0.5-bit/s/Hz fine-grained adaptive OFDM modulation for bandlimited underwater VLC.

In this paper, we propose and demonstrate a 0.5-bit/s/Hz fine-grained adaptive orthogonal frequency division multiplexing (OFDM) modulation scheme for bandlimited underwater visible light communication (UVLC) systems. Particularly, integer spectral efficiency is obtained by conventional OFDM with quadrature amplitude modulation (QAM) constellations, while fractional spectral efficiency is obtained by two newly proposed dual-frame OFDM designs. More specifically, OFDM with dual-frame binary phase-shift keying (DF-BPSK) is designed to achieve a spectral efficiency of 0.5 bit/s/Hz, while OFDM with dual-frame dual-mode index modulation (DF-DMIM) is designed to realize the spectral efficiencies of 0.5+n bits/s/Hz with n being a positive integer (i.e., n = 1, 2, …). The feasibility and superiority of the proposed 0.5-bit/s/Hz fine-grained adaptive OFDM modulation scheme in bandlimited UVLC systems are successfully verified by simulations and proof-of-concept experiments. Experimental results demonstrate that a significant achievable rate gain of 18.6 Mbps can be achieved by the proposed 0.5-bit/s/Hz fine-grained adaptive OFDM modulation in comparison to the traditional 1-bit/s/Hz granularity adaptive OFDM scheme, which corresponds to a rate improvement of 22.1%.

Triple combination targeting methyltransferase, BCL-2, and PD-1 facilitates antileukemia responses in acute myeloid leukemia.

BACKGROUND: A recent breakthrough therapy combining the BCL-2 inhibitor venetoclax with hypomethylating agents (HMAs) targeting DNA methyltransferase has improved outcomes for patients with acute myeloid leukemia (AML), but the responses and long-term survival in older/unfit patients and in patients with relapsed/refractory AML remain suboptimal. Recent studies showed that inhibition of BCL-2 or DNA methyltransferase modulates AML T-cell immunity. METHODS: By using flow cytometry and time-of-flight mass cytometry, the authors examined the effects of the HMA decitabine combined with the BCL-2 inhibitor venetoclax (DAC/VEN therapy) on leukemia cells and T cells in patients with AML who received DAC/VEN therapy in a clinical trial. The authors investigated the response of programmed cell death protein 1 (PD-1) inhibition in the DAC/VEN-treated samples in vitro and investigated the triple combination of PD-1 inhibition with HMA/venetoclax in the trial patients who had AML. RESULTS: DAC/VEN therapy effectively targeted leukemia cells and upregulated the expression of the immune checkpoint-inhibitory receptor PD-1 in T cells while preserving CD4-positive and CD8-positive memory T cells in a subset of patients with AML who were tested. In vitro PD-1 inhibition potentiated the antileukemia response in DAC/VEN-treated AML samples. The combined use of azacitidine, venetoclax, and nivolumab eliminated circulating blasts and leukemia stem cells/progenitor cells and expanded the percentage of CD8-positive memory T cells in an illustrative patient with relapsed AML who responded to the regimen in an ongoing clinical trial. CONCLUSIONS: Immunomodulation by targeting PD-1 enhances the therapeutic effect of combining an HMA and venetoclax in patients with AML.

XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia.

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease.

Discovery of a new polymorph of clotrimazole through melt crystallization: Understanding nucleation and growth kinetics.

Clotrimazole (CMZ) is a classical antifungal drug for studying crystallization. In this study, a new CMZ polymorph (Form 2) was discovered during the process of nucleation and growth rate determination in the melt. High-quality single crystals were grown from melt microdroplets to determine the crystal structure by x-ray diffraction. Form 2 is metastable and exhibits a disordered structure. The crystal nucleation and growth kinetics of the two CMZ polymorphs were systematically measured. Form 2 nucleates and grows faster than the existing form (Form 1). The maximum nucleation rate of Forms 1 and 2 was observed at 50 °C (1.07 Tg). The summary of the maximum nucleation rate temperature of CMZ and the other six organic compounds indicates that nucleation near Tg in the supercooled liquid is a useful approach to discovering new polymorphs. This study is relevant for the discovering new drug polymorphs through an understanding of nucleation and growth kinetics during melt crystallization.

