Marked Growth Inhibition Research Articles

212Pb α-Radioimmunotherapy Targeting CD38 in Multiple Myeloma: A Preclinical Study.

Multiple myeloma (MM) is a plasma cell cancer and represents the second most frequent hematologic malignancy. Despite new treatments and protocols, including high-dose chemotherapy associated with autologous stem cell transplantation, the prognosis of MM patients is still poor. α-radioimmunotherapy (α-RIT) represents an attractive treatment strategy because of the high-linear-energy transfer and short pathlength of α-radiation in tissues, resulting in high tumor cell killing and low toxicity to surrounding tissues. In this study, we investigated the potential of α-RIT with 212Pb-daratumumab (anti-hCD38), in both in vitro and in vivo models, as well as an antimouse CD38 antibody using in vivo models. Methods: Inhibition of cell proliferation after incubation of the RPMI8226 cell line with an increasing activity (0.185–3.7 kBq/mL) of 212Pb-isotypic control or 212Pb-daratumumab was evaluated. Biodistribution was performed in vivo by SPECT/CT imaging and after death. Dose-range–finding and acute toxicity studies were conducted. Because daratumumab does not bind the murine CD38, biodistribution and dose-range finding were also determined using an antimurine CD38 antibody. To evaluate the in vivo efficacy of 212Pb-daratumumab, mice were engrafted subcutaneously with 5 × 106 RPMI8226 cells. Mice were treated 13 d after engraftment with an intravenous injection of 212Pb-daratumumab or control solution. Therapeutic efficacy was monitored by tumor volume measurements and overall survival. Results: Significant inhibition of proliferation of the human myeloma RPMI8226 cell line was observed after 3 d of incubation with 212Pb-daratumumab, compared with 212Pb-isotypic control or cold antibodies. Biodistribution studies showed a specific tumoral accumulation of daratumumab. No toxicity was observed with 212Pb-daratumumab up to 370 kBq because of lack of cross-reactivity. Nevertheless, acute toxicity experiments with 212Pb-anti-mCD38 established a toxic activity of 277.5 kBq. To remain within realistically safe treatment activities for efficacy studies, mice were treated with 185 kBq or 277.5 kBq of 212Pb-daratumumab. Marked tumor growth inhibition compared with controls was observed, with a median survival of 55 d for 277.5 kBq of 212Pb-daratumumab instead of 11 d for phosphate-buffered saline. Conclusion: These results showed 212Pb-daratumumab to have efficacy in xenografted mice, with significant tumor regression and increased survival. This study highlights the potency of α-RIT in MM treatment.

Extracellular vesicle (ECV)-modified polyethylenimine (PEI) complexes for enhanced siRNA delivery in vitro and in vivo

Extracellular vesicles (ECVs) are secreted cell-derived membrane particles involved in intercellular signaling and cell-cell communication. By transporting various bio-macromolecules, ECVs and in particular exosomes are relevant in various (patho-) physiological processes. ECVs are also released by cancer cells and can confer pro-tumorigenic effects. Their target cell tropism, effects on proliferation rates, natural stability in blood and immunotolerance makes ECVs particularly interesting as delivery vehicles.Polyethylenimines (PEIs) are linear or branched polymers which are capable of forming non-covalent complexes with small RNA molecules including siRNAs or antimiRs, for their delivery in vitro and in vivo.This study explores for the first time the combination of PEI-based nanoparticles with naturally occurring ECVs from different cell lines, for the delivery of small RNAs. ECV-modified PEI/siRNA complexes are analyzed by electron microscopy vs. ECV or complex alone. On the functional side, we demonstrate increased knockdown efficacy and storage stability of PEI/siRNA complexes upon their modification with ECVs. This is paralleled by enhanced tumor cell-inhibition by ECV-modified PEI/siRNA complexes targeting Survivin. Pre-treatment with various inhibitors of cellular internalization reveals alterations in cellular uptake mechanisms and biological activities of PEI/siRNA complexes upon their ECV modification. Extending our studies towards PEI-complexed antimiRs against miR-155 or miR-1246, dose-dependent cellular and molecular effects are enhanced in ECV-modified complexes, based on the de-repression of direct miRNA target genes. Differences between ECVs from different cell lines are observed regarding their capacity of enhancing PEI/siRNA efficacies, independent of the target cell line for transfection. Finally, an in vivo therapy study in mice bearing s.c. PC3 prostate carcinoma xenografts reveals marked inhibition of tumor growth upon treatment with ECVPC3-modified PEI/siSurvivin complexes, based on profound target gene knockdown. We conclude that ECV-modification enhances the activity of PEI-based complexes, by altering pivotal physicochemical and biological nanoparticle properties.

Triple blockade of EGFR, MEK and PD-L1 has antitumor activity in colorectal cancer models with constitutive activation of MAPK signaling and PD-L1 overexpression

BackgroundMolecular mechanisms driving acquired resistance to anti-EGFR therapies in metastatic colorectal cancer (mCRC) are complex but generally involve the activation of the downstream RAS-RAF-MEK-MAPK pathway. Nevertheless, even if inhibition of EGFR and MEK could be a strategy for overcoming anti-EGFR resistance, its use is limited by the development of MEK inhibitor (MEKi) resistance.MethodsWe have generated in vitro and in vivo different CRC models in order to underline the mechanisms of MEKi resistance.ResultsThe three different in vitro MEKi resistant models, two generated by human CRC cells quadruple wild type for KRAS, NRAS, BRAF, PI3KCA genes (SW48-MR and LIM1215-MR) and one by human CRC cells harboring KRAS mutation (HCT116-MR) showed features related to the gene signature of colorectal cancer CMS4 with up-regulation of immune pathway as confirmed by microarray and western blot analysis. In particular, the MEKi phenotype was associated with the loss of epithelial features and acquisition of mesenchymal markers and morphology. The change in morphology was accompanied by up-regulation of PD-L1 expression and activation of EGFR and its downstream pathway, independently to RAS mutation status. To extend these in vitro findings, we have obtained mouse colon cancer MC38- and CT26-MEKi resistant syngeneic models (MC38-MR and CT26-MR). Combined treatment with MEKi, EGFR inhibitor (EGFRi) and PD-L1 inhibitor (PD-L1i) resulted in a marked inhibition of tumor growth in both models.ConclusionsThese results suggest a strategy to potentially improve the efficacy of MEK inhibition by co-treatment with EGFR and PD-L1 inhibitors via modulation of host immune responses.

TMOD-29. A MOUSE MODEL OF FGFR3-TACC3 UNCOVERS MECHANISMS OF GLIOMAGENESIS AND MAINTENANCE

Abstract Glioblastoma harboring Fibroblast Growth Factor Receptor 3 (FGFR3)-Transforming Acidic Coiled-Coil containing 3 (TACC-3) (FGFR3-TACC3) fusion is a distinct molecular subclass of tumors that can be targeted by FGFR kinase inhibitors. We generated a knock-in mouse in which the expression of FGFR3-TACC3 is doxycycline inducible and reversible. The tetO-FGFR3-TACC3-IRES-GFP cassette was placed into the Rosa26 locus and the FGFR3-TACC3 knock-in strain was intercrossed with GFAP-rtTA*M2 mice to trigger expression of the tetracycline transactivator (and the fusion gene) from the GFAP promoter. To recapitulate the genetic lesions that are more frequently associated with FGFR3-TACC3 in human glioblastoma, we also intercrossed the tetO-FGFR3-TACC3-IRES-GFP;GFAP-rtTA*M2 mice with TP53+/-or Ink4A+/-mouse strains. Both strains developed aggressive and infiltrating gliomas within 90 days after induction of FGFR3-TACC3 by doxycycline containing diet. Tumors occurred with a prevalence of ~50% and were found almost exclusively at two brain locations: the lateral septum and the neuro-epithelium adjacent to the fimbria of the hippocampus, thus recapitulating the restricted expression of endogenous FGFR3 in the ventral telencephalon and ganglionic eminence during development. The FGFR3-TACC3-positive glioma resembled human glioblastoma as they exhibited marked invasiveness and vascularization, elevated proliferative index and positivity for Nestin and GFAP. Rapid tumor progression caused appearance of neurological symptoms and death within few weeks. Doxycycline withdrawal from the diet of mice diagnosed with brain tumors by MRI resulted in rapid loss of FGFR3-TACC3 from glioma cells, marked inhibition of tumor growth with essentially complete glioma regression in some cases and recovery of general and neurological clinical symptoms. Our findings validate FGFR3-TACC3 as powerful oncogene that drives development of high-grade glioma from distinct areas of the brain. They also provide an accurate pre-clinical model in which glioma maintenance requires continuous expression of the fusion gene, thus supporting therapeutic targeting of the fusion protein in patients with FGFR3-TACC3-positive glioblastoma.

Antineoplastic Effect of a Combined Mitotane Treatment/Ionizing Radiation in Adrenocortical Carcinoma: A Preclinical Study.

Mitotane (MTT) is an adrenolytic drug used in adjuvant and advanced treatments of adrenocortical carcinoma (ACC). Ionizing radiation (IR) is also used in adrenal cancer treatment, even though its biological action remains unknown. To provide a reliable in vivo preclinical model of ACC, we used mouse xenografts bearing human ACC to test the effects of MTT and IR alone and in combination. We evaluated tumor growth inhibition by the RECIST criteria and analyzed the cell cycle by flow cytometry (FCM). In the xenograft ACC model treated with MTT/IR in combination, we observed a marked inhibition of tumor growth, with strong tumor regression (p < 0.0001) compared to MTT and IR given alone (p < 0.05). The MTT results confirm its antisteroidogenic activity (p < 0.05) in the xenograft ACC model, revealing its ability to render cancer cells more prone to radiotherapy treatment. In addition, to explain the biological effect of these treatments on the Mismatch Repair System (MMR), we interfered with the MSH2 gene expression in untreated and MTT/IR-treated H295R and SW13 cell lines. Moreover, we observed that upon treatment with MTT/IR to induce DNA damage, MSH2 gene inhibition in both the H295R and SW13 cell lines did not allow DNA damage repair, thus inducing cell death. In conclusion, MTT seems to have a radiosensitizing property and, when given in combination with IR, is able to promote neoplastic growth inhibition, leading to a significant reduction in tumor size due to cell death.

Tumor Priming by SMO Inhibition Enhances Antibody Delivery and Efficacy in a Pancreatic Ductal Adenocarcinoma Model.

Despite frequent overexpression of numerous growth factor receptors by pancreatic ductal adenocarcinomas (PDAC), such as EGFR, therapeutic antibodies have not proven effective. Desmoplasia, hypovascularity, and hypoperfusion create a functional drug delivery barrier that contributes to treatment resistance. Drug combinations that target tumor/stroma interactions could enhance tumor deposition of therapeutic antibodies, although clinical trials have yet to support this strategy. We hypothesize that macromolecular or nanoparticulate therapeutic agents may best exploit stroma-targeting "tumor priming" strategies, based on the fundamental principles of the Enhanced Permeability and Retention phenomenon. Therefore, we investigated the molecular and pharmacologic tumor responses to NVP-LDE225, an SMO inhibitor of sonic hedgehog signaling (sHHI), of patient-derived xenograft models that recapitulate the desmoplasia and drug delivery barrier properties of PDAC. Short-term sHHI exposure mediated dose- and time-dependent changes in tumor microvessel patency, extracellular matrix architecture, and interstitial pressure, which waned with prolonged sHHI exposure, and increased nanoparticulate permeability probe deposition in multiple PDAC patient-derived xenograft isolates. During sHHI-mediated priming, deposition and intratumor distribution of both a nontargeted mAb and a mAb targeting EGFR, cetuximab, were enhanced. Sequencing the sHH inhibitor with cetuximab administration resulted in marked tumor growth inhibition compared with cetuximab alone. These studies suggest that PDAC drug delivery barriers confound efforts to employ mAb against targets in PDAC, and that short-term, intermittent exposure to stromal modulators can increase tumor cell exposure to therapeutic antibodies, improving their efficacy, and potentially minimize adverse effects that may accompany longer-term, continuous sHHI treatment.

Influence of high-pressure processing on quality attributes of haddock and mackerel minces during frozen storage, and fishcakes prepared thereof

Abstract The study focused on assessing quality parameters of haddock and mackerel minces subjected to a high-pressure treatment (HP) at 200 and 300 MPa and frozen storage at −40 °C. Dry matter, water-holding capacity, protein solubility and oxidation, lipid oxidation, microbiological parameters, low molecular weight metabolites (LMW) and color parameters, were analyzed. The texture of fishcakes prepared on the basis of these fish minces was also studied, showing a decrease in firmness along with an increase in pressure. A marked inhibition of microbial growth was observed in fish minces when increasing the pressure level of HP-treatment. However, no significant effect (p Industrial relevance. The application of high pressure (HP) treatment of 200 and 300 MPa could be successfully applied to both lean and fatty fish species before freezing for reduction of microbial growth. The degree of lipid oxidation is decreasing with an increase in pressure as a result of inactivation of prooxidative endogenous enzymes. Fish minces become slightly lighter and softer after HP-treatment conducted at 200 MPa due to denaturation of proteins, thus enhancing sensory properties of fishcakes prepared thereof.

11P - Triple blockade of EGFR, MEK and PD-L1 as effective antitumor treatment in PD-L1 overexpressing, MEK inhibitor resistant colon cancer cells

BackgroundMolecular mechanisms driving acquired resistance to anti-EGFR therapies in metastatic Colorectal Cancer (mCRC) are complex but generally involve the activation of the downstream RAS-RAF-MEK-MAPK pathway. MethodsIn order to understand the mechanism underlying MEK inhibitor (MEKi) resistance, we generated three different MEKi resistant models, two all RAS WT (SW48-MR and LIM1215-MR) and one mutated in RAS (HCT116-MR). ResultsThese models showed features related to the gene signature of Colorectal Cancer CMS4 with upregulation of immune pathway as confirmed by Microarray and Western blot analysis. In particular, moving forward the MEKi phenotype we assisted to the loss of epithelial features and acquisition of mesenchymal markers and morphology. Moreover, this change in the morphology is accompanied by up-regulation of PD-L1 expression and activation of EGFR and its downstream pathway, independently of cell line status mutation. To extend these in vitro findings, we performed an in vivo study using MC38 and CT26 MEKi resistant syngeneic models that we have previously generated. Combined treatment of MEKi, EGFR inhibitor (EGFRi) and PD-L1inhibitor (PD-L1i) resulted in a marked inhibition of tumor growth in both MC38–MR and CT26-MR xenograft model. ConclusionsThese results suggest a strategy to potentially overcome immunotherapy resistance in CMS4-like tumors and to improve the efficacy of MEK inhibition by co-treatment with other agents providing an additional therapeutic strategy via modulation of host immune responses. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

&lt;i&gt;trans&lt;/i&gt;-2-Pentenal, an Active Compound in Cigarette Smoke, Identified &lt;i&gt;via&lt;/i&gt; Its Ability to Form Adducts with Glutathione

α,β-Unsaturated carbonyl compounds readily form adducts with SH or NH2 residues, which are nucleophilic agents, by Michael addition. Glutathione (GSH) is a tripeptide that contains an SH residue and functions as an antioxidant. We demonstrated previously that acrolein (ACR), crotonaldehyde (CA), and methyl vinyl ketone (MVK) are present in nicotine- and tar-removed cigarette smoke extract (CSE) and reacted with GSH in B16-BL6 mouse melanoma cells to form GSH-ACR, GSH-CA, and GSH-MVK adducts, suggesting a possible mechanism for CSE-induced cytotoxicity. In this study, we searched for novel α,β-unsaturated carbonyl compounds other than ACR, CA, and MVK. We selected candidate compounds in CSE based on accurate mass values generated using LC/MS analysis of products formed between CSE and GSH, and identified these using GC/MS analysis and library screening. As a result, we isolated trans-2-methyl-2-butenal, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural, which were poorly reactive with GSH and only very weakly inhibited growth of Colon-26 mouse carcinoma cells and BALB/3T3 clone A31 mouse normal cells. We also isolated 2-cyclopenten-1-one, trans-2-pentenal, 3-methyl-2-butenal and ethyl vinyl ketone, which were highly reactive with GSH and significantly inhibited the growth of both cell lines. Our data suggest that the reactivity of compounds in CSE with GSH may be positively correlated with the effect on inhibiting cell growth. Notably, trans-2-pentenal showed marked inhibition of carcinoma cells growth, whereas this compound exhibited little inhibitory effect on normal cells. trans-2-Pentenal may be a potent candidate or seed for antitumor agents.

Arsenic trioxide and tetraarsenic oxide induce cytotoxicity and have a synergistic effect with cisplatin in paclitaxel-resistant ovarian cancer cells

Background: Arsenic compounds (As2O3 and As4O6) have demonstrated anticancer effects in various malignancies. In this study, the cytotoxicity of arsenic compounds on ovarian cancer cell lines and the anticancer activity of the combination of arsenic compounds and cisplatin IN chemoresistant ovarian cancer cells were investigated.Methods: We investigated the cytotoxicity of As2O3 and As4O6 and their combinations with cisplatin in the paclitaxel-sensitive ovarian cancer cell lines SKOV3ip1 and HeyA8 and paclitaxel-resistant ovarian cancer cell lines SKOV3TRip2 and HeyA8-MDR. Growth and apoptosis were evaluated by MTT assay and annexin V assay using flow cytometry, respectively. For detection of apoptotic cells, immunofluorescence was performed using a cleaved caspase-3 antibody. Cell-cycle distribution was determined by propidium iodide staining and flow cytometry.Results: Treatment of each cell line with As2O3 or As4O6 led to a marked dose-dependent inhibition of cell growth. As2O3 and As4O6 treatment induced caspase-3-dependent apoptosis in all cell lines compared to the respective control groups (p < .05). As2O3 and As4O6 induced apoptosis of paclitaxel-sensitive and -resistant cancer cell lines following G2/M cell cycle arrest (p < .05). A synergistic effect was achieved by combining cisplatin with As2O3 or As4O6 in the paclitaxel-resistant ovarian cancer cell lines.Conclusions: As2O3 and As4O6 can inhibit cell growth and induce apoptosis in paclitaxel-sensitive and -resistant ovarian cancer cell lines. Their combination with cisplatin resulted in a synergistic effect in paclitaxel-resistant cancer cell lines. These results suggest that arsenic compounds may be given in monotherapy or combination therapy with cisplatin for treating paclitaxel-resistant ovarian cancer.

Abstract 3041: CD72 as a potential new therapeutic target in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL)

Abstract Cluster of differentiation 72 (CD72) is a 45 kDa type II transmembrane glycoprotein expressed primarily on the surface of B cells. In a search for a novel lymphoma specific target, public datasets were leveraged to assess mRNA expression. We investigated CD72 mRNA expression in cell lines (data from CCLE), patients (data from TCGA) and normal tissues (data from GTEx). The absence of CD72 in normal tissue and the presence in B cells and non-Hodgkin lymphomas (B-NHL) made CD72 a potential target for the treatment of B-NHL, mainly DLBCL and MCL. CD72 expression on the cell surface was tested by FACS in 15 DLBCL and 5 MCL cell lines. Of these, 12 DLBCL and 4 MCL were positive for CD72, while 3 DLBCL and 1 MCL showed no CD72 expression. In all cell lines tested, the cell surface expression of CD72 correlated with mRNA expression. To evaluate the potential of CD72 as an ADC target, 4 cell lines were treated in vitro with three commercial monoclonal anti-CD72 primary antibodies (clones J4-117, 3F3 and BU40), and MMAF-conjugated secondary antibody. The cell lines used were RL (DLBCL, CD72+), SCC-3 (DLBCL, CD72-), Jeko-1 and Mino (both MCL, CD72+). The cell growth inhibition after 96 hrs of treatment for for all control conditions; anti-CD72 primary antibody, IgG isotype, MMAF secondary antibody alone or combined with IgG isotype; tested in all cell lines, CD72+ or CD72- ranged from -3 to +4%. Conversely, anti-CD72 primary antibodies plus MMAF secondary antibody had marked growth inhibition effect ranged from 24 to 80% on the three CD72+ cell lines and no effect on cell growth in CD72- cell line, suggesting internalization of CD72 and growth inhibition dependent on the presence of CD72 on the cell surface. This is the first study demonstrating a potential role for CD72 as a novel target for DLBCL and MCL. These data suggest that CD72 may be a candidate for an ADC approach in DLBCL and MCL. Cell line% growth inhibition anti-CD72 + MMAF 2°Anti-CD72 clone J4-117Anti-CD72 clone 3F3Anti-CD72 clone BU40SCC-3 (CD72-)-31-5RL (CD72+)503330Jeko-1 (CD72+)433024Mino (CD72+)778066 Citation Format: Erika Von Euw, Eileen Taschereau, Da Ming Ou, Monica Mead, Sarah Larson, Dennis Slamon. CD72 as a potential new therapeutic target in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) [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 3041.

Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidine

Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines’ sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.

Metabolic reprogramming toward oxidative phosphorylation identifies a therapeutic target for mantle cell lymphoma.

Metabolic reprogramming is linked to cancer cell growth and proliferation, metastasis, and therapeutic resistance in a multitude of cancers. Targeting dysregulated metabolic pathways to overcome resistance, an urgent clinical need in all relapsed/refractory cancers, remains difficult. Through genomic analyses of clinical specimens, we show that metabolic reprogramming toward oxidative phosphorylation (OXPHOS) and glutaminolysis is associated with therapeutic resistance to the Bruton's tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma (MCL), a B cell lymphoma subtype with poor clinical outcomes. Inhibition of OXPHOS with a clinically applicable small molecule, IACS-010759, which targets complex I of the mitochondrial electron transport chain, results in marked growth inhibition in vitro and in vivo in ibrutinib-resistant patient-derived cancer models. This work suggests that targeting metabolic pathways to subvert therapeutic resistance is a clinically viable approach to treat highly refractory malignancies.

Naphthyl quinoxaline thymidine conjugate is a potent anticancer agent post UVA activation and elicits marked inhibition of tumor growth through vaccination

Anticancer anthracyclines are cytotoxic drugs that can induce antitumor immune response as a secondary effect through immunogenic cell death (ICD) mechanism. However, the immunogenic potency is quite limited, possibly due to that these chemotherapeutic agents are not specifically developed as ICD inducers. Thus, new drug entities through studies focusing on enhanced ICD induction would significantly promote antitumor immune response in the vaccination application. We report here a naphthyl quinoxaline thymidine conjugate as a new class of cytotoxic compounds that effectively induced in vivo antitumor activity through the vaccination application. Synthesized naphthyl quinoxaline conjugates were weak fluorescent thymidine analog yet exhibited a pronounced anticancer activity in the low nanomolar range post UVA activation. The potent activity of naphthyl conjugate was able to induce the marked detection of ICD markers including ATP and HMGB1 extracellular and calreticulin intracellularly at 2 h post UVA activation. Most importantly, mice vaccinated with cells treated with naphthyl conjugate plus UVA exhibited complete tumor growth inhibition in the tumor challenge study, and the induced immunogenic inhibition was much more effective than that of mitoxantrone anthracycline drug. All these results demonstrate the high potential of naphthyl quinoxaline conjugate for the cancer cell vaccine against tumor.

Synthetic miR-143 Exhibited an Anti-Cancer Effect via the Downregulation of K-RAS Networks of Renal Cell Cancer Cells InVitro and InVivo.

To understand the role of RAS-signaling networks in the pathogenesis of renal cell carcisnoma, we clarified the relationship between miR-143 and RAS. The expression of miR-143 was extremely downregulated in tumor tissues from renal cell carcinoma patients compared with that in the adjacent normal tissues and Caki-1 cells. We developed a synthetic miR-143#12, and we found that the ectopic expression of it inhibited cell growth with autophagy in Caki-1 cells. Also, the expression level of c-Myc was markedly decreased, resulting in the perturbation of cancer-specific energy metabolism by negatively modulating the expression of GLUT1 and the PTBP1/PKMs axis. A partial metabolic shift from glycolysis to oxidative phosphorylation induced autophagy through increasing the intracellular level of reactive oxygen species (ROS). In an in vivo study, the potent anti-tumor activity of polyion complex (PIC)-loaded miR-143#12 (miR-143#12/PIC) was shown by systemic administration of it to Caki-1 cell-xenografted mice. Higher levels of miR-143 were found in both blood and tumor tissues after the systemic administration with miR-143#12/PIC compared to those with lipoplexes in the xenografted mice. These findings indicated that this synthetic miR-143#12 induced a marked growth inhibition by impairing K-RAS-signaling networks in vitro and in vivo.

Anti-cancer Effects of a Chemically Modified miR-143 on Bladder Cancer by Either Systemic or Intravesical Treatment

We developed a novel chemically modified miR-143 (miR-143#12), and with it we investigated the contribution of miR-143 to the pathogenesis of bladder cancer (BC), in which miR-143 is extremely downregulated. Since miR-143 silenced K-RAS and RAS effector-signaling molecules Erk and Akt, we performed the ectopic expression of miR-143 in human BC 253J-BV cells, and we examined the growth inhibition and the mechanism of it in vitro and in orthotopic model mice. As a result, miR-143#12 induced a marked growth inhibition with apoptosis through impairing RAS-signaling networks, including SOS1, which exchanges guanosine diphosphate (GDP)/RAS for active guanosine triphosphate (GTP)/RAS. In the in vivo study, miR-143#12 exhibited a marked anti-tumor activity by either systemic or intravesical administration with polyionic copolymer (PIC) as the carrier, compared with the activity obtained by use of lipofection. These findings raised the possibility that the chemically modified miR-143#12 would be a candidate of microRNA (miRNA) medicine for BC delivered by intravesical infusion.

Kindlin-2 links mechano-environment to proline synthesis and tumor growth

Cell metabolism is strongly influenced by mechano-environment. We show here that a fraction of kindlin-2 localizes to mitochondria and interacts with pyrroline-5-carboxylate reductase 1 (PYCR1), a key enzyme for proline synthesis. Extracellular matrix (ECM) stiffening promotes kindlin-2 translocation into mitochondria and its interaction with PYCR1, resulting in elevation of PYCR1 level and consequent increase of proline synthesis and cell proliferation. Depletion of kindlin-2 reduces PYCR1 level, increases reactive oxygen species (ROS) production and apoptosis, and abolishes ECM stiffening-induced increase of proline synthesis and cell proliferation. In vivo, both kindlin-2 and PYCR1 levels are markedly increased in lung adenocarcinoma. Ablation of kindlin-2 in lung adenocarcinoma substantially reduces PYCR1 and proline levels, and diminishes fibrosis in vivo, resulting in marked inhibition of tumor growth and reduction of mortality rate. Our findings reveal a mechanoresponsive kindlin-2-PYCR1 complex that links mechano-environment to proline metabolism and signaling, and suggest a strategy to inhibit tumor growth.

Abstract P2-02-03: FASN inhibition as a potential treatment for therapy of endocrine resistant breast cancer

Abstract INTRODUCTION: Fatty acid synthase (FASN) is a key enzyme in tumor cell biology controlling endogenous lipid biosynthesis. It is overexpressed in a biologically aggressive subset of tumors, including breast carcinoma. We previously reported prolonged stabilization of disease with TVB-2640 in patients with advanced metastatic breast cancer, including some endocrine resistant ER+ tumors. Using in vitro and in vivo models, we assessed the role of FASN inhibition by TVB-3166 (preclinical version of TVB-2640) for treatment of endocrine resistant breast cancer. METHODS: Breast tumor cells were incubated with TVB-3166 (200nM), imaged and analyzed by automated Live-Cell analysis system (IncuCyte). For tumor growth inhibition, cells (2X106)were subcutaneously injected into SCID mice implanted with estrogen pellets. Once tumors were measurable, mice were divided into treatment groups: tamoxifen (4mg/kg), TVB-3166 (60mg/kg) and the combination. Patient tumor explants were incubated for 72h on gelatin sponges in culture medium in the absence or presence of 200nM TVB-3166. Tissue were fixed in 10% formalin and processed into paraffin blocks. Sections were stained with H&amp;E, ERα and Ki67. RESULTS: The effectiveness of FASN inhibition on the growth of tumor cells has been confirmed in a number of breast cancer cell lines such as MCF7, ZR75, MDA-MB-231 and others. TVB-3166 leads to a marked inhibition of growth in tamoxifen resistant (TamR) cells, which 15% greater than in the parent line. IHC and Western blot showed FASN inhibition leads to significantly reduction of ERα levels. Immunofluorescent confocal microscopy showed inhibition of FASN by TVB-3166 alters subcellular localization of ERα. TVB-3166 was able to significantly inhibit tamoxifen resistant breast tumor growth in mice (p&amp;lt;0.05). Additionally, TVB-3166 treatment of primary tumor explants decreased their proliferation (Ki67) compared to untreated controls (21% vs 38%, p&amp;lt;0.01). CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treating of endocrine resistant breast cancer. RNA sequencing of tumor explants is being performed to evaluate FASN inhibition impact on canonical and non-canonical ERα signaling pathways. Citation Format: Gruslova A, Sareddy GR, Vadlamudi RK, Viswanadhapalli S, Brenner A. FASN inhibition as a potential treatment for therapy of endocrine resistant breast cancer [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 P2-02-03.

Dimethyl fumarate and vitamin D derivatives cooperatively enhance VDR and Nrf2 signaling in differentiating AML cells in vitro and inhibit leukemia progression in a xenograft mouse model

Acute myeloid leukemia (AML) is one of the deadliest hematological malignancies without effective treatment for most patients. Vitamin D derivatives (VDDs) - active metabolites 1α,25-dihydroxyvitamin D2 (1,25D2) and 1α,25-dihydroxyvitamin D3 (1,25D3) and their analogs - are differentiation-inducing agents which have potential for the therapy of AML. However, calcemic toxicity of VDDs limits their clinical use at doses effective against cancer cells in vivo. Here, we demonstrate that in AML cell cultures, moderate pro-differentiation effects of low concentrations of VDDs can be synergistically enhanced by structurally distinct compounds known to activate the transcription factor Nuclear Factor (Erythroid-derived 2)-Like 2 (NFE2L2 or Nrf2). Particularly, dimethyl fumarate (DMF), which is clinically approved for the treatment of multiple sclerosis and psoriasis, strongly cooperated with 1,25D3, PRI-5100 (19-nor-1,25D2; paricalcitol) and PRI-5202 (a double-point modified 19-nor analog of 1,25D2). The pro-differentiation synergy between VDDs (1,25D3 or PRI-5202) and Nrf2 activators (DMF, tert-butylhydroquinone or carnosic acid) was associated with a cooperative upregulation of the protein levels of the vitamin D receptor (VDR) and Nrf2 as well as increased mRNA expression of their respective target genes. These data support the notion that VDDs and Nrf2 activators synergize in inducing myeloid cell differentiation through the cooperative activation of the VDR and Nrf2/antioxidant response element signaling pathways. We have previously reported that PRI-5202 is more potent by approximately two orders of magnitude than 1,25D3 as a differentiation inducer in AML cell lines. In this study, we found that PRI-5202 was also at least 5-fold less calcemic in healthy mice compared to both its direct precursor PRI-1907 and 1,25D3. In addition, PRI-5202 was remarkably more resistant against degradation by the human 25-hydroxyvitamin D3-24-hydroxylase than both 1,25D2 and 1,25D3. Importantly, using a xenograft mouse model we demonstrated that co-administration of PRI-5202 and DMF resulted in a marked cooperative inhibition of human AML tumor growth without inducing treatment toxicity. Collectively, our findings provide a rationale for clinical testing of low-toxic VDD/DMF combinations as a novel approach for differentiation therapy of AML.

