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Activation of the innate immune STimulator of INterferon Genes (STING) pathway potentiates antitumour immunity, but systemic delivery of STING agonists to tumours is challenging. We conjugated STING-activating cyclic dinucleotides (CDNs) to PEGylated lipids (CDN-PEG-lipids; PEG, polyethylene glycol) via a cleavable linker and incorporated them into lipid nanodiscs (LNDs), which are discoid nanoparticles formed by self-assembly. Compared to state-of-the-art liposomes, intravenously administered LNDs carrying CDN-PEG-lipid (LND-CDNs) exhibited more efficient penetration of tumours, exposing the majority of tumour cells to STING agonist. A single dose of LND-CDNs induced rejection of established tumours, coincident with immune memory against tumour rechallenge. Although CDNs were not directly tumoricidal, LND-CDN uptake by cancer cells correlated with robust T-cell activation by promoting CDN and tumour antigen co-localization in dendritic cells. LNDs thus appear promising as a vehicle for robust delivery of compounds throughout solid tumours, which can be exploited for enhanced immunotherapy.

Background Cytokine release syndrome (CRS) is a life-threatening complication of chimeric antigen receptor T cell (CAR-T) therapy. Macrophages/ monocytes are mediators of CRS. Tripterygium glycosides is an immunomodulator which could inhibit macrophages/ monocytes in animal models. Methods Two patients with relapsed and refractory hematological malignancies developed CRS after receiving CAR-T therapy. They received short-term tripterygium glycosides orally. Results Both patients showed rapid mitigation of fever with evidently decrease in elevated inflammatory cytokines within 72 hours. The patients' monocytes diminished remarkably, while CAR-T cells were neglectably affected. Treatment of 30 ng/mL triptolide in ex vivo cultured patients' blood for 24 hours selectively deplete over half of monocytes. Single cell RNA sequencing suggested selective depletion of CD14+CD16+ monocytes with decreased pro-inflammatory cytokines. Conclusions The low-cost and orally available tripterygium glycosides could be a promising alternative for CAR-T induced CRS, as well as other diseases complicated with CRS, e.g., coronavirus disease 2019.

Chimeric Antigen Receptor (CAR) T-cell therapy can be effective in treating human cancer but loss of the antigen recognized by the CAR poses a major obstacle. Here, we report an approach for vaccine boosting CAR T-cells, which triggers engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T-cells promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited priming of endogenous anti-tumor T-cells (antigen spreading). This process was accompanied by a shift in toward oxidative phosphorylation in CAR T-cells and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading induced by vaccine-boosted CAR-T enabled a proportion of complete responses even when the initial tumor was 50% CAR-antigen-negative, and heterogenous tumor control was further enhanced by genetically amplifying CAR-T IFN-γ expression. Thus, CAR T-cell-derived IFN-γ plays a critical role in promoting antigen spreading, and vaccine boosting provides a clinically-translatable strategy to drive such responses against solid tumors.

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.

The clinical application of cytokine therapies for cancer treatment remains limited due to severe adverse reactions and insufficient therapeutic effects. Although cytokine localization by intratumoral administration could address both issues, the rapid escape of soluble cytokines from the tumor invariably subverts this effort. We find that intratumoral administration of a cytokine fused to the collagen-binding protein lumican prolongs local retention and dramatically reduces systemic exposure. Combining local administration of lumican-cytokine fusions with systemic immunotherapies (tumor-targeting antibody, checkpoint blockade, cancer vaccine, or T cell therapy) improves efficacy without exacerbating toxicity in syngeneic tumor models and the Braf(V600E)/Pten(fl/fl) genetically engineered melanoma model. Notably, curative abscopal effects on non-cytokine-injected tumors were also observed as a result of a protective and systemic CD8+ T cell response primed by local therapy. Cytokine collagen-anchoring constitutes a facile, tumor-agnostic strategy to safely potentiate otherwise marginally effective systemic immunotherapies.