Constellation design of DFT-S-OFDM with dual-mode index modulation in VLC.

In this paper, we for the first time propose a novel partitioning-based constellation design approach for discrete Fourier transform-spread-orthogonal frequency division multiplexing modulation with dual-mode index modulation (DFT-S-OFDM-DM) in visible light communication (VLC) systems. Specifically, two partitioning-based constellation designs, i.e., block-based constellation partitioning and interleaving-based constellation partitioning, are proposed to generate two distinguishable constellation sets for DFT-S-OFDM-DM in VLC, by considering four 8-ary constellations including 8-ary quadrature amplitude modulation (8-QAM), 8-ary phase-shift keying (8-PSK), circular (4,4)-QAM, and circular (7,1)-QAM. The superiority of DFT-S-OFDM-DM using circular (7,1)-QAM constellation with interleaving-based constellation partitioning over other benchmark schemes has been successfully verified by both simulation and experimental results. It is shown by the experimental results that a significant distance extension of 44.6% is obtained by DFT-S-OFDM-DM using circular (7,1)-QAM constellation with interleaving-based constellation partitioning in comparison to DFT-S-OFDM with index modulation achieving the same spectral efficiency of 2.5 bits/s/Hz. It is also demonstrated that the proposed constellation design schemes are also generally applicable to the constellation with an arbitrary shape and an arbitrary size.

Bio-nanocomplexes with autonomous O2 generation efficiently inhibit triple negative breast cancer through enhanced chemo-PDT.

As one kind of aggressive cancer, triple-negative breast cancer (TNBC) has become one of the major causes of women mortality worldwide. Recently, combinational chemo-PDT therapy based on nanomaterials has been adopted for the treatment of malignant tumor. However, the efficacy of PDT was partly compromised under tumor hypoxia environment due to the lack of sustainable O2 supply. In this study, CeO2-loaded nanoparticles (CeNPs) with peroxidase activity were synthesized to autonomously generate O2 by decomposing H2O2 within tumor region and reprogramming the hypoxia microenvironment as well. Meanwhile, the compound cinobufagin (CS-1) was loaded for inhibiting TNBC growth and metastasis. Moreover, the hybrid membrane camouflage was adopted to improve the biocompatibility and targeting ability of nanocomplexes. In vitro assay demonstrated that decomposition of H2O2 by CeO2 achieved sustainable O2 supply, which accordingly improved the efficacy of PDT. In turn, the generated O2 improved the cytotoxicity and anti-tumor migration effect of CS-1 by downregulating HIF-1α and MMP-9 levels. In vivo assay demonstrated that the combination of CS-1 and PDT significantly inhibited the growth and distance metastasis of tumor in MDA-MB-231 bearing mice. Thus, this chemo-PDT strategy achieved satisfactory therapeutic effects by smartly utilizing the enzyme activity of nanodrugs and special micro-environment of tumor.

Effect of a TSPO ligand on retinal pigment epithelial cholesterol homeostasis in high-fat fed mice, implication for age-related macular degeneration.

