Angiokeratoma-like purpuric palmar nodules following chemotherapy.

We describe a patient with leukemia undergoing chemotherapy who developed painful purpuric nodules of the digits. These findings were concerning for endocarditis (clinically) and angiokeratomas on gross histology. After extensive evaluation, we report the development of painful purpuric nodules as a likely side effect of the patient's therapeutic regimen (hydroxyurea, danorubicin, cytarabine, and methotrexate).

Variability within rare cell states enables multiple paths toward drug resistance.

Molecular differences between individual cells can lead to dramatic differences in cell fate, such as death versus survival of cancer cells upon drug treatment. These originating differences remain largely hidden due to difficulties in determining precisely what variable molecular features lead to which cellular fates. Thus, we developed Rewind, a methodology that combines genetic barcoding with RNA fluorescence in situ hybridization to directly capture rare cells that give rise to cellular behaviors of interest. Applying Rewind to BRAFV600E melanoma, we trace drug-resistant cell fates back to single-cell gene expression differences in their drug-naive precursors (initial frequency of ~1:1,000-1:10,000 cells) and relative persistence of MAP kinase signaling soon after drug treatment. Within this rare subpopulation, we uncover a rich substructure in which molecular differences among several distinct subpopulations predict future differences in phenotypic behavior, such as proliferative capacity of distinct resistant clones after drug treatment. Our results reveal hidden, rare-cell variability that underlies a range of latent phenotypic outcomes upon drug exposure.

Genetic screening for single-cell variability modulators driving therapy resistance.

Cellular plasticity describes the ability of cells to transition from one set of phenotypes to another. In melanoma, transient fluctuations in the molecular state of tumor cells mark the formation of rare cells primed to survive BRAF inhibition and reprogram into a stably drug-resistant fate. However, the biological processes governing cellular priming remain unknown. We used CRISPR-Cas9 genetic screens to identify genes that affect cell fate decisions by altering cellular plasticity. We found that many factors can independently affect cellular priming and fate decisions. We discovered a new plasticity-based mode of increasing resistance to BRAF inhibition that pushes cells towards a more differentiated state. Manipulating cellular plasticity through inhibition of DOT1L before the addition of the BRAF inhibitor resulted in more therapy resistance than concurrent administration. Our results indicate that modulating cellular plasticity can alter cell fate decisions and may prove useful for treating drug resistance in other cancers.

Corrigendum: Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance.

This corrects the article DOI: 10.1038/nature22794.

Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance.

Therapies that target signalling molecules that are mutated in cancers can often have substantial short-term effects, but the emergence of resistant cancer cells is a major barrier to full cures. Resistance can result from secondary mutations, but in other cases there is no clear genetic cause, raising the possibility of non-genetic rare cell variability. Here we show that human melanoma cells can display profound transcriptional variability at the single-cell level that predicts which cells will ultimately resist drug treatment. This variability involves infrequent, semi-coordinated transcription of a number of resistance markers at high levels in a very small percentage of cells. The addition of drug then induces epigenetic reprogramming in these cells, converting the transient transcriptional state to a stably resistant state. This reprogramming begins with a loss of SOX10-mediated differentiation followed by activation of new signalling pathways, partially mediated by the activity of the transcription factors JUN and/or AP-1 and TEAD. Our work reveals the multistage nature of the acquisition of drug resistance and provides a framework for understanding resistance dynamics in single cells. We find that other cell types also exhibit sporadic expression of many of these same marker genes, suggesting the existence of a general program in which expression is displayed in rare subpopulations of cells.

Novel Gene Expression Profile of Women with Intrinsic Skin Youthfulness by Whole Transcriptome Sequencing.

While much is known about genes that promote aging, little is known about genes that protect against or prevent aging, particularly in human skin. The main objective of this study was to perform an unbiased, whole transcriptome search for genes that associate with intrinsic skin youthfulness. To accomplish this, healthy women (n = 122) of European descent, ages 18-89 years with Fitzpatrick skin type I/II were examined for facial skin aging parameters and clinical covariates, including smoking and ultraviolet exposure. Skin youthfulness was defined as the top 10% of individuals whose assessed skin aging features were most discrepant with their chronological ages. Skin biopsies from sun-protected inner arm were subjected to 3'-end sequencing for expression quantification, with results verified by quantitative reverse transcriptase-polymerase chain reaction. Unbiased clustering revealed gene expression signatures characteristic of older women with skin youthfulness (n = 12) compared to older women without skin youthfulness (n = 33), after accounting for gene expression changes associated with chronological age alone. Gene set analysis was performed using Genomica open-access software. This study identified a novel set of candidate skin youthfulness genes demonstrating differences between SY and non-SY group, including pleckstrin homology like domain family A member 1 (PHLDA1) (p = 2.4x10-5), a follicle stem cell marker, and hyaluronan synthase 2-anti-sense 1 (HAS2-AS1) (p = 0.00105), a non-coding RNA that is part of the hyaluronan synthesis pathway. We show that immunologic gene sets are the most significantly altered in skin youthfulness (with the most significant gene set p = 2.4x10-5), suggesting the immune system plays an important role in skin youthfulness, a finding that has not previously been recognized. These results are a valuable resource from which multiple future studies may be undertaken to better understand the mechanisms that promote skin youthfulness in humans.

Structural imprints in vivo decode RNA regulatory mechanisms.

Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.