Mapping transgender policies in the US 2017-2021: The role of geography and implications for health equity.

Transgender individuals globally face varying policy contexts that can influence their health. In the United States (US), a patchwork of exclusionary and inclusive policies exists, creating potentially different social and political contexts that shape transgender health depending on the state. In this article, we consider how recent legislation introduced in US states focused on transgender people may be a political determinant of health and affect health equity goals. To advance this aim, we employed the perspective of legal epidemiology to systematically search a US legal database (Westlaw) for policies focused on transgender individuals proposed between January of 2017 and September of 2021.698 policies were analyzed as affirming or exclusionary of transgender identities and categorized by content. We calculated a ratio of affirming versus exclusionary bills to create "exclusionary density" and "affirming density" measures. Those measures were used to calculate an inclusivity score and corresponding maps of inclusivity and exclusionary contexts by US state. Exclusionary and affirming density measures showed deeply polarized policy responses to transgender individuals depending on US state. Further, we observed differences in magnitude regarding the laws being proposed. Exclusionary laws largely focused on criminalization while inclusionary laws focused on representation in government agencies. These findings highlight that transgender individuals in the US can experience vastly different political contexts depending on where they live.

Sequential Administration of XPO1 and ATR Inhibitors Enhances Therapeutic Response in TP53-mutated Colorectal Cancer.

BACKGROUND & AIMS: Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. METHODS: To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. RESULTS: Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. CONCLUSIONS: Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.

High-throughput screening against protein:protein interaction interfaces reveals anti-cancer therapeutics as potent modulators of the voltage-gated Na+ channel complex.

Multiple voltage-gated Na+ (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex. Fibroblast growth factor 14 (FGF14) is a functionally relevant component of the Nav1.6 channel complex, a causative link to spinocerebellar ataxia 27 (SCA27) and an emerging risk factor for neuropsychiatric disorders. Yet, how this protein:channel complex is regulated in the cell is still poorly understood. To search for key cellular pathways upstream of the FGF14:Nav1.6 complex, we have developed, miniaturized and optimized an in-cell assay in 384-well plates by stably reconstituting the FGF14:Nav1.6 complex using the split-luciferase complementation assay. We then conducted a high-throughput screening (HTS) of 267 FDA-approved compounds targeting known mediators of cellular signaling. Of the 65 hits initially detected, 24 were excluded based on counter-screening and cellular toxicity. Based on target analysis, potency and dose-response relationships, 5 compounds were subsequently repurchased for validation and confirmed as hits. Among those, the tyrosine kinase inhibitor lestaurtinib was highest ranked, exhibiting submicromolar inhibition of FGF14:Nav1.6 assembly. While providing evidence for a robust in-cell HTS platform that can be adapted to search for any channelopathy-associated regulatory proteins, these results lay the potential groundwork for repurposing cancer drugs for neuropsychopharmacology.

Driver network as a biomarker: systematic integration and network modeling of multi-omics data to derive driver signaling pathways for drug combination prediction.

MOTIVATION: Drug combinations that simultaneously suppress multiple cancer driver signaling pathways increase therapeutic options and may reduce drug resistance. We have developed a computational systems biology tool, DrugComboExplorer, to identify driver signaling pathways and predict synergistic drug combinations by integrating the knowledge embedded in vast amounts of available pharmacogenomics and omics data. RESULTS: This tool generates driver signaling networks by processing DNA sequencing, gene copy number, DNA methylation and RNA-seq data from individual cancer patients using an integrated pipeline of algorithms, including bootstrap aggregating-based Markov random field, weighted co-expression network analysis and supervised regulatory network learning. It uses a systems pharmacology approach to infer the combinatorial drug efficacies and synergy mechanisms through drug functional module-induced regulation of target expression analysis. Application of our tool on diffuse large B-cell lymphoma and prostate cancer demonstrated how synergistic drug combinations can be discovered to inhibit multiple driver signaling pathways. Compared with existing computational approaches, DrugComboExplorer had higher prediction accuracy based on in vitro experimental validation and probability concordance index. These results demonstrate that our network-based drug efficacy screening approach can reliably prioritize synergistic drug combinations for cancer and uncover potential mechanisms of drug synergy, warranting further studies in individual cancer patients to derive personalized treatment plans. AVAILABILITY AND IMPLEMENTATION: DrugComboExplorer is available at https://github.com/Roosevelt-PKU/drugcombinationprediction. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comprehensive pharmacogenomic profiling of human papillomavirus-positive and -negative squamous cell carcinoma identifies sensitivity to aurora kinase inhibition in KMT2D mutants.

