Conflict of interest: D.J.R. and A.J.C. are co-founders in StemSynergy Therapeutics, a company commercializing small-molecule cell signaling inhibitors. W.G. and D.O. are employees of StemSynergy Therapeutics. William Guerrant and Luisana Astudillo contributed equally as second author.

No abstract

We report studies on loss of heme at or below pH 3.0 from two clinically important hemoglobin variants, HbE and HbS, in the presence and absence of phopholipid membranes. The kinetics of heme loss has been studied at pH 3.0 to simulate the same at a faster rate than at physiological pH, for spectroscopic investigation. Results obtained from the study clearly establish the probable fate of the lost heme to partition into the phospholipid bilayer independent of the pH range. This is also of particular importance to membranes containing the aminophospholipid and cholesterol which are predominantly localized in the inner leaflet of erythrocytes. Absorption measurements indicated such loss of heme when the Soret peak at 415 nm blue-shifted to 380 nm at pH 3.0. The extent of this blue shift decreased from 35 nm to (approx.) 15 nm in the presence of small unilammelar vesicles of both dimyristoyl- and dioleoyl-based phosphatidylcholine and phosphatidylethanolamine, indicating partitioning of the released heme in the membrane bilayer. The kinetics of heme loss was faster from HbE than HbA and HbS, obeying first-order reaction kinetics. Released heme could be involved in the premature destruction of erythrocytes in hemoglobin disorders.

SummaryHypoxia is a hall mark of solid tumor microenvironment and contributes to tumor progression and therapy failure. The developmentally important Notch pathway is implicated in cellular response of cancer cells to hypoxia. Yet, the mechanisms that potentiate Notch signaling under hypoxia are not fully understood. Hypoxia is also a stimulus for AMP-activated protein kinase (AMPK), a major cellular energy sensor. In this study, we investigated if AMPK interacts with the Notch pathway and influences the hypoxia-response of breast cancer cells. Activating AMPK with pharmacological agent or genetic approaches led to an increase in the levels of cleaved Notch1 and elevated Notch signaling in invasive breast cancer cell lines. In contrast, inhibition or depletion of AMPK reduced the amount of cleaved Notch1. Significantly, we show that the hypoxia-induced increase in cleaved Notch1 levels requires AMPK activation. Probing into the mechanism, we demonstrate that AMPK activation impairs the interaction between cleaved Notch1 and its ubiquitin ligase, Itch/AIP4 through the tyrosine kinase Fyn. Under hypoxia, the AMPK-Fyn axis promotes inhibitory phosphorylation of Itch which abrogates its interaction with substrates, thus stabilizing cleaved Notch1 by reducing its ubiquitination and degradation. We further show that inhibition of AMPK alleviates the hypoxia-triggered, Notch-mediated stemness and drug resistance phenotype. Breast cancer patient samples also showed co-expression of hypoxia/AMPK/Notch gene signature. Our work thus establishes AMPK as a key component in the adaptation of breast cancer cells to hypoxia, and proposes therapeutic inhibition of AMPK to mitigate the hypoxia-triggered aggressiveness.

Breast Cancer Stem Cells (BCSCs) are a major cause of therapy resistance and tumour progression. Currently, their regulation is not entirely understood. Previous work from our lab demonstrated context-specific pro-tumorigenic role of AMP-activated protein kinase (AMPK) in breast cancer cell survival under anchorage-deprivation and mammosphere formation hallmarks of BCSCs. We therefore investigated the role of AMPK in the maintenance of BCSC state/function. AMPK depletion reduces serial sphere formation in vitro and tumour initiation in vivo. Intriguingly, tumour-derived cell analysis using stem cell markers and functional assays revealed that AMPK is required for the maintenance of BCSC population in vivo. AMPK promotes the expression of stemness genes like Nanog, Sox2 and Bmi1 through the transcriptional upregulation of Twist via promoter acetylation. Further, AMPK-driven stemness plays a critical role in resistance to doxorubicin. Significantly, we found that AMPK activity increased after chemotherapy in patient-derived tumour samples alongside an increase in stemness markers. Importantly, AMPK depletion sensitises mice tumours to doxorubicin treatment. Our work indicates that targeting AMPK in conjunction with regular chemotherapy is likely to reduce the stem cell pool and improve chemosensitivity in breast cancers.

No abstract

Background Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. Methods Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. Results We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. Conclusions This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation.

<p>Off target profiling of NADI-351</p>