A major obstacle to curing chronic myeloid leukemia (CML) is the intrinsic resistance of CML stem cells (CMLSCs) to the drug imatinib mesylate (IM). Prosurvival genes that are preferentially expressed in CMLSCs compared with normal hematopoietic stem cells (HSCs) represent potential therapeutic targets for selectively eradicating CMLSCs. However, the discovery of such preferentially expressed genes has been hampered by the inability to completely separate CMLSCs from HSCs, which display a very similar set of surface markers. To overcome this challenge, and to minimize confounding effects of individual differences in gene expression profiles, we performed single-cell RNA-seq on CMLSCs and HSCs that were isolated from the same patient and distinguished based on the presence or absence of BCR-ABL. Among genes preferentially expressed in CMLSCs is PIM2 , which encodes a prosurvival serine-threonine kinase that phosphorylates and inhibits the proapoptotic protein BAD. We show that IM resistance of CMLSCs is due, at least in part, to maintenance of BAD phosphorylation by PIM2. We find that in CMLSCs, PIM2 expression is promoted by both a BCR-ABL–dependent (IM-sensitive) STAT5-mediated pathway and a BCR-ABL–independent (IM-resistant) STAT4-mediated pathway. Combined treatment with IM and a PIM inhibitor synergistically increases apoptosis of CMLSCs, suppresses colony formation, and significantly prolongs survival in a mouse CML model, with a negligible effect on HSCs. Our results reveal a therapeutically targetable mechanism of IM resistance in CMLSCs. The experimental approach that we describe can be generally applied to other malignancies that harbor oncogenic fusion proteins or other characteristic genetic markers.

The current research work demonstrates preparation and characterization of Reduced Graphene Oxide (rGO)@ High-Density Polyethylene (HDPE) aerogel coated Natural Rubber Latex foam (NRLF) with superhydrophobic and superoleophilic character, good sorption capacity for oil-water separation, high electrical conductivity and electromagnetic interference shielding effectiveness (EMI SE) of the same were studied. The coated NRLF was prepared by using a solution of rGO and HDPE in xylene solvent with cooling process to result into thermally induced phase separation of the graphene and Polyethylene molecular network. The rGO modified HDPE aerogel coated NRLF showed superhydrophobicity with static water contact angle > 150°. The rGO@HDPE aerogel coated NRLF with 2 wt% rGO has reached EMI SE value of 24 dB, meeting commercial requirements. The electrical conductivity, EMI SE and the hydrophobicity of the composites significantly improved after mixing rGO into HDPE sol. This prepared superhydrophobic NRLF with high electric conductivity may find attractive application in oil-water separation and EMI Shielding, which would also be economically favored option in the current scenario.

Self-Portrait on the Borderline Between Mexico and the United States by the Mexican artist Frida Kahlo reveals a multitude of insights. This paper assesses how the nuances embodied in the painting serve as critical clues to comprehend Frida Kahlo’s personal experiences, Mexican culture, and the approach of discovering the contextual background through the work of art. Beginning with a detailed formal analysis of the portrait, this paper further explores it by making connections with contextual evidence. Through timely reference to Frida Kahlo’s political stance, cultural identity, and health, the paper demonstrates how the painting proffers insight into both the artist’s life and Aztec culture.

Leukemia stem cells are a rare population with a primitive progenitor phenotype that can initiate, sustain, and recapitulate leukemia through a poorly understood mechanism of self-renewal. Here, we report that Kruppel-like factor 4 (KLF4) promotes disease progression in a murine model of chronic myeloid leukemia (CML)-like myeloproliferative neoplasia by repressing an inhibitory mechanism of preservation in leukemia stem/progenitor cells with leukemia-initiating capacity. Deletion of the Klf4 gene severely abrogated the maintenance of BCR-ABL1(p210)-induced CML by impairing survival and self-renewal in BCR-ABL1+ CD150+ lineage-negative Sca-1+ c-Kit+ leukemic cells. Mechanistically, KLF4 repressed the Dyrk2 gene in leukemic stem/progenitor cells; thus, loss of KLF4 resulted in elevated levels of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2), which were associated with inhibition of survival and self-renewal via depletion of c-Myc protein and p53 activation. In addition to transcriptional regulation, stabilization of DYRK2 protein by inhibiting ubiquitin E3 ligase SIAH2 with vitamin K3 promoted apoptosis and abrogated self-renewal in murine and human CML stem/progenitor cells. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling p53- and c-Myc-mediated regulation of survival and self-renewal in CML cells with leukemic-initiating capacity that can be targeted with small molecules.

