours. Using the catalytic Topo2a inhibitor ICRF193, we have performed a genome-wide siRNA screen in arrest-competent, non-transformed cells, to identify genes essential for this arrest mechanism. In addition, we have counter-screened several DNA-damaging agents and demonstrate that the Topo2a-dependent arrest is genetically distinct from DNA damage checkpoints. We identify the components of the SMC5/6 complex, including the activity of the E3 SUMO ligase NSE2, as non-redundant players that control the timing of the Topo2a-dependent arrest in G2. We have independently verified the NSE2 requirement in fibroblasts from patients with germline mutations that cause severely reduced levels of NSE2. Through imaging Topo2a-dependent G2 arrested cells, an increased interaction between Topo2a and NSE2 is observed at PML bodies, which are known SUMOylation hotspots. We demonstrate that Topo2a is SUMOylated in an ICRF193-dependent manner by NSE2 at a novel non-canonical site (K1520) and that K1520 sumoylation is required for chromosome segregation but not the G2 arrest.

Oncolytic properties of non-vaccinia poxviruses.

Vaccinia virus, a member of the Poxviridae family, has been extensively used as an oncolytic agent and has entered late stage clinical development. In this study, we evaluated the potential oncolytic properties of other members of the Poxviridae family. Numerous tumor cell lines were infected with ten non-vaccinia poxviruses to identify which virus displayed the most potential as an oncolytic agent. Cell viability indicated that tumor cell lines were differentially susceptible to each virus. Raccoonpox virus was the most potent of the tested poxviruses and was highly effective in controlling cell growth in all tumor cell lines. To investigate further the oncolytic capacity of the Raccoonpox virus, we have generated a thymidine kinase (TK)-deleted recombinant Raccoonpox virus expressing the suicide gene FCU1. This TK-deleted Raccoonpox virus was notably attenuated in normal primary cells but replicated efficiently in numerous tumor cell lines. In human colon cancer xenograft model, a single intratumoral inoculation of the recombinant Raccoonpox virus, in combination with 5-fluorocytosine administration, produced relevant tumor growth control. The results demonstrated significant antitumoral activity of this new modified Raccoonpox virus armed with FCU1 and this virus could be considered to be included into the growing armamentarium of oncolytic virotherapy for cancer.

Potential Anti-Inflammatory and Anti-Cancer Properties of Farnesol.

Farnesol, an acyclic sesquiterpene alcohol, is predominantly found in essential oils of various plants in nature. It has been reported to exhibit anti-cancer and anti-inflammatory effects, and also alleviate allergic asthma, gliosis, and edema. In numerous tumor cell lines, farnesol can modulate various tumorigenic proteins and/or modulates diverse signal transduction cascades. It can also induce apoptosis and downregulate cell proliferation, angiogenesis, and cell survival. To exert its anti-inflammatory/anti-oncogenic effects, farnesol can modulate Ras protein and nuclear factor kappa-light-chain-enhancer of activated B cells activation to downregulate the expression of various inflammatory mediators such as cyclooxygenase-2, inducible nitric oxide synthase, tumor necrosis factor alpha, and interleukin-6. In this review, we describe the potential mechanisms of action underlying the therapeutic effects of farnesol against cancers and inflammatory disorders. Furthermore, these findings support the clinical development of farnesol as a potential pharmacological agent in clinical studies.

Studies of the antitumor mechanism of action of dermaseptin B2, a multifunctional cationic antimicrobial peptide, reveal a partial implication of cell surface glycosaminoglycans.

