Rationale For Further Clinical Studies Research Articles

Abstract LB003: Combined BRAF, MEK, and PD-1 inhibition in BRAFV600E colorectal cancer patients: Correlative studies from a phase 2 trial

Abstract Background: Although BRAF inhibitors combined with EGFR and/or MEK inhibitors have improved efficacy in BRAFV600E colorectal cancer (CRC), response rates remain low and clinical benefit is not durable. Preclinical studies suggest that BRAF-targeted therapy in combination with immune checkpoint blockade could enhance anti-tumor activity. We have previously reported on the efficacy from an ongoing phase 2 clinical trial of anti-PD-1 antibody spartalizumab in combination with BRAF inhibitor dabrafenib and MEK inhibitor trametinib in BRAFV600E CRC patients. Of 26 patients, overall response rate (ORR) was 38% (10/26) and compares favorably to the historical controls in BRAFV600E CRC, yet the mechanisms of patient response in this trial need further investigation. Methods: Single-cell RNA-seq (scRNA-seq) was performed on 23 paired baseline and day 15 tumor biopsies. Patient-derived organoids (PDOs) were generated from baseline tumor biopsies. Results: scRNA-seq of paired biopsies revealed increased CD8+ T cell infiltration after treatment in patients with better clinical outcome (PFS > 6 months, n=11). From the on- versus pre-treatment differentially expression analysis and gene set enrichment analysis in the tumor epithelial compartment, we observed greater induction and enrichment of immune gene signatures such as antigen processing and presentation, type I and II interferon response, chemokine activity, as well as superior MAPK pathway inhibition with therapy in patients with PFS > 6 months. In comparison, patients with PFS < 6 months (n=12) showed less immune gene upregulation and MAPK pathway inhibition in tumor cells. PDOs treated with dabrafenib and trametinib exhibited gene expression changes that mirrored the changes observed in scRNA-seq of tumor cells in the same patients from which they were derived. BRAF/ERKi treatment in PDOs produced greater MAPK pathway inhibition and immune genes induction than BRAF/MEKi. Conclusion: Correlative studies of combined anti-PD-1 and BRAF/MEK inhibition suggest that the tumor-intrinsic immune response induced by MAPK pathway inhibition might underlie cooperativity between BRAF-targeted therapy and immune checkpoint blockade. A greater degree of immune gene induction in tumor cells could be enhanced by superior MAPK pathway inhibition, which provides rationale for further clinical studies. Citation Format: Jun Tian, Jonathan H. Chen, Sherry X. Chao, Karin Pelka, Vjola Jorgji, Islam Baiev, William B. Bradford, Edmond Wong, Princy Sindurakar, Tomonori Oka, Shadmehr Demehri, Nir Hacohen, Ryan B. Corcoran. Combined BRAF, MEK, and PD-1 inhibition in BRAFV600E colorectal cancer patients: Correlative studies from a phase 2 trial [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 LB003.

Safety, pharmacokinetics, and efficacy of BPI-15086 in patients with EGFR T790M-mutated advanced non-small-cell lung cancer: results from a phase I, single-arm, multicenter study

BackgroundEpidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance frequently occurs in patients with non-small-cell lung cancer (NSCLC). EGFR Thr790Met mutation (T790M+) is seen in ∼50% of patients. We assessed the safety, tolerability, and pharmacokinetics (PK) of BPI-15086, a novel, ATP-competitive, irreversible, third-generation, mutation-selective EGFR-TKI in patients with EGFR T790M-mutated NSCLC.Patients and methodsThis two-center, phase I, dose-escalation study included patients who were 18-65 years old, with an Eastern Cooperative Oncology Group performance status of 0-2, with histologically or cytologically confirmed locally advanced or metastatic T790M+ NSCLC who were not surgical or radiotherapy candidates, and had imaging-identified disease progression after prior EGFR-TKIs. This dose-escalation study enrolled patients using a 3 + 3 study design. Patients received 25, 50, 100, 200, and 300 mg/day orally in 21-day cycles. The primary endpoints were safety, tolerability, and PK. Secondary endpoints were objective response rate (ORR) and disease control rate (DCR). The dose-expansion study was not conducted.ResultsWe enrolled 17 patients from 29 December 2016 to 16 May 2018, in the safety and full analysis sets. All patients completed a single dosing trial, and no adverse events (AEs) causing drug discontinuation were seen. Grade 1-2 nausea, hypoalbuminemia, and decreased appetite were the most common treatment-related AEs. Grade 3 hyperglycemia was seen in one patient dosed at 300 mg/day. The ORR and DCR were 17.7% [95% confidence interval (CI) 3.8% to 43.4%] and 47.1% (95% CI 23.0% to 72.2%), respectively.ConclusionBPI-15086 is a safe and tolerable third-generation EGFR-TKI with a rationale for further clinical studies.