Age-related Macular Degeneration (AMD) is a major cause of sight impairment in the elderly with complex aetiology involving genetics and environment and with limited therapeutic options which have limited efficacy. We have previously shown in a mouse-model of the condition, induced by feeding a high fat diet, that adverse effects of the diet can be reversed by co-administration of the TSPO activator, etifoxine. We extend those observations showing improvements in retinal pigment epithelial (RPE) cells with decreased lipids and enhanced expression of cholesterol metabolism and transport enzymes. Further, etifoxine decreased levels of reactive oxygen species (ROS) in RPE and inflammatory cytokines in RPE and serum. With respect to gut microbiome, we found that organisms abundant in the high fat condition (e.g. in the genus Anaerotruncus and Oscillospira) and implicated in AMD, were much less abundant after etifoxine treatment. The changes in gut flora were associated with the predicted production of metabolites of benefit to the retina including tryptophan and other amino acids and taurine, an essential component of the retina necessary to counteract ROS. These novel observations strengthen earlier conclusions that the mechanisms behind improvements in etifoxine-induced retinal physiology involve an interaction between effects on the host and the gut microbiome.

High-throughput proteomic profiling reveals mechanisms of action of AMG925, a dual FLT3-CDK4/6 kinase inhibitor targeting AML and AML stem/progenitor cells.

FLT3 mutations, which are found in a third of patients with acute myeloid leukemia (AML), are associated with poor prognosis. Responses to currently available FLT3 inhibitors in AML patients are typically transient and followed by disease recurrence. Thus, FLT3 inhibitors with new inhibitory mechanisms are needed to improve therapeutic outcomes. AMG925 is a novel, potent, small-molecule dual inhibitor of FLT3 and CDK4/6. In this study. we determined the antileukemic effects and mechanisms of action of AMG925 in AML cell lines and primary samples, in particular AML stem/progenitor cells. AMG925 inhibited cell growth and promoted apoptosis in AML cells with or without FLT3 mutations. Reverse-phase protein array profiling confirmed its on-target effects on FLT3-CDK4/6-regulated pathways and identified unrevealed signaling network alterations in AML blasts and stem/progenitor cells in response to AMG925. Mass cytometry identified pathways that may confer resistance to AMG925 in phenotypically defined AML stem/progenitor cells and demonstrated that combined blockade of FLT3-CDK4/6 and AKT/mTOR signaling facilitated stem cell death. Our findings provide a rationale for the mechanism-based inhibition of FLT3-CDK4/6 and for combinatorial approaches to improve the efficacy of FLT3 inhibition in both FLT3 wild-type and FLT3-mutated AML.

Protection by vitamin D against high-glucose-induced damage in retinal pigment epithelial cells.

Diabetic retinopathy (DR) is a diabetes-associated complication characterized by irreversible deterioration of the microvessels within the retina, leading subsequently to severe retinal damage and vision loss. Vitamin D (VITD), a steroid hormone, plays multiple physiological functions in cellular homeostasis. Deficiency of VITD has been suggested to be associated with DR. To study the potential protective function of VITD in DR, high-glucose-treated ARPE-19 cells and STZ-induced diabetic mice were used as in vitro and in vivo models. The protective effects of VITD were assessed based on the changes of expression of antioxidant enzymes and cytokines in high-glucose-treated retinal pigment epithelial (RPE) cells and in the retina and RPE of diabetic and VITD-treated diabetic mice. The present study demonstrated that exposure to a high level of glucose caused upregulation of pro-inflammatory cytokines and a decrease in anti-oxidant enzyme expression in both in vitro and in vivo models. VITD treatment increased cell viability, reduced reactive oxygen species (ROS) production and caspase-3/7 activities in high-glucose-treated RPE cells. Our data suggest that VITD can protect the retina and RPE from high-glucose-induced oxidative damage and inflammation.

Gypenosides mediate cholesterol efflux and suppress oxidized LDL induced inflammation in retinal pigment epithelium cells.

