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The unprecedented medium-resolution ( R _λ ∼ 1500–3500) near- and mid-infrared (1–18 μ m) spectrum provided by JWST for the young (140 ± 20 Myr) low-mass (12–20 M _Jup ) L–T transition (L7) companion VHS 1256 b gives access to a catalog of molecular absorptions. In this study, we present a comprehensive analysis of this data set utilizing a forward-modeling approach applying our Bayesian framework, ForMoSA . We explore five distinct atmospheric models to assess their performance in estimating key atmospheric parameters: T _eff , log( g ), [M/H], C/O, γ , f _sed , and R . Our findings reveal that each parameter’s estimate is significantly influenced by factors such as the wavelength range considered and the model chosen for the fit. This is attributed to systematic errors in the models and their challenges in accurately replicating the complex atmospheric structure of VHS 1256 b, notably the complexity of its clouds and dust distribution. To propagate the impact of these systematic uncertainties on our atmospheric property estimates, we introduce innovative fitting methodologies based on independent fits performed on different spectral windows. We finally derived a T _eff consistent with the spectral type of the target, considering its young age, which is confirmed by our estimate of log( g ). Despite the exceptional data quality, attaining robust estimates for chemical abundances [M/H] and C/O, often employed as indicators of formation history, remains challenging. Nevertheless, the pioneering case of JWST’s data for VHS 1256 b has paved the way for future acquisitions of substellar spectra that will be systematically analyzed to directly compare the properties of these objects and correct the systematics in the models.

We present a performance analysis for the aperture masking interferometry (AMI) mode on board the James Webb Space Telescope Near Infrared Imager and Slitless Spectrograph (JWST/NIRISS). Thanks to self-calibrating observables, AMI accesses inner working angles down to and even within the classical diffraction limit. The scientific potential of this mode has recently been demonstrated by the Early Release Science (ERS) 1386 program with a deep search for close-in companions in the HIP 65426 exoplanetary system. As part of ERS 1386, we use the same data set to explore the random, static, and calibration errors of NIRISS AMI observables. We compare the observed noise properties and achievable contrast to theoretical predictions. We explore possible sources of calibration errors and show that differences in charge migration between the observations of HIP 65426 and point-spread function calibration stars can account for the achieved contrast curves. Lastly, we use self-calibration tests to demonstrate that with adequate calibration NIRISS F380M AMI can reach contrast levels of ∼9–10 mag at ≳ λ / D . These tests lead us to observation planning recommendations and strongly motivate future studies aimed at producing sophisticated calibration strategies taking these systematic effects into account. This will unlock the unprecedented capabilities of JWST/NIRISS AMI, with sensitivity to significantly colder, lower-mass exoplanets than lower-contrast ground-based AMI setups, at orbital separations inaccessible to JWST coronagraphy.

Summary: Combined BRAF and MEK inhibition is an effective treatment for BRAF-mutant cutaneous melanoma. However, most patients progress on this treatment due to drug resistance. Here, we applied the Sleeping Beauty transposon system to understand how melanoma evades MAPK inhibition. We found that the specific drug resistance mechanisms differed across melanomas in our genetic screens of five cutaneous melanoma cell lines. While drivers that reactivated MAPK were highly conserved, many others were cell-line specific. One such driver, VAV1, activated a de-differentiated transcriptional program like that of hyperactive RAC1, RAC1P29S. To target this mechanism, we showed that an inhibitor of SRC, saracatinib, blunts the VAV1-induced transcriptional reprogramming. Overall, we highlighted the importance of accounting for melanoma heterogeneity in treating cutaneous melanoma with MAPK inhibitors. Moreover, we demonstrated the utility of the Sleeping Beauty transposon system in understanding cancer drug resistance.

Abstract Rare gain-of-function mutations in RAC1 drive drug resistance to targeted BRAF inhibition in cutaneous melanoma. Here, we show that wildtype RAC1 is a critical driver of growth and drug resistance, but only in a subset of melanomas with elevated markers of de-differentiation. Similarly, SRC inhibition also selectively sensitized de-differentiated melanomas to BRAF inhibition. One possible mechanism may be the suppression of the de-differentiated state, as SRC and RAC1 maintained markers of de-differentiation in human melanoma cells. The functional differences between melanoma subtypes suggest that the clinical management of cutaneous melanoma can be enhanced by the knowledge of differentiation status. To simplify the task of classification, we developed a binary classification strategy based on a small set of ten genes. Using this gene set, we reliably determined the differentiation status previously defined by hundreds of genes. Overall, our study informs strategies that enhance the precision of BRAFi by discovering unique vulnerabilities of the de-differentiated cutaneous melanoma subtype and creating a practical method to resolve differentiation status.

