Robust Anti-proliferative Activity Research Articles

Abstract 1961: Discovery of a highly potent and brain penetrant fourth generation EGFR tyrosine kinase inhibitor

Abstract Third generation EGFR tyrosine kinase inhibitors (TKIs) such as Osimertinib have emerged as first-line standard of care therapy to treat non-small cell lung cancer (NSCLC) patients with activating EGFR mutations (del19, L858R or T790M). However, within 12 to 18 months following treatment, 15-20% of the patients develop C797S mutation, which drives resistance to these drugs; with the progression of disease brain metastasis is observed in 40-70% of the EGFRm+ NSCLC patients. Here, we report a highly potent and brain penetrant fourth generation EGFR-TKI ACE-34237307, designed to overcome C797S mutation (del19/C797S and L858R/C797S), and common single point mutations (del19 and L858R). ACE-34237307 is also designed to potently inhibit the more prevalent non-canonical EGFR mutations such as G719S and L861Q. ACE-34237307 showed robust anti-proliferative activity against EGFR double mutations in Ba/F3 (EGFR-del19/C797S and EGFR-L858R/C797S) cell lines with IC50 values of 0.1 and 0.23 nM, respectively. In phospho-EGFR assay, ACE-34237307 demonstrates over 79-fold selectivity against A431 (EGFR-WT) cell line when compared to PC-9 (EGFR-del 19) cell line. ACE-34237307 is highly brain penetrant with B/P ratio of 2.3 in rat and 1.2 in mouse. It has an excellent pharmacokinetic profile in rat, mouse and dog with good bioavailability, a flat PK curve and reduced Cmax, which will translate to a better safety profile. The in vivo anti-tumor efficacy was evaluated in Osimertinib resistant double mutant Ba/F3-EGFR-del19/C797S and L858R/C797S as well as del19 and L858R single mutant xenograft models. It has been shown that ACE-34237307 at doses of ≥ 0.6 mg/kg induces complete tumor regression or eradication in all tumor models tested. In brain tumor models with Ba/F3 (EGFR-del19/C797S) and PC-9 (EGFR-del19), ACE-34237307 showed complete inhibition or eradication of brain tumors and drastically extended animal survival at ≥ 0.6 mg/kg. In summary, ACE-34237307 is a potent fourth generation EGFR-TKI with high WT selectivity, excellent brain penetration, highly favorable DMPK profile, outstanding in vivo efficacy in subcutaneous and brain tumor models and low projected human dose amenable to QD dosing. Citation Format: sanjeev kumar, junmei wang, wenqian li, weitao pan, zhiming wen, Kuo-Long yu, Genshi Zhao, tinggui yin. Discovery of a highly potent and brain penetrant fourth generation EGFR tyrosine kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1961.

In vitro and vivo anti-tumor activity and mechanisms of the new cryptotanshinone derivative 11 against hepatocellular carcinoma

Global burden of hepatocellular carcinoma (HCC) is increasing. Chemotherapy and immunotherapy are the prevailing options for therapy. Developing new therapeutic strategies for HCC patients is still highly desirable. Recent studies demonstrate that cryptotanshinone is capable of inhibiting tumor growth in HCC and induces antitumor immunity in vitro. In our previous research, we discovered a new cryptotanshinone derivative 11 as an effective immunoregulatory enzyme indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitor. This study aims to evaluate its in vitro and in vivo antitumor activity against hepatocellular carcinoma. 11 displayed robust anti-proliferative activity against HCC cell lines and promoted apoptosis of HCC cell line through the mitochondrial-mediated apoptotic pathway. In H22 tumor-bearing mice models, 11 exhibited significant in vivo anti-tumor activity with different administration routes. And no obvious toxicity was observed. RNA-seq analysis demonstrated the differential expressed genes and alteration of key pathways associated with immune responses after administration of 11. Up-regulation of anti-tumor cytokines and down-regulation of cytokines that promote tumor growth were indicated and further validated. Our study demonstrates that 11 exhibits promising anti-tumor activity both in vitro and in vivo against hepatocellular carcinoma cancer. It is a lead compound for HCC immunotherapy and is worthy for further development.