Diosmetin exerts cardioprotective effect on myocardial ischaemia injury in neonatal rats by decreasing oxidative stress and myocardial apoptosis.

Myocardial injury caused by the myocardial ischaemia (MI) is still a troublesome condition in the clinic, including apoptosis, oxidative stress and inflammation. Diosmetin inhibits the cellular apoptosis and inflammatory response and enhances antioxidant activity. So, this study was designed to investigate the cardioprotective effects of diosmetin on MI model neonatal rats. Forty Sprague Dawley (SD) rats 7 days old were randomly divided into five groups. Four groups of rats received diosmetin (50, 100, and 200 mg/kg) or vehicle (MI group) after ischaemia. Another group received vehicle without ischaemia to serve as a control group. Rats were pretreated with diosmetin intraperitoneally for 7 days and intoxicated with isoproterenol (ISO, 85 mg/kg, sc) on the last 2 days. The expression of apoptotic molecules, myocardial systolic function index, antioxidant enzymes and myocardial enzyme was analyzed. Compared with the control group, the proliferation marker proteins of Ki67 were increased significantly (P < .05), the MI group significantly increased the cardiac apoptosis, oxidative stress and myocardial enzymes, and weakened myocardial contractility. The levels of p‐P65/P65 were increased significantly (P < .05) with decreased p‐AKT/AKT and p‐Nrf2/Nrf2 (P < .05). Nevertheless, pretreatment with diosmetin reversed these changes, especially high‐dose group. In summary, diosmetin has significant potential as a therapeutic intervention to ameliorate myocardial injury after MI and provides the rationale for further clinical studies.

Remifentanil attenuates cardiac dysfunction, lipid peroxidation and immune disorder in rats with isoproterenol-induced myocardial injury via JNK/NF-KB p65 inhibition.

BackgroundMyocardial injury caused by myocardial ischemia (MI) is still a severe condition that can result in apoptosis, oxidative stress, and inflammation. Remifentanil is a selective, ultra-short-acting, µ-opioid receptor agonist opioid. It can improve sinusoidal heart rate patterns in the fetus, for bupivacaine-induced cardiotoxicity, and with lipopolysaccharide (LPS)-induced cardiomyocytes injuries. This study aimed to explore the cardioprotective effects of remifentanil in MI model rats.MethodsSprague Dawley (SD) rats were split into five groups at random, including a control group, Isop group, low-dose remifentanil treatment group (10 µg/kg), medium-dose remifentanil treatment group (20 µg/kg), and a high-dose remifentanil treatment group (40 µg/kg). The MI model was achieved by subcutaneously injecting rats with isoproterenol (85 mg/kg) for two consecutive days. With the expression of apoptotic molecules, myocardial systolic function index, inflammation, antioxidant enzymes, and the myocardial enzyme taken into account, the data was analyzed.ResultsAfter treatment with remifentanil, the left ventricular wall thickness (LVWT), left ventricular end-systolic volume (LVESV), left ventricular ejection fraction (LVEF), fraction shortening (FS), and heart rate (HR) were significantly increased in comparison with the Isop group. Creatine kinase-MB (CK-MB), Mb, and cTnl expressions were decreased. Meanwhile, the levels of cleaved caspase-3 and caspase-9 were decreased. Remarkably, the levels of reactive oxidative species (ROS), malondialdehyde (MDA), and lactate dehydrogenase (LDH) were observed to be repressed, while the levels of superoxide dismutase (SOD) was significantly increased. More importantly, the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and interferon (IFN)-γ were decreased.ConclusionsRemifentanil has significant potential as a therapeutic intervention strategy for ameliorating myocardial injury after MI and these findings provide the rationale for further clinical studies.

