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Dysregulated gene programs are a hallmark of cancer, and oncogenic transcription factors (TFs) have emerged as key players that contribute to this cell state. Chordoma is a rare cancer derived from deregulation of the TF brachyury (encoded by the T gene). While brachyury is normally turned off in development and is minimally expressed in healthy tissues, it remains highly expressed and is required in chordoma. TFs like brachyury have been challenging to drug directly, and chordoma represents an ideal system for studying both the biology behind a transcriptional dependency in cancer and how it can therapeutically targeted. Given that chordomas lack targeted therapies, we aim to understand the role of brachyury in the disease, identify therapeutic strategies to target its genetic regulation, and ultimately translate these findings towards the clinic. To achieve this, we sought to compare the consequences of direct and indirect brachyury targeting via targeted protein degradation and transcriptional CDK inhibition. In the absence of a brachyury small molecule, we generated human chordoma cell lines that express only a degradable form of brachyury. With these cell lines, we have mapped the brachyury regulome and have used high resolution microscopy to visualise brachyury genetic regulation. We find that brachyury is an ultra-selective regulator in chordoma, and that brachyury autoregulates through a transcriptional condensate. Both targeted brachyury degradation and transcriptional inhibition with THZ1, a preclinical tool compound inhibiting CDKs 7/12/13, can selectively inhibit T transcription and disrupt the brachyury transcriptional condensate. While these direct and indirect approaches converge to target brachyury, they have distinct phenotypic outcomes; brachyury degradation induces senescence and transcriptional inhibition causes apoptosis. Specifically, brachyury degradation primes cells to treatment with senolytics, which selectively kill senescent chordoma cells. Targeting senescence represents a novel therapeutic strategy in chordoma. Our findings affirm that brachyury is the most promising target in chordoma. We show that while transcriptional inhibition can selectively target brachyury, it mechanistically it contrasts with direct brachyury inhibition. However, these dual therapeutic strategies show that is indeed possible to progress in targeting oncoproteins that have long been considered “undruggable”, and our efforts not only benefit chordoma patients but provide a framework for drugging other transcriptionally addicted cancers. Citation Format: Hadley E. Sheppard, Alessandra Dall’Agnese, Woojun D. Park, M. Shamim Shamim, Julien Dubrulle, Hannah L. Johnson, Fabio Stossi, Josh Sommer, Joan Levy, Tanaz Sharifnia, Mathias Wawer, Behnam Nabet, Paul A. Clemons, Stuart L. Schreiber, Paul Clarke, Rick Young, Charles Y. Lin, Paul Workman. A characterisation of direct and indirect transcription factor inhibition to target brachyury in chordoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6355.

Introduction Long noncoding RNAs (lncRNAs) are profoundly dysregulated in multiple myeloma (MM), yet their tumor-promoting features remain to be defined. Here, we have coupled extensive transcriptomic profiling of patient samples (n=360) with a genome-wide CRISPR interference (CRISPRi) viability screen (4 cell lines) to generate a map of lncRNA "dependencies" in MM; moreover, we describe the molecular and functional role – as well as its therapeutic potential – of the screen top hit RNA Regulator of Lipogenesis (RROL), a nuclear-retained lncRNA generated by alternative splicing of MIR17HG. Methods Loss-of-function (LOF) studies using antisense oligonucleotides (ASO) confirmed the RROL dependency in a large panel of MM cell lines and CD138+ patient cells, in vitro and in vivo in animal models. This effect was not antagonized in DROSHA KO MM cells or by ectopic expression of MIR17HG-derived miR-17-92, indicating a microRNA-independent role of RROL. Results Transcriptomic analysis after RROL depletion indicated a significant impact on the de novo lipogenesis (DNL) gene networks. RROL occupancy at DNL gene loci was detected by chromatin isolation by RNA precipitation followed by qRT-PCR (ChIRP-qPCR) and confirmed by DUAL RNA FISH analysis. At functional level, we demonstrated that RROL depletion reduces DNL in MM cells using an unbiased lipidomic profiling as well as by measuring the incorporation of C14-radiolabeled glucose into lipids. Importantly, we also proved that exogenous palmitate, the downstream product of DNL pathway, can significantly rescue the growth inhibitory effect of RROL depletion in MM cells; thus implicating a role for DNL in the growth promoting effect of this lncRNA. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to identify c-MYC (MYC) as a relevant protein interactor of RROL; a finding validated by RIP-qPCR. Coupling RNA FISH with immunofluorescence, we co-detected RROL and MYC at the ACC1 promoter. Importantly, neither RROL or MYC could occupy ACC1 promoter and exert regulatory control in the absence of the other factor. Moreover, using in vitro (Co-IP/MS) and in vivo (BioID) assays, we identified WDR82 as an RROL-dependent MYC partner implicated in the transcriptional control of ACC1 expression. Finally, to therapeutically antagonize RROL, we have screened >100 ASOs and provided the basis to develop a first-in-class RROL inhibitor. This inhibitor has shown a very strong anti-MM activity in vitro (IC50 Conclusion In conclusion, we here report a unique regulatory function of a novel lncRNA supporting MM cell growth via its control of the lipogenic metabolic axis. The ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM.

