phospho-Rb Expression Research Articles

OTHR-16. HYPERACTIVATION OF THE INTEGRATED STRESS RESPONSE VIA PROTEASOME INHIBITION KILLS DMG AND ATRT WHILE SPARING NORMAL IPSC BRAIN ORGANOIDS

Abstract BACKGROUND H3K27-altered diffuse midline glioma (DMG) and atypical teratoid/rhabdoid tumor (ATRT) have increased baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate stress. This activation is manifested by elevated levels of ATF4. Transient or low-level expression protects cells from stress, while persistent, high-level activation leads to cell death. Ixazomib is an orally bioavailable proteasome inhibitor that causes endoplasmic reticulum stress, which is a major upstream activator of the ISR. We hypothesized that the high baseline level of ATF4 in DMG and ATRT would make these tumors susceptible to ISR activators such as ixazomib. METHODS After determining the IC50, DMG and ATRT cell lines were treated with increasing concentrations of ixazomib. To evaluate if ixazomib selectively kills DMG and ATRT, we treated iPSC brain organoids in parallel with tumor cells. Combination treatment of ixazomib and idarubicin, a brain-penetrant anthracycline, was conducted to assess synergy. RESULTS Ixazomib significantly induced apoptosis and suppressed proliferation in multiple cell lines, as shown by western blots for cleaved PARP and phospho-Rb expression as well as cleaved caspase-3 and BrdU immunofluorescence (DMSO vs. 25nM p<0.0001 by ANOVA). Additionally, western blots detected elevated ATF4 and CHOP expression in ixazomib treated cells, indicating activation of the ISR. CC3 immunofluorescence and western blot for cleaved PARP showed significantly increased apoptosis in DMG and ATRT cell lines treated with ixazomib (JHHDIPG1 and CHLA06 – DMSO vs. 25nM p<0.0001 by ANOVA), while iPSC brain organoids were not affected. Synergy testing of ixazomib and idarubicin showed an overall zero-interaction-potency (ZIP) synergy score of 23.464 in CHLA06 and 19.084 in HSJD007 (scores of 10 or above indicate synergy). CONCLUSIONS By over-activating the ISR, ixazomib kills DMG and ATRT cells without impacting normal iPSC brain organoids. This effect may potentially be enhanced with the addition of idarubicin.

#2671 Renal tubular epithelial FFAR4 improves cellular senescence to alleviate interstitial fibrosis in CKD

Abstract Background and Aims Chronic kidney disease (CKD) is defined as an estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73 m2 or pathological albuminuria, which is known as one of the major contributors to worldwide mortality. Although there have been substantial advances in understanding CKD mechanisms, at present, there are no effective therapies to treat it. We had formerly found that TUG891, a free fatty acid receptor 4 (FFAR4) (a member of the G protein-coupled receptor (GPCR) family) agonist, alleviated tubular damage as well as interstitial fibrosis in mice with CKD. Nevertheless, the multifunctional effects of FFAR4 in the kidney have not been well described. Method In the current research, the FFAR4 expression was abnormally reduced in tubular epithelial cells (TECs) of unilateral ureteral obstruction (UUO) and adenine-induced CKD mice. Systemic and conditional TEC-specific knockout of FFAR4 aggravated renal function, pathological damage and interstitial fibrosis, whereas FFAR4 activation by TUG-891 alleviated the severity of disease in UUO and adenine-induced CKD mice. Results Notably, FFAR4, as a key determinant, was firstly explored to regulate the cellular senescence in TECs and CKD mice injured kidneys, as represented through the activity of senescence associated β-galactosidase (SA-β-gal), marker protein p21, p53, Lamin B1, phospho-histone H2A.X, phospho-Rb expression, and secretory phenotype IL-6 level. Mechanistically, pharmacological activation and overexpression of FFAR4 reversed the decrease of aging-related SirT3 protein, where FFAR4 regulated SirT3 expression to exhibit anti-senescent effect via Gq subunit-mediated CaMKKβ/AMPK signaling in UUO and adenine-induced CKD mice. Conclusion These results underline the primordial role of renal tubular FFAR4 in the cellular senescence through AMPK/SirT3 signaling and define FFAR4 as a target for underlying drugs against CKD.

#6747 ACTIVATION OF GPR120 IMPROVES THE SENESCENCE OF TUBULAR EPITHELIAL CELLS IN SEPTIC ACUTE KIDNEY INJURY

Abstract Background and Aims Acute kidney injury (AKI) is a serious clinical complication that has high morbidity and mortality. Although there have been substantial advances in understanding AKI mechanisms, at present, there are no effective therapies to treat or prevent it. Method We had formerly found that TUG891, a GPR120 (a member of the G protein-coupled receptor (GPCR) family) agonist, alleviated tubular damage as well as renal dysfunction in mice with AKI. Nevertheless, the multifunctional effects of FFAR4 in the kidney have not been well described. Results In the current research, the GPR120 expression was abnormally reduced in tubular epithelial cells (TECs) of cecal ligation/perforation induced AKI mice, respectively. Systemic and conditional TEC-specific knockout of GPR120 aggravated renal function and pathological damage, whereas GPR120 activation by TUG-891 alleviated the severity of disease in AKI mice induced with cisplatin. Notably, GPR120, as a key determinant, was firstly explored to regulate the cellular senescence in TECs and AKI mice injured kidneys, as represented through the activity of senescence associated β-galactosidase (SA-β-gal), marker protein p21, p53, Lamin B1, phospho-histone H2A.X, phospho-Rb expression, and secretory phenotype IL-6 level. Mechanistically, pharmacological activation and overexpression of FFAR4 reversed the decrease of aging-related SirT3 protein, where GPR120 regulated SirT3 expression to exhibit anti-senescent effect via Gq subunit-mediated CaMKKβ/AMPK signaling in cisplatin-induced mice and tubular epithelial cells. Conclusion These results underline the primordial role of renal tubular GPR120 in the cellular senescence through AMPK/SirT3 signaling and define GPR120 as a target for underlying drugs against septic AKI.

A phase I study of the combination of palbociclib and dexamethasone for the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia

PurposeDespite advances in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), outcomes for relapsed/refractory (R/R) disease remain poor. Preclinical studies suggest that the combination of the CDK4/6 inhibitor palbociclib and dexamethasone may be effective in targeting leukemic cell growth. We conducted a phase I study of escalating doses of palbociclib in combination with dexamethasone in adults with R/R B-ALL. MethodsCycle 1 consisted of single agent palbociclib given for 7 days and continued for 28 additional days in combination with dexamethasone 20 mg daily. Palbociclib dosing began at 100 mg daily. Patients with a response were eligible for maintenance consisting of 1 week of palbociclib plus dexamethasone (20 mg daily × 2 days, 16 mg daily × 2 days, 12 mg daily × 2 days, 6 mg daily × 1 day), followed by 3 weeks of palbociclib alone. Safety, efficacy, and the expression of phospho-RB and c-MYB/BCL-2 were measured. ConclusionsSeven patients were treated on study before it was closed early due to slow accrual. No dose limiting toxicities were identified. One patient had a complete response with incomplete hematologic recovery, suggesting possible efficacy of the treatment. Reduction in CD34+ cells, p-RB, c-MYB, and BCL-2 expression also suggested on-target therapy effects.

Abstract 2136: Designing an immunofluorescence-based biomarker assay for detecting Rb and Phospho-Rb expression in HR+/HER2-breast cancer

Abstract Introduction: Cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) have emerged as a major advance in treating hormone receptor-positive/human epidermal growth factor receptor-2-negative breast cancer (HR+/HER2- BC), which accounts for over 60% of all breast tumors. However, response rate to CDK4/6i combined with endocrine therapy (ET) is only 50% or less, with de novo resistance seen in 15-30% in patients with HR+/HER2- metastatic BC. Therefore, predictive biomarkers that can help select patients who are likely to derive clinical benefit from adding CDK4/6i to ET are urgently needed to spare non-responders from toxicities and cost. The long-term objective of our study is to design and develop a predictive biomarker assay that can be used on circulating tumor cells for evaluating tumor response to CDK4/6i [palbociclib, ribociclib, and abemaciclib]. In this research, we aimed to select internal controls that can be used for the immunofluorescence (IF) assay to evaluate the expression of Rb and phospho-Rb as potential predictive biomarkers for CDK4/6i therapy. Methods: MCF7 and T47D parental (P) cells were treated with various concentrations of abemaciclib and palbociclib to determine their effects on cell growth by automated cell counting using Ensight® Multimode Plate Reader and on Rb and phospho-Rb expression by western blotting (WB) and IF using Leica SP8 STED confocal microscope. We also utilized derivatives of MCF7 and T47D cells resistant to estrogen deprivation (EDR) and/or Palbociclib (PalboR). Results: Abemaciclib and palbociclib treatment resulted in a concentration-dependent decrease in cell growth of MCF7 P (IC50: 37.4 and 254.7 nM, respectively) and T47D P (IC50: 2.7 and 59.1 nM, respectively) cells. Abemaciclib also reduced the expression phospho/total-Rb in a concentration-dependent manner by both WB and IF. The IC50 for abemaciclib was 19 nM in MCF7 P and 24 nM in T47D P cells when detected by WB. Similarly, there was a concentration-dependent decrease in the phospho/total-Rb with abemaciclib treatment as detected by IF. Expression of both total Rb and phospho-Rb was undetectable in the P/PalboR T47D cells compared to T47D P cells by both WB and IF. Therefore, this cell line can be used as a control for lack of Rb expression. EDR/PalboR derivative of MCF7 had ~50% reduction in Rb and ~70% reduction in phospho-Rb by IF, supporting its use as a control for reduced Rb expression. Conclusion: In summary, we have identified cell line models that can be used as internal controls for detecting the expression of total and phospho-Rb as predictive biomarkers of CDK4/6i treatment response in HR+/HER2- MBC patients. Ongoing performance validation studies are underway, including automated quantitation using digital whole slide fluorescent imaging to optimize the throughput and dynamic range of the assay. Citation Format: Mantasha Tabassum, Alexis Ruiz, Noor M. Abdulkareem, Mothaffar F. Rimawi, George E. Miles, Meghana V. Trivedi. Designing an immunofluorescence-based biomarker assay for detecting Rb and Phospho-Rb expression in HR+/HER2-breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2136.