Age-related macular degeneration (AMD) is a predominant cause of visual deficit in aged population. Abnormal accumulation of cholesterol, including oxidized low-density lipoprotein (oxLDL), underneath the retinal pigment epithelium (RPE) cells contributes to the development of AMD. Gypenosides (Gyp) are glycosides extracted from Gynostemma pentaphyllum and have demonstrated protective effects against inflammation and oxidative stress. To determine the therapeutic potential of Gyp for AMD, we investigated its effect on cholesterol trafficking and metabolism and assessed the protective function of Gyp against oxLDL-induced damage in RPE cells. Cholesterol efflux to high-density lipoprotein (HDL) and human serum was significantly increased in RPE cells treated with Gyp when compared to untreated control cells. Expression of cholesterol metabolism (CYP27A1, CYP46A1) and trafficking (TSPO, ABCA1 and ABCG1) genes was also markedly increased in Gyp-treated RPE cells. OxLDL-treated RPE cells had significantly increased cholesterol accumulation and lipid droplet formation. There were marked increases in reactive oxygen species (ROS) generation and proinflammatory cytokines via NF-κB activation in RPE cells treated with oxLDL, while incubation with Gyp rectified these changes. These findings provide pharmacological evidence that Gyp has the potential to treat patients with early onset AMD by promoting cellular cholesterol removal from RPE cells and inhibiting inflammation and oxidative stress.

Gypenosides attenuate retinal degeneration in a zebrafish retinitis pigmentosa model.

Retinitis pigmentosa (RP) is a collection of heterogenous genetic retinal disorders resulting in cumulative retinal deterioration involving progressive loss of photoreceptors and eventually in total blindness. Oxidative stress plays a central role in this photoreceptor loss. Gypenosides (Gyp) are the main functional component isolated from the climbing vine Gynostemma pentaphyllum and have been shown to defend cells against the effects of oxidative stress and inflammation, providing protection in experimentally-induced optic neuritis. The zebrafish model has been used to investigate a range of human diseases. Previously we reported early retinal degeneration in a mutant zebrafish line carrying a point-nonsense mutation in the retinitis pigmentosa GTPase regulator interacting protein 1 (rpgrip1) gene that is mutated in RP patients. The current study investigated the potential protective effects of Gyp against photoreceptor degeneration in the Rpgrip1 deleted zebrafish. Rpgrip1 mutant zebrafish were treated with 5 µg/ml of Gyp in E3 medium from 6 h post fertilization (hpf) till 1 month post fertilization (mpf). Rpgrip1 mutant zebrafish treated with 5 µg/ml of Gyp showed a significant decrease by 68.41% (p = 0.0002) in photoreceptor cell death compared to that of untreated mutant zebrafish. Expression of antioxidant genes catalase, sod1, sod2, gpx1, gclm, nqo-1 and nrf-2 was significantly decreased in rpgrip1 mutant zebrafish eyes by 61.51%, 77.40%, 60.11%, 81.17%, 72.07%, 78.95% and 85.42% (all p < 0.0001), respectively, when compared to that of wildtype zebrafish; superoxide dismutase and catalase activities, and glutathione levels in rpgrip1 mutant zebrafish eyes were significantly decreased by 87.21%, 21.55% and 96.51% (all p < 0.0001), respectively. There were marked increases in the production of reactive oxygen species (ROS) and malondialdehyde (MDA) by 2738.73% and 510.69% (all p < 0.0001), respectively, in rpgrip1 mutant zebrafish eyes; expression of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α was also significantly increased by 150.11%, 267.79% and 190.72% (all p < 0.0001), respectively, in rpgrip1 mutant zebrafish eyes, compared to that of wildtype zebrafish. Treatment with Gyp significantly counteracted these effects. This study indicates that Gyp has a potential role in the treatment of RP.

Phase 1 study of combinatorial sorafenib, G-CSF, and plerixafor treatment in relapsed/refractory, FLT3-ITD-mutated acute myelogenous leukemia patients.

