Key Points• Loss of Bim contributes to adaptive rather than intrinsic bortezomib resistance in multiple myeloma.• A Bim-targeting strategy combining an HDACI with a BH3 mimetic overcomes such resistance through a new link between autophagy and apoptosis.

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AZD1775 targets the cell cycle checkpoint kinase Wee1 and potentiates genotoxic agent cytotoxicity through p53-dependent or -independent mechanisms. Here, we report that AZD1775 interacted synergistically with histone deacetylase inhibitors (HDACIs e.g., Vorinostat), which interrupt the DNA damage response (DDR), to kill p53-wild type or -deficient as well as FLT3-ITD leukemia cells in association with pronounced Wee1 inhibition and diminished cdc2/Cdk1 Y15 phosphorylation. Similarly, Wee1 shRNA knock-down significantly sensitized cells to HDACIs. While AZD1775 induced Chk1 activation, reflected by markedly increased Chk1 S296/S317/S345 phosphorylation leading to inhibitory T14 phosphorylation of cdc2/Cdk1, these compensatory responses were sharply abrogated by HDACIs. This was accompanied by premature mitotic entry, multiple mitotic abnormalities, and accumulation of early S-phase cells displaying increased newly replicated DNA, culminating in robust DNA damage and apoptosis. The regimen was active against patient-derived AML cells harboring either wild type or mutant p53, and various NGS-defined mutations. Primitive CD34+/CD123+/CD38− populations enriched for leukemia-initiating progenitors, but not normal CD34+ hematopoietic cells, were highly susceptible to this regimen. Finally, combining AZD1775 with Vorinostat in AML murine xenografts significantly reduced tumor burden and prolonged animal survival. A strategy combining Wee1 with HDACI inhibition warrants further investigation in AML with poor prognostic genetic aberrations.

eIn selective autophagy, the adaptor protein SQSTM1/p62 plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation. Here we report that whereas SQSTM1/p62 levels fluctuated in a timedependent manner during autophagy, inhibition or knockdown of Cdk9/cyclin T1 transcriptionally downregulated SQSTM1/ p62 but did not affect autophagic flux. These interventions, or short hairpin RNA (shRNA) directly targeting SQSTM1/p62, resulted in cargo loading failure and inefficient autophagy, phenomena recently described for Huntington's disease neurons. These events led to the accumulation of the BH3-only protein NBK/Bik on endoplasmic reticulum (ER) membranes, most likely by blocking loading and autophagic degradation of NBK/Bik, culminating in apoptosis. Whereas NBK/Bik upregulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine (CQ) or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., genetically by shRNA targeting Ulk1, beclin-1, or Atg5 or pharmacologically by 3-methyladenine [3-MA] or spautin-1), arguing that NBK/Bik accumulation stems from inefficient autophagy. Finally, NBK/Bik knockdown markedly attenuated apoptosis in vitro and in vivo. Together, these findings identify novel cross talk between autophagy and apoptosis, wherein targeting SQSTM1/p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation, which triggers apoptosis.A utophagy is an evolutionarily conserved process by which damaged organelles and unneeded proteins are degraded by lysosomes to maintain intracellular homeostasis and to recycle cellular nutrients. While autophagy can promote cell death (1), in most cases, it is cytoprotective and contributes to drug resistance (2). In response to chemotherapeutic agents, apoptosis (type I) and autophagy (type II) represent two major forms of programmed cell death (3). Autophagy and apoptosis share molecular regulatory mechanisms governed by Bcl-2 family proteins (3, 4). Specifically, Bcl-2 and Bcl-x L prevent both apoptosis and autophagy by sequestering different BH3-only proteins (e.g., proapoptotic Bim and Bid [5] and proautophagic beclin-1 [6,7]). As a result, antagonism of Bcl-2/Bcl-x L function releases and activates these BH3-only proteins, leading to apoptosis and autophagy, respectively (8). While apoptosis represents a well-established mechanism of action of conventional and targeted anticancer agents (3), autophagy may play both positive and negative roles in tumorigenesis and cancer treatment (9, 10). Consequently, whether autophagy should be inhibited or activated remains the subject of debate. Accordingly, both autophagy inhibitors and inducers are currently undergoing clinical evaluation (11).Protection of cells from injury by harmful macromolecules or damaged organelles through autophagy as a quality control (QC) mechanism (11, 12) involves the sequestration and tran...

