A Randomized, Double-Blinded, Phase II Trial of Gemcitabine and Nab-Paclitaxel Plus Apatorsen or Placebo in Patients with Metastatic Pancreatic Cancer: The RAINIER Trial.

LESSONS LEARNED: The addition of the heat shock protein 27 (Hsp27)-targeting antisense oligonucleotide, apatorsen, to a standard first-line chemotherapy regimen did not result in improved survival in unselected patients with metastatic pancreatic cancer.Findings from this trial hint at the possible prognostic and predictive value of serum Hsp27 that may warrant further investigation. BACKGROUND: This randomized, double-blinded, phase II trial evaluated the efficacy of gemcitabine/nab-paclitaxel plus either apatorsen, an antisense oligonucleotide targeting heat shock protein 27 (Hsp27) mRNA, or placebo in patients with metastatic pancreatic cancer. METHODS: Patients were randomized 1:1 to Arm A (gemcitabine/nab-paclitaxel plus apatorsen) or Arm B (gemcitabine/nab-paclitaxel plus placebo). Treatment was administered in 28-day cycles, with restaging every 2 cycles, until progression or intolerable toxicity. Serum Hsp27 levels were analyzed at baseline and on treatment. The primary endpoint was overall survival (OS). RESULTS: One hundred thirty-two patients were enrolled, 66 per arm. Cytopenias and fatigue were the most frequent grade 3/4 treatment-related adverse events for both arms. Median progression-free survival (PFS) and OS were 2.7 and 5.3 months, respectively, for arm A, and 3.8 and 6.9 months, respectively, for arm B. Objective response rate was 18% for both arms. Patients with high serum level of Hsp27 represented a poor-prognosis subgroup who may have derived modest benefit from addition of apatorsen. CONCLUSION: Addition of apatorsen to chemotherapy does not improve outcomes in unselected patients with metastatic pancreatic cancer in the first-line setting, although a trend toward prolonged PFS and OS in patients with high baseline serum Hsp27 suggests this therapy may warrant further evaluation in this subgroup.

Defining a Novel Role for the Pdx1 Transcription Factor in Islet ß-Cell Maturation and Proliferation During Weaning.

The transcription factor encoded by the Pdx1 gene is a critical transcriptional regulator, as it has fundamental actions in the formation of all pancreatic cell types, islet ß-cell development, and adult islet ß-cell function. Transgenic- and cell line-based experiments have identified 5'-flanking conserved sequences that control pancreatic and ß-cell type-specific transcription, which are found within areas I (bp -2694 to -2561), II (bp -2139 to -1958), III (bp -1879 to -1799), and IV (bp -6200 to -5670). Because of the presence in area IV of binding sites for transcription factors associated with pancreas development and islet cell function, we analyzed how an endogenous deletion mutant affected Pdx1 expression embryonically and postnatally. The most striking result was observed in male Pdx1ΔIV mutant mice after 3 weeks of birth (i.e., the onset of weaning), with only a small effect on pancreas organogenesis and no deficiencies in their female counterparts. Compromised Pdx1 mRNA and protein levels in weaned male mutant ß-cells were tightly linked with hyperglycemia, decreased ß-cell proliferation, reduced ß-cell area, and altered expression of Pdx1-bound genes that are important in ß-cell replication, endoplasmic reticulum function, and mitochondrial activity. We discuss the impact of these novel findings to Pdx1 gene regulation and islet ß-cell maturation postnatally.

MLL3 and MLL4 Methyltransferases Bind to the MAFA and MAFB Transcription Factors to Regulate Islet ß-Cell Function.

Insulin produced by islet ß-cells plays a critical role in glucose homeostasis, with type 1 and type 2 diabetes both resulting from inactivation and/or loss of this cell population. Islet-enriched transcription factors regulate ß-cell formation and function, yet little is known about the molecules recruited to mediate control. An unbiased in-cell biochemical and mass spectrometry strategy was used to isolate MafA transcription factor-binding proteins. Among the many coregulators identified were all of the subunits of the mixed-lineage leukemia 3 (Mll3) and 4 (Mll4) complexes, with histone 3 lysine 4 methyltransferases strongly associated with gene activation. MafA was bound to the ∼1.5 MDa Mll3 and Mll4 complexes in size-fractionated ß-cell extracts. Likewise, closely related human MAFB, which is important to ß-cell formation and coproduced with MAFA in adult human islet ß-cells, bound MLL3 and MLL4 complexes. Knockdown of NCOA6, a core subunit of these methyltransferases, reduced expression of a subset of MAFA and MAFB target genes in mouse and human ß-cell lines. In contrast, a broader effect on MafA/MafB gene activation was observed in mice lacking NCoA6 in islet ß-cells. We propose that MLL3 and MLL4 are broadly required for controlling MAFA and MAFB transactivation during development and postnatally.