Stroma-leukemia interactions mediated by CXCR4, CD44, VLA4, and their respective ligands contribute to therapy resistance in FLT3-ITD-mutated acute myelogenous leukemia (AML). We conducted a phase 1 study with the combination of sorafenib (a FLT3-ITD inhibitor), plerixafor (a SDF-1/CXCR4 inhibitor), and G-CSF (that cleaves SDF-1, CD44, and VLA4). Twenty-eight patients with relapsed/refractory FLT3-ITD-mutated AML were enrolled from December 2010 to December 2013 at three dose levels of sorafenib (400, 600, and 800 mg twice daily) and G-CSF and plerixafor were administered every other day for seven doses starting on day one. Sorafenib 800 mg twice daily was selected for the expansion phase. While no dose-limiting toxicities (DLT) were encountered in the four-week DLT window, hand-foot syndrome and rash were seen beyond the DLT window, which required dose reductions in most patients. The response rate was 36% (complete response (CR) = 4, complete remission with incomplete platelet recovery (CRp) = 4, complete remission with incomplete hematologic recovery (CRi) = 1, and partial response (PR) = 1) for the intention to treat population. Treatment resulted in 58.4 and 47 mean fold mobilization of blasts and CD34 /38- stem/progenitor cells, respectively, to the circulation. Expression of the adhesion molecules CXCR4, CD44, and VLA4 on circulating leukemia cells correlated negatively with the mobilization of CD34+/38-, CD34+/38-/123+ "progenitor" cells (all P ≤ .002). Mass cytometry analysis of sequential samples from two patients demonstrated resistance emerging early on from sub-clones with persistent Akt and/or ERK signaling. In conclusion, the strategy of combined inhibition of FLT3 kinase and stromal adhesive interactions has promising activity in relapsed/refractory, FLT3-ITD-mutated AML, which warrants further evaluation in the front-line setting.

Role of MSC-derived galectin 3 in the AML microenvironment.

In acute myeloid leukemia (AML), high Galectin 3 (LGALS3) expression is associated with poor prognosis. The role of LGALS3 derived from mesenchymal stromal cells (MSC) in the AML microenvironment is unclear; however, we have recently found high LGALS3 expression in MSC derived from AML patients is associated with relapse. In this study, we used reverse phase protein analysis (RPPA) to correlate LGALS3 expression in AML MSC with 119 other proteins including variants of these proteins such as phosphorylated forms or cleaved forms to identify biologically relevant pathways. RPPA revealed that LGALS3 protein was positively correlated with expression of thirteen proteins including MYC, phosphorylated beta-Catenin (p-CTNNB1), and AKT2 and negatively correlated with expression of six proteins including integrin beta 3 (ITGB3). String analysis revealed that proteins positively correlated with LGALS3 showed strong interconnectivity. Consistent with the RPPA results, LGALS3 suppression by shRNA in MSC resulted in decreased MYC and AKT expression while ITGB3 was induced. In co-culture, the ability of AML cell to adhere to MSC LGALS3 shRNA transductants was reduced compared to AML cell adhesion to MSC control shRNA transductants. Finally, use of novel specific LGALS3 inhibitor CBP.001 in co-culture of AML cells with MSC reduced viable leukemia cell populations with induced apoptosis and augmented the chemotherapeutic effect of AraC. In summary, the current study demonstrates that MSC-derived LGALS3 may be critical for important biological pathways for MSC homeostasis and for regulating AML cell localization and survival in the leukemia microenvironmental niche.

Proteomic Profiling of Signaling Networks Modulated by G-CSF/Plerixafor/Busulfan-Fludarabine Conditioning in Acute Myeloid Leukemia Patients in Remission or with Active Disease prior to Allogeneic Stem Cell Transplantation.

To characterize intracellular signaling in peripheral blood (PB) cells of acute myeloid leukemia (AML) patients undergoing pretransplant conditioning with CXCR4 inhibitor plerixafor, granulocyte colony-stimulating factor (G-CSF), and busulfan plus fludarabine (Bu+Flu) chemotherapy, we profiled 153 proteins in 33 functional groups using reverse phase protein array. CXCR4 inhibition mobilized AML progenitors and clonal AML cells, and this was associated with molecular markers of cell cycle progression. G-CSF/plerixafor and G-CSF/plerixafor/Bu+Flu modulated distinct signaling networks in AML blasts of patients undergoing conditioning with active disease compared to nonleukemic PB cells of patients in remission. We identified AML-specific proteins that remained aberrantly expressed after chemotherapy, representing putative chemoresistance markers in AML.

Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin.

A critical step of DNA single-strand break repair is the rapid recruitment of the scaffold protein XRCC1 that interacts with, stabilizes and stimulates multiple enzymatic components of the repair process. XRCC1 recruitment is promoted by PARP1, an enzyme that is activated following DNA damage and synthesizes ADP-ribose polymers that XRCC1 binds directly. However, cells possess two other DNA strand break-induced PARP enzymes, PARP2 and PARP3, for which the roles are unclear. To address their involvement in the recruitment of endogenous XRCC1 into oxidized chromatin we have established 'isogenic' human diploid cells in which PARP1 and/or PARP2, or PARP3 are deleted. Surprisingly, we show that either PARP1 or PARP2 are sufficient for near-normal XRCC1 recruitment at oxidative single-strand breaks (SSBs) as indicated by the requirement for loss of both proteins to greatly reduce or ablate XRCC1 chromatin binding following H2O2 treatment. Similar results were observed for PNKP; an XRCC1 protein partner important for repair of oxidative SSBs. Notably, concentrations of PARP inhibitor >1000-fold higher than the IC50 were required to ablate both ADP-ribosylation and XRCC1 chromatin binding following H2O2 treatment. These results demonstrate that very low levels of ADP-ribosylation, synthesized by either PARP1 or PARP2, are sufficient for XRCC1 recruitment following oxidative stress.

Conditional reprogrammed human limbal epithelial cells represent a novel in vitro cell model for drug responses.

In this study, we established human limbal epithelial cells (LECs) from normal limbal tissues by using Conditional Reprogramming (CR) technology (refer to CR-LEC cells in this study). We have successfully established CR-LEC cell strains from three human donors (3 out of 3), and normal rabbits (2 out of 2) and pig (1 out of 1) as well. CR-LEC cells sustained a continuous and stable proliferation status with a normal karyotype, normal response to DNA damage, well-defined structured spheres in matrigel 3D culture. Responses of CR-LEC cells to IFN α2b, Ganciclovir and 5-Fluorouracil were different, suggesting that these drugs had different toxicities to these cells as expected. More important, there was no significant difference of responses to drugs between early and late passages of CR-LEC cells (p>0.05), indicating CR-LEC cells can serve a stable normal human cell model for toxicity assessment. Toxicity tests with monolayer cultures of CR-LEC cells were measured by staining the F-actin and Dsg-1 expression. Toxicity of three drugs at LD50 concentration resulted in a gradually increased destruction of monolayer, which is, in accordance with the irritation grade of three drugs on human cornea epithelium. Therefore, CR-LEC cells provide a novel and reliable in vitro physiological cell model for corneal toxicity assessment.

Distinct protein signatures of acute myeloid leukemia bone marrow-derived stromal cells are prognostic for patient survival.

Mesenchymal stromal cells (MSC) support acute myeloid leukemia (AML) cell survival in the bone marrow (BM) microenvironment. Protein expression profiles of AML-derived MSC are unknown. Reverse phase protein array analysis was performed to compare expression of 151 proteins from AML-MSC (n=106) with MSC from healthy donors (n=71). Protein expression differed significantly between the two groups with 19 proteins over-expressed in leukemia stromal cells and 9 over-expressed in normal stromal cells. Unbiased hierarchical clustering analysis of the samples using these 28 proteins revealed three protein constellations whose variation in expression defined four MSC protein expression signatures: Class 1, Class 2, Class 3, and Class 4. These cell populations appear to have clinical relevance. Specifically, patients with Class 3 cells have longer survival and remission duration compared to other groups. Comparison of leukemia MSC at first diagnosis with those obtained at salvage (i.e. relapse/refractory) showed differential expression of 9 proteins reflecting a shift toward osteogenic differentiation. Leukemia MSC are more senescent compared to their normal counterparts, possibly due to the overexpressed p53/p21 axis as confirmed by high ß-galactosidase staining. In addition, overexpression of BCL-XL in leukemia MSC might give survival advantage under conditions of senescence or stress and overexpressed galectin-3 exerts profound immunosuppression. Together, our findings suggest that the identification of specific populations of MSC in AML patients may be an important determinant of therapeutic response.

APLF promotes the assembly and activity of non-homologous end joining protein complexes.

Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein-DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.

Antileukemia activity of the novel peptidic CXCR4 antagonist LY2510924 as monotherapy and in combination with chemotherapy.

Targeting the stromal cell-derived factor 1α (SDF-1α)/C-X-C chemokine receptor type 4 (CXCR4) axis has been shown to be a promising therapeutic approach to overcome chemoresistance in acute myeloid leukemia (AML). We investigated the antileukemia efficacy of a novel peptidic CXCR4 antagonist, LY2510924, in preclinical models of AML. LY2510924 rapidly and durably blocked surface CXCR4 and inhibited stromal cell-derived factor 1 (SDF-1)α-induced chemotaxis and prosurvival signals of AML cells at nanomolar concentrations more effectively than the small-molecule CXCR4 antagonist AMD3100. In vitro, LY2510924 chiefly inhibited the proliferation of AML cells with little induction of cell death and reduced protection against chemotherapy by stromal cells. In mice with established AML, LY2510924 caused initial mobilization of leukemic cells into the circulation followed by reduction in total tumor burden. LY2510924 had antileukemia effects as monotherapy as well as in combination with chemotherapy. Gene expression profiling of AML cells isolated from LY2510924-treated mice demonstrated changes consistent with loss of SDF-1α/CXCR4 signaling and suggested reduced proliferation and induction of differentiation, which was proved by showing the attenuation of multiple prosurvival pathways such as PI3K/AKT, MAPK, and ß-catenin and myeloid differentiation in vivo. Effective disruption of the SDF-1α/CXCR4 axis by LY2510924 may translate into effective antileukemia therapy in future clinical applications.

High-throughput profiling of signaling networks identifies mechanism-based combination therapy to eliminate microenvironmental resistance in acute myeloid leukemia.

The bone marrow microenvironment is known to provide a survival advantage to residual acute myeloid leukemia cells, possibly contributing to disease recurrence. The mechanisms by which stroma in the microenvironment regulates leukemia survival remain largely unknown. Using reverse-phase protein array technology, we profiled 53 key protein molecules in 11 signaling pathways in 20 primary acute myeloid leukemia samples and two cell lines, aiming to understand stroma-mediated signaling modulation in response to the targeted agents temsirolimus (MTOR), ABT737 (BCL2/BCL-XL), and Nutlin-3a (MDM2), and to identify the effective combination therapy targeting acute myeloid leukemia in the context of the leukemia microenvironment. Stroma reprogrammed signaling networks and modified the sensitivity of acute myeloid leukemia samples to all three targeted inhibitors. Stroma activated AKT at Ser473 in the majority of samples treated with single-agent ABT737 or Nutlin-3a. This survival mechanism was partially abrogated by concomitant treatment with temsirolimus plus ABT737 or Nutlin-3a. Mapping the signaling networks revealed that combinations of two inhibitors increased the number of affected proteins in the targeted pathways and in multiple parallel signaling, translating into facilitated cell death. These results demonstrated that a mechanism-based selection of combined inhibitors can be used to guide clinical drug selection and tailor treatment regimens to eliminate microenvironment-mediated resistance in acute myeloid leukemia.