Despite advances in precision medicine, the clinical prospects for patients with ovarian and uterine cancers have not substantially improved. Here, we analyzed genome-scale CRISPR/Cas9 loss-of-function screens across 851 human cancer cell lines and found that frequent overexpression of SLC34A2 – encoding a phosphate importer – is correlated to sensitivity to loss of the phosphate exporter XPR1 in vitro and in vivo. In patient-derived tumor samples, we observed frequent PAX8-dependent overexpression of SLC34A2 , XPR1 copy number amplifications, and XPR1 mRNA overexpression. Mechanistically, in SLC34A2 -high cancer cell lines, genetic or pharmacologic inhibition of XPR1-dependent phosphate efflux leads to the toxic accumulation of intracellular phosphate. Finally, we show that XPR1 requires the novel partner protein KIDINS220 for proper cellular localization and activity, and that disruption of this protein complex results in acidic vacuolar structures preceding cell death. These data point to the XPR1:KIDINS220 complex and phosphate dysregulation as a therapeutic vulnerability in ovarian cancer.
Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.
The opioid epidemic led to an increase in the number of Neonatal Opioid Withdrawal Syndrome (NOWS) cases in infants born to opioid-dependent mothers. Hallmark features of NOWS include weight loss, severe irritability, respiratory problems, and sleep fragmentation. Mouse models provide an opportunity to identify brain mechanisms that contribute to NOWS. Neonatal outbred Swiss Webster Cartworth Farms White (CFW) mice were administered morphine (15mg/kg, s.c.) twice daily for postnatal days (P) 1-14, an approximate of the third trimester of human gestation. Female and male mice underwent behavioral testing on P7 and P14 to determine the impact of opioid exposure on anxiety and pain sensitivity. Ultrasonic vocalizations (USVs) and daily body weights were also recorded. Brainstems containing pons and medulla were collected during morphine withdrawal on P14 for RNA-sequencing. Morphine induced weight loss from P2-14, which persisted during adolescence (P21) and adulthood (P50). USVs markedly increased at P7 in females, emerging earlier than males. On P7 and P14, both morphine-exposed female and male mice displayed hyperalgesia on the hot plate and tail flick assays, with females showing greater hyperalgesia than males. Morphine-exposed mice exhibited increased anxiety-like behavior in the open-field arena on P21. Transcriptome analysis of the brainstem, an area implicated in opioid withdrawal and NOWS, identified pathways enriched for noradrenergic signaling in females and males. We also found sex-specific pathways related to mitochondrial function and neurodevelopment in females and circadian entrainment in males. Sex-specific transcriptomic neuroadaptations implicate unique neurobiological mechanisms underlying NOWS-like behaviors. 3 SIGNIFICANCE STATEMENTNeonatal opioid withdrawal syndrome (NOWS) is a poorly understood condition that has both a genetic and environmental component and is thought to be mechanistically distinct from opioid withdrawal in adults. The development of murine models for measuring neurobehavioral responses is critical for informing the neurobiological adaptations underlying NOWS. Using outbred mice that more closely model human genetic variation, we discovered several sex differences in behavioral timing and severity of NOWS-model behaviors as well as transcriptomic adaptations in brain tissue that together suggest distinct mechanisms and sex-specific therapeutics for reversing withdrawal symptoms and restoring brain function.
Rationale. Addiction to methamphetamine (MA) is a public health issue in the United States. While psychostimulant use disorders are heritable, their genetic basis remains poorly understood. We previously identified heterogeneous nuclear ribonucleoprotein H1 (Hnrnph1; H1) as a quantitative trait gene underlying sensitivity to MA-induced locomotor activity. Mice heterozygous for a frameshift deletion in the first coding exon of H1 (H1 +/-) showed reduced MA-induced locomotor activity, dopamine release, and dose-dependent differences in MA conditioned place preference. However, the effects of H1 +/on innate and MA-modulated reward sensitivity are not known.Objectives. We examined innate reward sensitivity and modulation by MA in H1 +/mice via intracranial selfstimulation (ICSS).Methods. We used intracranial self-stimulation (ICSS) of the medial forebrain bundle to assess shifts in reward sensitivity following acute, ascending doses of MA (0.5-4.0 mg/kg, i.p.) at 10 min or 2 h post-MA. We also assessed video-recorded behaviors during ICSS testing sessions.Results. Ten min post-MA, H1 +/mice displayed reduced maximum response rates, H1 +/females had lower M50 values than wild-type females, and H1 +/influenced ICSS responding relative to maximum control rates (MCR). Two h post-MA, higher response rates were observed in females, irrespective of genotype. There was a dose-dependent reduction in distance to the response wheel 10 min post-MA and reduced immobility time in the perimeter corners both 10 min and 2 h post-MA.Conclusions. H1 +/mice displayed altered MA-induced reward modulation in a time-, sex-, and dose-dependent manner. This expands the set of MA-induced phenotypes observed in H1 +/mice.
The opioid epidemic led to an increase in the number of Neonatal Opioid Withdrawal Syndrome (NOWS) cases in infants born to opioid-dependent mothers. Hallmark features of NOWS include weight loss, severe irritability, respiratory problems, and sleep fragmentation. Mouse models provide an opportunity to identify brain mechanisms that contribute to NOWS. Neonatal outbred Swiss Webster Cartworth Farms White (CFW) mice were administered morphine (15mg/kg, s.c.) twice daily for postnatal days (P) 1-14, an approximate of the third trimester of human gestation. Male and female mice underwent behavioral testing on P7 and P14 to determine the impact of opioid exposure on anxiety and pain sensitivity. Ultrasonic vocalizations (USVs) and daily body weights were also recorded. Brainstems containing pons and medulla were collected during morphine withdrawal on P14 for RNA-sequencing. Morphine induced weight loss from P2-14, which persisted during adolescence (P21) and adulthood (P50). USVs markedly increased at P7 in females, emerging earlier than males. On P7 and P14, both morphine exposed female and male mice displayed hyperalgesia on the hot plate and tail flick assays, with females having greater hyperalgesia than males. Morphine-exposed mice exhibited increased anxiety-like behavior in the open-field arena at P21. Transcriptome analysis of the brainstem (medulla plus pons), an area implicated in opioid withdrawal and NOWS, identified pathways enriched for noradrenergic signaling in females and males. We also found sex-specific pathways related to mitochondrial function and neurodevelopment in females and circadian entrainment in males. Sex-specific transcriptomic neuroadaptations implicate unique neurobiological mechanisms underlying NOWS-like behaviors.
Clinical outcomes for patients with ovarian and uterine cancers have not improved greatly in the past twenty years. To identify ovarian and uterine cancer vulnerabilities, we analyzed genome-scale CRISPR/Cas9 loss-of-function screens across 739 human cancer cell lines. We found that many ovarian cancer cell lines overexpress the phosphate importer SLC34A2, which renders them sensitive to loss of the phosphate exporter XPR1. We extensively validated the XPR1 vulnerability in cancer cell lines and found that the XPR1 dependency was retained in vivo. Overexpression of SLC34A2 is frequently observed in tumor samples and is regulated by PAX8 - a transcription factor required for ovarian cancer survival. XPR1 overexpression and copy number amplifications are also frequently observed. Mechanistically, SLC34A2 overexpression and impaired phosphate efflux leads to the accumulation of intracellular phosphate and cell death. We further show that proper localization and phosphate efflux by XPR1 requires a novel binding partner, KIDINS220. Loss of either XPR1 or KIDINS220 results in acidic vacuolar structures which precede cell death. These data point to the XPR1:KIDINS220 complex - and phosphate dysregulation more broadly - as a therapeutic vulnerability in ovarian cancer.
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