Developing tools to accurately predict the clinical prevalence of drug-resistant mutations is a key step toward generating more effective therapeutics. Here we describe a high-throughput CRISPR-Cas9–based saturated mutagenesis approach to generate comprehensive libraries of point mutations at a defined genomic location and systematically study their effect on cell growth. As proof of concept, we mutagenized a selected region within the leukemic oncogene BCR-ABL1. Using bulk competitions with a deep-sequencing readout, we analyzed hundreds of mutations under multiple drug conditions and found that the effects of mutations on growth in the presence or absence of drug were critical for predicting clinically relevant resistant mutations, many of which were cancer adaptive in the absence of drug pressure. Using this approach, we identified all clinically isolated BCR-ABL1 mutations and achieved a prediction score that correlated highly with their clinical prevalence. The strategy described here can be broadly applied to a variety of oncogenes to predict patient mutations and evaluate resistance susceptibility in the development of new therapeutics. Refereed/Peer-reviewed

Lycopene Beta-cyclase (LCY-B) is thought to play a critical role in Beta‐carotene synthesis in fruit. A full-length cDNA clone encoding Lycopene Beta-cyclase was isolated from muskmelon (Cucumis melo L.) by RT-PCR and RACE. The clone, designated CmLcyb1, contains 1871 nucleotides, with an open reading frame of 1512 nucleotides. The deduced 504-amino-acid sequence showed high identities with other plant Lycopene Beta-cyclases. Real time quantitative RT-PCR analysis indicated that CmLcyb1 was expressed in all tissues and organs of muskmelon inbred M01-3 with white mesocarp and, ‘Homoka’, an orange mesocarp cultivar. The expression levels of CmLcyb1 in roots, stems, leaves and flowers in the two genotypes differed little. The expression level was highest in mature fruit of ‘Homoka’ and was much higher than that in mature fruit of M01-3. Moreover, the mRNA level of CmLcyb1 was very low in fruits before fruit-size fixation and increased dramatically in the size-fixed fruits of these two genotypes. The mRNA levels of CmLcyb1 during fruit development of ‘Homoka’ were all higher than those of M01-3. Interestingly, Beta‐carotene content showed almost the same change trend as mRNA levels during fruit development in these two genotypes, suggesting that Beta‐carotene accumulation may be linked to the CmLcyb1 transcript level in muskmelon fruit.

The oncoprotein BCR-ABL transforms myeloid progenitor cells and is responsible for the development of chronic myeloid leukemia (CML). In transformed cells, BCR-ABL suppresses apoptosis as well as autophagy, a catabolic process in which cellular components are degraded by the lysosomal machinery. The mechanism by which BCR-ABL suppresses autophagy is not known. Here we report that in both mouse and human BCR-ABL–transformed cells, activating transcription factor 5 (ATF5), a prosurvival factor, suppresses autophagy but does not affect apoptosis. We find that BCR-ABL, through PI3K/AKT/FOXO4 signaling, transcriptionally up-regulates ATF5 expression and that ATF5, in turn, stimulates transcription of mammalian target of rapamycin (mTOR; also called mechanistic target of rapamycin), a well-established master negative-regulator of autophagy. Previous studies have shown that the BCR-ABL inhibitor imatinib mesylate induces both apoptosis and autophagy, and that the resultant autophagy modulates the efficiency by which imatinib kills BCR-ABL–transformed cells. We demonstrate that imatinib-induced autophagy is because of inhibition of the BCR-ABL/PI3K/AKT/FOXO4/ATF5/mTOR pathway that we have identified in this study.

