Arhgef2 regulates mitotic spindle orientation in hematopoietic stem cells and is essential for productive hematopoiesis.

How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell-driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2-/- fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.

Author Correction: On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

The inaccessibility of living bone marrow (BM) hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human BM-on-a-chip (BM chip) supports the differentiation and maturation of multiple blood cell lineages over 4 weeks while improving CD34+ cell maintenance, and that it recapitulates aspects of BM injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation, as well as BM recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34+ cells and BM-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that BM chips containing cells from patients with the rare genetic disorder Shwachman-Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil maturation abnormality. As an in vitro model of haematopoietic dysfunction, the BM chip may serve as a human-specific alternative to animal testing for the study of BM pathophysiology.

Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126.

CAG/CTG trinuncleotide repeats are fragile sequences that when expanded form DNA secondary structures and cause human disease. We evaluated CAG/CTG repeat stability and repair outcomes in histone H2 mutants in S. cerevisiae. Although the two copies of H2A are nearly identical in amino acid sequence, CAG repeat stability depends on H2A copy 1 (H2A.1) but not copy 2 (H2A.2). H2A.1 promotes high-fidelity homologous recombination, sister chromatid recombination (SCR), and break-induced replication whereas H2A.2 does not share these functions. Both decreased SCR and the increase in CAG expansions were due to the unique Thr126 residue in H2A.1 and hta1Δ or hta1-T126A mutants were epistatic to deletion of the Polδ subunit Pol32, suggesting a role for H2A.1 in D-loop extension. We conclude that H2A.1 plays a greater repair-specific role compared to H2A.2 and may be a first step towards evolution of a repair-specific function for H2AX compared to H2A in mammalian cells.

TGFß signaling underlies hematopoietic dysfunction and bone marrow failure in Shwachman-Diamond Syndrome.

Shwachman-Diamond Syndrome (SDS) is a rare and clinically-heterogeneous bone marrow (BM) failure syndrome caused by mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene. Although SDS was described over 50 years ago, the molecular pathogenesis is poorly understood due, in part, to the rarity and heterogeneity of the affected hematopoietic progenitors. To address this, we used single cell RNA sequencing to profile scant hematopoietic stem and progenitor cells from SDS patients. We generated a single cell map of early lineage commitment and found that SDS hematopoiesis was left-shifted with selective loss of granulocyte-monocyte progenitors. Transcriptional targets of transforming growth factor-beta (TGFß) were dysregulated in SDS hematopoietic stem cells and multipotent progenitors, but not in lineage-committed progenitors. TGFß inhibitors (AVID200 and SD208) increased hematopoietic colony formation of SDS patient BM. Finally, TGFß3 and other TGFß pathway members were elevated in SDS patient blood plasma. These data establish the TGFß pathway as a novel candidate biomarker and therapeutic target in SDS and translate insights from single cell biology into a potential therapy.

Therapeutic discovery for marrow failure with MDS predisposition using pluripotent stem cells.

Monosomy 7 or deletion of 7q (del(7q)) are common clonal cytogenetic abnormalities associated with high grade myelodysplastic syndrome (MDS) arising in inherited and acquired bone marrow failure. Current non-transplant approaches to treat marrow failure may be complicated by stimulation of clonal outgrowth. To study the biological consequences of del(7q) within the context of a failing marrow, we generated induced pluripotent stem cells (iPSCs) derived from patients with Shwachman Diamond Syndrome (SDS), a bone marrow failure disorder with MDS predisposition, and genomically engineered a 7q deletion. The TGFß pathway was the top differentially regulated pathway in transcriptomic analysis of SDS versus SDSdel(7q) iPSCs. SMAD2 phosphorylation was increased in SDS relative to wild type cells consistent with hyperactivation of the TGFbeta pathway in SDS. Phospho-SMAD2 levels were reduced following 7q deletion in SDS cells and increased upon restoration of 7q diploidy. Inhibition of the TGFbeta pathway rescued hematopoiesis in SDS-iPSCs and in bone marrow hematopoietic cells from SDS patients while it had no impact on the SDSdel(7q) cells. These results identified a potential targetable vulnerability to improve hematopoiesis in an MDS-predisposition syndrome, and highlight the importance of the germline context of somatic alterations to inform precision medicine approaches to therapy.

The Chromatin Remodeler Isw1 Prevents CAG Repeat Expansions During Transcription in Saccharomyces cerevisiae.

CAG/CTG trinucleotide repeats are unstable sequences that are difficult to replicate, repair, and transcribe due to their structure-forming nature. CAG repeats strongly position nucleosomes; however, little is known about the chromatin remodeling needed to prevent repeat instability. In a Saccharomyces cerevisiae model system with CAG repeats carried on a YAC, we discovered that the chromatin remodeler Isw1 is required to prevent CAG repeat expansions during transcription. CAG repeat expansions in the absence of Isw1 were dependent on both transcription-coupled repair (TCR) and base-excision repair (BER). Furthermore, isw1∆ mutants are sensitive to methyl methanesulfonate (MMS) and exhibit synergistic MMS sensitivity when combined with BER or TCR pathway mutants. We conclude that CAG expansions in the isw1∆ mutant occur during a transcription-coupled excision repair process that involves both TCR and BER pathways. We observed increased RNA polymerase II (RNAPII) occupancy at the CAG repeat when transcription of the repeat was induced, but RNAPII binding did not change in isw1∆ mutants, ruling out a role for Isw1 remodeling in RNAPII progression. However, nucleosome occupancy over a transcribed CAG tract was altered in isw1∆ mutants. Based on the known role of Isw1 in the reestablishment of nucleosomal spacing after transcription, we suggest that a defect in this function allows DNA structures to form within repetitive DNA tracts, resulting in inappropriate excision repair and repeat-length changes. These results establish a new function for Isw1 in directly maintaining the chromatin structure at the CAG repeat, thereby limiting expansions that can occur during transcription-coupled excision repair.

PRMT1-Mediated Translation Regulation Is a Crucial Vulnerability of Cancer.

Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control. In particular, loss of Prmt1 led to a decrease in arginine methylation of the translation initiation complex, thereby disrupting its assembly and inhibiting translation. p53/Rb-null cells were sensitive to p53-induced translation stress, and analysis of human cancer cell line data from Project Achilles further revealed that Prmt1 and translation-associated pathways converged on the same functional networks. We propose that targeted therapy against Prmt1 and its associated translation-related pathways offer a mechanistic rationale for treatment of osteosarcomas and other cancers that exhibit dependencies on translation stress response. Cancer Res; 77(17); 4613-25. ©2017 AACR.

Differential Regulation of the Melanoma Proteome by eIF4A1 and eIF4E.

Small molecules and antisense oligonucleotides that inhibit the translation initiation factors eIF4A1 and eIF4E have been explored as broad-based therapeutic agents for cancer treatment, based on the frequent upregulation of these two subunits of the eIF4F cap-binding complex in many cancer cells. Here, we provide support for these therapeutic approaches with mechanistic studies of eIF4F-driven tumor progression in a preclinical model of melanoma. Silencing eIF4A1 or eIF4E decreases melanoma proliferation and invasion. There were common effects on the level of cell-cycle proteins that could explain the antiproliferative effects in vitro Using clinical specimens, we correlate the common cell-cycle targets of eIF4A1 and eIF4E with patient survival. Finally, comparative proteomic and transcriptomic analyses reveal extensive mechanistic divergence in response to eIF4A1 or eIF4E silencing. Current models indicate that eIF4A1 and eIF4E function together through the 5'UTR to increase translation of oncogenes. In contrast, our data demonstrate that the common effects of eIF4A1 and eIF4E on translation are mediated by the coding region and 3'UTR. Moreover, their divergent effects occur through the 5'UTR. Overall, our work shows that it will be important to evaluate subunit-specific inhibitors of eIF4F in different disease contexts to fully understand their anticancer actions. Cancer Res; 77(3); 613-22. ©2016 AACR.

Bidirectional cross talk between patient-derived melanoma and cancer-associated fibroblasts promotes invasion and proliferation.

Tumor-stroma interactions are critical for epithelial-derived tumors, and among the stromal cell types, cancer-associated fibroblasts (CAFs) exhibit multiple functions that fuel growth, dissemination, and drug resistance. However, these interactions remain insufficiently characterized in non-epithelial tumors such as malignant melanoma. We generated monocultures of melanoma cells and matching CAFs from patients' metastatic lesions, distinguished by oncogenic drivers and immunoblotting of characteristic markers. RNA sequencing of CAFs revealed a homogenous epigenetic program that strongly resembled the signatures from epithelial cancers, including enrichment for an epithelial-to-mesenchymal transition (EMT). Melanoma CAFs in monoculture displayed robust invasive behavior while patient-derived melanoma monocultures showed very little invasiveness. Instead, melanoma cells showed increased invasion when co-cultured with CAFs. In turn, CAFs showed increased proliferation when exposed to melanoma conditioned media (CM), mediated in part by melanoma-secreted transforming growth factor-alpha that acted on CAFs via the epidermal growth factor receptor. This study provides evidence that bidirectional interactions between melanoma and CAFs regulate progression of metastatic melanoma.

The lncRNA SLNCR1 Mediates Melanoma Invasion through a Conserved SRA1-like Region.

Long non-coding RNAs (lncRNAs) have been implicated in numerous physiological processes and diseases, most notably cancers. However, little is known about the mechanism of many functional lncRNAs. We identified an abundantly expressed lncRNA associated with decreased melanoma patient survival. Increased expression of this lncRNA, SLNCR1, mediates melanoma invasion through a highly conserved sequence similar to that of the lncRNA SRA1. Using a sensitive technique we term RATA (RNA-associated transcription factor array), we show that the brain-specific homeobox protein 3a (Brn3a) and the androgen receptor (AR) bind within and adjacent to SLNCR1's conserved region, respectively. SLNCR1, AR, and Brn3a are specifically required for transcriptional activation of matrix metalloproteinase 9 (MMP9) and increased melanoma invasion. Our observations directly link AR to melanoma invasion, possibly explaining why males experience more melanoma metastases and have an overall lower survival in comparison to females.

Rps14 haploinsufficiency causes a block in erythroid differentiation mediated by S100A8 and S100A9.

Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8--whose expression was increased in mutant erythroblasts, monocytes and macrophages--is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

Noncanonical microRNAs and endogenous siRNAs in normal and psoriatic human skin.

Noncanonical microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes. Noncanonical miRNAs, which bypass part of the canonical miRNA biogenesis pathway, can originate from a variety of genomic loci, which include small nucleolar RNAs (snoRNAs), transfer RNAs (tRNAs) and introns, whereas endo-siRNAs can arise from repetitive elements, some of which are transposable. The roles of noncanonical miRNAs and endo-siRNAs in complex diseases have yet to be characterized. To investigate their potential expression and function in psoriasis, we carried out a comprehensive, genome-wide search for noncanonical miRNAs and endo-siRNAs in small RNA deep-sequencing data sets from normal and psoriatic human skin. By analyzing more than 670 million qualified reads from 67 small RNA libraries, we identified 21 novel, noncanonical miRNAs (3 snoRNA-derived and 2 tRNA-derived miRNAs and 16 miRtrons) and 39 novel endo-siRNAs that were expressed in skin. The expression of four novel small RNAs was validated by qRT-PCR in human skin, and their Argonaute association was confirmed by co-immunoprecipitation of ectopic small RNAs in HEK293 cells. Fifteen noncanonical miRNAs or endo-siRNAs were significantly differentially expressed in psoriatic-involved versus normal skin, including an Alu-short interspersed element-derived siRNA which was 17-fold up-regulated in psoriatic-involved skin. These and other differentially expressed small noncoding RNAs may function as regulators of gene expression in skin and potentially play a role in psoriasis pathogenesis.

PSORS2 is due to mutations in CARD14.

Psoriasis is a common, immune-mediated genetic disorder of the skin and is associated with arthritis in approximately 30% of cases. Previously, we localized PSORS2 (psoriasis susceptibility locus 2) to chromosomal region 17q25.3-qter after a genome-wide linkage scan in a family of European ancestry with multiple cases of psoriasis and psoriatic arthritis. Linkage to PSORS2 was also observed in a Taiwanese family with multiple psoriasis-affected members. In caspase recruitment domain family, member 14 (CARD14), we identified unique gain-of-function mutations that segregated with psoriasis by using genomic capture and DNA sequencing. The mutations c.349G>A (p.Gly117Ser) (in the family of European descent) and c.349+5G>A (in the Taiwanese family) altered splicing between CARD14 exons 3 and 4. A de novo CARD14 mutation, c.413A>C (p.Glu138Ala), was detected in a child with sporadic, early-onset, generalized pustular psoriasis. CARD14 activates nuclear factor kappa B (NF-kB), and compared with wild-type CARD14, the p.Gly117Ser and p.Glu138Ala substitutions were shown to lead to enhanced NF-kB activation and upregulation of a subset of psoriasis-associated genes in keratinocytes. These genes included chemokine (C-C motif) ligand 20 (CCL20) and interleukin 8 (IL8). CARD14 is localized mainly in the basal and suprabasal layers of healthy skin epidermis, whereas in lesional psoriatic skin, it is reduced in the basal layer and more diffusely upregulated in the suprabasal layers of the epidermis. We propose that, after a triggering event that can include epidermal injury, rare gain-of-function mutations in CARD14 initiate a process that includes inflammatory cell recruitment by keratinocytes. This perpetuates a vicious cycle of epidermal inflammation and regeneration, a cycle which is the hallmark of psoriasis.

A subset of methylated CpG sites differentiate psoriatic from normal skin.

Psoriasis is a chronic inflammatory immune-mediated disorder affecting the skin and other organs including joints. Over 1,300 transcripts are altered in psoriatic involved skin compared with normal skin. However, to our knowledge, global epigenetic profiling of psoriatic skin is previously unreported. Here, we describe a genome-wide study of altered CpG methylation in psoriatic skin. We determined the methylation levels at 27,578 CpG sites in skin samples from individuals with psoriasis (12 involved, 8 uninvolved) and 10 unaffected individuals. CpG methylation of involved skin differed from normal skin at 1,108 sites. Twelve mapped to the epidermal differentiation complex, upstream or within genes that are highly upregulated in psoriasis. Hierarchical clustering of 50 of the top differentially methylated (DM) sites separated psoriatic from normal skin samples with uninvolved skin exhibiting intermediate methylation. CpG sites where methylation was correlated with gene expression are reported. Sites with inverse correlations between methylation and nearby gene expression include those of KYNU, OAS2, S100A12, and SERPINB3, whose strong transcriptional upregulation is an important discriminator of psoriasis. Pyrosequencing of bisulfite-treated DNA from skin biopsies at three DM loci confirmed earlier findings and revealed reversion of methylation levels toward the non-psoriatic state after 1 month of anti-TNF-α therapy.

Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome.

Psoriasis is a chronic and complex inflammatory skin disease with lesions displaying dramatically altered mRNA expression profiles. However, much less is known about the expression of small RNAs. Here, we describe a comprehensive analysis of the normal and psoriatic skin miRNAome with next-generation sequencing in a large patient cohort. We generated 6.7 × 10(8) small RNA reads representing 717 known and 284 putative novel microRNAs (miRNAs). We also observed widespread expression of isomiRs and miRNA*s derived from known and novel miRNA loci, and a low frequency of miRNA editing in normal and psoriatic skin. The expression and processing of selected novel miRNAs were confirmed with qRT-PCR in skin and other human tissues or cell lines. Eighty known and 18 novel miRNAs were 2-42-fold differentially expressed in psoriatic skin. Of particular significance was the 2.7-fold upregulation of a validated novel miRNA derived from the antisense strand of the miR-203 locus, which plays a role in epithelial differentiation. Other differentially expressed miRNAs included hematopoietic-specific miRNAs such as miR-142-3p and miR-223/223*, and angiogenic miRNAs such as miR-21, miR-378, miR-100 and miR-31, which was the most highly upregulated miRNA in psoriatic skin. The functions of these miRNAs are consistent with the inflammatory and hyperproliferative phenotype of psoriatic lesions. In situ hybridization of differentially expressed miRNAs revealed stratified epidermal expression of an uncharacterized keratinocyte-derived miRNA, miR-135b, as well as the epidermal infiltration of the hematopoietic-specific miRNA, miR-142-3p, in psoriatic lesions. This study lays a critical framework for functional characterization of miRNAs in healthy and diseased skin.