Rescued Growth Inhibition Research Articles

The PRC2.1 Subcomplex Opposes G1 Progression through Regulation of CCND1 and CCND2.

Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenetic approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued proliferation inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in antagonizing G1 progression in a diversity of cell linages, including CML, breast cancer and immortalized cell lines.

Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target.

Fusobacterium nucleatum, a pathobiont inhabiting the oral cavity, contributes to opportunistic diseases, such as periodontal diseases and gastrointestinal cancers, which involve microbiota imbalance. Broad-spectrum antimicrobial agents, while effective against F. nucleatum infections, can exacerbate dysbiosis. This necessitates the discovery of more targeted narrow-spectrum antimicrobial agents. We therefore investigated the potential for the fusobacterial enoyl-ACP reductase II (ENR II) isoenzyme FnFabK (C4N14_ 04250) as a narrow-spectrum drug target. ENRs catalyze the rate-limiting step in the bacterial fatty acid synthesis pathway. Bioinformatics revealed that of the four distinct bacterial ENR isoforms, F. nucleatum specifically encodes FnFabK. Genetic studies revealed that fabK was indispensable for F. nucleatum growth, as the gene could not be deleted, and silencing of its mRNA inhibited growth under the test conditions. Remarkably, exogenous fatty acids failed to rescue growth inhibition caused by the silencing of fabK. Screening of synthetic phenylimidazole analogues of a known FabK inhibitor identified an inhibitor (i.e., 681) of FnFabK enzymatic activity and F. nucleatum growth, with an IC50 of 2.1 μM (1.0 μg/mL) and a MIC of 0.4 μg/mL, respectively. Exogenous fatty acids did not attenuate the activity of 681 against F. nucleatum. Furthermore, FnFabK was confirmed as the intracellular target of 681 based on the overexpression of FnFabK shifting MICs and 681-resistant mutants having amino acid substitutions in FnFabK or mutations in other genetic loci affecting fatty acid biosynthesis. 681 had minimal activity against a range of commensal flora, and it was less active against streptococci in physiologic fatty acids. Taken together, FnFabK is an essential enzyme that is amenable to drug targeting for the discovery and development of narrow-spectrum antimicrobial agents.

Suppression of Super-Enhancer-Driven TAL1 Expression By KLF4 in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by differentiation arrest and clonal proliferation of immature thymocytes. Many oncogenes in T-ALL are genes encoding transcription factors. TAL bHLH transcription factor 1 ( TAL1) is one of the most frequently dysregulated transcription factors in T-ALL and is ectopically overexpressed in approximately 50% of T-ALL cases, owing to chromosome translocations, SIL (SCL-interrupting locus) -TAL1 fusion, and 5‘ super-enhancer (SE)-generating mutations. TAL1 overexpression caused by 5‘ TAL1 SE is found in 5% of patients with T-ALL and is associated with unfavorable clinical outcomes. However, no clinically available drugs specifically inhibit TAL1 or TAL1 SE components. Kruppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors that exhibits both oncogenic and tumor-suppressive functions in a context-dependent manner. In T-cell malignancies, KLF4 is regarded as a tumor suppressor because KLF4 expression is silenced by promoter methylation, and the induction of KLF4 promotes apoptosis through the BCL-2/BCL-XL pathway. However, whether KLF4 affects the expression of T-ALL-related transcription factors remains unclear. We first analyzed three independent previously reported microarray datasets to explore the essential downstream targets of KLF4 in T-ALL cells. The following groups of genes were extracted: genes upregulated in patients with T-ALL compared to normal thymocytes (GSE46170), genes upregulated in Klf4-deficient T-ALL mice (GSE75663), and genes downregulated in KLF4-overexpressing human T-ALL cells (Li W et al. Mol. Cancer. 2015). Overlapping genes in these groups were identified as potential downstream targets of KLF4. Among these, we focused on TAL1, a well-known oncogenic transcription factor gene in T-ALL. We performed a functional analysis of KLF4 using TAL1-positive T-ALL cell lines (Jurkat, MOLT-3, CCRF-CEM, and RPMI-8402). Genetic induction of KLF4 showed stronger anti-leukemic effects in Jurkat and MOLT-3 cells, 5‘ TAL1 SE-positive T-ALL cell lines, compared with CCRF-CEM and RPMI-8402 cells, SIL-TAL1-positive T-ALL cell lines. KLF4 overexpression significantly reduced mRNA expression levels of TAL1 in Jurkat and MOLT-3 cells but not in CCRF-CEM and RPMI-8402 cells. Thus, we hypothesized that KLF4 specifically suppressed SE-driven TAL1 expression by impairing mutated TAL1 enhancer activity. The luciferase reporter assay confirmed that the activity of the mutated TAL1 enhancer was significantly reduced by the exogenous expression of KLF4, whereas that of the wild-type reporter was not affected. According to previous studies, mutations in TAL1 enhancers in Jurkat and MOLT-3 cells create MYB primary motifs, and MYB binding at this location is essential for the formation of 5‘ TAL1 SE. Therefore, we examined whether KLF4 represses MYB expression. The chromatin immunoprecipitation assay and qPCR analysis revealed that KLF4 bound to the proximal gene promoter of MYB and directly downregulated its expression. The luciferase reporter assay using the MYB promoter showed a significant reduction of reporter activity upon KLF4 overexpression. Finally, we investigated whether pharmacological induction of KLF4 using APTO-253, a small-molecule inducer ofKLF4, could control leukemia. APTO-253 inhibited the growth of all TAL1-positive T-ALL cell lines at nanomolar concentrations. The GI 50 values of APTO-253 for Jurkat and MOLT-3 cells were lower than those for CCRF-CEM and RPMI-8402 cells, which was consistent with the KLF4-overexpression-induced growth inhibition of T-ALL cell lines. APTO-253 induced KLF4 expression and decreased TAL1 and MYB expression in Jurkat and MOLT-3 cells. Furthermore, exogenous MYB expression rescued growth inhibition by APTO-253. Collectively, these results indicated that APTO-253 suppressed the growth of T-ALL cells by targeting SE-driven TAL1 expression via the induction of KLF4. In summary, we revealed that KLF4 suppressed SE-driven TAL1 expression via the direct inhibition of MYB. Moreover, pharmacological induction of KLF4 demonstrated anti-leukemic activity in T-ALL cells. These findings propose a promising strategy for patients with T-ALL and 5' TAL1 SE.

Loss of allosteric regulation in α-isopropylmalate synthase identified as an antimicrobial resistance mechanism

Despite our best efforts to discover new antimicrobials, bacteria have evolved mechanisms to become resistant. Resistance to antimicrobials can be attributed to innate, inducible, and acquired mechanisms. Mycobacterium abscessus is one of the most antimicrobial resistant bacteria and is known to cause chronic pulmonary infections within the cystic fibrosis community. Previously, we identified epetraborole as an inhibitor against M. abscessus with in vitro and in vivo activities and that the efficacy of epetraborole could be improved with the combination of the non-proteinogenic amino acid norvaline. Norvaline demonstrated activity against the M. abscessus epetraborole resistant mutants thus, limiting resistance to epetraborole in wild-type populations. Here we show M. abscessus mutants with resistance to epetraborole can acquire resistance to norvaline in a leucyl-tRNA synthetase (LeuRS) editing-independent manner. After showing that the membrane hydrophobicity and efflux activity are not linked to norvaline resistance, whole-genome sequencing identified a mutation in the allosteric regulatory domain of α-isopropylmalate synthase (α-IPMS). We found that mutants with the α-IPMSA555V variant incorporated less norvaline in the proteome and produced more leucine than the parental strain. Furthermore, we found that leucine can rescue growth inhibition from norvaline challenge in the parental strain. Our results demonstrate that M. abscessus can modulate its metabolism through mutations in an allosteric regulatory site to upregulate the biosynthesis of the natural LeuRS substrate and outcompete norvaline. These findings emphasize the antimicrobial resistant nature of M. abscessus and describe a unique mechanism of substrate-inhibitor competition.

Compounds originating from the edible mushroom Auricularia auricula-judae inhibit tropomyosin receptor kinase B activity

Tropomyosin receptor kinase B (TrkB) serves as a pivotal factor in various cancers. To identify novel natural compounds with TrkB-inhibiting properties, a screening approach was applied using extracts from a collection of wild and cultivated mushroom fruiting bodies, and Ba/F3 cells that ectopically express TrkB (TPR-TrkB). We selected mushroom extracts that selectively inhibited proliferation of the TPR-TrkB cells. We then evaluated the ability of exogenous interleukin 3 to rescue growth inhibition by the selected TrkB-positive extracts. An ethyl acetate extract of Auricularia auricula-judae actively inhibited auto-phosphorylation of TrkB. LC–MS/MS analysis of this extract revealed substances that might be responsible for the observed activity. This screening approach demonstrates, for the first time, that extracts originating from the mushroom A. auricula-judae exhibit TrkB-inhibition properties that might hold therapeutic potential for TrkB-positive cancers.

Abstract 90: STAT3-driven MAPK activation represents a therapeutic vulnerability in ASCL1 high SCLC

Abstract Background Small cell lung cancer (SCLC) is an aggressive high grade neuroendocrine tumor (Hann et al. 2019; Bernhardt et al. 2016). Whilst MAPK mutations can be found in roughly 30% of human cancers (Schubbert et al. 2007) including non-small cell lung cancer (NSCLC), genomic and proteomic analyses have indicated suppression of MAPK pathway activity in SCLC (Cerami et al. 2012; Gao et al. 2013). Previous attempts to determine whether this might be therapeutically important (Ravi et al, 1998; Cristea et al. 2016/2020) have had conflicting conclusions. SCLC has recently been defined by the relative expression of four major transcriptional regulators (ASCL1, NEUROD1, POU2F3, YAP1) (Rudin et al., 2019). In this study we aimed to elucidate the effect of MAPK activation in these different SCLC subtypes and explore its therapeutic vulnerability. Results ASCL1-, NEUROD1- and POU2F3- driven SCLC cell lines were transduced with a doxycycline-inducible vector for expression of MEKDDS217D/S221D (MEK1). Activation through MEK1 in ASCL1-driven SCLC cell lines resulted in a significant decrease in cell growth over 9 days. This was associated with G2 cell-cycle arrest and senescence. Expression of MEK1 in the cells of other SCLC subtypes and NSCLC failed to show any appreciable changes in cell growth. Remarkably, athymic mice injected with a MEK1 expressing ASCL1-driven cell line showed significantly slower tumor formation and longer survival than when MEK1 was not expressed. Notably, we observed the opposite when MEK1 expressing NEUROD1-driven cells were injected into athymic mice. We observed strong upregulation of negative feedback regulators DUSP6 and SPRY2 upon MAPK activation which has previously been described in solid tumors and pre-B ALL (Courtois-Cox et al 2006; Shojaee et al. 2015). Interestingly, phosphokinase array demonstrated that, almost exclusively, STAT3 through phosphorylation at S727 was strongly upregulated in the ASCL1-driven subtype after MEK1 expression. This prompted us to look at other regulators of the JAK-STAT pathway and found an increase in phosphorylation of the inhibitory phosphatase PTPN6 (SHP1) in the ASCL1-driven subtype and no STAT5 phosphorylation in any of the subtypes. We next examined whether these cells were sensitive to STAT3 inhibition. Upon treatment with a STAT3 inhibitor, stattic, ASCL1-driven SCLC cells reached their IC50 after 3-5 days in comparison to 9 days for other SCLC subtypes. MEK inhibition through PD0325901 rescued growth inhibition upon MAPK activation and was further associated with a decrease in DUSP6, SPRY2 and pS727 STAT3. JAK1/2 inhibition through ruxolitinib had no effect on cell viability. Summary These findings suggest that ASCL1-driven SCLC in vitro and in vivo is sensitive to activation of MAPK signaling. Our data provides additional understanding of SCLC biology and its complex signaling networks and potential subtype-specific drug susceptibilities. Citation Format: Rebecca Caeser, Christopher Hulton, Emily Costa, Vidushi Durani, Megan Little, Xiaoping Chen, Sam E. Tischfield, Marina Asher, Faruk Erdem Kombak, Shweta S. Chavan, Nisargbhai S. Shah, Metamia Ciampricotti, Elisa de Stanchina, John T. Poirier, Charles M. Rudin, Triparna Sen. STAT3-driven MAPK activation represents a therapeutic vulnerability in ASCL1 high SCLC [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 90.

CRAF dimerization with ARAF regulates KRAS-driven tumor growth

KRAS, which is mutated in ∼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulatingMAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.

Interaction of BTB-TAZ protein MdBT2 and DELLA protein MdRGL3a regulates nitrate-mediated plant growth.

Nitrate acts as a vital signal molecule in the modulation of plant growth and development. The phytohormones gibberellin (GA) is also involved in this process. However, the exact molecular mechanism of how nitrate and GA signaling pathway work together in regulating plant growth remains poorly understood. In this study, we found that a nitrate-responsive BTB/TAZ protein MdBT2 participates in regulating nitrate-induced plant growth in apple (Malus × domestica). Yeast two-hybridization, protein pull-down, and bimolecular fluorescence complementation (BiFC) assays showed that MdBT2 interacts with a DELLA protein MdRGL3a, which is required for the ubiquitination and degradation of MdRGL3a proteins via a 26S proteasome-dependent pathway. Furthermore, heterologous expression of MdBT2 partially rescued growth inhibition caused by overexpression of MdRGL3a in Arabidopsis. Taken together, our findings indicate that MdBT2 promotes nitrate-induced plant growth partially through reducing the abundance of the DELLA protein MdRGL3a.

MCHM Acts as a Hydrotrope, Altering the Balance of Metals in Yeast

While drugs and other industrial chemicals are routinely studied to assess risks, many widely used chemicals have not been thoroughly evaluated. One such chemical, 4-methylcyclohexane methanol (MCHM), is an industrial coal-cleaning chemical that contaminated the drinking water supply in Charleston, WV, USA in 2014. While a wide range of ailments was reported following the spill, little is known about the molecular effects of MCHM exposure. We used the yeast model to explore the impacts of MCHM on cellular function. Exposure to MCHM dramatically altered the yeast transcriptome and the balance of metals in yeast. Underlying genetic variation in the response to MCHM, transcriptomics and, mutant analysis uncovered the role of the metal transporters, Arn2 and Yke4, to MCHM response. Expression of Arn2, which is involved in iron uptake, was lower in MCHM-tolerant yeast and loss of Arn2 further increased MCHM tolerance. Genetic variation within Yke4, an ER zinc transporter, also mediated response to MCHM, and loss of Yke4 decreased MCHM tolerance. The addition of zinc to MCHM-sensitive yeast rescued growth inhibition. In vitro assays demonstrated that MCHM acted as a hydrotrope and prevented protein interactions, while zinc induced the aggregation of proteins. We hypothesized that MCHM altered the structures of extracellular domains of proteins, and the addition of zinc stabilized the structure to maintain metal homeostasis in yeast exposed to MCHM.

Abstract 2322: Ferroptosis induced by erastin in RAS mutant ovarian cancer cells

Abstract Although apoptosis has been thought as the principal mechanism of programed cell death in chemotherapy, other forms of non-apoptotic death also has been noted. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of cell death termed ferroptosis. This mechanism is expected to induce eradication of chemotherapeutic (e.g. platinum) resistant cancer cells. In this study we demonstrate this new form of cell death in ovarian cancer cells. The clear cell epithelial ovarian cancer cell lines, TOV-21G (KRAS_G13C) and KOC-7C (RAS wild type) were used in this study. Erastin dose-dependently inhibited growth in TOV-21G cells, but not KOC-7C cells. Treatment of TOV-21G cells with erastin resulted in increase of ROS. Iron chelator deferoxamine (DFO) rescued growth inhibition induced by erastin in TOV-21G cells. Although cisplatin inhibited growth in TOV-21G cells, this inhibition was not rescued by DFO. The GSH level was reduced by erastin, but this reduction was not rescued by DFO. In addition, the basal GSH level was higher and the basal ROS level was lower in KOC-7C cells than those in TOV-21G cells. Erastin induced ferroptosis by reducing the GSH level in RAS mutant ovarian cancer cells. Although DFO rescued growth inhibition induced by erastin, the pathway was not on System Xc-. The sensitivity to erastin might depends on the basal level of GSH in cells. Ferroptosis had great potential to become a new approach in anti-tumor therapies in ovarian cancer. Citation Format: Motoki Takenaka, Noriko Suzuki, Minako Mori, Hitomi Aoki, Tasuku Hirayama, Hideko Nagasawa, Ken-ichirou Morishige. Ferroptosis induced by erastin in RAS mutant ovarian cancer cells [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 2322.

G-protein coupled receptor 15 mediates angiogenesis and cytoprotective function of thrombomodulin

Thrombomodulin (TM) stimulates angiogenesis and protects vascular endothelial cells (ECs) via its fifth epidermal growth factor-like region (TME5); however, the cell surface receptor that mediates the pro-survival signaling activated by TM has remained unknown. We applied pull-down assay followed by MALDI-TOF MS and western blot analysis, and identified G-protein coupled receptor 15 (GPR15) as a binding partner of TME5. TME5 rescued growth inhibition and apoptosis caused by calcineurin inhibitor FK506 in vascular ECs isolated from wild type (WT) C57BL/6 mice. On the other hand, TME5 failed to protect ECs isolated from GPR15 knockout (GPR15 KO) mice from FK506-caused vascular injury. TME5 induced activation of extracellular signal-regulated kinase (ERK) and increased level of anti-apoptotic proteins in a GPR15 dependent manner. In addition, in vivo Matrigel plug angiogenesis assay found that TME5 stimulated angiogenesis in mice. TME5 promoted endothelial migration in vitro. Furthermore, TME5 increased production of NO in association with activated endothelial NO synthase (eNOS) in ECs. All these pro-angiogenesis functions of TME5 were abolished by knockout of GPR15. Our findings suggest that GPR15 plays an important role in mediating cytoprotective function as well as angiogenesis of TM.

NEK2 Promotes Aerobic Glycolysis in Multiple Myeloma Through Regulating Splicing of Pyruvate Kinase

BackgroundAerobic glycolysis, a hallmark of cancer, is characterized by increased metabolism of glucose and production of lactate in normaxia. Recently, pyruvate kinase M2 (PKM2) has been identified as a key player for regulating aerobic glycolysis and promoting tumor cell proliferation and survival.MethodsTandem affinity purification followed up by mass spectrometry (TAP-MS) and co-immunoprecipitation (Co-IP) were used to study the interaction between NIMA (never in mitosis gene A)-related kinase 2 (NEK2) and heterogeneous nuclear ribonucleoproteins (hnRNP) A1/2. RNA immunoprecipitation (RIP) was performed to identify NEK2 binding to PKM pre-mRNA sequence. Chromatin-immunoprecipitation (ChIP)-PCR was performed to analyze a transcriptional regulation of NEK2 by c-Myc. Western blot and real-time PCR were executed to analyze the regulation of PKM2 by NEK2.ResultsNEK2 regulates the alternative splicing of PKM immature RNA in multiple myeloma cells by interacting with hnRNPA1/2. RIP shows that NEK2 binds to the intronic sequence flanking exon 9 of PKM pre-mRNA. Knockdown of NEK2 decreases the ratio of PKM2/PKM1 and also other aerobic glycolysis genes including GLUT4, HK2, ENO1, LDHA, and MCT4. Myeloma patients with high expression of NEK2 and PKM2 have lower event-free survival and overall survival. Our data indicate that NEK2 is transcriptionally regulated by c-Myc in myeloma cells. Ectopic expression of NEK2 partially rescues growth inhibition and cell death induced by silenced c-Myc.ConclusionsOur studies demonstrate that NEK2 promotes aerobic glycolysis through regulating splicing of PKM and increasing the PKM2/PKM1 ratio in myeloma cells which contributes to its oncogenic activity.

Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis

Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis.

423 The intracellular domain of amphiregulin localizes to the nucleus and rescues growth inhibition in response to EGF receptor blockade

423 The intracellular domain of amphiregulin localizes to the nucleus and rescues growth inhibition in response to EGF receptor blockade

Abstract 721: Mevalonate pathway mediates acquired anti-HER2 treatment resistance in HER2+ breast cancer

Abstract Background: Compelling evidence suggests that a more complete blockade of the HER receptor layer and its signaling, by combining anti-HER2 drugs such as Trastuzumab (T) and Lapatinib (L), is highly effective. However, resistance remains a challenge. To understand resistance mechanisms, we established a broad panel of L, T, and L+T resistant cell line models. Initial mRNA expression profiling identified upregulation or restoration of the mevalonate (MVA) pathway in some models where HER signaling is completely and sustainably blocked, suggesting a possible role of the MVA pathway in resistance. This pathway is a biosynthetic process for cholesterol and isoprenoid intermediates, particularly farnesyl and geranylgeranyl pyrophosphates (FPP and GGPP). Coordinating gene expression of this pathway is regulated by the master transcription factor, SREBP. Statins, cholesterol-lowering drugs, block this pathway via inhibiting the rate-limiting enzyme, HMG-CoA reductase. While a role of the MVA pathway in tumor initiation and progression was reported, its role in anti-HER2 resistance has remained elusive. Methods: SKBR3, AU565, and UACC812 parental HER2+ cells and their T, L, and L+T resistant derivatives (TR, LR, and LTR) were used. SREBP activity was determined by a reporter luciferase assay. Cell growth was assayed by methylene blue staining. Apoptosis was determined by Annexin V staining and the protein level of cleaved PARP. Molecular signaling analysis was performed by western blotting (WB) and reverse phase protein array (RPPA). Results: Reporter assays showed the inhibition of the SREBP activity upon LT treatment and its restoration at resistance. Blocking the MVA pathway with statins led to a marked growth inhibition or apoptosis in LR/LTR models, in which the HER signaling remains sustainably inhibited, while cognate parental cells and TR cells, in which HER is (re)activated, were slightly inhibited. Prevention of statin-induced growth inhibition or apoptosis by exogenous MVA indicated the specific role of this pathway in resistance. Cholesterol or its precursor squalene could not rescue growth inhibition. In contrast, both FPP and GGPP reversed the growth inhibition or apoptosis in SKBR3 and AU565 LR/LTR models, while in the UACC812LTR model only GGPP rescued. WB showed that mTOR and estrogen receptor (ER) are downstream effectors mediating the MVA pathway to anti-HER2 resistance. Proteomic analysis revealed additional novel MVA effectors which are currently under biochemical and functional validation. Conclusion: The MVA pathway plays a key role as an escape pathway by activating alternative signaling, including mTOR and ER pathways, in acquired resistance to potent HER2 inhibition in a cholesterol-independent but FPP/GGPP-dependent manner. Targeting the MVA pathway or its downstream effectors might provide a novel therapeutic strategy to overcome anti-HER2 resistance. Citation Format: Huizhong Hu, Lukas Simon, Lanfang Qin, Agostina Nardone, Chad Shaw, Gary Chamness, Laura Heiser, Nicholas Wang, Joe W. Gray, C. Kent Osborne, Rachel Schiff. Mevalonate pathway mediates acquired anti-HER2 treatment resistance in HER2+ breast 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 721. doi:10.1158/1538-7445.AM2015-721

Abstract P5-05-01: Targeting the mevalonate pathway to overcome acquired anti-HER2 treatment resistance

Abstract Background: Compelling preclinical and clinical evidence suggests that a more complete blockade of the HER receptor layer and its signaling, by combining anti-HER2 drugs, such as Trastuzumab (T) and Lapatinib (L), is highly effective. However, resistance is still common and remains a challenge. To understand resistance mechanisms and further to identify novel therapeutic strategies, we established a broad panel of L, T, and L+T resistant cell line models. Initial mRNA expression profiling identified upregulation or restoration of the mevalonate (MVA) pathway in some models where HER signaling is completely and sustainably blocked. The MVA pathway is commonly considered as a biosynthetic process primarily for cholesterol and isoprenoid intermediates, particularly farnesyl and geranylgeranyl pyrophosphates (FPP and GGPP, respectively). Statins, widely-used cholesterol-lowering drugs, block this pathway via inhibition of the rate-limiting enzyme, HMG-CoA reductase. While accumulating evidence also suggests a role of the MVA pathway in tumor initiation and progression, its role in anti-HER2 resistance remains elusive. Methods: SKBR3, AU565, and UACC812 parental HER2+ cells and their T, L, and L+T resistant (TR, LR, and LTR respectively) derivatives were used in this study. Cell growth after treatment with statins in the presence or absence of MVA, cholesterol, squalene, FPP, or GGPP was measured by methylene blue staining. Apoptosis was determined by Annexin V staining and the protein level of cleaved PARP. Parallel analysis of molecular signaling was done by western blotting. Results: Blocking the MVA pathway with lipophilic statins, simvastatin or atorvastatin, led to a marked growth inhibition or apoptosis in LR/LTR models, in which the HER signaling remains sustainably inhibited, while cognate parental cells and TR cells, in which HER is (re)activated, were only slightly inhibited. Interestingly, only lipophilic statins (which can be taken up by cancer cells), but not hydrophilic statins such as pravastatin (whose primary target is liver cells), conveyed the inhibitory effect. Prevention of statin-induced apoptosis by adding exogenous MVA indicated that the cell death caused by statin treatment was via its specific blockade of the MVA pathway. Cholesterol or its precursor squalene could not rescue growth inhibition. In contrast, both FPP and GGPP reversed the growth inhibition or apoptosis in SKBR3 and AU565 LR/LTR models, while in the UACC812LTR model only GGPP rescued. Interestingly, mTOR was identified as the downstream signaling target of the MVA pathway in SKBR3 and AU565LTR models, while in the UACC812LTR model, the growth inhibition by statin was due to substantial estrogen receptor (ER) protein reduction. Conclusion: The MVA pathway plays a key role as an escape pathway by activating alternative signaling, including mTOR and ER pathways, in acquired resistance to potent HER2 inhibition in a cholesterol-independent but FPP/GGPP-dependent manner. Targeting the MVA pathway or its downstream effectors could provide a novel therapeutic strategy to overcome anti-HER2 resistance. Citation Format: Huizhong Hu, Lukas M Simon, Agostina Nardone, Chad A Shaw, Gary C Chamness, Laura M Heiser, Nicholas Wang, C Kent Osborne, Rachel Schiff. Targeting the mevalonate pathway to overcome acquired anti-HER2 treatment resistance [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-01.

The effect of 17-allylamino-17-demethoxygeldanamycin alone or in combination with paclitaxel on anaplastic thyroid carcinoma cells.

The effect of 17-allylamino-17-demethoxygeldanamycin (17-AAG), an hsp90 inhibitor, alone or in combination with paclitaxel on survival of anaplastic thyroid carcinoma (ATC) was evaluated. In 8505C and CAL62 cells, after treatment of 17-AAG, cell viability decreased, and the percentage of dead cells increased. 17-AAG did not cause cleavage of caspase-3 protein, and change expression of IAPs. Pretreatment of z-VAD-fmk did not alter cell viability and the percentage of dead cells. In 17-AAG-treated cells, knockdown of p53 rescued growth inhibition, while cycloheximide attenuated cell death. When cells were treated with both 17-AAG and paclitaxel, all of the combination index values were higher than 1, indicating antagonism between 17-AAG and paclitaxel. In 17-AAG- and paclitaxel-treated cells, compared with paclitaxel alone-treated cells, the protein levels of hsp90, hsp70, and hsc70 increased. In conclusion, our results suggest that 17-AAG induces non-apoptotic cell death requiring de novo protein synthesis in ATC cells. Moreover, these results demonstrate that 17-AAG antagonizes paclitaxel with concomitant alterations in hsp90 client proteins in ATC cells.

Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen

BackgroundHIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex.ResultsNef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades.ConclusionsOur findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.

EZH2 overexpression in natural killer/T-cell lymphoma confers growth advantage independently of histone methyltransferase activity

AbstractThe role of enhancer of zeste homolog 2 (EZH2) in cancer is complex and may vary depending on the cellular context. We found that EZH2 is aberrantly overexpressed in the majority of natural killer/T-cell lymphoma (NKTL), an aggressive lymphoid malignancy with very poor prognosis. We show that EZH2 upregulation is mediated by MYC-induced repression of its regulatory micro RNAs and EZH2 exerts oncogenic properties in NKTL. Ectopic expression of EZH2 in both primary NK cells and NKTL cell lines leads to a significant growth advantage. Conversely, knock-down of EZH2 in NKTL cell lines results in cell growth inhibition. Intriguingly, ectopic EZH2 mutant deficient for histone methyltransferase activity is also able to confer growth advantage and rescue growth inhibition on endogenous EZH2 depletion in NKTL cells, indicating an oncogenic role of EZH2 independent of its gene-silencing activity. Mechanistically, we show that EZH2 directly promotes the transcription of cyclin D1 and this effect is independent of its enzymatic activity. Furthermore, depletion of EZH2 using a PRC2 inhibitor 3-deazaneplanocin A significantly inhibits growth of NK tumor cells. Therefore, our study uncovers an oncogenic role of EZH2 independent of its methyltransferase activity in NKTL and suggests that targeting EZH2 may have therapeutic usefulness in this lymphoma.

Caffeic Acid Phenethyl Ester Suppresses Proliferation and Survival of TW2.6 Human Oral Cancer Cells via Inhibition of Akt Signaling

Caffeic acid phenethyl ester (CAPE) is a bioactive component extracted from honeybee hive propolis. Our observations indicated that CAPE treatment suppressed cell proliferation and colony formation of TW2.6 human oral squamous cell carcinoma (OSCC) cells dose-dependently. CAPE treatment decreased G1 phase cell population, increased G2/M phase cell population, and induced apoptosis in TW2.6 cells. Treatment with CAPE decreased protein abundance of Akt, Akt1, Akt2, Akt3, phospho-Akt Ser473, phospho-Akt Thr 308, GSK3β, FOXO1, FOXO3a, phospho-FOXO1 Thr24, phospho-FoxO3a Thr32, NF-κB, phospho-NF-κB Ser536, Rb, phospho-Rb Ser807/811, Skp2, and cyclin D1, but increased cell cycle inhibitor p27Kip. Overexpression of Akt1 or Akt2 in TW2.6 cells rescued growth inhibition caused by CAPE treatment. Co-treating TW2.6 cells with CAPE and 5-fluorouracil, a commonly used chemotherapeutic drug for oral cancers, exhibited additive cell proliferation inhibition. Our study suggested that administration of CAPE is a potential adjuvant therapy for patients with OSCC oral cancer.