A comprehensive single-cell breast tumor atlas defines epithelial and immune heterogeneity and interactions predicting anti-PD-1 therapy response.

We present an integrated single-cell RNA sequencing atlas of the primary breast tumor microenvironment (TME) containing 236,363 cells from 119 biopsy samples across eight datasets. In this study, we leverage this resource for multiple analyses of immune and cancer epithelial cell heterogeneity. We define natural killer (NK) cell heterogeneity through six subsets in the breast TME. Because NK cell heterogeneity correlates with epithelial cell heterogeneity, we characterize epithelial cells at the level of single-gene expression, molecular subtype, and 10 categories reflecting intratumoral transcriptional heterogeneity. We develop InteractPrint, which considers how cancer epithelial cell heterogeneity influences cancer-immune interactions. We use T cell InteractPrint to predict response to immune checkpoint inhibition (ICI) in two breast cancer clinical trials testing neoadjuvant anti-PD-1 therapy. T cell InteractPrint was predictive of response in both trials versus PD-L1 (AUC = 0.82, 0.83 vs. 0.50, 0.72). This resource enables additional high-resolution investigations of the breast TME.

Arthroscopic Surgery Versus Nonoperative Treatment for Calcific Tendinitis of the Shoulder: A Retrospective Cohort Study.

BACKGROUND: Treatment options for calcific tendinitis (CT) of the shoulder remain controversial. A consensus for an operative indication for this condition is lacking. PURPOSE: To compare nonoperative versus operative treatment for shoulder CT and analyze factors affecting the prognosis after treatment. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A total of 180 patients diagnosed with symptomatic CT between January 2017 and September 2021 were evaluated in this retrospective cohort study. There were 103 patients treated nonoperatively at our institution, which included the use of nonsteroidal anti-inflammatory drugs, acupuncture, steroid injections, extracorporeal shock wave therapy, and ultrasound-guided needle aspiration/percutaneous irrigation. However, 77 patients were treated with arthroscopic surgery after 6 months of failed nonoperative treatment. The visual analog scale (VAS) for pain, the Constant-Murley score, and imaging were used to assess and evaluate outcomes. Descriptive data, functional outcomes, and imaging findings were compared between the operative and nonoperative groups before and after propensity score matching. Additionally, prognostic factors including calcium deposit size, tendon infiltration by calcium deposits, involvement of single or multiple tendons, and occurrence of rotator cuff tears were analyzed by comparing the groups to determine their effect on treatment options and recovery. RESULTS: Magnetic resonance imaging showed that the supraspinatus tendon (66.7%) was most commonly involved, followed by the infraspinatus (42.8%) and subscapularis (21.1%) tendons. Tendon infiltration by calcium deposits was observed in 84.4% of the patients, and rotator cuff tears occurred in 30.0% of the patients. After propensity score matching, there was no significant difference in changes in the Constant-Murley score (48.1 ± 25.4 vs 49.0 ± 22.8, respectively; P = .950) and VAS score (4.9 ± 2.3 vs 4.5 ± 1.9, respectively; P = .860) between the operative and nonoperative groups at the final follow-up. However, for patients with shoulder CT and without rotator cuff tears, there was a significant difference in changes in the Constant-Murley score (52.93 ± 25.18 vs 42.13 ± 22.35, respectively; P = .012) and VAS score (5.21 ± 2.06 vs 3.81 ± 1.98, respectively; P < .001) between the operative and nonoperative groups, but the recovery time in the operative group was longer than that in the nonoperative group (86.92 ± 138.56 vs 30.42 ± 54.97 days, respectively; P = .016). The results also showed that calcium deposit size, involvement of multiple tendons, and tendon infiltration by calcium deposits did not affect the recovery time after treatment. The survival analysis showed that rotator cuff tears affected the complete recovery of shoulder function. CONCLUSION: This study demonstrated no significant difference between nonoperative and operative treatment for patients with shoulder CT, on the whole. However, for patients with shoulder CT and without rotator cuff tears, the effect of operative treatment was better than that of nonoperative treatment; yet, operative treatment was shown to prolong the recovery time. Calcium deposit size, tendon infiltration by calcium deposits, and involvement of multiple tendons did not correlate with recovery time or the recovery of function. A rotator cuff tear was the only factor affecting the complete recovery of shoulder function.