Knowledge, Attitudes, and Practices (KAP) Relating to Avian Influenza (H10N8) among Farmers' Markets Workers in Nanchang, China.

Three cases of avian influenza virus H10N8 were reported in Nanchang, China, as of April 2014. To identify the knowledge, attitudes, and practices (KAP) related to H10N8 among farmers' market workers, a cross-sectional survey was conducted in 63 farmers' markets in Nanchang. Using the resulting data, characteristics of poultry and non-poultry workers' knowledge, attitudes, and practice were described. Results suggest that interventions targeting high-risk workers should be developed and implemented by public health agencies to prevent the spread of H10N8. Additionally policies that encourage farmers' market workers to receive influenza vaccine should be developed, adopted, and enforced.

Coexistence of Avian Influenza Virus H10 and H9 Subtypes among Chickens in Live Poultry Markets during an Outbreak of Infection with a Novel H10N8 Virus in Humans in Nanchang, China.

Infection with the novel H10N8 virus in humans has raised concerns about its pandemic potential worldwide. We report the results of a cross-sectional study of avian influenza viruses (AIVs) in live poultry markets (LPMs) in Nanchang, China, after the first human case of H10N8 virus infection was reported in the city. A total of 201 specimens tested positive for AIVs among 618 samples collected from 24 LPMs in Nanchang from December 2013 to January 2014. We found that the LPMs were heavily contaminated by AIVs, with H9, H10, and H5 being the predominant subtypes and more than half of the LPMs providing samples that were positive for the H10 subtype. Moreover, the coexistence of different subtypes was common in LPMs. Of the 201 positive samples, 20.9% (42/201) had mixed infections with AIVs of different HA subtypes. Of the 42 mixed infections, 50% (21/42) showed the coexistence of the H9 and H10 subtypes, with or without H5, and were from chicken samples. This indicated that the H10N8 virus probably originated from segment reassortment of the H9 and H10 subtypes.

Inactivation of specific ß cell transcription factors in type 2 diabetes.

Type 2 diabetes (T2DM) commonly arises from islet ß cell failure and insulin resistance. Here, we examined the sensitivity of key islet-enriched transcription factors to oxidative stress, a condition associated with ß cell dysfunction in both type 1 diabetes (T1DM) and T2DM. Hydrogen peroxide treatment of ß cell lines induced cytoplasmic translocation of MAFA and NKX6.1. In parallel, the ability of nuclear PDX1 to bind endogenous target gene promoters was also dramatically reduced, whereas the activity of other key ß cell transcriptional regulators was unaffected. MAFA levels were reduced, followed by a reduction in NKX6.1 upon development of hyperglycemia in db/db mice, a T2DM model. Transgenic expression of the glutathione peroxidase-1 antioxidant enzyme (GPX1) in db/db islet ß cells restored nuclear MAFA, nuclear NKX6.1, and ß cell function in vivo. Notably, the selective decrease in MAFA, NKX6.1, and PDX1 expression was found in human T2DM islets. MAFB, a MAFA-related transcription factor expressed in human ß cells, was also severely compromised. We propose that MAFA, MAFB, NKX6.1, and PDX1 activity provides a gauge of islet ß cell function, with loss of MAFA (and/or MAFB) representing an early indicator of ß cell inactivity and the subsequent deficit of more impactful NKX6.1 (and/or PDX1) resulting in overt dysfunction associated with T2DM.

The B55α-containing PP2A holoenzyme dephosphorylates FOXO1 in islet ß-cells under oxidative stress.