To address the unmet need for effective biomarker-driven targeted therapy for human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) and cervical cancer, we conducted a high-throughput drug screen using 1122 compounds in 13 HPV-positive and 11 matched HPV-negative cell lines. The most effective drug classes were inhibitors of polo-like kinase, proteasomes, histone deacetylase, and Aurora kinases. Treatment with a pan-Aurora inhibitor, danusertib, led to G2M arrest and apoptosis in vitro. Furthermore, danusertib decreased tumor size compared with controls in patient derived xenograft models of HNSCC. To identify biomarkers predicting response, we determined associations between mutations and drug sensitivity. Our data and the Genomics of Drug Sensitivity in Cancer database showed that cancer cells with KMT2D mutations were more sensitive to Aurora kinase inhibitors than were cells without mutations. Knockdown of KMT2D in wild-type cells led to increased Aurora kinase inhibitor-induced apoptosis. We identified Aurora kinase inhibitors as effective and understudied drugs in HNSCC and CESC. This is the first published study to demonstrate that mutations in KMT2D, which are common in many cancers, correlate with drug sensitivity in two independent datasets.

The polo-like kinase 1 inhibitor volasertib synergistically increases radiation efficacy in glioma stem cells.

BACKGROUND: Despite the availability of hundreds of cancer drugs, there is insufficient data on the efficacy of these drugs on the extremely heterogeneous tumor cell populations of glioblastoma (GBM). RESULTS: The PKIS of 357 compounds was initially evaluated in 15 different GSC lines which then led to a more focused screening of the 21 most highly active compounds in 11 unique GSC lines using HTS screening for cell viability. We further validated the HTS result with the second-generation PLK1 inhibitor volasertib as a single agent and in combination with ionizing radiation (IR). In vitro studies showed that volasertib inhibited cell viability, and high levels of the anti-apoptotic protein Bcl-xL expression were highly correlated with volasertib resistance. Volasertib sensitized GSCs to radiation therapy by enhancing G2/M arrest and by inducing apoptosis. Colony-formation assay demonstrated that volasertib plus IR synergistically inhibited colony formation. In intracranial xenograft mouse models, the combination of volasertib and radiation significantly inhibited GSC tumor growth and prolonged median survival compared with radiation treatment alone due to inhibition of cell proliferation, enhancement of DNA damage, and induction of apoptosis. CONCLUSIONS: Our results reinforce the potential therapeutic efficacy of volasertib in combination with radiation for the treatment of GBM. METHODS: We used high-throughput screening (HTS) to identify drugs, out of 357 compounds in the published Protein Kinase Inhibitor Set, with the greatest efficacy against a panel of glioma stem cells (GSCs), which are representative of the classic cancer genome atlas (TCGA) molecular subtypes.

Xenotransplantation of pediatric low grade gliomas confirms the enrichment of BRAF V600E mutation and preservation of CDKN2A deletion in a novel orthotopic xenograft mouse model of progressive pleomorphic xanthoastrocytoma.

To identify cellular and molecular changes that driver pediatric low grade glioma (PLGG) progression, we analyzed putative cancer stem cells (CSCs) and evaluated key biological changes in a novel and progressive patient-derived orthotopic xenograft (PDOX) mouse model. Flow cytometric analysis of 22 PLGGs detected CD133+ (<1.5%) and CD15+ (20.7 ± 28.9%) cells, and direct intra-cranial implantation of 25 PLGGs led to the development of 1 PDOX model from a grade II pleomorphic xanthoastrocytoma (PXA). While CSC levels did not correlate with patient tumor progression, neurosphere formation and in vivo tumorigenicity, the PDOX model, IC-3635PXA, reproduced key histological features of the original tumor. Similar to the patient tumor that progressed and recurred, IC-3635PXA also progressed during serial in vivo subtransplantations (4 passages), exhibiting increased tumor take rate, elevated proliferation, loss of mature glial marker (GFAP), accumulation of GFAP-/Vimentin+ cells, enhanced local invasion, distant perivascular migration, and prominent reactive gliosis in normal mouse brains. Molecularly, xenograft cells with homozygous deletion of CDKN2A shifted from disomy chromosome 9 to trisomy chromosome 9; and BRAF V600E mutation allele frequency increased (from 28% in patient tumor to 67% in passage III xenografts). In vitro drug screening identified 2/7 BRAF V600E inhibitors and 2/9 BRAF inhibitors that suppressed cell proliferation. In summary, we showed that PLGG tumorigenicity was low despite the presence of putative CSCs, and our data supported GFAP-/Vimentin+ cells, CDKN2A homozygous deletion in trisomy chromosome 9 cells, and BRAF V600E mutation as candidate drivers of tumor progression in the PXA xenografts.

Studies on the metabolism and biological activity of the epimers of sulindac.

Sulindac is a nonsteroidal, anti-inflammatory drug (NSAID) that has also been studied for its anticancer activity. Recent studies suggest that sulindac and its metabolites act by sensitizing cancer cells to oxidizing agents and drugs that affect mitochondrial function, resulting in the production of reactive oxygen species and death by apoptosis. In contrast, normal cells are not killed under these conditions and, in some instances, are protected against oxidative stress. Sulindac has a methyl sulfoxide moiety with a chiral center and was used in all of the previous studies as a mixture of the R- and S-epimers. Because epimers of a compound can have very different chemical and biological properties, we have separated the R- and S-epimers of sulindac, studied their individual metabolism, and performed preliminary experiments on their effect on normal and lung cancer cells exposed to oxidative stress. Previous results had indicated that the reduction of (S)-sulindac to sulindac sulfide, the active NSAID, was catalyzed by methionine sulfoxide reductase (Msr) A. In the present study, we purified an enzyme that reduces (R)-sulindac and resembles MsrB in its substrate specificity. The oxidation of both epimers to sulindac sulfone is catalyzed primarily by the microsomal cytochrome P450 (P450) system, and the individual enzymes responsible have been identified. (S)-Sulindac increases the activity of the P450 system better than (R)-sulindac, but both epimers increase primarily the enzymes that oxidize (R)-sulindac. Both epimers can protect normal lung cells against oxidative damage and enhance the killing of lung cancer cells exposed to oxidative stress.

A high-throughput screening compatible assay for activators and inhibitors of methionine sulfoxide reductase A.

The methionine sulfoxide reductase (Msr) system has been shown to play an important role in protecting cells against oxidative damage. This family of enzymes can repair damage to proteins resulting from the oxidation of methionine residues to methionine sulfoxide, caused by reactive oxygen species. Previous genetic studies in animals have shown that increased levels of methionine sulfoxide reductase enzyme A (MsrA), an important member of the Msr family, can protect cells against oxidative damage and increase life span. A high-throughput screening (HTS) compatible assay has been developed to search for both activators and inhibitors of MsrA. The assay involves a coupled reaction in which the oxidation of NADPH is measured by either spectrophotometric or fluorometric analysis. Previous studies had shown that MsrA has a broad substrate specificity and can reduce a variety of methyl sulfoxide compounds, including dimethylsulfoxide (DMSO). Since the chemicals in the screening library are dissolved in DMSO, which would compete with any of the standard substrates used for the determination of MsrA activity, an assay has been developed that uses the DMSO that is the solvent for the compounds in the library as the substrate for the MsrA enzyme. A specific activator of MsrA could have important therapeutic value for diseases that involve oxidative damage, especially age-related diseases, whereas a specific inhibitor of MsrA would have value for a variety of research studies.

Selenocompounds can serve as oxidoreductants with the methionine sulfoxide reductase enzymes.

In a recent study on the reducing requirement for the methionine sulfoxide reductases (Msr) (Sagher, D., Brunell, D., Hejtmancik, J. F., Kantorow, M., Brot, N. & Weissbach, H. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 8656-8661), we have shown that thioredoxin, although an excellent reducing system for Escherichia coli MsrA and MsrB and bovine MsrA, is not an efficient reducing agent for either human MsrB2 (hMsrB2) or human MsrB3 (hMsrB3). In a search for another reducing agent for hMsrB2 and hMsrB3, it was recently found that thionein, the reduced, metal-free form of metallothionein, could function as a reducing system for hMsrB3, with weaker activity using hMsrB2. In the present study, we provide evidence that some selenium compounds are potent reducing agents for both hMsrB2 and hMsrB3.

Thionein can serve as a reducing agent for the methionine sulfoxide reductases.

It has been generally accepted, primarily from studies on methionine sulfoxide reductase (Msr) A, that the biological reducing agent for the members of the Msr family is reduced thioredoxin (Trx), although high levels of DTT can be used as the reductant in vitro. Preliminary experiments using both human recombinant MsrB2 (hMsrB2) and MsrB3 (hMsrB3) showed that although DTT can function in vitro as the reducing agent, Trx works very poorly, prompting a more careful comparison of the ability of DTT and Trx to function as reducing agents with the various members of the Msr family. Escherichia coli MsrA and MsrB and bovine MsrA efficiently use either Trx or DTT as reducing agents. In contrast, hMsrB2 and hMsrB3 show <10% of the activity with Trx as compared with DTT, raising the possibility that, in animal cells, Trx may not be the direct hydrogen donor or that there may be a Trx-independent reducing system required for MsrB2 and MsrB3 activity. A heat-stable protein has been detected in bovine liver that, in the presence of EDTA, can support the Msr reaction in the absence of either Trx or DTT. This protein has been identified as a zinc-containing metallothionein (Zn-MT). The results indicate that thionein (T), which is formed when the zinc is removed from Zn-MT, can function as a reducing system for the Msr proteins because of its high content of cysteine residues and that Trx can reduce oxidized T.