Leukemia stem cells (LSCs) are a rare population able to sustain and recapitulate leukemia through a poorly understood mechanism of self-renewal. Because more than half of patients relapse after the cessation of TKI therapy, it is clear that a cure is not possible with TKIs alone, and LSC-specific drugs are urgently needed to simultaneously eliminate bulk leukemia with TKIs and LSCs. Here we report that KLF4 promotes disease progression in chronic myeloid leukemia (CML) by repressing an inhibitory mechanism in LSCs that can be activated with small molecules. Deletion of the Klf4 gene severely abrogated maintenance of BCR-ABL(p210)-induced CML by impairing survival and self-renewal in LSCs whereas increased self-renewal was observed in hematopoietic stem cells during serial transplantation. Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated levels of the DYRK2 kinase in LSCs, which was associated with p53-mediated apoptosis and inhibition of self-renewal through depletion of c-Myc protein. Supporting this model, stabilization of DYRK2 protein, by inhibiting the ubiquitin E3 ligase SIAH2 with vitamin K3, promoted apoptosis in a panel of CML cell lines (K562, KU-812, and KCL-22) by inducing DYRK2 expression. Knocking out the DYRK2 gene in K562 cells by Cas9/CRISPR abrogated cytotoxicity induced by vitamin K3 and the presence of p53 significantly lowered IC50. In vivo treatment of CML mice diminished the number of LSCs evaluated in secondary transplants. In humans, vitamin K3 induced apoptosis in bone marrow cells from CML patients but not in healthy individuals by inducing DYRK2, p53 phosphorylation, and c-Myc depletion; furthermore, vitamin K3 abrogated capacity of CD34+ cells to generate colonies in methylcellulose only in CML. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling both p53 and c-Myc mediated regulation of survival and self-renewal in CML LSCs that can be activated pharmacologically. Citation Format: Chun Shik Park, Ye Shen, Andrew Lewis, Koramit Suppipat, Monica Puppi, Julie Tomolonis, Taylor Chen, Paul Pang, Toni-Ann Mistretta, Leyuan Ma, Michael Green, Rachel Rau, Daniel Lacorazza. Pharmacologic inhibition of SIAH2 stabilizes DYRK2 and inhibits survival and self-renewal in chronic myeloid leukemia (CML) leukemic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 145.

Tetraploid muskmelon plants were induced successfully from diploid Cucumis melo inbred M01-3 (2n = 24) by colchicine. The morphological characteristics and fruit qualities of diploid and tetraploid muskmelons were investigated. The results showed that the leaves and flowers of the tetraploid plants were markedly larger, the plants were obviously higher, and the stems were thicker than those of the diploid plants. Transmission electron microscope observation revealed that the numbers of chloroplast, granule and grana, and the length of chloroplast and granule of the tetraploid plant leaves were significantly more or longer than those of the diploid plants. The soluble solid, soluble sugar and vitamin C contents in the tetraploid fruit were distinctly higher than those in the diploid fruit. The weight of the tetraploid fruit was 30% heavier than that of the diploid fruit. And the seeds from the tetraploid plants were broader and thicker than those from the diploid plants. Together, our results suggest that the tetraploid muskmelon exhibited better agronomical characteristics than the diploid muskmelon and the tetraploid muskmelon could be used as the improved variety and a potential germplasm for the development of triploid fruit.

To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

Infectious bursal disease virus (IBDV) is a non-enveloped double-stranded RNA virus belonging to the Birnaviridae family. It shows substantial variation in the major antigen region of the viral capsid protein VP2, where a hypervariable region plays a key role in the virulence of IBDV and its epitope. This study identifies several putative recombinants from previously published data to suggest that homologous recombination may naturally occur between different IBDV strains. In addition, a novel very virulence sublineage emerges in the VP2 phylogenic tree, comprising three putative recombination strains isolated in Korea and China, KSH, KK1 and SH-h. The major putative parents of the three mosaics are descended from the vaccine lineage while their hypervariable regions from vvIBDV. These findings also suggest that vaccine coverage may have influence on the evolution and genetic diversity of IBDV, resulting in a novel group with vvIBDV phenotype through recombination with wild IBDV.