The FOXO1 (forkhead box O1) transcription factor influences many key cellular processes, including those important in metabolism, proliferation and cell death. Reversible phosphorylation of FOXO1 at Thr(24) and Ser(256) regulates its subcellular localization, with phosphorylation promoting cytoplasmic localization, whereas dephosphorylation triggers nuclear import and transcriptional activation. In the present study, we used biochemical and molecular approaches to isolate and link the serine/threonine PP2A (protein phosphatase 2A) holoenzyme containing the B55α regulatory subunit, with nuclear import of FOXO1 in pancreatic islet ß-cells under oxidative stress, a condition associated with cellular dysfunction in Type 2 diabetes. The mechanism of FOXO1 dephosphorylation and nuclear translocation was investigated in pancreatic islet INS-1 and ßTC-3 cell lines subjected to oxidative stress. A combined chemical cross-linking and MS strategy revealed the association of FOXO1 with a PP2A holoenzyme composed of the catalytic C, structural A and B55α regulatory subunits. Knockdown of B55α in INS-1 cells reduced FOXO1 dephosphorylation, inhibited FOXO1 nuclear translocation and attenuated oxidative stress-induced cell death. Furthermore, both B55α and nuclear FOXO1 levels were increased under hyperglycaemic conditions in db/db mouse islets, an animal model of type 2 diabetes. We conclude that B55α-containing PP2A is a key regulator of FOXO1 activity in vivo.

Phosphorylation within the MafA N terminus regulates C-terminal dimerization and DNA binding.

Phosphorylation regulates transcription factor activity by influencing dimerization, cellular localization, activation potential, and/or DNA binding. Nevertheless, precisely how this post-translation modification mediates these processes is poorly understood. Here, we examined the role of phosphorylation on the DNA-binding properties of MafA and MafB, closely related transcriptional activators of the basic-leucine zipper (b-Zip) family associated with cell differentiation and oncogenesis. Many common phosphorylation sites were identified by mass spectrometry. However, dephosphorylation only precluded the detection of MafA dimers and consequently dramatically reduced DNA-binding ability. Analysis of MafA/B chimeras revealed that sensitivity to the phosphorylation status of MafA was imparted by sequences spanning the C-terminal dimerization region (amino acids (aa) 279-359), whereas the homologous MafB region (aa 257-323) conveyed phosphorylation-independent DNA binding. Mutational analysis showed that formation of MafA dimers capable of DNA binding required phosphorylation within the distinct N-terminal transactivation domain (aa 1-72) and not the C-terminal b-Zip region. These results demonstrate a novel relationship between the phosphoamino acid-rich transactivation and b-Zip domains in controlling MafA DNA-binding activity.

The stability and transactivation potential of the mammalian MafA transcription factor are regulated by serine 65 phosphorylation.

The level of the MafA transcription factor is regulated by a variety of effectors of beta cell function, including glucose, fatty acids, and insulin. Here, we show that phosphorylation at Ser(65) of mammalian MafA influences both protein stability and transactivation potential. Replacement of Ser(65) with Glu to mimic phosphorylation produced a protein that was as unstable as the wild type, whereas Asp or Ala mutation blocked degradation. Analysis of MafA chimeric and deletion constructs suggests that protein phosphorylation at Ser(65) alone represents the initial degradation signal, with ubiquitinylation occurring within the C terminus (amino acids 234-359). Although only wild type MafA and S65E were polyubiquitinylated, both S65D and S65E potently stimulated transactivation compared with S65A. Phosphorylation at Ser(14) also enhanced activation, although it had no impact on protein turnover. The mobility of MafA S65A was profoundly affected upon SDS-PAGE, with the S65E and S65D mutants influenced less due to their ability to serve as substrates for glycogen synthase kinase 3, which acts at neighboring N-terminal residues after Ser(65) phosphorylation. Our observations not only illustrate the sensitivity of the cellular transcriptional and degradation machinery to phosphomimetic mutants at Ser(65), but also demonstrate the singular importance of phosphorylation at this amino acid in regulating MafA activity.

Inositol hexakisphosphate and Gle1 activate the DEAD-box protein Dbp5 for nuclear mRNA export.