Small planets ( R _p ≤ 4 R _⊕ ) are divided into rocky super-Earths and gaseous sub-Neptunes separated by a radius gap, but the mechanisms that produce these distinct planet populations remain unclear. Binary stars are the only main-sequence systems with an observable record of the protoplanetary disk lifetime and mass reservoir, and the demographics of planets in binaries may provide insights into planet formation and evolution. To investigate the radius distribution of planets in binary star systems, we observed 207 binary systems hosting 283 confirmed and candidate transiting planets detected by the Kepler mission, then recharacterized the planets while accounting for the observational biases introduced by the secondary star. We found that the population of planets in close binaries ( ρ ≤ 100 au) is significantly different from the planet population in wider binaries ( ρ > 300 au) or single stars. In contrast to planets around single stars, planets in close binaries appear to have a unimodal radius distribution with a peak near the expected super-Earth peak of R _p ∼ 1.3 R _⊕ and a suppressed population of sub-Neptunes. We conclude that we are observing the direct impact of a reduced disk lifetime, smaller mass reservoir, and possible altered distribution of solids reducing the sub-Neptune formation efficiency. Our results demonstrate the power of binary stars as a laboratory for exploring planet formation and as a controlled experiment of the impact of varied initial conditions on mature planet populations.

We present the most precise constraints to date for the mass and age distributions of single ultracool dwarfs in the solar neighborhood, based on an updated volume-limited sample of 504 L, T, and Y dwarfs within 25 pc. We develop a Monte Carlo approach using the $\langle V/{V}_{\max }\rangle $ statistic to correct for incompleteness and obtain a space density of $({1.83}_{-0.15}^{+0.16})\times {10}^{-2}$ pc ^−3 for spectral types L0–Y2. We calculate bolometric luminosities for our sample, using an updated “super-magnitude” method for the faintest objects. We use our resulting luminosity function and a likelihood-based population synthesis approach to simultaneously constrain the mass and age distributions. We employ the fraction of young L0–L7 dwarfs as a novel input for this analysis that is crucial for constraining the age distribution. For a power-law mass function $\tfrac{{dN}}{{dM}}\propto {M}^{-\alpha }$ , we find $\alpha ={0.58}_{-0.20}^{+0.16}$ , indicating an increase in numbers toward lower masses, consistent with measurements in nearby star-forming regions. For an exponential age distribution b ( t ) ∝ e ^− ^β ^t we find β = −0.44 ± 0.14, i.e., a population with fewer old objects than often assumed, which may reflect dynamical heating of the Galactic plane as much as the historical brown dwarf birthrate. We compare our analysis to that of Kirkpatrick et al., who used a similar volume-limited sample. Although our mass function measurements are numerically consistent, their assumption of a flat age distribution is disfavored by our analysis, and we identify several important methodological differences between our two studies. Our calculation of the age distribution of solar neighborhood brown dwarfs is the first based on a volume-limited sample.

We report dynamical mass measurements of the individual stars in the most luminous and massive stellar member of the nearby Ophiuchus star-forming region, the young tight binary system S1. We combine 28 archival data sets with seven recent proprietary Very Long Baseline Array (VLBA) observations obtained as part of the Dynamical Masses of Young Stellar Multiple Systems with the VLBA project (DYNAMO–VLBA), to constrain the astrometric and orbital parameters of the system, and recover high-accuracy dynamical masses. The primary component, S1A, is found to have a mass of 4.11 ± 0.10 M _⊙ , significantly lower than the typical value ∼6 M _⊙ previously reported in the literature. We show that the spectral energy distribution (SED) of S1A can be reproduced by a reddened blackbody with a temperature between roughly 14,000 and 17,000 K. According to evolutionary models, this temperature range corresponds to stellar masses between 4 M _⊙ and 6 M _⊙ , so the SED is not a priori inconsistent with the dynamical mass of S1A. The luminosity of S1 derived from SED fitting, however, is only consistent with models for stellar masses above 5 M _⊙ . Thus, we cannot reconcile the evolutionary models with the dynamical mass measurement of S1A: The models consistent with the location of S1A in the Hertzsprung-Russel diagram correspond to masses higher by 25% at least than the dynamical mass. For the secondary component, S1B, a mass of 0.831 ± 0.014 M _⊙ is determined, consistent with a low-mass young star. While the radio flux of S1A remains roughly constant throughout the orbit, the flux of S1B is found to be higher near apastron.

