Quantum pBac: An effective, high-capacity piggyBac-based gene integration vector system for unlocking gene therapy potential.

Recent advances in gene therapy have brought novel treatment options for cancer. However, the full potential of this approach has yet to be unlocked due to the limited payload capacity of commonly utilized viral vectors. Virus-free DNA transposons, including piggyBac, have the potential to obviate these shortcomings. In this study, we improved a previously modified piggyBac system with superior transposition efficiency. We demonstrated that the internal domain sequences (IDS) within the 3' terminal repeat domain of hyperactive piggyBac (hyPB) donor vector contain dominant enhancer elements. Plasmid-free donor vector devoid of IDS was used in conjunction with a helper plasmid expressing Quantum PBase™ v2 to generate an optimal piggyBac system, Quantum pBac™ (qPB), for use in T cells. qPB outperformed hyPB in CD20/CD19 CAR-T production in terms of performance as well as yield of the CAR-T cells produced. Furthermore, qPB also produced CAR-T cells with lower donor-associated variabilities compared to lentiviral vector. Importantly, qPB yielded mainly CD8+ CAR-TSCM cells, and the qPB-produced CAR-T cells effectively eliminated CD20/CD19-expressing tumor cells both in vitro and in vivo. Our findings confirm qPB as a promising virus-free vector system with an enhanced payload capacity to incorporate multiple genes. This highly efficient and potentially safe system will be expected to further advance gene therapy applications.

Comparison of chimeric antigen receptor-T cell-mediated cytotoxicity assays with suspension tumor cells using plate-based image cytometry method.

In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. The GFP-based method demonstrated higher sensitivity in detecting CAR-T cell-mediated cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions.

The MKK-Dependent Phosphorylation of p38α Is Augmented by Arginine Methylation on Arg49/Arg149 during Erythroid Differentiation.

The activation of p38 mitogen-activated protein kinases (MAPKs) through a phosphorylation cascade is the canonical mode of regulation. Here, we report a novel activation mechanism for p38α. We show that Arg49 and Arg149 of p38α are methylated by protein arginine methyltransferase 1 (PRMT1). The non-methylation mutations of Lys49/Lys149 abolish the promotive effect of p38α on erythroid differentiation. MAPK kinase 3 (MKK3) is identified as the major p38α upstream kinase and MKK3-mediated activation of the R49/149K mutant p38α is greatly reduced. This is due to a profound reduction in the interaction of p38α and MKK3. PRMT1 can enhance both the methylation level of p38α and its interaction with MKK3. However, the phosphorylation of p38α by MKK3 is not a prerequisite for methylation. MAPK-activated protein kinase 2 (MAPKAPK2) is identified as a p38α downstream effector in the PRMT1-mediated promotion of erythroid differentiation. The interaction of MAPKAPK2 with p38α is also significantly reduced in the R49/149K mutant. Together, this study unveils a novel regulatory mechanism of p38α activation via protein arginine methylation on R49/R149 by PRMT1, which impacts partner interaction and thus promotes erythroid differentiation. This study provides a new insight into the complexity of the regulation of the versatile p38α signaling and suggests new directions in intervening p38α signaling.

HDAC8 regulates long-term hematopoietic stem-cell maintenance under stress by modulating p53 activity.

The maintenance and functional integrity of long-term hematopoietic stem cells (LT-HSCs) is critical for lifelong hematopoietic regeneration. Histone deacetylases (HDACs) modulate acetylation of lysine residues, a protein modification important for regulation of numerous biological processes. Here, we show that Hdac8 is most highly expressed in the phenotypic LT-HSC population within the adult hematopoietic hierarchy. Using an Hdac8-floxed allele and a dual-fluorescence Cre reporter allele, largely normal hematopoietic differentiation capacity of Hdac8-deficient cells was observed. However, the frequency of phenotypic LT-HSC population was significantly higher shortly after Hdac8 deletion, and the expansion had shifted to the phenotypic multipotent progenitor population by 1 year. We show that Hdac8-deficient hematopoietic progenitors are compromised in colony-forming cell serial replating in vitro and long-term serial repopulating activity in vivo. Mechanistically, we demonstrate that the HDAC8 protein interacts with the p53 protein and modulates p53 activity via deacetylation. Hdac8-deficient LT-HSCs displayed hyperactivation of p53 and increased apoptosis under genotoxic and hematopoietic stress. Genetic inactivation of p53 reversed the increased apoptosis and elevated expression of proapoptotic targets Noxa and Puma seen in Hdac8-deleted LT-HSCs. Dramatically compromised hematopoietic recovery and increased lethality were seen in Hdac8-deficient mice challenged with serial 5-fluorouracil treatment. This hypersensitivity to hematopoietic ablation was completely rescued by inactivation of p53. Altogether, these results indicate that HDAC8 functions to modulate p53 activity to ensure LT-HSC maintenance and cell survival under stress.