Regulation of nuclear mRNA export is critical for proper eukaryotic gene expression. A key step in this process is the directional translocation of mRNA-ribonucleoprotein particles (mRNPs) through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope. Our previous studies in Saccharomyces cerevisiae defined an in vivo role for inositol hexakisphosphate (InsP6) and NPC-associated Gle1 in mRNA export. Here, we show that Gle1 and InsP6 act together to stimulate the RNA-dependent ATPase activity of the essential DEAD-box protein Dbp5. Overexpression of DBP5 specifically suppressed mRNA export and growth defects of an ipk1 nup42 mutant defective in InsP6 production and Gle1 localization. In vitro kinetic analysis showed that InsP6 significantly increased Dbp5 ATPase activity in a Gle1-dependent manner and lowered the effective RNA concentration for half-maximal ATPase activity. Gle1 alone had minimal effects. Maximal InsP6 binding required both Dbp5 and Gle1. It has been suggested that Dbp5 requires unidentified cofactors. We now propose that Dbp5 activation at NPCs requires Gle1 and InsP6. This would facilitate spatial control of the remodelling of mRNP protein composition during directional transport and provide energy to power transport cycles.

Regulation of replication protein A functions in DNA mismatch repair by phosphorylation.

Replication protein A (RPA) is involved in multiple stages of DNA mismatch repair (MMR); however, the modulation of its functions between different stages is unknown. We show here that phosphorylation likely modulates RPA functions during MMR. Unphosphorylated RPA initially binds to nicked heteroduplex DNA to facilitate assembly of the MMR initiation complex. The unphosphorylated protein preferentially stimulates mismatch-provoked excision, possibly by cooperatively binding to the resultant single-stranded DNA gap. The DNA-bound RPA begins to be phosphorylated after extensive excision, resulting in severalfold reduction in the DNA binding affinity of RPA. Thus, during the phase of repair DNA synthesis, the phosphorylated RPA readily disassociates from DNA, making the DNA template available for DNA polymerase delta-catalyzed resynthesis. These observations support a model of how phosphorylation alters the DNA binding affinity of RPA to fulfill its differential requirement at the various stages of MMR.

Evidence for involvement of HMGB1 protein in human DNA mismatch repair.

Defects in human DNA mismatch repair predispose to cancer, but many components of the pathway have not been identified. We report here the identification and characterization of a novel component required for mismatch repair in human cells. A 30-kDa protein was purified to homogeneity by virtue of its ability to complement a depleted HeLa extract in repair of mismatched heteroduplexes. The complementing activity was identified as HMGB1 (the high mobility group box 1 protein), a non-histone chromatin protein that facilitates protein-protein interactions and recognizes DNA damage. Evidence is also presented that HMGB1 physically interacts with MutSalpha and is required at a step prior to the excision of mispaired nucleotide in mismatch repair.

Differential requirement for proliferating cell nuclear antigen in 5' and 3' nick-directed excision in human mismatch repair.

Proliferating cell nuclear antigen (PCNA) is involved in mammalian mismatch repair at a step prior to or at mismatch excision, but the molecular mechanism of this process is not fully understood. To examine the role of PCNA in mismatch-provoked and nick-directed excision, orientation-specific mismatch removal of heteroduplexes with a pre-existing nick was monitored in human nuclear extracts supplemented with the PCNA inhibitor protein p21. We show here that, whereas 3' nick-directed mismatch excision was completely inhibited by low concentrations of p21 or a p21 C-terminal fusion protein, 5' nick-directed excision was only partially blocked under the same conditions. No further reduction of the 5' excision was detected when a much higher concentration of p21 C-terminal protein was used. These results suggest the following. (i) There is a differential requirement for PCNA in 3' and 5' nick-directed excision; and (ii) 5' nick-directed excision is conducted by a manner either dependent on or independent of PCNA. Our in vitro reconstitution experiments indeed identified a 5' nick-directed excision pathway that is dependent on PCNA, hMutSalpha, and a partially purified fraction from a HeLa nuclear extract.

Partial reconstitution of human DNA mismatch repair in vitro: characterization of the role of human replication protein A.

DNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many of the components have not yet been identified. Using a functional in vitro reconstitution system, this study identified three HeLa cell fractions essential for in vitro MMR. These fractions divide human MMR into two distinct stages: mismatch-provoked excision and repair synthesis. In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR. Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis. It is anticipated that further analysis of these fractions will identify additional MMR components and enable the complete reconstitution of the human MMR pathway with purified proteins.