Abstract Background The mechanism of action for most cancer drugs is not clear. Large-scale pharmacogenomic cancer cell line datasets offer a rich resource to obtain this knowledge. Here, we present an analysis strategy for revealing biological pathways that contribute to drug response using publicly available pharmacogenomic cancer cell line datasets. Methods We present a custom machine-learning based approach for identifying biological pathways involved in cancer drug response. We test the utility of our approach with a pan-cancer analysis of ML210, an inhibitor of GPX4, and a melanoma-focused analysis of inhibitors of BRAF V600 . We apply our approach to reveal determinants of drug resistance to microtubule inhibitors. Results Our method implicated lipid metabolism and Rac1/cytoskeleton signaling in the context of ML210 and BRAF inhibitor response, respectively. These findings are consistent with current knowledge of how these drugs work. For microtubule inhibitors, our approach implicated Notch and Akt signaling as pathways that associated with response. Conclusions Our results demonstrate the utility of combining informed feature selection and machine learning algorithms in understanding cancer drug response.

We present JWST Early Release Science coronagraphic observations of the super-Jupiter exoplanet, HIP 65426b, with the Near-Infrared Camera (NIRCam) from 2 to 5 μ m, and with the Mid-Infrared Instrument (MIRI) from 11 to 16 μ m. At a separation of ∼0.″82 (87 ${}_{-31}^{+108}$ au), HIP 65426b is clearly detected in all seven of our observational filters, representing the first images of an exoplanet to be obtained by JWST, and the first-ever direct detection of an exoplanet beyond 5 μ m. These observations demonstrate that JWST is exceeding its nominal predicted performance by up to a factor of 10, depending on separation and subtraction method, with measured 5 σ contrast limits of ∼1 × 10 ^−5 and ∼2 × 10 ^−4 at 1″ for NIRCam at 4.4 μ m and MIRI at 11.3 μ m, respectively. These contrast limits provide sensitivity to sub-Jupiter companions with masses as low as 0.3 M _Jup beyond separations of ∼100 au. Together with existing ground-based near-infrared data, the JWST photometry are fit well by a BT-SETTL atmospheric model from 1 to 16 μ m, and they span ∼97% of HIP 65426b's luminous range. Independent of the choice of model atmosphere, we measure an empirical bolometric luminosity that is tightly constrained between $\mathrm{log}\left({L}_{\mathrm{bol}}/{L}_{\odot }\right)$ = −4.31 and −4.14, which in turn provides a robust mass constraint of 7.1 ± 1.2 M _Jup . In totality, these observations confirm that JWST presents a powerful and exciting opportunity to characterize the population of exoplanets amenable to high-contrast imaging in greater detail.

We present the direct-imaging discovery of a giant planet orbiting the young star AF Lep, a 1.2 M _⊙ member of the 24 ± 3 Myr β Pic moving group. AF Lep was observed as part of our ongoing high-contrast imaging program targeting stars with astrometric accelerations between Hipparcos and Gaia that indicate the presence of substellar companions. Keck/NIRC2 observations in $L^{\prime} $ with the vector vortex coronagraph reveal a point source, AF Lep b, at ≈340 mas, which exhibits orbital motion at the 6 σ level over the course of 13 months. A joint orbit fit yields precise constraints on the planet’s dynamical mass of ${3.2}_{-0.6}^{+0.7}$ M _Jup , semimajor axis of ${8.4}_{-1.3}^{+1.1}$ au, and eccentricity of ${0.24}_{-0.15}^{+0.27}$ . AF Lep hosts a debris disk located at ∼50 au, but it is unlikely to be sculpted by AF Lep b, implying there may be additional planets in the system at wider separations. The stellar inclination ( i _* = ${54}_{-9}^{{+11}^\circ} $ ) and orbital inclination ( i _o = ${50}_{-12}^{{+9}^\circ} $ ) are in good agreement, which is consistent with the system having spin–orbit alignment. AF Lep b is the lowest-mass imaged planet with a dynamical mass measurement and highlights the promise of using astrometric accelerations as a tool to find and characterize long-period planets.

We present the highest fidelity spectrum to date of a planetary-mass object. VHS 1256 b is a

Transposon based screens carried out in mice can identify genes critical for tumourigensis. Here, the authors describe transposon screens in mouse models of breast cancer and highlight a large group of tumour suppressors that could underlie selection for common chromosome arm losses in cancer.

We derive the bolometric luminosities ( L _bol ) of 865 field-age and 189 young ultracool dwarfs (spectral types M6–T9, including 40 new discoveries presented here) by directly integrating flux-calibrated optical to mid-infrared (MIR) spectral energy distributions (SEDs). The SEDs consist of low-resolution ( R ∼ 150) near-infrared (NIR; 0.8–2.5 μ m) spectra (including new spectra for 97 objects), optical photometry from the Pan-STARRS1 survey, and MIR photometry from the CatWISE2020 survey and Spitzer/IRAC. Our L _bol calculations benefit from recent advances in parallaxes from Gaia, Spitzer, and UKIRT, as well as new parallaxes for 19 objects from CFHT and Pan-STARRS1 presented here. Coupling our L _bol measurements with a new uniform age analysis for all objects, we estimate substellar masses, radii, surface gravities, and effective temperatures ( T _eff ) using evolutionary models. We construct empirical relationships for L _bol and T _eff as functions of spectral type and absolute magnitude, determine bolometric corrections in optical and infrared bandpasses, and study the correlation between evolutionary model-derived surface gravities and NIR gravity classes. Our sample enables a detailed characterization of BT-Settl and ATMO 2020 atmospheric model systematics as a function of spectral type and position in the NIR color–magnitude diagram. We find the greatest discrepancies between atmospheric and evolutionary model-derived T _eff (up to 800 K) and radii (up to 2.0 R _Jup ) at the M/L spectral type transition boundary. With 1054 objects, this work constitutes the largest sample to date of ultracool dwarfs with determinations of their fundamental parameters.

We present the latest and most precise characterization of the architecture for the ancient (≈11 Gyr) Kepler-444 system, which is composed of a K0 primary star (Kepler-444 A) hosting five transiting planets and a tight M-type spectroscopic binary (Kepler-444 BC) with an A–BC projected separation of 66 au. We have measured the system’s relative astrometry using the adaptive optics imaging from Keck/NIRC2 and Kepler-444 A’s radial velocities from the Hobby-Eberly Telescope and reanalyzed relative radial velocities between BC and A from Keck/HIRES. We also include the Hipparcos-Gaia astrometric acceleration and all published astrometry and radial velocities in an updated orbit analysis of BC’s barycenter. These data greatly extend the time baseline of the monitoring and lead to significant updates to BC’s barycentric orbit compared to previous work, including a larger semimajor axis ( $a={52.2}_{-2.7}^{+3.3}$ au), a smaller eccentricity ( e = 0.55 ± 0.05), and a more precise inclination ( $i=85\buildrel{\circ}\over{.} {4}_{-0\buildrel{\circ}\over{.} 4}^{+0\buildrel{\circ}\over{.} 3}$ ). We have also derived the first dynamical masses of B and C components. Our results suggest that Kepler-444 A’s protoplanetary disk was likely truncated by BC to a radius of ≈8 au, which resolves the previously noticed tension between Kepler-444 A’s disk mass and planet masses. Kepler-444 BC’s barycentric orbit is likely aligned with those of A’s five planets, which might be primordial or a consequence of dynamical evolution. The Kepler-444 system demonstrates that compact multiplanet systems residing in hierarchical stellar triples can form at early epochs of the universe and survive their secular evolution throughout cosmic time.

Benchmark brown dwarf companions with well-determined ages and model-independent masses are powerful tools to test substellar evolutionary models and probe the formation of giant planets and brown dwarfs. Here, we report the independent discovery of HIP 21152 B, the first imaged brown dwarf companion in the Hyades, and conduct a comprehensive orbital and atmospheric characterization of the system. HIP 21152 was targeted in an ongoing high-contrast imaging campaign of stars exhibiting proper-motion changes between Hipparcos and Gaia, and was also recently identified by Bonavita et al. (2022) and Kuzuhara et al. (2022). Our Keck/NIRC2 and SCExAO/CHARIS imaging of HIP 21152 revealed a comoving companion at a separation of 0.″37 (16 au). We perform a joint orbit fit of all available relative astrometry and radial velocities together with the Hipparcos-Gaia proper motions, yielding a dynamical mass of ${24}_{-4}^{+6}\,{M}_{\mathrm{Jup}}$ , which is 1–2 σ lower than evolutionary model predictions. Hybrid grids that include the evolution of cloud properties best reproduce the dynamical mass. We also identify a comoving wide-separation (1837″ or 7.9 × 10 ^4 au) early-L dwarf with an inferred mass near the hydrogen-burning limit. Finally, we analyze the spectra and photometry of HIP 21152 B using the Saumon & Marley (2008) atmospheric models and a suite of retrievals. The best-fit grid-based models have f _sed = 2, indicating the presence of clouds, T _eff = 1400 K, and $\mathrm{log}g=4.5\,\mathrm{dex}$ . These results are consistent with the object’s spectral type of T0 ± 1. As the first benchmark brown dwarf companion in the Hyades, HIP 21152 B joins the small but growing number of substellar companions with well-determined ages and dynamical masses.

Orbital eccentricities directly trace the formation mechanisms and dynamical histories of substellar companions. Here, we study the effect of hyperpriors on the population-level eccentricity distributions inferred for the sample of directly imaged substellar companions (brown dwarfs and cold Jupiters) from hierarchical Bayesian modeling (HBM). We find that the choice of hyperprior can have a significant impact on the population-level eccentricity distribution inferred for imaged companions, an effect that becomes more important as the sample size and orbital coverage decrease to values that mirror the existing sample. We reanalyze the current observational sample of imaged giant planets in the 5–100 au range from Bowler et al. and find that the underlying eccentricity distribution implied by the imaged planet sample is broadly consistent with the eccentricity distribution for close-in exoplanets detected using radial velocities. Furthermore, our analysis supports the conclusion from that study that long-period giant planets and brown dwarf eccentricity distributions differ by showing that it is robust to the choice of hyperprior. We release our HBM and forward-modeling code in an open-source Python package, ePop! , and make it freely available to the community.

Abstract Background The introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods. Results Here we describe a simple, reproducible approach using the SB transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells. Conclusions Collectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.

Patients with malignant melanoma have a 5-year survival rate of only 15–20% once the tumor has metastasized to distant tissues. While MAP kinase pathway inhibitors (MAPKi) are initially effective for the majority of patients with melanoma harboring BRAFV600E mutation, over 90% of patients relapse within 2 years. Thus, there is a critical need for understanding MAPKi resistance mechanisms. In this manuscript, we performed a forward genetic screen using a whole genome shRNA library to identify negative regulators of vemurafenib resistance. We identified loss of NF1 and CUL3 as drivers of vemurafenib resistance. NF1 is a known driver of vemurafenib resistance in melanoma through its action as a negative regulator of RAS. However, the mechanism by which CUL3, a key protein in E3 ubiquitin ligase complexes, is involved in vemurafenib resistance was unknown. We found that loss of CUL3 was associated with an increase in RAC1 activity and MEKS298 phosphorylation. However, the addition of the Src family inhibitor saracatinib prevented resistance to vemurafenib in CUL3KD cells and reversed RAC1 activation. This finding suggests that inhibition of the Src family suppresses MAPKi resistance in CUL3KD cells by inactivation of RAC1. Our results also indicated that the loss of CUL3 does not promote the activation of RAC1 through stabilization, suggesting that CUL3 is involved in the stability of upstream regulators of RAC1. Collectively, our study identifies the loss of CUL3 as a driver of MAPKi resistance through activation of RAC1 and demonstrates that inhibition of the Src family can suppress the MAPKi resistance phenotype in CUL3KD cells by inactivating RAC1 protein.

Summary: Given its role as the source of definitive hematopoietic cells, we sought to determine whether mutations initiated in the hemogenic endothelium would yield hematopoietic abnormalities or malignancies. Here, we find that endothelium-specific transposon mutagenesis in mice promotes hematopoietic pathologies that are both myeloid and lymphoid in nature. Frequently mutated genes included previously recognized cancer drivers and additional candidates, such as Pi4ka, a lipid kinase whose mutation was found to promote myeloid and erythroid dysfunction. Subsequent validation experiments showed that targeted inactivation of the Pi4ka catalytic domain or reduction in mRNA expression inhibited myeloid and erythroid cell differentiation in vitro and promoted anemia in vivo through a mechanism involving deregulation of AKT, MAPK, SRC, and JAK-STAT signaling. Finally, we provide evidence linking PI4KAP2, previously considered a pseudogene, to human myeloid and erythroid leukemia. : Using transposon mutagenesis that targets the endothelium, Ziyad et al. identify Pi4ka as an important regulator of hematopoiesis. Loss of Pi4ka inhibits myeloid and erythroid cell differentiation. Previously considered a pseudogene in humans, PI4KAP2 is shown to be protein-coding and a negative regulator of PI4KA signaling. Keywords: erythropoiesis, myelopoiesis, hematopoiesis, leukemia, hemogenic endothelium, lipid kinase, Pi4ka, PI4KAP2, Akt, Erk

Objective: Excessive hepatic gluconeogenesis is a defining feature of type 2 diabetes (T2D). Most gluconeogenic flux is routed through mitochondria. The mitochondrial pyruvate carrier (MPC) transports pyruvate from the cytosol into the mitochondrial matrix, thereby gating pyruvate-driven gluconeogenesis. Disruption of the hepatocyte MPC attenuates hyperglycemia in mice during high fat diet (HFD)-induced obesity but exerts minimal effects on glycemia in normal chow diet (NCD)-fed conditions. The goal of this investigation was to test whether hepatocyte MPC disruption provides sustained protection from hyperglycemia during long-term HFD and the differential effects of hepatocyte MPC disruption on TCA cycle metabolism in NCD versus HFD conditions. Method: We utilized long-term high fat feeding, serial measurements of postabsorptive blood glucose and metabolomic profiling and 13C-lactate/13C-pyruvate tracing to investigate the contribution of the MPC to hyperglycemia and altered hepatic TCA cycle metabolism during HFD-induced obesity. Results: Hepatocyte MPC disruption resulted in long-term attenuation of hyperglycemia induced by HFD. HFD increased hepatic mitochondrial pyruvate utilization and TCA cycle capacity in an MPC-dependent manner. Furthermore, MPC disruption decreased progression of fibrosis and levels of transcript markers of inflammation. Conclusions: By contributing to chronic hyperglycemia, fibrosis, and TCA cycle expansion, the hepatocyte MPC is a key mediator of the pathophysiology induced in the HFD model of T2D.

Summary: Hepatocellular carcinoma (HCC) is a devastating cancer increasingly caused by non-alcoholic fatty liver disease (NAFLD). Disrupting the liver Mitochondrial Pyruvate Carrier (MPC) in mice attenuates NAFLD. Thus, we considered whether liver MPC disruption also prevents HCC. Here, we use the N-nitrosodiethylamine plus carbon tetrachloride model of HCC development to test how liver-specific MPC knock out affects hepatocellular tumorigenesis. Our data show that liver MPC ablation markedly decreases tumorigenesis and that MPC-deficient tumors transcriptomically downregulate glutathione metabolism. We observe that MPC disruption and glutathione depletion in cultured hepatomas are synthetically lethal. Stable isotope tracing shows that hepatocyte MPC disruption reroutes glutamine from glutathione synthesis into the tricarboxylic acid (TCA) cycle. These results support a model where inducing metabolic competition for glutamine by MPC disruption impairs hepatocellular tumorigenesis by limiting glutathione synthesis. These findings raise the possibility that combining MPC disruption and glutathione stress may be therapeutically useful in HCC and additional cancers. : Tompkins et al. utilize stable glutamine isotope tracers in vivo and ex vivo to demonstrate hepatocyte MPC disruption increases TCA cycle glutamine utilization at the expense of glutathione synthesis and decreases hepatocellular tumorigenesis. Keywords: cancer, glutamine, glutathione, hepatocellular carcinoma, metabolomics, liver, Mitochondrial Pyruvate Carrier, stable isotope tracing, synthetic lethality

The Sleeping Beauty (SB) transposon insertional mutagenesis system offers a streamlined approach to identify genetic drivers of cancer. With a relatively random insertion profile, SB is uniquely positioned for conducting unbiased forward genetic screens. Indeed, SB mouse models of cancer have revealed insights into the genetics of tumorigenesis. In this review, we highlight experiments that have exploited the SB system to interrogate the genetics of cancer in distinct biological contexts. We also propose experimental designs that could further our understanding of the relationship between tumor microenvironment and tumor progression.

Genetic mouse models of soft tissue sarcoma provide critical insights into disease pathophysiology, which are oftentimes unable to be extracted from human tumor samples or xenograft models. In this study we describe a mouse model of soft tissue sarcoma mediated by adenoviral-Cre recombinase injection into Trp53fl/fl/Ptenfl/fl lox-stop-lox luciferase mice. Injection of adenovirus expressing Cre recombinase, either subcutaneously or intramuscularly in two experimental groups, results in viral infection and gene recombination with 100% penetrance within the first 24 hours following injection. Luciferase expression measured by real-time bioluminescence imaging increases over time, with an initial robust increase following viral injection, followed by a steady rise over the next several weeks as primary tumors develop and grow. Intramuscular injections were more commonly associated with evidence of systemic viral distribution than subcutaneous injections. All mice developed soft tissue sarcomas at the primary injection site, with histological examination identifying 93% of tumors as invasive pleomorphic sarcomas based on microscopic morphology and immunohistochemical expression of sarcoma markers. A lymphocytic infiltrate was present in 64% of the sarcomas in this immunocompetent model and 71% of tumors expressed PD-L1. This is the first report of a viral-Cre mediated Trp53/Pten mouse model of undifferentiated pleomorphic sarcoma. The bioluminescence imaging feature, along with high penetrance of the model and its immunological characteristics, makes it suited for pre-clinical studies of soft tissue sarcoma.

Recently we described skin tumors driven by skin-specific expression of Zmiz1 and here we define keratoacanthoma pathobiology in this mouse model. Similar to human keratoacanthoma development, we were able to segregate murine keratoacanthomas into three developmental phases: growth, maturation, and regression. These tumors had areas with cellular atypia, high mitotic rate, and minor local invasion in the growth phase, but with development they transitioned to maturation and regression phases with evidence of resolution. The early aggressive appearance could easily be misdiagnosed as a malignant change if the natural pathobiology was not well-defined in the model. To corroborate these findings in the Zmiz1 model, we examined squamous skin tumors from another tumor study in aging mice, and these tumors followed a similar biological progression. Lastly, we were able to evaluate the utility of the model to assess immune cell infiltration (F4/80, B220 Granzyme B, CD3 cells, arginase-1) in the regression phase; however, because inflammation was present at all phases of development, a more comprehensive approach will be needed in future investigations. Our study of keratoacanthomas in selected murine models suggests that these squamous tumors can appear histologically aggressive during early development, but with time will enter a regression phase indicating a benign biology. Importantly, studies of squamous skin tumor models should be cautious in tumor diagnosis as the early growth distinction between malignant versus benign based solely on histopathology may not be easily discerned without longitudinal studies to confirm the tumor pathobiology.

We previously utilized a Sleeping Beauty (SB) transposon mutagenesis screen to discover novel drivers of HCC. This approach identified recurrent mutations within the Dlk1-Dio3 imprinted domain, indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis. For the current study, we performed transcriptome and small RNA sequencing to profile gene expression in SB-induced HCCs in an attempt to clarify the genetic element(s) contributing to tumorigenesis. We identified strong induction of Retrotransposon-like 1 (Rtl1) expression as the only consistent alteration detected in all SB-induced tumors with Dlk1-Dio3 integrations, suggesting that Rtl1 activation serves as a driver of HCC. While previous studies have identified correlations between disrupted expression of multiple Dlk1-Dio3 domain members and HCC, we show here that direct modulation of a single domain member, Rtl1, can promote hepatocarcinogenesis in vivo. Overexpression of Rtl1 in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant (86%) tumor formation. Additionally, we detected overexpression of RTL1 in 30% of analyzed human HCC samples, indicating the potential relevance of this locus as a therapeutic target for patients. The Rtl1 locus is evolutionarily derived from the domestication of a retrotransposon. In addition to identifying Rtl1 as a novel driver of HCC, our study represents one of the first direct in vivo demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis.

Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma (HCC). Although these conditions also lead to integrated stress response (ISR) or unfolded protein response (UPR) activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is up-regulated in liver tumors in human HCC and two mouse models thereof. Chop-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. Chop-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases.