SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

3D Functional Genomics Screens Identify CREBBP as a Targetable Driver in Aggressive Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo-like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. SIGNIFICANCE: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.

A mouse SWATH-mass spectrometry reference spectral library enables deconvolution of species-specific proteomic alterations in human tumour xenografts.

SWATH-mass spectrometry (MS) enables accurate and reproducible proteomic profiling in multiple model organisms including the mouse. Here, we present a comprehensive mouse reference spectral library (MouseRefSWATH) that permits quantification of up to 10,597 proteins (62.2% of the mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high sequence similarity between mouse and human proteins, facilitating the study of host microenvironment-tumour interactions from 'bulk tumour' measurements. We apply the XenoSWATH pipeline to characterize an intraductal xenograft model of breast ductal carcinoma in situ and uncover complex regulation consistent with stromal reprogramming, where the modulation of cell migration pathways is not restricted to tumour cells but also operates in the mouse stroma upon progression to invasive disease. MouseRefSWATH and XenoSWATH open new opportunities for in-depth and reproducible proteomic assessment to address wide-ranging biological questions involving this important model organism.

Sox9 regulates cell state and activity of embryonic mouse mammary progenitor cells.

Embryonic mammary cells are a unique population comprised of undifferentiated, highly plastic progenitor cells that create normal mammary tissues. The mammary gland continues to develop after birth from descendants of embryonic mammary cells. Here, we establish cell lines from mouse mammary organs, immediately after they formed during prenatal development, to facilitate studies of primitive mammary cells, which are difficult to isolate in sufficient quantities for use in functional experiments. We show that some lines can be induced to secrete milk, a distinguishing feature of mammary epithelial cells. Targeted deletion of Sox9, from one clone, decreases the ability to respond to lactogenic stimuli, consistent with a previously identified role for Sox9 in regulating luminal progenitor function. Sox9 ablation also leads to alterations in 3D morphology and downregulation of Zeb1, a key epithelial-mesenchymal transition regulator. Prenatal mammary cell lines are an invaluable resource to study regulation of mammary progenitor cell biology and development.

SWATH mass spectrometry as a tool for quantitative profiling of the matrisome.

Proteomic analysis of extracellular matrix (ECM) and ECM-associated proteins, collectively known as the matrisome, is a challenging task due to the inherent complexity and insolubility of these proteins. Here we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins in both non-enriched and ECM enriched tissue without the need for prior fractionation. Utilising a spectral library containing 201 matrisomal proteins, we compared the performance and reproducibility of SWATH MS over conventional data-dependent analysis mass spectrometry (DDA MS) in unfractionated murine lung and liver. SWATH MS conferred a 15-20% increase in reproducible peptide identification across replicate experiments in both tissue types and identified 54% more matrisomal proteins in the liver versus DDA MS. We further use SWATH MS to evaluate the quantitative changes in matrisome content that accompanies ECM enrichment. Our data shows that ECM enrichment led to a systematic increase in core matrisomal proteins but resulted in significant losses in matrisome-associated proteins including the cathepsins and proteins of the S100 family. Our proof-of-principle study demonstrates the utility of SWATH MS as a versatile tool for in-depth characterisation of the matrisome in unfractionated and non-enriched tissues. SIGNIFICANCE: The matrisome is a complex network of extracellular matrix (ECM) and ECM-associated proteins that provides scaffolding function to tissues and plays important roles in the regulation of fundamental cellular processes. However, due to its inherent complexity and insolubility, proteomic studies of the matrisome typically require the application of enrichment workflows prior to MS analysis. Such enrichment strategies often lead to losses in soluble matrisome-associated components. In this study, we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins. We show that SWATH MS provides a more reproducible coverage of the matrisome compared to data-dependent analysis (DDA) MS. We also demonstrate that SWATH MS is capable of accurate quantification of matrisomal proteins without prior ECM enrichment and fractionation, which may simplify sample handling workflows and avoid losses in matrisome-associated proteins commonly linked to ECM enrichment.

Corrigendum: Probing the early stages of shock-induced chondritic meteorite formation at the mesoscale.

This corrects the article DOI: 10.1038/srep45206.

Giant convecting mud balls of the early solar system.

