Chem-CRISPR/dCas9FCPF: a platform for chemically induced epigenome editing.

Epigenetic aberration is one of the major driving factors in human cancer, often leading to acquired resistance to chemotherapies. Various small molecule epigenetic modulators have been reported. Nonetheless, outcomes from animal models and clinical trials have underscored the substantial setbacks attributed to pronounced on- and off-target toxicities. To address these challenges, CRISPR/dCas9 technology is emerging as a potent tool for precise modulation of epigenetic mechanism. However, this technology involves co-expressing exogenous epigenetic modulator proteins, which presents technical challenges in preparation and delivery with potential undesirable side effects. Recently, our research demonstrated that Cas9 tagged with the Phe-Cys-Pro-Phe (FCPF)-peptide motif can be specifically targeted by perfluorobiphenyl (PFB) derivatives. Here, we integrated the FCPF-tag into dCas9 and established a chemically inducible platform for epigenome editing, called Chem-CRISPR/dCas9FCPF. We designed a series of chemical inhibitor-PFB conjugates targeting various epigenetic modulator proteins. Focusing on JQ1, a panBET inhibitor, we demonstrate that c-MYC-sgRNA-guided JQ1-PFB specifically inhibits BRD4 in close proximity to the c-MYC promoter/enhancer, thereby effectively repressing the intricate transcription networks orchestrated by c-MYC as compared with JQ1 alone. In conclusion, our Chem-CRISPR/dCas9FCPF platform significantly increased target specificity of chemical epigenetic inhibitors, offering a viable alternative to conventional fusion protein systems for epigenome editing.

Culture expansion of CAR T cells results in aberrant DNA methylation that is associated with adverse clinical outcome.

Chimeric antigen receptor (CAR) T cells provide new perspectives for treatment of hematological malignancies. Manufacturing of these cellular products includes culture expansion procedures, which may affect cellular integrity and therapeutic outcome. In this study, we investigated culture-associated epigenetic changes in CAR T cells and found continuous gain of DNAm, particularly within genes that are relevant for T cell function. Hypermethylation in many genes, such as TCF7, RUNX1, and TOX, was reflected by transcriptional downregulation. 332 CG dinucleotides (CpGs) showed an almost linear gain in methylation with cell culture time, albeit neighboring CpGs were not coherently regulated on the same DNA strands. An epigenetic signature based on 14 of these culture-associated CpGs predicted cell culture time across various culture conditions. Notably, even in CAR T cell products of similar culture time higher DNAm levels at these CpGs were associated with significantly reduced long-term survival post transfusion. Our data demonstrate that cell culture expansion of CAR T cells evokes DNA hypermethylation at specific sites in the genome and the signature may also reflect loss of potential in CAR T cell products. Hence, reduced cultivation periods are beneficial to avoid dysfunctional methylation programs that seem to be associated with worse therapeutic outcome.

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

Anti-CD19 CARs displayed at the surface of lentiviral vector particles promote transduction of target-expressing cells.

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.

A Novel Siglec-4 Derived Spacer Improves the Functionality of CAR T Cells Against Membrane-Proximal Epitopes.

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.

Modeling the process of childhood ETV6-RUNX1 B-cell leukemias.

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. Pre-leukaemic clones carrying ETV6-RUNX1 oncogenic lesions are frequently found in neonatal cord blood, but only few ETV6-RUNX1 carriers develop pB-ALL. The highly demanding and pending challenge is to reveal the multistep natural history of ETV6-RUNX1 pB-ALL, because it can offer non-toxic prophylactic interventions to preleukemic carriers. However, the lack of a genetically engineered ETV6-RUNX1 mouse model mimicking the human pB-ALL has hampered our understanding of the pathogenesis of this disease. This rule has now been broken in a study of the effect of the ETV6-RUNX1 oncogene in cancer development in a mouse model in which oncogene expression is restricted to the stem cell compartment. In this article, we review the different attempts to model this disease, including the recent representative success stories and we discuss its potential application to both identify etiologic factors of childhood ETV6-RUNX1 pB-ALL and prevent the conversion of a preleukemic clone in an irreversible transformed state.

Infection Exposure Promotes ETV6-RUNX1 Precursor B-cell Leukemia via Impaired H3K4 Demethylases.