Abstract 6720: Using proteasome inhibition to hyperactivate the integrated stress response in aggressive pediatric brain tumors

Abstract H3K27-altered diffuse midline glioma (DMG) and atypical teratoid/rhabdoid tumor (AT/RT) are aggressive pediatric brain tumors with dismal outcomes. DMG and AT/RT have increased baseline activation of the integrated stress response (ISR), an evolutionarily conserved system that enables cells to tolerate various forms of stress. This activation is manifested by elevated levels of ATF4 and NRF2 in DMG and AT/RT, respectively. Transient or low-level expression of these transcription factors protect cells from stress, while sustained, high-level activation leads to cell death. Ixazomib is an orally bioavailable proteasome inhibitor that causes endoplasmic reticulum stress, which is a major upstream activator of the ISR. We hypothesized that the high baseline level of ATF4 in DMG and NRF2 in AT/RT would make these tumors susceptible to ISR activators such as ixazomib. After determining the IC50 of ixazomib, DMG and AT/RT cell lines (JHH-DIPG1, JHH-DIPG16A, and CHLA06) were treated with increasing concentrations of the drug. Cleaved caspase 3 immunofluorescence showed a significant increase of apoptosis in all cell lines (JHH-DIPG16A and CHLA06 p<0.0001, JHH-DIPG1 p=0.0008 by ANOVA). Additionally, western blots for cleaved PARP and phospho-Rb expression detected induction of apoptosis and suppression of cell proliferation in JHH-DIPG1 treated with ixazomib. We are currently testing ixazomib in combination with idarubicin, a brain-penetrant anthracycline, and gemcitabine, a brain-penetrant nucleoside analog. Synergy testing in CHLA06 showed an overall zero-interaction-potency (ZIP) synergy score of 23.464 with inhibitory concentrations of ixazomib and idarubicin in the low nanomolar range (scores of 10 or above indicate synergy). Similarly, ixazomib and gemcitabine synergized to suppress CHLA06 growth and induce apoptosis (67% Annexin V+ cells in combination compared to 14 and 24% Annexin V+ with single agent treatment). In the future, we will determine if ixazomib selectively kills DMG and AT/RT while sparing normal cells in iPSC brain organoids. We will also test ixazomib singly and in combination with traditional chemotherapy in orthotopic xenografts of AT/RT. These results suggest that ixazomib, especially in combination with idarubicin, gemcitabine or other ISR activators, has the potential to serve as an effective therapy for aggressive pediatric brain tumors. Citation Format: Orlandi Valencia Novak, Tyler Findlay, Jeffrey Rubens, Charles Eberhart, Eric H. Raabe. Using proteasome inhibition to hyperactivate the integrated stress response in aggressive pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6720.

FFAR4 improves the senescence of tubular epithelial cells by AMPK/SirT3 signaling in acute kidney injury

Acute kidney injury (AKI) is a serious clinical complication with high morbidity and mortality rates. Despite substantial progress in understanding the mechanism of AKI, no effective therapy is available for treatment or prevention. We previously found that G protein-coupled receptor (GPCR) family member free fatty acid receptor 4 (FFAR4) agonist TUG891 alleviated kidney dysfunction and tubular injury in AKI mice. However, the versatile role of FFAR4 in kidney has not been well characterized. In the study, the expression of FFAR4 was abnormally decreased in tubular epithelial cells (TECs) of cisplatin, cecal ligation/perforation and ischemia/reperfusion injury-induced AKI mice, respectively. Systemic and conditional TEC-specific knockout of FFAR4 aggravated renal function and pathological damage, whereas FFAR4 activation by TUG-891 alleviated the severity of disease in cisplatin-induced AKI mice. Notably, FFAR4, as a key determinant, was firstly explored to regulate cellular senescence both in injured kidneys of AKI mice and TECs, which was indicated by senescence-associated β-galactosidase (SA-β-gal) activity, marker protein p53, p21, Lamin B1, phospho-histone H2A.X, phospho-Rb expression, and secretory phenotype IL-6 level. Mechanistically, pharmacological activation and overexpression of FFAR4 reversed the decrease of aging-related SirT3 protein, where FFAR4 regulated SirT3 expression to exhibit anti-senescent effect via Gq subunit-mediated CaMKKβ/AMPK signaling in cisplatin-induced mice and TECs. These findings highlight the original role of tubular FFAR4 in cellular senescence via AMPK/SirT3 signaling and identify FFAR4 as a potential drug target against AKI.

Pterostilbene Sensitizes Cisplatin-Resistant Human Bladder Cancer Cells with Oncogenic HRAS

Simple SummaryRAS oncoproteins are considered undruggable cancer targets. Nearly 15% of cases of bladder cancer have a mutation of HRAS. The active HRAS contributes to the tumor progression and the risk of recurrence. Using our novel gene expression screening platform, pterostilbene was identified to sensitize cisplatin-resistant bladder cancer cells with HRAS alterations via RAS-related autophagy and cell senescence pathways, suggesting a potentially chemotherapeutic role of pterostilbene for cisplatin treatment of human bladder cancer with oncogenic HRAS. Pterostilbene is a safe and readily available food ingredient in edible plants worldwide. Exploiting the principle of combination therapy on pterostilbene-enhanced biosensitivity to identify undruggable molecular targets for cancer therapy may have a great possibility to overcome the cisplatin resistance of bladder cancer. Our data make HRAS a good candidate for modulation by pterostilbene for targeted cancer therapy in combination with conventional chemotherapeutic agents cisplatin plus gemcitabine.Analysis of various public databases revealed that HRAS gene mutation frequency and mRNA expression are higher in bladder urothelial carcinoma. Further analysis revealed the roles of oncogenic HRAS, autophagy, and cell senescence signaling in bladder cancer cells sensitized to the anticancer drug cisplatin using the phytochemical pterostilbene. A T24 cell line with the oncogenic HRAS was chosen for further experiments. Indeed, coadministration of pterostilbene increased stronger cytotoxicity on T24 cells compared to HRAS wild-type E7 cells, which was paralleled by neither elevated apoptosis nor induced cell cycle arrest, but rather a marked elevation of autophagy and cell senescence in T24 cells. Pterostilbene-induced autophagy in T24 cells was paralleled by inhibition of class I PI3K/mTOR/p70S6K as well as activation of MEK/ERK (a RAS target) and class III PI3K pathways. Pterostilbene-induced cell senescence on T24 cells was paralleled by increased pan-RAS and decreased phospho-RB expression. Coadministration of PI3K class III inhibitor 3-methyladenine or MEK inhibitor U0126 suppressed pterostilbene-induced autophagy and reversed pterostilbene-enhanced cytotoxicity, but did not affect pterostilbene-elevated cell senescence in T24 cells. Animal study data confirmed that pterostilbene enhanced cytotoxicity of cisplatin plus gemcitabine. These results suggest a therapeutic application of pterostilbene in cisplatin-resistant bladder cancer with oncogenic HRAS.

Abstract P5-21-15: The synergistic antitumor activity of entinostat (MS-275) in combination with palbociclib (PD 0332991) in estrogen receptor-positive and triple-negative breast cancer

Abstract BACKGROUND: CDK4/6 regulates the G1-S phase transition by phosphorylating the retinoblastoma protein (Rb). Given their potent clinical efficacy, CDK4/6 inhibitors used in combination with hormone receptor (HR) blockade (with an aromatase inhibitor or fulvestrant) are emerging as the standard of care for patients with metastatic HR-positive breast cancers. The CDK4/6 inhibitors palbociclib and ribociclib are FDA-approved for use in HR-positive breast cancer patients, and abemaciclib is currently in phase III trials. We observed that approximately 74% (25/34) of breast cancer cell lines had high phosphorylated Rb (phospho-Rb) expression levels and that triple-negative breast cancer (TNBC) cell lines often expressed phospho-Rb, suggesting that targeting phospho-Rb via CDK4/6 inhibition may be effective against TNBC. The histone deacetylase (HDAC) inhibitors increase p21Cip1 levels, promoting proteasomal degradation of cyclin B1 and resulting in G2/M arrest. Entinostat is an oral, class 1, selective HDAC inhibitor currently in phase III testing in HR-positive breast cancer. Preclinical and clinical data demonstrate that entinostat, in combination with HR blockade, has anticancer activity. Our group recently reported that entinostat combined with other anticancer drugs induced apoptosis via induction of proapoptotic proteins such as Noxa and Bim in breast cancer cell lines. Based on these findings, we hypothesized that entinostat-induced apoptosis and palbociclib-induced cell cycle arrest synergize to produce enhanced antitumor effects in estrogen receptor (ER)-positive breast cancer and TNBC cell lines with high phospho-Rb expression levels. METHODS: We assessed the combination antitumor effects and their mechanisms via CellTiter Blue and sulforhodamine B assays, flow cytometry, apoptosis (caspase 3/7) assays, anchorage-independent growth assays, Western blotting, reverse phase protein array (RPPA), and mammary fat pad xenograft mouse models. RESULTS: RPPA data showed that ER-positive and TNBC cell lines more often expressed phospho-Rb than did other breast cancer cell subtypes (7/10 and 8/17 cell lines, respectively). We found that the combination of entinostat and palbociclib synergistically inhibited tumor cell proliferation (combinational index less than 1.0), reduced in vitro colony formation (P < 0.05), inhibited in vivo tumor growth in ER-positive MCF-7 breast cancer cells (P < 0.05), and inhibited tumor growth in TNBC xenograft mouse models (MDA-MB-231) more effectively than did either drug alone. CONCLUSION: Taken together, our data provide evidence that combining entinostat with palbociclib enhances the antitumor effects of these drugs. Along with our continued effort to determine predictive biomarkers, our findings justify conducting a clinical trial of combination treatment with entinostat and palbociclib in patients with ER-positive breast cancer or TNBC. Citation Format: Lee J, Lim B, Pearson T, Tripathy D, Ordentlich P, Ueno NT. The synergistic antitumor activity of entinostat (MS-275) in combination with palbociclib (PD 0332991) in estrogen receptor-positive and triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-15.

Knockdown of TGIF attenuates the proliferation and tumorigenicity of EC109 cells and promotes cisplatin-induced apoptosis.

A previous study has reported that frequent amplifications of the TG-interacting factor (TGIF) were observed in esophageal squamous cell carcinoma. The aim of the present study was to investigate the potential role of TGIF in the proliferation and tumorigenicity of the esophageal cancer cell line EC109 and cisplatin-induced apoptosis. Stable TGIF-knockdown EC109 cell line was established by infecting short hairpin RNA (shRNA) lentiviral particles. Soft agar and tumor xenograft assays were applied in nude mice. Flow cytometry was employed to evaluate the cell cycle and apoptosis. Western blot analysis was used to detect the expression of proteins. TGIF knockdown suppressed EC109 cell proliferation, colony formation in soft agar and tumor growth in nude mice, induced cell cycle arrest in the G1 phase, and promoted cisplatin-induced apoptosis. In addition, TGIF knockdown significantly reduced the expression of phospho-Rb in EC109 cells. The reduced level of full length PARP expression and the increased level of cleaved caspase-3 expression were observed in EC109 cells with the treatment of cisplatin and TGIF knockdown. The results suggest that knockdown of TGIF attenuated the proliferation and tumorigenicity of EC109 cells, and promoted cisplatin-induced apoptosis.

Expression of pRb and p16INK4 in human thymic epithelial tumors in relation to the presence of human polyomavirus 7.

BackgroundWe have recently reported the presence of the Human polyomavirus 7 (HPyV7) in human thymic epithelial tumors as assessed by diverse molecular techniques. Here we report on the co-expression of p16, retinoblastoma protein (pRb) and phosphorylated retinoblastoma protein (phospho-Rb) in human thymic epithelial tumors in relation to HPyV7.MethodsPRB, phospho-RB and p16 expression was assessed by immuno-histochemistry in 37 thymomas and 2 thymic carcinomas. 17 thymomas (46 %) and 1 thymic carcinoma (50 %) were recently tested positive for HPyV7. In addition, 20 follicular hyperplasias were tested.ResultsExpression of pRb was observed in 35 thymomas (94.6 %), in 16 thymomas (43.2 %) the expression was strong. Phospho-Rb was observed in 31 thymomas (83.8 %). 19 thymomas (51.4 %) showed immunoreactivity for p16 of which 8 thymomas revealed very strong p16 expression. No p16 expression was detected in thymic carcinomas. In addition, no significant correlation between the presence of HPyV7 and pRb-, phospho-Rb- and p16-expression could be established. No correlation between pRb, phospho-Rb, p16 and WHO staging, Masaoka-Koga staging or the presence of MG was found. All 20 follicular hyperplasias showed expression of pRb and less expression of phospho-Rb.ConclusionsAlthough polyomaviruses have been shown to interact with cell cycle proteins no correlation between the presence of HPyV7 and the expression of pRb, phospho-Rb and p16 in human thymic epithelial tumors was observed. In as much HPyV7 contributes to human thymomagenesis remains to be established. Our data indicate pRb, phospho-Rb and p16 expression are rather unlikely to be involved in HPyV7 related thymomagenesis.

Abstract 4710: Evaluation of the glutaminase inhibitor CB-839 in non-small cell lung cancer

Abstract Glutamine is an abundant nutrient used by cancer cells to support energetic and biosynthetic pathways. Given that many cancer cells exhibit glutamine dependence, there has been interest in developing inhibitors targeting glutaminase, which catalyzes conversion of glutamine to glutamate. CB-839 is a glutaminase inhibitor with preclinical efficacy in suppressing cancer cell growth in culture and in vivo. It is now in Phase I clinical trials for solid and hematological malignancies, including non-small cell lung cancer (NSCLC). Here we set out to identify biomarkers predicting sensitivity of NSCLC cells to CB-839, using a highly annotated panel of 81 cell lines. These lines had heterogeneous oncogenotypes and displayed about a 5-fold range in their ability to tolerate glutamine deprivation. Assessing CB-839 sensitivity in ∼40 lines revealed a 5-fold difference in growth suppression among cell lines. There was a strong correlation between CB-839 sensitivity and both glutamine dependence and glutamate secretion. In a subset of 4 representative cell lines, CB-839 suppressed [U-13C]glutamine metabolism in both sensitive and insensitive cells suggesting other mechanisms contribute to CB-839 sensitivity. To identify potential biomarkers predicting CB-839 sensitivity, the glutamine dependence and drug sensitivity phenotypes were correlated with several orthogonal high-content data sets including mutational analysis of commonly-mutated oncogenes and tumor suppressors; sensitivity to chemotherapeutic agents; gene expression arrays; and reverse phase protein arrays (RPPA). Although we did not identify a strong correlation between mutational status and CB-839 sensitivity, expression of phospho-Rb (S807/811) and Smad3 did correlate with drug sensitivity. In addition, sensitivity to glutamine withdrawal correlated with expression of c-Myc and MEK2 protein. While further work is necessary to examine the importance of these proteins in glutamine metabolism, we hypothesize that one or more of these may represent novel biomarkers predicting sensitivity to glutaminase inhibition. Citation Format: Lindsey K. Boroughs, Pei-Hsuan Chen, Ling Cai, Mirna Rodriguez, Winter Zhang, Kenneth E. Huffman, Lauren A. Byers, Luc Girard, Adi F. Gazdar, John V. Heymach, Michael A. White, John D. Minna, Ethan Emberley, Alison Pan, Frank Parlati, Ralph J. DeBerardinis. Evaluation of the glutaminase inhibitor CB-839 in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4710. doi:10.1158/1538-7445.AM2015-4710

Nano‐formulation Enhances the Anti‐angiogenic Potential of Tunicamycin

Breast cancer is characterized by the progressive accumulation of mutations and chromosomal aberrations leading to altered growth properties and oncogenic transformations of cells. Both endogenous and exogenous factors contribute. Current understanding on the blood vessel formation has developed therapies to interrupt the angiogenic process. But, due to poor performance and inefficacy, many anti‐angiogenic/anti‐cancer treatments have been called into question. After achieving a success with tunicamycin (a biologic) in inhibiting angiogenesis in vitro and in vivo (Matrigel™ implants in nude mice) and consequently the prevention of breast tumor progression ~55‐65% in 3 weeks in athymic nude mice we have developed a nano‐formulation to increase efficacy. Tunicamycin gold nano‐particles inhibited cellular proliferation ~50% (MTT assay) within one hour of treatment. Cell cycle progression was blocked due to inhibition of either both cyclin D1 and cdk4, or cyclin D1, or the cdk4 expression as well as the expression of phospho Rb. Phosphorylation of p53 (serine‐392) was also down‐regulated. Increased expression of GRP‐78/Bip identified “ER stress”. Upregulated expression (1.6‐5.5 folds) of phospho‐PERK supported induction of unfolded protein response (upr) signaling but no apoptosis. Supported in part by grants from Susan G. Komen for Cure BCTR0600582 (DKB) and NIH/NIMHD 2G12MD007583 (KB).

Abstract 1007: Nanoformulation enhances anti-angiogenic efficacy of Tunicamycin

Abstract The breast cancer landscape indicates it is a global problem and accounts for nearly a quarter of all cancers in women. In 2008, 1.4 million women were diagnosed with the disease worldwide and 438,000 died globally in 2010. In the United States, it is estimated that 296,000 women and 2,240 men will be diagnosed with breast cancer in 2013 and 39,620 women and 410 men will die from the disease. In 2030, with no major changes in prevention or treatment 747,802 women are estimated to die worldwide. Breast cancer treatment includes surgery, radiation therapy, chemotherapy and/or hormonal therapy, and within the past 15 years, targeted antibody therapy. Unfortunately, de novo and acquired resistance are major issues with all known targeted therapies. Tumor microenvironment (TME) is immensely complex and contributes to chemoresistance. Using Tunicamycin, a potent inhibitor of asparagine-linked (N-linked) protein glycosylation we have established earlier that Tunicamycin inhibits (i) angiogenesis in vitro by arresting cells in G1; (ii) in vivo angiogenesis in Matrigel™ implant in nude mice; and (iii) prevents the progression of double- and triple-negative breast tumors in athymic nude mice by inducing “ER stress”. Nanoparticles (<100 nm) evade the immune system's clearing mechanisms long enough to reach the targeted disease tissue efficiently. We have, therefore, hypothesized that nano-formulated Tunicamycin would have a better efficacy in treating breast cancer in the clinic. The objective was to evaluate nano-formulated Tunicamycin, and we have used Tunicamycin encapsulated in peptide nanotubes, nanotubes bound to gold nanoparticles (Au NPs) conjugated with Tunicamycin, Tunicamycin conjugated with nanotubes, Au NPs bound to tubes and conjugated with Tunicamycin, and Au NPs conjugated with Tunicamycin. The MTT assay indicated that nanoparticles (1 μg/mL) inhibited capillary endothelial cells proliferation ∼50% within one hour of treatment whereas the native Tunicamycin had no effect. The nanoformulated Tunicamycin blocked the cell cycle progression by inhibiting either both cyclin D1 and CDK4, or cyclin D1, or the CDK4 expression as well as the expression of phospho Rb (serine-229/threonine-252). Phosphorylation of p53 at serine-392 was down-regulated but not the total p53. Increased expression of GRP-78/Bip identified “ER stress”. Upregulated expression (1.6-5.5 folds) of phopsho-PERK supported induction of unfolded protein response (upr) signaling. Down regulated expression of caspase-9 and caspase-3 proposes a non-canonical pathway of cell death during “ER stress” induced by nano-formulated Tunicamycin. Citation Format: Aditi Banerjee, Dipak K. Banerjee. Nanoformulation enhances anti-angiogenic efficacy of Tunicamycin. [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 1007. doi:10.1158/1538-7445.AM2014-1007

Abstract 1327: Targeting the RB-pathway in sarcoma: Utility of CDK4/6 inhibitors

Abstract Background: Sarcomas are rare neoplasms of mesenchymal origin. Survival rates have not improved with standard chemotherapy; however, molecular insights have posed new drug targets. Aberrations in the RB-pathway, including loss of RB, CDK4 and cyclin D1 overexpression and p16 loss, have been identified in sarcomas. PD 03322991 (PD) is a second generation CDK4/6 inhibitor with the potential to reactivate the RB-pathway and suppress cell cycle progression. We evaluated the efficacy of PD in a panel of sarcoma cell lines. Methods: Sarcoma cell lines (HT-1080, SK-LMS-1 and SaOS-2) were evaluated for RB-pathway alterations using western blot. Cells were treated with PD for 1, 2, 4, 6 and 8 days to determine IC50 values by MTT assay. Following the same treatment protocol at IC50 concentrations of PD, cells were harvested for cell cycle analysis, western blot and annexin cell death assay. Results: HT-1080 and SK-LMS-1 were RB-positive with high expression of phospho-RB; SaOS-2 was RB-negative. CDK4/6 were uniformly expressed. HT-1080 and SK-LMS-1 had p16 loss while SaOS-2 overexpressed cyclin D1. PD was effective in all cell lines. Increasing treatment duration led to reduction of IC50 values. PD caused marked G1 arrest as early as day 1 in RB-positive HT-1080 (86.2% vs 74.2% [vehicle]) and SK-LMS-1 (91.4% vs 67.8% [vehicle]) but not RB-negative SaOS-2 (52.9-59.4% vs 57.0% [vehicle]). Annexin assay demonstrated minimal apoptosis with PD (HT-1080: 1.4-3.0% vs 41.2% [control], SK-LMS-1: 0.6-12.0% vs 23.4% [control], SaOS-2: 3.6-7.3% vs 78.7% [control]). Conclusions: PD appears to have a cytostatic rather than cytotoxic effect in sarcoma. RB-negativity does not preclude PD efficacy but may require higher IC50 values. In RB-negative sarcomas, PD does not cause cell cycle arrest suggesting an alternate mechanism from reactivation of the RB-pathway is present. PD may serve as a potential therapy for sarcoma and should be studied in combination with other targeted therapies that can take advantage of its ability to cause G1 arrest. Cell LinePD 991 IC50 Concentration (uM)Day 1Day 2Day 4Day 6Day 8HT-1080∼∼4.31.51.1SK-LMS-1∼∼∼2.41.2SaOS-2∼15.510.17.06.0 Citation Format: Ashleigh M. Francis, Jason P. Carey, Angela Alexander, Khandan Keyomarsi, Kelly K. Hunt. Targeting the RB-pathway in sarcoma: Utility of CDK4/6 inhibitors. [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 1327. doi:10.1158/1538-7445.AM2014-1327

P0168 Cotargeting MAPK and CDK4 signaling with concurrent radiotherapy as a strategy for the treatment of non-small-cell lung cancer

<h3>Background</h3> Non-small-cell lung cancer frequently exhibits mutational activation of the Ras/MAPK pathway, which is implicated in resistance to ionising radiation and chemotherapy. There is an urgent need for the development of novel therapies to treat KRAS-mutant lung cancer. Activation through Mek-Erk-dependent signaling occurs through cyclin D1 induction and subsequent assembly of the cyclin D1-CDK4/CDK6 complex, which results in Rb phosphorylation and Rb-E2F dissociation and G1-S cell cycle. MEK inhibitors do not engage the cell-cycle checkpoint, resulting in tumour stasis but not regression, despite robust apoptosis. The aim of the current study was to test the hypothesis that MAP kinase inhibitors and CDK4 inhibitors will offer clear therapeutic benefit when integrated into radiotherapy for treatment of KRAS-mutant lung cancer. <h3>Methods</h3> Clonogenic survival assays were done with the H358, A549, and H460 cell lines using the MEK inhibitor trametinib and the CDK4 inhibitor PD033291. Cell viability was assessed by tetrazolium-based CellTiter 96 aqueous non-radioactive cell proliferation assay (MTS). DNA repair was analysed using immunocytochemistry. Western blots were used to investigate the effects on downstream signaling. Flow cytometry was used to analyse cell cycle and apoptosis. Growth delay was used to determine the effects of trametinib and PD0332991 on in vivo tumour radiosensitivity. <h3>Findings</h3> Preradiation treatment with trametinib and PD0332991 significantly decreased clonogenic survival compared with either agent alone. Dose modifying factors at a surviving fraction for trametinib and PD0332991 were 2.38, 1.62, and 1.77 for H358, A549, and H460, respectively. Cell proliferation was decreased by treatment with trametinib and PD0332991 as compared with either agent alone. Flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest and enhanced apoptosis in these cell lines. The expression of phospho-Rb and phospho-Erk1/2 were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumour growth delay in A549 xenografts. <h3>Interpretation</h3> These data suggest that trametinib can be used with CDK inhibitors to augment the radiation response in KRAS-mutant non-small-cell lung cancer.

Nanoformulation enhances anti-angiogenic efficacy of tunicamycin.

Nanoparticles (<100 nm) evades the immune system's clearing mechanisms long enough to reach the targeted disease tissue efficiently. We have, therefore, hypothesized that nano-formulated Tunicamycin would have a better efficacy and consequently it will be a better candidate for treating solid tumor including breast cancer in the clinic. Tunicamycin, a potent inhibitor of asparagine-linked (N-linked) protein glycosylation has been found earlier (I) inhibits angiogenesis in vitro by arresting cells in G1; (II) in vivo angiogenesis in Matrigel™ implant in nude mice; and (III) prevents the progression of a double- and a triple-negative breast tumor in athymic nude mice by inducing "ER stress" in tumor microvasculature. Tunicamycin could work alone or in combination with radiation/radiotherapy. To evaluate nano-formulated Tunicamycin, we have synthesized Tunicamycin encapsulated in peptide nanotubes, nanotubes bound to gold nanoparticles (Au NPs) conjugated with Tunicamycin, Tunicamycin conjugated with nanotubes, Au NPs bound to tubes and conjugated with Tunicamycin, and Au NPs conjugated with Tunicamycin. Functionalization of the nanoparticles was characterized by transmission electron microscopy (TEM), Fourier Transformed Infrared (FTIR) Spectroscopy, dynamic light scattering, atomic force microscopy (AFM), and absorbance spectroscopy. The 3-(4,5-methylthiazol-2-yl)-2,5-dipheyl-tetrazolium bromide (MTT) assay indicated that nanoparticles (1 μg/mL) inhibited capillary endothelial cells proliferation, i.e., angiogenesis ~50% within one hour of treatment whereas the native Tunicamycin had no effect. The nano-formulated Tunicamycin blocked the cell cycle progression by inhibiting either both cyclin D1 and CDK4, or cyclin D1, or the CDK4 expression as well as the expression of phospho Rb (serine-229/threonine-252). Phosphorylation of p53 at serine-392 was down-regulated but not the total p53. Increased expression of GRP-78/Bip identified "ER stress". Upregulated expression (1.6-5.5 fold) of phopsho-PERK and significant reduction of mannosylphospho dolichol synthase (DPMS) expression supported induction of unfolded protein response (upr) signaling. Down regulated expression of caspase-9 and caspase-3 proposes a non-canonical pathway of cell death during "ER stress" induced by nano-formulated Tunicamycin.

The aqueous extract of Ficus religiosa induces cell cycle arrest in human cervical cancer cell lines SiHa (HPV-16 Positive) and apoptosis in HeLa (HPV-18 positive).

Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FRaq) bark in human cervical cancer cell lines, SiHa and HeLa. FRaq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G1/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FRaq induced apoptosis through an increase in intracellular Ca2+ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FRaq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FRaq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer.

The role of β-adrenergic receptor signaling in the proliferation of hemangioma-derived endothelial cells

BackgroundInfantile hemangioma (IH) is a benign vascular neoplasm that arises from the abnormal proliferation of endothelial cells and enhanced angiogenesis. Recently, propranolol has been found to be effective in the management of IH, suggesting that β-adrenergic receptors (β-ARs) may play an important role in the pathogenesis of IH.ResultsIn the present study, we investigated the β-adrenergic signaling that is associated with hemangioma-derived endothelial cell (HemEC) proliferation. The results showed that both β1- and β2-ARs were expressed in HemECs. Stimulation of the β-ARs by isoprenaline induced cell proliferation and elevation of second messenger cAMP levels. The proliferation-promoting action of isoprenaline was abolished by a β1-selective antagonist and was more effectively abolished by a β2-selective antagonist; the mechanism for the action of the antagonists was a G0/G1 phase cell cycle arrest which was associated with decreased cyclin D1, CDK-4, CDK-6 and phospho-Rb expression. Pre-treatment of the cells with VEGFR-2 or ERK inhibitors also prevented the isoprenaline-mediated proliferation of cells. In agreement with the involvement of β-ARs and VEGFR-2 in the HemEC response, β-AR antagonists and the VEGFR-2 inhibitor significantly attenuated isoprenaline-induced ERK phosphorylation. Moreover, treating the cells with isoprenaline markedly increased VEGF-A expression and VEGFR-2 activity in a β2-AR-dependent manner.ConclusionsWe have demonstrated that the activation of the β-ARs in the ERK pathway may be important mechanisms in promoting HemEC growth. Furthermore, stimulation of the β-AR may transactivate VEGFR-2 signaling and further increase HemEC proliferation.

Bortezomib induces tumor-specific cell death and growth inhibition in hepatocellular carcinoma and improves liver fibrosis

Human hepatocellular carcinoma (HCC) is highly ubiquitinated. The ubiquitination is important to the generatation of HCC. The antitumor and antifibrosis effects of an ubiquitin-proteasome system inhibitor, bortezomib, on HCC with liver cirrhosis (LC) were analyzed in vitro and in vivo. The effect of bortezomib was analyzed in the rat hepatocarcinogenesis model using a DEN and CDAA diet (DEN/CDAA model), which shows severe LC and generation of HCC. The decrease of GST-P-positive foci and HCC were analyzed in vivo. Cell death was analyzed by cell death detection kit. Liver fibrosis was checked by sirius-red staining and α-smooth muscle actin staining. The in vitro study involved 3 HCC cell lines (HepG2, HuH7, and HLF) and primary rat and human hepatocytes. The proliferation rate of the HCC cell line was analyzed using the MTT assay and FACS analysis. The toxicity of bortezomib was checked using the LDH release assay for primary human and rat hepatocytes. In the rat hepatocarcinogenesis model, bortezomib prevented the development of preneoplastic lesions during the early stages of hepatocarcinogenesis and specifically induced cell death in HCC. Furthermore, bortezomib inhibited cell proliferation and induced tumor-specific cell death in HCC cell lines with decrease of cyclin D1 and phospho-Rb expression. Further, bortezomib showed no hepatotoxicity of primary rat and human hepatocytes, suggesting that it might be an HCC-specific drug. Bortezomib also prevented the activation of hepatic stellate cells and inhibited the liver fibrosis of the DEN/CDAA model. Bortezomib appears to be an ideal target drug for HCC with LC.