CBFß-SMMHC creates aberrant megakaryocyte-erythroid progenitors prone to leukemia initiation in mice.

Acute myeloid leukemia (AML) arises through multistep clonal evolution characterized by stepwise accumulation of successive alterations affecting the homeostasis of differentiation, proliferation, self-renewal, and survival programs. The persistence and dynamic clonal evolution of leukemia-initiating cells and preleukemic stem cells during disease progression and treatment are thought to contribute to disease relapse and poor outcome. Inv(16)(p13q22) or t(16;16)(p13.1;q22), one of the most common cytogenetic abnormalities in AML, leads to expression of a fusion protein CBFß-SMMHC (CM) known to disrupt myeloid and lymphoid differentiation. Anemia is often observed in AML but is presumed to be a secondary consequence of leukemic clonal expansion. Here, we show that CM expression induces marked deficiencies in erythroid lineage differentiation and early preleukemic expansion of a phenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitor population. Using dual-fluorescence reporter mice in lineage tracking and repopulation assays, we show that CM expression cell autonomously causes expansion of abnormal Pre-Meg/E progenitors with compromised erythroid specification and differentiation capacity. The preleukemic Pre-Meg/Es display dysregulated erythroid and megakaryocytic fate-determining factors including increased Spi-1, Gata2, and Gfi1b and reduced Zfpm1, Pf4, Vwf, and Mpl expression. Furthermore, these abnormal preleukemic Pre-Meg/Es have enhanced stress resistance and are prone to leukemia initiation upon acquiring cooperative signals. This study reveals that the leukemogenic CM fusion protein disrupts adult erythropoiesis and creates stress-resistant preleukemic Pre-Meg/E progenitors predisposed to malignant transformation. Abnormality in Meg/E or erythroid progenitors could potentially be considered an early predictive risk factor for leukemia evolution.

Manufacturing CD20/CD19-targeted iCasp9 regulatable CAR-TSCM cells using a Quantum pBac-based CAR-T engineering system.

CD19-targeted chimeric antigen receptor (CAR) T cell therapies have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However, >50% of CD19-CAR-T-treated patients experience progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore, it remains a challenge to efficiently, cost-effectively, and consistently manufacture clinically relevant numbers of virally engineered CAR-T cells. Using a highly efficient piggyBac transposon-based vector, Quantum pBac™ (qPB), we developed a virus-free cell-engineering system for development and production of multiplex CAR-T therapies. Here, we demonstrate in vitro and in vivo that consistent, robust and functional CD20/CD19 dual-targeted CAR-T stem cell memory (CAR-TSCM) cells can be efficiently produced for clinical application using qPB™. In particular, we showed that qPB™-manufactured CAR-T cells from cancer patients expanded efficiently, rapidly eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. Therefore, the simplicity of manufacturing multiplex CAR-T cells using the qPB™ system has the potential to improve efficacy and broaden the accessibility of CAR-T therapies.

Elevation of protein kinase Cα stimulates osteogenic differentiation of mesenchymal stem cells through the TAT-mediated protein transduction system.

Mesenchymal stem cells (MSCs) can differentiate toward various lineages, including the osteogenic lineage, and thus hold great potential for clinic purposes. By using pharmacological inhibitors, protein kinase C (PKC) signaling has been shown to either negatively or positively regulate differentiation of bone, however, due to the low transfection efficiency in MSCs, the role of individual PKC isoforms is still not fully understood. In this study, we established a TAT peptide-mediated transduction system that efficiently delivered PKCα proteins into MSCs in a non-invasive fashion. The increased PKCα protein levels significantly promoted osteogenic differentiation in the murine mesenchymal C3H10T1/2 cells and in primary MSCs from both human and mouse, as demonstrated by the enhanced activity of the osteoblast marker, alkaline phosphatase, and the enhanced expression of the key transcription factor runx2. Mineralization is an important functional indication for bone differentiation. Our results further showed that PKCα promoted expression of the important osteocalcin gene and the accumulation of calcium minerals. Taken together, this study provides direct evidence showing that PKCα positively regulates osteogenic differentiation and demonstrates that the TAT peptide-mediated method enables functional study of specific PKC isoforms in MSCs without using viral infection. This may promote the application of PKCs in therapeutic treatment.

Protein-arginine methyltransferase 1 suppresses megakaryocytic differentiation via modulation of the p38 MAPK pathway in K562 cells.

Protein-arginine methyltransferase 1 (PRMT1) plays pivotal roles in various cellular processes. However, its role in megakaryocytic differentiation has yet to be investigated. Human leukemia K562 cells have been used as a model to study hematopoietic differentiation. In this study, we report that ectopic expression of HA-PRMT1 in K562 cells suppressed phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation as demonstrated by changes in cytological characteristics, adhesive properties, and CD41 expression, whereas knockdown of PRMT1 by small interference RNA promoted differentiation. Impairment of the methyltransferase activity of PRMT1 diminished the suppressive effect. These results provide evidence for a novel role of PRMT1 in negative regulation of megakaryocytic differentiation. Activation of ERK MAPK has been shown to be essential for megakaryocytic differentiation, although the role of p38 MAPK is still poorly understood. We show that knockdown of p38alpha MAPK or treatment with the p38 inhibitor SB203580 significantly enhanced PMA-induced megakaryocytic differentiation. Further investigation revealed that PRMT1 promotes activation of p38 MAPK without inhibiting activation of ERK MAPK. In p38alpha knockdown cells, PRMT1 could no longer suppress differentiation. In contrast, enforced expression of p38alpha MAPK suppressed PMA-induced megakaryocytic differentiation of parental K562 as well as PRMT1-knockdown cells. We propose modulation of the p38 MAPK pathway by PRMT1 as a novel mechanism regulating megakaryocytic differentiation. This study thus provides a new perspective on the promotion of megakaryopoiesis.

Identification of the methylation preference region in heterogeneous nuclear ribonucleoprotein K by protein arginine methyltransferase 1 and its implication in regulating nuclear/cytoplasmic distribution.

Protein arginine methylation plays crucial roles in numerous cellular processes. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein participating in a variety of cellular functions including transcription and RNA processing. HnRNP K is methylated at multiple sites in the glycine- and arginine-rich (RGG) motif. Using various RGG domain deletion mutants of hnRNP K as substrates, here we show by direct methylation assay that protein arginine methyltransferase 1 (PRMT1) methylated preferentially in a.a. 280-307 of the RGG motif. Kinetic analysis revealed that deletion of a.a. 280-307, but not a.a. 308-327, significantly inhibited rate of methylation. Importantly, nuclear localization of hnRNP K was significantly impaired in mutant hnRNP K lacking the PRMT1 methylation region or upon pharmacological inhibition of methylation. Together our results identify preferred PRMT1 methylation sequences of hnRNP K by direct methylation assay and implicate a role of arginine methylation in regulating intracellular distribution of hnRNP K.

Targeting miR-126 in inv(16) acute myeloid leukemia inhibits leukemia development and leukemia stem cell maintenance.

Acute myeloid leukemia (AML) harboring inv(16)(p13q22) expresses high levels of miR-126. Here we show that the CBFB-MYH11 (CM) fusion gene upregulates miR-126 expression through aberrant miR-126 transcription and perturbed miR-126 biogenesis via the HDAC8/RAN-XPO5-RCC1 axis. Aberrant miR-126 upregulation promotes survival of leukemia-initiating progenitors and is critical for initiating and maintaining CM-driven AML. We show that miR-126 enhances MYC activity through the SPRED1/PLK2-ERK-MYC axis. Notably, genetic deletion of miR-126 significantly reduces AML rate and extends survival in CM knock-in mice. Therapeutic depletion of miR-126 with an anti-miR-126 (miRisten) inhibits AML cell survival, reduces leukemia burden and leukemia stem cell (LSC) activity in inv(16) AML murine and xenograft models. The combination of miRisten with chemotherapy further enhances the anti-leukemia and anti-LSC activity. Overall, this study provides molecular insights for the mechanism and impact of miR-126 dysregulation in leukemogenesis and highlights the potential of miR-126 depletion as a therapeutic approach for inv(16) AML.

Calcium-dependent methylation by PRMT1 promotes erythroid differentiation through the p38α MAPK pathway.

Protein arginine methyltransferase 1 (PRMT1) stimulates erythroid differentiation, but the signaling events upstream are yet to be identified. Ca2+ plays crucial roles during erythroid differentiation. Here, we show that Ca2+ enhances methylation during induced erythroid differentiation and that Ca2+ directly upregulates the catalytic activity of recombinant PRMT1 by increasing Vmax toward the substrate heterogeneous nuclear ribonucleoprotein A2. We demonstrate that PRMT1 is essential and responsible for the effect of Ca2+ on differentiation. Depletion of Ca2+ suppresses PRMT1-mediated activation of p38α and p38α-stimulated differentiation. Furthermore, Ca2+ stimulates methylation of p38α by PRMT1. This study uncovers a novel regulatory mechanism for PRMT1 by Ca2+ and identifies the PRMT1/p38α axis as an intracellular mediator of Ca2+ signaling during erythroid differentiation.

State-Transition Analysis of Time-Sequential Gene Expression Identifies Critical Points That Predict Development of Acute Myeloid Leukemia.

Temporal dynamics of gene expression inform cellular and molecular perturbations associated with disease development and evolution. Given the complexity of high-dimensional temporal genomic data, an analytic framework guided by a robust theory is needed to interpret time-sequential changes and to predict system dynamics. Here we model temporal dynamics of the transcriptome of peripheral blood mononuclear cells in a two-dimensional state-space representing states of health and leukemia using time-sequential bulk RNA-seq data from a murine model of acute myeloid leukemia (AML). The state-transition model identified critical points that accurately predict AML development and identifies stepwise transcriptomic perturbations that drive leukemia progression. The geometry of the transcriptome state-space provided a biological interpretation of gene dynamics, aligned gene signals that are not synchronized in time across mice, and allowed quantification of gene and pathway contributions to leukemia development. Our state-transition model synthesizes information from multiple cell types in the peripheral blood and identifies critical points in the transition from health to leukemia to guide interpretation of changes in the transcriptome as a whole to predict disease progression. SIGNIFICANCE: These findings apply the theory of state transitions to model the initiation and development of acute myeloid leukemia, identifying transcriptomic perturbations that accurately predict time to disease development.See related commentary by Kuijjer, p. 3072 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/15/3157/F1.large.jpg.

SIRT1 Activation Disrupts Maintenance of Myelodysplastic Syndrome Stem and Progenitor Cells by Restoring TET2 Function.

Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is derived from aberrant clonal hematopoietic stem/progenitor cells (HSPCs) that persist after conventional therapies. Defining the mechanisms underlying MDS HSPC maintenance is critical for developing MDS therapy. The deacetylase SIRT1 regulates stem cell proliferation, survival, and self-renewal by deacetylating downstream proteins. Here we show that SIRT1 protein levels were downregulated in MDS HSPCs. Genetic or pharmacological activation of SIRT1 inhibited MDS HSPC functions, whereas SIRT1 deficiency enhanced MDS HSPC self-renewal. Mechanistically, the inhibitory effects of SIRT1 were dependent on TET2, a safeguard against HSPC transformation. SIRT1 deacetylated TET2 at conserved lysine residues in its catalytic domain, enhancing TET2 activity. Our genome-wide analysis identified cancer-related genes regulated by the SIRT1/TET2 axis. SIRT1 activation also inhibited functions of MDS HSPCs from patients with TET2 heterozygous mutations. Altogether, our results indicate that restoring TET2 function through SIRT1 activation represents a promising means to target MDS HSPCs.

HDAC8 Inhibition Specifically Targets Inv(16) Acute Myeloid Leukemic Stem Cells by Restoring p53 Acetylation.

Acute myeloid leukemia (AML) is driven and sustained by leukemia stem cells (LSCs) with unlimited self-renewal capacity and resistance to chemotherapy. Mutation in the TP53 tumor suppressor is relatively rare in de novo AML; however, p53 can be regulated through post-translational mechanisms. Here, we show that p53 activity is inhibited in inv(16)(+) AML LSCs via interactions with the CBFß-SMMHC (CM) fusion protein and histone deacetylase 8 (HDAC8). HDAC8 aberrantly deacetylates p53 and promotes LSC transformation and maintenance. HDAC8 deficiency or inhibition using HDAC8-selective inhibitors (HDAC8i) effectively restores p53 acetylation and activity. Importantly, HDAC8 inhibition induces apoptosis in inv(16)(+) AML CD34(+) cells, while sparing the normal hematopoietic stem cells. Furthermore, in vivo HDAC8i administration profoundly diminishes AML propagation and abrogates leukemia-initiating capacity of both murine and patient-derived LSCs. This study elucidates an HDAC8-mediated p53-inactivating mechanism promoting LSC activity and highlights HDAC8 inhibition as a promising approach to selectively target inv(16)(+) LSCs.

Gene Expression Profiling and Pathway Network Analysis Predicts a Novel Antitumor Function for a Botanical-Derived Drug, PG2.

PG2 is a botanical drug that is mostly composed of Astragalus polysaccharides (APS). Its role in hematopoiesis and relieving cancer-related fatigue has recently been clinically investigated in cancer patients. However, systematic analyses of its functions are still limited. The aim of this study was to use microarray-based expression profiling to evaluate the quality and consistency of PG2 from three different product batches and to study biological mechanisms of PG2. An integrative molecular analysis approach has been designed to examine significant PG2-induced signatures in HL-60 leukemia cells. A quantitative analysis of gene expression signatures was conducted for PG2 by hierarchical clustering of correlation coefficients. The results showed that PG2 product batches were consistent and of high quality. These batches were also functionally equivalent to each other with regard to how they modulated the immune and hematopoietic systems. Within the PG2 signature, there were five genes associated with doxorubicin: IL-8, MDM4, BCL2, PRODH2, and BIRC5. Moreover, the combination of PG2 and doxorubicin had a synergistic effect on induced cell death in HL-60 cells. Together with the bioinformatics-based approach, gene expression profiling provided a quantitative measurement for the quality and consistency of herbal medicines and revealed new roles (e.g., immune modulation) for PG2 in cancer treatment.

Protein arginine methyltransferase 1 interacts with and activates p38α to facilitate erythroid differentiation.

Protein arginine methylation is emerging as a pivotal posttranslational modification involved in regulating various cellular processes; however, its role in erythropoiesis is still elusive. Erythropoiesis generates circulating red blood cells which are vital for body activity. Deficiency in erythroid differentiation causes anemia which compromises the quality of life. Despite extensive studies, the molecular events regulating erythropoiesis are not fully understood. This study showed that the increase in protein arginine methyltransferase 1 (PRMT1) levels, via transfection or protein transduction, significantly promoted erythroid differentiation in the bipotent human K562 cell line as well as in human primary hematopoietic progenitor CD34(+) cells. PRMT1 expression enhanced the production of hemoglobin and the erythroid surface marker glycophorin A, and also up-regulated several key transcription factors, GATA1, NF-E2 and EKLF, which are critical for lineage-specific differentiation. The shRNA-mediated knockdown of PRMT1 suppressed erythroid differentiation. The methyltransferase activity-deficient PRMT1G80R mutant failed to stimulate differentiation, indicating the requirement of arginine methylation of target proteins. Our results further showed that a specific isoform of p38 MAPK, p38α, promoted erythroid differentiation, whereas p38ß did not play a role. The stimulation of erythroid differentiation by PRMT1 was diminished in p38α- but not p38ß-knockdown cells. PRMT1 appeared to act upstream of p38α, since expression of p38α still promoted erythroid differentiation in PRMT1-knockdown cells, and expression of PRMT1 enhanced the activation of p38 MAPK. Importantly, we showed for the first time that PRMT1 was associated with p38α in cells by co-immunoprecipitation and that PRMT1 directly methylated p38α in in vitro methylation assays. Taken together, our findings unveil a link between PRMT1 and p38α in regulating the erythroid differentiation program and provide evidence suggesting a novel regulatory mechanism for p38α through arginine methylation.