Carbonaceous asteroids may have been the precursors to the terrestrial planets, yet despite their importance, numerous attempts to model their early solar system geological history have not converged on a solution. The assumption has been that hydrothermal alteration was occurring in rocky asteroids with material properties similar to meteorites. However, these bodies would have accreted as a high-porosity aggregate of igneous clasts (chondrules) and fine-grained primordial dust, with ice filling much of the pore space. Short-lived radionuclides melted the ice, and aqueous alteration of anhydrous minerals followed. However, at the moment when the ice melted, no geological process had acted to lithify this material. It would have been a mud, rather than a rock. We tested the effect of removing the assumption of lithification. We find that if the body accretes unsorted chondrules, then large-scale mud convection is capable of producing a size-sorted chondrule population (if the body accretes an aerodynamically sorted chondrule population, then no further sorting occurs). Mud convection both moderates internal temperature and reduces variation in temperature throughout the object. As the system is thoroughly mixed, soluble elements are not fractionated, preserving primitive chemistry. Isotopic and redox heterogeneity in secondary phases over short length scales is expected, as individual particles experience a range of temperature and water-rock histories until they are brought together in their final configuration at the end of convection. These results are consistent with observations from aqueously altered meteorites (CI and CM chondrites) and spectra of primitive asteroids. The "mudball" model appears to be a general solution: Bodies spanning a ×1000 mass range show similar behavior.

SOX11 promotes invasive growth and ductal carcinoma in situ progression.

Here, we show that SOX11, an embryonic mammary marker that is normally silent in postnatal breast cells, is expressed in many oestrogen receptor-negative preinvasive ductal carcinoma in situ (DCIS) lesions. Mature mammary epithelial cells engineered to express SOX11 showed alterations in progenitor cell populations, including an expanded basal-like population with increased aldehyde dehydrogenase (ALDH) activity, and increased mammosphere-forming capacity. DCIS.com cells engineered to express SOX11 showed increased ALDH activity, which is a feature of cancer stem cells. The CD44+/CD24-/ALDH+ cell population was increased in DCIS.com cells that expressed SOX11. Upregulating SOX11 expression in DCIS.com cells led to increased invasive growth both in vitro and when they were injected intraductally in a mouse model of DCIS that recapitulates human disease. Invasive lesions formed sooner and tumour growth was augmented in vivo, suggesting that SOX11 contributes to the progression of DCIS to invasive breast cancer. We identified potential downstream effectors of SOX11 during both microinvasive and invasive tumour growth stages, including several with established links to regulation of progenitor cell function and prenatal developmental growth. Our findings suggest that SOX11 is a potential biomarker for DCIS lesions containing cells harbouring distinct biological features that are likely to progress to invasive breast cancer. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Probing the early stages of shock-induced chondritic meteorite formation at the mesoscale.

Chondritic meteorites are fragments of asteroids, the building blocks of planets, that retain a record of primordial processes. Important in their early evolution was impact-driven lithification, where a porous mixture of millimetre-scale chondrule inclusions and sub-micrometre dust was compacted into rock. In this Article, the shock compression of analogue precursor chondrite material was probed using state of the art dynamic X-ray radiography. Spatially-resolved shock and particle velocities, and shock front thicknesses were extracted directly from the radiographs, representing a greatly enhanced scope of data than could be measured in surface-based studies. A statistical interpretation of the measured velocities showed that mean values were in good agreement with those predicted using continuum-level modelling and mixture theory. However, the distribution and evolution of wave velocities and wavefront thicknesses were observed to be intimately linked to the mesoscopic structure of the sample. This Article provides the first detailed experimental insight into the distribution of extreme states within a shocked powder mixture, and represents the first mesoscopic validation of leading theories concerning the variation in extreme pressure-temperature states during the formation of primordial planetary bodies.

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials. Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Novel ABCA12 mutations in harlequin ichthyosis: a journey from photo diagnosis to prenatal diagnosis.

Harequin ichthyosis is a severe autosomal recessive ichthyosis of congenital onset caused by biallelic mutations in the ABCA12 gene. We report two neonates of Indian origin with harlequin ichthyosis. The parents were retrospectively found to have novel mutations in ABCA12 gene after neonatal demise, which helped in providing prenatal diagnosis in subsequent pregnancies.

Mutations in CSTA, encoding Cystatin A, underlie exfoliative ichthyosis and reveal a role for this protease inhibitor in cell-cell adhesion.

Autosomal-recessive exfoliative ichthyosis presents shortly after birth as dry, scaly skin over most of the body with coarse peeling of nonerythematous skin on the palms and soles, which is exacerbated by excessive moisture and minor trauma. Using whole-genome homozygosity mapping, candidate-gene analysis and deep sequencing, we have identified loss-of-function mutations in the gene for protease inhibitor cystatin A (CSTA) as the underlying genetic cause of exfoliative ichthyosis. We found two homozygous mutations, a splice-site and a nonsense mutation, in two consanguineous families of Bedouin and Turkish origin. Electron microscopy of skin biopsies from affected individuals revealed that the level of detachment occurs in the basal and lower suprabasal layers. In addition, in vitro modeling suggests that in the absence of cystatin A protein, there is a cell-cell adhesion defect in human keratinocytes that is particularly prominent when cells are subject to mechanical stress. We show here evidence of a key role for a protease inhibitor in epidermal adhesion within the lower layers of the human epidermis.

An anomalous basaltic meteorite from the innermost main belt.

Triangulated observations of fireballs allow us to determine orbits and fall positions for meteorites. The great majority of basaltic meteorites are derived from the asteroid 4 Vesta. We report on a recent fall that has orbital properties and an oxygen isotope composition that suggest a distinct parent body. Although its orbit was almost entirely contained within Earth's orbit, modeling indicates that it originated from the innermost main belt. Because the meteorite parent body would likely be classified as a V-type asteroid, V-type precursors for basaltic meteorites unrelated to Vesta may reside in the inner main belt. This starting location is in agreement with predictions of a planetesimal evolution model that postulates the formation of differentiated asteroids in the terrestrial planet region, with surviving fragments concentrated in the innermost main belt.

The impact rate on Earth.

Recent data, and modelling of the interaction between asteroids and the atmosphere, has defined a complete size-frequency distribution for terrestrial impactors, from meteorite-sized objects up to kilometre-sized asteroids, for both the upper atmosphere and the Earth's surface. Although there remain significant uncertainties in the incidence of specific size-fractions of impactors, these estimates allow us to constrain the threat posed by impacts to human populations. It is clear that impacts remain a significant natural hazard, but uniquely, they are a threat that we can accurately predict, and take steps to avoid.

Volatile fractionation in the early solar system and chondrule/matrix complementarity.

Bulk chondritic meteorites and terrestrial planets show a monotonic depletion in moderately volatile and volatile elements relative to the Sun's photosphere and CI carbonaceous chondrites. Although volatile depletion was the most fundamental chemical process affecting the inner solar nebula, debate continues as to its cause. Carbonaceous chondrites are the most primitive rocks available to us, and fine-grained, volatile-rich matrix is the most primitive component in these rocks. Several volatile depletion models posit a pristine matrix, with uniform CI-like chemistry across the different chondrite groups. To understand the nature of volatile fractionation, we studied minor and trace element abundances in fine-grained matrices of a variety of carbonaceous chondrites. We find that matrix trace element abundances are characteristic for a given chondrite group; they are depleted relative to CI chondrites, but are enriched relative to bulk compositions of their parent meteorites, particularly in volatile siderophile and chalcophile elements. This enrichment produces a highly nonmonotonic trace element pattern that requires a complementary depletion in chondrule compositions to achieve a monotonic bulk. We infer that carbonaceous chondrite matrices are not pristine: they formed from a material reservoir that was already depleted in volatile and moderately volatile elements. Additional thermal processing occurred during chondrule formation, with exchange of volatile siderophile and chalcophile elements between chondrules and matrix. This chemical complementarity shows that these chondritic components formed in the same nebula region.

The evolutionary and ecological benefits of asteroid and comet impacts.

Commonly viewed solely as agents of destruction, asteroid and comet impact events can also have a beneficial influence on processes from the molecular to the evolutionary scale. On the heavily bombarded early Earth, impacts might have delivered and caused the synthesis of prebiotic compounds that eventually led to life. At the organismal and ecosystem level, impact events can provide new habitats through the shock processing of target materials and by enhancing water availability, such as within intracrater lakes. At the evolutionary level, by destroying entire groups of organisms, impacts might have been instrumental in enabling the rise of new groups, such as the dinosaurs and mammals. Here, we synthesize the emerging literature on the beneficial effects of impacts to provide a novel perspective on these extraterrestrial agents of biological change.