Dermaseptin-B2 (DRS-B2) is a multifunctional cationic antimicrobial peptide (CAP) isolated from frog skin secretion. We previously reported that DRS-B2 possesses anticancer and antiangiogenic activities in vitro and in vivo. In the present study, we evaluated the antiproliferative activity of DRS-B2 on numerous tumor cell lines, its cell internalization and studies of its molecular partners as well as their influences on its structure. Confocal microscopy using ([Alexa594]-(Cys0)-DRS-B2) shows that in sensitive human tumor cells (PC3), DRS-B2 seems to accumulate rapidly at the cytoplasmic membranes and enters the cytoplasm and the nucleus, while in less sensitive tumor cells (U87MG), DRS-B2 is found packed in vesicles at the cell membrane. Furthermore FACS analysis shows that PC3 cells viability decreases after DRS-B2 treatment while U87 MG seems to be unaffected. However, "pull down" experiments performed with total protein pools from PC3 or U87MG cells and the comparison between the antiproliferative effect of DRS-B2 and its synthetic analog containing all D-amino acids suggest the absence of a stereo-selective protein receptor. Pretreatment of PC3 cells with sodium chlorate, decreases the antiproliferative activity of DRS-B2. This activity is partially restored after addition of exogenous chondroitin sulfate C (CS-C). Moreover, we demonstrate that at nanomolar concentrations CS-C potentiates the antiproliferative effect of DRS-B2. These results highlight the partial implication of glycosaminoglycans in the mechanism of antiproliferative action of DRS-B2. Structural analysis of DRS-B2 by circular dichroism in the presence of increasing concentration of CS-C shows that DRS-B2 adopts an α-helical structure. Finally, structure-activity-relationship studies suggest a key role of the W residue in position 3 of the DRS-B2 sequence for its antiproliferative activity.

Serotonergic system antagonists target breast tumor initiating cells and synergize with chemotherapy to shrink human breast tumor xenografts

Breast tumors comprise an infrequent tumor cell population, termed breast tumor initiating cells (BTIC), which sustain tumor growth, seed metastases and resist cytotoxic therapies. Hence therapies are needed to target BTIC to provide more durable breast cancer remissions than are currently achieved. We previously reported that serotonergic system antagonists abrogated the activity of mouse BTIC resident in the mammary tumors of a HER2-overexpressing model of breast cancer. Here we report that antagonists of serotonin (5-hydroxytryptamine; 5-HT) biosynthesis and activity, including US Federal Food and Drug Administration (FDA)-approved antidepressants, targeted BTIC resident in numerous breast tumor cell lines regardless of their clinical or molecular subtype. Notably, inhibitors of tryptophan hydroxylase 1 (TPH1), required for 5-HT biosynthesis in select non-neuronal cells, the serotonin reuptake transporter (SERT) and several 5-HT receptors compromised BTIC activity as assessed by functional sphere-forming assays. Consistent with these findings, human breast tumor cells express TPH1, 5-HT and SERT independent of their molecular or clinical subtype. Exposure of breast tumor cells ex vivo to sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), reduced BTIC frequency as determined by transplanting drug-treated tumor cells into immune-compromised mice. Moreover, another SSRI (vilazodone; Viibryd) synergized with chemotherapy to shrink breast tumor xenografts in immune-compromised mice by inhibiting tumor cell proliferation and inducing their apoptosis. Collectively our data suggest that antidepressants in combination with cytotoxic anticancer therapies may be an appropriate treatment regimen for testing in clinical trials.

Survivin does not influence the anti-apoptotic action of XIAP on caspase-9

Survivin inhibits apoptosis in numerous tumor cell lines and has emerged as promising target for cancer therapy. The anti-apoptotic effect of survivin was attributed to a direct interaction with XIAP (X-linked inhibitor of apoptosis) and to an indirect effect, where survivin antagonizes the anti-XIAP action of Smac. The direct interaction is thought to lead to synergistic inhibition of caspase-9 and, at the same time, to enhanced stability of XIAP by reducing its auto-ubiquitination. Using recombinant proteins, we have investigated the influence of survivin on the inhibition of caspase-9 by XIAP in vitro. With a fluorescence-based assay for the apoptosome-stimulated activity of caspase-9, we show that survivin has no effect on the inhibition of caspase-9 by XIAP, neither in the presence nor in the absence of Smac. Employing analytical size exclusion chromatography (SEC) and analytical ultracentrifugation, we show that survivin does not physically interact with XIAP. We confirm in vitro that XIAP ubiquitinates itself in the presence of the appropriate recombinant enzymes and Mg2+-ATP and could show that survivin neither influences the kinetics nor the extent of XIAP's self-ubiquitination. Our results call for a revision of the current view of how survivin interferes with the mitochondrial pathway of apoptosis.

