A genome-wide single-cell 3D genome atlas of lung cancer progression.

Alterations in three-dimensional (3D) genome structures are associated with cancer1-5. However, how genome folding evolves and diversifies during subclonal cancer progression in the native tissue environment remains unknown. Here, we leveraged a genome-wide chromatin tracing technology to directly visualize 3D genome folding in situ in a faithful Kras-driven mouse model of lung adenocarcinoma (LUAD)6, generating the first single-cell 3D genome atlas of any cancer. We discovered stereotypical 3D genome alterations during cancer development, including a striking structural bottleneck in preinvasive adenomas prior to progression to LUAD, indicating a stringent selection on the 3D genome early in cancer progression. We further showed that the 3D genome precisely encodes cancer states in single cells, despite considerable cell-to-cell heterogeneity. Finally, evolutionary changes in 3D genome compartmentalization - partially regulated by polycomb group protein Rnf2 through its ubiquitin ligase-independent activity - reveal novel genetic drivers and suppressors of LUAD progression. Our results demonstrate the importance of mapping the single-cell cancer 3D genome and the potential to identify new diagnostic and therapeutic biomarkers from 3D genomic architectures.

Perturb-tracing enables high-content screening of multiscale 3D genome regulators.

Three-dimensional (3D) genome organization becomes altered during development, aging, and disease1-23, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multiscale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screen, a new cellular barcode readout method (BARC-FISH), and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their genome organization from 13,000 imaging target-perturbation combinations, alongside perturbation-paired barcode readout in the same single cells. Using 1.4 million 3D positions along chromosome traces, we discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop-extrusion and A-B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. We found that the ATP-dependent helicase CHD7, the loss of which causes the congenital neural crest syndrome CHARGE24 and a chromatin remodeler previously shown to promote local chromatin openness25-27, counter-intuitively compacts chromatin over long range in different genomic contexts and cell backgrounds including neural crest cells, and globally represses gene expression. The DNA compaction effect of CHD7 is independent of its chromatin remodeling activity and does not require other protein partners. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome functions, such as global gene and nuclear regulation, in health and disease.

Differential expression of mTOR related molecules in the placenta from gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia patients.

The mechanistic target of rapamycin (mTOR) pathway is involved in the function and growth of the placenta during pregnancy. The mTOR pathway responds to nutrient availability and growth factors that regulate protein expression and cell growth. Disrupted mTOR signaling is associated with the development of several obstetric complications. The purpose of this study was to identify the differential placental expression of various mTOR-associated proteins in the placenta during normal gestation (Control), gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia (PE). Immunohistochemistry localized activated proteins (phospho; p) mTOR, pp70, p4EBP1, pAKT and pERK. Real-time PCR array was performed to show differing placental expression of additional mTOR-associated genes. Western blot was performed for pAMPK protein. We observed: 1) increased pmTOR during GDM and decreased pmTOR during IUGR and PE, 2) increased pp70 during IUGR and decreased pp70 during GDM and PE, 3) increased p4EBP1 during GDM, IUGR, and PE, 4) increased pAKT during GDM, 5) increased pERK during IUGR, 6) differential placental expression of mTOR pathway associated genes and increased pAMPK during GDM and PE. We conclude that regulation of the mTOR pathway is uniquely involved in the development of these obstetric complications. Insights into this pathway may provide avenues that if modify may help alleviate these diseases.

Lung injury in axolotl salamanders induces an organ-wide proliferation response.

BACKGROUND: Ambystoma mexicanum, the axolotl salamander, is a classic model organism used to study vertebrate regeneration. It is assumed that axolotls regenerate most tissues, but the exploration of lung regeneration has not been performed until now. RESULTS: Unlike the blastema-based response used during appendage regeneration, lung amputation led to organ-wide proliferation. Pneumocytes and mesenchymal cells responded to injury by increased proliferation throughout the injured lung, which led to a recovery in lung mass and morphology by 56 days post-amputation. Receptors associated with the Neuregulin signaling pathway were upregulated at one and 3 weeks post lung amputation. We show expression of the ligand, neuregulin, in the I/X cranial nerve that innervates the lung and cells within the lung. Supplemental administration of Neuregulin peptide induced widespread proliferation in the lung similar to an injury response, suggesting that neuregulin signaling may play a significant role during lung regeneration. CONCLUSION: Our study characterizes axolotl lung regeneration. We show that the lung responds to injury by an organ-wide proliferative response of multiple cell types, including pneumocytes, to recover lung mass.

A novel, rapid, and low cost method for preparing tissues with metallic stents for routine histology.

BACKGROUND: Coronary artery stenting has become a common procedure and cardiovascular pathology specimens containing these metallic stents are accordingly becoming common. Histologic examination of stented vessels is imperative, but special techniques are needed due to the presence of metal within the tissue. We describe a rapid and inexpensive method for preparing stented vascular specimens for routine histology suitable for use in almost any histology laboratory. DESIGN: After formalin fixation and decalcification, stented vascular segments were freeze-embedded and sectioned using a handheld power micro cutoff wheel tool into ~1 mm slices. Sections were allowed to thaw and the strut shards removed with fine forceps. No longer containing metal, the sections were processed for routine paraffin embedding, microtomy and staining. RESULTS: Histologic sections showed only minor tissue disruption around the stent struts. In our experience with 25 stented arteries (mean interval from implantation 5.6 years), the mean subjective section quality score was 4.1 out of 5. The position of each strut could easily be determined, along with neointimal in-stent restenosis and thrombosis. Local reaction to each strut could be surmised even if minor tissue disruption occurred. The entire process was completed in 2-3 days. The incremental cost over that of routine histology is nominal. CONCLUSION: This method for examining stented vascular segments histologically could readily be applied in most pathology laboratories and serves as a highly practical solution to dilemma of examining stents histologically.

Transcriptional correlates of proximal-distal identify and regeneration timing in axolotl limbs.

Cells within salamander limbs retain memories that inform the correct replacement of amputated tissues at different positions along the length of the arm, with proximal and distal amputations completing regeneration at similar times. We investigated the possibility that positional memory is associated with variation in transcript abundances along the proximal-distal limb axis. Transcripts were deeply sampled from Ambystoma mexicanum limbs at the time they were administered fore arm vs upper arm amputations, and at 19 post-amputation time points. After amputation and prior to regenerative outgrowth, genes typically expressed by differentiated muscle cells declined more rapidly in upper arms while cell cycle transcripts were expressed more highly. These and other expression patterns suggest upper arms undergo more robust tissue remodeling and cell proliferation responses after amputation, and thus provide an explanation for why the overall time to complete regeneration is similar for proximal and distal amputations. Additionally, we identified candidate positional memory genes that were expressed differently between fore and upper arms that encode a surprising number of epithelial proteins and a variety of cell surface, cell adhesion, and extracellular matrix molecules. Also, genes were discovered that exhibited different, bivariate patterns of gene expression between fore and upper arms, implicating dynamic transcriptional regulation for the first time in limb regeneration. Finally, 43 genes expressed differently between fore and upper arm samples showed similar transcriptional patterns during retinoic acid-induced reprogramming of fore arm blastema cells into upper arm cells. Our study provides new insights about the basis of positional information in regenerating axolotl limbs.

Optimizing the Use of Gene Expression Profiling in Early-Stage Breast Cancer.

Purpose Gene expression profiling assays are frequently used to guide adjuvant chemotherapy decisions in hormone receptor-positive, lymph node-negative breast cancer. We hypothesized that the clinical value of these new tools would be more fully realized when appropriately integrated with high-quality clinicopathologic data. Hence, we developed a model that uses routine pathologic parameters to estimate Oncotype DX recurrence score (ODX RS) and independently tested its ability to predict ODX RS in clinical samples. Patients and Methods We retrospectively reviewed ordered ODX RS and pathology reports from five institutions (n = 1,113) between 2006 and 2013. We used locally performed histopathologic markers (estrogen receptor, progesterone receptor, Ki-67, human epidermal growth factor receptor 2, and Elston grade) to develop models that predict RS-based risk categories. Ordering patterns at one site were evaluated under an integrated decision-making model incorporating clinical treatment guidelines, immunohistochemistry markers, and ODX. Final locked models were independently tested (n = 472). Results Distribution of RS was similar across sites and to reported clinical practice experience and stable over time. Histopathologic markers alone determined risk category with > 95% confidence in > 55% (616 of 1,113) of cases. Application of the integrated decision model to one site indicated that the frequency of testing would not have changed overall, although ordering patterns would have changed substantially with less testing of estimated clinical risk-high or clinical risk-low cases and more testing of clinical risk-intermediate cases. In the validation set, the model correctly predicted risk category in 52.5% (248 of 472). Conclusion The proposed model accurately predicts high- and low-risk RS categories (> 25 or ≤ 25) in a majority of cases. Integrating histopathologic and molecular information into the decision-making process allows refocusing the use of new molecular tools to cases with uncertain risk.

Exploring drug delivery for the DOT1L inhibitor pinometostat (EPZ-5676): Subcutaneous administration as an alternative to continuous IV infusion, in the pursuit of an epigenetic target.

Protein methyltransferases are emerging as promising drug targets for therapeutic intervention in human cancers. Pinometostat (EPZ-5676) is a small molecule inhibitor of the DOT1L enzyme, a histone methyltransferase that methylates lysine 79 of histone H3. DOT1L activity is dysregulated in the pathophysiology of rearranged mixed lineage leukemia (MLL-r). Pinometostat is currently in Phase 1 clinical trials in relapsed refractory acute leukemia patients and is administered as a continuous IV infusion (CIV). The studies herein investigated alternatives to CIV administration of pinometostat to improve patient convenience. Various sustained release technologies were considered, and based on the required dose size as well as practical considerations, subcutaneous (SC) bolus administration of a solution formulation was selected for further evaluation in preclinical studies. SC administration offered improved exposure and complete bioavailability of pinometostat relative to CIV and oral administration. These findings warranted further evaluation in rat xenograft models of MLL-r leukemia. SC dosing in xenograft models demonstrated inhibition of MLL-r tumor growth and inhibition of pharmacodynamic markers of DOT1L activity. However, a dosing frequency of thrice daily (t.i.d) was required in these studies to elicit optimal inhibition of DOT1L target genes and tumor growth inhibition. Development of an extended release formulation may prove useful in the further optimization of the SC delivery of pinometostat, moving towards a more convenient dosing paradigm for patients.