An Epithelial-to-Mesenchymal Transcriptional Switch Triggers Evolution of Pulmonary Sarcomatoid Carcinoma (PSC) and Identifies Dasatinib as New Therapeutic Option.

Pulmonary sarcomatoid carcinoma (PSC) is a rare and aggressive form of NSCLC. Rarity and poor characterization have limited the development of PSC-tailored treatment protocols, leaving patients with inadequate therapeutic options. In this study, we investigated the gene expression profile of PSCs, with the aim to characterize the molecular mechanisms responsible for their evolution and to identify new drugs for their treatment. A training set of 17 biphasic PSCs was selected and tested for the expression of a large panel of 770 genes related to cancer progression using NanoString technology. Computational analyses were used to characterize a PSCs-gene specific signature from which pathways and drivers of PSC evolution were identified and validated using functional assays in vitro. This signature was validated in a separate set of 15 PSCs and 8 differentiated NSCLC and used to interrogate the cMAP database searching for FDA-approved small molecules able to counteract PSC phenotype. We demonstrated that the transcriptional activation of an epithelial mesenchymal transition (EMT) program drives PSC phylogeny in vivo. We showed that loss of the epithelial-associated transcription factor (TF) OVOL2 characterizes the transition to sarcomatoid phenotype triggering the expression of EMT promoting TFs, including TWIST and ZEB and the expression of the membrane kinase DDR2. Finally, using a drug repurposing approach, we identified dasatinib as potential inhibitor of the PSC-gene expression signature and we confirmed in vitro that this drug efficiently restrains proliferation and reverts the sarcomatoid-associated phenotype. Our data provide new insights into PSC evolution and provide the rationale for further clinical studies with dasatinib.

Trifluoperazine, a novel autophagy inhibitor, increases radiosensitivity in glioblastoma by impairing homologous recombination

BackgroundResistance to adjuvant radiotherapy is a major cause of treatment failure in patients with glioblastoma (GBM). Autophagy inhibitors have been shown to enhance the efficacy of radiotherapy for certain solid tumors. However, current inhibitors do not penetrate the blood-brain-barrier (BBB). Here, we assessed the radiosensitivity effects of the antipsychotic drug trifluoperazine (TFP) on GBM in vitro and in vivo.MethodsU251 and U87 GBM cell lines as well as GBM cells from a primary human biopsy (P3), were used in vitro and in vivo to evaluate the efficacy of TFP treatment. Viability and cytotoxicity was evaluated by CCK-8 and clonogenic formation assays. Molecular studies using immunohistochemistry, western blots, immunofluorescence and qPCR were used to gain mechanistic insight into the biological activity of TFP. Preclinical therapeutic efficacy was evaluated in orthotopic xenograft mouse models.ResultsIC50 values of U251, U87 and P3 cells treated with TFP were 16, 15 and 15.5 μM, respectively. TFP increased the expression of LC3B-II and p62, indicating a potential disruption of autophagy flux. These results were further substantiated by a decreased Lysotracker Red uptake, indicating impaired acidification of the lysosomes. We show that TFP and radiation had an additive effect when combined. This effect was in part due to impaired TFP-induced homologous recombination. Mechanistically we show that down-regulation of cathepsin L might explain the radiosensitivity effect of TFP. Finally, combining TFP and radiation resulted in a significant antitumor effect in orthotopic GBM xenograft models.ConclusionsThis study provides a strong rationale for further clinical studies exploring the combination therapy of TFP and radiation to treat GBM patients.

Pregestational diabetes induces fetal coronary artery malformation via reactive oxygen species signaling.