• The NAE inhibitor pevonedistat induces Chk1/Wee1 activation and the intra-S checkpoint, limiting its anti-AML efficacy.• The HDAC inhibitor belinostat potentiates the in vitro and in vivo activity of pevonedistat in AML by disrupting the DDR.Two classes of novel agents, NEDD8-activating enzyme (NAE) and histone deacetylase (HDAC) inhibitors, have shown single-agent activity in acute myelogenous leukemia (AML)/myelodysplastic syndrome (MDS). Here we examined mechanisms underlying interactions between the NAE inhibitor pevonedistat (MLN4924) and the approved HDAC inhibitor belinostat in AML/MDS cells. MLN4924/belinostat coadministration synergistically induced AML cell apoptosis with or without p53 deficiency or FLT3-internal tandem duplication (ITD), whereas p53 short hairpin RNA (shRNA) knockdown or enforced FLT3-ITD expression significantly sensitized cells to the regimen. MLN4924 blocked belinostat-induced antiapoptotic gene expression through nuclear factor-kB inactivation. Each agent upregulated Bim, and Bim knockdown significantly attenuated apoptosis. Microarrays revealed distinct DNA damage response (DDR) genetic profiles between individual vs combined MLN4924/belinostat exposure. Whereas belinostat abrogated the MLN4924-activated intra-S checkpoint through Chk1 and Wee1 inhibition/downregulation, cotreatment downregulated multiple homologous recombination and nonhomologous end-joining repair proteins, triggering robust double-stranded breaks, chromatin pulverization, and apoptosis. Consistently, Chk1 or Wee1 shRNA knockdown significantly sensitized AML cells to MLN4924. MLN4924/belinostat displayed activity against primary AML or MDS cells, including those carrying next-generation sequencingdefined poor-prognostic cancer hotspot mutations, and CD34

To investigate the effect of salt and sugar on the physicochemical properties of flaxseed polysaccharidepotato starch (FG-PS) complexes we measured pasting, gelling, swelling properties, and freeze-thaw stability by methods of rapid viscosity analysis, texture analysis, centrifugation, and freeze-thaw storage, respectively. In the presence of NaCl, the pasting temperature, peak viscosity, final viscosity, breakdown value, and swelling power of FG-PS complexes increased as the NaCl level increased. The hardness of FG-PS complexes gradually reduced with increasing NaCl level. Syneresis of FG-PS complexes was the lowest with addition of high levels of NaCl. These results indicate that both starch-salt and hydrocolloid-salt interactions might govern the physicochemical properties of FG-PS complexes in the presence of NaCl. In the presence of sugar, an increase in pasting temperature, peak viscosity, final viscosity, breakdown value, and gel hardness of FG-PS complexes was observed with increasing added level of sucrose and glucose, while swelling power and syneresis decreased.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-b1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. STEM CELLS 2017;35:1719-1732 SIGNIFICANCE STATEMENTWe established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection and demonstrated an underlying mechanism for the immunosuppressive effect of iPSC-MSCs through inhibition of the cleavage of caspases, which was inhibited by a series of paracrine factors secreted by iPSC-MSCs. Inhibition of caspases suppressed T cell responses, decreased Th1 and Th2 frequency, and increased CD4 1 CD25 1 Foxp3 1 regulatory T cells. These data indicate a novel mechanism for the immunomodulation of MSC via regulation of caspases.

Antiretroviral therapy (ART) has been introduced recently and has significantly impacted morbidity and mortality, but can also engender drug resistance. To identify the prevalence of HIV-1 drug resistance (HIVDR) among patients with antiretroviral therapy failure in Sichuan during the period from 2010 to 2016, we carried out a longitudinal study in Sichuan, a province with the highest HIV/AIDS prevalence in China. The data and blood samples were collected from HIV/AIDS patients who received ART for more than half a year. Overall 5,512 sequences were completed from 7,059 ART-failure patients, and 2,499 individuals were identified as drug resistant. Among those with HIVDR mutations identified, 25.37% were against non-nucleoside reverse transcriptase inhibitors (NNRTIs), and 1.60% was against nucleoside reverse transcriptase inhibitors (NRTIs). NRTI-resistant drugs were mainly lamivudine (3TC) (57.77%) and emtricitabine (FTC), while NNRTI-resistant drugs were mainly nevirapine (NVP) (91.13%) and efavirenz (EFV) (72.81%). The most common recombination subtypes of HIV-1 in sequenced samples were CRF07_ BC (circulating recombinant form, CRF) (41.42%), followed by CRF01_AE (40.77%). Moreover, drug resistance rate increased with the prolongation of treatment time (χ 2 = 14.758, P < 0.05). The overall prevalence of acquired drug resistance in HIV-1 infected patients in Sichuan was 5.47%, which has remained relatively stable from 2010 to 2016. HIV-1 CRF01_AE and CRF07_BC subtypes were the main epidemic strains, and the possibility of resistance was higher in CRF01_AE subtypes. The current study highlights the importance of acquired drug resistance surveillance over a long period.