Effects of flavopiridol on critical regulation pathways of CD133high/CD44high lung cancer stem cells.

Background:Flavopiridol a semisynthetic flavone that inhibits cyclin-dependent kinases (CDKs) and has growth-inhibitory activity and induces a blockade of cell-cycle progression at G1-phase and apoptosis in numerous human tumor cell lines and is currently under investigation in phase II clinical trials. Cancer stem cells (CSCs) are comprised of subpopulation of cells in tumors that have been proposed to be responsible for recurrence and resistance to chemotherapy. The aim of the present study was to investigate the effects of flavopiridol in cancer stem cell cytoskeleton, cell adhesion, and epithelial to mesenchymal transition in CSCs.Methods:The cells were treated with flavopiridol to determine the inhibitory effect. Cell viability and proliferation were determined by using the WST-1 assay. Caspase activity and immunofluorescence analyses were performed for the evaluation of apoptosis, cell cytoskeleton, and epithelial-mesenchymal transition (EMT) markers. The effects of flavopiridol on the cell cycle were also evaluated. Flow cytometric analysis was used to detect the percentages of CSCs subpopulation. We analyzed the gene expression patterns to predict cell cycle and cell cytoskeleton in CSCs by RT-PCR.Results:Flavopiridol-induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspases activity. Cell cycle analyses revealed that flavopiridol induces G1 phase cell cycle arrest. Flavopiridol significantly decreased the mRNA expressions of the genes that regulate the cell cytoskeleton and cell cycle components and cell motility in CSCs.Conclusion:Our results suggest that Flavopiridol has activity against lung CSCs and may be effective chemotherapeutic molecule for lung cancer treatment.

Abstract 2319: Selinexor, a selective inhibitor of nuclear export (SINE), enhances the in vivo efficacy of checkpoint blockade with antibodies targeting CTLA4 or PD-1/PD-L1 in melanoma

Abstract Selinexor is a SINE (Selective Inhibitor of Nuclear Export) compound that has been administered to >1000 cancer patients in Phase I and II trials to date, with evidence of efficacy and tolerability. This small molecule targets exportin-1 (XPO1), a key nuclear export protein with >200 cargo proteins which include both tumor suppressors and cell cycle modulators. As a result, selinexor blocks nuclear export of proteins including IκB, NFAT1c, STAT1 and STAT3, which regulate expression of the inhibitory T cell receptors CTLA4, PD1 and its ligand, PD-L1. We hypothesized that selinexor would upregulate T cell checkpoint molecule expression, and thereby enhance the anti-tumor activity of antibodies targeting PD-1/PD-L1 or CTLA4. Human (A375, CHL-1) and murine (B16F10) melanoma cell lines expressed high levels of PD-L1 protein at baseline, and PD-L1 expression was induced following selinexor treatment in numerous other tumor cell lines (including HCT-116, MDA-MB-468, MV-4-11, OVCAR-8, and PC-3). Examination of lymphocytes revealed that selinexor also increased expression of PD-1 and CTLA4 by ∼2-fold. Mice bearing syngeneic B16F10 melanoma tumors treated with selinexor (15 mg/kg 2 x weekly) and anti-CTLA4 (250 μg, 2 x weekly) demonstrated a significant reduction in tumor growth rate (p = 0.0065) while monotherapy had no significant effect on tumor growth. Similar results were obtained in mice bearing B16F10 melanoma treated with the combination of selinexor + anti-PD-1 (200 μg, 2 x weekly, p < 0.034) or selinexor + anti-PD-L1 (100-200 μg, 2 x weekly, p < 0.001). Importantly, no weight loss or signs of toxicity were evident in any in vivo study. Further immunophenotypic analyses have been completed in animals receiving selinexor alone or in combination with anti-PD-L1. In combination treated mice, we observed a significantly increased percentage of splenic NK cells (p ≤ 0.050), and a significantly increased percentage of splenic Th1 T cells (p≤0.011), all compared to vehicle treated mice. Interestingly, combining selinexor with anti-PD-L1 significantly decreased the percentage of splenocytes that expressed PD-L1 (p<0.001). These changes are indicative of increased anti-tumor immune activity; however, they were accompanied by significantly increased percentages of myeloid cell subsets in combination treated mice (p ≤ 0.050). The immunologic significance of this myeloid cell expansion is currently under investigation. These data indicate that the efficacy of selinexor may be enhanced by disrupting immune checkpoints in effector cells (T and NK cells). This provides data in support of novel, evidenced-based combinations involving immunotherapy with XPO1 inhibition that deserve further investigation for advanced cancer. Citation Format: Matthew R. Farren, Reena Shakya, Rebecca Hennessey, Thomas Mace, Jennifer Yang, Omar Elnaggar, Gregory Young, Yosef Landesman, Robert Carlson, Sivan Elloul, Marsha Crochiere, Christin Burd, Gregory B. Lesinski. Selinexor, a selective inhibitor of nuclear export (SINE), enhances the in vivo efficacy of checkpoint blockade with antibodies targeting CTLA4 or PD-1/PD-L1 in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2319.

Trichosanthin reduces the viability of SU‑DHL‑2 cells via the activation of the extrinsic and intrinsic apoptotic pathways.

Previous studies have indicated that trichosanthin (TCS) exerts antitumor activity by inducing apoptosis in numerous tumor cell lines. However, the effects of TCS on lymphoma remain to be elucidated. The current study demonstrated that TCS inhibited the proliferation of thirteen lymphoma cell lines in a dose‑dependent manner, with SU‑DHL‑2 cells exhibiting the greatest sensitivity to TCS. Treatment of SU‑DHL‑2 cells with TCS led to cell cycle arrest at the S to G2/M phase transition. Furthermore, flow cytometric analysis, Hoechst 33258 staining and western blotting indicated that TCS induced the apoptosis of SU‑DHL‑2 cells in a time‑ and concentration‑dependent manner. In addition, the activation of caspase‑3 and ‑7 and poly (ADP‑ribose) polymerase were observed. Pharmacological pan-caspase inhibition was observed to reduce TCS‑induced apoptosis. Inhibition of caspase‑8 or ‑9 alone was observed to partially reverse the effect of TCS on apoptosis. In conclusion, the current study indicates that TCS may induce apoptosis in SU‑DHL‑2 cells via the extrinsic and intrinsic pathways.

Preclinical activity of SL-801, a reversible inhibitor of Exportin-1 (XPO1)/Chromosome Region Maintenance-1 (CRM1) in solid and hematologic cancers.

e13543 Background: XPO1 controls key cellular processes by regulating nuclear export of proteins and RNA. XPO1 is overexpressed in many cancers, causing dysregulated protein localization, aberrant growth, and resistance to apoptosis. Recent work has revealed XPO1 to be a clinically relevant target, and nuclear export inhibitors have emerged as a new class of anti-cancer agents with clinical activity in multiple indications. SL-801 is a structurally novel small molecule that binds covalently but reversibly to XPO1, blocking binding of substrate cargos. The kinetics of target binding and preclinical activity of SL-801 in multiple tumor types are reported here. Methods: SL-801 activity was evaluated against 240 tumor cell lines using the OncoPanel platform and in human tumor xenografts in SCID mice. Pull-down assays with biotinylated SL-801 were used to assess SL-801 binding to XPO1. Results: SL-801 demonstrated broad and potent activity across numerous tumor cell lines. GI50s in hematologic cancers ranged from 3-93 nM in leukemias, 1-103 nM in lymphomas, and 3-11 nM in multiple myelomas. SL-801 also inhibited solid tumor growth, with GI50s ≤ 10 nM in breast, brain, cervical, ovarian, gastric, kidney, liver, lung, melanoma, prostate, and sarcoma lines. SL-801 exhibited substantial activity in vivo. In an ARH-77 multiple myeloma xenograft, 90% of mice treated with SL-801 in a multi-dose regimen survived 150 days, while the median survival of vehicle-treated mice was 40 days. SL-801 was also active in human acute lymphoblastic leukemia, non-small cell lung cancer, and prostate carcinoma xenograft models. Pull-down assays showed that SL-801 binding to XPO1 decreased after 2 hours, whereas the prototypical XPO1 inhibitor leptomycin B bound tightly to XPO1 for at least 8 hours. Conclusions: SL-801 is a promising clinical candidate that inhibits a novel target and possesses potent and broad anti-cancer activity. The reversible binding of SL-801 to XPO1 may offer the potential to develop dosing schedules to enable recovery in normal tissues, broadening the therapeutic index of this class of agents. IND-enabling work is underway to support entry into clinical trials.