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches. Cancer Res; 77(16); 4365-77. ©2017 AACR.

Metformin-Derived Growth Inhibition in Renal Cell Carcinoma Depends on miR-21-Mediated PTEN Expression.

PURPOSE: Metformin (MF) acts as a tumour-suppressor in renal cell carcinoma (RCC) by inhibiting the AKT/mTOR pathway via AMPK activation. Here, we explore the influence of miR-21 and its target gene PTEN on MF effects in CAKI-1 and CAKI-2 cells. METHODS: Proliferation assays (MTS) and qRT-PCR after transient transfection with pre- and anti-miR-21 and MF treatment were conducted. AMPK-dependency was assessed via transfection of siAMPK. The expression of PTEN, AKT and miR-21 after transient pre-miR-21 transfection and MF treatment was analysed. RESULTS: We demonstrate that CAKI-1 cells, which were found to be less sensitive towards MF, showed a significant higher miR-21 and lower PTEN expression than CAKI-2. This was confirmed in a primary RCC collective (n = 28): miR-21 and PTEN expression correlated negatively. MF treatment lowered miR-21 AMPK-dependently and increased PTEN expression in the cell lines. Ectopic miR-21 regulation modulated MF sensitivity. Western blot analysis showed that pre-miR-21 transfection and MF treatment regulated PTEN expression with impact on pAKT levels in the cells. CONCLUSIONS: We show that differing MF sensitivity in RCC cells is associated with and mediated through the regulation of miR-21/PTEN expression with an impact on subsequent AKT signalling. This provides imaginable clinical implications regarding MF therapy of RCC patients for the future.

Infection Exposure is a Causal Factor in B-cell Precursor Acute Lymphoblastic Leukemia as a Result of Pax5-Inherited Susceptibility.

UNLABELLED: Earlier in the past century, infections were regarded as the most likely cause of childhood B-cell precursor acute lymphoblastic leukemia (pB-ALL). However, there is a lack of relevant biologic evidence supporting this hypothesis. We present in vivo genetic evidence mechanistically connecting inherited susceptibility to pB-ALL and postnatal infections by showing that pB-ALL was initiated in Pax5 heterozygous mice only when they were exposed to common pathogens. Strikingly, these murine pB-ALLs closely resemble the human disease. Tumor exome sequencing revealed activating somatic, nonsynonymous mutations of Jak3 as a second hit. Transplantation experiments and deep sequencing suggest that inactivating mutations in Pax5 promote leukemogenesis by creating an aberrant progenitor compartment that is susceptible to malignant transformation through accumulation of secondary Jak3 mutations. Thus, treatment of Pax5(+/-) leukemic cells with specific JAK1/3 inhibitors resulted in increased apoptosis. These results uncover the causal role of infection in pB-ALL development. SIGNIFICANCE: These results demonstrate that delayed infection exposure is a causal factor in pB-ALL. Therefore, these findings have critical implications for the understanding of the pathogenesis of leukemia and for the development of novel therapies for this disease.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Cytoplasmic replication of Staphylococcus aureus upon phagosomal escape triggered by phenol-soluble modulin α.

Staphylococcus aureus is a Gram-positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non-professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of the innate immune system and may also lead to dissemination within migrating phagocytes. Further, S. aureus efficiently lyses host cells with a battery of cytolytic toxins. Recently, phenol-soluble modulins (PSM) have been identified to comprise a genus-specific family of cytolytic peptides. Of these the PSMα peptides have been implicated in killing polymorphonuclear leucocytes after phagocytosis. We questioned if the peptides were active in destroying endosomal membranes to avoid lysosomal killing of the pathogen and monitored integrity of infected host cell endosomes by measuring the acidity of the intracellular bacterial microenvironment via flow cytometry and by a reporter recruitment technique. Isogenic mutants of the methicillin-resistant S. aureus (MRSA) strains USA300 LAC, USA400 MW2 as well as the strongly cytolytic methicillin-sensitive strain 6850 were compared with their respective wild type strains. In all three genetic backgrounds, PSMα mutants were unable to escape from phagosomes in non-professional (293, HeLa, EAhy.926) and professional phagocytes (THP-1), whereas mutants in PSMß and δ-toxin as well as ß-toxin, phosphatidyl inositol-dependent phospholipase C and Panton Valentine leucotoxin escaped with efficiencies of the parental strains. S. aureus replicated intracellularly only in presence of a functional PSMα operon thereby illustrating that bacteria grow in the host cell cytoplasm upon phagosomal escape.

True versus Pseudo-Intestinal Malrotation: Case Series and Review.

Intestinal malrotation is an anomaly of fetal intestinal rotation that can present with symptoms after birth or in early childhood, but is rarely diagnosed in adults. Patients who have symptomatic presentations require surgery. Other entities may mimic intestinal malrotation and respond to non-surgical management. We present 2 adult cases with the radiological diagnosis of intestinal malrotation: one with true malrotation presenting as a duodenal mass, and another with "pseudo-malrotation" due to altered anatomy. These cases illustrate the importance of recognizing and differentiating these rare adult presentations of true malrotation from "pseudo-malrotation" in regards to their acute management.

Phagolysosomal integrity is generally maintained after Staphylococcus aureus invasion of nonprofessional phagocytes but is modulated by strain 6850.

Staphylococcus aureus is a major cause of a variety of both local and systemic infections. It can invade human host cells, a process that may account for disseminated and recurrent infections. S. aureus postinvasion events in nonprofessional phagocytes are only partially understood. While morphological data suggest a phagosomal escape, there is a lack of corroborating functional data. Using a combination of pH determination and morphological techniques, we have tested the integrity of Staphylococcus-containing phagosomes in 293 (HEK-293), HeLa, and EA.hy926 cells over time. Rapid acidification of S. aureus-containing phagosomes occurred and was sustained for up to 24 h. All S. aureus strains tested displayed equally sustained intraphagosomal pH levels without exhibiting any correlation with pH level and hemolytic activity. The membrane morphology of the phagosomal compartment was heterogeneous, even under conditions where acidic pH was fully maintained, an observation incompatible with phagolysosomal membrane destruction. As an exception, S. aureus strain 6850 showed a reduced phagosomal acidification signal 6 h after invasion. Additionally, only strain 6850 failed to localize to LAMP-1-positive vesicles in HeLa cells, although this was observed only rarely. Several other strongly beta-hemolytic strains did not modulate phagolysosomal pH, suggesting that S. aureus alpha-toxin and beta-toxin are not sufficient for this process. Taken together, our data suggest that S. aureus-containing phagolysosomes generally remain functionally intact in nonprofessional phagocytes, thereby contrasting with transmission electron micrographic results.

Some statistical implications of dose uncertainty in radiation dose-response analyses.

Statistical dose-response analyses in radiation epidemiology can produce misleading results if they fail to account for radiation dose uncertainties. While dosimetries may differ substantially depending on the ways in which the subjects were exposed, the statistical problems typically involve a predominantly linear dose-response curve, multiple sources of uncertainty, and uncertainty magnitudes that are best characterized as proportional rather than additive. We discuss some basic statistical issues in this setting, including the bias and shape distortion induced by classical and Berkson uncertainties, the effect of uncertain dose-prediction model parameters on estimated dose-response curves, and some notes on statistical methods for dose-response estimation in the presence of radiation dose uncertainties.

A reanalysis of thyroid neoplasms in the Israeli tinea capitis study accounting for dose uncertainties.

In the 1940s and 1950s, children in Israel were treated for tinea capitis by irradiation to the scalp to induce epilation. Follow-up studies of these patients and of other radiation- exposed populations show an increased risk of malignant and benign thyroid tumors. Those analyses, however, assume that thyroid dose for individuals is estimated precisely without error. Failure to account for uncertainties in dosimetry may affect standard errors and bias dose-response estimates. For the Israeli tinea capitis study, we discuss sources of uncertainties and adjust dosimetry for uncertainties in the prediction of true dose from X-ray treatment parameters. We also account for missing ages at exposure for patients with multiple X-ray treatments, since only ages at first treatment are known, and for missing data on treatment center, which investigators use to define exposure. Our reanalysis of the dose response for thyroid cancer and benign thyroid tumors indicates that uncertainties in dosimetry have minimal effects on dose-response estimation and for inference on the modifying effects of age at first exposure, time since exposure, and other factors. Since the components of the dose uncertainties we describe are likely to be present in other epidemiological studies of patients treated with radiation, our analysis may provide a model for considering the potential role of these uncertainties.