Background With the deluge of available genomic data in recent years, it is becoming evident that MM cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We here elucidated the biological role of CDK7 and explored the functional consequence of its inhibition in MM using chemical and genetic approaches, including a recently reported selective CDK7 covalent inhibitor YKL-5-124, and engineered systems for rapid CDK7 protein degradation (dTAG). As previously shown with non-selective inhibitors, CDK7 inhibition via YKL-5-124 was active against a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. The efficacy of YKL-5-124 was confirmed in vivo in several murine MM models. Selective pharmacological degradation of endogenously tagged CDK7 and inducible KO/KD cell systems confirmed impact of CDK7 inhibition on MM cell proliferation supporting the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data support the notion of CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient cells. Conclusion Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1 respectively, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs. Isobologram analysis revealed strong synergism with a combination index (CI)

In this paper, we describe an analysis for data collected on a three-dimensional spatial lattice with treatments applied at the horizontal lattice points. Spatial correlation is accounted for using a conditional autoregressive model. Observations are defined as neighbours only if they are at the same depth. This allows the corresponding variance components to vary by depth. We use the Markov chain Monte Carlo method with block updating, together with Krylov subspace methods, for efficient estimation of the model. The method is applicable to both regular and irregular horizontal lattices and hence to data collected at any set of horizontal sites for a set of depths or heights, for example, water column or soil profile data. The model for the three-dimensional data is applied to agricultural trial data for five separate days taken roughly six months apart in order to determine possible relationships over time. The purpose of the trial is to determine a form of cropping that leads to less moist soils in the root zone and beyond.We estimate moisture for each date, depth and treatment accounting for spatial correlation and determine relationships of these and other parameters over time.

Modern technology now has the ability to generate large datasets over space and time. Such data typically exhibit high autocorrelations over all dimensions. The field trial data motivating the methods of this paper were collected to examine the behaviour of traditional cropping and to determine a cropping system which could maximise water use for grain production while minimising leakage below the crop root zone. They consist of moisture measurements made at 15 depths across 3 rows and 18 columns, in the lattice framework of an agricultural field. Bayesian conditional autoregressive (CAR) models are used to account for local site correlations. Conditional autoregressive models have not been widely used in analyses of agricultural data. This paper serves to illustrate the usefulness of these models in this field, along with the ease of implementation in WinBUGS, a freely available software package. The innovation is the fitting of separate conditional autoregressive models for each depth layer, the ‘layered CAR model’, while simultaneously estimating depth profile functions for each site treatment. Modelling interest also lies in how best to model the treatment effect depth profiles, and in the choice of neighbourhood structure for the spatial autocorrelation model. The favoured model fitted the treatment effects as splines over depth, and treated depth, the basis for the regression model, as measured with error, while fitting CAR neighbourhood models by depth layer. It is hierarchical, with separate conditional autoregressive spatial variance components at each depth, and the fixed terms which involve an errors-in-measurement model treat depth errors as interval-censored measurement error. The Bayesian framework permits transparent specification and easy comparison of the various complex models compared.

The use of hole opening tools in conjunction with Rotary Steerable Systems (RSS) has increased dramatically in the past few years. Although excellent performance has been delivered with the mainstream commercial tools, alternate options have been developed to enable an RSS to drill the pilot hole in conjunction with a string tool to open the hole in a single run. The most popular current option involves a weight or hydraulic activated underreamer. However, fixed blade, multi-diameter reaming tools have recently been developed for use within Rotary Steerable (RS) assemblies: An eccentric string reamer that will pass through a small pilot hole but then drill and produce a larger hole. This provides a significantly lower-cost hole opening option.A concentric string reamer that utilizes a tapered design incorporating a mid and main reamer section. This innovative design uses a mid-reamer that enlarges the pilot hole to an intermediate size and stabilizes the main reamer above it on its gauge pads. Because of its unique design, the mid reamer is able to stabilize the tool even if the pilot hole is of poor quality or is overgauge. This is particularly beneficial with RS tools in soft formations, leading to improved borehole quality and BHA stabilization. It also enables the use of smaller RS tools, thus providing greater directional potential. Additionally, a single RSS can be used to drill multiple hole size intervals, minimizing BHA changes, which in turn increases rig floor safety and lowers costs. Several distinct applications are documented where these fixed bladed solutions, in combination with both Push and Point RS tools, have proven extremely successful. This has provided greater flexibility with regard to tool selection and well planning options, delivering lower cost per foot in RS projects. Case studies are presented from the Far East, Norway, and the Gulf of Mexico.

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Management of grazing lands for the accumulation of soil carbon stocks (CS) has been proposed as an effective way to reduce net greenhouse gas emissions from agriculture. However, there are conflicting reports on the effects of grazing management on soil carbon. Most comparisons have involved some combination of no grazing, rotational grazing and set stocking. In the present study we compared two adjacent commercial grazing systems, distinguished on the basis of inputs and livestock productivity, located on New England basaltic landscapes experiencing a cool temperate climate. The high-intensity (H) system sustains an average stocking rate of 18 dry sheep equivalents (dse) ha–1, with a turnoff rate of 9dseha–1year–1, with high levels of investment in assets, management and fertiliser. The low-intensity (L) system, with less intensive management and half the fertiliser of the H system, sustains a stocking rate of 9dseha–1, with a turnoff rate of 3dseha–1year–1, which is slightly higher than the regional average. Pasture biomass production was inferred (back-calculated) from stocking rates and animal feed requirements using published data. From the H and L systems, seven paired landscapes from valley floor to upper hillslopes and plateaux were selected. The seventh included a forest reserve. One hundred and eighty-six undisturbed soil cores (0–0.5m depth) were assessed for bulk density, total C and N, particulate C and a range of plant nutrients. There were few differences in CS, soil pH and nutrient levels between H and L grazing systems. Average CS (0–0.3m) in pasture soils was 103Mgha–1, but was higher in the forest soil at 190Mgha–1. Regression of CS versus soil mass was a satisfactory method of dealing with the bias introduced by the higher soil bulk density in perennial pasture systems compared with the forest. The similarity of CS in H and L pasture soils was despite inferred net primary production being 1.9–3.6MgCha–1year–1 greater in H than L systems, implying higher rates of C turnover in the former. The global warming potential of the inferred annual emissions of CH4 and N2O in the H and L systems was equivalent to approximately 19% and 13% of the cycling atmospheric–plant CO2 carbon respectively.

While a variety of statistical models now exist for the spatio-temporal analysis of two-dimensional (surface) data collected over time, there are few published examples of analogous models for the spatial analysis of data taken over four dimensions: latitude, longitude, height or depth, and time. When taking account of the autocorrelation of data within and between dimensions, the notion of closeness often differs for each of the dimensions. Here, we consider a number of approaches to the analysis of such a dataset, which arises from an agricultural experiment exploring the impact of different cropping systems on soil moisture. The proposed models vary in their representation of the spatial correlation in the data, the assumed temporal pattern and choice of conditional autoregressive (CAR) and other priors. In terms of the substantive question, we find that response cropping is generally more effective than long fallow cropping in reducing soil moisture at the depths considered (100 cm to 220 cm). Thus, if we wish to reduce the possibility of deep drainage and increased groundwater salinity, the recommended cropping system is response cropping.