Newcastle disease (ND), caused by ND virus (NDV), is one of the most serious illnesses of birds, particularly chickens, and has been one of the major causes of economic losses in the poultry industry. Live vaccines are widely used to prevent chicken from NDV all over the world. Given the implications that recombination has for RNA virus evolution, it is clearly important to determine the extent to which recombination plays a role in NDV evolution. In this study, we performed the phylogenetic and recombination analysis on complete NDV genomes. A natural multi-recombinant cockatoo/Indonesia/14698/90 (AY562985) was identified. Its two minor parental-like strains might be from the NDV vaccine lineage and anhinga/U.S.(Fl)/44083/93 lineage, respectively. Our study suggests that recombination plays a role in NDV evolution. Especially, the study also suggests that live vaccines have capacity to play roles in shaping NDV evolution by homologous recombination with circulating virus.

Genome-wide RNA interference (RNAi) screening in mammalian cells has proven to be a powerful tool for identifying new genes and molecular pathways relevant to many cellular processes and diseases. For example, screening for genes that, when inactivated, lead to resistance to cancer therapeutic drugs can reveal new mechanisms for how resistance develops, and identify potential targetable strategies to overcome drug resistance. Here, we describe a detailed procedure for performing a high throughput RNAi screen using a genome-wide human short hairpin RNA (shRNA) library for identifying tyrosine kinase inhibitor (TKI)-resistance genes in a human CML cell line model.

Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances, there is no mutation in BCR-ABL, and the basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed a large-scale RNA interference screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL(+) cells IM-resistant. In these IMSG knockdown cells, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling is sustained after IM treatment because of up-regulation of PRKCH, which encodes the protein kinase C (PKC) family member PKCη, an activator of CRAF. PRKCH is also up-regulated in samples from CML patients with BCR-ABL-independent IM resistance. Combined treatment with IM and trametinib, a U.S. Food and Drug Administration-approved MEK inhibitor, synergistically kills BCR-ABL(+) IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL-independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels of PRKCH, and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent IM resistance in CML and CML stem cells.

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. Transformation of HSCs by the breakpoint cluster region-ABL tyrosine kinase (BCR-ABL) oncogene causes chronic myelogenous leukemia (CML). The E-twenty six (ets) transcription factor GA binding protein (GABP) is a tetrameric transcription factor complex that contains GABPα and GABPβ proteins. Deletion in bone marrow of Gabpa , the gene that encodes the DNA-binding component, caused cell cycle arrest in HSCs and profound loss of hematopoietic progenitor cells. Loss of Gabpα prevented development of CML, although mice continued to generate BCR-ABL–expressing Gabpα-null cells for months that were serially transplantable and contributed to all lineages in secondary recipients. A bioinformatic screen identified the serine-threonine kinase protein kinase D2 (PRKD2) as a potential effector of GABP in HSCs. Prkd2 expression was markedly reduced in Gabpα-null HSCs and progenitor cells. Reduced expression of PRKD2 or pharmacologic inhibition decreased cell cycling, and PRKD2 rescued growth of Gabpα-null BCR-ABL–expressing cells. Thus, GABP is required for HSC cell cycle entry and CML development through its control of PRKD2. This offers a potential therapeutic target in leukemia.

Chronic myeloid leukemia (CML) is the first blood cancer known to originate from a single hematopoietic stem cell (HSC) by expression of BCR-ABL, a product of the chromosomal translocation t(9;22), that slowly progress to a lethal fast-growing leukemia caused by malignant reprogramming of progenitor cells (blast crisis). Although CML can be successfully managed with targeted therapy by suppressing BCR-ABL kinase activity with tyrosine kinase inhibitors (TKI), patients remain in remission as long as they adhere to a lifelong treatment. The leukemic stem cell (LSC) population emerges as a key ‘CML reservoir’ that escapes TKI therapy by developing BCR-ABL-independent mechanisms of self-renewal and survival. LSC still remains an elusive target because of our poor understanding of specific self-renewal mechanisms and inability to selectively eliminate LSC without damaging normal hematopoiesis. Thus, there is a need for alternative drugs for relapse patients to prevent reactivation of BCR-ABL-positive LSC after stopping chemotherapy or emergence of chemoresistance and as frontline therapy to achieve treatment-free remission. We found that somatic deletion of the transcriptional factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. This data suggest that KLF4 promotes self-renewal of LSCs whereas serial transplantation indicates that KLF4 restricts stress self-renewal of normal HSCs and thus inhibition of KLF4 function would impair LSC self-renewal without altering blood production. Analyses of global gene expression in purified LSCs and genome-wide binding of KLF4 in a murine CML cell line revealed that KLF4 represses the gene encoding for the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). Immunoblots revealed elevated levels of DYRK2 protein in KLF4-deficient LSCs purified from the bone marrow of CML mice. Because phosphorylation of c-Myc and p53 by DYRK2 induces proteosomal degradation and cell death, respectively, we found that DYRK2 upregulation in KLF4-deficient LSCs was associated with a reduction of c-Myc protein and increased cleavage of PARP. As a proof-of-principle of the therapeutic potential of this finding, we explored the efficacy of vitamin K3 to eradicate LSCs by inhibiting the ubiquitin E3 ligase SIAH2 in charge of DYRK2 degradation. Vitamin K3 efficiently reduced cell viability in a panel of human-derived CML cell lines by inducing Dyrk2 expression and apoptosis. The identification of Dyrk2 as a critical mediator of LSC downfall is an innovative paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in CML patients. Citation Format: Chun Shik Park, Ye Shen, Koramit Suppipat, Julie Tomolonis, Monica Puppi, Toni-Ann Mistretta, Leyuan Ma, Michael Green, Daniel Lacorazza. KLF4 promotes self-renewal by repressing DYRK2-mediated degradation of c-Myc in leukemic stem cells: development of targeted therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3334.

Avian influenza A viruses (AIVs), including the H5N1, H9N2, and H7N7 subtypes, have been directly transmitted to humans, raising concerns over the possibility of a new influenza pandemic. To prevent a future avian influenza pandemic, it is very important to fully understand the molecular basis driving the change in AIV virulence and host tropism. Although virulent variants of other viruses have been generated by homologous recombination, the occurrence of homologous recombination within AIV segments is controversial and far from proven. This study reports three circulating H9N2 AIVs with similar mosaic PA genes descended from H9N2 and H5N1. Additionally, many homologous recombinants are also found deposited in GenBank. Recombination events can occur in PB2, PB1, PA, HA, and NP segments and between lineages of the same/different serotype. These results collectively demonstrate that intragenic recombination plays a role in driving the evolution of AIVs, potentially resulting in effects on AIV virulence and host tropism changes.

Resistance to imatinib (IM) has posed a great obstacle in treating Chronic Myeloid Leukemia(CML). New generations of tyrosine kinase inhibitors (TKIs) have been developed for BCR-ABL dependent IM resistance, which is commonly due to BCR-ABL kinase-domain mutations. However, in 50% or more of IM-resistant CML patients there is no mutation in BCR-ABL, and intrinsic resistance of CML stem cells often causes disease relapse. Understanding the mechanism of BCR-ABL independent IM resistance is therefore essential for devising strategies to eradicate resistant and residual leukemia. Through a genome-wide shRNA screen in a IM-sensitive human CML cell line, K562 cells, we identified 16 imatinib-sensitizing genes (IMSGs), knockdown of which increased IM resistance of multiple human CML cell lines (e.g., K562, KYO-1 cells. >2 folds increment), and primary CML mice bone marrow cells (> 2 folds of CFUs). Knocking down 11 IMSGs increased IC50IM by more than 5 folds, and also conferred equivalent level of resistance to the second generation TKI, Dasatinib. Extensive signaling pathway analysis by immune-blotting for characteristic protein phosphorylation markers revealed co-activation of PKC and MEK/ERK pathway in majority of IMSG knockdown K562 cell lines. qRT-PCR analysis in IMSG knockdown cells identified PRKCH as the predominant deregulated PKC family member. Mild overexpression of PRKCH increased IC50IM by 10-20 folds, and in vitro phosphorylation confirmed that PRKCH could directly phosphorylate and activate CRAF, therefore MEK/ERK pathway. PRKCH is highly expressed in IM-resistant samples with wildtype BCR-ABL compared to those with mutant BCR-ABL (p To investigate whether simultaneous inhibition of BCR-ABL and MEK/ERK signaling could more efficiently kill BCR-ABL-independent IM-resistant CML cells, we analyzed the effect of combining IM treatment with a FDA-approved MEK inhibitor, Trametinib, in multiple BCR-ABL independent IM-resistant CML mouse models, and primary resistant CML cells. Treatment with both drugs showed a substantially greater effect than either drug alone, and in many instances, the effect of combined drug treatment was synergistic. qRT-PCR analysis showed that PRKCH expression in IM-resistant CML stem cells is higher than that in IM-sensitive CML progenitors (4~25 folds, p In addition to MEK/ERK pathway, we found PRKCH could activate PKD2, a downstream effector of novel PKCs. PRKCH expression increases upon IM treatment, along with increased PKD2 activity (S916 phosphorylation) in multiple CML cell lines(e.g., K562, KYO-1, LAMA84). PKD2 expression was also induced by IM in primary human CML stem cells. PKD2 promotes cell survival by activating NF-kB pathway, and IM treatment increased p65/RelA phosphorylation at S536 and its nuclear translocation, together with over 4 folds increase in expression of anti-apoptotic p65 target genes (e.g., CIAP2, Bfl-1/A1). Pharmacological inhibition using pan-PKD inhibitor, CRT0066101, completely abolishes IM induced p65 phosphorylation and increased the killing of CML cells by IM. This study for the first time reveals the critical role of PKC pathway in BCR-ABL independent IM resistance in CML. The validation of MEK/ERK and PKD/NF-kB pathway as critical downstream effectors provided specific targets for small molecule therapy in TKI-resistant CML and other malignancies caused by deregulated PKC pathways. Disclosures Cerny: Cellerant Therapeutics: Honoraria, Research Funding.

Abstract 1672 Chronic Myelogenous Leukemia (CML) is a myeloproliferative neoplasm (MPN) caused by transformation of hematopoietic stem cells (HSCs) by the BCR-ABL oncogene. CML is associated with excessive proliferation of HSCs and massive expansion of the myeloid cell pool. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPα, along with various GABPβ partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. In a mouse model of CML, animals transplanted with BCR-ABL-infected bone marrow developed massive myeloid cell expansion and died with a MPN. Induced deletion of Gabpa prevented development of CML, yet mice continued to produce mature BCR-ABL-expressing granulocytes for months without apparent illness. BCR-ABL+ cells were transplantable into secondary recipients without development of CML, and contributed to all hematopoietic lineages, thereby confirming expression of BCR-ABL by long-term HSCs. We used a bioinformatic approach to analyze GABP-bound genes that are upregulated in both human and mouse LSCs compared to normal HSCs. Among 115 GABP-bound, CML-associated genes, we identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase that has been implicated in cancer, but has not previously been associated with HSC functions or CML. Deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In vitro growth of BCR-ABL+ bone marrow cells was prevented by Gabpa deletion, but growth was partially rescued by forced expression of PRKD2. Knockdown and pharmacologic inhibition of PRKD2 blocked cell cycle entry of BCR-ABL+ cells. We conclude that Gabp is required for HSC cell cycle entry and for development of CML, and that these effects of GABP are mediated, in part, by PRKD2. These findings suggest that PRKD2 kinase may serve as a novel therapeutic target in leukemia. Disclosures: No relevant conflicts of interest to declare.