Further insights into the SAR of α-substituted cyclopropylamine derivatives as inhibitors of histone demethylase KDM1A.

Epigenetics alterations including histone methylation and acetylation, and DNA methylation, are thought to play important roles in the onset and progression of cancer in numerous tumour cell lines. Lysine-specific demethylase 1 (LSD1 or KDM1A) is highly expressed in different cancer types and inhibiting KDM1A activity seems to have high therapeutic potential in cancer treatment. In the recent years, several inhibitors of KDM1A have been prepared and disclosed. The majority of these derivatives were designed based on the structure of tranylcypromine, as the cyclopropane core is responsible for the covalent interaction between the inhibitor and the catalytic domain of KDM proteins. In this study, we have further extended the SAR regarding compounds 1a-e, which were recently found to inhibit KDM1A with good activity. The decoration of the phenyl ring at the β-position of the cyclopropane ring with small functional groups, mostly halogenated, and in particular at the meta position, led to a significant improvement of the inhibitory activity against KDM1A, as exemplified by compound 44a, which has a potency in the low nanomolar range (31nM).

V‑set and transmembrane domain‑containing 1 is silenced in human hematopoietic malignancy cell lines with promoter methylation and has inhibitory effects on cell growth.

Numerous leukocyte differentiation antigens act as important markers for research, diagnosis, triage and eventually treatment targets for hematopoietic malignancies. V‑set and transmembrane domain‑containing 1 (VSTM1) was identified by immunogenomic analysis as a potential leukocyte differentiation antigen gene. VSTM1 is located at 19q13.4 on human chromosomes, an important genomic region prone to genetic and epigenetic modifications in numerous hematopoietic malignancies. VSTM1‑v1, a primary splicing form encoded by VSTM1, is a type I transmembrane molecule with an extracellular immunoglobulin V‑like domain and two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. In the present study, VSTM1 expression was examined in normal human peripheral leukocytes and hematopoietic tumor cell lines; in addition, the aberrant methylation of the VSTM1 gene was evaluated using methylation‑specific polymerase chain reaction (MSP). The results of the present study demonstrated that VSTM1 was widely expressed in normal human peripheral blood leukocytes, including granulocytes and monocytes, in concurrence with previous studies, as well as lymphocytes; in addition, the molecular size and expression levels of VSTM1 varied considerably between leukocytes. However, VSTM1 was undetectable in numerous hematopoietic tumor cell lines following promoter hypermethylation. The effects of pharmacologically‑induced demethylation of the VSTM1 gene and promoter region were analyzed using MSP and biosulfite genomic sequencing, and the results revealed that VSTM1 expression was restored in methylation‑silenced Jurkat cells. In addition, CKK‑8 assays revealed that VSTM1‑v1 overexpression in Jurkat cells resulted in growth suppression. Furthermore, the inhibitory effect on cell growth was enhanced following antibody‑induced cross‑linking of VSTM1‑v1. In conclusion, the results of the present study indicated that promoter methylation silenced VSTM1 and negatively regulated cell growth in human hematopoietic malignancy cell lines.