PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in human and murine acute myeloid leukemia.

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

CLEAR: coverage-based limiting-cell experiment analysis for RNA-seq.

BACKGROUND: Direct cDNA preamplification protocols developed for single-cell RNA-seq have enabled transcriptome profiling of precious clinical samples and rare cell populations without the need for sample pooling or RNA extraction. We term the use of single-cell chemistries for sequencing low numbers of cells limiting-cell RNA-seq (lcRNA-seq). Currently, there is no customized algorithm to select robust/low-noise transcripts from lcRNA-seq data for between-group comparisons. METHODS: Herein, we present CLEAR, a workflow that identifies reliably quantifiable transcripts in lcRNA-seq data for differentially expressed genes (DEG) analysis. Total RNA obtained from primary chronic lymphocytic leukemia (CLL) CD5+ and CD5- cells were used to develop the CLEAR algorithm. Once established, the performance of CLEAR was evaluated with FACS-sorted cells enriched from mouse Dentate Gyrus (DG). RESULTS: When using CLEAR transcripts vs. using all transcripts in CLL samples, downstream analyses revealed a higher proportion of shared transcripts across three input amounts and improved principal component analysis (PCA) separation of the two cell types. In mouse DG samples, CLEAR identifies noisy transcripts and their removal improves PCA separation of the anticipated cell populations. In addition, CLEAR was applied to two publicly-available datasets to demonstrate its utility in lcRNA-seq data from other institutions. If imputation is applied to limit the effect of missing data points, CLEAR can also be used in large clinical trials and in single cell studies. CONCLUSIONS: lcRNA-seq coupled with CLEAR is widely used in our institution for profiling immune cells (circulating or tissue-infiltrating) for its transcript preservation characteristics. CLEAR fills an important niche in pre-processing lcRNA-seq data to facilitate transcriptome profiling and DEG analysis. We demonstrate the utility of CLEAR in analyzing rare cell populations in clinical samples and in murine neural DG region without sample pooling.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ß is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit ß and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

PERSIST platform provides programmable RNA regulation using CRISPR endoRNases.

Regulated transgene expression is an integral component of gene therapies, cell therapies and biomanufacturing. However, transcription factor-based regulation, upon which most applications are based, suffers from complications such as epigenetic silencing that limit expression longevity and reliability. Constitutive transgene transcription paired with post-transcriptional gene regulation could combat silencing, but few such RNA- or protein-level platforms exist. Here we develop an RNA-regulation platform we call "PERSIST" which consists of nine CRISPR-specific endoRNases as RNA-level activators and repressors as well as modular OFF- and ON-switch regulatory motifs. We show that PERSIST-regulated transgenes exhibit strong OFF and ON responses, resist silencing for at least two months, and can be readily layered to construct cascades, logic functions, switches and other sophisticated circuit topologies. The orthogonal, modular and composable nature of this platform as well as the ease in constructing robust and predictable gene circuits promises myriad applications in gene and cell therapies.

Leukemia-initiating HSCs in chronic lymphocytic leukemia reveal clonal leukemogenesis and differential drug sensitivity.

The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2high CLL-HSCs that exhibit increased sensitivity to lenalidomide and resistance to fludarabine compared to GATA2lowCLL-HSCs. These results highlight the existence of therapeutically targetable disease precursors in CLL.

NF-κB perturbation reveals unique immunomodulatory functions in Prx1+ fibroblasts that promote development of atopic dermatitis.

Skin is composed of diverse cell populations that cooperatively maintain homeostasis. Up-regulation of the nuclear factor κB (NF-κB) pathway may lead to the development of chronic inflammatory disorders of the skin, but its role during the early events remains unclear. Through analysis of single-cell RNA sequencing data via iterative random forest leave one out prediction, an explainable artificial intelligence method, we identified an immunoregulatory role for a unique paired related homeobox-1 (Prx1)+ fibroblast subpopulation. Disruption of Ikkb-NF-κB under homeostatic conditions in these fibroblasts paradoxically induced skin inflammation due to the overexpression of C-C motif chemokine ligand 11 (CCL11; or eotaxin-1) characterized by eosinophil infiltration and a subsequent TH2 immune response. Because the inflammatory phenotype resembled that seen in human atopic dermatitis (AD), we examined human AD skin samples and found that human AD fibroblasts also overexpressed CCL11 and that perturbation of Ikkb-NF-κB in primary human dermal fibroblasts up-regulated CCL11. Monoclonal antibody treatment against CCL11 was effective in reducing the eosinophilia and TH2 inflammation in a mouse model. Together, the murine model and human AD specimens point to dysregulated Prx1+ fibroblasts as a previously unrecognized etiologic factor that may contribute to the pathogenesis of AD and suggest that targeting CCL11 may be a way to treat AD-like skin lesions.

Targeted degradation of the enhancer lysine acetyltransferases CBP and p300.

The enhancer factors CREB-binding protein (CBP) and p300 (also known as KAT3A and KAT3B) maintain gene expression programs through lysine acetylation of chromatin and transcriptional regulators and by scaffolding functions mediated by several protein-protein interaction domains. Small molecule inhibitors that target some of these domains have been developed; however, they cannot completely ablate p300/CBP function in cells. Here we describe a chemical degrader of p300/CBP, dCBP-1. Leveraging structures of ligand-bound p300/CBP domains, we use in silico modeling of ternary complex formation with the E3 ubiquitin ligase cereblon to enable degrader design. dCBP-1 is exceptionally potent at killing multiple myeloma cells and can abolish the enhancer that drives MYC oncogene expression. As an efficient degrader of this unique class of acetyltransferases, dCBP-1 is a useful tool alongside domain inhibitors for dissecting the mechanism by which these factors coordinate enhancer activity in normal and diseased cells.

The ROR1 antibody-drug conjugate huXBR1-402-G5-PNU effectively targets ROR1+ leukemia.

Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody-based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.

Genomic analysis of cellular hierarchy in acute myeloid leukemia using ultrasensitive LC-FACSeq.

Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.

Mobile apps for professional dermatology education: an objective review.

Mobile dermatology applications (apps) created for the purpose of educating students and trainees present convenient supplemental learning opportunities. Before these apps can be widely utilized, there must be a method to assess educational objectives, quality, comprehensiveness of content, evidence-based accuracy, user-friendly design, and potential for bias. Herein, an established rubric was used to conduct a graded review of apps spanning general dermatology, skin cancer, and cosmetics, with an additional emphasis on affordability and accessibility for the user.

LC-FACSeq is a method for detecting rare clones in leukemia.

Detecting, characterizing, and monitoring rare populations of cells can increase testing sensitivity, give insight into disease mechanism, and inform clinical decision making. One area that can benefit from increased resolution is management of cancers in clinical remission but with measurable residual disease (MRD) by multicolor FACS. Detecting and monitoring genomic clonal resistance to treatment in the setting of MRD is technically difficult and resource intensive due to the limited amounts of disease cells. Here, we describe limited-cell FACS sequencing (LC-FACSeq), a reproducible, highly sensitive method of characterizing clonal evolution in rare cells relevant to different types of acute and chronic leukemias. We demonstrate the utility of LC-FACSeq for broad multigene gene panels and its application for monitoring sequential acquisition of mutations conferring therapy resistance and clonal evolution in long-term ibrutinib treatment of patients with chronic lymphocytic leukemia. This technique is generalizable for monitoring of other blood and marrow infiltrating cancers.

Metabolic versatility in a modern lineage of cyanobacteria from terrestrial hot springs.

The extent of oxygenated environments on the early Earth was much lower than today, and cyanobacteria were critical players in Earth's shift from widespread anoxia to oxygenated surface environments. Extant cyanobacteria that aggregate into cones, tufts and ridges are used to understand the long record of photosynthesis and microbe-mineral interactions during times when oxygen was much lower, i.e., the Archean and the Proterozoic. To better understand the metabolic versatility and physiological properties of these organisms, we examined publicly available genomes of cyanobacteria from modern terrestrial hydrothermal systems and a newly sequenced genome of a cyanobacterium isolated from conical and ridged microbialites that grow in occasionally sulfidic hydrothermal springs in Yellowstone National Park, USA. Phylogenomic analyses reveal that cyanobacteria from globally distributed terrestrial and shallow marine hydrothermal systems form a monophyletic clade within the Cyanobacteria phylum. Comparative genomics of this clade reveals the genetic capacity for oxygenic photosynthesis that uses photosystems I and II, and anoxygenic photosynthesis that uses a putative sulfide quinone reductase to oxidize sulfide and bypass photosystem II. Surprisingly large proportions of the newly sequenced genome from Yellowstone National Park are also dedicated to secondary metabolite production (15.1-15.6%), of which ∼6% can be attributed to antibiotic production and resistance genes. All this may be advantageous to benthic, mat-forming photosynthesizers that have to compete for light and nutrients in sporadically or permanently sulfidic environments, and may have also improved the tolerance of ancient counterparts of these cyanobacteria to sulfidic conditions in benthic communities that colonized the coastal margins in the Archean and the Proterozoic.

PI3K p110δ inactivation antagonizes chronic lymphocytic leukemia and reverses T cell immune suppression.

Targeted therapy with small molecules directed at essential survival pathways in leukemia represents a major advance, including the phosphatidylinositol-3'-kinase (PI3K) p110δ inhibitor idelalisib. Here, we found that genetic inactivation of p110δ (p110δD910A/D910A) in the Eµ-TCL1 murine chronic lymphocytic leukemia (CLL) model impaired B cell receptor signaling and B cell migration, and significantly delayed leukemia pathogenesis. Regardless of TCL1 expression, p110δ inactivation led to rectal prolapse in mice resembling autoimmune colitis in patients receiving idelalisib. Moreover, we showed that p110δ inactivation in the microenvironment protected against CLL and acute myeloid leukemia. After receiving higher numbers of TCL1 leukemia cells, half of p110δD910A/D910A mice spontaneously recovered from high disease burden and resisted leukemia rechallenge. Despite disease resistance, p110δD910A/D910A mice exhibited compromised CD4+ and CD8+ T cell response, and depletion of CD4+ or CD8+ T cells restored leukemia. Interestingly, p110δD910A/D910A mice showed significantly impaired Treg expansion that associated with disease clearance. Reconstitution of p110δD910A/D910A mice with p110δWT/WT Tregs reversed leukemia resistance. Our findings suggest that p110δ inhibitors may have direct antileukemic and indirect immune-activating effects, further supporting that p110δ blockade may have a broader immune-modulatory role in types of leukemia that are not sensitive to p110δ inhibition.

The Role of an Integrated Multidisciplinary Clinic in the Management of Patients with Cutaneous Lymphoma.

The clinical benefit of a multidisciplinary clinic practice model has been well described in a variety of medical specialties and cancer types. It proves particularly valuable when an integrated team is needed to optimally manage patients with rare or complex neoplasms. However, the ideal implementation of an integrated multidisciplinary care program for translational research and education has not been well reported. Herein, we propose how a multimodality cutaneous lymphoma (CL) clinic model can optimally manage CL patients. We offer our perspective on this model as an efficient means for delivering patient care, a continuing education resource for referring physicians, a conduit for translational and clinical research, and an educational tool for medical students, house staff, and fellows.