Hypoplastic coronary artery disease is a congenital coronary artery malformation associated with a high risk of sudden cardiac death. However, the etiology and pathogenesis of hypoplastic coronary artery disease remain undefined. Pregestational diabetes increases reactive oxygen species (ROS) levels and the risk of congenital heart defects. We show that pregestational diabetes in mice induced by streptozotocin significantly increased 4-hydroxynonenal production and decreased coronary artery volume in fetal hearts. Pregestational diabetes also impaired epicardial epithelial-to-mesenchymal transition (EMT) as shown by analyses of the epicardium, epicardial-derived cells, and fate mapping. Additionally, the expression of hypoxia-inducible factor 1α (Hif-1α), Snail1, Slug, basic fibroblast growth factor (bFgf), and retinaldehyde dehydrogenase (Aldh1a2) was decreased and E-cadherin expression was increased in the hearts of fetuses of diabetic mothers. Of note, these abnormalities were all rescued by treatment with N-acetylcysteine (NAC) in diabetic females during gestation. Ex vivo analysis showed that high glucose levels inhibited epicardial EMT, which was reversed by NAC treatment. We conclude that pregestational diabetes in mice can cause coronary artery malformation through ROS signaling. This study may provide a rationale for further clinical studies to investigate whether pregestational diabetes could cause hypoplastic coronary artery disease in humans.

Abstract 1747: BGB324, a selective small molecule Axl kinase inhibitor to overcome EMT-associated drug resistance in carcinomas: Therapeutic rationale and early clinical studies

Abstract Axl is a member of the TAM (Tyro3, Axl and Mer) family of receptor tyrosine kinases that regulate multiple cellular responses including cell survival, proliferation, and migration. Axl expression is predictive of poor patient overall survival in a variety of human cancers including triple negative breast (TNBC), pancreatic ductal adenocarcinoma (PDA) and non-small cell lung cancer (NSCLC). Axl expression is induced by the epithelial-to-mesenchymal transition (EMT) gene program in cancer cells and Axl signaling is required to maintain EMT-associated features including invasiveness, metastasis, stem cell-like traits and resistance to targeted inhibitors and other chemotherapeutic agents. BGB324/R428 is an oral, selective small molecule inhibitor of Axl that recently was evaluated in early clinical safety studies in healthy volunteers. Treatment with BGB324 up to and including 1.5 gms daily (per os) was established as being safe and well tolerated. The endpoints of the study included pharmacokinetics and safety. Bioavailability was increased in the presence of food and systemic exposure increased dose proportionately. At the highest dose the apparent elimination half-life approached four days presenting a range of dosing options. We evaluated the effects of BGB324 in preclinical models of TNBC, PDA and NSCLC, including 2D/3D cell culture and mouse xenograft models, in combination with targeted and chemotherapeutic agents. BGB324 treatment of mesenchymal carcinoma cells blocked invasiveness and enhanced chemotherapeutic efficacy. BGB324 abrogated the tumor initiation capacity of TNBC cells, an activity associated with cancer stem cells. BGB324 treatment blocked the emergence of EMT-associated acquired resistance to erlotinib in human NSCLC xenografts. Furthermore, combination treatment of BGB324 with chemotherapy inhibited the growth of human NSCLC xenografts and significantly prolonged survival in orthotopic and genetically engineered mouse models of PDA. Collectively, these data suggest that the first-in-class selective Axl inhibitor BGB324 can overcome EMT-related acquired therapeutic resistance and enhance the efficacy of multiple anti-cancer strategies. Together with the results of results of the early clinical safety studies, this provides a rationale for further clinical studies. Citation Format: Katarzyna Wnuk-Lipinska, Crina Tiron, Gro Gausdal, Tone Sandal, Robin Frink, Stefan Hinz, Monica Hellesøy, Lavina Ahmed, Hallvard Haugen, Xiao Liang, Magnus Blø, David Micklem, Murray Yule, John Minna, Longen Zhou, Rolf Brekken, James Lorens. BGB324, a selective small molecule Axl kinase inhibitor to overcome EMT-associated drug resistance in carcinomas: Therapeutic rationale and early clinical studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1747. doi:10.1158/1538-7445.AM2014-1747

Activity of the Hypoxia-Activated Prodrug, TH-302, in Preclinical Human Acute Myeloid Leukemia Models

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm. Recent evidence has shown the bone marrow microenvironment in patients with AML to be intrinsically hypoxic. Adaptive cellular responses by leukemia cells to survive under low oxygenation also confer chemoresistance. We therefore asked whether therapeutic exploitation of marrow hypoxia via the hypoxia-activated nitrogen mustard prodrug, TH-302, could effectively inhibit AML growth. We assessed the effects of hypoxia and TH-302 on human AML cells, primary samples, and systemic xenograft models. We observed that human AML cells and primary AML colonies cultured under chronic hypoxia (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic controls. TH-302 treatment resulted in dose- and hypoxia-dependent apoptosis and cell death in diverse AML cells. TH-302 preferentially decreased proliferation, reduced HIF-1α expression, induced cell-cycle arrest, and enhanced double-stranded DNA breaks in hypoxic AML cells. Hypoxia-induced reactive oxygen species by AML cells were also diminished. In systemic human AML xenografts (HEL, HL60), TH-302 [50 mg/kg intraperitoneally (i.p.) 5 times per week] inhibited disease progression and prolonged overall survival. TH-302 treatment reduced the number of hypoxic cells within leukemic bone marrows and was not associated with hematologic toxicities in nonleukemic or leukemic mice. Later initiation of TH-302 treatment in advanced AML disease was as effective as earlier TH-302 treatment in xenograft models. Our results establish the preclinical activity of TH-302 in AML and provide the rationale for further clinical studies of this and other hypoxia-activated agents for leukemia therapy.

Type 2 Diabetes Relevant Bioactive Potential of Freshly Harvested and Long-Term Stored Pears Usingin vitroAssay Models

ABSTRACT Fresh pear consumption could provide health benefits by reducing the risk of chronic diseases such as hyperglycemia linked to type 2 diabetes. This in vitro study investigated the phenolic-linked antihyperglycemia bioactive factors in aqueous and ethanolic extracts of peel and pulp from eight different freshly harvested and long-term stored pear varieties. Total soluble phenolics, 2,2-diphenyl-1-picrylhydrazyl radical scavenging-based antioxidant activity and associated in vitro alpha-glucosidase, alpha-amylase and angiotensin I-converting inhibitory activities were analyzed. Peel extracts had higher total soluble phenolic content and related antioxidant capacity than pulp extracts. Comice variety had the highest total phenolic contents with positive correlation to total antioxidant activity. Aqueous pulp extracts had high alpha-amylase inhibitory activities with no correlation to phenolic content. However, the peel ethanolic extracts had the highest alpha-glucosidase inhibitory activity with positive correlation to total phenolics. This research provides the biochemical rationale for further clinical studies to include pear as part of healthy diet. PRACTICAL APPLICATIONS Using in vitro structure-function relevant bioassays to screen a range of fruits and vegetables helps to develop biochemical rationale to target and strategize the increased use of these fresh fruits and vegetables for better health and wellness. Pears, because of their phenolic bioactives, have potential to be included as a part of a comprehensive diet for managing early stages of hyperglycemia linked to type 2 diabetes. This study provides foundation for screening of pears for improving dietary strategies for managing this chronic disease and can be the biochemical rationale for further clinical studies.

Sa2048 Predicting Regional Colonic Concentrations of 5-Aminosalicylic Acid (5-ASA) From Oral and Topical Therapy With Dynamic Modeling in Ulcerative Colitis

Background 5-aminosalicylate (5-ASA) formulations are approved for treatment andmaintenance of remission in mild-moderate ulcerative colitis (UC). Determination of the colonic distribution of 5-ASA from various formulations is challenging, since estimates depend on 5-ASA measurements in the serum and the urine. A dynamic model of colonic concentrations of 5-ASA after oral pH dependent delayed-release mesalamine, oral pH dependent extended delayed-release MultiMatrixSystem (MMX) mesalamine, 5-ASA enema, foam and suppositories was developed to determine the colonic pharmacokinetics of these agents. Methods Using published data, we created a computer model with STELLA software (isee, Hanover, NH). Colonic 5-ASA in the right, transverse, descending, sigmoid colon, and rectumwere estimated after once daily doses of the above formulations individually and in combination. Simulations of active mild-moderate UC, (defined as 6 bowel movements (BMs)/day), as well as UC in remission, (defined as 1 BM/day), were performed. Gastrointestinal transit times were determined using published literature modified for periods of sleep. Random distributions were used to model variations in colonic emptying. Data obtained was compared using paired Student's T Tests for difference in mean concentrations with SPSS software (IBM, Armonk, NY, USA.) Results Uneven distribution of 5-ASA was seen with both oral preparations. For UC in remission, the highest levels of 5-ASA in the right colon and transverse colon, (p-value < .01), were from oral delayed-release mesalamine (4.8 gm), in the descending colon and sigmoid colon (p-value < .01) from MMX mesalamine (4.8 gm), and from 5-ASA enema (4g) in the rectum (p-value < .01) (table 1). With active UC, sigmoid and rectal levels from both oral preparations were markedly decreased. Sigmoid levels were highest with foam (4g) (p-value < .01), and rectal levels highest with 5-ASA enema (p-value < .01) (table 1). Differences in recto-sigmoid levels of 5-ASA from enemas and suppositories (1.5g) were markedly influenced by time of BM occurrence (figure 1). For UC in remission, increasing MMX mesalamine dose to 4.8 gm daily resulted in the higher sigmoid-rectal levels compared to combination therapy of 2.4g MMX mesalamine (dose approved for maintenance of remission) with 1g/day 5-ASA enema. Conclusions This is the first dynamic model designed to overcome the difficulties of predicting colonic concentrations of oral and rectally administered 5-ASA formulations. The model suggests that compared to oral delayedrelease mesalamine, MMX mesalamine treatment results in higher 5-ASA levels in the left colon. Higher colonic levels of 5-ASA are achieved when insertion of topical 5-ASAs takes place shortly after a bowel movement. Dynamic modeling provides a rationale for further clinical studies to optimize 5-ASA treatment. Table 1: Mean colonic concentrations of 5-Aminosalicylic acid from oral and topical therapies in ulcerative colitis

Effects of phenylbutyrate on proliferation and apoptosis in human prostate cancer cells in vitro and in vivo.

Phenylbutyrate (PB) is a potent differentiating agent and currently under investigation for the treatment of prostate cancer (CaP) and other malignancies. We have studied the impact of PB in vitro and in vivo on differentiation, proliferation and apoptosis in the LNCaP and LuCaP 23.1 prostate cancer xenograft models. In vitro we found that i) PB increased PSA secretion/cell, ii) inhibited cell proliferation in a time- and dose-dependent manner resulting in a cell cycle arrest in G1-phase and iii) induced apoptosis at concentrations of 2.5 mM after 3 days of treatment. In PB treated animals tumor growth stabilized or regressed. Combination of castration and PB treatment had a synergistic antiproliferative effect. The growth-inhibitory and differentiating properties and a low toxicity profile of PB provide rationale for further clinical studies in patients with CaP.

Oral dehydroepiandrosterone in physiologic doses modulates immune function in postmenopausal women

This study tests the hypothesis that dehydroepiandrosterone or its metabolic products are immunomodulatory in postmenopausal women with relative adrenal androgen deficiency. A prospective, randomized, double-blind, crossover study of 11 subjects with 3-week treatment arms separated by a 2-week washout period was performed. Immunologic evaluation at the beginning and end of the treatment arms consisted of flow cytometry to delineate T-cell populations, in vitro T-cell mitogenic response and cytokine production, and natural killer cell cytotoxicity. Statistical analysis was based on a split-plot design with analysis of variance with repeated measures. Dehydroepiandrosterone supplementation decreased CD4+ (helper) T cells and increased CD8+/CD56+ (natural killer) cells. Although T-cell mitogenic and interleukin-6 responses were inhibited, natural killer cell cytotoxicity increased dramatically. These data provide the first in vivo evidence in human for an immunomodulatory effect of dehydroepiandrosterone. The salutary immune changes could account for clinical and experimental evidence of antioncogenic effects of this steroid. This study provides a strong rationale for further clinical studies on dehydroepiandrosterone supplementation in adrenal androgen-deficient states.