CD73 contributes to the pathogenesis of fusion-negative rhabdomyosarcoma through the purinergic signaling pathway.

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and adolescents. Fusion-negative RMS (FN-RMS) accounts for more than 80% of all RMS cases. The long-term event-free survival rate for patients with high-grade FN-RMS is below 30%, highlighting the need for improved therapeutic strategies. CD73 is a 5' ectonucleotidase that hydrolyzes AMP to adenosine and regulates the purinergic signaling pathway. We found that CD73 is elevated in FN-RMS tumors that express high levels of TWIST2. While high expression of CD73 contributes to the pathogenesis of multiple cancers, its role in FN-RMS has not been investigated. We found that CD73 knockdown decreased FN-RMS cell growth while up-regulating the myogenic differentiation program. Moreover, mutation of the catalytic residues of CD73 rendered the protein enzymatically inactive and abolished its ability to stimulate FN-RMS growth. Overexpression of wildtype CD73, but not the catalytically inactive mutant, in CD73 knockdown FN-RMS cells restored their growth capacity. Likewise, treatment with an adenosine receptor A2A-B agonist partially rescued FN-RMS cell proliferation and bypassed the CD73 knockdown defective growth phenotype. These results demonstrate that the catalytic activity of CD73 contributes to the pathogenic growth of FN-RMS through the activation of the purinergic signaling pathway. Therefore, targeting CD73 and the purinergic signaling pathway represents a potential therapeutic approach for FN-RMS patients.

A telomere-targeting drug depletes cancer initiating cells and promotes anti-tumor immunity in small cell lung cancer.

There are few effective treatments for small cell lung cancer (SCLC) underscoring the need for innovative therapeutic approaches. This study focuses on exploiting telomerase, a critical SCLC dependency as a therapeutic target. A prominent characteristic of SCLC is their reliance on telomerase activity, a key enzyme essential for their continuous proliferation. Here we utilize a nucleoside analog, 6-Thio-2'-deoxyguanosine (6TdG) currently in phase II clinical trials, that is preferentially incorporated by telomerase into telomeres leading to telomere dysfunction. Using preclinical mouse and human derived models we find low intermittent doses of 6TdG inhibit tumor growth and reduce metastatic burden. Anti-tumor efficacy correlates with a reduction in a subpopulation of cancer initiating like cells (CICs) identified by their expression of L1CAM/CD133 and highest telomerase activity. 6TdG treatment also leads to activation of innate and adaptive anti-tumor responses. Mechanistically, 6TdG depletes CICs and induces type-I interferon signaling leading to tumor immune visibility by activating tumor cell STING signaling. We also observe increased sensitivity to irradiation after 6TdG treatment in both syngeneic and humanized SCLC xenograft models both of which are dependent on the presence of host immune cells. This study underscores the immune-enhancing and metastasis-reducing effects of 6TdG, employing a range of complementary in vitro and in vivo SCLC preclinical models providing a potential therapeutic approach to SCLC.

CRISPR-Cas9 base editing of pathogenic CaMKIIδ improves cardiac function in a humanized mouse model.

Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.

Elimination of CaMKIIδ Autophosphorylation by CRISPR-Cas9 Base Editing Improves Survival and Cardiac Function in Heart Failure in Mice.

BACKGROUND: Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. METHODS: To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. RESULTS: Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+ transients and an increased frequency of arrhythmias after chronic ß-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. CONCLUSIONS: Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.

The MYC-YBX1 Circuit in Maintaining Stem-like Vincristine-Resistant Cells in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that causes significant devastation, with no effective therapy for relapsed disease. The mechanisms behind treatment failures are poorly understood. Our study showed that treatment of RMS cells with vincristine led to an increase in CD133-positive stem-like resistant cells. Single cell RNAseq analysis revealed that MYC and YBX1 were among the top-scoring transcription factors in CD133-high expressing cells. Targeting MYC and YBX1 using CRISPR/Cas9 reduced stem-like characteristics and viability of the vincristine-resistant cells. MYC and YBX1 showed mutual regulation, with MYC binding to the YBX1 promoter and YBX1 binding to MYC mRNA. The MYC inhibitor MYC361i synergized with vincristine to reduce tumor growth and stem-like cells in a zebrafish model of RMS. MYC and YBX expression showed a positive correlation in RMS patients, and high MYC expression correlated with poor survival. Targeting the MYC-YBX1 axis holds promise for improving survival in RMS patients.

Single-swap editing for the correction of common Duchenne muscular dystrophy mutations.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease of progressive muscle weakness and wasting caused by the absence of dystrophin protein. Current gene therapy approaches using antisense oligonucleotides require lifelong dosing and have limited efficacy in restoring dystrophin production. A gene editing approach could permanently correct the genome and restore dystrophin protein expression. Here, we describe single-swap editing, in which an adenine base editor edits a single base pair at a splice donor site or splice acceptor site to enable exon skipping or reframing. In human induced pluripotent stem cell-derived cardiomyocytes, we demonstrate that single-swap editing can enable beneficial exon skipping or reframing for the three most therapeutically relevant exons-DMD exons 45, 51, and 53-which could be beneficial for 30% of all DMD patients. Furthermore, an adeno-associated virus delivery method for base editing components can efficiently restore dystrophin production locally and systemically in skeletal and cardiac muscles of a DMD mouse model containing a deletion of Dmd exon 44. Our studies demonstrate single-swap editing as a potential gene editing therapy for common DMD mutations.

TWIST2-mediated chromatin remodeling promotes fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a common soft tissue sarcoma in children that resembles developing skeletal muscle. Unlike normal muscle cells, RMS cells fail to differentiate despite expression of the myogenic determination protein MYOD. The TWIST2 transcription factor is frequently overexpressed in fusion-negative RMS (FN-RMS). TWIST2 blocks differentiation by inhibiting MYOD activity in myoblasts, but its role in FN-RMS pathogenesis is incompletely understood. Here, we show that knockdown of TWIST2 enables FN-RMS cells to exit the cell cycle and undergo terminal myogenesis. TWIST2 knockdown also substantially reduces tumor growth in a mouse xenograft model of FN-RMS. Mechanistically, TWIST2 controls H3K27 acetylation at distal enhancers by interacting with the chromatin remodelers SMARCA4 and CHD3 to activate growth-related target genes and repress myogenesis-related target genes. These findings provide insights into the role of TWIST2 in maintaining an undifferentiated and tumorigenic state of FN-RMS and highlight the potential of suppressing TWIST2-regulated pathways to treat FN-RMS.

Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy.

Mutations in RNA binding motif protein 20 (RBM20) are a common cause of familial dilated cardiomyopathy (DCM). Many RBM20 mutations cluster within an arginine/serine-rich (RS-rich) domain, which mediates nuclear localization. These mutations induce RBM20 mis-localization to form aberrant ribonucleoprotein (RNP) granules in the cytoplasm of cardiomyocytes and abnormal alternative splicing of cardiac genes, contributing to DCM. We used adenine base editing (ABE) and prime editing (PE) to correct pathogenic p.R634Q and p.R636S mutations in the RS-rich domain in human isogenic induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Using ABE to correct RBM20R634Q human iPSCs, we achieved 92% efficiency of A-to-G editing, which normalized alternative splicing of cardiac genes, restored nuclear localization of RBM20, and eliminated RNP granule formation. In addition, we developed a PE strategy to correct the RBM20R636S mutation in iPSCs and observed A-to-C editing at 40% efficiency. To evaluate the potential of ABE for DCM treatment, we also created Rbm20R636Q mutant mice. Homozygous (R636Q/R636Q) mice developed severe cardiac dysfunction, heart failure, and premature death. Systemic delivery of ABE components containing ABEmax-VRQR-SpCas9 and single-guide RNA by adeno-associated virus serotype 9 in these mice restored cardiac function as assessed by echocardiography and extended life span. As seen by RNA sequencing analysis, ABE correction rescued the cardiac transcriptional profile of treated R636Q/R636Q mice, compared to the abnormal gene expression seen in untreated mice. These findings demonstrate the potential of precise correction of genetic mutations as a promising therapeutic approach for DCM.

Blocking LAIR1 signaling in immune cells inhibits tumor development.

The current immune checkpoint blockade therapy has been successful in treating some cancers but not others. New molecular targets and therapeutic approaches of cancer immunology need to be identified. Leukocyte associated immunoglobulin like receptor 1 (LAIR1) is an immune inhibitory receptor expressing on most immune cell types. However, it remains a question whether we can specifically and actively block LAIR1 signaling to activate immune responses for cancer treatment. Here we report the development of specific antagonistic anti-LAIR1 monoclonal antibodies and studied the effects of LAIR1 blockade on the anti-tumor immune functions. The anti-LAIR1 antagonistic antibody stimulated the activities of T cells, natural killer cells, macrophages, and dendritic cells in vitro. The single-cell RNA sequencing analysis of intratumoral immune cells in syngeneic human LAIR1 transgenic mice treated with control or anti-LAIR1 antagonist antibodies indicates that LAIR1 signaling blockade increased the numbers of CD4 memory T cells and inflammatory macrophages, but decreased those of pro-tumor macrophages, regulatory T cells, and plasmacytoid dendritic cells. Importantly, the LAIR1 blockade by the antagonistic antibody inhibited the activity of immunosuppressive myeloid cells and reactivated T cells from cancer patients in vitro and impeded tumor metastasis in a humanized mouse model. Blocking LAIR1 signaling in immune cells represents a promising strategy for development of anti-cancer immunotherapy.

The histone reader PHF7 cooperates with the SWI/SNF complex at cardiac super enhancers to promote direct reprogramming.

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through overexpression of the transcription factors Gata4, Mef2c and Tbx5; later, Hand2 and Akt1 were found to further enhance this process1-5. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogramme adult fibroblasts6,7. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor8. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, it increases chromatin accessibility and transcription factor binding at these sites. Furthermore, PHF7 recruits cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 achieves efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic readers, such as PHF7, in harnessing chromatin remodelling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

Mice With Increased Numbers of Polyploid Hepatocytes Maintain Regenerative Capacity But Develop Fewer Hepatocellular Carcinomas Following Chronic Liver Injury.

BACKGROUND & AIMS: Thirty to 90% of hepatocytes contain whole-genome duplications, but little is known about the fates or functions of these polyploid cells or how they affect development of liver disease. We investigated the effects of continuous proliferative pressure, observed in chronically damaged liver tissues, on polyploid cells. METHODS: We studied Rosa-rtTa mice (controls) and Rosa-rtTa;TRE-short hairpin RNA mice, which have reversible knockdown of anillin, actin binding protein (ANLN). Transient administration of doxycycline increases the frequency and degree of hepatocyte polyploidy without permanently altering levels of ANLN. Mice were then given diethylnitrosamine and carbon tetrachloride (CCl4) to induce mutations, chronic liver damage, and carcinogenesis. We performed partial hepatectomies to test liver regeneration and then RNA-sequencing to identify changes in gene expression. Lineage tracing was used to rule out repopulation from non-hepatocyte sources. We imaged dividing hepatocytes to estimate the frequency of mitotic errors during regeneration. We also performed whole-exome sequencing of 54 liver nodules from patients with cirrhosis to quantify aneuploidy, a possible outcome of polyploid cell divisions. RESULTS: Liver tissues from control mice given CCl4 had significant increases in ploidy compared with livers from uninjured mice. Mice with knockdown of ANLN had hepatocyte ploidy above physiologic levels and developed significantly fewer liver tumors after administration of diethylnitrosamine and CCl4 compared with control mice. Increased hepatocyte polyploidy was not associated with altered regenerative capacity or tissue fitness, changes in gene expression, or more mitotic errors. Based on lineage-tracing experiments, non-hepatocytes did not contribute to liver regeneration in mice with increased polyploidy. Despite an equivalent rate of mitosis in hepatocytes of differing ploidies, we found no lagging chromosomes or micronuclei in mitotic polyploid cells. In nodules of human cirrhotic liver tissue, there was no evidence of chromosome-level copy number variations. CONCLUSIONS: Mice with increased polyploid hepatocytes develop fewer liver tumors following chronic liver damage. Remarkably, polyploid hepatocytes maintain the ability to regenerate liver tissues during chronic damage without generating mitotic errors, and aneuploidy is not commonly observed in cirrhotic livers. Strategies to increase numbers of polypoid hepatocytes might be effective in preventing liver cancer.

Dual ARID1A/ARID1B loss leads to rapid carcinogenesis and disruptive redistribution of BAF complexes.

SWI/SNF chromatin remodelers play critical roles in development and cancer. The causal links between SWI/SNF complex disassembly and carcinogenesis are obscured by redundancy between paralogous components. Canonical cBAF-specific paralogs ARID1A and ARID1B are synthetic lethal in some contexts, but simultaneous mutations in both ARID1s are prevalent in cancer. To understand if and how cBAF abrogation causes cancer, we examined the physiologic and biochemical consequences of ARID1A/ARID1B loss. In double knockout liver and skin, aggressive carcinogenesis followed de-differentiation and hyperproliferation. In double mutant endometrial cancer, add-back of either induced senescence. Biochemically, residual cBAF subcomplexes resulting from loss of ARID1 scaffolding were unexpectedly found to disrupt polybromo containing pBAF function. 37 of 69 mutations in the conserved scaffolding domains of ARID1 proteins observed in human cancer caused complex disassembly, partially explaining their mutation spectra. ARID1-less, cBAF-less states promote carcinogenesis across tissues, and suggest caution against paralog-directed therapies for ARID1-mutant cancer.

Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix.

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.

Twist2 amplification in rhabdomyosarcoma represses myogenesis and promotes oncogenesis by redirecting MyoD DNA binding.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of TWIST2 in fusion-negative RMS. Knockdown of TWIST2 in RMS cells results in up-regulation of MYOGENIN and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.

Active enhancer and chromatin accessibility landscapes chart the regulatory network of primary multiple myeloma.

Multiple myeloma (MM) is an aggressive cancer that originates from antibody-secreting plasma cells. Although genetically and transcriptionally well characterized, the aberrant gene regulatory networks that underpin this disease remain poorly understood. Here, we mapped regulatory elements, open chromatin, and transcription factor (TF) footprints in primary MM cells. In comparison with normal antibody-secreting cells, MM cells displayed consistent changes in enhancer activity that are connected to superenhancer (SE)-mediated deregulation of TF genes. MM cells also displayed widespread decompaction of heterochromatin that was associated with activation of regulatory elements and in a major subset of patients' deregulation of the cyclic adenosine monophosphate pathway. Finally, building SE-associated TF-based regulatory networks allowed identification of several novel TFs that are central to MM biology. Taken together, these findings significantly add to our understanding of the aberrant gene regulatory network that underpins MM.

Identification of key factors regulating self-renewal and differentiation in EML hematopoietic precursor cells by RNA-sequencing analysis.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient's hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study. RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment. In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.