The BET Inhibitor INCB054329 Primes AML Cells for Venetoclax-Induced Apoptosis

Bromodomain and extra-terminal (BET) inhibitors may be efficacious for treatment of acute myeloid leukemia because they attenuate the expression of critical oncogenes including MYC and BCL2. These BET inhibitors (BETi) disrupt the transcriptional elongation process by displacing BET family members BRD2,3, and 4 off of chromatin, and causing RNA polymerase promoter-proximal pausing. We used precision nuclear run-on transcription sequencing (PROseq) to directly measure the effects of INCB054329, a potent BETi, on RNA polymerase II pausing and elongation. We found dramatic reductions on the elongation of key oncogenes such as MYC and BCL2 within 15 min of adding the drug. These effects became more significant over time, eventually affecting nearly two thousand genes. By four hours after drug addition, we found a loss of ribosomal gene expression and a loss of mitochondrial gene expression that is characteristic of genes regulated by MYC, suggesting that these were secondary to turning off MYC expression. When we examined the potential of the BETi INCB054329 for therapeutic efficacy in AML using Alamar Blue assays, which measure cellular redox potential, we noted marked growth inhibition of AML cell lines. However, growth assays and measurements of apoptosis using Annexin V staining found that BETi induced minimal apoptosis and cells were largely cytostatic. BrdU incorporation assays showed that INCB054329 caused the cells to accumulate in the G0/G1 phase of the cell cycle. Metabolic studies indicated that INCB054329 treatment for 48 hours caused disruption of mitochondrial respiration rate and severely reduced glycolytic capacity. Taken together, the growth inhibition, cell cycle arrest and reduced metabolic rate suggests that INCB054329 promoted quiescence in AML cells, but that this is reversible, consistent with the clinical experience of single-agent treatment of hematologic malignancies with BETi.MLL fusion proteins enhance transcription by stimulating RNA polymerase elongation, suggesting INCB054329may provide a therapeutic option to reverse this effect. However, the cell cycle arrest suggested that a second compound may be needed to trigger cell death. We first performed in vivo studies with INCB054329 using a systemic AML xenograft model of MV4-11 cells that express MLL-AF4. Engrafted NSGS mice received INCB054329 in 3 different doses (vehicle vs 10, 30 and 75mg/kg q.d) daily. During treatment, the kinetics of MV-4-11 expansion was monitored via flow cytometry for the detection of human AML in the blood. At approximately 4 weeks after transplant, the vehicle mice became moribund, and all experimental groups were sacrificed for analysis of chimerism. Significant decreases in leukemic expansion were evident in the bone marrow (vehicle vs75mg/kg, p<.001) and spleen (vehicle vs. 75mg/kg, p <.001) of treated mice. As BETi decreases expression of BCL2, we posited that BH3 directed therapy with the BCL-2 inhibitor venetoclax (VEN) could be enhanced by INCB054329. In vitro, we found that the combination of INCB054329 and VEN resulted in significant growth inhibition and apoptosis of treated AML cells. This finding prompted us to test the combination of INCB054329 with VEN in vivo. Mice engrafted with human AML cells received INCB054329 (50mg/kg q.d), VEN (25mg/kg q.d) or the combination. Four weeks after transplant, analyses by flow cytometric measurement of human CD45 of combination treated mice revealed significant decreases of AML cells in the bone marrow (vehicle vs. BRDi/VEN p = 0.004) and spleen (vehicle vs.BRDi/VEN, p = 0.001). Further studies are underway to test this combination in both VEN sensitive and resistant AML primary xenograftmodels. These preliminary data suggest that INCB054329 may serve as a non-cytotoxic priming agent for BH3 directed therapy, and the combination of INCB054329 +VEN may provide a potent therapy in a variety of genetically distinct subtypes of AML. DisclosuresStubbs:Incyte: Employment. Liu:Incyte: Employment. Rathmell:Calithera: Research Funding. Hiebert:Incyte: Research Funding. Savona:Boehringer Ingelheim: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding.