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Mutations in SPOP , the gene encoding a component of the E3 ubiquitin ligase complex, impair ubiquitination-dependent degradation of BRD2, BRD3 and BRD4 proteins and result in activation of ATK–mTORC1 signaling and resistance to BET inhibitors. Pharmacological blockade of AKT represents a viable strategy to restore the sensitivity of SPOP-mutant prostate tumors to BET inhibitors. These results, together with findings by Dai et al . and Janouskova et al ., uncover a new nongenetic mechanism of resistance to BET inhibition involving cancer-type-specific mutations in SPOP , and support the evaluation of SPOP mutation status to inform the administration of BET inhibitors in the clinic. Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anticancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is the most frequently mutated in primary prostate cancer. Here we demonstrate that wild-type SPOP binds to and induces ubiquitination and proteasomal degradation of BET proteins (BRD2, BRD3 and BRD4) by recognizing a degron motif common among them. In contrast, prostate cancer–associated SPOP mutants show impaired binding to BET proteins, resulting in decreased proteasomal degradation and accumulation of these proteins in prostate cancer cell lines and patient specimens and causing resistance to BET inhibitors. Transcriptome and BRD4 cistrome analyses reveal enhanced expression of the GTPase RAC1 and cholesterol-biosynthesis-associated genes together with activation of AKT–mTORC1 signaling as a consequence of BRD4 stabilization. Our data show that resistance to BET inhibitors in SPOP -mutant prostate cancer can be overcome by combination with AKT inhibitors and further support the evaluation of SPOP mutations as biomarkers to guide BET-inhibitor-oriented therapy in patients with prostate cancer.

Background: Oestrogen receptor-negative (ER−) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer. Methods: Gene and protein expression profiles were analysed in a panel of ER− breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively. Results: The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN -inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN -inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN- γ secretion by tumour cells. Conclusions: Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER− breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.

BCL-2 family proteins are central regulators of mitochondrial apoptosis and validated anti-cancer targets. Using small cell lung cancer (SCLC) as a model, we demonstrated the presence of differential addiction of cancer cells to anti-apoptotic BCL-2, BCL-X L or MCL-1, which correlated with the respective protein expression ratio. ABT-263 (navitoclax), a BCL-2/BCL-X L inhibitor, prevented BCL-X L from sequestering activator BH3-only molecules (BH3s) and BAX but not BAK. Consequently, ABT-263 failed to kill BCL-X L -addicted cells with low activator BH3s and BCL-X L overabundance conferred resistance to ABT-263. High-throughput screening identified anthracyclines including doxorubicin and CDK9 inhibitors including dinaciclib that synergized with ABT-263 through downregulation of MCL-1 . As doxorubicin and dinaciclib also reduced BCL-X L , the combinations of BCL-2 inhibitor ABT-199 (venetoclax) with doxorubicin or dinaciclib provided effective therapeutic strategies for SCLC. Altogether, our study highlights the need for mechanism-guided targeting of anti-apoptotic BCL-2 proteins to effectively activate the mitochondrial cell death programme to kill cancer cells. Small cell lung cancer cells (SCLC) are differentially sensitive to inhibitors of the BCL-2 family. Here the authors analyse the response to BH3 mimetics in SCLC, delineate patterns of expression of apoptotic proteins correlated with differential sensitivities and demonstrate a synergistic anti-tumour activity between ABT-199 and anthracyclines or CDK9 inhibitors.

A new mass-spectrometry method has been developed to obtain high-resolution spectra of folded proteins bound to lipids; using this technique as well as X-ray crystallography provides evidence for membrane protein conformational change as a result of lipid–protein interaction. Lipid bound to influence protein structure Many of the high-resolution membrane protein structures published recently are notable for the presence of lipids closely associated with the protein, prompting the question, how are these lipids influencing membrane complex structure? Carol Robinson and colleagues have developed a new ion mobility mass spectrometry (IM-MS) method that enabled them to obtain mass spectra of folded protein conformations bound to lipids. Using this method they identified lipids that altered the stability of MscL (mechanosensitive channel of large conductance), aquaporin Z and the ammonia channel. They then determined the X-ray crystal structure of the ammonia channel bound to one of these lipids (phosphatidylglycerol), which revealed how a conformational change in a specific loop led to the formation of a phosphatidylglycerol-binding site. The major conclusion from this work is that an individual lipid-binding event can change the stability of a membrane complex. On the cover, IM-MS captures a native membrane protein complex emerging from an ion mobility cell. Shown is the ammonia channel in apo, one- and two-lipid bound states. Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments 1 , 2 , 3 , 4 , 5 , 6 , 7 and that lipids can bind to specific sites, for example, in potassium channels 8 . Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli , using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation 9 . AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates. p62/SQSTM1 acts as a key mediator in the selective autophagy of protein aggregates, or aggrephagy. Here the authors identify the prefoldin-like chaperone UXT as an autophagy adaptor of p62 dependent aggrephagy and show that ectopic UXT expression delays motor neuron degeneration in a Xenopus model.

Most patients with anaplastic lymphoma kinase (ALK)-rearranged or ROS proto-oncogene 1 (ROS1)-rearranged non-small-cell lung cancer (NSCLC) are sensitive to tyrosine kinase inhibitor (TKI) therapy, but resistance invariably develops, commonly within the CNS. This study aimed to analyse the safety, efficacy, and pharmacokinetic properties of lorlatinib, a novel, highly potent, selective, and brain-penetrant ALK and ROS1 TKI with preclinical activity against most known resistance mutations, in patients with advanced ALK-positive or ROS1-positive NSCLC. In this international multicentre, open-label, single-arm, first-in-man phase 1 dose-escalation study, eligible patients had advanced ALK-positive or ROS1-positive NSCLC and were older than 18 years, with an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Lorlatinib was administered orally to patients at doses ranging from 10 mg to 200 mg once daily or 35 mg to 100 mg twice daily, with a minimum of three patients receiving each dose. For some patients, tumour biopsy was done before lorlatinib treatment to identify ALK resistance mutations. Safety was assessed in patients who received at least one dose of lorlatinib; efficacy was assessed in the intention-to-treat population (patients who received at least one dose of study treatment and had either ALK or ROS1 rearrangement). The primary endpoint was dose-limiting toxicities during cycle 1 according to investigator assessment; secondary endpoints included safety, pharmacokinetics, and overall response. This study is ongoing and is registered with ClinicalTrials.gov, number NCT01970865. Between Jan 22, 2014, and July 10, 2015, 54 patients received at least one dose of lorlatinib, including 41 (77%) with ALK-positive and 12 (23%) with ROS1-positive NSCLC; one patient had unconfirmed ALK and ROS1 status. 28 (52%) patients had received two or more TKIs, and 39 (72%) patients had CNS metastases. The most common treatment-related adverse events among the 54 patients were hypercholesterolaemia (39 [72%] of 54 patients), hypertriglyceridaemia (21 [39%] of 54 patients), peripheral neuropathy (21 [39%] of 54 patients), and peripheral oedema (21 [39%] of 54 patients). One dose-limiting toxicity occurred at 200 mg (the patient did not take at least 16 of 21 prescribed total daily doses in cycle 1 because of toxicities attributable to study drug, which were grade 2 neurocognitive adverse events comprising slowed speech and mentation and word-finding difficulty). No maximum tolerated dose was identified. The recommended phase 2 dose was selected as 100 mg once daily. For ALK-positive patients, the proportion of patients who achieved an objective response was 19 (46%) of 41 patients (95% CI 31–63); for those who had received two or more TKIs, the proportion of patients with an objective response was 11 (42%) of 26 patients (23–63). In ROS1-positive patients, including seven crizotinib-pretreated patients, an objective response was achieved by six (50%) of 12 patients (95% CI 21–79). In this phase 1, dose-escalation study, lorlatinib showed both systemic and intracranial activity in patients with advanced ALK-positive or ROS1-positive NSCLC, most of whom had CNS metastases and had previously had two or more TKI treatments fail. Therefore, lorlatinib might be an effective therapeutic strategy for patients with ALK-positive NSCLC who have become resistant to currently available TKIs, including second-generation ALK TKIs, and is being investigated in a phase 3 randomised controlled trial comparing lorlatinib to crizotinib (ClinicalTrials.gov, NCT03052608). Pfizer.

Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typically, hydrotropes are amphiphilic molecules and differ from classical surfactants in that they have low cooperativity of aggregation and work at molar concentrations. Here, we show that adenosine triphosphate (ATP) has properties of a biological hydrotrope. It can both prevent the formation of and dissolve previously formed protein aggregates. This chemical property is manifested at physiological concentrations between 5 and 10 millimolar. Therefore, in addition to being an energy source for biological reactions, for which micromolar concentrations are sufficient, we propose that millimolar concentrations of ATP may act to keep proteins soluble. This may in part explain why ATP is maintained in such high concentrations in cells.

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors. Multidrug efflux pumps actively expel a wide range of toxic substrates from bacteria and play a major role in drug resistance. Here authors show the in situ structure of the efflux pump AcrAB-TolC obtained by electron cryo-tomography and subtomogram averaging.

Rajasekhar Suragani et al . show that a modified activin receptor IIB ligand trap increases red blood cell numbers in mice, rats and monkeys and ameliorates anemia in mice and rats, including in a mouse model of myelodysplastic syndromes. The ligand trap binds to the cytokine GDF11 and acts by inhibiting Smad2/3 signaling, thereby reversing ineffective erythropoiesis. Also in this issue, Michael Dussiot et al . show related findings using a wild-type activin receptor IIA ligand trap. Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis 1 , 2 , which is EPO independent 2 , 3 . Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo . Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo . Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-β (TGF-β) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

The cancer drug rocaglamide A cements the RNA helicase eIF4A on polypurine sequences and thereby prevents scanning of the 43S subunit along the messenger RNA, highlighting how a drug can act by stabilizing sequence-selective RNA–protein interactions. Mechanism of action of rocaglate drugs The cancer drug rocaglamide A (RocA) inhibits translation of a subclass of RNA transcripts. Because it targets the RNA helicase eIF4A, the specificity of RocA was thought to reside in the highly structured 5′ untranslated region of messenger RNAs that require eIF4A activity. Nicholas Ingolia and colleagues now show that the mechanism of inhibition has a different basis. RocA cements eIF4A on polypurine sequences and thereby prevents scanning of the 43S subunit along the messenger RNA. This report highlights how a drug can act by stabilizing sequence-selective RNA–protein interactions. Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs 1 , 2 , 3 , 4 . RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5′ untranslated regions that depend strongly on eIF4A-mediated unwinding 5 . However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA 1 , 2 , 6 . Here we show that secondary structure in 5′ untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA–eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA–protein interactions.