Abstract A016: NXP800, a novel, small molecule GCN2 kinase activator, demonstrates potent single-agent activity in ARID1A and ARID1B-deficient endometrial cancer xenograft models

Abstract Background: NXP800 is a clinical stage, antineoplastic, oral, small molecule GCN2 kinase activator that was discovered in a phenotypic screen for inhibitors of the heat shock factor 1-regulated stress response. In a panel of human carcinoma cell lines NXP800 induced the expression of genes associated with activation of the integrated stress response and demonstrated robust antiproliferative activity, particularly in ARID1a-mutated ovarian carcinoma cells lines. In ARID1a-mutated ovarian carcinoma xenografts, treatment with NXP800 resulted in substantial tumor growth inhibition and tumor regression. NXP800 is currently being investigated in a Phase 1b clinical trial in patients with ARID1a-mutated ovarian carcinoma. Here we describe an in vivo study of NXP800 in ARID1a/ARID1b-deficient endometrial cancer xenografts, supporting the clinical development of NXP800 in this indication. Methods: Animal strain: CD1 Nude mice (nu/nu, Charles River). Human endometrial cancer cell lines: RL95-2 (ARID1a mutated, ATCC) and KLE (ARID1a wildtype, ARID1b mutated, ATCC), SNG-M (ARID1a mutated, Creative Bioarray). Xenograft tumors were generated by subcutaneous implantation on the right lower flank of the thigh at a cell density of 2 × 106 cells/mouse, at 0.1 ml Matrigel dilution volume/injection. Experiment groups: Vehicle, NXP800 (35 mg/kg); treatment: QD on days 0-4, 7-11, 14-18, 21-25, 28-30. Loss of ARID1A protein expression was confirmed by western blots. Results: In the SNG-M model, baseline tumor volume for the vehicle and NXP800 groups were 130.2, and 139.7 mm3, respectively, and on Day 32, average tumor volumes were 420.4, and 85.4 mm3, respectively, representing TGI of 81%. In the RL-95 model, baseline tumor volume for the vehicle and NXP800 groups were 124.5, and 129.9 mm3, respectively, and on Day 32, average tumor volumes were 773.8, and 190.3 mm3, respectively, representing TGI of 76%. In the KLE model, baseline tumor volume for both the vehicle and NXP800 groups were 88.1 mm3, respectively, and on Day 32, average tumor volumes were 216.4, and 18.9 mm3, for the vehicle and NXP800 groups, respectively, representing TGI of 91.3%. Conclusions: ARID1A and ARID1B genes encode the alternate, but obligatory, DNA-targeting subunit of the switch/sucrose non-fermentable (SWI/SNF) complex. ARID1A is the most frequently mutated SWI/SNF subunit across cancer types and is mutated in approximately 35% of endometrial carcinomas. NXP800 demonstrated robust antitumor activity in xenografts of endometrial carcinoma, a serious condition for which new treatment options are needed, including sustained tumor growth inhibition both in ARID1A and ARID1B mutated models, supporting the clinical development of NXP800 in endometrial cancer. Citation Format: Ramez N. Eskander, Bradley J. Monk, Brian M. Slomovitz, Enrique Poradosu, Allison Woods, Shay Shemesh, Paul Clarke, Robert Te Poele, Marissa Powers, Paul Workman, Shannon N. Westin. NXP800, a novel, small molecule GCN2 kinase activator, demonstrates potent single-agent activity in ARID1A and ARID1B-deficient endometrial cancer xenograft models [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A016.

Celmod Induced Disruption of Granulopoiesis Continuum Identified Using Humanized Cereblon Mice

Introduction: CRBN mediated degradation of Ikaros by Pomalidomide (Pom) and Mezigdomide (Mezi) results in robust antiproliferative activity against myeloma cells, with concomitant activation of immune cells such as T and NK cells. However, degradation of Ikaros has been shown to mediate maturation arrest of neutrophils leading to neutropenia. Unlike chemotherapy drugs, CELMoD driven neutropenia is an on-target adverse effect. Although the next generation cereblon E3 ligase modulators are potent and highly selective, neutrophil biased adverse effects remain persistent. Here, we sought to define the impact of Ikaros degradation through a longitudinal assessment of granulopoiesis in the bone marrow of humanized CRBN mice treated with Pom and Mezi. Methods: We have performed multi-parametric studies leveraging a humanized CRBN (hCRBN) C57BL/6 mouse model. Driven by the endogenous mouse CRBN promoter, human CRBN was inserted into the mouse CRBN gene, preventing expression of mCRBN. These hCRBN mice are capable of specific degradation of CELMoD mediated substrates, thus enabling an in vivo model to understand pathophysiological mechanisms. Homozygous hCRBN mice were treated daily with Pom and Mezi via oral gavage until days 7, 14 and 21 when mice were euthanized. Peripheral blood and bone marrow were collected for flow cytometric and molecular analyses. Critical population classes such as multipotent progenitors, lineage committed progenitors, pre-myelocytes, immature neutrophils and mature neutrophils were defined by immunophenotypes, and pharmacodynamic parameters such as Aiolos/Ikaros degradation were evaluated. All data is represented as fold change over median of vehicle control. Results: The hCRBN mouse model was able to recapitulate Pom and Mezi driven clinical neutropenia. Peripheral absolute neutrophil count gradually decreased with increased dosing (QD7, QD14 and QD21) in mice treated with Pom (26%, 45% and 74%) and Mezi (35%, 57% and 70%). Treatment with Pom and Mezi resulted in disruption of early hematopoietic multi-progenitor populations leading to a widespread effect at multiple stages of haematopoiesis. Intriguingly, in bone marrow these effects were both temporally differential for multi-potent progenitors, and across various stages of neutrophil maturation. Myeloid biased, MPP3 population increased to ~55% and ~103% at QD7, followed by a steady decline, renormalizing to baseline at QD14 and QD21 for Pom and Mezi respectively. The first myeloid lineage committed progenitors, CMP population, reduced to 21% (Pom) and 42% (Mezi) at QD7 stabilizing at ~17-21% below baseline by QD21. In contrast GMP population, descendants from CMP, increased to ~50% (Pom) and ~117% (Mezi) at QD7. For Pom treatment (but not with Mezi), GMP proportions renormalized to baseline at QD21. Pre-myelocytes, one of the earliest neutrophil maturation stages, shrunk to ~24-30% at QD21 without any differences between Pom and Mezi. On the other hand, immature neutrophils steadily diminished to 51% (Pom) and 70% (Mezi) at QD21. This effect was more pronounced in mice treated with Mezi. Mature neutrophils in bone marrow, part of a highly dynamic reservoir that extravasates into blood, increased to 10-18% at all the time points for both Pom and Mezi. Overall, impairment of granulopoiesis is more profound in hCRBN mice treated with Mezi than Pom, owing to the more potent Ikaros degradation. Mechanistically, degradation of neo-substrates such as Ikaros affect the myelopoiesis and neutrophil maturation but are likely not the sole drivers. Addition of corticosteroids such as dexamethasone have been shown to mitigate IMiD/CELMoD mediated neutropenia in the clinical setting. Mezi treated animals who were co-administered dexamethasone demonstrated partially enhanced early progenitor populations compared to single agent Mezi. To investigate patterns of Ikaros transcriptional control, specific populations were flow sorted in parallel, for focused molecular analyses such as RNAseq using the SMARTseq platform and will be included in the presentation. Conclusion: These studies delineate previously unreported effects of CELMoDs on a multifaceted continuum of myelopoiesis. These insights will help rationally design dose and schedule considerations for CELMoD treatment regimens alone, or in combination with other therapeutic agents to alleviate CELMoD induced neutropenia.

Design of novel analogues of t-DPH1 with reduced cytotoxicity, taking the three conserved characteristics of the dermaseptin family as the feasible starting point

In recent years, there has been growing scientific interest in balancing the bioactivity and cytotoxicity of antimicrobial peptides (AMPs) for more comprehensive research and application. In this study, we focused on t-DPH1, a representative member of the dermaseptin family, as a template. We synthesized five analogues were synthesised to to assess the impact of three conserved features of the dermaseptin family (1. the tryptophan residue at 3rd position, 2. the consensus motif at mid-region, and 3. C-terminal amidation) on the bioactivity of t-DPH1 and their potential to reduce cytotoxicity. Our results demonstrated that analogues lacking an amino group at the C-terminus, specifically t-DPH1-NH2, exhibited potent antimicrobial activity against Gram-negative bacteria, including Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). Furthermore, t-DPH1-NH2 retained its robust antiproliferative activity against the human non-small lung cancer cell line H157. Regarding cytotoxicity, t-DPH1-NH2 displayed lower levels of haemolytic activity and cytotoxicity against two normal cell lines. Notably, we employed C. elegans as an in vivo model to assess the cytotoxicity of peptides, and the results revealed no induction of in vivo toxicity by t-DPH1-NH2. Collectively, t-DPH1-NH2 provides valuable insights for further investigation as promising bioactive peptide templates in the development of antibiotic and anticancer prototype drugs. The modifications implemented based on the conserved structure of the peptide family offer a new direction for reducing the cytotoxicity of peptides.

Abstract P2-24-04: The ER antagonist giredestrant induces profound chromatin remodeling including activation of cis-regulatory elements bound by FOXA1 and GATA3 in HR+ breast cancer models

Abstract The progression of HR+ breast cancers is governed by ER signaling which makes the development of optimized ER-targeting agents a high priority. Among current therapeutics, selective ER antagonists and degraders (SERDs) have emerged as a particularly attractive class. Fulvestrant is the only SERD that is currently approved, but its clinical potential is hypothesized to be somewhat limited by poor physicochemical properties, dosage and exposure. We identified giredestrant as an orally bioavailable, highly potent non-steroidal ER antagonist, immobilizer and degrader with robust anti-proliferative activity. Giredestrant is currently being evaluated in phase III clinical trials for the treatment of ER+ breast cancer, after showing evidence of clinical activity in phase I and II trials. Here, we set out to investigate the impact of giredestrant on the transcriptome and chromatin landscape of ER+ breast cancer cells in vitro and in vivo. RNA-seq data from MCF-7 cells in vitro and three patient-derived xenograft (PDX) models in vivo showed that giredestrant inversely regulates the RNA levels of E2-regulated genes. Transcriptome wide, the expression of genes affected by giredestrant are strongly correlated (r2=0.8) with those affected by siRNA-mediated silencing of ESR1, consistent with on-target ER antagonism. Performing ATAC-seq in the three PDX models, including one ESR1 mutant model, reveals that giredestrant reduces accessibility in up to 18,173 chromatin regions that are associated with genes induced by E2. These regions are also enriched in the ER binding motif. In addition, we found up to 17,127 chromatin regions with increased accessibility upon giredestrant treatment. These regions are associated with genes suppressed by E2, and are enriched in the FOXA1 binding motif. To probe giredestrant’s role in functional modulation of cis-regulatory elements (CREs), we performed ChIP-seq targeting FOXA1 and GATA3, as key pioneer factors for ER, as well as ER itself and H3K27ac as a marker of active enhancers in MCF-7 cells. We identified 3,195 regions with significantly reduced H3K27ac signals that also exhibit significant reduction of both ER and FOXA1 binding upon giredestrant treatment, indicating that giredestrant deactivates these CREs concomitantly with suppressing ER and FOXA1 binding. ChIP-seq analysis further revealed 2,292 regions with a significant increase in H3K27ac signals upon giredestrant treatment. Interestingly, the GATA3 motif was identified as the most enriched motif in these activated CREs. Further, GATA3 and FOXA1 ChIP-seq signals were stronger in the activated CREs compared to the deactivated ones. Similar profiling of both tamoxifen, an approved selective ER modulator (SERM), as well an GNE-858, a SERM from the same chemical series as giredestrant, showed their epigenetic effects to be highly distinct from those observed for giredestrant, suggesting that the high degree of chromatin remodeling is likely related to giredestrant’s mechanism of action. Our studies here show that giredestrant profoundly alters ER binding and remodels the chromatin landscape, thus changing the transcriptional profile of cells in a manner that is associated with an anti-proliferative response. While deactivation of CREs with ER binding is perhaps in line with expectations, giredestrant treatment also leads to the activation of a number of regions associated with pioneer factors, potentially through their redistribution. This raises the question of the functional consequences of CRE activation and if this is relevant for the anti-proliferative effects of giredestrant. Citation Format: Musaddeque Ahmed, Jane Guan, Wei Zhou, Xiaosai Yao, Ciara Metcalfe, Marc Hafner. The ER antagonist giredestrant induces profound chromatin remodeling including activation of cis-regulatory elements bound by FOXA1 and GATA3 in HR+ breast cancer models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-04.

Design, synthesis, and discovery of Eudistomin Y derivatives as lysosome-targeted antiproliferation agents

Eudistomin Y is a novel class of β-carbolines of marine origin with potential antiproliferation activity against MDA-MB-231 cells (triple-negative breast carcinoma). However, the subcellular target or the detailed mechanism against cancer cell proliferation has not yet been identified. In this study, based on its special structure, a novel series of Eudistomin Y fluorescent derivatives were designed and synthesized by enhancing the electron-donor effect of N-9 to endow it with fluorescent properties through N-alkylation. The structure–activity relationships against the proliferation of cancer cells were also analyzed. A quarter of Eudistomin Y derivatives showed much higher potency against cancer cell proliferation than the original Eudistomin Y1. Fluorescent derivative H1k with robust antiproliferative activity could arrest MDA-MB-231 cells in the G2–M phase. The subcellular localization studies of the probes, including H1k, and Eudistomin Y1 were performed in MDA-MB-231 cells, and the co-localization and competitive inhibition assays revealed their lysosome-specific localization. Moreover, H1k could dose-dependently increase the autophagy signal and downregulate the expression of cyclin-dependent kinase (CDK1) and cyclin B1 which principally regulated the G2–M transition. Furthermore, the specific autophagy inhibitor 3-methyladenine significantly inhibited the H1k-triggered antiproliferation of cancer cells and the downregulation of CDK1 and cyclin B1. Overall, the lysosome is identified as the subcellular target of Eudistomin Y for the first time, and derivative H1k showed robust antiproliferative activity against MDA-MB-231 cells by decreasing Cyclin B1–CDK1 complex via a lysosome-dependent pathway.

95 (PB085) - Preliminary results from FLAGSHP-1: A Phase I dose escalation study of ERAS-601, a potent SHP2 inhibitor, in patients with previously treated advanced or metastatic solid tumors

Background: SHP2 is an oncogenic tyrosine phosphatase that transduces receptor tyrosine kinase signaling to the RAS/MAPK pathway via its phosphatase-mediated regulation of guanine nucleotide exchange factors. ERAS-601 is a potent, selective, and orally bioavailable allosteric inhibitor of SHP2 that has demonstrated robust anti-proliferative activity in vitro and in vivo as monotherapy and in combination in preclinical models of cancer harboring EGFR, KRAS, BRAF class III and NF1LOF mutations.

Novel biphenyl-based scaffold as potent and selective histone deacetylase 6 (HDAC6) inhibitors: Identification, development and pharmacological evaluation

A series of novel biphenyl-based scaffold derivatives were identified as selective histone deacetylase 6 (HDAC6) inhibitors through an in-house compound library screening approach. The biological evaluation indicated that most of target compounds exhibited moderate to good inhibitory activity and selectivity against HDAC6. Especially, compound C10 was identified as a potent and highly selective HDACs inhibitor, with HDAC1 IC50 value of 3600 nM, HDAC6 IC50 value of 23 nM, and the HDAC1/6 selectivity index of 157. Moreover, C10 displayed robust anti-proliferative activity, induced cancer cells apoptosis, increased the level of acetylated α-tubulin and inhibited cancer cells migration in vitro. C10 showed significant antitumor efficacy (TGI: 75%) in CT26 colon carcinoma xenograft model in mice with no considerable toxicity in vivo. More importantly, C10 could also activate antitumor immunity so as to synergistically exert antitumor effects in vivo. Overall, our findings have provided a new avenue for design, development and investigation into the mechanism underlying the antitumor efficacy of selective HDAC6 inhibitors.

A Novel Compound Plumercine from Plumeria alba Exhibits Promising Anti-Leukemic Efficacies against B Cell Acute Lymphoblastic Leukemia

The current study was focused to evaluate the antioxidant and the anti-cancerous properties of Plumeria alba. Plumeria alba was chosen due to its existing medicinal values. Antioxidant assays like Superoxide radical scavenging assay, Nitrous Oxide radical scavenging assay, were performed, on the methanolic and ethyl acetate extracts of the plant, that depicts the pro-oxidant nature of the extract. Further, they were tested to check cell viability on B cell Acute Lymphoblastic Leukemia (ALL) Cell line (NALM 6), human lung cancer cell line (A549), T cell Acute Lymphoblastic Leukemia cell line (MOLT4), and PBMC isolated from normal donors utilizing MTT assay. Robust anti-proliferative activity was observed in the case of NALM 6 followed by A549, MOLT4, whereas negligible activity was observed in the case of PBMC. Intrigued by this finding, in silico docking was performed using three bioactive compounds namely Plumericine, Isoplumericine, and 13-O-p-Coumaroylplumieride, unique to Plumeria sp. They were docked against five different cyclins and Cdk proteins responsible for ALL. The compounds have shown satisfactory results and their druggability and ADMET properties were checked further. Plumercine turned out to be the most competent compound and hence can be considered as a potential leukemic drug candidate in the future.

Antiproliferative activity of two copper (II) complexes on colorectal cancer cell models: Impact on ROS production, apoptosis induction and NF-κB inhibition

The main goal of this work was to screen the antiproliferative activity and mechanism of actions of two copper complexes: [Cu(dmp)2(CH3CN)]2+ (1) and [Cu(phen)2(CH3CN)]2+ (2) on 2D and 3D colorectal cancer cells models. Cell viability studies on three colorectal cancer cell lines (HT-29, LS174T, Caco-2) displayed that 1 showed more robust antiproliferative activity than 2 and cisplatin. Intracellular copper content (63.24% and 48.06% for 1 and 2, respectively) can explain the differences in the cytotoxicity assay.ROS production is the primary mechanism of action involved in the antiproliferative activity of 1 showing 4-, 70- and 2.5- fold increased values of ROS level for HT-29, LS174T, Caco-2 cancer cell lines, respectively. This effect takes place along with the depolarization of the mitochondrial membrane at 2 µM. Besides, both complexes increased apoptosis on three cancer cell lines at low micromolar concentrations (0.5–2.5 μM). Moreover, 1 and 2 inhibited NF-κB pathway both in HT-29-NF-kB-hrGFP monolayer (0.5 to 1 μM) and spheroids HT-29 GFP (5 to 10 μM). This inhibitory effect leads to an inactivation of the MMP-9 expression on HT-29 cell line.Altogether, these results showed that 1 exhibits antiproliferative activity on human colorectal cancer cells in the monolayer and the 3D model.

Study of the antilymphoma activity of pracinostat reveals different sensitivities of DLBCL cells to HDAC inhibitors

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

GC-MS Analysis and Screening of Anti-Proliferative Potential of Methanolic Extract of Garcinia cowa on Different Cancer Cell Lines

Introduction: Garcinia cowa (Clusiaceae) is popular among integrative medicine in several communities. This study undertook to evaluate the anti-proliferative activity on cancer cells and its cytotoxic effect on normal cells. Here we are reporting for the first time the metabolomic profiling of G. cowa leaf. Methods: Anti-proliferative potential of ethyl acetate and methanol extract of Garcinia cowa leaf assessed by MTT assay. Metabolomic profiling obtained by GC/ MS analysis. Nuclear morphology visualized by DAPI staining. Caspase activation analysed through spectrophotometric assay. Results: The study reveals, that the methanolic extract is more potential in inducing anti-proliferative activity than ethyl acetate extract. Robust antiproliferative activity of the methanolic extract evidenced in lung cancer cell line, A549 followed by MCF–7, HepG2, MOLT – 4, MDA-MB-468 cells. The anti-proliferative effect was negligible in normal PBMC. Further, a dose-dependent increase of nuclear fragmentation visualized in A549 cells treated with the methanolic extract. Post methanolic extract treatment upregulation of caspase-3 and caspase-9 also evidenced in A549 cells. GC/MS analysis revealed the presence of phytoconstituents of different phytochemical groups comprising of 3.45% diterpenoid, 5.45% triterpenoid, 11.24% steroid, 2.03% phytosterol, etc. in methanol extract, as well as 4.53% diterpenoid, 2.88% triterpenoid, 1.09% steroid, 2.11% phytosterol, etc. in ethyl acetate extract with considerable biological importance. Conclusion: This is the maiden report of the metabolomic profiling of leaf extracts of Garcinia cowa which possess a good repository of potentially bioactive molecules that holds a great promise as a future therapeutic agent in combating lung cancer.

Abstract B009: The histone deacetylase inhibitor pracinostat and its metabolite SB991 are active as single agents and in combination with 5-azacitidine in acute myeloid leukemia cells

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

Euphosantianane A⁻D: Antiproliferative Premyrsinane Diterpenoids from the Endemic Egyptian Plant Euphorbia Sanctae-Catharinae.

Euphorbia species are rich in diterpenes. A solvent extraction of Euphorbia sanctae-catharinae, a species indigenous to the Southern Sinai of Egypt, afforded several premyrsinane diterpenoids (1–4) as well as previously reported metabolites (5–13) that included three flavonoids. Isolated compounds were chemically characterized by spectroscopic analysis. Identified compounds were bioassayed for anti-proliferative activity in vitro against colon (Caco-2) and lung (A549) tumor cell lines. Compound 9 exhibited robust anti-proliferative activity against A549 cells (IC50 = 3.3 µM). Absolute configurations for 8 versus 9 were determined by experimental and TDDFT-calculated electronic circular dichorism (ECD) spectra.

Abstract 5792: Preclinical antitumor activity of BMS-986158, an oral BET inhibitor, for the treatment of cancer

Abstract Background: Regulation and recognition of covalent chromatin modifications by cellular proteins are key determinants of gene expression. Bromodomain and extra-terminal (BET) proteins, including BRD2, BRD3, BRD4, and BRDT, bind directly to acetylated lysine on histone tails to promote gene transcription. Oncogenes such as c-MYC, BCL2, and ASCL1, are directly regulated by BET proteins. Here, we report the preclinical evaluation of BMS-986158, an orally bioavailable, potent, and selective BET inhibitor. Methods: BMS-986158 activity was assessed in tumor growth assays across solid and hematologic cancer cell lines in vitro and in vivo in patient-derived xenografts (PDX). Dose-response gene expression profiling of ex vivo blood from healthy donors was used to identify transcripts exhibiting a pharmacodynamic effect with BMS-986158 treatment. Results: Tumor growth inhibition (TGI) with BMS-986158 was observed across different solid and hematologic cancer cell lines. Mutations in chromatin regulators were identified among BMS-986158–sensitive cell lines. BMS-986158 also demonstrated antitumor activity (TGI ≥ 70%) in 24 of 82 PDX models tested (Table). Genetic alterations that correlate with BMS-986158 responsiveness will be presented. Finally, an ex vivo dose-response profiling study in healthy human blood identified 124 genes responsive to BMS-986158, including chemokines and chemokine receptors. Conclusions: BMS-986158, a potent and selective BET inhibitor, demonstrated robust anti-proliferative activity in vitro across a broad range of cancer cell lines. Consistent with the in vitro data, preclinical antitumor activity was observed in various PDX tumor models. Furthermore, gene expression profiling studies in human blood samples identified select target genes responsive to BMS-986158 treatment that may be useful as clinical pharmacodynamic markers. TGI assay in PDX modelsTumor TypeTotal sample, nResponders, naAdenocarcinoma319Lung squamous cell carcinoma217Breast cancer82Brain cancer10Colorectal cancer63Esophageal cancer31Gallbladder cancer21Head and neck cancer31Mixedb70Total8224a Responders were defined as having > 70% TGIb Mixed tumor types include large cell neuroendocrine carcinoma of the lung, liver, liver metastases from rectum, mixed adenocarcinoma, neuroendocrine tumor, and non-small cell lung cancer Citation Format: Susan Wee, Donald Jackson, Heshani Desilva, Maya Dajee, Julie Carman, Petra Ross-MacDonald, Jinping Gan, Richard A. Westhouse, Christine Huang, Zheng Yang, Michael Poss, John T. Hunt, Gregory D. Vite, Ashvinikumar V. Gavai. Preclinical antitumor activity of BMS-986158, an oral BET inhibitor, for the treatment of 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 5792.

Abstract 1944: Tazemetostat displays synergistic antiproliferative activity with backbone therapies in preclinical models of AT/RT and MRT

Abstract Malignant Rhabdoid Tumors (MRT) and Atypical Teratoid Rhabdoid Tumors (AT/RT) are typically pediatric cancers which are rare and aggressive with extremely high unmet medical need. At a molecular level MRT and AT/RT tumors are almost universally characterized by loss of the INI1 protein (also known as SNF5 or SMARCB1), a component of the Switch Sucrose Non-fermentable (SWI/SNF) multimeric chromatin modifying complex. In many cell types, the SWI/SNF complex and the PRC2 complex have an antagonistic relationship in the regulation of tumor suppressor genes, cell cycle checkpoints, hedgehog and myc pathway genes, among others [Wilson et al, Cancer Cell. 2010 Oct 19;18(4):316-28.]. This explicated the possibility of a novel treatment modality for these tumors, based on inhibition of EZH2 - the catalytic subunit of PRC2. Tazemetostat (EPZ-6438) is a potent and orally bioavailable small molecule inhibitor of EZH2 currently in phase 2 clinical trials in adult patients with non-Hodgkin lymphoma, INI1-negative tumors and mesothelioma. Dependency of INI1-negative tumors on EZH2 catalytic activity was demonstrated in preclinical models of MRT, where robust tumor regressions were induced with tazemetostat treatment [Knutson et al, Proc Natl Acad Sci USA. 2013 May 7;110(19):7922-7]. Consistent with this, tazemetostat demonstrated activity in relapsed or refractory patients with INI1-negative tumors enrolled in the adult phase 1 clinical trial. In addition, a phase 1 dose escalation study in pediatric patients with INI1-negative solid tumors is currently ongoing. Backbone therapies for MRT and AT/RT are comprised of cytotoxic chemotherapy which may or may not be administered in the context of stem cell transplant, in addition to surgical resection and radiation therapy. In support of combination clinical scenarios including EZH2 inhibition in this setting, we sought to explore the antiproliferative effects of combining tazemetostat with current small molecule treatment therapies in cell line models of AT/RT and MRT. Synergistic activity was observed when tazometostat was combined with individual components of chemotherapeutic regimens and targeted therapies such as vincristine, doxorubicin, alisertib and HDAC inhibitors.Further, we investigated the effects of ionizing radiation together with tazemetostat treatment. Application of X-ray irradiation concomitantly or after tazemetostat treatment, induced robust antiproliferative activity and reduction in clonogenic potential of both AT/RT cell lines tested.Taken together these results suggest that pharmacological inhibition of EZH2 enhances the activity of backbone therapy and may have an advantage over monotherapy in INI1-negative cancers supporting the therapeutic potential of combination regimens that include tazemetostat in these tumors. Citation Format: Christine R. Klaus, Jeffrey A. Keats, Jesse J. Smith, Richard Chesworth, Robert A. Copeland, Scott Ribich, Alejandra Raimondi. Tazemetostat displays synergistic antiproliferative activity with backbone therapies in preclinical models of AT/RT and MRT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2017-1944

Biological activities of frankincense essential oil in human dermal fibroblasts

Although frankincense essential oil (FREO) has become increasingly popular in skin care, research on its biological activities in human skin cells is scarce, if not completely absent. In the current study, we explored the biological activities of FREO in pre-inflamed human dermal fibroblasts by analyzing the levels of 17 important protein biomarkers pertinent to inflammation and tissue remodeling. FREO exhibited robust anti-proliferative activity in these skin cells. It also significantly inhibited collagen III, interferon gamma-induced protein 10, and intracellular cell adhesion molecule 1. We also studied its effect in regulating genome-wide gene expression. FREO robustly modulated global gene expression. Furthermore, Ingenuity® Pathway Analysis showed that FREO affected many important signaling pathways that are closely related to inflammation, immune response, and tissue remodeling. This study provides the first evidence of the biological activities of FREO in human dermal fibroblasts. Consistent with existing studies in other models, the current study suggests that FREO possesses promising potential to modulate the biological processes of inflammation and tissue remodeling in human skin. Further research into the biological mechanisms of action of FREO and its major active components is recommended.

Anti-inflammatory activity of Juniper (Juniperus communis) berry essential oil in human dermal fibroblasts

Although juniper (Juniperus communis) berry essential oil (JEO) has been used in skin care products, research on its biological activity in human skin cells is scarce. In the current study, we explored the biological activity of JEO (with alpha-pinene as the major active component) in pre-inflamed human dermal fibroblasts, which were designed to mimic the disease biology of chronic inflammation and fibrosis. We analyzed the levels of 17 important protein biomarkers pertinent to inflammation and tissue remodeling. JEO exhibited robust antiproliferative activity and significantly inhibited the increased production of the proinflammatory chemokines interferon gamma-induced protein 10 (IP-10) and interferon-inducible T-cell alpha chemoattractant (I-TAC). Additionally, JEO significantly inhibited tissue remodeling biomarkers, namely collagen I, collagen III, and plasminogen activator inhibitor 1 (PAI-I). Macrophage colony-stimulating factor (M-CSF), an immunomodulatory protein molecule, was also significantly downregulated by JEO. Moreover, we found that JEO robustly modulated global gene expression. Ingenuity Pathway Analysis also showed that JEO affected many important signaling pathways that are closely related to metabolism, inflammation, immune response, wound healing, and cancer biology. This study provides the first evidence of the biological activity of JEO in human dermal fibroblasts. Thus, JEO is a promising therapeutic candidate for inflammatory conditions in the skin.

Melaleuca (Melaleuca alternifolia) essential oil demonstrates tissue-remodeling and metabolism-modulating activities in human skin cells

Melaleuca (Melaleuca alternifolia) essential oil (MEO), commonly known as tea tree oil, is popularly used in skincare products. In the current study, we investigated the biological activity of a commercially available MEO (with terpinen-4-ol as the major active component) in pre-inflamed human dermal fibroblasts, which were designed to simulate chronic inflammation. We analyzed the levels of seventeen biomarkers that are important in inflammation and tissue remodeling. Additionally, we studied the effect of MEO on genome-wide gene expression. MEO showed a robust antiproliferative activity against the cells. It also increased the levels of monocyte chemoattractant protein 1, an inflammatory chemokine, and several tissue remodeling molecules such as epidermal growth factor receptor, matrix metalloproteinase 1, and tissue inhibitor of metalloproteinase-1 and -2. It was also noted that MEO diversely modulated global gene expression. Furthermore, Ingenuity Pathway Analysis showed that MEO affects many important signaling pathways that are closely related to metabolism, which suggests its potential modulation of metabolism. The results provide an important evidence of the biological activity of MEO in human dermal fibroblasts. They also suggest that MEO plays useful roles in tissue remodeling and metabolism; however, further research is needed to explore the mechanisms underlying these actions.